Advances in Applied Microbiology, Volume 40

ADVANCES IN

Applied Microbiology VOLUME 40

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ADVANCES IN

Applied Microbiology Edited by

SAUL NEIDLEMAN Vacaville, California

ALLEN I. LASKIN Somerset, New Jersey

VOLUME 40

Academic Press San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper.

@

Copyright 0 1995 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495

United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW 1 7DX International Standard Serial Number: 0065-2 164 International Standard Book Number: 0-12-002640-6 PRINTED IN THE UNITED STATES OF AMERICA 95 96 9 7 9 8 99 0 0 B B 9 8 7 6

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CONTENTS

Microbial Cellulases: Protein Architecture. Molecular Properties. and Biosynthesis

AJAYSINGH AND KIYOSHIHAYASHI I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Microbial Sources of Cellulolytic Enzymes ........................... I11. Protein Architecture and Molecular Properties ........................

IV. Structural and Catalytic Properties .................................. V Cellulase Biosynthesis ............................................. VI . Future Prospects .................................................. References., ......................................................

.

1 2 6 15 26 34 35

Factors Inhibiting and Stimulating Bacterial Growth in Milk: An Historical Perspective

D . K . O’TOOLE I . Introduction ...................................................... I1. Early Developments ............................................... 111. Lactenin .......................................... ........... IV. Studies with Lactic Acid Bacteria ................................... V. Effect of Cow Ration on Milk as a Growth Medium for Bacteria . . . . . . . . . VI . Role of Inhibitors in Milk Quality ................................... VII. Stimulators of the Growth of Lactic Acid Bacteria ..................... VIII . Growth Inhibitors of Other Bacteria .................................. IX. Applications of the Antimicrobial Systems in Milk .................... X . Conclusion .......................... Appendix A ......................... Appendix B ......................... ........................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

45 47 48 53 69 71 72 77 81 82 83 84 85

Challenges in Commercial Biotechnology . Part I. Product. Process. and Market Discovery

ALE^

PROKOP

I . Introduction

...................................................... .................................. I11. Primary Market Classification ....................................... I1. Biocatalysts: Sources for Discovery

V

95 108 113

vi

CONTENTS

IV. Pathway to Commercialization: Discovery and Development (D & D) Cycle ..................................................... V. Product Discovery ................................................. VI . Process Discovery ................................................. VII. Market Discovery ................................................. VIII . Interactions of Discovery Aspects within the D & D Cycle . . . . . . . . . . . . . . References........................................................

114 115 141 148 149 152

Challenges in Commercial Biotechnology. Part II. Product. Process. and Market Development ALE; PROKOP

I . Introduction

......................................................

I1. Product Development ..............................................

111. Process Development and Scale-Up .................................. IV. Market Development ............................................... V. Outlook .......................................................... References........................................................

155 156 173 209 227 233

Effects of Genetically Engineered Microorganisms on Microbial Populations and Processes in Natural Habitats JACK

D. DOYLE.GUENTHER STOTZKY. GWENDOLYN MCCLUNG. AND CHARLES W . HENDRICKS

I . Introduction ...................................................... I1 Aquatic Environments ............................................. I11 Activated Sludge .................................................. IV Soil V. Plants ........................................................... VI Discussion ....................................................... References ........................................................

. . . .

.............................................................

237 245 248 249 261 267 280

Detection. Isolation. and Stability of Megaplasmid-Encoded Chloroaromatic Herbicide-Degrading Genes within Pseudomonas Species

DOUGLASJ . CORK AND AMJADKHALIL I . Introduction ...................................................... I1. Selected Model Haloaromatic and Aromatic Compounds of Agricultural and Industrial Importance ............................... I11. Detection and Isolation of Large Plasmids from Pseudomonos spp . . . . . . .

289 290 301

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IV. Effect of Alternative Carbon Sources on the Stability and Curing of a Large Molecular Weight Plasmid. pDKl .......................... V . Summary ........................................................ References........................................................

308

INDEX.................................................................

323

CONTENTSOFPREVIOUSVOLUMES .........................................

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Microbial CelI ulases: Protein Arch itect ure, Molecular Properties, and Biosynthesis AJAYSINGH AND KIYOSHIHAYASHI Biomaterials Conversion Laboratory National Food Research lnstitute Tsukuba, Ibaraki 305, Japan

I. Introduction 11. Microbial Sources of Cellulolytic Enzymes

111.

IV.

V.

V1.

A. Fungi B. Bacteria Protein Architecture and Molecular Properties A. Cellobiohydrolases B. Endoglucanases C. P-Glucosidases Structural and Catalytic Properties A. Structural Organization B. Glycosylation C. Synergistic Mechanism D. Catalytic Mechanism Cellulase Biosynthesis A. Induction and Regulation B. Localization C. Biosynthesis in Recombinant Cells Future Prospects References

I. Introduction

Cellulose is the most abundant carbohydrate polymer in the biosphere. An estimated synthesis rate of cellulose is approximately 4 X lo7tons per year. For a long-range solution to our resource problems of energy, chemicals, and food, cellulose is the most promising renewable carbon source that is available in large quantities. Today, the need for oil substitutes appears to be acute mainly because of an increase in oil prices and a higher risk of shortage. Enzymatic hydrolysis of cellulose has been widely studied during the last 20 years as a way to provide fermentable sugars, which, in turn, could be converted into various value-added chemicals and fuels. However, because of the physical nature of the cellulose molecule and the fact that its natural occurrence is always accompanied by associated materials, hemicellulose and lignin, the efficiency of cellulolytic enzymes deserves improvement in most cases. 1 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 40 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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AJAY SINGH AND KIYOSHI HAYASHI

Enzymatic hydrolysis of cellulose, although not yet commercially attractive, is expected to be feasible in the long run, as has been the case with starch hydrolysis (Bisaria and Mishra, 1989). The inherent complexity of cellulolytic enzyme mixtures, in terms of the number of enzyme components and their specificity, has caused difficulty in our understanding of the cellulase enzyme system. Considerable progress in our understanding of the role of various enzyme components in cellulose degradation has recently been provided by studies involving the powerful tools of molecular biology, genetic engineering techniques, monoclonal antibodies, and protein chemistry (BBguin, 1990; Kubicek, 1992). In recent years, recombinant DNA technology has provided a means for isolating, characterizing, and manipulating the genes for a large number of different proteins (Glick and Pasternak, 1989). Therefore, the use of r-DNA technology applied to the cellulase system will facilitate not only a better understanding of catalytic functioning, cooperative interactions between different enzyme components, and regulation of these enzymes, but also the development of practical systems for the utilization of native cellulose. The cellulase-cellulose system and the importance of cellulose bioconversion research have already been subjects of detailed studies including cellulose structure and pretreatment (Chang et al., 1981; Young and Rowell, 1986; Fan et al., 1982; Lipinsky, 1979; Ghosh and Singh, 1993),cellulose bioconversion (Mandels, 1982; Bisaria and Ghose, 1980; Klyosov, 1986; Singh and Kumar, 1991; Singh et al., 1992a; Mishra and Singh, 1993), cellulase purification and properties (Ghose, 1987; Wood, 1985; Enari and Niku-Paavola, 1987; Merivuori et al., 1985a; Gong et al., 1979), cellulase production and applications (Lee et al., 1980; Fan et al., 1987; Ryu and Mandels, 1980; Ladisch et al., 1983; Mandels, 1985; Lachke and Deshpande, 1988; Srinivasan and Seeta Laxman, 1988; Whitaker, 1990; Vallander and Eriksson, 1990), and the genetics of cellulolytic microorganisms (Durand et al., 1988; Orpin, 1988; Shoemaker et al., 1981; Knowles et al., 1988; Pentilla et a]., 1988; Pasternak and Glick, 1987; Kuhad and Singh, 1993a). Recent advances in the nature, protein architecture, and regulation of biosynthesis and secretion of the cellulase enzyme system will be discussed in the present article. II. Microbial Sources of Cellulolytic Enzymes

Cellulases are synthesized by a large number of microorganisms (Table I). These organisms have in common the production of extracellular hydrolytic enzymes that attack the cellulose polymer. However, rela-

MICROBIAL CELLULASES

3

TABLE I WITH RESPECT TO MOSTWIDELY STUDIED MICROORGANISMS CELLULOLYTIC POTENTIAL

Fungi

Bacteria

White-rot Phanerochaete chrysosporiurn Coriolus versicolor Brown-rot Poria placenta Lanzitus trabeurn Soft-rot Trichoderma reesei Penicillium funiculosum Fusariurn oxysporum Aspergillus niger Anaerobic Neocalimastix frontalis

Aerobic Cellulomonas fimi Pseudomonas fluorescens Bacilus subtilis Cellvibrio gilvus Cytophaga hutchinsonii Anaerobic Clostridiurn thermocellurn Acetovibrio cellulolyticus Ruminococcus albus Actinomycetes Therrnoactinomyces curvata Streptornyces flavogriseus

tively few microorganisms are able to produce the necessary group of enzymes for degradation of crystalline cellulose (Mandels and Weber, 1969).

A. FUNGI Fungi are the most studied organisms with respect to cellulose degradation and cellulase production. Fungi producing the necessary enzymes for the cell-free degradation of crystalline cellulose generally belong to the Ascomycetes and Deuteromycetes groups or to the whiterot Basidiomycetes (Eriksson and Johnsrud, 1982). Brown-rot Basidiomycetes also degrade cellulose but they seem to lack exo-1,4-Pglucanases (Ljungdahl and Eriksson, 1985). The white-rot group of fungi is rather heterogenous; however, they have in common the capability to degrade lignin as well as other lignocellulose components (Eriksson, 1981).The most studied white-rot fungus was first isolated from wood chip piles and was given the name Chrysosporium lignorum (Bergman and Nilsson, 1966). The name was later changed to Sporotrichum pulverulentum for its imperfect stage (von Hofsten and von Hofsten, 1974) and then to Phanerochaete chrysosporium (Burdsall and Eslyn, 1974) for its perfect stage. The fungus is characterized as thermotolerant and has an optimum growth rate around 38" C (Eriksson and Pettersson, 1975a). It produces five

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AJAY SINGH AND KIYOSHI HAYASHI

endo-l,4-/3-glucanases (EC 3.2.1.4), one exo-1,4-/3-glucanase (EC 3.2.1.91), and two 1.4-/3-glucosidases (EC 3.2.1.21) (Eriksson and Pettersson, 1975b; Deshpande et al., 1978). Brown-rot fungi, which rapidly depolymerize cellulose, seem to utilize a mechanism of cellulose degradation different from that operating in P. chrysosporium. Brown-rot fungi apparently lack the synergistic endo-exo-glucanase cooperation to degrade crystalline cellulose because they lack exo-1,4-/3-glucanase activity (Highley, 1975, 1980, 1987a,b; Highley and Murmanis, 1985). Among brown-rot fungi, Poria placenta, Tyromyces palustris, and Lenzites trabeum are the best known examples of cellulolytic organisms (Eriksson et a]., 1990). Soft-rot fungi have the capability to deplete both polysaccharides and lignin; however, the polysaccharides are the main targets for this group. The ability of soft-rot fungi to produce all the necessary extracellular enzymes for degradation of crystalline cellulose varies. The best known of those producing a complete enzyme system is Trichoderma reesei (Mandels and Reese, 1964; Mandels, 1975; Gritzali and Brown, 1979; Fagerstam and Pettersson, 1980; Bhikhabhai et al., 1984). Trichoderma koningii (Wood and Phillips, 1969; Wood and McCrae, 1972, 1975, 1986a; Fan et a]., 1987), Penicillium funiculosum (Wood et al., 1980; Wood and McCrae, 1982, 1986b,c), Penicillium citrinum (Kuhad and Singh, 1993b), Fusarium solani (Wood, 1969, 1971, 1975; Wood and McCrae, 1977), Fusarium oxysporum (Kumar et al., 1991, 1992; Singh et al., 1992a,b; Christakopoulos et al., 1989, 1990), and Aspergillus niger (Singh et al., 1988a,b, 1989a,b, 1990a,b,c, 1991a,b) are other well known examples of cellulolytic soft-rot fungi. Trichoderma reesei secretes large amounts of different cellulases that solubilize crystalline cellulose efficiently by their combined action. It produces at least three endo-1,4-p-glucanases (EG I-111), two exo-l,4-/3-glucanases (cellobiohydrolases, CBH-I and CBH-11), and 1,4-P-glucosidases (Stahlberg et al., 1988; Teeri et al., 1987; Enari and Niku-Paavola, 1987; Enari et al., 1981).

The anaerobic rumen-inhabiting fungi also have the capability to degrade cellulose to glucose. The fermentation of cellulose by a rumen anaerobic fungus results in the formation of acetate. CO,, formate, acetate, lactate, and hydrogen (Orpin, 1977; Bauchop, 1981). The cellulolytic enzyme system in one of the rumen anaerobic fungi, Neocalimastix frontalis, has been studied by several groups (Pearce and Bauchop, 1985; Wood et al., 1986). The major digestion product by the action of N. frontalis cellulase on Avicel (microcrystalline cellulose) is glucose and not cellobiose (Pearce and Bauchop, 1985).This suggests that excess P-glucosidase is produced relative to cellulase by N. frontalis. This

MICROBIAL CELLULASES

5

contrasts with the situation in T. reesei, which produces only small quantities of P-glucosidase (Mandels, 1982). Wood et al. (1986) isolated an extracellular enzyme system from N. frontalis that was several fold more active in solubilizing cotton fibres per unit of endo-l,4-P-glucanase than the cellulase of T. reesei mutant strain Rut C-30, one of the most active cellulases.

B. BACTERIA

Degradation of cellulose by bacterial systems occurs both aerobically and anaerobically. The cellulase systems of bacteria are not directly comparable to those of fungi. Bacterial degradation of cellulose seems to take place by a cluster of enzymes rather than by individual endoand exoglucanases as in the case of fungi (Ljungdahl and Eriksson, 1985). Many bacteria lack 1,4-P-glucosidases and employ phosphorylases for the degradation of cellobiose. Cellulases of aerobic bacteria, such as the Cellulomonas species, appear to be tightly bound to their cellulose substrates and appear in the culture medium only in the stationary phase (B6guin et al., 1977). Langsford et al. (1984) reported up to 10 components with carboxymethyl cellase (CMCase)activity as determined by nondenaturing polyacrylamide gel electrophoresis (PAGE). Various cellulolytic species of Bacillus, such as B. subtilis, B. polymyxa, B. brevis, B. licheniformis, and B. cereus, have been described (Priest, 1977; Knosel, 1971; Robson and Chambliss, 1984, 1986, 1987; Thayer, 1978). All of these species are able to hydrolyze carboxymethyl cellulose with cellobiose as the main product. The cellulolytic Pseudomonas species has been studied by Ueda et al. (1952) and the best-studied strain was Pseudomonas fluorescens var. cellulosa (Yamane et al., 1970; Gilbert et al., 1987). Cellvibrio gilvus, which grows well on cellulose, cellobiose, and other cellodextrins, has a complex extracellular cellulase system similar to P. fluorescens. Degradation products are chiefly cellobiose and cellotriose (Storvick et a]., 1963; Alaexander, 1968; Sasaki et al., 1983; Blackall et al., 1985; Wynn and Pemberton, 1986). Actinomycetes form an important part of the microbial community responsible for lignocellulose degradation. Generally, actinomycetes cellulases are inducible extracellular enzymes, which appear to attack cellulose in a way similar to fungal cellulases (Stutzenberger and Kahler, 1986; Stutzenberger and Lupo, 1986; Hagerdal et a]., 1978; Calza et a]., 1985; Bartley et al., 1984; MacKenzie et al., 1984, 1987; MacKenzie, 1986). The thermophillic species of Thermomonspora and Thermoacti-

6

AJAY SINGH AND KIYOSHI HAYASHI

nomyces and the mesophilic species of Streptomyces are the most studied organisms. The cellulolytic system of anaerobic bacteria has gained considerable interest mainly because of the possibility of use in industrial processes. Clostridium thermocellum produces a multicomponent cellulolytic complex termed “cellulosome” (Lamed et al., 1983a,b; Lamed and Bayer, 1988).In young culture of C. thermocellum, cellulosome is tightly bound to the substrate. After the consumption of cellulose, the bound enzymes are released in to the culture medium (Lamed et al., 1985; Cornet et al., 1983). Several endo-l,4-/3-glucanases with M, values ranging from 56 to 125 kDa have been purified from this organism (Wu and Demain, 1988; Ng and Zeikus, 1981). Several other anaerobic bacteria including Acetovibrio cellulolyticus (MacKenzie et al., 1987; Saddler and Khan, 1981), Ruminococcus flavefaciens (Pettipher and Latham, 1979; Latham et al., 1978), Ruminococcus albus (Wood and Wilson, 1984; Stack and Cotta, 1986), and Bacteriodes succinogens (Groleau and Forsberg, 1983) have also been studied with respect to cellulose degradation and cellulase production. Ill. Protein Architecture and Molecular Properties

Cellulose is a homopolymer of about 8000-12,000 glucose units linked linearly by /3-1,4-glucosidic linkages, forming a crystalline unit held together by hydrogen bonding (Brown, 1982). Based on this structure, Reese et al. (1950) postulated the involvement of two different types of enzymes in the degradation of natural cellulose. According to this hypothesis, a C, enzyme renders the crystalline cellulose accessible for hydrolytic attack and a C, enzyme then hydrolyzes the modified cellulose. However, evidence for the existence of the C, enzyme is still obscure. The cellulolytic enzymes have been defined as the enzymes hydrolyzing cellulose, thereby yielding soluble sugars small enough to pass through microbial cell walls (Eriksson, 1967). While considerable progress has been made in the understanding of the nature and function of cellulolytic enzymes, a modification of this definition seems necessary today. In addition to the hydrolytic enzymes, oxidative and phosphorolytic enzymes also participate in cellulose degradation in some microorganisms. There are at least three major types of hydrolytic enzymes involved in the degradation of native cellulose to glucose (Mandels and Weber, 1969). 1. Endoglucanases (endo-l,4-/3-~-glucan-4-glucanohydrolase, EC 3.2.1.4) that randomly attack the cellulose chain and split B-1,4glucosidic linkages.

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MICROBIAL CELLULASES

2. Exoglucanases, present as cellobiohydrolases (exo-l,4-/3-~-glucan 4-cellobiohydrolase, EC 3.2.1.91) that release either cellobiose or glucose from the nonreducing end of cellulose. 3. P-Glucosidase or cellobiase (EC 3.2.1.21) that hydrolyzes cellobiose and other water soluble cellodextrins to glucose.

Phanerochaete chrysosporium produces two types of oxidative enzymes, cellobiose : quinone oxidoreducatase (cellobiose dehydrogenase, EC 1.1.5.1) and cellobiose oxidase for oxidation of cellobiose (Westermark and Eriksson, 1974b; Ayers et a]., 1978; Dekker, 1980; Coudray et al., 1982; Morpeth and Jones, 1986). Some aerobic and anaerobic bacteria produce phosphorylases that are also involved in cellulose degradation. A wide variety of substrates (Wood, 1988) have been used to study the specificity of cellulolytic enzymes (Table 11). The most commonly used substrates have been cotton fiber, Avicel (microcrystalline cellulose), phosphoric acid-swollen cellulose, carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), cellobiose, p-nitro-phenyl-P-Dglucoside, and cellodextrins. Properties of individual enzymes are discussed in the following sections. A. CELLOBIOHYDROLASES Cellobiohydrolases degrade cellulose by hydrolyzing cellobiose units from the nonreducing end of the chain and have very limited action on substituted celluloses such as CMC and HEC (Bisaria and Mishra, 1989). They are very active on swollen, partially degraded, amorphous TABLE I1 ASSAYOF CELLULOLYTIC ENZYMESINVOLVEDIN CELLULOSE DEGRADATION Enzyme Endo-l,4-P-glucanase Exo-l,4-P-glucanase

1,4-p-Glucosidase Cellobiose: quinone oxidoreductase Cellobiose oxidase

Substrate Cellulose derivatives (CMC, HEC), cellodextrins Amorphous cellulose, cellodextrins, pnitrophenyl-P-cellobioside, p-nitrophenyl-P-lactoside p-Nitrophenyl-P-D-glucoside, cellobiose, cellodextrins 2,6-Dichlorophenol indophenol (DCIP), cellobiose Cellobiose, cellodextrins

Product Reducing sugars Reducing sugars,cellobiose pnitrophenol p-Nitrophenol, glucose reducing sugars Decrease in absorbance at specific wave length Onic acid

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AJAY SINGH AND KIYOSHI HAYASHI

cellulose; however, oh soluble cello-oligosaccharides, cellotriose to cellohexose, their activity decreases with small chain length. They are not very active against crystalline cellulose but exhibit highly cooperative synergistic action in the presence of endoglucanases (Enari and NikuPaavola, 1987; Bisaria and Mishra, 1989).Avicel, with a degree of polymerization (DP) of about 200 has more chain ends accessible to the enzyme and is degraded more rapidly than the cotton fiber [DP 10,000). Cellulases are characterized by a multiplicity of enzyme components whose exact number varies from one organism to another. Heterogeneity of the cellobiohydrolase component from T. reesei and Penicillium pinophilum has been studied in detail [Wood et al., 1988; Wood and McCrae, 1986a). Two immunologically distinct cellobiohydrolases, CBH I and CBH 11, have been detected in the extracellular medium of T. reesei using polyclonal antibodies (Fagerstam and Pettersson, 1979). Both are glycoproteins differing in the amount of carbohydrate attached to the protein and also in amino acid composition (Enari and NikuPaavola, 1987; Wood et al., 1988). The nature and origin of these isoenzymic forms are not yet completely understood. However, these differences could be due to genetic factors, partial proteolysis, or differential glycosylation (Coughlan, 1985). CBH I composes the major part [about 60%) of the cellulolytic enzyme mixture synthesized by T. reesei. Its gene has been cloned and sequenced [Teeri et al., 1983; Chen et al., 1990). It codes for a polypeptide with a corresponding M, of 58 kDa. When two different mutants, T. reesei L27 and T. reesei QM 9414, were compared, the data for gene sequence were consistent with the primary structure of CBH I protein [Fagerstam et al., 1984). Purification of CBH I has been attempted by several researchers (Nummi et al., 1983; Beldman et al., 1985; Okada eta]., 1986; Hostomska and Mikos, 1984; Odegaard et al., 1984).Purified CBH I has been found to exhibit pronounced heterogeneity (Gum and Brown, 1977), which has been specified as 0- and N-linked glycosylation (Salovuori et al., 1987; Perera et al., 1990). Purification data also indicate considerable differences in the M, of the purified proteins. However, relative proportions of amino acids present [Table 111) in these proteins are quite comparable (Gum and Brown, 1977; Shoemaker et al., 1983; Odegaard et al., 1984). Molecular weight and carbohydrate content of cellulases and P-glucosidase from T. reesei are presented in Table IV. The second cellobiohydrolase, CBH 11, which splits cellobiose from cellulose, has also been purified and characterized (Fagerstam and Pettersson, 1979). Its physicochemical properties, such as the isoelectric point and M,, are similar to those of EG I, a form of endoglucanase.

9

MICROBIAL CELLULASES TABLE 111 AMINOACIDCOMPOSITION OF CELLULASE COMPONENTS FROM Trichoderma reesei CBH I Amino acid Ala Arg Asx CYS Glx GlY His Ile Leu LYS Met Phe Pro Ser Thr TYr TrP Val

EG I

EG 111

A

B

C

D

E

D

5.8 1.8 11.3 4.8 8.2 12.5 1.0 2.4 5.6 2.6 1.2 3.0 5.6 11.3 11.5 4.8 1.8 4.6

7.1 2.1 12.4 4.7 10.0 15.1 1.0 2.1 6.1 2.9 1.5 3.4 5.0 10.8 12.7 5.0 2.4 5.3

5.9 1.8 12.7 4.7 7.0 11.0 2.0 2.9 5.4 2.1 2.2 2.2 5.6 13.5 10.3 5.3 1.6 4.5

7.3 1.4 12.7 3.3 6.6 10.7 1.2 3.8 5.7 1.6 1.2 2.1 7.6 11.8 11.1 4.0 2.6 5.2

7.0 2.5 12.3 3.0 8.0 10.6 1.2 5.3 5.8 1.5 1.0 3.2 4.7 10.5 11.0 3.5 2.8 5.5

8.0 2.8 12.3 3.0 8.5 11.8 1.5 4.8 5.2 1.7 0.7 3.0 5.8 9.6 10.0 3.3 2.5 5.3

Note. Values of amino acids are given as percentage of total residues. Reference A (Bhikhabhai et al., 1984),B (Odegaardet al., 1984),C (Shoemakeret al., 1983),D (Shoemakerand Brown, 1978),E (Saloheimo et al., 1988).

Thus, a methodological problem has long hindered the purification and characterization of this enzyme. This problem has been solved by using affinity chromatography on thiocellobiose coupled to Affigel (van TilTABLE IV MOLECULAR WEIGHT AND CARBOHYDRATE CONTENT OF CELLULASES AND p-GLUCOSIDASES FROM Trichoderma Reesei Enzyme CBH I EG I EG 111 p-Glucosidase

Mr ( m a 1

Carbohydrate (%]

64.0 50.3 55.0 44.7 34.5 42.2 76.6 79.7

6.0 7.2 11.0 12.0 15.0 18.0 0 1.3

Reference Bhikhabhai et al. (1984) Odegaard et al. (1984) Shoemaker et 01. (1983) Hakansson et al. (1979) Odegaard et al. (1984) Salovuori et al. (1987) Odegaard et al. (1984) Chirico and Brown (1987)

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AJAY SINGH AND KIYOSHI HAYASHI

beurgh et al., 1984) or immunoadsorption (Niku-Paavola et al., 1986). Its gene, cbh2, has also been cloned and sequenced and codes for a 471-amino acid protein whose composition is identical to that of purified CBH I1 (Bhikhabhai et al., 1984; Teeri et al., 1987; Chen et al., 1987). CBH I and CBH I1 lack any apparent homology in their amino acid sequences (Chanzy et al., 1983; Teeri et al., 1987), and while CBH I binds preferentially to crystalline regions, CBH I1 binds to both the crystalline and amorphous regions of cellulose. This reflects the differences in the active surfaces of the enzymes. Although CBH I and CBH I1 from T. reesei QM9414 show some common features, such as binding to the same affinity column and high activity toward microcrystalline cellulose (van Tilbeurgh et a]., 1984; Fagerstam and Pettersson, 1980), the modes of action of the two enzymes seem to differ considerably. The reaction patterns toward 4-methylumbellyferyl-~-~-glucoside, MeUmb(Glc),, and MeUmB(Glc),, are totally different from the two enzymes. With MeUmb(Glc), as substrate no MeUmb(Glc), could be observed as a reaction product from CBH I hydrolysis (van Tilbeurgh et al., 1982, 1986),while it is the main product from CBH I1 hydrolysis. Further studies are needed to reveal the nature and functions of CBH I and CBH 11. Characterization studies require pure enzymes because of strong synergistic interactions between different types of cellulases, and even traces of activities other than those expected can obscure the results.

B. ENDOGLUCANASES Endoglucanases (CMCases)hydrolyze CMC or swollen-cellulose in a random fashion; as a result, there is a rapid decrease in chain length and a slow increase in reducing groups. They also act on cellodextrins; their action on cellodextrins decreases as the chain length decreases (Wood, 1985; Bisaria and Mishra, 1989). Three different endoglucanases, i.e., EG I, EG 11, and EG 111, are produced by T. reesei, which also occur in multiple forms (Biely et al., 1985; Kammel and Kubicek, 1985; Labudova and Farkas, 1983; Messner et al., 1988; Hrmova et al., 1986). However, there are different views regarding the classification, nature, and functions of various isoenzymic forms of endoglucanases because of controversies in the values of M,, and carbohydrate contents of different enzymes present in any one cellulase system (Bbguin, 1990). The major endoglucanase component, EG I (previously known as Endo-11; Hakansson et al., 1979), represents approximately 5-10% of the secreted proteins in T. reesei QM9414 cultures. The enzyme has

MICROBIAL CELLULASES

11

been isolated and characterized in several laboratories (Henrissat et al., 1985; Bhikhabhai et al., 1984; Niku-Paavola et al., 1985; Penttila et al., 1986; Saloheimo et al., 1988; Nieves et al., 1990; Luderer et al., 1991). The M, of EG I glycoprotein has been reported to be from 43 to 55 kDa by different researchers (Henrissat et al., 1985; Niku-Paavola et al., 1985). The nature of carbohydrate residue is still unclear. According to the amino acid (aa)sequence, six putative N-glycosylation sites would be present (at aa 78,164,204,208,281,and 394). The size of glycosylated fragments was not compatible with the usual (Man) ,(GlcNAc), or (Man),(GlcNAc), structure (Salovuori et al., 1987). It has also been reported that 70% of EG I carbohydrate was 0-glycosidically linked (Salovuori, 1987), which indicates less occupation of the resulting Nglycosylation sites in EG I. The N-terminal sequence of EG I indicates considerable sequence homology between EG I and CBH I in spite of the clear differences in substrate specificity (Bhikhabhai and Pettersson, 1984). Since the enzyme is comparatively resistant to proteolysis (Luderer et al., 1991) and enzymatic deglycosylation (Kubicek et al., 1987), postsecretional modification appears to contribute little to the high number of multiple forms of endoglucanases (Sheir-Neiss and Montenecourt, 1984; Farkas et al., 1982; Biely and Markovic, 1988; Hayn and Esterbauer, 1985).However, Hakansson et al. (1979) found no detectable glycosylation of purified 50-kDa EG I, thus pointing to a possible postsecretional modication. A second endoglucanase, EG 111, has also been characterized by several researchers (Saloheimo et al., 1988; Beldman et al., 1985; Bhikhabhai et al., 1984; Stahlberg et al., 1988). Multiple forms of EG I11 have been purified exhibiting M, values of 48, 48, and 37 kDa with corresponding PIS of 5.4, 5.7, and 4.8 (Stahlberg et al., 1988; Saloheimo et al., 1988).Two other proteins exhibited amino acid composition closely resembling those reported for EG I1 and EG IV (Shoemaker and Brown, 1978). The nature of third EG 111 (M, 48 kDa, PI 5.1) has not yet been identified. However, a fingerprint very similar to that of EG I11 (M, 48 kDa, PI 5.7) has been obtained using CNBr-peptide mapping (Kubicek, 1992). This indicates that it is a very closely related subspecies. A number of other endoglucanases have also been isolated from T. reesei cultures that could not be identified as either EG I or EG 111. Bhikhabhai et al. (1984) purified a 48-kDalpI 4.5 endoglucanase which differs from EG I11 with respect to its amino acid composition and Nterminal sequence. Its carbohydrate content and amino acid composition are sufficiently unique to suggest that this protein is not related to either EG I11 or EG I. It could be the product of an additional EG gene

12

AJAY SINGH AND KIYOSHI HAYASHI

whose isolation is required to characterize this protein. Similarly, NikuPaavola et al. (1985) purified a 43-kDa/pI 4.0 endoglucanase with low (2-7%) carbohydrate content and postulated that it constitutes a proteolytic fragment. Other researchers (Gong et al., 1979; Hakansson et al., 1978; Hong et al., 1986; Ulker and Sprey, 1990) have also purified lowmolecular-weight endoglucanases (20-25 kDa) which lack glycosylation. Their formation appeared to depend strictly on the presence of cellulose in the medium, unlike other cellulases [Messner et al., 1988; Hrmova et al., 1986). However, the nature of these proteins is still unclear, These enzymes are rapidly degraded by fungal extracellular proteases and constitute only a very small portion of the secreted proteins (Kubicek, 1992). Further studies are needed in order to explore the nature of these enzymes. The cellulolytic enzyme system of bacteria seems to be quite complex. Yamane et al. [1970) identified two extracellular (A and B) and one cell-bound (C) endoglucanase components of bacteria. P. fluorescens var. cellulosa, which were purified and characterized. All three of these endoglucanases hydrolyzed a variety of substances, including crystalline and amorphous cellulose and cellodextrins. The mode of action toward CMC appears to be similar to that shown by fungal endoglucanases. Yoshikawa et al. (1974) later reported that components A and B were not homogeneous. In subsequent work by Gilbert et al. (1987) on the molecular biology of the cellulase complex from P. fluorescens, it was shown that four distinct isolated endoglucanase genes exhibited no homology with one another. On the other hand, Wolff et al. (1986) could isolate only one endoglucanse gene from this organism, suggesting that the multiplicity of endoglucanases from this species obtained in earlier studies is a result of post-translational modification of a single gene product. In another widely studied bacterial species, Cellulomonas fimi, Langsford et al. (1984) noted up to 10 components with CMCase (endoglucase) activity as determined by nondenaturing polyacrylamide gel electrophoresis. Some of the active components were glycosylated. The glycosylated enzymes bind strongly to Avicel and are stabilized by this substrate, whereas enzymes free in solution are unstable and are altered by proteolysis and deglycosylation. Studies were focused on the two tightly substrate-bound enzymes designated CB 1 and CB 2 (Gilkes et al., 1984). Both components were glycoproteins with mannose as the sole glycosyl component (BBguin et al., 1987). However, the mode of action and substrate specificities were found to be different. Both hydrolyze CMC but with different specific activities (370 and 85 units/ mg protein for CB 1 and CB 2, respectively). This finding led researchers

MICROBIAL CELLULASES

13

to characterize CB 1 as an endoglucanse and CB 2 as an exoglucanase (Gilkes et al., 1984; Warren et aJ., 1987). There are relatively few reports (Sashihara et al., 1984) of Bacillus sp. producing endoglucanses. The best characterized example is B. subtilis DLG (Robson and Chambliss, 1987). The endoglucanse was confirmed by measuring the ability of the enzyme to reduce the viscosity of a CMC solution, which was similar in fashion to T. reesei EG I. However, the B. subtilis enzyme exhibited almost twice the specific activity of T. reesei EG I when trinitrophenyl CM was used as the substrate. The DNA sequence of B. subtilis DLG endoglucanase gene indicates that the enzyme is coded for a single sequence at the Nterminal end (Robson and Chambliss, 1987). Such a signal is believed to be necessary for an efficient export of protein in most organisms, and is usually composed of a short sequence of charged amino acids followed by a longer stretch of strongly hydrophobic amino acids (Perlman and Halvorson, 1983). The DNA sequence also revealed that the endoglucanase gene of B. subtilis has a coding capacity for an approximately 55-kDa protein. However, the isolated extracellular endoglucanase was only 35.2 kDa. The apparent differnece wasmost likely due to post-translational modification (Robson and Chambliss, 1986). C. P-GLUCOSIDASES The least studied of the enzymes involved in cellulose hydrolysis are P-glucosidases. They represent only a very small portion (about 1%) of the total extracellular proteins secreted by T. reesei. p-Glucosidases hydrolyze degradation products of cellulose to glucose. They have maximum activity toward cellobiose and their action on cellodextrins decreases as chain length increases. p-Glucosidases are characterized by their transferase activity, retention of the P-configuration of glucose on hydrolysis, inhibition by low concentrations of gluconolactone, and high activity toward cellotriose and less activity toward larger cellodextrins (Bisaria and Mishra, 1989). The purification of p-glucosidases from extracellular culture filtrates, intracellular extracts, and plasma membranes of organisms has been reported by several groups (Hofer et al., 1991; Bergham and Pettersson, 1974; Enari et al., 1981; Gong et al., 1977; Chirico and Brown, 1987; Schmid and Wandrey, 1987; Jackson and Talburt, 1988; Wilhelm and Sahm, 1986; Singh et al., 1990d; Umile and Kubicek, 1986). A comparison of the amino acid composition of P-glucosidases from T. reesei is given in Table V. Two immunologically distinct forms (I,Mr 50 m a , pl 6.1; II,Mr 35 kDa, pl 5.7) of extracellular P-glucosidases have been re-

AJAY SINGH AND KIYOSHI HAYASHI

14

TABLE V OF @-GLUCOSIDASES FROM Trichoderma reesei AMINOACIDCOMPOSITION

Amino acid Ala Asx CYS Glx GlY His Ile Leu LYS

Met Phe Pro Ser Thr TYr TrP Val

Bergham and Pettersson (1974) 10.1 3.4 12.5 1.3 6.7 10.8 1.2 4.8 7.1 3.0 1.3 2.6 5.6 8.2 7.1 3.5 2.1 8.2

Odegaard et al. (1984) 10.3 3.3 13.4 0.6 7.2 11.4 1.4 4.3 6.6 2.9 1.0 3.9 7.0 9.5 7.8 3.7

ND 5.1

Chirico and Brown (1987) 10.0 3.0 12.5 0.8 6.9 10.5 1.2 4.3 7.1 3.3 1.0 2.5 5.0 9.6 6.8 3.4 3.0 9.0

Note. Values of amino acids given as percentage of total residues.

ported by Hofer et al. (1991).They provided evidence that partial proteolysis of the 80-kDa P-glucosidase results in the formation of two different protein fragments. It is also very likely that some of the pglucosidases described earlier (Bergham and Pettersson 1974; and Enari et al., 1981) are proteolytic fragments of the 80-kDa P-glucosidase. Intracellular and plasma membrane P-glucosidases have also been identified. However, the exact genetic and biochemical relationship among these different P-glucosidases is not yet clear. It has been suggested that extracellular enzyme may result from the release of intracellular or cell wall-bound enzyme to the outside medium upon autolysis. Although the role and intracellular location of this enzyme are still unclear, it has been proposed to function in controlling the accumulation of cellulase inducers (Inglin et a]., 1980; Loewenberg, 1984). Enari et al. (1981) purified the P-glucosidase of T. reesei that showed an apparent molecular weight of 35 kDa on gel chromatography. The M, calculated from SDS electrophoresis of the purified P-glucosidase was 73 kDa. The discrepancy was most likely due to the glycoprotein nature of the enzyme (0.1 mg sugar/mg protein). Glycoproteins are known to behave abnormally in SDS electrophoresis. Another p-

MICROBIAL CELLULASES

15

glucosidase component of 130 kDa was also observed, but this component represented only 2% of the total p-glucosidase activity. Similar results have been reported for T. viride p-glucosidases (Bergham and Pettersson, 1974; Gong et al., 1977). Species of Aspergillus and Sporotrichum seem to produce pglucosidases of higher molecular weight than does Trichoderma. Purified p-glucosidase from A. niger has a molecular weight of 150 kDa and consists of two polypeptide chains that can be separated by SDS-PAGE (Enari et al., 1981). Aspergillus oryzae has been reported to form an enzyme with M, 218 kDa, which also consists of two polypeptide chains (Mega and Matsushima, 1979). Meier and Canevascini (1981) isolated two different p-glucosidases from S. thermophile, one with M, 440 kDa, having only aryl-P-glucosidase activity] while the other, with M, 40 kDa, having cellobiase activity with only low activity toward arylP-glucosides. Kashiwagi et al. (1991) reported the isolation of a p-glucosidase gene from C. gilvus and the purification and properties of the cloned pglucosidase. The purified enzyme has M, 82 kDa and PI 6.0. It attacked cellobiose very slowly, compared to its activity toward higher cellooligosaccharides. The same research group recently reported the nucleotide sequence of the p-glucosidase gene from C. gilvus (Kashiwagi et al., 1993). The P-glucosidase from C. gilvus was cloned into Escherichia coli and sequenced. The structural gene consisted of 2565 base pairs encoding 854 amino acid residues with a characteristic signal peptide. The p-glucosidase of C. thermocellum was shown to be very heat stable (Ait et al., 1979). The purified enzyme has M, 50 kDa and PI 4.6. However, the affinity of the enzyme for cellobiose and p-nitrophenylP-D-glucoside (PNPG) was very low with a K, value of 2 X lop3M for PNPG and 8.3 x low2M for cellobiose. It was postulated that the pglucosidase has an unspecific action and may participate in hydrolyzing saccharides having other types of p-structure linkages. The cellobiose and cellodextrins produced may instead enter the cell where they are metabolized by the specific cellodextrin and cellobiose phosphorylases to yield glucose-1-phosphate and free glucose as has been demonstrated by Alaexander (1968). IV. Structural and Catalytic Properties

A. STRUCTURAL ORGANIZATION A comparison of the sequence of about 50 fungal and bacterial cellulase genes and of some other polysaccharide hydrolases has offered evidence that enzyme proteins acting on insoluble substrates like cellu-

AJAY SINGH AND KIYOSHI HAYASHI

16

lose are composed of separate domains, which allow a spacial separation of the sites involved in substrate recognition and enzyme activity (Beguin, 1990; Kubicek, 1992). A remarkable sequence homology (Fig. I) can be detected between the N-terminus of EG I11 and the N- or Ctermini of three other T. reesei cellulases (Teeri et al., 1987). Perhaps the most interesting feature emerging from the sequence analysis of four cellulase genes in T. reesei (Knowles et al., 1988) is the presence of a short conserved region at either the N-terminal (CBH I, EG 111) or C-terminal (CBH I, EG I) end of the corresponding protein (blocks A and B, Fig. 2). The finding that partial proteolysis by papain can cleave CBH I and CBH I1 into an enzymatic active “core,” and a cellulose adsorbing “tail” domain, gave evidence for the domain organization of the T. ressei cellulases (van Tilbeurgh et a)., 1986; Tomme et al., 1988). Small-angle X-ray scattering studies revealed a rather unusual tadpolelike shape of both CBH I and CBH 11, with a large isomorphic head and a flexible terminal extension (Schmuck et al., 1986; Abuja et al., 1988a,b). Block A (about 30 aa) is rich in glycine residues, which are often found in turns in protein structures and cysteine residues, the positions of which are strictly conserved in all four cellulases (Knowles et al., 1988). These features indicate that Block A might fold into a small separate domain stabilized by disulfide bridges. On the other hand, the block B sequence, which always joins the A block to the main body of the enzyme, is rich in hydroxyl amino acids and proline (Lehtovaara et al., 1986; Knowles et al., 1987). In CBH I, this region has been shown to be heavily glycosylated (Allen, 1983). There is also evidence that in CBH I1 most of the glycans are located in this particular region (Fagerstam et al., 1984). Crystallization of either of these enzymes has not yet been possible. However, CBH I1 was crystallized after proteolytic removal of the AB region (Bergfors et a]., 1989). The removal of the AB region from CBH I and CBH I1 has been shown to decrease significantly

8

1

7

n

n

n

n -

CBH I ( C ) H Y l G Q C G G I 1 G Y l S G l Q T V l C l A S l G l T T l C l Q V L l N l P l Y Y l S I

I

I

I

I

I

I

I

I

I

I

l

l

I

C B H I I ( N ) V W I G Q C G G I 0 N WIS G l P T C l C l A S l G l S T l C l V Y S l N l D l Y Y l S I

EG I

I

I

I

I I

I I

I I

I

l

l

I

( C ) H WIG Q C G G I 1 G Y l S G I C K T I C I T S l G l T T l C l Q Y S l N l D l Y Y l S I

I

I

I

I

I

I

I

I

I

I

l

l

I

EG I I I C N ) V W I G Q C G G I 1 G WIS G l P T N I C I A P I G I S A l C l S T L l N l p l y Y l A I

u

u

u

u -

FIG. 1. Comparison of amino acid sequence homology among four cellulase components of Trichoderma reesei. The residues conserved in all four proteins are blocked.

17

MICROBIAL CELLULASES Trichoderma reesei

CBH

I

lntron

ss H2N I I 1

CBHll

EG 1

1

-I+

H

m?j+:l H

I

B A

ism

1

H

E G 111

1

Clostridi um thcrmocell u m

EG A EG 0 EG

COOH

Ser pro

-,-I Thrid; ... ...

r [

D

Cellulomonas fimi

50% homologous Pro-Thr rich

-

EN G EX G

I

*. ......

Aspergillus nigpr ( Glucoamylase)

Ser -Thr -rich G1

[

62

1

.

Starch binding domain

I ,.,...:.'i FH C...

..... ::: r:.

FIG.2. Structural organization of the cellulase genes from Trichoderma reesei, Clostridiurn thermocellum, and Cellulornonas fimi and glucoamylase genes from Aspergillus niger. The terminal domains and the putative hinge regions rich in hydroxyl amino acids and proline are indicated. The catalytic domains are represented by open boxes and the intron positions are shown by solid bars.

their affinity to microcrystalline cellulose, Avicel, while the activity on soluble substrates is retained (van Tilbeurgh et a]., 1986).These results suggest that the terminal regions are at least in part responsible for the binding of the enzyme to insoluble, crystalline cellulose and that the hydrolytic site is located in the larger domain (Knowles et a]., 1988). Thus, the AB region forms a functional domain wherein the A part interacts with cellulose and the B part provides a flexible arm connect-

18

AJAY SINGH AND KIYOSHI HAYASHI

ing the catalytic and adsorptive regions of the enzyme (Tomme et al., 1988;Johansson et al., 1989). The results obtained by Stahlberg et al. (1988)show that this also was the case with EG 111. While the binding domain is composed of two clearly distinguishable regions, A and B, the main part of the enzyme is the core protein containing the active site. According to van Tilbeurgh et al. (1985),the specificity of the EG I11 shows some similarity in the fact that both EG I11 and CBH I1 have their homologous domains at the N-termini, in contrast to CBH I and EG I, which have similar sequences at the C-termini. In contrast to EG I, no transferase activity was observed for EG 111.Differences in transferase activities among the five endoglucanases isolated from S. pulverulentum were also demonstrated by Streamer et al. (1975). Detailed knowledge of the three-dimensional structure of cellulases is not available, mainly because of the failure to crystallize these proteins. However, the crystallization of the catalytic core protein of CBH I1 of T. reesei (Bergfors et al., 1989)has revealed that it consists of a sevenstrand, singly wound parallel alp barrel. Extended loops from the barrel produce a large channel for cellulose binding. The active site is located at the C-terminus of the p-sheet and could be identified clearly using data collected with an inhibitor diffused into the crystal. It is present in the tunnel, through which the cellulose threads and two aspartic acid residues form the catalytic residue. Comparable investigations for other cellulase components are still lacking. It has been proposed that the various cellulases secreted by T. reesei may differ in the number of surface loops forming the active site (Kraulis et al., 1989).The tunnellike structure may be typical of exoglucanases (cellobiohydrolases) because it allows an attack only from the end of the cellulose chain. The primary structure of cellulases from various bacteria and fungi, deduced from the nucleotide sequences, is diverse. However, the structural organization of the Cellulomonas fimi cellulases closely resembles that of the T. reesei cellulases (Fig. 2). A region of about 100 aa at the N-terminus of the endoglucanase (ENG) and at the C-terminus of exoglucanase (EXG) is 50% conserved in the two enzymes and is linked to the rest of the protein by a strictly conserved stretch of alternating Pro and Thr residues (O’Neill et al., 1986;Wong et al., 1986).Extensive amino acid homologies among C. thermocellum endoglucanases EG A, EG B, and EG D are also found in the terminal region. A direct repeat of 24 aa is found in the C-termini in all three enzymes. The proline-hydroxy amino acid sequences have also been shown to be conserved in the ENG gene of Bacillus sp. (Fukumori et al., 1986a,b)and CEL I

MICROBIAL CELLULASES

19

of Microbispora bispora (Yablonsky et al., 1988). The function of the terminal repeats in these C. thermocellum endoglucanases is not clear; however, they may be related to the binding of enzymes to the large glycosylated subunits of cellulosome (S, or SL),which may also have a role similar to that of the T. reesei block A tail (Bbguin et al., 1985; Grepinet and Bbguin, 1986; Joliff et al., 1986a,b). Fungal glucoamylases have been shown to bear functional similarity to cellulases in terms of cleavage of glucosidic linkages (Ueda, 1981; Svensson et al., 1983). Glucoamylase G 1 from A. niger (Fig. 2) has a region of approximately 100 aa at its C-terminus that is responsible for binding to insoluble starch but apparently is not required for the hydrolysis of small soluble substrates (Svensson et al., 1986). This terminal domain is linked to the rest of the protein by a glycosylated region rich in hydroxyl amino acids and proline similar to the putative hinge region in the T. reesei cellulases. The small G 2 form of this glucoamylase is generated by the proteolytic removal of the starch binding domain from G 1. It seems that the proteolytic removal of the substrate binding domain from both cellulase and glucoamylase may be an important mechanism for the modification of their substrate specificities during the course of hydrolysis of complex insoluble substrates (Knowles et al., 1988).Once the insoluble substrate has been degraded to short, soluble oligosaccharides, enzymes with higher specific activities toward the more soluble substrates would be available. In this way the microorganisms can increase the diversity of enzymes encoded by a limited number of genes. B. GLYCOSYLATION

Most of the fungal and a number of bacterial cellulases are glycoproteins with sugars attached at asparagine (N-linked)or serine and threonine (0-linked) residues. Most of the 0-glycosylation occurs in the hinge region of the protein, whereas N-glycosylation is restricted to the core domain (Kubicek, 1992). The carbohydrate content of cellulases varies from 1 to 10% (Enari and Niku-Paavola, 1987) and the principal sugar in all the cases studied is mannose, but other sugars such as glucose, galactose, xylose, N-acetyl glucosamine, and galactosamine have also been detected (Salovuori et al., 1987). The presence of carbohydrate moieties in microbial cellulases suggests their functional significance in cellulolysis. Because of the many hydroxyl groups available, the carbohydrates can hydrogen bond with the substrate and may also increase the solubility of the protein (Darnel1et al., 1986). Affinity

20

AJAY SINGH AND KIYOSHI HAYASHI

of mannan polymer to cellulose crystals has been demonstrated, which suggests the functional role of mannose-rich carbohydrates of cellulases in binding of crystalline cellulose (Chanzy et al., 1979, 1984). When produced in E. coli in nonglycosylated form, the CBH I of T. reesei (Knowles et al., 1988) and the two cellulases of C. fimi (Gilkes et a]., 1984) retained their activities on soluble substrates. The core proteins of CBH I and CBH I1 lacking the heavily 0-glycosylated terminal tail were found still active on the cello-oligosaccharides (van Tilbeurgh et al., 1986). These results demonstrate that glycosylation is not required for the hydrolysis of small soluble substrates. The heavily glycosylated region is localized near the C-terminus of CBH I and N-terminus of CBH I1 (Tomme et al., 1988; McHale and Coughlan, 1981). EG I and EG I1 also contain N- and 0-linked carbohydrates (Bhikhabhai and Pettersson, 1984; Pentilla et al., 1986). In CBH I, three N-glycosylation sites are occupied (Gum and Brown, 1977; Salovuori et al., 1987) and the N-linked carbohydrate resembles the highly conserved (Man),(ManNAc), structure found in other organisms (Salovuori et al., 1987). 0-Glycosylation lacks defined target sequence. The presence of mono-, di-, tri-, and tetrasaccharide chains has been revealed by several researchers (Kruszewska et al., 1989).In microsomes of T. reesei, attachment of these oligosaccharides to protein has also been observed. The function of 0-glycosylation has been discussed with respect to protection against proteolysis or adsorption to cellulose; however, it may also be required for efficient secretion of cellulases (Messner and Kubicek, 1988; Kubicek et al., 1987). Glycosylation has also been reported to play an important role in the stability of extracellular cellulases in T. reesei. N-Linked glycosylation appears to stabilize the structure of the cellulases and thereby protect them from enzymatic attack during secretion (Merivuori et al., 1985b; Sanchez et al., 1982; Onishi et al., 1979; Speake et al., 1981). An optimum level of N-linked glycosylation is required for efficient binding to and subsequent hydrolysis of crystalline cellulose (Pentilla et al., 1988). It has also been shown by these authors that native CBH I1 of T. reesei bound more effectively onto crystalline cellulose than the highly N-glycosylated enzyme produced by a recombinant yeast (Pentilla et al., 1988). While native CBH I and CBH I1 hydrolyzed highly crystalline cellulose, the recombinant enzymes were able to degrade less crystalline cellulose with a reduced efficiency. While N-linked glycosylation appears to be involved in imparting a specific conformation to the cellulases for binding to and hydrolysis of crystalline cellulose, 0-linked glycosylation seems to be required for secretion of the active cellulase.

21

MICROBIAL CELLULASES

C. SYNERGISTIC MECHANISM

It is now a well-established fact that degradation of crystalline cellulose by microorganisms is carried out by a multicomponent enzyme complex wherein the individual components interact in a synergistic way to convert the insoluble substrate into glucose. Individual enzyme components have been considered unable to break down the crystalline cellulose to any substantial extent. However, when the components are recombined they act in a synergistic manner to render the cellulose soluble (Coughlan, 1985; Finch and Roberts, 1985). Eriksson and Wood (1985) have shown that the individual exoglucanases, endoglucanases, and P-glucosidases degrade practically none of the crystalline cellulose, while a mixture of the three enzymes causes extensive hydrolysis. Wood and McCrae (1979) have demonstrated the synergistic effect beween cellobiohydrolase and endoglucanases and also between cellobiohydrolase and P-glucosidase (Table VI). A similar synergism has been demonstrated between exo- and endoglucanses from F. solani (Wood and McCrae, 1979; Wood, 1980), S. pulverulentum (Streamer et al., 1975), Trichoderma viride and P. funiculosum (Selby, 1969), Taleromyces emersonii (McHale and Coughlan, 1980),and Sclerotium rolfsii (Sadana and Patil, 1985). Cross-synergism between cellobiohydrolase from one fungus with endoglucanse to another has also been demonstrated (Wood, 1975). However, the extent of cooperation seems to vary considerably (Table VII). Thus endo- and exoglucanses from the fungi F. solani, P. funiculosum, and T. koningii can be exchanged and give rise to extensive solubi-

TABLE VI COTTON-SOLUBILIZING ACTIVITY OF CELLOBIOHYDROLASE, AND p-GLUCOSIDASE OF ENDOGLUCANASE, Trichoderma reesei Cotton solubilization Enzyme

(%I

Culture filtrate Cellobiohydrolase (CBH) Endoglucanase (EG) p-Glucosidase (BG) CBH + EG CBH + BG CBH + EG + BG

71 1 1 0 53 20 72

22

AJAY SINGH AND KIYOSHI HAYASHI TABLE VII SYNERGISTICEFFECTSOF CELLOBIOHYDROLASEAND ENDOGLUCANASE FROM DIFFERENT SOURCES IN SOLUBILIZING COTTON Source of cellobiohydrolase

Source of endoglucanase

Solubilization of cotton (oh]

F. solani F. solani T. koningii T. koningii P. funiculosum T. koningii P. funiculosum

F. solani T. koningii T. koningii F. solani T. koningii M. verrucario M.verrucaria

59 51 54 79 51 20 11

lization of cellulose. However, when exoglucanase from these fungi acted together with the endoglucanases of rumen anaerobic bacteria Ruminococcus albus and B. succinogenes, no synergistic effect was obtained (Wood, 1983).Wood (1988)demonstrated a synergistic action between cellulase components from the anaerobic thermophillic bacterium C. thermocellum and T. koningii (Table VIII). Clostridium thermocellum cellulase cannot extensively break down cotton cellulose in the absence Ca2+or dithiothreitol (DTT). However, a combination of C. thermocellum cellulase and T. koningii cellobiohydrolase can break down cellulose even in the absence of Ca2+or DTT. Apart from endo/exo-type synergism, two unusual types of synergism have also been reported. Combined action of Irpex lacteus endoglucanases causes considerable hydrolysis of crystalline cellulose (Kanda et al., 1976).The two endoglucanases differ in the way they attack soluble cellulose. Another type of synergistic action exo/exo-type was reported TABLE VIII HYDROLYSIS OF COTTON BY CELLULASES OF Clostridium thermocellum AND Trichoderma koningii C. thermocellum cellulase Addition

C. thermocellum cellulase

None DTTa CaCI, DTT + CaCl, DTT,dithiothreitol.

8 0 20

35

+

T. koningii cellulase 40 35 55 68

MICROBIAL CELLULASES

23

by Fagerstam and Pettersson (1980).They found that the hydrolysis of crystalline cellulose by the combined action of CBH I and CBH I1 of T. reesei was more than twice that calculated from the extent of hydrolysis by each of the separate enzymes. Brown and Gritzali (1984)also found that the exo/exo-synergism is important in the intact cellulase system as it degrades crystalline cellulose. The synergistic action of cellulases has been found to depend on the ratio of the individual enzymes, the degree of substrate saturation, and the type of substrate (Henrissat et al., 1985;Woodward et al., 1988). The action of CBH I and CBH I1 and the endoglucanases EG I and EG I1 purified from T. reesei was evaluated against different substrates (Henrissat et al., 1985).Synergism between the endoglucanases and CBH I1 follows the normal patterns for endo/exo type synergism. CBH I degrades the P-D-glucan from barley in a typical endo-patter. With cellulose as substrate, the synergism between EG I or EG I1 and CBH I depends on the structural and ultrastructural features of the substrate. CBH I demonstrates an unusually strong and irreversible binding to cellulose (Reese, 1982;Chanzy et al., 1983).Sprey and Lambert (1983) even suggest that there is a lectin-type binding between cellulases and cellulose substrate. The synergistic action exhibited by CBH I and CBH I1 seems to be due to the mixture of both endo- and exo-type character of the functions of both of these enzymes. A scheme for the hydrolysis of cellulose was suggested by Enari and Niku-Paavola (1987).Accordingly, hydrolysis of insoluble native cellulose is accomplished by the synergistic action of the two enzymes CBH I and CBH I1 (Fig. 3). The endoglucanases participate in the hydrolysis only at a later stage when soluble cellodextrins are hydrolyzed. The synergistic action of purified cellulases from T. reesei in the hydrolysis of cellulose was found to decrease with increasing substrate concentration, depending strongly on the type of cellulose used, and was maximum on crystalline cellulose (Nidetzky et al., 1993).However, the activity of individual enzymes was greatest on amorphous cellulose. The binary combinations CBH I/EG I11 and CBH I/CBH I1 exhibited a greater degree of synergism on crystalline cellulose, Synergistic action between two immunologically distinct cellobiohydrolases (I and 11) from P. pinophilum has also been demonstrated (Wood and McCrae, 1986a).The maximum rate of attack on Avicel was exhibited when the two enzymes were mixed in the ratio 1 :1. It was suggested that they may be two stereospecific enzymes concerned with the hydrolysis of the two different configurations of nonreducing groups that would exist in cellulose. Only CBH I1 from P. pinophilum acts synergistically with cellobiohydrolases of T. koningii and F. solani

24

AJAY SINGH AND KIYOSHI HAYASHI

CBH I

+ CBH I 1

b GLUCOSE

FIG.3. Suggested scheme for the enzymatic hydrolysis of cellulose.

to solubilize Avicel. CBH I1 showed no synergistic action with the endoglucanases of T. koningii or F. solani, but CBH I did.

D. CATALYTIC MECHANISM Cellulases catalyze the breakdown of the p-1,4-glucosidic linkage. It has been proposed that cellulose hydrolysis occurs by an acid catalysis mechanism at the active site (Knowles et al., 1987).Attempts to identify the active sites of the cellulases have been carried out by chemical modification and inhibition studies as well as sequence homologies among related enzymes (Paice et al., 1984; Paice and Jurasek, 1979; Dinur et a]., 1986). The active sites of many p-glucosidases have been found to involve aspartic acid residues. The results of Clarke and Yaguchi (1985) on the effects of chemical modifications of Schizophyllum commune EG I supported the hypothesis that carboxyl groups are involved in the hydrolysis of cellulose. Limited homologies in the cellulase primary structures to the active sites of hen egg white lysozyme (HEWL) have been noted (Paice et al., 1984; Teeri et a]., 1987). Figure 4 shows the weak homologies that have been proposed between lysozymes and cellulases. A comparison of amino acid sequences between T. reesei cellulase and HEWL indicates little sequence homology; how-

25

MICROBIAL CELLULASES

HEWL S.C.

EG I

C. f . EXG C. f . ENG

I35 I FI E S NIF N T Q A n 133 rJ NI E SIC A ElFlG N 136 SI E I F l N l L v V ~ A ~ E + I 13181 P I E lY A V G I F A I L

+

- -

N T D G S I T D Yl I I P G V K N I T D Yl I +

M K

W D AlTlE P I

I

S N Y Q TlTlA E U

-

HEWL S.C.

EG I

T.r.

CBH I1

135 I n FI E SIN F N T Q A T - 1 N I R N T D G S T - I D Yl 133 1 n 1 I - n I I N I E S I C A E F G N l Q l - I N I I l P l Q l V l K N T -ID Yl I 244 + I I I I I I I I I + LI E I C I N Y A V T l Q l L l N l L l P l N l V l A M Y L l D l A

-

u u u u

U

FIG.4. Comparison of amino acid homologies between hen egg white lysozyme (HEWL) and cellulases. S.C., Schizopyllum commune; C.f., Cellulomonas fimi; T.r., Trichoderma reesei.

ever, the EG I of S. commune has been found to bear a considerable sequence homology with the HEWL and it has been suggested that the catalytic mechanisms are likely to be similar in the two cases (Yaguchi et a]., 1983). Lysozyme is, however, the smallest of the carbohydrate-binding proteins and only one-third of the size of a typical cellulase. Thus it cannot be the only possible model for cellulases. Sequence analysis of different cellulases has revealed that the primary structures of S. commune EG I and T. reesei EG I11 are similar, although the aa sequence at their Ntermini are totally different (Saloheimo et ~ l . 1988). , Currently, in the absence of any definitive evidence amylases provide at least as good a model for the active site of cellulases. The localization of the active site of cellulases is a difficult task in the absence of solved three-dimensional structures. Crystallization of only two cellulases EG D of C. thermocellum (Joliff et a]., 1986b) and the catalytic domain of CBH 11 of T. reesei (Knowles et a]., 1987) have so far been successful. Some recent techniques such as computer-aided modeling and site-directed mutagenesis would also be useful in predicting the location of the active site (van Custem et al., 1987).Availability of such information is expected to lead to the design of newer enzymes with superior rates of catalysis.

26

AJAY SINGH AND KIYOSHI HAYASHI

Although considerably detailed information is now available on the structure and molecular properties of the cellulase components of T. reesei and other organisms, our understanding about the degradation of crystalline cellulose is less complete. Firm knowledge of the kinetics and substrate specificities of individual cellulase components is required to postulate a model for cellulose degradation. The introduction of a low-molecular-weight chromogenic 4-methylumbelliferyl-~-glycoside has eliminated the problem of cross-contamination of enzymes purified to homogeneity (van Arsdell et al., 1987;Pentilla et a]., 1987). There are pronounced differences in the kinetics of glycoside hydrolysis even between CBH I and CBH 11, and EG I and EG 111.In the case of CBHs, few differences are apparent between two enzymes, but differences do occur with higher homologues. CBH I attacks at sites other than the nonreducing end in the higher homologues and hydrolyzes both cellobiosides and lactosides. On the other hand, CBH I1 shows strict substrate specificity, three to four contiguous p-1&linked glycosyl residues being required (Claeyssens et al., 1989;van Tilbeurgh et al., 1989). It has also been deduced that CBH I acts with overall retention of configuration, whereas CBH I1 acts with overall reversion (Claeyssens et a]., 1990).

Endoglucanases act via rentention of their configuration (Claeyssens et a]., 1990).EG I has been classified as a nonspecific endoglucanase because it hydrolyzes not only cellulose but also xylan (Biely and Markovic, 1988).Its hydrolytic reaction proceeds via transfer reactions and the apparent hydrolytic parameters (Kcat,&/Km) increase steadily as a function of the number of glucose residues (Claeyssens et al., 1990).It probably possesses several subunits, each capable of binding a glucose molecule, and the glycosyl intermediates formed during hydrolytic reactions are then transferred to acceptor molecules. V. Cellulase Biosynthesis

A. INDUCTION AND REGULATION

Biosynthesis of cellulases is generally assumed to be subject to biochemical and genetic regulation. Cellulases are inducible enzymes, the best inducers being cellulose, cellobiose, lactose, and sophorose (2-0P-D-glycopyranosyl-D-glucose).Synthetic compounds such as palmitate and acetate esters of disaccharides and thiocellobiose have also been shown to function as inducers of cellulases (Reese et al., 1969;Rho et a]., 1982).In virtually all the microorganisms examined, the synthesis of cellulases is induced by the presence of cellulose and repressed in the presence of glucose. The fact that cellulose is insoluble in water and

MICROBIAL CELLULASES

27

cannot enter the microbial cell suggests that it cannot be the inducing component. This appears to be logical in view of the differences in structures of different natural cellulosic substrates. The most generally accepted view of the induction process is that the organisms produce a basic level or a constitutive amount of cellulase. This gives rise to soluble hydrolysis products of cellulose, which enter the microbial cells and function as inducers (Eriksson et al., 1990). Trichoderma species contain cellulases located on the surface of conidia, which appear to be involved in the initial attack on cellulose (Kubicek, 1987; Kubicek et al., 1987). CBH I1 has been found to be the major conidial bound cellulase in some hyperproducing strains (Messner et al., 1991). A putative cyclic AMP-dependent transcription activator binding region is about 400 base pairs upstream of the start point of the cbh2 gene, and a correlation between cAMP levels and sporulation in T. viride has been reported (Chen et al., 1987; Farkas et al., 1990). The induction by cellobiose appears to be concentration dependent, with high concentration having an inhibitory effect due to the build up of intracellular glucose (Coughlan, 1985; Hrmova et al., 1986). Cellobiose has been found to be an inducer of cellulases in S. pulverulentum, S. thermophile, and Neurospora crassa (Eriksson and Hamp, 1978; Canevascini et al., 1979; Eberhart et al., 1977). However, it does not seem to be an inducer of cellulases in T. reesei, at least not with washed mycelium (Eriksson and Hamp, 1978; Sternberg and Mandels, 1979). A considerable difference between T. viride and S. pulverulentum in regulating the production of cellulase was observed by Eriksson and Hamp (1978). In S. pulverulentum, endoglucanase activity was induced by cellobiose at low concentrations such as 1 mg/liter, while cellobiose at the same or even higher concentrations did not induce endoglucanase activity in T. viride QM6a. Sophorose is the most potent inducer of both exo- and endocellulase synthesis in T. reesei and its mutants (Nisizawa et al., 1971; Mandels et al., 1962; Sternberg and Mandels, 1979). However, the enzyme activity was about half that of the cellulose-induced enzyme. While sophorose is a very efficient inducer in low concentrations, in high concentration it inhibits production of cellulases. This has been explained by assuming that sophorose is enzymatically hydrolyzed to glucose, which inhibits cellulase synthesis (Nisizawa et al., 1972). The de novo induction of cellulase synthesis by sophorose has been proven by the inhibitory effect of the translational inhibitor puromycin on enzyme synthesis (Nisizawa et al., 1971, 1972). Montenecourt (1983) found no particular differences in intracellular cAMP levels between induced and noninduced cultures following exposure to sophorose and the induction of endoglucanase .

28

AJAY SINGH AND KIYOSHI HAYASHI

The intracellular level of some key metabolites at the time of cellulase induction in a T. reesei mutant was investigated by Farkas et al. (1987). The period of cellulase induction was preceded by a transient peak of ATP concentration and accompanied by an increased cAMP level. Addition of substrate, lactose, briefly stopped endoglucanase synthesis, increased ATP and glucose-6-phosphate concentration, and decreased the cAMP levels. Endoglucanase secretion again resumed after 2 hr. The increased levels of cAMP at the time of cellulase synthesis indicate that it may play a role in modulating catabolite repression (Cochet et al., 1984). Interestingly, T. reesei strains synthesizing higher cellulase activity in the medium display higher steady-state levels of CBH I- and CBH 11-mRNA in the mycelia, suggesting that transcription may limit cellulase production in the lower producer strain (Messner and Kubicek, 1991; El-Gogary et al., 1989).The mechanism of activation of cellulase gene transcription is a challenge in our understanding of how cellulase biosynthesis is regulated. Cellulase synthesis is generally considered to be subject to carbon catabolite repression (Nisizawa et al., 1971; Canevascini et al., 1979). Almost no cellulase is formed during growth on glucose, glycerol, and other carbon sources related to glycolytic metabolism (Nisizawa et al., 1972). The derepression of enzyme synthesis occurs on the exhaustion of glucose in Myrothecium verrucaria cultures (Hulme and Stranks, 1970). The catabolite repression of cellulase synthesis has also been shown in T. reesei and other fungi and in some bacteria (Steward and Leatherwood, 1976; Eriksson and Hamp, 1978; Canevascini et al., 1979). No correlation was found between cellulase repression and intracellular cAMP levels in fungi (Montenecourt et al., 1981);however, cAMP regulates the biosynthesis of cellulases in bacteria (W. E. Wood et al., 1984). It was recently shown that the addition of glucose to T. reesei cultures producing cellulases only decreased the rate of synthesis of CBH I by 40% and the fungus continued to produce cellulase up to 40 hr (Messner and Kubicek, 1991). The results were similar when a number of other glucose analogues or inhibitors of glucose uptake or metabolism were used. There was no evidence that glucose or a catabolite thereof in fact controls the transcription of the cellulase gene (Kubicek, 1992). Alterations detected at the level of the endoplasmic reticulum in some T. reesei mutants suggest that glucose may affect the development of the secretory pathway or some of its components (Ghosh et a]., 1982; Glenn et al., 1985). The decreased level of a key enzyme (dolicholphosphate-mannose synthase) in the 0-glycosylation pathway in T. reesei has been found during growth in glucose (Kruszewska et al., 1989). The above observations clearly indicate that the role of glucose in cellulase synthesis requires further investigations.

MICROBIAL CELLULASES

29

The role of L-sorbose in the induction and regulation of cellulase and P-glucosidase biosynthesis and secretion in fungi is well documented (Nanda et al., 1982, 1986; Bisaria et al., 1986; Kubicek, 1983; Sahoo et al., 1986; Duff et al., 1985; Kawamori et al., 1986). L-Sorbose inhibits P-1,3-glucan synthetase, with the result that P-glucan content decreases in the cell wall (Kubicek, 1983) and enhanced levels of P-glucosidase, which are closely associated with P-1,3-glucan, are released into the culture medium. Kawamori et al. (1986) suggested sorbose as an inducer of cellulase synthesis in a mutant strain, T. reesei PC-3-7,because endoglucanse synthesis on sorbose was inhibited in the presence of cycloheximide. The addition of sorbose to cellulose/cellobiose-grown cultures of T. reesei QM 9414 resulted in a sevenfold increase in extracellular cellulase activity (Nanda et al., 1986). Such activity could not be explained by sorbose-induced changes in cell wall composition since cellnlases are predominently extracellular (Vaheri et al., 1979; Nanda et al., 1986). The specific consumption rate of cellobiose in strain QM 9414 was also reduced in the presence of sorbose. A molecular model based on the above mentioned rate of sorbose was proposed by Bisaria and Mishra (1989). The effect of sorbose on synthesis of P-glucosidase and cellulase is given in Table IX. The synthesis of cellulases in fungi can be hampered by factors other than catabolite repression. A wide variety of phenols can repress the synthesis of both cellulases and xylanases in Chaetomium globosum and S. commune (Varadi, 1972). The enzyme synthesis was considerably repressed even at concentrations less than 1mM of vanillin, vanillic acid, and vanillyl alcohol. Production of endoglucanase was drastically repressed in a phenol oxidase-less mutant (Phe 3) of S. pulverulentum in the presence of kraft lignin and phenols even at a concentration of 1 mM. However, Miiller et al. (1988) demonstrated that cellulase production in white-rot fungi is stimulated by lignin-

TABLE IX EFFECTOF L-SORFJOSE ON CELLULASE BIOSYNTHESIS IN Trichodermo reesei

Extensive branching, septation, and increase in the number of hyphal tips. Inhibition of p-1,3-glucan synthetase. Decrease in the level of cell-wall p-1,3-glucan. Reduced level of intracellular p-glucosidase. 5. Release of cell-wall-bound p-glucosidase. 6. Increase in extracellular enzyme secretion. 7. Reduction of the cellobiose uptake into the cell. 8. Reduction of intracellular breakdown of an active inducer, sophorose. 9. Minimized catabolite repression.

1. 2. 3. 4.

30

AJAY SINGH AND KIYOSHI HAYASHI

related phenolic compounds. They suggested that stimulation of cellulase synthesis was due to an indirect rather than a direct induction by the phenols themselves. Phenol oxidases produced by the white-rot fungi oxidize phenolic compounds to their corresponding quinones. These quinones are further reduced to phenols by cellobiose : quinone oxidoreductase (Westermark and Eriksson, 1974a). Cellobiose is oxidized to cellobionolactone in this reaction, which is a powerful inducer of cellulases (Bruchmann, 1978). The extracellular cellulase activity is dependent not only on mechanisms regulating their biosynthesis but also on the presence of specific inhibitors regulating the activity of enzymes themselves. The role of one such inhibitor, gluconolactone, has been studied for the regulation of P-glucosidase of S. pulverulentum. Gluconolactone is produced either by oxidation of glucose by glucose oxidase or by hydrolysis of cellobionolactone. The K, values for gluconolactone as a competitive inhibitor of the two extracellular P-glucosidases from S. pulverulentum were 3.5 X and 15 X lo-’ M (Deshpande et al., 1978). B. LOCALIZATION Generally, microbial cellulases are regarded as extracellular enzymes because of their appearance in the culture medium during growth. However, some of the cellulase components have been demonstrated to be cell-wall or membrane bound. In T. reesei QM 9414 large amounts of cellulases are secreted into the medium when grown at a low concentration of cellobiose. Under induced conditions, P-glucosidase was mainly detected in cell debris and was not released unless cell autolysis took place (Vaheri et al., 1979). However, Kubicek (1981) noticed an appreciable association of endoglucanase with the cell wall fraction of T. reesei QM 9414 during its growth on cellulose and cellobiose. The cell-bound endoglucanse was bound to the mannoproteins located at the outer region of cell walls and could be released effectively by treatment with a-mannanase or trypsin (Gander, 1974). Although membrane-bound P-glucosidases have been reported in T. reesei and M. bispora they are predominently cell wall associated in most of the fungi (Kubicek, 1982, 1983; Dekker, 1981; Sprey, 1986; Umile and Kubicek, 1986; Yablonsky et al., 1988). Cell-wall-bound Pglucosidase is released into the medium after the completion of exponential phase. T. reesei is known to contain a most efficeint cellulase system but it is deficient in P-glucosidase activity. Since the major portion of P-glucosidase activity in T. reesei is cell-wall associated, various methods to release it from cell walls have been investigated by

MICROBIAL CELLULASES

31

several research groups (Kubicek, 1981; Nanda et al., 1982; Kubicek and Pitt, 1982; Bisaria et al., 1986). Lytic enzymes such as chitinase, p-1, 3-glucanse, a-1, 3-glucanase, and p-1, 6-glucanase have been observed in the culture filtrate of T. viride grown on S. commune cell walls (de Vries and Wessels, 1972, 1973). Cultures supplemented with chitin resulted in a higher release of P-glucosidase (Nanda et al., 1982). Chitin has also been found to co-induce p-1, 3-glucanase along with chitinase in T. reesei (Kubicek, 1981; Dekker, 1981).P-Glucosidase has been suggested to be located within the intramural space (Kubicek, 1981), and it can be released by treatment of cell walls with chitinase and p-1, 3-glucanase. On the other hand endoglucanase is released by treatment with a-mannanase or trypsin. The p-1, 3-glucan of the cell wall plays an important role in the release of P-glucosidase (Kubicek, 1983). Mutants of T. pseudokoningii, having low levels of wall-bound p-1, 3 -glucanase, released almost no wall-bound P-glucosidase. LSorbose, known to cause paramorphogenesis in fungi, inhibits pl, 3-glucanase synthetase and increases extracellular 6-glucosidase activity (Mishra and Tatum, 1972; Bisaria et al., 1986). In sorbosesupplemented cultures of T. reesei, peripheral hyphae of the mycelial clumps were highly branched with stubby tips and short internodal distances (Bisaria et al., 1986).On the other hand, in cellobiose medium, the mycelium grew as long, tapering hyphae with sparse branching. Similar results on localization and secretion of p-glucosidase from T. pseudokoningii have been reported. Sprey (1986) used ferritinconjugated antibodies in electron microscopic studies to show that p-glucosidase was localized mainly in the outermost exopolysaccharide layer, in the plasma membrane, and, to a lesser extent, in the carbohydrate portions of the cell wall. A cellulose degradation mechanism was proposed by Sprey (1986) in which a sequential degradation of cellooligomers is accomplished by @-glucosidaseattached to an exopolysaccharide layer, followed by glucan and plasma membrane-located pglucosidase action on cello-oligomers of successively lower degrees of polymerization. However, it is still not certain if the same or different forms of p-glucosidases are present in the plasma membrane and the cell walls.

IN RECOMBINANTCELLS C. BIOSYNTHESIS

The number of structural genes for each cellulase type (except CBH 1/11 and EG I/III) and the regulatory genes have not been completely identified because of the lack of a well defined genetic cycle in T. reesei.

32

AJAY SINGH AND KIYOSHI HAYASHI

Although a sexual stage has been reported in Trichoderma, mating types analogous to S. cerevisiae and Neurospora crassa are not available (Webster, 1964;Mortimer and Hawthorne, 1966).Because traditional methods of mapping the structural and regulatory genes are not available the mechanism of regulation at the molecular level has been studied by isolating mutants with altered physiology (Gallo et al., 1978;Kawamori et al., 1985;Nevalainen et al., 1980;Ghosh et al., 1982).In a number of hyperproducing mutants there has been a coordinated increase in all the components of cellulases, indicating involvement of a major regulatory gene (Sheir-Neiss and Monenecourt, 1984; Ghosh et al., 1982). Furthermore, the concomitant loss of crystalline and soluble substrate-hydrolyzing activity, and mannase and xylanase activities in some cellulase-negative mutants, also indicated that the biosynthesis of these enzymes may be regulated by a single gene (Nevalainen and Palva, 1978).Advances in genetic engineering and molecular biological techniques and successful cloning (Table X) of some fungal and bacterial cellulase genes have made it possible to identify and understand the role of structural and regulatory genes involved in cellulase biosynthesis. Escherichia coli has been the most common host for cloning of different cellulase genes. However, cellulase genes have also been efficiently cloned and expressed in Zyrnomonas mobilis (Misawa et al., 1988; Lejeune et al., 1988),B. subtilis, B. megatarium (Lee and Pack, 1987), and S.cerevisiae (Skipper et al., 1985;Shoemaker et a]., 1983;Pentilla et al., 1987). The expression of cellulase genes in heterologous hosts must ensure transcription of the cloned gene through cloned natural promotors or the control of the promotors of the vector system, efficient translation of the gene, and subsequent export of the cellulase protein to the extracellular medium (Bisaria and Mishra, 1989).The expressed gene product has been identified either by CMCase plate assay (Teather and Wood, 1982) or by immunoprecipitation of enzymes (Whittle et al., 1982;Gilkes et a]., 1984;Bdguin et al., 1985;Teeri et al., 1987; Chen et al., 1987).The genomic library constructed in the phage lambda vector has been screened for cellulase gene by hybridizing with two distinct cDNA probes: one synthesized from mRNA formed during growth of T. reesei on glucose, and the other from mRNA induced on cellulose (Skipper et al., 1985;Pentilla et al., 1987;Shoemaker et al., 1983;Teeri et al., 1983).The relevant cDNA sequences were identified by hybrid selection to the corresponding mRNAs and immunoprecipitation of proteins synthesized in vitro. The secretion of cloned gene product in bacteria and yeast appears to be governed by the same molecular mechanisms as in the native

MICROBIAL CELLULASES

33

TABLE X MOLECULAR CLONING OF CELLULASE GENE Donor cell Bacillus subtilis Cellulomonas fimi C. fimi

C. uda Clostridium thermocellum

EIulinia chrysanthemi Microbispora bispora Pseudomonas fluorescens Ruminococcus flavefaciens Thermomonospora sp. YX Aspergillus niger Candida pelliculosa Schizophyllum commune Trichoderma reesei

Host cell Escherichia coli (PBC51 E. coli (pBR322) E. coli (pBR322)

Cellulase gene type

Reference Nakamura et al. (1986)

Exoglucanase (ExGI Endoglucanase (ENGI Endoglucanase

O’Neill et al. (1986) Wong et al. (1986)

Zymomonas rnobilis (pZA22) E. coli (pBR322) (PHC791

Endoglucanase cel A cel B

(hLIC7) (h1059) (pBR325) E. coli (pMMB34)

cel C cel D P-Glucosidase Endoglucanase

Romaneic et al. (1987) GrBpinet and BBguin (1986) Joliff et al. (1986a) Joliff et al. (1986b) Kadam et al. (1988) Barras et al. (1984)

E. coli (pBR322)

Endoglucanase

Yablonsky et al. (1988)

E. coli (pBR322)

Endoglucanase

Wolff et al. (1986)

E. coli (pMEB2OO)

Cellulase

E. coli (pBR322)

Endoglucanase

E. coli ( ~ 3 0 3 0 )

P-Glucosidase

Barros and Thomson (1987) Collmer and Wilson (1983) Pentilla et al. (1984)

E. coli (YEp13)

p-Glucosidase

Kohchi and Toh (1985)

E. coli (hgtll, pBR328) E. coli (h1059) (h1059) (A1059) (h1059)

Endoglucanase

Willick et al. (1987)

Cellulases CBH I CBH I1 EG I EG 111

Teeri et al. (1983) Teeri et al. (1983) Pentilla et a]. (1986) Saloheimo et al. (1988)

Misawa et al. (1988)

host system. The N-terminal nucleotide sequences of both the cloned bacterial and fungal cellulases predict an initial sequence of 25 to 34 amino acids with a charged amino acid at the N-terminus and lengthy hydrophobic sequences (Watson, 1984; Wickner and Lodish, 1985; Pugsley and Schwartz, 1985). A complete secretion of enzymes to the

34

AJAY SINGH AND KIYOSHI HAYASHI

extracellular medium occurs when the cellulase genes are cloned in Bacillus sp. or S. cerevisiae (Robson and Chambliss, 1986; Lee and Pack, 1987; van Arsdell et al., 1987; Shoemaker et al., 1983). On the other hand, the presence of the N-terminal sequence directs its export to the periplasm when E. coli is the host (Sharma et al., 1987).Trichoderma reesei cellulases are generally overglycosylated in yeast, which indicates that post-translational modifications in yeast and filamentous fungi are different. VI. Future Prospects

Trichoderma reesei is one of the most efficient producers of extracellular enzymes; more than 50% of all proteins produced by this organism are secreted into the extracellular medium. Although our understanding of the molecular biology and enzymology of cellulase biosynthesis has advanced rapidly in recent years, more detailed knowledge in this area may be necessary in order to increase the production of cellulolytic enzymes by this fungus. The most,exciting aspect of cellulase biochemistry currently being examined is the discovery of how the threedimensional structure of cellulases brings about the degradation of cellulose. These studies are required in order to improve cellulases by modifying their active center or the structures involved, and also to explain the need for different ratios of individual cellulase components for the optimal hydrolysis of cellulose. From an application point of view, the desirable properties of cellulases are high catalytic efficiency, high thermal stability, and low end-product inhibition (Bissetand Sternberg, 1978; Reese and Mandels, 1980;Ryu and Mandels, 1980).Adsorption of cellulases on cellulose is another critical factor governing hydrolysis of amorphous and crystalline cellulose (Klyosov et al., 1982,1986; Singh et al., 1 9 9 2 ~ )The . role of glycosylation in the binding efficiency of cellulases onto crystalline cellulose needs to be further investigated so that the cellulases with high binding efficiency can be developed. The advent of recombinant DNA technology has dramatically accelerated research in the field of cellulase enzyme systems. Efforts have been directed to clone genes from cellulolytic organisms with the desired molecular properties. In the past few years a large number of cellulase genes have been isolated, characterized, and expressed in a variety of hosts. Cloning of genes for individual components of the cellulase system would allow the biosynthesis of pure components and also provide information on their relative importance in the enzymatic hydrolysis of cellulose. Understanding the enzymology of cellulose hydrolysis appears to be more difficult than anticipated earlier. This is mainly

MICROBIAL CELLULASES

35

because of the very complex and variable nature of the natural substrate cellulose. The development of an efficient transformation system opens the possibility of constructing improved strains producing cellulases with higher specific activities on particular substrates. An equally important aspect would be improvement in the thermoresistance of these enzymes. Although protein engineering offers the necessary technology, the isolation and characterization of thermostable cellulase and their genes are required. With this work as a foundation, the development of new cellulolytic organisms that can be used to exploit the enormous amount of waste cellulose may be feasible. ACKNOWLEDGMENT STA Fellowship provided to AS. by JRDC and JISTEC, Japan, is gratefully acknowledged.

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Factors Inhibiting and Stimulating Bacterial Growth in Milk: An Historical Perspective D. K. O’TOOLE Department of Biology and Chemistry City University of Hong Kong Kowloon, Hong Kong

I. 11. 111. IV.

V. VI.

VII.

VIII.

IX. X.

Introduction Early Developments Lactenin Studies with Lactic Acid Bacteria A. Agglutinin for Lactic Acid Bacteria B. The Lactoperoxidase System C. Autoinhibition by Hydrogen Peroxide D. Thiocyanate in Milk E. Lactoferrin F. Other Possible Inhibitors for Lactic Acid Bacteria in Milk G. Seasonal Variation in Bacterial Activity in Milk H. Heated Milk as a Growth Medium for Lactic Acid Bacteria I. Effect of Culture Manipulations J. Effect of Milk on Morphology of Starter Cultures Effect of Cow Ration on Milk as a Growth Medium for Bacteria Role of Inhibitors in Milk Quality A. Raw Milk Quality Stimulators of the Growth of Lactic Acid Bacteria A. Investigations of Milk and Milk Components B. Stimulation by Minerals C. Complex Substances as Stimulators D. Commercial Stimulators Growth Inhibitors of Other Bacteria A. Inhibitors for Enterobacteria B. Inhibitors for Bacilli C. Inhibitory Factor for Propionibacteria D. Inhibition of the Staphylococci E. Inhibition of Leptospiras F. Inhibition of Mycoplasmas Applications of the Antimicrobial Systems in Milk Conclusion Appendix A Appendix B References

I. Introduction

Milk is a complex biological fluid which is secreted by female mammals from their mammae and which contains liberal quantities of fat, 45 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 40 Copyright 8 1995 by Academic Press. Inc. All rights of reproduction in any form reserved.

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carbohydrate, protein, minerals, and vitamins, which are ideal for the growth of the mammal’s offspring but which also supply many of the nutrient requirements of bacteria. Consequently, many types of bacteria grow rapidly in milk and milk products. This fact motivates the technologically advanced industry practices developed by the dairy industry to protect milk and dairy products from deterioration during processing, storage, and distribution. Thus, the growth-supporting properties of milk are well known and appreciated; not as well appreciated and understood are the growth inhibiting properties of milk. In addition, some suggest that the growth of bacteria in milk is further controlled by stimulating factors which promote bacterial growth, as suggested for cheese starter bacteria (Czulak and Meanwell, 1951). Thus the growth we see may be the net response of the bacteria to two different forces, an inhibiting force which retards the growth of or destroys bacterial cells and a stimulating force which enhances bacterial growth under the conditions of the test. The source of these inhibiting and stimulating factors is inherent; that is, they are part of the natural protective systems in the milk, but they can be influenced by the female’s diet which can be deficient in essential constituents that would normally be transmitted to milk. Cow milk has been most extensively studied, but the inhibiting properties have been noted in milk from buffaloes (Natarajan and Dudani, 1961; Patel, 1969), camels (El Agamy et a]., 1992), goats (Hammer and Babel, 1957), and humans. Milk from all mammals probably shows these properties. Most of the work done in this area has been concerned with the effect of milk on particular organisms with few attempts, with the exception of the lactoperoxidase-thiocyanate-hydrogen peroxide system, to link the observations with those found with other organisms. The main concern of this review is the lactic acid bacteria and the effect of milk on their growth, but propionibacteria, coliforms, bacilli, and others are also dealt with because it cannot be assumed that these separate agents bear no relationship to those factors inhibiting the lactic acid bacteria. The growth of many other organisms is known to be inhibited in milk (see Appendix A). Reviews include those of Jones and Little (1927), Reiter and Moller-Madsen (1963), Reiter and Oram (1967), and Reiter (1978, 1985). By far the most prolific worker in this field within the last 30 years has been Dr. Bruno Reiter. Interest in these substances has waxed and waned over the decades. There seem to be four main reasons for interest in their presence. The first important studies celntered on the possibility of using the inhibitory properties to control mastitis and began before the antibiotic era. The

BACTERIAL GROWTH IN MILK

47

second and most fruitful studies began when their effects on lactic acid bacteria, the cheese-making bacteria as well as mastitis bacteria, were studied. The third series of studies began to investigate their role in controlling and influencing milk quality. The fourth strand of study was concerned with the effect of milk on the growth of organisms pathogenic to young mammals, distinct from the mastitis organisms which infected the mammary glands, and includes the protective properties of milk for infants. II. Early Developments

Fokker’s research in 1890 is thought to be the first to note the germicidal properties of milk; he found that raw goat milk soured more slowly than heated goat milk that was similarly inoculated (Hammer and Babel, 1957). Jones and Little (1927) reported Heinemann’s conclusion that milk contains a bactericidal substance, specific for certain organisms, which was destroyed by heating at 60°C for 30 min. They added that Rosenan and McCoy, after noting that bacterial numbers declined during the first 8-10 hr postmilking, suggested that agglutination and phagocytosis caused the decline. However, Chambers found no relationship between agglutination and growth inhibitory properties except for their equal susceptibility to heat, 80°Cfor 2 min, and reported specificity with regard to the cow and bacterial species employed. Jones and Little (1927), on the contrary, reported that Sherman and Curran found that young cultures of Streptococcus lacticus inoculated into fresh milk had a 30-min lag period compared to controls. (Note that many early names are now invalid; readers are directed to Index Bergyana (Buchanan et al., 1966) and the latest edition of Bergey’s Manual of Systematic Bacteriology to check the fate of these taxons; the lactic acid streptococci are now placed in the genus Lactococcus). Hanssen (1924) noted that in fresh milk the maximum decline in numbers of Salmonella typhi and Salmonella paratyphi B took place within 30 min. The milk retained this activity for 24 hr during storage in ice; it seemed that when the cow was receiving, among other things, fresh grass, turnip, and cabbage, the bactericidal activity was greatest, and that bactericidal activity was the least when receiving no fresh grass but only ensilage (Hanssen, 1924). He thought that the bactericidal property of milk was closely related to oxidases and peroxidases, as both had a similar temperature limitation of 75°C for 15 min. He proposed that the enzymes came from the feed (Hanssen, 1924). The first systematic studies were those of Jones and associates, who found that milk and whey had bacteriostatic activity for a nonhemolytic

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mastitis streptococcus; it failed to grow 4-6 hr postinoculation (Jones and Little, 1927), and it did not use the substance in the lag period; rather, the organism adapted itself to the inhibitory principle (Jones and Simms, 1929). The activity was natural, inherent in cow milk, and specific in that Bacterium coli and Bacterium bovisepticus cultures were not inhibited by milk inhibitory for streptococcus but B. bovisepticus could absorb some of the activity for the streptococcus from milk (Jones and Little, 1927);on the contrary, cells of a scarlet fever streptococcus, live or dead, did not absorb the principle (Jones and Simms, 1929) even though the substance killed the organism (Jones, 1929). Colostrum contained less “principle” than normal milk, and charcoal removed some, and Kieselguhr none, of the principle which was destroyed by heat (80°C for 20 min) but not by drying (Jones, 1928). The substance was not concentrated in the rennet whey as blood proteins are, and it had a different heat inactivation pattern from that of the bactericidal or bacteriostatic property of cow serum. Their technique ruled out the agglutination theory and they concluded that the substance was not “alexin” (complement) (Jones and Little, 1927). Jones and Simms (1930) proposed the name “lactenin” for this substance(s), a name applied for many years to any unknown substance that inhibited bacterial growth in milk, and suggested that it was a protein even though unaffected by trypsin. It existed as a salt of whatever cation was present, it had a low isoelectric point, it contained only C, H, 0, and N and no reducing sugars, and its natural advantages and possible uses were unknown (Jones and Simms, 1930). Ill. Lactenin

Further investigations began in the 1950s, the first studies being those of Wilson and Rosenblum. They tested streptococci for their sensitivity to lactenin and obtained the results shown in Table I. Wilson and Rosenblum (1952a) used a group A streptococcus (327W) and found that milk retained a bactericidal effect for up to 24 hr irrespective of inoculum size. Growth improved but no inactivation of lactenin occurred when 5% normal human, rabbit, sheep, or horse serum was added to milk. Lactenin was partially inactivated in milk when defibrinated human, rabbit, or sheep blood was added. Their most important observation (Wilson and Rosenblum, 1952b) was that rigid exclusion of oxygen prevented the activity of lactenin. They examined substances for their ability to inactivate lactenin (Wilson and Rosenblum, 1952b), the results of which are in Appendix B, and found that cysteine (100 ppm), sodium thioglycollate (100 ppm),

49

BACTERIAL GROWTH IN MILK TABLE I SEROLOGICAL GROUPSOF STREPTOCOCCI SENSITIVE TO LACTENIN IN RAW MILK Group A B C, D,E, H,

F G K

L

Number of strains

Strains sensitive

477

477 1

25 31, 25, 4, 4,10 3 25 3

0 1

5 2

Note. From Wilson and Rosenblum (1952a).

glutathione (100 ppm), and 2,3-dithiopropanol(lo ppm) were the most effective. Auclair began similar investigations (Auclair and Hirsch, 1953) with the conception that he was dealing with a very complex phenomenon. He confirmed an earlier observation (Wilson and Rosenblum, 1952b) that the antistreptococcal properties of milk were not due to lysozyme. He quantified the bactericidal properties of milk using dilution assays which were prepared in sterile skim milk adjusted to pH 7.0 to which was added an equal volume of glucose-peptone-broth inoculated with 5% v/v of an 18-hr serum-broth culture of Streptococcus pyogenes, strain “Richards.” After 6 hr at 37”C, distilled water (5x test volume) was added and the pH determined. The response curve obtained (Auclair and Hirsch, 1953) (Fig. 1)was explained as follows: milk contains a small quantity of stimulator and at least two separate inhibitors which they called “lactenin 1” and “lactenin 2” (Fig. 2). Lactenin 1 (Ll) occurs mainly in colostrum, is most stable at pH 6.0-6.5, and is 90% destroyed at 68°C. Lactenin 2 (L2) occurs mainly in milk, is most stable at pH 7.0, and is 95% destroyed at 74°C. L1 and L2 act synergistically, each having low potency in the absence of the other (Auclair and Berridge, 1953). Anaerobiosis and cysteine abolish the inhibitory activity of both lactenins (Auclair and Hirsch, 1953) but do not destroy the lactenins, but L2 is destroyed by H,O, and L1 remains relatively unaffected (Auclair, 1954b). Occurrence of the lactenins was not correlated with udder infection nor with stage of lactation, age of cow, or the season of the year (Auclair, 1954b). Auclair (1954b) found that Streptococcus lactis was more seriously inhibited by L1 and L2 together than L2 alone but Streptococcus agalactiae was more sensitive to L2 than L1 and L2 together, and that Staphylo-

D. K. O'TOOLE

50

7.0

6.5

PH 6.0

5.5

If2

1/4

1/8

1/16

1032

1/64

1/128 If256 1/512 1/1024 If2048 1/4096 1/8192

Control

Dilution of sample FIG.1. The dose-response curve showing the pH change in midlactation milk and colostrum inoculated with S. pyogenes strain 'Richards'. ( X ) Standard, (0)milk from cow Pandora in midlactation, (A)colostrum from cow Rosalie. From Auclair and Hirsch (1953).Reprinted with the permission of Cambridge University Press.

coccus aureus was only slightly inhibited by L1 and L2. L1 and L2 could be partially separated using acetone fractionation in small steps (Auclair and Berridge, 1953), whereas on electrophoresis, which gave good separation of L2, the activity of neither lactenin could be linked with a corresponding protein brand. Auclair (1954b) concluded that variation in the activity of lactic acid bacteria in milk was due to variations in natural stimulators in milk. At that time Jag0 (1954)noted that the growth of lactic starter cultures in pasteurized milk varied. He concluded there was an inhibitor associated with the fat globule because buttermilk was inhibitory but butter granules were not, and as the concentration of cream added to raw milk increased, so did inhibition of the cultures. Heated separator slime (primarily white blood cells and manure) neutralized the inhibitor. He suggested (Jago,1954) the inhibitor was an enzyme and that it interfered with the utilization of growth-promoting substances normally present in milk. Jago's studies were instigated because of variations in the activity of lactic cultures in cheese making, as reflected in activity tests done by

51

BACTERIAL GROWTH IN MILK

7.0

6.5

PH 6.0

5.5

If2

1/4

1/77

1/16

1/32

1/64

Ill28

1,256 1412 1/1024 1,2048

Control

Dilution of milk FIG.2. The hypothetical effects of decreasing concentrations of two inhibitory substances and one stimulatory substance on the acid production of S.pyogenes as it relates to the standard response curve of the organism to milk. (0) Dose-response of the test organism to raw milk, ( x ) inhibitory substance I,, (0)inhibitory substance I,, [A) stimulatory substance S. From Auclair and Hirsch (1953). Reprinted with the permission of Cambridge University Press.

inoculating 9 ml of whole milk in a test tube and measuring acidity after 6 hr at 30°C. By such tests lactic cultures are classed as either fast, producing about 0.6% acid in this time, or slow, producing less acid in this time. Wright and Tramer (1957)pointed out, however, that fast cultures produced acid throughout the tube but slow cultures (e.g., Streptococcus cremoris 60, 803) often produced acid only in the cream layer. Prolific growth of slow strains occurred in the cream layer, as observed microscopically, but not in the bulk of the milk (Wright and Tramer, 1957). Slow cultures also showed improved acid production when tubes were inverted every half-hour but only if air was excluded by placing wax on top of the milk, as air reduced acid production by fast and slow cultures (Wright and Tramer, 1957). Elimination of creaming by homogenization or heating (82°C) or addition of rennet or agar improved acid production by slow starters, and their growth was better in skim milk than in whole milk. They showed that if milk was separated, the milk and cream were separately heated for 30 min at either 63 or 76"C, and the milk was reconstituted from portions sub-

52

D. K. O’TOOLE

jected to different heat treatments, the activity of the cultures and the cream-rising properties of the milk depended on the heat treatment of the skim milk. In skim milk heated at 63°C for 30 min, slow starters formed clusters that deposited on the bottom of tubes; in whey from such milk the organisms formed floccules of massed agglutinated cells. Growth was normal in whey from milk heated to 76°C for 30 min. Activity and chain length of the organisms were not related as even slow cultures formed shorter chains in milk heated to 76°C for 30 min. Wright and Tramer (1957) named the substance responsible for this activity “agglutinin.” Wright and Tramer (1958) noted a second type of slow starter, of which S. cremoris 972 was one; even when creaming was prevented, this type showed increased activity only when the milk used was heated to a high temperature. Streptococcus cremoris 803 was affected by the agglutination effect and its maximum activity occurred in whole milk heated to 80°C for 24 sec, but 90°C for 24 sec was necessary for the maximum activity of 972. The addition of 2-10% milk to autoclaved or steamed milk, and to wheys from these milks, inhibited the 972 culture, but dilutions of milk heated to 63°C in milk heated to 85°C gave a different dose-response curve. This difference was due to the presence of reducing compounds in milk heated to 85-88”C, measurable with the sodium nitroprusside test; these compounds could be destroyed by H,O, treatment (30-100 ppm) followed by catalase treatment. Wright and Tramer (1958) found a relationship between milk peroxidase and the inhibitory properties of milk as Hanssen (1924) had suggested. Culture activity and peroxidase activity behaved similarly in whey heated to various temperatures. In addition, whey was not affected by exposure to pH 2.82, but below this pH culture activity increased and peroxidase activity decreased. Milk heated to 82°C contained residual peroxidase of about 1%of that originally present in the milk. There was an interaction between the inhibitor in whey and H,O,, as whey treated with 10 ppm H,O, showed little adverse effect on peroxidase but at 20 ppm peroxidase was destroyed and culture activity improved. The inhibitor in a mixture of homogenized and commercially sterilized milk was overcome with cysteine but excess of cysteine was inhibitory, i.e., there was a critical level of cysteine needed to neutralize the inhibitor in high-temperature short-time pasteurized milk. Sodium hydrosulfite (Na,S2O,.2H,O) also counteracted the inhibition caused by peroxidase, but not that caused by agglutinin, and overcame a partial bacteriostatis of 972 in homogenized, pasteurized milk. Sodium azide, which inactivates catalase (critical concentration about 1 mM), also inactivated peroxidase and the inhibitor. A similarity was shown between the lactenin of Auclair which af-

BACTERIAL GROWTH IN MILK

53

fected S. lactis M1 and that affecting strain 972 (Wright and Tramer, 1958). They found colostrum to be low in peroxidase compared to milk; Auclair found colostrum poor in lactenin 2. They tested colostrum for agglutinin and found correspondence between the creaming properties of milk induced by colostrum and the activity of agglutinin-sensitive organisms; they suggested that colostrum contains 10 times as much agglutinin as normal milk. They supported the concept that the removal of inhibition can be achieved by either inactivation of peroxidase or reduction of a postulated inhibitory oxidation product. They also noted that although many studies of slow cheese starters have found that the inhibitory effect of milk disappears when it is heated to 71°C for 30 min, this treatment was also just sufficient to inactivate peroxidase in milk. Wright and Tramer (1958) concluded that L1 was equivalent to agglutinin and L2 was equivalent to peroxidase, i.e., lactoperoxidase. Auclair and Portmann (1959) reexamined the question and found that the substance inhibiting 760 was different from L l ; they named it L3 and reported that it was different from the substance causing agglutination of fat globules. Since about 1959, investigations into the inhibitory properties of milk are confounded by separate studies of peroxidase and agglutinin and further studies of the general growth-supporting properties of milk for various bacteria, I shall deal with the agglutinin and peroxidase investigations separately as far as possible. Most studies deal with single strains of streptococci, but mixed cultures are also inhibited by milk (Randolph and Gould, 1966). IV. Studies with Lactic Acid Bacteria

A. AGGLUTININ FOR LACTICACIDBACTERIA

The observations of Wright and Tramer were not only quickly confirmed but also extended, in that it was shown that 803 not only rose with the fat globules but also deposited to the bottom of the culture (Stadhouders, 1963). Keogh (1958) showed that there was generally a relationship between inhibition in raw whole milk and a positive ring test; she also noted excessive numbers of organisms in the cream layer when compared with the skim milk layer and prevented inhibition by skimming and by adding agar. She further showed (Keogh, 1958) that some cultures were consistently faster in milk heated to 98°C for 15 min than in raw milk (strains K, DR7, R6, US3, R1, DRC1, DRC2, DRC3) but others were as slow or slower in heated milk as in raw milk (strains C1, C2, C3, Clo). The capability of skimming to remove the inhibitory effect of raw milk depended on milk supply for some cultures (strains DR7, K, US3, DRC3); the removal of inhibition by agar also

54

D. K. O’TOOLE

depended on milk supply, but a slightly different group of strains showed the variation (strains DR7, HP, R1, DRC1). The acetoneextracted and -dialyzed precipitate produced by Auclair and Berridge (1953) which contained L1 and L2 also contained agglutinin, as it produced a positive ring test and caused microscopic agglutination of bacteria and contained peroxidase (Keogh, 1958). When added to heated milk the extract also stimulated some bacteria in a manner similar to that found in raw milk. Gillies (1961a) showed that inhibition of HP and R6 was prevented in whole, pasteurized milk constantly rotated in a bottle from which air was excluded. HP was agglutinated around fat globules. As the percentage cream added to sterile reconstituted skim milk increased, the susceptible strain HP showed a rapid decrease in acid production followed by a gradual increase and then a final decrease (Fig. 3). This final decrease as the percentage cream approached 1OOO/” was shown also by a resistant strain C13. The inhibitory property attached to the fat globule was destroyed by heating at 76°C for 30 min (Gillies, 196lb). The inhibitory factor for R6 could be washed out of cream after two 50

Suscepuble strain HP Resistant strain C13

0

- - - -.

I

I

I

I

20

40

60

80

100

Percentage cream added FIG.3. The effect of increasing concentration of cream in milk on the acid production of lactic cultures resistant and susceptible to inhibitors in milk. From Gillies (1961bj. This figure provided by the Department of Primary Industries, Queensland, from their Journal QJAAS published by the DPI Queensland.

BACTERIAL GROWTH IN MILK

55

washings in sterile tap water, but the cream was still inhibitory after one washing and agglutinated organisms were present in the fat layer. When the water from the second washing was added to sterile skim milk, R6 was inhibited. There was no agglutination of the organism. The addition to pasteurized whole milk of homologous rabbit antisera to resistant strains caused those strains to act like susceptible strains (Gillies, 196lb). By the acriflavine test all resistant strains were smooth and all susceptible strains were rough (Gillies, 196lb). Gillies (196lb) concluded there was no firm proof that the inhibition of susceptible strains in milk was due to specific antibodies, but Randolph (1963) found that R1 was inhibited by a factor associated with the immune globulin fraction in milk, which suggested an antibody as the cause of inhibition. Specific antibodies in milk inhibiting lactic streptococci (strains ML1, KH, DR7) had previously been demonstrated by McPhillips (1958). Portmann and Auclair (1959) found that of 15 strains of lactic streptococci, 11were sensitive to both inhibition and agglutination, and could be placed in five groups according to how they behaved in raw skimmed milk absorbed with suspensions of the organisms. These groups were: 760, DR7, K; E8,972; 803, R1, US3; KH, ML1; and HP. Strains C3, C10, T7, and DRC3 were unaffected. However, a sensitive strain of ClO was derived from a resistant culture of ClO and shown to be in a group different from these five groups (Auclair and Vassal, 1963). Portmann and Auclair (1959) suggested that inhibition and agglutination may be due to the same substance. Stadhouders (1963),in addition to confirming the prevention of inhibition by rennet, showed that agglutination of the bacteria before removal into the cream layer was not necessary. The agglutinins were inactivated in homogenized skim milk and were present in whey at the same concentration as that in milk, i.e., they were whey proteins. The euglobulin fraction of milk promoted agglutination and inhibited acid production by 803, whereas the pseudoglobulin fraction was without effect (Stadhouders, 1963). The distribution of the agglutinins between the cream and skim milk fractions of milk were dependent on temperature (Stadhouders, 1963). Stadhouders and Hup (1970) distinguished within the euglobulin antibodies a group called cryoglobulins which not only agglutinated bacteria and fat globules separately but also attached bacteria to fat globules. The antibodies which attach bacteria to fat globules are very specific, and the binding sites on the antibodies for bacteria are the same as those which agglutinate fat globules and bacteria. Goat milk fat globules removed the antibodies which agglutinate fat globules in cow milk. They thought that L1 and L3 were equivalent (Stadhouders and Hup, 1970).

56

D. K. O’TOOLE

An investigation of the agglutination phenomenon (Emmons et al., 1966a) using a sensitive test (Emmons et a]., 1965) for agglutinating antibodies to streptococci was prompted by problems associated with cottage cheese manufacture (Emmons et al., 1966b, 1967). Instead of rising on fat globules into the cream layer (Emmons et al., 1966a) the organisms agglutinated and settled to the bottom of the vat (Emmons et al., 1966b)(cottagecheese is made from skimmed milk). This produced a bottom layer 0.5-1 cm thick with a very low pH and generally slow acid development in the rest of the vat; the curd tended to shatter easily and was “mealy.” Absorption of the milk with saline-washed heatkilled cells reduced the sediment, but even so there was no absolute relationship between agglutination titres in whey and the extent of settling in skim milk (Emmons et a]., 1966b). Old milk and excessive Ca ions may be linked with the problem (Emmons et al., 1966b) but pasteurization at 71-74OC for 30 min virtually eliminated it (Emmons et al., 1967). Using two strains sensitive (Rl, DR7) and one resistant (C2) to agglutination, Emmons et al. (1966a)showed that for R 1 and DR7 colostrum was high in agglutinating antibodies whose concentration rapidly decreased to a relatively constant level within 2 weeks of parturition, and that antibodies to C2 were virtually nonexistent. The concentration of antibodies in milk was independent of breed and age of cow and of antibody titre in blood serum in which concentrations were higher than in milk. The agglutinating antibodies for C2, R1, and DR7 showed similar results for milk and blood from Short Horn beef cattle, swine, and Shropshire sheep, and similar results for blood from Clydesdale horses, Holstein bulls, dairy calves, unbred dairy heifers, and rabbits (Emmons et al., 1966a).Blood serum from hens also showed agglutination of lactic streptococci (McPhillips, 1960). Heat-killed cells of R1 removed agglutination and inhibitory principles for R1, but not for KH and HP, from skim milk and whey (Randolph and Gould, 1968). The inhibitor was bound firmly to R1 cells but some could be removed by 0.8% NaCl at 45°C for 2 hr and 15% NaCl at 37°C for 1 2 hr (Randolph and Gould, 1968). The inhibitory principle for R1 was associated with the immunoglobulins in milk (Randolph and Gould, 1968). It had been concluded earlier (Portmann et al., 1960) that lactenins were very similar to antibodies because vaccination of cows with sensitive and resistant lactic acid bacteria had resulted in increased inhibition of the organisms by the milk. B. THELACTOPEROXIDASE SYSTEM

The identification of L2 with lactoperoxidase (LP) was further strengthened when it was shown that both were destroyed by similar

BACTERIAL GROWTH IN MILK

57

processes and at a similar rate (Portmann et al., 1959). LP occurs in saliva, uterine fluid, and white blood cells (Reiter, 1973), and has since been found in goat milk (Zapico et al., 1990), ewe milk (Medina et al., 1989),and guinea pig milk and saliva (Stephens et al., 1979). It appears to exert a greater inhibitory effect in winter milk than in milk produced at other times of the year (Stadhouders, 1961) but more than LP is involved in the inhibition of microorganisms. Hydrogen peroxide (H202),the second component in the system, was discovered when it was shown that LP, but not horseradish peroxidase, inhibited S. cremoris 972 under aerobic conditions and catalase reversed this inhibition (Jago and Morrison, 1962). In a study (Pickering et al., 1962) involving S . lactis (C6, C10, 712, ML3) and S. crernoris (C7, 927, 972, 803), a wide range of hydrogen donors were oxidized by LP in the presence of a limited range of H,02 concentrations. H202 generating systems such as ascorbic acid, glucose plus glucose oxidase, and the hypoxanthine plus xanthine oxidase system added to the LP system inhibited lactic acid bacteria (Klebanoff et al., 1966). In milk, xanthine oxidase and ascorbic acid (Reiter and Oram, 1967) as well as H202produced by the organisms during aerobic growth (Jago and Morrison, 1962; Klebanoff et a]., 1966) could well be the source of H202 needed for the LP system to function. The microbial H202is generated by NADH oxidase, and if the organism produces insufficient NADH peroxidase, then H202builds up in the medium (Hogg and Jago, 1970a,d). Thiocyanate, the third component in the system, was discovered during studies on the antilactobacillus system in saliva (Klebanoff and Luebke, 1965).LP could replace a heat-labile, nondialysable component of the antilactobacillus system in which thiocyanate was already known to be a component, Iodide (Hogg and Jago, 1970c; Klebanoff and Luebke, 1965; Reiter and Oram, 1967), indican (Reiter and Oram, 1967), and bromide (Hogg and Jago, 1970c) can replace the thiocyanate in the system. Strain 972 but not 803 was inhibited by 2 p M KSCN in the presence of other components of the LP system and both strains were inhibited by 10 p M KSCN alone (Oram and Reiter, 1966a; Reiter and Oram, 1967). When thiocyanate was removed from milk with ion exchange resins 972 could grow normally in the cheese-making process (Reiter, 1973). With the recognition of H202as a component of the system, H202generating systems have been used to activate the system to control microbial growth. These H202-generatingsystems include urea peroxide (Earnshaw et al., 1989, 1990), the glucose oxidase system (Dionysius et al., 1992; Earnshaw et a]., 1989),and sodium carbonate peroxyhydrate (Dionysius et al., 1992) as well as pure H202(Siragusa and Johnson, 1989).

58

D. K. O'TOOLE

What is the inhibitor? It was suggested (Jago and Morrison, 1962) that an oxidized product of a substrate present in milk was the cause. Attempts (Oram and Reiter, 1966b) to isolate an intermediate failed but there were similarities between the activities of the product causing inhibition and sulfur dicyanide (S(CN),).However, the inhibitor (Hogg and Jago, 1970b) was pH dependent (the system is inactivated in milk by raising pH to 7.5), was unstable to heating at 60°C or above for 20 min, was stable for several days at a low temperature, had a pK, of about 5.2, and was influenced by excess thiocyanate in the medium. From a study (Maguire and Dunford, 1971) of the kinetics of formation of the primary compound from the LP-catalyzed oxidation of thiocyanate by H,Oz, it was found that the second-order rate constant, k,,was pH independent (range 3.0-10.8) and had a value of (9.2 k 0.9) x lo6 M-' sec-'. Either cyanosulfurous or cyanosulfuric acid was suggested (Hogg and Jago, 1970b) as the likely inhibitor, but hypothiocyanite (OSCN)was subsequently suggested (Aune and Thomas, 1977) and confirmed as the inhibitor (Pruitt and Tenovuo, 1982).Jones and Simms (1930) were proved wrong in that S was also involved in the inhibitor. The LP system was neutralized by catalase (Mickelson, 1966) when the organism was grown under aerobic conditions (Jago and Morrison, 1962) and was inhibited by thiosulfate, CSH, thiourea, methimazole, cysteine, ergothioneine, azide, ascorbic acid (Klebanoff et al., 1966), fluoride, cyanide (Pickering et al., 1962),and thioglycollic acid (Mickelson, 1966). Some thought (Stadhouders and Veringa, 1962) that the inhibition of lactic acid bacteria by LP and the prevention of that inhibition by cysteine were related. Cysteine was proposed to act as an H donor in a mixture with H,O, and LP, whereas in the absence of H,Oz it formed an irreversible compound with LP. Sensitive organisms may have an absolute requirement for free cysteine (Stadhouders and Veringa, 1962). The sensitivity of lactic streptococci to LP in milk was related to their capacity to inactivate LP (Stadhouders and Veringa, 1962). Pickering et al. (1962)found that lactic streptococci modified LP, and that all of the cultures of S. lactis and S . cremoris tested for sensitivity to LP showed a response similar to 803. Natural and modified LP differed in that natural LP showed a peak of activity at 0.1 mM H,O,, while modified LP showed activity over a broad range of H,Oz concentrations with peak activity at 30 mM H,O,; KF and KCN inactivated both forms but each form showed a different percentage inactivation by a given concentration of the inactivators; natural LP was more resistant to heat inactivation of 64°C for 20 min at pH 6.0 than modified LP, and velocity constants and activation energies of heat denaturation

BACTERIAL GROWTH IN MILK

59

differed for the two forms. Further studies showed that with strain C10 inactivation of LP was due to a buildup of excessive H,O, in the medium during aerobic growth; up to 0.11 mM H,O, was measured in the culture (Hogg and Jago, 1970a). Preincubation of 972 with LP and subsequent transfer of the cells to an LP-free medium containing the other constituents of the LP system resulted in inhibition of the organisms, which was counteracted by adding catalase to the LP-free medium (Steele and Morrison, 1969). In addition to modifying LP and producing excessive quantities of H,O,, the lactic streptococci produce an NADH and NADPH oxidizing enzyme which catalyzes the reduction of the inhibitor to a noninhibiting product (Hogg and Jago, 1970d; Oram and Reiter, 1966a). This enzyme was present in extracts of 803 and could neutralize the inhibitor for 972 (Oram and Reiter, 1966a). A schematic representation (Hogg and Jago, 1970d) of the action of LP on lactic streptococci is shown in Fig. 4.

FIG.4 . The postulated sites of activity of the lactoperoxidase-thiocyanate-hydrogen peroxide-generated inhibitor in lactic cultures. From Hogg and Jago (1970d).

60

D. K. O’TOOLE

How does the inhibitor influence the streptococci? Because the LP system prevented lactic acid formation and oxygen uptake in 972, it was suggested (Oram and Reiter, 1966a) that the system acts on the energy-generating process, i.e., the Embden-Meyerhof-Parnas pathway. The activities of enzymes in inhibited cells were examined and compared with those in uninhibited cells of two strains, 972 and 803 (Oram and Reiter, 1966a). Inhibited cocci of both strains had no hexokinase activity. Hexokinase in cell-free extracts was inhibited by the system and the inhibition was not reduced by dialysis or the addition of reducing substances (Oram and Reiter, 1966a). Thiocyanate alone did not inhibit hexokinase in extracts. Whole inhibited cells washed in Ringer phosphate showed normal hexokinase activity (Oram and Reiter, 1966a). Lactic dehydrogenase, NADH oxidase, and NADH peroxidase had normal activity in cell-free extracts of inhibited cocci, but some inhibited cocci (Oram and Reiter, 1966a) had higher phosphohexose isomerase activity [972), phosphohexokinase activity [803), and glyceraldehyde-%phosphate dehydrogenase activity (972, 803). Inhibited cocci also have partially inhibited fructose diphosphate aldolase activity (Hogg and Jago, 1970d). In resting cells of Lactobacillus acidophilus, though, the LP system inhibited the accumulation of glutamic acid and certain other amino acids (Clem and Klebanoff, 1966). In addition to the lactic starter organisms S. cremoris and S. lactis, and L. acidophilus, other organisms inhibited by the LP system are Streptococcus faecalis, Staphylococcus albus, Escherichia coli, Bacillus megatherium (Klebanoff et al., 1966),S. pyogenes, and S. agalactiae, which showed a delay of up to 6 hr before growth initiation that could be abolished with catalase (Mickelson, 1966). Recent reviews of the LP system include those of Pruitt and Reiter (1985) and Wolfson and Sumner (1993).

c. AUTOINHIBITION BY HYDROGEN PEROXIDE Another explanation offered for inhibition of lactic acid bacteria in milk is that they produce autoinhibitory levels of H,O, (Gilliland and Speck, 1969). Cultures were grown in reconstituted skim milk containing 0.01 mg/ml of catalase and H,O, levels were measured. Cultures containing catalase reduced the pH to 5.0 some 2 1 to 54 min faster than controls in which H,O, accumulated (Gilliland and Speck, 1969).There was no accumulation of H,O, in tubes containing catalase, whereas the addition of pyruvate reduced H,O, accumulation with no effect on acid production (Gilliland and Speck, 1969). Reduced glutathione was a

BACTERIAL GROWTH IN MILK

61

little more effective at preventing H,O, production and stimulated acid production slightly, but 0.5 mM FeSO, prevented H,O, production and greatly increased acid production, even more than catalase boosted acid production (Gilliland and Speck, 1969). Autoclaved catalase had no effect. A micrococcus isolate, F4, and a preparation of its capsular material increased growth and acid production of S. lactis, S. cremoris, Streptococcus thermophilus, Lactobacillus bulgaricus, and Lactobacillus casei var. casei (Nath and Wagner, 1973). Some of this stimulation was due to catalase but some other factor was also involved, as pure catalase could not restore the maximum stimulation obtained with the F4 associated material. The maximum accumulation of H,O, occurred during the most rapid decrease in pH (Nath and Wagner, 1973). The autoinhibitory levels of H,O, in milk could be modified by the natural levels of catalase in milk, which vary with the severity of subclinical mastitis in the cows producing the milk.

D.THIOCYANATE IN MILK In view of the importance of thiocyanate in the lactoperoxidase system, the concentrations that occur in milk and how they come to be there are of interest. Two precursors of this substance in milk isolated from plants of the Brassica family are glucobrassicin (Virtanen, 1961) and neoglucobrassicin (Reiter and Moller-Madsen, 1963), the former being a thioglucoside containing an indole group (Virtanen, 1961).Thiocyanate can be formed by enzymatic processes from mustard oil glucosides (Virtanen and Cmelin, 1960). Seasonal variations in the thiocyanate content of milk have been noted. In France a mean maximum of about 10 ppm NaSCN was obtained in summer, with a mean minimum of about 4 ppm NaSCN in winter (Boulange, 1959). Results from Victoria were similar; there was a yearly average of 4.9 ppm (range 1.4 to 10.8 ppm), the minimum values being obtained in August (Lawrence, 1971). In feeding experiments (Virtanen and Cmelin, 1960) a mixture of ammonium and potassium thiocyanate was fed to individual cows. This caused a jump in the thiocyanate content of milk from 0.24 to 1.4 mg Yo within 24 hr in one case, and in another case a jump to the same level from 0.12 mg Yo was observed. There was a lag period of 10 to 28 days before the thiocyanate content of the milk dropped to the pretreatment level (Virtanen and Cmelin, 1960). Clearly female diet can influence the thiocyanate content of milk.

62

D. K. O'TOOLE

E. LACTOFERRIN A red protein isolated from milk in 1960 (Groves,1960)is now known as lactoferrin. Studies showed that this protein had iron-binding properties (Gordon et al., 1962)and that bovine milk contained low levels of Fe (Oram and Reiter, 1968).Many bacteria require relatively high levels of Fe in their growth medium, such as species of Bacillus (Oram and Reiter, 1968) and E. coli (Reiter, 1978),and when they are inoculated into milk containing high levels of lactoferrin their growth is retarded or death occurs. Thus the activity of lactoferrin is antinutritional in character. However, the antibacterial effect of lactoferrin is neutralized by citrate in the milk (Bishop et al., 1976). Levels of lactoferrin in bovine milk vary from 0.1 to 0.35 mg/ml (Gaunt et al., 1980),but in human milk levels are very high (greater than 2 mg/ml) (Reiter, 1978). In bovine milk the level of lactoferrin is inversely related to the milk production levels of individual cows (Thomas and Fell, 1985). F. OTHER POSSIBLE INHIBITORS FOR LACTIC ACIDBACTERIAIN MILK Other inhibitors for bacterial growth in milk are possible but have not yet been recognized. The fatty acids caprylic, capric, lauric, and myristic showed some inhibition for S. cremoris, S. lactis and Streptococcus diacetilactis, whereas oleic acid was more selective, mainly affecting S.cremoris C13 (Anders and Jago, 1964).The oleic acid altered the metabolism of pyruvate by C13 by inhibiting the enzyme involved in the formation of acetate (Anders and Jago, 1970). Butyric, caproic, palmitic, stearic, arachidic, linoleic, and linolenic acids were ineffective (Anders and Jago, 1964). Various milk-associated volatile compounds (Table 11) were found by the disc assay method to be inhibitory for strains of S. lactis, S . cremoris, S . diacetilactis, Leuconostoc citrovorum, and S. aureus (Kulshrestha and Marth, 1970). The influence of fatty acids in milk may be complicated by an interaction with Ca2+ions (Galbraith et al., 1971). Streptococcus faecium is inhibited by 1 mM sodium lauryl sulfate, 0.35 mM linoleic acid, and lauric acid; however, the activity of the latter compound was counteracted by 1 mM CaZ+and 1 mM Mgz+,and that of linoleic by 2.0 mM Ca2+but not by Mg2+(Galbraith et al., 1971). Of 42 compounds tested for their inhibitory activity against S . agalactiae (see Appendix B) 8 were inhibitory at 200 pg/ml with serine, tyrosine, and glycyl-1-glutamic acid being least inhibitory (Brown,

63

BACTERIAL GROWTH IN MILK TABLE I1

SOME MILKASSOCIATEDVOLATILECOMPOUNDSTHATCANCAUSEINHIBITIONOF BACTERIAL GROWTH Substance (100 ppm)

S. lactis

S . cremoris

S . diacetilactis

L. citrovorum

Formaldehyde Glyoxal Decanoic acid Butyric acid Formic acid Hexanoic acid Diacetyl

+ + +"

+ + (1 strain] +" +a +a +a

+ + + ( 1 strain]

S. aureus _____

~~

+ + +

+ +

+

+

When tested at 100 ppm these substances caused no inhibition. Note. From Kulshrestha and Marth (1970).

1974).The most inhibitory were L-asparagine, L-isoleucine, L-leucine, L-methionine, and L-valine. The D-isomers of isoleucine and leucine were inactive up to 100 pg/ml. Only D-a-keto-p-methyl-N-valeric acid, of the precursors of isoleucine, leucine, and valine, was active at 1 mM or less (Brown, 1974).L-Cystine, which could neutralize to some extent the natural inhibitors of milk, could not counteract the effect of Lisoleucine (Brown, 1974). It is possible that some of the above free amino acids may be the cause of inhibition of lactic acid bacteria in milk under natural conditions. However, of these substances, it is more likely that the fatty acids are a cause of inhibition because of the phenomenon of lipolysis in raw milk. Under natural conditions and in some circumstances the natural lipase in some milk supplies can be activated and a reasonable amount of free fatty acid produced before the milk is processed. These residual fatty acids can cause defects in milk and may also interfere with the activity of bacteria in milk.

G. SEASONAL VARIATIONIN BACTERIALACTIVITY IN MILK

Bacterial activity in milk apparently varies with the seasons (Czulak and Meanwell, 1951;Hunter, 1949).This seasonal variation could be due to either the practice of seasonal calving, which is accompanied by physiological changes in the milk associated with the lactation of the cow, or the availability of pastures for the animals. No studies have set out to separate these two factors. Brown and McDonald (1982)used acid production and plate counts to measure the effect of lactation of individual cows on the growth of

64

D. K. O’TOOLE

S. agalactiae. They found that over the lactation as the yield of milk dropped the suitability of milk for the growth of the culture increased. In the milk from one cow, counts after growth in milk from early versus late lactation varied from 0.4 to 1.6 x lo9 CFU per milliliter. Bactericidal and bacteriostatic activity of milk for S. aureus var. aviarius was less apparent in February (Northern Hemisphere) than at other times of the year (Bergaminiand Solinas, 1950).The most notable effect is on starter activity. A low period of activity was noted in England from December to March (Czulak and Meanwell, 1951) and a similar activity pattern (Fig. 5) was noticed in Australia (Jago, 1954). In New Zealand when seasonal calving was practiced, one fast strain, C13, showed decreasing activity as the season progressed but a slow strain, AM1, showed an increase in activity (Lawrence and Pearce, 1972). Season, particularly spring, had a significant effect on gas, acid production, and multiplication of S. diacetilactis and Streptococcus paracitrovorus, the latter being quite sensitive (Gibahman, 1960). Inhibition seemed greatest when pastures were in a state of decline (Jago and Swinbourne, 1959). Others have noted no seasonal effects (McEwen

0.8

0.7 U .-

m U

U .-”

m

0

; 0.6 ” 2 8

‘ 2

0.5

0.4

I 1 Sept.

I

I

4

I

I

4

Oct.

Nov.

Dec.

Jan.

Feb.

Mar.

I

I

I

I

I

Apr.

May

June

July

Aug.

Months of the year (1952-3) FIG.5. Variation over 1 year (Southern hemisphere) in lactic acid production by lactic cultures resistant and suseptible to the natural inhibitors in raw milk when grown in boiled and raw milk. Curves A and C show results from susceptible strains in boiled (0) and raw [O)milk, and curves B and D are results from resistant strains in boiled [A)and raw (A) milk. There were eight susceptible and two resistant strains. From Jag0 (1954). Reprinted with permission of Cambridge University Press.

BACTERIAL GROWTH IN MILK

65

and White, 1950), and Auclair found no correlation between season and lactenin content of bulk milk (Berridge, 1955). But the ability of certain strains of S. Iactis to ferment normally in spring season milk was linked with the organism’s greater proteolytic activity, its ability to synthesize nicotinic acid, and its requirement for more biotin but less riboflavin (Speck, 1962). An attempt (Reiter, 1973) to correlate seasonal variations in free and protein bound amino acids with the occurrence of difficulties in cheese and butter manufacture, associated with slow starter growth, ended in failure despite a sophisticated approach. In an earlier attempt (Anderson et al., 1955) to correlate the peptide, peptone, and proteose peptone content of milk with activity of lactic acid bacteria, a correlation between increased peptide content and lactic acid production was reported. The evidence, however, was not very strong. This slowness of starter activity, particularly in winter months, was thought to be linked with availability of nitrogen in the milk (Garvie and Mabbitt, 1956). Czulak and Meanwell (1951) originally postulated that there was a constant concentration of inhibitor which was counteracted by a growth factor in milk which varied with the season, being lowest in winter. They thought that uninhibited cultures could synthesize the growth factor. Stadhouders (1961) noted higher inhibitory activity due to peroxidase in winter. He suggested that a thermolabile stimulator inactivated peroxidase partially but irreversibly in summer milk, and that either thermostable substances or stimulative substances formed on heating were also involved. Despite this he did not detect a particularly noticeable seasonal variation in starter activity, although he did note a seasonal variation in agglutinins (Stadhouders, 1963). LP system-associated susceptibility of Streptococcus uberis, another mastitis-causing organism, to late lactation milk, secretions obtained after drying off and up to 21 days postcalving, was positively related to the susceptibility of cows to challenge from the organism (Marshall et al., 1986).

H. HEATED MILKAS A GROWTHMEDIUM FOR LACTICACIDBACTERIA In many of the studies of the inhibitors in milk, heated milk of some description has been used as a diluent. It is assumed that heating to kill extraneous organisms has no effect on the growth-supporting or -inhibiting properties of milk, but this assumption is not valid as Brown (1967) has shown (Fig. 6). Whether the diluent is steamed or autoclaved can have a significant influence on results. A summary of studies of

D. K. O'TOOLE

66 7.0 .8

.6 .4 .2

6.0 .8

pH

.6 .4

.2 5.0

,..

.8

.6 .4

4.2

*

.. .. .. .. .. ..

t I

I

I

I

I

I

I

0

2

4

8

16

32

64

'

I

'

128

256

512

Dilutions FIG.6. The effect of different heat treatments of the diluent milk on the inhibitory activity of pasteurized milk for S . agalactiae 660. Diluent milks were heated at (1) 80°C for 30 min, (2) 90°C for 30 min, (3) 100°C for 30 min, (4) 121OC for 15 min. From Brown (1967).

the effect of heating on the growth-supporting properties of milk are tabulated in the brief review of Feldstein and Westhoff (1979). More specifically, the ability of lactic acid bacteria to grow in milk heated at various temperatures and times has been studied. Acid production by s. lactis and s. cremoris was more rapid in milk heated to 76.7"C momentarily than in milk heated at 62.8"Cfor 30 min (Rice, 1942).A culture of S. lactis grew better in autoclaved skim milk (115°C for 15 min) than in low heat milk (80°Cfor 10 min) although autoclaved milk showed greater variability in its growth-supporting properties; with increased time of heating at 80 and 100°C there was increased stimulation of starter growth (Foster, 1952). The beneficial effect of heating milk strongly was lost when the casein was removed (Foster,

BACTERIAL GROWTH IN MILK

67

1952). Autoclaved milk stimulated acid production in low heat milk; one part to two parts low heat milk as well as autoclaved milk alone supported growth, and peptonized milk (100 pg/ml) stimulated acid production in low heat milk in a similar manner (Foster, 1952). Cycles of stimulation and inhibition of lactic starter cultures, linked with the presence of denatured serum protein cysteine, were observed in milk heated to various temperatures (Greene and Jezeski, 1956). In milk heated for 30 min there was a primary period of stimulation up to 72°C inhibition at 80 and 9O"C, and further stimulation at 95 and 120°C. Inhibition caused by heating at a temperature below 100°C was a unique function of the available cysteine concentration in milk. Volatile sulfides arising from heated sulfydryl compounds were also implicated (Greene and Jezeski, 1956), evidence for which is provided by Henningson and Kosikowski (1957) who found that if S. pyogenes grew in whey heated to 80°C for 5 or 10 min they could demonstrate the presence of free sulfydryl groups. As the maximum growth of the organism in whey heated at 100°C for various periods decreased, there was a corresponding decrease in the concentration of sulfydryl groups as shown by a decrease in the reaction intensity of the nitroprusside test (Henningson and Kosikowski, 1957). Heated, i.e., denatured, lactalbumin had a stimulating effect similar to that of heated milk (Henningson and Kosikowski, 1957). A study (Lyster, 1964) of the effect of heating skim milk at various temperatures for 15 min on the liberation of sulfydryl groups has shown (Fig. 7) that as milk is heated above 70°C there is a rapid increase in the concentration of free sulfydryl groups in the milk. A similar response pattern was obtained when the milk was heated for 1 min at similar temperatures (Kirchmeier et al., 1984). These observations can explain some of the variation in the ability of heated milk to support the growth of lactic acid bacteria.

I. EFFECTOF CULTURE MANIPULATIONS The sensitivity of some strains to the inhibitors in milk is affected by the maintenance procedures adopted for the culture before testing. Some strains, e.g., S. lactis ClO, although insusceptible to inhibitors in raw and pasteurized milk, developed susceptibility on prolonged daily subculture in autoclaved milk (Jago and Swinbourne, 1959). This was due to the presence in the ClO culture of cells resistant and sensitive to both lactoperoxidase (LP) and agglutinin (Auclair and Vassal, 1963). Those sensitive to agglutinin were also sensitive to LP, and those sensitive to LP were not necessarily sensitive to agglutinin. Streptococcus

D. K. O'TOOLE

68 0.18

1

0.16 0.14 0.12

5z-

0.10

Y 0.08 0.06 0.04 0.02

40

50

60

70

80

90

100

Temperature,'C FIG. 7. The -SH content of skim milk heat for 15 min at various temperatures. (0) Free -SH,(0)total -SH.From Lyster (1964).Reprinted with permission of Cambridge University Press.

cremoris 972 contained cells sensitive to both inhibitors but the agglutinin sensitive 760 contained cells (100%) sensitive to agglutinin and cells (48%) sensitive to LP (Auclair and Vassal, 1963). Auclair and Vassal (1963) suggested that subculturing in autoclaved milk unmasked a specific antigen on the surface of the cells for which a homologous antibody exists normally in cow milk. More recently, growth conditions for a Listeria monocytogenes Scott A culture, a nonlactic organism, were shown to influence the length of lag periods induced by the lactoperoxidase system. Culture inocula prepared from shaken cultures began growth sooner than statically grown cells (Siragusa and Johnson, 1989). Apparently aerobic growth prepared the organisms better for growth in the presence of the LP system than anaerobically grown cells.

J. EFFECTOF MILKON MORPHOLOGY OF STARTER CULTURES In a comparison (Morris and Edwards, 1949) of growth rates of S. Iactis, as measured by the number of colony forming units (CFU) per milliliter and methylene blue reduction times, it was found that CFU per milliliter was lower in raw milk than in heated milk (70°C for 1hr)

BACTERIAL GROWTH IN MILK

69

even though methylene blue reduction times were similar. Cultures could be placed in three groups: (a) those forming long chains in raw milk and short chains and diplococci in heated milk; (b) those forming clumps in raw milk; and (c) those forming short chains in raw and heated milk. The ratio of the average numbers of cocci per chain in raw and heated milk for six strains were 21.25 : 1.7; 2.5 : 1.4; 6.9 : 1.0; 3.6 : 1.0; 11.0 : 1.75; and 1.0 : 1.2. A protein was thought to cause the phenomenon, but the clumping was not due to agglutination and the chain inducing ability of milk was destroyed when milk was heated at 68 and 70°C for 1 hr (Morris and Edwards, 1949). Peptone added to milk (Garvie and Mabbitt, 1956) changed the morphology of lactic streptococci; in its presence the long axis of the organism was along the chain, and in its absence the long axis of the cocci was across the chain. V. Effect of Cow Ration on Milk as a Growth Medium for Bacteria

Because the thiocyanate concentration in milk can be increased by altering the cow’s ration, one could expect a cow’s ration to influence bacterial growth in her milk as cows graze different pastures and consume different rations. Pounden and associates investigated aspects of the effect of cow rations on the growth of S. agalactiae, an important lactic acid bacterium that causes mastitis. They wanted to measure the relative resistance of animals to S. agalactiae without actually exposing the animals to the organism, so they measured acid production by the organism in the milk-the less acid produced the more resistant the milk, and perhaps the cow, to the organism (Pounden, 1952). Among factors examined were the feeding of vitamin A, citrates, and phosphates, intravenous injections of vitamin E, phosphates, and ascorbic acid, and the roughage fed (Pounden et al., 1952). Only roughage seemed to have any effect, but other factors could not be ruled out (Pounden, 1952).

Milk from a group of Holstein-Friesian and Jersey cows receiving high-quality roughage (alfalfa hay and silage) was consistently more inhibitory for S . agalactiae than milk from a group fed low-quality timothy hay (Pounden et a ] . ,1952). In another experiment three groups of cows were selected and put on blue grass pasture, on a legume and brome grass pasture, and on a low-quality mixed hay, mostly timothy, which had an average protein content of 7.16%; alfalfa hay had an average protein content of 17.51% (Pounden, 1952). During three seasons milk from the group fed low-quality hay was significantly (P < 0.01) more inhibitory for the microorganism than milk from cows on higher-quality pasture (Pounden et a]., 1956a). Introduction to pas-

70

D. K. O’TOOLE

ture after feeding stored food during winter was correlated with increased incidence of streptococcal mastitis (Pounden et al., 1956a). The addition of corn silage to a timothy hay and grain ration resulted in greater acid production in milk (Pounden et al., 1956b). When changing from pasture (alfalfa, blue grass) to a forage crop silage there was a slight increase in the inhibition of organisms in the milk, but a transfer from pasture to a hay, grain, and silage (corn or forage crop) ration seemed to have no effect on acid production by the organism (Pounden et al., 1956b). Cows on a ration of fresh-cut pasture of alfalfa, Ladino clover, and grass (soilage) or a ration of silage made from that pasture (plus a grain mixture in both cases) showed a difference in resistance to streptococcal mastitis; the silage-fed group had a lower incidence of mastitis (Pounden et al., 1958) and their milk had greater inhibitory activity for S. agalactiae (Pounden et al., 1960).The activity of a hemolytic staphylococcus was also lower in milk from silage-fed cows, as the organism took longer to reduce methylene blue in such milk than to reduce methylene blue in milk from silage-fed cows. Pounden’s main concern was mastitis; he presents evidence of changes in the growth-supporting properties of milk as cow ration changes, and he notes flare-ups of mastitis which occur coincidentally with these ration changes (Pounden and Frank, 1961; Pounden et al., 1958). Pounden et al. (1958) noted, for example, one herd in which 5% of cows had mastitis in the month before going on to pasture and 28% had mastitis in the month following the ration change. Changes in the growth-supporting properties of milk tended to be inversely related to level of milk production, i.e., low milk production was related to increased bacterial inhibition (Pounden et al., 1957), which is contrary to the results of Brown and McDonald (1982). When cows were placed on pasture there was an initial decrease in bacterial inhibition which was followed by a partial recovery of the inhibitory properties of milk (Schalm et al., 1971). One explanation (Schalm et al., 1971) offered for Pounden’s observations is that pasture increased milk secretion, thus diluting leucocyte numbers and humoral factors which normally act as bactericidal agents in milk. Schalm et al. (1971) suggested that the partial increase in inhibitory factors is due to subsequent reestablishment of the disturbed equilibrium of the defence mechanisms. No inhibitory effecton yoghurt cultures, S . lactis or S. thermophilus, was noted in milk from cows fed a weed, Lepidium bonariense (Luck et al., 1966). The activity of a lactic streptococcus, ML, was greatly reduced during a drought and improved markedly immediately after rain (Hunter, 1949). The addition of 0.01% tryptone to cheese milk counteracted the adverse effect of drought on milk (Hunter, 1949).Speck

BACTERIAL GROWTH IN MILK

71

(1962) noted variations in milk composition, including proteose-peptone content, due to pasture and dry feeding but found no correlations with culture activity. Differences in starter growth were more dependent on culture characteristics than cow ration (Speck, 1962).

VI. Role of Inhibitors

in Milk Quality

Inhibitors in milk have played a number of roles. First, they have been found to influence tests for coliforms and for antibiotic content of milk. Second, they have been used to manipulate the keeping quality of milk, as outlined later in this review. A. RAW MILKQUALITY Milk quality depends on its microbiological status which is shown by various traditional tests. Trout et al. (1951) found that the inhibitory properties of milk were instrumental in maintaining the bacteriological and organoleptic properties of raw milk. Although they were thought to improve the keeping quality of milk from tropical countries, no proof of this was found in lactenin levels (Auclair, 1968). The inhibitors may also mask the true bacteriological condition of raw milk (Morris, 1945). Milk containing large numbers of coliforms but having long methylene blue reduction times, that is, apparently of good quality, contained substances which were specifically bactericidal and bacteriostatic for coliform organisms (Morris, 1943).In addition, Clegg et al. (1956) found that they retard the growth of thermoduric bacteria in raw milk but do not improve the keeping quality of pasteurized milk, a view contradicted by recent studies which showed that activation of the LP system in milk improved the keeping quality of pasteurized milk (Kamau et al., 1991). When testing milk for the presence of Campylobacter jejuni the rate of isolation was improved by inactivating the LP system by raising the pH to 7.5 (Beumer et al., 1988). During one test for antibiotic residues in milk a culture of Bacillus stearothermophilus var calidolactis is used (other cultures are also used) and false positives are obtained, sometimes due to the natural inhibitors in milk. Lysozyme, lactoferrin, and the LP system inhibit the organism at levels found in milk, and there was a synergistic effect between lysozyme and lactotransferrin (Carlsson and Bjorck, 1987). It was suggested that with elucidation of the LP system in milk, the system could be activated to improve the keeping quality of refrigerated milk in view of its activity against pseudomonad bacteria (Bjorck, 1978);

72

D. K. O’TOOLE

this idea was extended to short time keeping quality of milk in tropical countries with low-technology dairy farming environments (Bjorck et al., 1979a). By adding 10 ppm thiocyanate and 8.5 ppm hydrogen peroxide, the level of bacteria in milk at 30°C remained static for 7-8 hr (Bjorck et al., 1979b). Tests in Sri Lanka (Harnulv and Kandasamy, 1982) and Kenya (Ridley and Shalo, 1990) showed that the method could be used to extend the keeping quality of water-cooled evening milk. The treatment did not affect the quality of the milk for cheddar and cottage cheese manufacture (Zall et al., 1983), an important point in view of the effect of natural inhibitors on the growth of lactic acid bacteria. However, acid production and cell growth by yoghurt cultures were delayed in milk in which the LP system was activated (Basaga and Dik, 1994). VII. Stimulators of the Growth of Lactic Acid Bacteria

Although a stimulator in milk is postulated, its identity is not known. The most common approach to the problem has been to test compounds at random for their stimulative properties in milk. In addition, many compounds have been found which are essential or stimulate the growth of lactic acid bacteria under certain conditions. A. INVESTIGATIONS OF MILKAND MILKCOMPONENTS Auclair (1954a) noted the stimulation of S. agalactiae by colostrum, an observation made earlier with S. lactis and S. cremoris for which the addition of 10-25% colostrum induced more rapid acid production (Rice, 1942). Among the substances tested by Auclair which might replace the activity of colostrum, it was found (Auclair, 1954a) that only sulfur-containing compounds such as NaS and thioglycollic acid (1 mM) and cysteine and cystine (0.01 mM) were effective. Auclair (1954a) suggested that the concentration of certain sulfur compounds normally present in milk was responsible for marked differences in the growth rate of some organisms. The rate of acid production by lactic streptococci varied in different milk fractions (Gilliland and Olson, 1963), whole milk, skim milk, buttermilk, whey, whey from buttermilk, and buttermilk from washed cream. Acid production was more rapid (Gilliland and Olson, 1963) in whole milk and buttermilk than in skim milk, and even more rapid in a 1: 1 mixture of the skim milk and buttermilk. Brown (1974) studied 110 low-molecular-weight compounds (see Appendix B) for possible stimulators of S. agalactiae with the aim of

BACTERIAL GROWTH IN MILK

73

finding what may be the natural stimulators of milk. All compounds were tested in a 1 : 3 2 dilution of pasteurized skim milk (60°C for 30 min) in steamed skim milk (100°C for 45 min) supplemented with 300 pg/ml of NH,SCN. The only stimulators (at an initial concentration of 1pg/ml) found were hydrolyzed cephalin, cytidine-5'-triphosphate, and sulfur-containing substances which, in descending order of stimulative activity, were L-cystine, L-cysteine, DL-homocystine, reduced glutathione, DL-homocysteine, dithiothreitol, NaS, and sodium thioglycollate; DL + allo-cystathionine was initially inhibitory but became stimulative on dilution.

B. STIMULATION BY MINERALS Mineral requirements and their interaction with other requirements of the organisms have been studied. Streptococcus cremoris HP and S. lactis C10 show a requirement for Fe and V, the latter metal replacing the requirement for the former, but Mo was without effect (Reiter and Oram, 1968). The quantities for maximum growth of the organisms were 0.002 ppm (ClO),0.001 ppm (HP) of Fe, and 0.01 ppm (ClO), 0.04 ppm (HP) of V (Reiter and Oram, 1968). Fe activated aldolase in cell-free extracts of lactic starters, and the sequestration of Fe in milk reduced lactic acid production by S. thermophilus (Reiter and Moller-Madsen, 1963). Strains of S. cremoris and S. lactis were stimulated by the addition of 0.4% sea salt or 0.4 ml seaweed (kelp) extract per 10 ml of pasteurized skim milk (Olsen and Qutub, 1970). Of 1 2 trace elements present in seaweed, Fe, Mg, and Mo, each at 2 ppm, Se at 1 ppm, 2-4 ppm Zn, and 2 ppm Co stimulated acid production; B, Pb, and Li (at 8 ppm) had no effect or were slightly inhibitory, but Cu, I, and Hg at >4 ppm were inhibitory. A mixture of Fe (2 ppm) + Mg (4 ppm) + Se (0.5 ppm) significantly (P < 0.01) increased the rate of acid production in milk (Olsen and Qutub, 1970). A stimulative component of tomato juice was replaceable with Mn and 63 of 71 strains of lactic acid bacteria required this mineral (Stamer et al., 1964). Lactobacillus acidophilus and Lactobacillus bifidus were stimulated when 0.1-0.3% hexametaphosphate was added to a modified milk, presumably due to the removal of Ca2+ions (Sasaki et al., 1959). The amino acid requirements of s. thermophilus were partially dependent on and affected by the presence of Ca2+in the medium (Reiter and Moller-Madsen, 1963). This organism requires K+ and Mg2+;the latter is not replaceable with Ca2+ (Reiter and Moller-Madsen, 1963). Streptococcus lactis and S. cremoris responded to acetate and citrate, a reaction that may be related to Ca2+and Mg2+requirements of the organisms (Reiter and MollerMadsen, 1963).

74

D. K. O’TOOLE

C. COMPLEX SUBSTANCES AS STIMULATORS

In addition to simple organic compounds there are some complex substances which stimulate lactic acid bacteria and which have been studied to determine their active constituents. Some of these stimulators are pancreas extract (Koburger et al., 1963; Sandine et al., 1956),tomato juice (Stamer et al., 1963),corn steep liquor (Johnson et al., 1971; Sasaki et al., 1959), a liver extract (Garvie and Mabbitt, 1956; Sasaki et aJ., 1959), an aqueous extract from thermophilic methane fermentations (Khandak et al., 1972), chitin hydrolysate (Sasaki et al., 1959), peptone (Wilson and Rosenblum, 1952a), and tryptone (Hunter, 1949). Tryptone stimulated the growth in milk of the slow strain S. cremoris ML, and cystine, cysteine, and glutathione, but not tryptophan, produced stimulation similar to that produced by tryptone (Hunter, 1949). However, tryptophan and threonine were stimulative for strains of S. cremoris in another study (Reiter, 1973). Generally, strains of S. lactis grow adequately using sodium caseinate as the sole nitrogen source; S. cremoris is more fastidious and requires alanine and proline at concentrations similar to those found in milk, and some strains require tyrosine and lysine (Reiter, 1973). Pancreas extract (Dahiya and Speck, 1963) improved acid production by lactic streptococci in fresh milk so that it compared favorably to reconstituted skim milk. This extract could be replaced by inosine, the best of the purines and purine ribosides tested, but the availability of various nitrogen sources, such as amino acids and peptides, also seemed important (Dahiya and Speck, 1963). In an earlier study (Sandine et aJ., 1956) two stimulants for L. casei and S. lactis were found in pancreas extract. They were active in the presence of “Streptogenin” (a term coined by Woolley to describe peptides and crude extracts containing peptides that stimulate bacteria of the family Lactobacteriaceae). One appeared to be a peptide containing lysine, aspartic acid, serine, glycine, glutamic acid, threonine, alanine, proline, valine, and probably leucine and isoleucine. A peptide (MW ~ 4 7 0 0 produced ) by S. Jactis which stimulated L. casei had similar composition but contained, in addition to the above, arginine, histidine, ornithine, phenylalanine, and tyrosine; none of these are sulfur amino acids (Branen and Keenan, 1970). Johnson et al. (1971) implicated inosine (1 pg/ml) and hypoxanthine adenine in pancreas extract as the stimulants for S. Jactis. Two strains of S. cremoris (KH and HC) produced a substance stimulating acid production by R6 which was not a peptide, amino acid, or an amine; by UV spectral analysis, it appeared to be guanine which, like adenine and guanosine, stimulated growth

BACTERIAL GROWTH IN MILK

75

when added to milk (Kothari and Nambudripad, 1973). Adenine and adenosine were the active constituents in tomato juice stimulating L. bulgaricus (Cogan et al., 1968a) but an inhibitor also present for this organism was a nucleotide containing xylose and adenine (Cogan et al., 1968b). For L. citrovorum tomato juice contained the stimulants fructose and CO,. Corn steep liquor (Johnson et al., 1971) contained four constituents that stimulated S . lactis and S . cremoris, one of which was a peptide containing an aromatic amino acid; the others were not nucleotides or nucleosides, but could have been purine or pyrimidine bases. When the growth of streptococcal starter cultures were compared in fresh skim milk and reconstituted skim milk, growth in the fresh skim milk was reduced but could be enhanced by the addition of inosine; however, purines and purine ribosides were not as effective (Dahiya and Speck, 1964). Hydrolyzed casein and yeast extract stimulated L. casei (Hutchings et al., 1941). There were two separate substances, one of which was biotin. The other was unknown but contained an acid and probably an amino group, and was not a nucleotide (Hutchings et al., 1941). This substance stimulated Lactobacillus helvecticus, Lactobacillus delbruckii, S. lactis, and Propionibacterium pentosaceum (Hutchings et al., 1941).Under defined conditions, p-aminobenzoic acid, methionine, adenine, guanine, hypoxanthine, and xanthine were growth factors for Lactobacillus arabinosus, and Lactobacillus pentosus, S . lactis, and Leuconostoc mesenteroides responded to purine bases but not to the other substances (except that adenine did not stimulate L. mesenteroides) (Snell and Mitchell, 1942). A L. bulgaricus strain requiring lactose, but not growing on glucose or galactose, was stimulated by pyruvate (Snell et al,, 1948). When added to media containing all known growth essentials, peptone, and plant and animal tissue extracts, increased early growth of L. casei. The growth factor (Pollack and Lindner, 1943) neither was a peptide nor was it glutamic acid which did cause a strong growth response. The vitamins required by S. lactis, S . cremoris, S. diacetilactis, and S . thermophilus are nicotinic acid, pantothenate, and biotin, but pyridoxal stimulates growth (Reiter and Moller-Madsen, 1963). Menadione (2-methyl-1&naphthoquinone), related to vitamin K, has been suggested (Berridge, 1955) as the inhibitor associated with cream in milk ( Jago, 1954). When menadione and menadione diphosphate (tetrasodium 2-methyl-l,4-naphthohydroquinone diphosphate acid ester) were fed to cows it had no effect on starter culture activity even though menadione added to milk was inhibitory (Wilkowske et al., 1955).

76

D. K. O’TOOLE

In synthetic media glutamine and glutamic acid stimulated the growth of S. lactis, L. pentosus, L. arabinosus, and L. casei (Pollackand Lindner, 1942). Another growth factor for S. Jactis was not a known vitamin, nor could it be replaced with a combination of 23 amino acids (Smith, 1943). Folic acid, however, was suggested as a growth factor for this species (Keresstesy et a]., 1943).A substance in yeast extract stimulating the oxidation of pyruvate by S. faecalis was not leucine, phenylalanine, methionine, cysteine, glutathione, folic acid, or vitamins (O’Kane and Gunsalus, 1948), but it was suggested (Snell and Brequist, 1949) that it may be the same as a factor replacing acetate which stimulated L. casei. Acetate is also essential for lactic acid streptococci, but in S. faecalis it was replaceable with lipoic acid or mevalonic acid (Reiter and Moller-Madsen, 1963). Sodium inocinate was sufficiently stimulative for L. bulgaricus to reduce the manufacturing time for yoghurt by 1 hr. This organism and S. crernoris were also slightly stimulated by lacticol monostearate (Tsugo and Tanigucki, 1962). N-Acetyl-glucosomine, 1-amino lactose, lactulose, and N-acetyl lactosamine had no effect on various lactic acid bacteria (Tsugo and Tanigucki, 1962).Formic acid, up to 50 ppm, stimulates the growth of L. bulgaricus when growing with S. therrnophilus (Bottazzi et al., 1971). The growth of lactic streptococci was stimulated in milk to which pgalactosidase was added (Gilliland et al., 1972). This was apparently due to the organisms ability to grow faster on the glucose released by the enzyme than on lactose (Gilliland et al., 1972). D. COMMERCIAL STIMULATORS

Several commercial stimulators have been developed, particularly to overcome periods of low culture activity in milk such as spring and autumn (Hylmar et al., 1969).There was “stimulac” from East Germany, and “starter” composed of (Hylmar et al., 1969) yeast extract (10 g), casein acid hydrolysate (15 g), casein enzyme hydrolysate (15 g), peptone (15 g), calf heart infusion (150 ml), pancreas infusion (150 ml), calf muscle infusion (150 ml), 85% lactic acid (20 ml), distilled water (500 ml); an inoculum of 0.1%in milk was recommended. Yeast autolysate or yeast hydrolysate at 0.05-0.02% was recommended (Gibahman, 1960). Another commercial agent, “Lac-Val” (essentially a soluble yeast extract), stimulated (Weber, 1971) S. lactis, S. therrnophilus, S. diacetilactis, and L. helveticus at concentrations of 0.3-0.67%. The agent is thought to stimulate through reducing the lag phase (Weber, 1971).

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VIII. Growth Inhibitors of Other Bacteria

A. INHIBITORS FOR ENTEROBACTERIA The enterobacteria comprise a wide range of organisms including the coliforms. Milk samples containing large numbers of coliforms but exhibiting long methylene blue reduction times led to the observation that milk contained substances which were specifically bactericidal and bacteriostatic for coliform organisms [Morris, 1943). When inoculated into raw milk at 37°C the coliform count dropped from about 400,000 to 80 per ml within 6 hr [Morris, 1943). The substance is present in milk and whey in equal concentrations; in milk serum, 0.25% gave 99% kill of the organisms in sterile milk [Morris and Edwards, 1950). The concentration varied with the source of cow milk, just as it did in goat milk, and it was destroyed after 30 min at 53°C but was not destroyed within a similar time at 52°C [Morris and Edwards, 1950). Suspensions of heat-killed coliform organisms could absorb the factor but the effectiveness of the absorption depends on the temperature and time combination used to kill the organisms. Dead cells of S. lactis were also effective as absorbents of the factor [Morris and Edwards, 1950). Agglutination of coliform organisms by raw milk has been demonstrated [Portmann et al., 1962). Milk taken 1 2 hr after the induction of the inflammatory response in a cow udder by the injection of distilled water caused a sharp decline in numbers of Aerobacter aerogenes [ Jain et a]., 1967). Heating at 56°C for 30 min removed the effect, and 1% guinea pig serum added to the heated milk and to normal milk with no bacteriostatic properties resulted in reduced A. aerogenes counts [ Jain et al., 1967). This same organism added to normal milk and mastitic milk [somatic cell counts of >500,00O/ml) showed a range of responses. Some normal milks supported growth but others, and mastitic milks, either inhibited the growth of the organism or killed it [Carrol and Jain, 1969). Colostrum had low bactericidal activity which was enhanced in some cases by guinea pig serum. Heat-killed A. aerogenes removed the activity from milk and serum [Carrol and Jain, 1969). These observations can be explained by the presence of complement in milk which has been detected using guinea pig erythrocytes and a conglutin test (Reiter and Oram, 1967). It is regularly found in bulk milk, in colostrum, and toward the end of lactation but is irregularly present in normal cow milk [Reiter and Oram, 1967). Reiter and Oram (1967) suggest that complement and the formation of antibody-antigen complexes with gram-negative organisms are the cause, in many in-

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stances, of the bactericidal activity of milk against gram-negative organisms. Species of Yersinia (Farrag and Marth, 1992) are also killed by milk and can be controlled by the activation of the LP system. B. INHIBITORS FOR BACILLI An inhibitor which specifically inhibits Bacillus species was reported (Cheeseman and Jayne-Williams, 1964). First found affecting B. stearothermophilus, 23 of 60 spored thermophiles were inhibited by the substance. Other species inhibited are Bacillus coagulans (Ashton and Busta, 1968),Bacillus subtilis (Oram and Reiter, 1968; Pugh, 1970),and Bacillus calidolactis (Pugh, 1970), a variety of B. stearothermophilus used in tests to detect antibiotics in milk. The inhibitor is present in raw, pasteurized, and dried milk and is associated with the 30% (NH,),SO, precipitate from milk serum at pH 6.5 (Cheeseman and Jayne-Williams, 1964). It is stable on heating to 80-90°C for 60 min but is destroyed by 121°C for 15 min. The factor is not dialysable, separates with milk protein when filtered on Sephadex C-25, and is destroyed by trypsin and chymotrypsin (Cheeseman and Jayne-Williams, 1964). About 20 mM FeSO, counteracts it (Ashton and Busta, 1968; Cheeseman and Jayne-Williams, 1964; Pugh, 1970),as does MgSO,, but the latter is not as effective as FeSO, (Ashton and Busta, 1968; Cheeseman and Jayne-Williams, 1964). It is now thought to be lactotransferrin (Reiter and Oram, 1967), but whether there is more than one inhibitor for bacilli was disputed (Ashton and Busta, 1968).The concentration of a substance in milk inhibiting B. stearothermophilus increased as the protein content of the milk increased (Ashton and Busta, 1968). It was associated with the casein fraction of the milk of which it was a constant property. It was similar to lactotransferrin in that iron and magnesium counteracted its effect but dissimilar in that it showed greater heat stability (Ashton and Busta, 1968). The evidence for a second substance is that the secretion from nonlactating cows contained a substance not suppressed by Fe ions, with greater heat stability than lactotransferrin (Oram and Reiter, 1968), which was not inactivated by antiserum. Another inhibitor for bacilli, neutralized by Fe ions and probably lactotransferrin, was found whose concentration varied inversely with milk yield, maximum titres being present in the dry period secretion (Pugh, 1970). The mechanism by which this substance acts on the organism is unknown but Ashton and Busta (1968)suggested that the inhibitor may bind iron essential for the growth of the organism. Hence the addition

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of excess Fe ions saturates the sites on the inhibitor and the essential Fe ions become available for microbial growth (Ashton and Busta, 1968). It is known that lactotransferrin from bovine, human, and goat sources, when not fully saturated with Fe, has no effect on the activation of spores of B. stearothermophilus but it inhibits their germination (Oram and Reiter, 1968; Reiter and Oram, 1967). The bacteriostatic activity of lactotransferrin is also enhanced by Zn2+,Co2+,Mn2+,Ni2+,and Cuz+ (Oram and Reiter, 1968). c.

INHIBITORY

FACTOR FOR PROPIONIBACTERIA

The inability of Propionibacterium shermanii to grow in whey from a cheese factory was noted during a search for bacteriophage to these organisms (Vedamuthu et al., 1968).Similar activity was noted in whey from Cheddar and Colby cheese manufacturing plants and in whey obtained by direct acidification and by renneting. The factor also inhibited Propionibacterium arabinosum, Propionibacterium fruedenreichii, Propionibacterium jensenii, Propionibacterium pentosaccum, Propionibacterium peterssonii, E. coli, Micrococcus varians, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putrefaciens, and S. faecalis (Vedamuthu et al., 1968). It occurred at all times of the year, stored well at 5°C for many months, was stable in the range pH 4-7, was destroyed by boiling (Vedamuthu et al., 1968, 1971) and by 82°C for 16 sec, and was nondialysable (Vedamuthu et al., 1968).The activity was associated with the immunoglobulin portion of milk whey, presumably the pseudoglobulin fraction (Vedamuthu et al., 1971). There was some correlation between hemolytic activity of the organisms (eight species of propionibacteria tested) and susceptibility to bactericidal activity (Vedamuthu et al., 1971). Heating the whey for 10 min destroyed a band on disc gel electrophoresis associated with the activity (Vedamuthu et a]., 1971).

D. INHIBITIONOF THE STAPHYLOCOCCI Some (Baumgartner et al., 1965; Meeder, 1964a,b) have denied that raw milk is lethal for bacteria and for staphylococci in particular. The coagulation times for 31 strains of S. aureus var. aviarius were longer for 84% of the organisms in milk heated to 100°C than in milk heated to 65°C (Bergamini and Solinas, 1950).This property of milk was stable at 4°C.In Russian work (Mutevin, 1963),milk was shown to have strong antistaphylococcal activity. After some 10,000 tests on milk and udder secretions, four lysozymes or antibacterial substances were proposed

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as the cause. They were called lysozyme of the udder (U), lysozyme of milk (M), thermostatic lysozyme (T), and basic lysozyme (B). The characteristics of these substances, adapted from two tables in the paper of Mutevin (1963),are shown in Table 111.

E. INHIBITION OF LEPTOSPIRAS In addition to type-specific antibodies, milk contains a heat-stable factor which inactivates and lyses leptospiras (Kaplan et al., 1962).This factor has been demonstrated in milk from cows, goats, and humans, and in human colostrum. Virulent and avirulent strains of Leptospira hyos, Leptospira icterohaemorrhagiae, and Leptospira pomona are dissolved by the factor. The factor persists in milk stored at 4OC for 2 months and survives pasteurization at 80°C for 5 min, but is destroyed by boiling (Kaplan et al., 1962).

F. INHIBITION OF MYCOPLASMAS Experimental mastitis could be induced in cows with T-mycoplasmas (Gourlay et a]., 1972) but strains varied in their virulence and cows showed differences in susceptibility. Endotoxin-stimulated mastitic milk had lower mycoplasmacidal activity, but activity varied from cow to cow. This contrasted with pyrogen-stimulated mastitis, which gave TABLE 111 CHARACTERISTICS OF FOURANTISTAPHYLOCOCCAL SUBSTANCES IN MILK Group Characteristic ~

~~~

Diffusion in agar Delay growth of “lachrymalmicrococcus” Delay growth of staphylococcus Presence in normal milk Presence in colostrum Resists heating to 60°C Resists 65°C for 30 min Resists 70°C for 30 min Resists 75°C for 30 min Resists 100°C Udder lysozyme, U; milk lysozyme, M; thermostatic lysozyme, T; basic lysozyme, B. Note. Adapted from Mutevin (1963).

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milk with higher bactericidal activity. The activity was present in the whey from the milk; when the whey was fractionated it was associated with the immunoglobulin fraction but complement was not involved. Activity was lost on dialysis (Brownlie et al., 1974).The active principle could be absorbed by heterologous and homologous mycoplasmas and erythrocytes (Howard et al., 1975). IX. Applications of the Antimicrobial Systems in Milk

Jones and Simms (1930) concluded that the possible uses of lactenin were unknown but in the last 15 years a number of applications have been tested. First, Bjorck et al., (1979a,b) demonstrated the use of the LP system for short-term preservation of raw milk before processing. Activation of the LP system retarded the growth of gram-negative bacteria and allowed storage at relatively elevated temperatures without the milk deteriorating in quality, When stored at 5°C the growth of gramnegative psychrotrophic organisms was suppressed so that it took 4 days for LP-activated raw milk to reach the same count as that of the control milk at 1 day (Swart et al., 19901. This application was further tested in Kenya (Ridley and Shalo, 1990) and Sri Lanka (Harnulv and Kandasamy, 1982), and was shown to work with buffalo milk in India (Chakraborty et al., 1986). Kamau et al. (1990) showed that the growth of L. monocytogenes in raw milk could be controlled for 5 days at 10°C and for 8 hr at 35"C, and for S. aureus growth at both temperatures. In addition, Zapico et al. (1993) showed that the effect of the LP activated system in goat's milk depended on the strain of L. monocytogenes so that growth by 2 of 3 strains was prevented over 10 days at 4°C. The shelf life of pasteurized milk at 10°C was increased by 20 days after activation of the LP system by addition of 2.4 M SCN and 0.6 M H,O, followed immediately by holder pasteurization of 63°C for 30 min (Kamau et al., 1991). Second, L. monocytogenes was eliminated from French style fresh cheese made from milk contaminated with the organism by spreading the LP system on the surface of the cheese (Denis and Ramet, 1989). Similar experiments were done with cottage cheese during which it was shown that E. coli and S. typhimurium were killed by the LP system (Earnshaw et al., 1989). The LP system could provide a further safety feature for these cheeses to eliminate important pathogens as well as by extending shelf life. Third, the rearing of calves from dairy cows requires the feeding of milk replacers and consequently calves are often susceptible to infection with enterotoxigenic coli. Through activation of the LP system in the

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calf replacers and addition of lactoferrin, infections were reduced in severity and duration, even in those animals already infected, when fed LP activated milk replacer (Still et al., 1990),and calf weight gains were greater (Reiter et al., 1981).A dose of 4000 units of lactoperoxidase per liter of milk replacer prevented development of diarrhea in calves due to enterotoxigenic E. coli and was even sufficient to treat the disease (Prieels et aI., 1989). The addition of acetic or butyric acid to milk fat substitutes in milk replacer contributed to a reduction in the enterobacteria (gram negative) flora in the abomasum of calves (Glas and Schaafsma, 1988). X. Conclusion

The growth of bacteria in milk, because of its complex nature, is not as simple as it may at first appear. It seems that the net bacterial growth measured is the result of the organisms resisting inhibitory systems present in the milk and being stimulated by some unknown substances also present. Interest in this phenomenon stems from three important aspects of milk. First, there is the industrial use of milk. Milks from cows, goats, buffaloes, and camels are important industrial commodities. Anything which enhances or detracts from the usefulness of milk for this purpose is important. Second, when farmers are harvesting milk from their farm animals the animals are prone to mastitis or infection of the udder. Mastitis lowers productivity, reduces milk quality, and decreases the income of the farmer. Third, milk is the food of young mammals, human babies in particular, and anything which will enhance the growth and health of a human body, as well as that of other young mammals, is considered desirable. The relationship of the inhibitory systems to what was originally called lactenin does not appear to have been established. The lactoperoxidase-thiocynate-hydrogen peroxide system has been most thoroughly studied and could easily be what Auclair called L2, except for the fact that L1 and L2 act synergistically (Auclair and Berridge, 1953) and L1 appears to be an antibody (Portmann et al., 1960). So far there seems to have been no attempt to investigate how an antibody could influence the LP system. Although L1 may be an antibody, no explanation as to how an antibody could be inactivated by anaerobiosis has been offered. Further, lactenin is not inactivated by cyanide, yet the LP/SCN-/H,O, system is. Growth inhibition in milk of lactic cultures could not be explained by the LP system or by the H,O, concentration (Roginski et al., 1984).

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The role of antibodies in the inhibition of lactic acid bacteria is not clear. I suggest, however, that the role that they play in milk is similar to that played by antibodies to groups A, B, C, and D streptococci in rabbit serum. Larson et al. (1974) showed that human serum, as it was diluted, had the effect of exhibiting bactericidal activity when in concentrated solution but was bacteriostatic for the organism as the serum was diluted. It may be that a complement-like reaction is involved with the inhibition of milk. That is, once the specific antibodies attach to the organism other substances in the milk then attach to the antigen-antibody complex. Another possible mechanism causing the inhibition of lactic acid bacteria involves a lack of Fez+.Lactic cultures, in the absence of sufficient Fez+ may not be able to produce sufficient NADH peroxidase, or other enzymes necessary to decompose hydrogen peroxide. Hence, sufficient hydrogen peroxide could build up in the milk to allow the oxidation of thiocyanate by LP. The depletion of the Fez+could be due to lactotransferrin, the inhibitor of bacilli. One further inhibitor of bacteria in milk is the presence of free fatty acids in the milk due to the activation of the lipase in milk. Activation of lipase is variable and depends on the machinery being used to handle the milk, as well as the diet of the cow. The natural stimulator(s) in milk for lactic acid bacteria is unknown. An essential nitrogen source has been suggested as the stimulator, as have sulfydryl-containing substances. Whatever its identity, there appears to be a stimulator associated with the fat globule membrane. This is indicated by the work of Jag0 and Gillies, which shows that the growth-supporting properties of cream varied as the cream was diluted in skim milk. Further work is required to determine what the nature of the stimulator is in the cream fraction in milk. A fully integrated understanding of the growth of bacteria in milk awaits elucidation. Appendix A Species of bacteria inhibited by milk include: A. aerogenes, B. subtilis, B. coagulans, B. stearothermophilus,E. coli, species of Flavobacterium, L. lactis, Lactobacillus jugurti, L. helveticus, L. bulgaricus, L. hyos, L. icterohaernorrhagiae, L. pomona, M. varians, Mycoplasma bovigenitalium, M. agalactiae, M. bovoculi, P. arabinosum, P. fruedenreichii, P. jensenii, P. pentosaceum, P. peterssonii, P. aeruginosa, P. fhorescens, P. putrefaciens, S. typhi, S. paratyphi B, Shigella dysenteriae, Shigella paradysenteriae, S. aureus, S. albus, S. lactis, S. cremoris, S. diacetilactis, S. dysagalactiae, S. faecalis, S. citrophilus, S. liquefaciens, S. uberis, S. pyogenes, S. thermophilus, and S. agalactiae.

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Appendix B The following substances were tested for their interaction with lactenin by Wilson and Rosenblum (1952b). Inactivated lactenin: sodium thioglycollate (100 ppm), cysteine (100 ppm), glutathione (100 pprn), 2,3-dithiopropanol (1 ppm). Did not inactivate lactenin but did inhibit lactenin-sensitive organisms: hydroxylamine, sodium fluoride, sodium azide, sodium pyrophosphate, sodium selenite. Did not inactivate lactenin but inhibited sensitive and resistant organism: urethane, 2,4dinitrophenol, methionine, carbon monoxide. Did not inactivate lactenine: sodium cyanide. Did not interfere with lactenin: glutamine, riboflavin, pyridoxine, biotin, folic acid, calcium pantothenate, nicotinic acid, guanine, xanthine, p-aminobenzoic acid. Sodium ascorbate had no effect. Substances tested for inhibitory and stimulative activity in milk by Brown (1974) were as follows. Compounds tested for inhibitory activity: those indicated by an asterisk below and DLtryptophan. Compounds tested for stimulative activity: Vitamins: Ascorbic acid, biotin, choline, cyanocobalamin, folic acid, inositol, nicotinic acid, pantothenic acid, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, thiamine monophosphate chloride, thiamine pyrophosphate chloride. Amino acids and related compounds: L-alanine*, L-arginiine*, L-asparagine*, L-aspartic acid*, L-citrulline*, DL + allo-cystahionine*, L-cysteine, L-cystine, L-glutamic acid*, L-glutamine*, glycine*, L-histidine*, DL-homocysteine, m-homocystine, m-allo-dhydroxylysine* , L-hydroxyproline*, L-isoleucine*, L-leucine*, L-lysine*, L-methionine*, L-ornithine*, L-phenylalanine*, L-proline*, L-serine*, L-threonine*, L-tyrosine*, L-valine*, p-alanine*, P-alanyl-histidine (L-carnosine), P-alanyl-L-methyl histidine (Lanserine), 3-anthraniloylalanine (m-kynurenine)* , m-a-amino-N-butyric acid*, yamino-butyric acid*, 2-aminoethanesulfonic acid (Taurine)*, L-cysteic acid*, glycylL-glutamic acid*, glutathionine (reduced and oxidized), m-methionine-m.-sulfoxide*, phosphoserine*. N-methyl-glycine (Sarcosine)*, N-a-phenylacetyl-L-glutamine*, Phospholipids, fatty acids and related compounds: glycerol, P-glycero-phosphate*, DLa-glycerophosphate*, glycerophosphoryl-ethanolamine*,DL-3-phospho-glyceraldehyde,3-phospho-~-glyceric acid, 2-phosphoglyceric acid, 2-phosphoenol-pyruvic acid, 0-phospho-ethanolamine*, potassium isobutyrate, potassium isovalerate, potassium N-valerate, potassium 2-methylbutyrate, a-keto-isocaproic acid*, a-keto-isovaleric acid*. acid*, DL-a-keto-p-methyl-N-valeric Cephalin, lecithin, lysophosphatidyl ethanolamine, phosphatidyl ethanolamine, and phosphatidyl serine were hydrolyzed by the method of DawsonlSGO.After evaporation to dryness, the water-soluble material was tested. Nucleosides and nucleotides: Adenosine-5’-mono- and diphosphate, cytidine, cytidine5‘-monophosphate, cytidine-5’-diphosphate, cytidine-5’-triphosphate, guanosine, guanosine-5’-monophosphate,guanosine-5’-diphosphate, guanosine-5’-triphosphate, inosine, inosine-5’-monophosphate, inosine-5’-diphosphate, inosine-5‘-triphosphate, uridine, uridine-5‘-monophosphate,uridine-5‘-diphosphate, uridine-5’-triphosphate, guanosine-5’-diphosphomannose,uridine-5‘-diphosphoglucose,uridine-5’-diphosphogalactose.

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Other compounds: Sodium citrate, creatinine*, dithiothreitol, ethanolamine (Seramine)*, D-glucosamine, hippuric acid, N-acetyl-neuraminic acid, neuramine-lactose, orotic acid, sodium sulfide, sodium thioglycollate, urea*, uric acid.

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Anders, R. F., and Jago, G. R. (1970). The effect of fatty acids on the metabolism of pyruvate in lactic acid streptococci. J. Dairy Res. 37, 445-456. Anderson, A. W., Parker, R. B., and Elliker, P. R. (1955). The nutritional requirements of lactic streptococci isolated from starter cultures. 111. Variation in growth-promoting properties of fresh whole milk. J. Dairy Sci. 38,1083-1088. Ashton, D. H., and Busta, F. F. (1968).Milk components inhibitory to Bacillus stearothermophilus. J. Dairy Sci. 51, 842-847. Auclair, J. E. (1954a). Stimulation of certain bacteria by raw milk. Nature (London] 173, 491.

Auclair, J. E. (1954b). The inhibition of micro-organisms by raw milk. 111. Distribution and properties of two inhibitory substances, lactenin 1 and lactenine 2.1. Dairy Aes. 21, 323-336.

Auclair, J. E. (1968). Bacteriostatic-properties of milk from tropical countries. Ann. Bull.-Int. Dairy Fed. 4, 7-44. Auclair, J. E., and Berridge, N. J. (1953). The inhibition of micro-organisms by raw milk. 11. The separation and electrophoretic examination of two different inhibitory fractions. J. Dairy Aes. 20, 370-374. Auclair, J. E., and Hirsch, A. (1953). The inhibition of micro-organisms by raw milk. I. The occurrence of inhibitory and stimulatory phenomena. Methods of estimation. J. Dairy Res. 20, 45-59. Auclair, J.E., and Portmann, A. (1959). Inhibitory action of the lactenins on the lactic streptococci starters of cheese. Int. Dairy Cong. Proc., 15th, London, 1959, Vol. 2, pp. 580-586. Auclair, J. E., and Vassal, Y. (1963). Occurrence of variants sensitive to agglutinins and to lactoperoxidase in a lactenin-resistant strain of Streptococcus lactis. J. Dairy Res. 30, 345-349.

Aune, T. M., and Thomas, E. L. (1977). Accumulation of hypothiocyanate ion during peroxidase-catalyzed oxidation of thiocyanate ion. Eur. J. Biochem. 80, 209-214. Basaga, H., and Dik, T. (1994). Effect of the lactoperoxidase system on the activity of starter cultures for yogurt production. Milchwissenschaft 49, 144-146. Baumgartner, H., Kastli, P., Walser, V., and Needer, K. (1965).Experiments on the bactericidal properties of milk. Pathol. Microbiol. 28, 3-11; cited in Dairy Sci. Abstr. 27, 2857.

Bergamini, L., and Solinas, J. (1950). Research on some properties of lactenin and the sensitivity to it of some strains of Staphylococcus aureus type aviarius. Boll. SOC. Ital. Biol. Sper. 35, 805-808; cited in Dairy Sci. Abstr. 22, 745. Berridge, N. J, (1955). Inhibitory substances of bacterial and other origins in milk and milk products. J. Sci. Food Agric. 6, 65-72. Beumer, R. R., Cruysen, J. J. M., and Birtantie, I. R. K. (1988). The occurrence of Campylobacter jejuni in raw cows’ milk. J. Appl. Bacteriol. 65, 93-96.

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Bishop, J. G., Schanbacher, F. L., Ferguson, L. C., and Smith, K. L. (1976). In vitro growth inhibition of mastitis-causing coliform bacteria by bovine apo-lactoferrin and reversal of inhibition by citrate and high concentrations of apo-lactoferrin. Infect. Immun. 14, 911-918. Bjorck, L. (1978). Antibacterial effect of the lactoperoxidase system on psychrotrophic bacteria in milk. J. Dairy Res. 45, 109-118. Bjorck, L., Claesson, O., and Schulthes, W. (1979a). Antibacterial effect of the lactoperoxide system on psychrotrophic bacteria in milk. J. Dairy Res. 45, 109-118. Bjorck, L., Claesson, O., and Schulthes, W. (1979b). The lactoperoxidase/thiocyanate/ hydrogen peroxide system as a temporary preservative for raw milk in developing countries. Milchwissenschaft 34, 726-729. Bottazzi, V., Battistotti, B., and Vescove, M. (1971). Continuous production of yoghurt cultures and stimulation of Lactobacillus bulgaricus by formic acid. Milchwissenschaft 26,214-219. Boulange, M. (1959). Fluctuation saisonniere du taux des thiocyanates dans le lait frais de vache. C. R. Seances SOC.Biol. Ses Fil. 153, 2019-2020. Branen, A. L., and Keenan, T. W. (1970). Identification of a stimulant for Lactobacillus casei produced by Streptococcus lactis. Appl. Microbiol. 20, 757-760. Brown, R. W. (1967). Factors affecting growth of Streptococcus agalactiae in milk. J. Dairy Sci. 50, 1572-1584. Brown, R. W. (1974). Compounds affecting Streptococcus agalactiae growth in milk. J. Dairy Sci. 57, 797-802. Brown, R. W., and McDonald, T. J. (1982). Effect of time of sample collection, milk composition, and stage of lactation on the growth-promoting properties of milk for Streptococcus agalactiae. Am. J. Vet. Res. 43, 973-978. Brownlie, J., Howard, C. J., and Gourlay, R. N. (1974). Mycoplasmacidal activity of bovine milk for T-mycoplasmas. J. Hyg. 73, 415-423. Buchanan, R. E., Holt, J. G., and Lessel, Jr., E. F. (1966). “Index Bergeyana: An Annotated Alphabetic Listing of Names of the Taxa of Bacteria.” Williams and Wilkins, Baltimore, MI). Carlsson, A., and Bjorck, L. (1987). The effect of some indigenous antibacterial factors in milk on the growth of Bacillus stearothermophilus var calidolactis. Milchwissenschaft 42,282-285. Carrol, E. J., and Jain, N. C. (1969). Bactericidal a-‘ivity of normal milk, mastitic milk, and colstrum against Aerobacter aerogenes. Am. J. Vet. Res. 30, 1123-1132. Chakraborty, B. K., Chaudry, S. S., Alex, K. A., Jacob, G., and Soni, G. J. (1986).Application of the lactoperoxidase system for preserving buffalo milk produced in Indian villages. Milchwissenschaft 41, 16-19. Cheeseman, C. C., and Jayne-Williams, D. J. (1964). An inhibitory substance present in milk. Nature (London) 204, 688-689. Clegg, L. F. L., Sergeant, V., Franklin, J. C., and Auclair, J. E. (1956). The effect of lactenin on the growth of thermoduric bacteria in milk, with some observations on “bitty cream.” Dairy Ind. 21, 128-133. Clem, W. H., and Klebanoff, S. J. (1966). Inhibitory effect of saliva on glutamic acid accumulation by Lactobacillus acidophilus and the role of the lactoperoxidasethiocyanate system. J. Bacteriol. 91, 1848-1853; cited in Dairy Sci. Abstr. 28, 2471. Cogan, T. M., Gilliland, S. E., and Speck, M. L. (1968a). Identification of stimulants for Lactobacillus bulgaricus in tomato juice. Appl. Microbiol. 16, 1215-1219. Cogan, T. M., Gilliland, S. E., and Speck, M. L. (1968b). Characterization of a inhibitor for Lactobacillus bulgaricus in tomato juice. Appl. Microbiol. 16, 1220-1224.

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Wilson, A. T., and Rosenblum, H. (1952a). The antistreptococcal property of milk. I. Some characteristics of the activity of lactenin in vitro. The effect of lactenin on the hemolytic streptococci of the several serological groups. J. Exp. Med. 95, 25-38. Wilson, A. T., and Rosenblum, H. (1952b). The antistreptococcal property of milk. 11. The effects of anaerobiosis, reducing agents, thiamine, and other chemicals on lactenin action. J. Exp. Med. 95, 39-50. Wolfson, L. M. and Sumner, S. S. (1993). Antibacterial activity of the lactoperoxidase system: a review. J. Food Prot. 56, 887-892. Wright, R. C., and Tramer, J. (1957). The influence of cream rising upon the activity of bacteria in heat-treated milk. J. Dairy Res. 24, 174-183. Wright, R. C., and Tramer, J. (1958). Factors influencing the activity of cheese starters. The role of milk peroxidase. J. Dairy Res. 25, 104-118. Zall, R. R., Chen, J. H., and Dzurec, D. J. (1983). Effect of thiocynate-lactoperoxidasehydrogen peroxide system and farm heat treatment on the manufacturing of cottage cheese and cheddar cheese. Milchwissenschaft, 38, 203-206. Zapico, P., Gaya, P., Nuiiez, M., and Medina, M. (1990). Lactoperoxidase and thiocyanate contents of goats’ milk during lactation. Lett. Appl. Microbiol. 11,90-92. Zapico, P., Gaya, P., Nubez, M., and Medina, M. (1990). Goats’ milk lactoperoxidase system against Listeria monocytogenes. J. Food Prot. 56, 988-990.

Challenges in Commercial Biotechnology. Part 1. Product, Process, and Market Discovery ALESPROKOP Department of Chemical Engineering Vanderbilt University Nashville, Tennessee 37235

I. Introduction A. Biotechnology, Process, and Process Systems B. Interdisciplinary Nature of Biotechnology C. Industrial Aspects of Biotechnology D. Systems Approach 11. Biocatalysts: Sources for Discovery A. Definitions and Classification B. Organismic Biocatalysts C. Enzyme-Based Biocatalysts D. Biocatalyst Processing and Reactors III. Primary Market Classification IV. Pathway to Commercialization: Discovery and Development (D&D)Cycle V. Product Discovery A. Classification of Products and Screens B. Examples of Screens C. Route of Discovery vs Route of Manufacturing D. Example of Discovery of a New Chemical Class (and of Product Development) E. Example of Discovery from Plant Biotechnology F. Special Considerations for Discovering (and Developing) Products of Different Industrial Sectors G. Approaches to Discovery H. Role of Serendipity in Product Discovery I. How the Top 20 Drugs Were Discovered VI. Process Discovery A. Approaches to Process Discovery B. Biotransformations C. Examples of Bioprocess Discovery VII. Market Discovery WI. Interactions of Discovery Aspects within the D&D Cycle A. Product Discovery vs Product Development B. Product Discovery vs Process DiscoverylDevelopment C. Market Discovery vs Development References

1. Introduction

A. BIOTECHNOLOGY, PROCESS, AND PROCESS SYSTEMS The word biotechnology has various meanings. In the United States (and in some western European countries) it has frequently become 95 ADVANCES IN APPLIED MICROBIOLOGY. VOLUME 40 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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restricted to, and almost synonymous with, the application of genetic engineering. In the rest of the world biotechnology has retained its original definition, so that it comprises all the technical aspects of cultivating cells or tissues and the products and processes derived from such cultures, including enzyme technology (Finn, 1988).A definition by the Office of Technology Assessment (OTA) of the U.S.Congress states that “biotechnology, broadly defined, includes any technique that uses living organisms (or parts of organisms) to make or modify products, to improve plants or animals, or to develop microorganisms for specific use” (Office of Technology Assessment, 1984). Mimicking of naturally occurring materials should be added to the above definition. Implicit in the above definitions is a processing aspect of handling biological material (process engineering) and the real object of our investigation-process. A process is a sequence of operations or steps resulting in the production of a certain product or in a particular treatment or raw materials. A technical field dealing with bioprocessing is called bioprocess engineering or bioprocessing. Most biotechnological processes consist of a chemical reaction process section (fermentation, or bioreaction section) and of a subsequent product recovery section. Sometimes, operations preceeding and including fermentation are called upstream processing (e.g., media preparation, sterilization) and those of product recovery are called downstream processing. A process system can be defined by addressing the interrelated and interdependent nature of elements of a process. It can be arbitrarily defined as consisting of the upstream and downstream segments, including those of fermentation, cell recovery, and product recovery processing. Each process system interacts with its environment by means of mass, energy, and information flows. A set of elements and flows determines the internal structure of the process system. Such elements are then described as structural elements. Time and space conglomerates of elements, having a certain common similarity, are called subsystems.

B. INTERDISCIPLINARY NATURE OF BIOTECHNOLOGY Bioprocesses are systems in which complete living cells, tissues, or their components (e.g.,organelles) are used to effect desired physical or chemical changes. Bioprocess systems (biocatalysts) could potentially replace some current chemical catalysts; they include enzymes, microbial cells, and plant/animal tissue cells, and they draw from many disciplines of science and engineering. Table I summarizes some major

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TABLE I INTERACI'ION OF DISCIPLINE-SYSTEM-PROCESS IN BIOTECHNOLOGY Discipline Microbiology, ecology, taxonomy Chemistry, enzymology Genetics, molecular biology, genetic engineering Physiology, cell biology, plant sciences Process engineering physical sciences Economics, law, social sciences Systems engineering

Biocatalyst system"

Process aspects

M, P/A

Culture choice

E, M, P/A E, M, P/A

E, M, P/A

Reactions, product recovery Product discovery Process discovery Process operation: response to environment Enzyme reactors, process operation optimization, control and design Business and regulatory aspects

M, P/A

Systems analysis and synthesis

M, P/A E, M, P/A

E,enzyme; M, microbial; PIA, plantlanimal.

interactions between systems, processes, and disciplines in biotechnology and provides a foundation for industrial applications of biotechnol-

ogy in several sectors: 1. Pharmaceuticals, including medical diagnostics and biosensing 2. Animal and plant agriculture 3. Specialty chemicals and food additives 4. Commodity chemicals and energy production 5 . Services within the environmental area.

From these it is thus clear that biotechnology is a multidisciplinary field and has multiindustry applicability. Two categories of biotechnology are sometimes distinguished: 1. Traditional (before the invention of genetic engineering) 2. New (after the invention of genetic engineering).

It is predicted that new biotechnology, related to genetic engineering and hybridoma technologies, will account for a large fraction of the total biotechnology industry in the future, although both the traditional and the new will coexist for a long time.

c . INDUSTRIAL ASPECTSOF BIOTECHNOLOGY The central problem here is a definition of research and developmental aspects (R&D)that must be completed to bring a product to market.

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Other terms often used are discovery and development. Definitions are given in Table 11, which also includes regulatory, legal, and social constraints imposed on a product (“soft constraints”; they are denoted as such because they are hard to quantify and solutions are not obvious). The general scheme of R&Dactivity in an industrial setting is depicted in Fig. 1,indicating the long path to market and the tremendous cost and time involved. Besides soft constraints, economic constraints also play a decisive role. The process must be designed to give the private sector an advantage in the marketplace. The optimal process must be defined within the context of competing goals or means of competitive advantage [Wheelwright, 1991): 1. First to market [strong proprietary position, safe, and efficacious product) 2. High quality 3. Low cost. TABLE I1

INDUSTRIAL (RBrD) ASPECTSOF BIOTECHNOLOGY Term Discovery Product discovery Process discovery Market discovery Development Product development Process development and scale-up Market development Regulatory aspects Health, safety, and environment Industrial Legal aspects Social and ethical

Description Product screen via genetic, biological, chemical, and molecular means Process discovery via catalyst selection, metabolic pathway optimization and statistical improvement Market forecasting, strategic planning and market analysis Product improvement via product formulation and delivery, via improved processing and manufacturing Inoculum development, process scale-up, control, optimization, and process and plant design Product uniqueness, market orientation, and technical factors FDA, USDA, EPA, NIH, etc. GLP, GMP, QC, validation Trade secrets, patents, licensing Policy, technology transfer, public perception, ethical issues

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I

I

Processdiscovery

Forecasting

1

Optimization

Economics I

I

'-I I

I I

I I

99

Process Development and Scale-up

i-

-hi

11-4 I

I 4 I

Manufacturing

Product in Market Market Development I

t - _ _ - _ _ _ - - _ - , - - L - _ - - FIG.1. R&D in perspective (---, information transfer; -product transfer].

Each of these offer advantages that can be realized only at the expense of the others. In choosing between them, the best market performance must be sought. The first to market typically requires a strong patent (license) position, granted approval (safety and efficacy), and short process development times. High quality requires high purity and activity, as well as consistent production (process validation). The cost is related to the complexity of processing, the efficiency of each step, the scale of operation, and the degree of manufacturing and equipment flexibility. The product uniqueness and its life cycle expectancy are also important. Once the competitive advantage is identified it must be maintained because of product specifications and available resources.

D. SYSTEMS APPROACH The differentiation of a system into subsystems allows for an investigation of an organization of a system at different levels of hierarchy. The general system is then composed of elements, subsystems, and levels of hierarchy, bound by the corresponding relations within each system. Structural elements, also called elementary events (processes), then play certain functions. The hierarchy of living systems and of a

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hypothetical biotechnological process is depicted in Table 111, presenting a relationship between hierarchy, structure, and function (elementary events). Table IV lists examples of individual subsystems with corresponding elementary events. Such decomposition of a system via breakdown into its components involves not only an analytical approach but also a synthetic view, both germane to a systems approach (general systems approach). Such an approach attempts to point out related properties of the investigated phenomena by selecting relevant (systemic) properties, relations, and functions. The process of synthesis is based on a reduction of information at each level of hierarchy by means of purposeful selection of important systemic phenomena and by identification of interactions among them. Note that the complex metabolic network presented for L-lysine biosynthesis is already highly simplified, featuring only important elements and interactions. The population level is treated as a quasi-homogeneous system composed of identical cells (another oversimplification) and the reactor level (not shown) is featured by perfect reactor mixedness and oxygen balances. The synthesis or transition to the higher levels of hierarchy (also denoted as scale-up) can be thus carried out by lumping of lower specific levels into higher (systemic) levels of hierarchy, since it may be difficult to incorporate all properties of specific levels. The selection of systemic properties and reactants is not governed by strict rules and is a matter

TABLE 111 HIERARCHY OF A BIOTECHNOLOGICAL PROCESS Hierarchy level

Structural element

Molecular molecules Organelles

Atoms, macromolecules Macromolecules

Cell

Organelles

Population

Cells

Reactor

Elementary zones

Biotechnological process

Unit equipment

Note. Adapted from Table VII of Prokop (1982).

Function (elementary event) Synthesis/decay of epigenetic control Duplication of organelles, metabolic manifestations of cells Translocation of substances, complex biochemical reactions, morphological differentiation, cell cycle control Growth and multiplication, interaction between cells Reactions, mass transfer, hydrodynamics, heat exchange, diffusion, etc. Unit operations: upstream, reactor, downstream

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TABLE IV LISTOF SUBSYSTEMS AND ELEMENTARY EVENTS OF L-LYSINEBIOSYNTHESIS Subsystems 1. Maintenance 2. Glycolysis/energy

3. Lysine

4. Protein 5. Threonine/homoserine

Elementary events Oxygen and substrate uptake, maintenance of cellular functions Control of glycolysis, energy generation and control, provision of building blocks for biosynthesis Lysine biosynthesis pathway, positive glycolytic flux control, negative feedback control, metabolic block at homoserine dehydrogenase, negative feedback by oxygen excess Protein synthesis, growth, negative feedback by oxygen excess Threonine/homoserine pathway, two metabolic blocks (homoserine dehydrogenase, homoserine kinase),two auxotrophic mutations (homoserine, threonine)

Note. Adapted from Fig. 6 of Prokop (1982).

of art exercised by scientists and engineers. At a cellular level, typically, systemic events and reactants are usually those at the branching points of metabolism, undergoing feedback and feedforward control. At the level of reactor, typically, mass transfer (oxygen)may control the general reaction scheme and is thus typically chosen as an elementary event (for economic reasons). A major postulate of the systems approach is a recognition that the performance of the whole is not the sum of individual parts, but a consequence of the relationship of the performance between the parts (levels of hierarchy). For example, oxygen uptake of cells (in a population) is coupled with oxygen transfer and oxygen balance at the reactor level, giving rise to a reactor model. A further step in the systems approach is separation of conditions leading to a limitation of either biological or physical conditions (regime analysis, Part I1 of this series). The last step is usually construction of a mathematical model of the system and simulation by means of a model. The model is conceived as a formal description of a real process and reflects a relationship between input, state, and output variables of the system. Simulation is then an investigation of a system by means of mathematical manipulation of a model. The systems approach thus embodies two faceted view: analytical and synthetic. Tools of analysis are most often discussed in chemical reaction engineering textbooks; methods of synthesis are typical for

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discovery and developmental aspects. An effective biotechnology R&D thus uses two components of investigative study (Fig. 2): 1, Specific knowledge of disciplines as a structural basis for development of different application fields 2. General problem-solving strategies represented in the integrated systems approach, as a basis of “knowledge pathway.”

As such, the first component represents a specialized view, the second one a generalists view (Table V). In fact, in real life, the problem-solving

(7 Tools of analysis

Specific knowledge: Microbiology Biochemistry Chemistry Physics Genetics, etc.

(r> Methods of synthesis

General problem strategies: speculative thinking personalizedthinking data-bound thinking relational thinking

Product discovery Product development and scale-up Plant design Market development Societal effects

a FIG.2. Components of effective R&D activity.

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TABLE V CONCEPTUAL DIFFERENCES BETWEEN SPECIALISTSAND GENERALISTS AND TYPEOF SOLUTIONS AND METHODS Specialists 1. A whole to be divided into

components 2. The dimension of depth 3. Exclusive postures and intentions 4. Deduced principles 5. An accurate view 6. A sole expert 7. Narrow skill 8. Solution seeker close-ended problems 9. Application-oriented 10. Well-defined or definable limits 11. Usually one correct answer 12. Rigorous techniques available

Generalists Components associated into a whole The dimension of breadth Mutual concerns/interactions Induced concepts A representative view (scheme, analogy) An interdisciplinary team Broad views Problem definition solutions are openended Systems/concepts awaiting applications Ill-defined or nonexistent limits Multiplicity of correct answer Generahague techniques available

Note. Adapted from Gilbreath (1987,p. 89).

approaches do not always fall into only two such categories. According to Lefton et al. (1980) each analytical and synthetic view can be of two types (Tl-T2 and T3-T4)(Fig. 3). From an individual perspective, it may be useful to identify and be aware of what kind of thinking mode one is using. An example of Tl-T4 types drawn from biotechnological R&D is given in Table VI. More examples will be presented while discussing scale-up methods (Part I1 of this series). A general systems researcher starts with the laws of different disciplines (analysis), searches for similarities among them, and then announces to the world a new “law about laws” using generalization (synthesis)(Weinberg,1975,1982).The power of generalization through induction is that we can use the general laws to draw conclusions about cases not yet observed. Yet such a situation may not be always right. This leaping from discipline to discipline does not always work. Most of the time errors are due to either improper use of assumptions of oversimplifications of the real world at the problem formulation stage. The question is, then, why not to wait for more evidence (data) before generalization? The answer lies in the explosive growth of knowledge. We just cannot afford to wait for more specifics to come. The world is pressing us to contribute and to solve problems. The general systems researcher is constantly jumping to conclusions on insufficient evidence; this is a prerequisite for rapid learning. There is a danger in-

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intuitive

Conceptual

Speculative thinking: generalization based on theoly and intuition rules of thumb

Relational thinking: logical analysis of data via relationships and generalizations

Personalized specific and personal decisions based on past experience and

Data-bound thinking: statistical and pattern analysis of data

Systematic

Concrete

Information evaluation Information gathering FIG.3. Types of problem-solving approaches (adapted with permission from Lefton et al. (19801, Fig. 42, p. 445, copyright 1980 by Ballinger-Harper Business). TABLE VI EXAMPLES OF DIFFERENTMETHODS OF THINKING IN R&D Method of thinking TI. Relational T2. Data-bound T3. Personalized

T4. Speculative

Example Specific rate analysis, regime analysis, morphological analysis Statistical analysis, pattern analysis Origins of scale-up problems, personnel experience with facilities/equipment,microbiologist’s knowledge of culture characteristics, chemist’s knowledge of product Rule of thumb in engineering analysis, “gut” feelings of specialists on biologicalkhemical characteristics

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volved, in that some generalizations may not be correct. Why do we need such general systems thinking in biotechnology? The answer is related to the nature of the inherent complexity of industrial R&D problems. Solutions to industrial biotechnology need to come from the contributions of multidisciplinary specialists. Therefore, one needs to provide a conceptual framework by which the multidisciplinary specialists can have a common representation of their latest understanding of the biotechnical systems. In general, types of systems with respect to thinking and their complexity are of three kinds (Fig. 4). Examples taken from biotechnology are listed in Table VII. Obviously, a systems approach is useful for systems of Type 111, since the problem is too diverse for analysis and too structural for a statistical treatment (typically in the development area). It represents an attempt to organize “pockets of knowledge” of the system and subsystems. The position of Randomness (diversity)

Most complex

111 Organized complexity

Complexity (number of hierarchies, subsystems, elementary processes)

FIG.4. Types of systems with respect to methods of thinking (adaptedwith permission from “Introduction to General Systems Thinking,” Weinberg (1975), Fig. 1.9, p. 18, copyright 1975 by John Wiley, reprinted with permission of John Wiley & Sons).

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EXAh.lF'LES OF COMPLEXITY OF BIOTECHNOLOGICAL SYSTEMS AND INVESTIGATIVE TOOLS

Type of system

I. Organized simplicity

11. Unorganized complexity

111. Organized complexity

Tools used

Example

Analytical/deterministic reaction mechanisms, Michaelis-Menten equation, enzyme inhibitiodactivation Statistical/stochastic, stochastic: factorial experimental design screening

Single enzyme reacting system Microbial biotransformations (e.g., steroids]

Systems approachanalysis and synthesis: specific rate analysis, pattern analysis checklist

Secondary metabolite production in complex media (e.g., antibiotics) Bioremediation Cell culture in serum media Biocorrosion/biofouling Microbial and cell culture in chemically defined media Microbial growth on single limiting substrate Sterility problems in biotechnology and food processing

specific examples in Table VII will move as our understanding and knowledge increase and more synthetic tools become available. Figure 5 depicts a multiplicity in pathways of the discovery (product or process) procedure. This is the most significant finding of this series of two articles: the multiplicity of solutions in practical biotechnology, requiring a general systems (global,holistic) view. There are many ways a product can be manufactured, and different alternatives are to be evaluated using industrial constraints: 1. Total discovery and development time 2. Cost (process economy, market position) 3. Quality (product quality, consistency, stability, etc.).

As a rule, many industrial (biotechnology) problems are open-ended, and multiple solutions are available on both the chemical and biological processing sides. These include, naturally, nonprofitable tracks, fallible tracks, and back-tracking. Different questions may be posed regarding how to produce a given product: 1. What to use-Chemical vs enzymatic vs fermentation route? 2. What analoghariant molecule?

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Pathway number of R&D

Multiple solutions to industrial problems

Time, cost & quality dependent

FIG.5. Multiple pathways to open-ended problem solutions.

3. What is the best therapeutic formulation/delivery? 4. What are the process variants available once a route is fixed?

The systems approach provides the best available methodology to guide us in finding an effective solution of complex biotechnological problems. A summary of strategies involved in R&D in part at discovery, mostly at product and process development and scale-up, is shown in Table VIII. All of the above material presented in the Introduction merely defines the status of biotechnology and, most importantly, explains the importance of the systems approach. Specific examples will be presented below. TABLE VIII STRATEGIES INVOLVEDIN R&D Strategy Empirical SystemsIEngineering Principles Simplification Interactions (feedback) Separation Model and simulation

Example Trial and error, statistically bound approach Systematic (rational) process development Reduction of information Identification of relationships between subsystems Regime analysis [biological vs. physical phenomena) Structural model of metabolism coupled with a reactor model

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II. Biocatalysts: Sources for Discovery A. DEFINITIONS AND CLASSIFICATION A biocatalyst in biotechnology represents a laboratory observation on an organism or an isolated enzyme activity having the potential to be used in developing a marketable product. For product definition and its interrelationship to biocatalysts see Section IV. Compared to chemical processing, the use of biocatalysts in biotechnological processing offers many advantages (Table IX). The same table also lists disadvantages. Biocatalysis enables one to carry single- or multistep reactions with high stereo- and regiospecificity under mild conditions. The chirality effects in drugs and agricultural chemicals present new challenges and the need to isolate a single isomer from a racemic mixture. Novel chemistries via enzymatic catalysis lead to new routes for manufacturing of novel molecules, to novel routes for existing chemicals, and to new technologies adding value to feedstocks and by-products. The emergence of biotechnology-based technologies is due to:

The explosive increase in understanding of biological systems The development of large-scale bioprocessing techniques The maturity of the petrochemical-based chemical industry The increasing environmental pressures and restrictions on conventional chemical technology. 1. 2. 3. 4.

TABLE IX COMPARISON OF BIOTECHNOLOGICAL AND CONVENTIONAL CHEMICAL PROCESSES Advantages

Disadvantages

Multistep enzymatic conversions in one processing step

Dilute reactant conditions resulting in low productivity

Structural and stereochemical specificity

Large equipment and capital costs

Mild conditions of temperature, pressure, and pH

Complex purification because of complex reaction conditions

High conversion efficiency when optimized

Complexity of multiphase reaction conditions

New routes to novel molecules

Protein denaturation and cell lysis

Use of renewable resources

Low health and environmental risk

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Besides the use of different intact natural organisms as biocatalysts, recent developments in biotechnology allow for advanced tools to be applied in biocatalysis. 1. Advanced breeding and genetic engineering tools (Protoplast fusion, Plasmid transfer, Recombinant DNA technology), leading to the creation of new traits at the cellular level in plants, animals, microorganisms, and man 2. Hybridoma technology, leading to the production of pure and specific antibodies (Liddell and Cryer, 1991) 3. Plant tissue culturing, providing for manipulation and propagation of plants and of their genetic traits (Stafford and Warren, 1991) 4. Whole-cell and organelle immobilization, providing for practical exploitation of catalytic properties in specially designed reactors (Hartmeier, 1986) 5. Enzyme immobilization, providing for their more extended practical use (in enzyme reactors)(Hartmeier, 1986) 6. Protein engineering, allowing the modification of protein (enzyme) structure and activity (Moody and Wilkinson, 1990) 7. Advanced methods of culturing cells in bioreactors, of product separation, and of purification (Ho and Wang, 1991:Wheelwright, 1991) 8. Purely chemical methods leading to mimicking of biological entities and phenomena (Nakanishi et al., 1993; Kahn et al., 1988) 9. Combinations of the above.

There are many ways in which biocatalysts can be classified. One possible way based on the above advanced tools of biotechnology is listed in Table X. The two terms used in each pair in the table do TABLE X POSSIBLE CLASSIFICATION BASISFOR BI~CATALYSTS Cells vs. enzymes Free cells (suspended) vs immobilized cells Free enzymes (soluble) vs immobilized enzymes Natural vs modified (recombinant) biocatalysts (cells, proteins) Natural catalysts vs mimetic catalysts Single cells vs multicellular (or organ) culture Growing vs nongrowing cells (maintained) Microbial vs other cells (plant, animal, human) Nonspecific antibodies vs monoclonal antibodies Monoclonal antibodies vs catalytic or hybrid antibodies Batch vs continuous (or semibatch) processing mode-reactor Plug flow vs continuous-flow mixed reactor (CSTR) processing mode

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not necessarily represent opposites, but certain specific differences are introduced. One major distinction regarding any decision on biocatalyst selection is the question of the use of cells or enzymes. Table XI contrasts these two groups of biocatalysts. One conclusion can be easily reached: cells will be used for products requiring multistep reactions, and enzymes will be used for single-step reactions not requiring coenzymes regeneration. The spectrum of products exploited by organismic systems is quite broad (TableXII),biotransformation being more effectively carried out by enzyme systems. B. ORGANISMIC BIOCATALYSTS There is no need to classify organismic biocatalysts or to list their nutritional requirements. In addition to natural organisms, developments in molecular biology and genetics during the last two decades allow for the employment of genetically engineered (recombinant) organisms toward our applied goals. C. ENZYME-BASEDBIOCATALYSTS Enzymes can be used in two basic ways: as free enzymes (in solution) and as immobilized enzymes. The immobilization techniques include the following (Hartmeier, 1986): 1. Covalent attachment (to a carrier) via a spacer linker 2. Physical adsorption (to a carrier) 3. Covalent crosslinking (of enzyme molecules by a crosslinking

agent, e.g., by glutaraldehyde, or with an inert protein) 4. Entrapment in a macro- or micromembrane or gel (e.g., in a hollow fiber filtration module or in a microcapsule). A comparison of the different methods is attempted in Table XIII. The additional limitations, in addition to the those listed in the Table XIII, are: 1. Purification of enzymes is difficult and costly 2. Cannot generally be used for reaction systems requiring coen-

zymes, as their regeneration is difficult (e.g., a conversion of reduced coenzyme NADPH+ to the oxidized state, NADP). Most of the above immobilization methods can also be applied to cell immobilization. A good example of cell entrapment is the formation of alginate/Ca2 beads with cells entrapped inside. Immobilized enzymes and cells often exhibit better biocatalyst stability (enzyme activ+

TABLE XI

BIOPRCGESSING COMPARISON BETWEEN h'fiCROORGAh'ISMS AND ENZYMES Cells Advantages Low cost

Self-reproducing capability Size-dependent mass transport limitation Coenzyme regeneration capability Multireaction chain possible

Enzymes Disadvantages

Secondary reactions (incl. biomass) Possible mass transport limitation at immobilization Limited space-time domain

Advantages

Disadvantages

No secondary reactions

High cost

Coenzyme regeneration No mass transport limitation (in solution) High space-time yields

Possible mass transport limitation at immobilization

Suitable for only a few reaction steps Difficult downstream processing (e.g., pyrogens) Limited reproducibility (unless altered genetically)

Simple downstream processing Good reproducibility

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TABLE XI1 MAJORPROCESS APPLICATIONS FOR CELLULAR ACTIVITY Production of cell biomass Production of enzymes Production of primary metabolites Production of secondary metabolites Bioconversion of chemicals Production of proteins in nonnatural host by means of genetically altered organisms

ity) over an extended period of time. There are many examples of the use of immobilized biocatalysts (both enzymes and cells) having industrial significance. D. BIOCATALYST PROCESSING AND REACTORS

Physical and processing requirements for two major biocatalyst groups are often quite similar. Although many modifications or additional steps can be included, biotechnological processes generally share many common unit operations. A unit operation is an abstracted handling of material common to many chemical and biotechnological proTABLE XI11 COMPARISON OF ENZYME IMMOBILIZATION METHODS Method Covalent attachment

Covalent crosslinking

Adsorption

Entrapment

Advantages Immobilization may be affected by pH, ionic strength, or substrate concentration Enzyme strongly bound

Enzyme does not need a chemical modification; carrier can be reused; cheap process Enzyme does not require modification; enzyme protected from microbial degradation

Disadvantages Activity may be modified; costly process

Loss of enzyme activity during immobilization; carrier regeneration not possible Desorption may occur due to changes in ionic strength; microbial inactivation may occur Diffusional limitation of substrate to and product from, depending on particle size; enzyme inactivation during preparation possible

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cesses (e.g., a centrifuge is used in liquid/solid separations). Table XIV lists some typical unit operations encountered in bioprocessing. Biocatalyst preparation at the organismic level may include (to be expanded in the following sections) pure culture (organism) isolation, strain development, culture preservation, and inoculum (seed) development; at the enzyme level, preparation may include enzyme (protein) isolation, enzyme immobilization, and reactor design for soluble and immobilized enzymes. The central equipment in the biocatalyst processing scheme is the bioreactor. The concept of containment of biocatalysts in a reaction vessel originated from chemical reactors, the most well-known example being the application of reactors for submerged penicillin production by means of Penicillium fungi during World War 11. The reactor is a vessel, traditionally fitted with a mixer (this requirement has been amended) and with control and monitoring equipment to carry chemical reactions. Bioreactor classification can be based on the type of energy introduced for mixing (and aeration for aerobic processes): 1. mechanically mixed bioreactors 2. Pneumatically driven bioreactors (also called air-lift bioreactors,

a combination of a bubble column with internal or external loop) 3. Hydraulically driven bioreactors (e.g., a nonmixed vessel fitted with an external loop and a pump for liquid circulation; the Vogelbusch deep-jet bioreactor is a good example). 111. Primary Market Classification

For the purpose of this chapter (and the following one) we will adopt a classification of market on the basis of the primary market which they serve (Burrill and Lee, 1992). Market definitions are as follows. TABLE XIV

TYPICAL UNIT OPERATIONS AT BIOPROCESSING Raw material handling (solid, liquid) Media preparation [dissolution) Biocatalyst preparation Media and equipment sterilization [steam) Bioconversion Biocatalyst separation [centrifuge) Volume reduction (protein salting-out) Purification (extraction, chromatography) Product formulation

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1. Human health care: diagnostics and therapeutics 2. Agricultural biotechnology (Ag-Bio): animal and plant 3. Suppliers to the industry 4. Chemical, environmental, and services

According to 1992 data, the majority (83%)of public companies specialize in human health care (66% of the total biotechnology industry). In addition, the majority of newly created companies are active in this market segment. The ag-bio segment (9% of public vs 10% of the total) involves microbial crop protectants, animal growth hormones, plant and animal transgenics, food processing and diagnostics, and animal health (diagnostics and vaccines). The suppliers to the industry provide instrumentation and software, bioreactors and instrumentation equipment, supplies, and reagents (7% of public vs 16% of the total). The chemical, environmental, and services segment, comprising fine and commodity chemicals and environmental services (monitoring, bioremediation) and other services (analytical)(9Yoof public vs 8% of the total), is the fastest growing segment. IV. Pathway to Commercialization: Discovery and Development (D&D) Cycle

Figure 1 (R&Dscheme), otherwise denoted as the D&D cycle, presented the main pathway toward the marketplace as being the following (each having a discovery and development component). Product + Process + Market Some useful definitions are given below. Product in biotechnology signifies a chemical substance generated in a biotechnology-related manufacturing process, a medical device (e.g., DNA hybridization probe, MAbbased detection kit) for carrying out bioassay or chemical speciedorganism identification, or services delivered, as at an environmental cleanup. The definition of process as mentioned above, will be expanded to include: 1. An industrial/commercial route to manufacture a given product, 2. An industrial/commercial method to recover, remove, and detox-

ify compounds for environmental clean-up or in mining (e.g., soil bioremediation). Market is represented by beneficiaries of the products. Discovery is a process leading to a new product or process (for the first time). A more narrow definition comprises the time when a new biological or clinical

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use of a compound is identified or a new service established. Development is meant as a technical improvement or modification (innovation) of an existing product or process. Alternatively, the term may refer to the entire process of taking a newly discovered product through regulatory approval and to the point of marketing. V. Product Discovery

A. CLASSIFICATION OF PRODUCTS AND SCREENS The concept of biocatalysis, introduced in Section I1 as a laboratory rather than an industrially explored tool, may serve as a basis for demonstrating product potential from biotechnology. A product, a result of industrial manufacturing, originates from the application of different biological and chemical principles and screens, against the diversity of materials of an organismic or chemical nature. Table XV presents product categories according to industrial sectors. A more narrow classification, only for microbial products, is presented in Table XVI. Before product discovery begins, the target area must be designated. With input from marketing, research, and planning teams, the management outlines target areas of research as a clear commitment of the corporation. In a pharmaceutical industry, specific subclasses of the drug classification system may be chosen: central nervous system agents (phychopharmaceuticals), pharmacodynamic agents, chemotherapeutic agents, and metabolic diseases and endocrine function agents. The actual product discovery process involves several steps (Fig. 6). One can start by identifying a candidate (lead compound or lead structure). A lead compound is one that shows some biological activity or product application promise and is the direct bioactive chemical ancestor of a drug (deStevens, 1990). It can be derived from experience (known active compound), it may be found serendipitously, or it may arise from a structure-activity relationship (SAR, see below). Once a lead compound has been identified it may be subjected to structural variation, resulting in many test compounds. Screening of new products is a very time-consuming and expensive activity. Some screening services are available commercially. Only a small number of the test compounds will enter a final stage, leading to a better and safer drug. Typical sources available to an R&D organization are listed in Table XVII. Before attempting to classify screens, it will be useful to analyze the types of biocatalysts and how products can be derived from them. Different organisms are typically considered as a source of useful activi-

116

ALES PROKOP TABLE XV PRODUCT CATEGORIES ACCORDING TO INDUSTRIALSECTORS

Pharmaceuticals Diagnostics Monoclonal antibodies (diagnostic, preventive, and therapeutic products) DNA hybridization probes Therapeutics Regulatory proteins (insulin, interferons, human growth hormone, lymphokines, neuroactive peptides) Blood products (human serum albumin, antihemophilic factor, thrombolytic and fibrinolytic enzymes) Vaccines (viral, bacterial, parasitic) Antibiotics Alkaloids Ag-bio Animal Diagnosis, prevention and control of animal diseases Animal nutrition and growth promotion (feed supplements, rumen inoculum, antibiotics, growth hormone) Genetic improvement of breeds Plant Improvement of specific plant characteristics (herbicide resistance, insecticide resistance) Crop improvement (plant growth stimulation, nitrogen fixation) Crop protection (insecticides-toxins, infective agents) Plant pathology (phytotoxins, phytoalexins, plant disease) Suppliers to the industry Instrumentation Control software Bioreactors Chemical, environmental, and services Specialty chemicals and food additives Amino acids (glutamic acid, methionine, lysine, tryptophan, aspartic acid, phenylalanine) Enzymes (amylases, isomerases, rennin, cellulases, proteases, glucoamylase) Vitamins (B2, B12,C, E) Nucleotides and nucleosides Biomass products (bakers’ yeast, fodder yeast) Lipids (fatty acids, fatty alcohols, microbial oils, sophorolipids, surfactants) Steroids Aromatic chemicals Polysaccharides (alginates, pullulan, dextrans, xanthan) Nonnutritive sweeteners Fat replacers Flavors and fragrances Dyes (pigments) Starter cultures Continued

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TABLE XV (Continued) Commodity chemicals Alcohols and solvents (ethanol, glycerol, butanol, isopropanol, 2,3-butandiol, acetone) Organic acids (acetic acid, citric acid, lactic acid) Monosaccharides (glucose, xylose, sorbose,high-fructose corn syrup) Environmental applications Monitoring Bioremediation Pollution control and toxic waste management Microbial mining (leaching, accumulation of metals) Microbial-enhanced oil recovery Specific target molecules (synthetic) Specific target molecules (synthetic) Other services (analytical) Note. Adapted from Office of Technology Assessment (1984).

ties and products. A screening of organisms should be followed by their improvement by genetic means. Products produced by different organisms or isolate from them can be tested using different receptor/ ligand (recognition) mechanisms. Similarly, biocatalysts of a nonorganismic nature should be tested. This includes the variety of chemical species available in nature or man-made or modified chemicals. The systematic molecular modification of drug candidates relies on a structure-activity relationship. The drug discovery rationale is summarized in Fig. 7, showing three major components: cellular and molecular TABLE XVI MAJORPROCESS APPLICATIONS FOR MICROBIAL ACTIVITY Production of whole cells (biomass) Production of low-molecular-weight compounds Production of primary metabolites Production of secondary metabolites Transformation of organic compounds by nongrowing cells Production of high-molecular-weight compounds Polysaccharides Lipids Proteins Process dependent on general microbial metabolism Degradationloxidation of effluents and noxious wastes Mineral extraction Note. Reprinted with permission from Primrose (1987),Table 6.1, p. 63. Copyright 1987 by Blackwell Scientific Publications Ltd.

118

ALES PROKOP Phase

Lead finding

f l

Q 7n(/) Test compound

Better and safer

Lead optimization (Structure-activity relationship)

Drug development

FIG.6. Empirical phases of drug discovery.

diversity, cellular and molecular screens, and therapeutic screens. The modern cost-effective drug discovery program should start with the first two components, the third one being added only after the preliminary screen is completed. However, the time to develop and implement therapeutic screens requires an early start, before random screening begins. This is all in contrast to the traditional drug discovery approach. Until the past two decades, the effects of chemicals were measured in whole animals, isolated organs, tissues, or bacteria, e.g., using traditional in vivo pharmacological models. The endpoint measured was a functional response of the animal (e.g., a change in heart rate) or tissue (e.g., contraction) when the tested compound was administered to the animal or added to the fluid surrounding the tissue. This traditional approach of screening thousands of compounds for biological activity is being gradually replaced by more rational approaches based on cellular and molecular mechanisms, allowing more detailed understanding of the factors that govern the interaction of the compound with the target macromolecule. Similar screening programs exist for products of TABLE XVII SOURCES OF NEWPRODUCT LEADS Internal research and development programs Collaborative research programs with universities In-licensing from an outside company

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Sources of cellular and molecular diversity 1. Organismic world

collection of microorganisms mammalian cells plant cells other organisms *enrichment from nature 2. Strain/cell line

improvement traditional genetics genetic engineering

3.Natural products isolated (pure) compounds culture extracts extracts of organisms traditional medicines 4. Synthetic products bank of chemical species subunits of existing species breakdown products of existing species

Cellular and molecular screens Culture screen: selective pressure random screen

Genetic improvement:

Molecular recognition:

mutation 8 ligandlreceptor selection recombination antibody/antigen genetic engineering other recognition mechanisms

Chemical structure & function natural motifs chemical diversity modifying specificity mimetics

FIG.7.

Rationale for drug discovery (loosely adapted f r o m Affymax (1989),P. 29). * Shows some possible interactions.

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is orally active, and (2) it can be made via economic chemical synthesis (not via biotechnology). Captopril is thus an example of replacement of injectable proteins (having therapeutic activity through agonist activity or blockage of a key receptor site) by smaller patentable molecules capable of oral or transdermal administration. A well-tested model of the active site of angiotensin-converting enzyme has allowed the rational design of captopril, a novel and highly effective drug that combats high blood pressure (Ondetti et a]., 1993).

D. EXAMPLEOF DISCOVERY OF A NEWCHEMICAL CLASS (AND OF PRODUCT DEVELOPMENT) Captopril as discussed above is the first member of its class, representing a new chemical class of therapeutic drugs. Ivermectin (Merck) is another one. The product discovery part in this example is rather small; however, the extent of the whole story as published (Campbell et a]., 1983) is interesting for demonstrating the amount of effort and cost involved in development of a new drug. Losses of $3 billion per year in U.S. are attributed to parasitic infections of livestock. Most of the economic losses are due to parasitic worms (nematodes) and arthropods (insects, ticks, mites). The limitations of chemotherapeutic control of parasites are precluding their effective management: (1)There is a need to integrate treatment with epidemiological factors and management practices; (2) Different drugs must be used for different kinds of parasites; (3) The emergence of drugresistance in parasites. Ivermectins belong to the family of avermectins which constitute a new chemical class of drugs having a novel mode of action against a broad spectrum of nematode and arthropod parasites. It cures River Blindness, a highly prevalent disease in tropical and subtropical countries. Employment of microbiological methods resulted in the discovery of a new natural antibiotic activity. The assay, based on an in vitro test, proved to lead to nothing of potential interest. In vivo tests on mice infected with nematodes lead to the discovery of avermectins. The microbial producer was identified as an actinomycete (Streptomyces avermitilis) and was obtained from a soil sample after an extensive screening of 40,000 cultures. Initial fermentation yield was 9 pg/ml of broth. A 9-fold yield improvement was obtained through better medium formulation. Further mutation and selection lead to mutants exhibiting rapid growth rates, high avermectin production rates, and a longer production period. Media and mutant selection resulted in a 50-fold increase in yield to 1-5 g/liter.

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antibiotics or proteins (e.g., the antibiotic erythromycin inhibits an enzyme that is unique to bacteria, not man). A group of p-lactam antibiotics, including modified molecules as well as their analogs, exhibit a number of target-site reactions involved in the cell wall biosynthesis of gram-positive microorganisms, mostly in the final steps of a unique cell wall peptidoglycan (Fleming et a]., 1982). Two major cross-linking reactions are utilized to synthesize peptidoglycan: (1)glycan chain elongation to form a glycan backbone, and (2)transpeptidation resulting in protein backbone formation. The formation for the peptide bond between two pentapeptide units is accompanied by the release of a D-alanine residue. In the presence of penicillin, the second reaction is inhibited, causing a drastic disorganization of the cell envelope of growing cells. In addition, the terminal D-Ala group can be transformed using water as the acceptor in a reaction, referred to as the DD-carboxypeptidasereaction, which is also sensitive to penicillin. Both transpeptidase and carboxypeptidase reactions are depicted in Fig. 9. The DD-carboxypeptidase penicillin-sensitive activity has been used to screen for p-lactam antibiotic producers and can be assayed using the cell wall precursor UDP-MurNAc-L-Ala-D-Ala-Dap-D-Ala-D-Ala as substrate (UDP is undecapronyl-P-P, MurNAc is N-acetyl muramic acid, Dap is diamino pimelic acid). The DD-carboxypeptidase reaction may be monitored by determination of the liberated D-alanine,requiring complicated preparative and reaction systems. A new assay has been developed using an isolated target enzyme and HPLC of reaction products and substrates in an automated mode. This inhibition assay proved to be useful for screening and further detection of known j3-lactams (Fig. 8) as well as for discovering new ones. A great number of actinomycete isolates were screened and a few (0.04% of total screened) new isolates were found to produce new p-lactam antibiotics (Table XIX).

c. ROUTE OF DISCOVERYVS ROUTEOF MANUFACTURING The case of the discovery of captopril (Squibb) will serve as an example of a biotechnology route of discovery (ROD) as opposed to chemical route of manufacturing (ROM) of this prdduct. The most rewarding starting point of a search and discovery has been the diversity of organismic nature and the plethora of biological activities present there, whether they are of microbial, plant, insect, of mammalian origin. The example of captopril draws from the reptile area and clearly demonstrates that other limitations (delivery mode, economy, etc.) may prevent a biological route of manufacturing. Instead, a purely synthetic (chemi-

122

p-lactam type

Penicillin

ALES PROKOP

Example

Structure

-

Penicillin N COOH

R1 = a-amino-adipic acid (D)

Cephalosporin C Carbamate

Cephalosporin

'COOH

R1 = a-amino-adipic R2 = CHz-O-CO-NH2

acid (D)

Cephamycin C

Cephamycin

R1 = a-amino-adipic acid (D) Rz = CH2-O-CO-NHz

' COOH Clavams

Clavulanic acid R1=

Carbapenems

CH.CH20H

Thienamycin (trans p-lactam) R1= CH(OH)2 CH3 R2 = S-CHz-CHz-NH2

-

MM22380 (cis p-lactam) R1 = CH(OH)-CHs Rz = S-CHz-CH2-NHCOCH3 Nocardicin

Nocardicin A R1=

FIG. 8. B-Lactamantibiotics produced by Actinomycetes. The four-memberheterocycle with nitrogen atom distinguishes this group of chemical species (reprinted with permission from Fleming et al. (1982),Fig. 4,p. 126, copyright 1982 by Butterworth-Heinemann).

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I €.cob

I

123

Transpeptidase Activity

-

GIC NAc

I

Mur-NAc-L-Ala-D-Glu-mesoDpm-D-Ala-D-Ala

I

GlC - NAc

I

+

Glc-NAc I D-Ala-D-Ala-mesoDpm-D-Glu-L-Ala-Mur-NAc

I

Glc-NAc

I

GIC - NAc

I Mur-NAc-L-Ala-D-Glu-mesoDpm-D-Ala

I

-

GIC NAc

Glc-NAc

I

I

mesoDpm-D-Glu-L-Ala-Mur-NAc

I

+

D-Ala

I

I D-Ala

Glc-NAc

I D-Ala

€.co/i Carboxypeptidase Activity

-

GIC NAc

I

Mur-NAc-L-Ala-D-Glu-mesoDpm-D-Ala-D-Ala

I

+ H20

-

Glc NAc

-

GIC NAc I Mur-NAc-L-Ala-D-Glu-mesoDpm-D-Ala

I

+

D-Ala

-

GIC NAc FIG.9. Transpeptidase and carboxypeptidase reactions (reprinted with permission from Fleming et al. (1982),Fig. 2, p. 121, copyright 1982 by Butterworth-Heinemann).

cal) route or a mimetic product route (derived from the original biologically derived chemical entity) is used. An angiotensin-converting enzyme (ACE) is involved in the conversion Angiotensin I + Angiotensin 11,

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ALES PROKOP TABLE XIX TYPICAL RESULTS OF SCREENING OF @-LACTAMS Total number of isolates Number of positives (DD-carboxypeptidase) Thienamycins Penicillin N Cephamycins/cephalosporins Novel B-lactams

10,047 394 302 3 5 4

Note. Reprinted with permission from Fleming et al. (1982). Table 9, p. 128. Copyright 1982 by Academic Press.

cleaving a dipeptide (His-Leu) off the carboxylic terminus of angiotensin I. The product of this reaction is a peptide hormone exhibiting a vasoconstrictor activity leading to hypertension and congestive heart failure (Cushman et a]., 1980; Navia and Murcko, 1992). The key therapeutic goal is to control angiotensin I1 activity. Since ACE is a carboxypeptidase, it was hypothesized that its catalytic site might be similar to that of carboxypeptidase A, an enzyme that removes one amino acid from the carboxylic end of its peptide substrates. For carboxypeptidase A the three-dimensional structure of the enzyme-substrate complex is known. The most important features of this active site are a positive charge that interacts with the C-terminal carboxylic group on the enzyme active site of the substrate and a Zn2+ ion that activates the carbonyl group on the side chain of the substrate. On the basis of this model a nonapeptide, SQ 20,881 (a venom peptide), was proposed as an inhibitor of ACE: Glu-Trp-Pro- Arg-Pro-Gln-IIe-Pro-Pro. Two problems associated with SQ 20,881 precluded Squibb from further development of this peptide: (1) its administration is feasible only via injection, and (2) the high cost of its synthesis (note that, at present, recombinant DNA technology offers a relatively cost-effective method of manufacturing). Further refinement of the binding site model lead to the identification of the proline analog captopril as a potent inhibitor of the enzyme Zn2++ HS-CH,-CH(CH,)-CO-N-COO-

+

+,

featuring a negatively charged moiety as a counterion to the positive charge of the active site and a mercapto group that can function as a ligand for Zn2+ ions. The advantages of captopril are as follows: (1) it

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I

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is orally active, and (2) it can be made via economic chemical synthesis (not via biotechnology). Captopril is thus an example of replacement of injectable proteins (having therapeutic activity through agonist activity or blockage of a key receptor site) by smaller patentable molecules capable of oral or transdermal administration. A well-tested model of the active site of angiotensin-converting enzyme has allowed the rational design of captopril, a novel and highly effective drug that combats high blood pressure (Ondetti et a]., 1993).

D. EXAMPLEOF DISCOVERY OF A NEWCHEMICAL CLASS (AND OF PRODUCT DEVELOPMENT) Captopril as discussed above is the first member of its class, representing a new chemical class of therapeutic drugs. Ivermectin (Merck) is another one. The product discovery part in this example is rather small; however, the extent of the whole story as published (Campbell et a]., 1983) is interesting for demonstrating the amount of effort and cost involved in development of a new drug. Losses of $3 billion per year in U.S. are attributed to parasitic infections of livestock. Most of the economic losses are due to parasitic worms (nematodes) and arthropods (insects, ticks, mites). The limitations of chemotherapeutic control of parasites are precluding their effective management: (1)There is a need to integrate treatment with epidemiological factors and management practices; (2) Different drugs must be used for different kinds of parasites; (3) The emergence of drugresistance in parasites. Ivermectins belong to the family of avermectins which constitute a new chemical class of drugs having a novel mode of action against a broad spectrum of nematode and arthropod parasites. It cures River Blindness, a highly prevalent disease in tropical and subtropical countries. Employment of microbiological methods resulted in the discovery of a new natural antibiotic activity. The assay, based on an in vitro test, proved to lead to nothing of potential interest. In vivo tests on mice infected with nematodes lead to the discovery of avermectins. The microbial producer was identified as an actinomycete (Streptomyces avermitilis) and was obtained from a soil sample after an extensive screening of 40,000 cultures. Initial fermentation yield was 9 pg/ml of broth. A 9-fold yield improvement was obtained through better medium formulation. Further mutation and selection lead to mutants exhibiting rapid growth rates, high avermectin production rates, and a longer production period. Media and mutant selection resulted in a 50-fold increase in yield to 1-5 g/liter.

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Chemical investigation lead to isolation and structure determination: 1, Silica gel chromatograms indicated UV-absorbing bands 2. Mass spectra lead to identification of two series of compounds

3. Reverse-phase HPLC facilitated separation of compounds 4. Mass spectrometry in l 3 C mode lead to structure determination 5. X-ray crystallography lead to crystal structure

6. 300 MHz 'H NMR helped to determine solution conformation 7. Molecular modeling lead to determination of stereochemistry 8. Use of radiolabeled amino acids and organic acids helped to elucidate mechanism of formation and biosynthetic pathway. Combined chemical and biological findings are briefly reviewed below. 1. Compounds of the a-series (methoxy group of 5-position) were less effective than those of the b-series (hydroxyl group) 2. Avermectin B, (olefinic bond between carbons 22 and 23) is more active orally 3. Avermectin B, (hydrated bond between carbons 22 and 23 with hydroxyl at 23-position) is more active parenterally 4. The objective was to prepare compounds with the conformation of the two series but lacking the 23-hydroxyl to retain desirable features of both series. This was achieved by selective dehydrogenation of avermectin B, to result in ivermectin (22,23-dihydroavermectinB,) (Fig. 10).

The efficacy of ivermectin rests in broad spectrum activity, both oral and parenteral administration, and in potency much greater than other antihelmintics (10-1000 times more potent). The mode of action is through a y-aminobutyric acid (GABA)block, interfering in signal transmission from interneurons to excitatory motorneurons. The product is safe as long as it does not cross the blood-brain barrier. There is no toxicity in test animals at 30 times the recommended dose in cattle (subcutaneous injection), or at 20,000 times the recommended dose in dogs (orally). In addition, metabolic disposition in host animals has been carefully checked using tritium labeling (no residues in tissues or bodily fluids) and showing complete recovery of unaltered drug. The extent of tests and investigations reported for this drug is considerable even though it is applied as a veterinary drug. At the time of writing it was in commercial use in various countries and was made available free to many developing countries, as a result of a top-level decision at the manufacturing company, in exchange for free clinical trials in Egypt.

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A1 R5=CH3 B1

R5-

H

22, 23-Dihydroavermectin B1 (ivermectin)

FIG.10. Chemical structure of the avermectins (reprintedwith permission from Campbell et al. (1983), Fig. 2,p. 825, copyright 1983 by the AAAS).

E. EXAMPLEOF DISCOVERY FROM PLANTBIOTECHNOLOGY

In this example, the product is a herbicide tolerance in plants (Shah et al., 1986). Glyphosate [N-(phosphonomethyl)glycine]is a potent,

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broad-spectrum herbicide that inhibits the growth of both weed and crop plants. Glyphosate interferes with aromatic amino acid biosynthesis by inhibiting the enzyme 5-enolpyruvylshikimate-%phosphate (EPSP) synthase of the shikimate pathway. Inhibition of this enzyme prevents the synthesis of chlorismate-derived aromatic amino acids and secondary metabolites in plants. Glyphosate-tolerant Petunia hybrid cell culture was established after stepwise selection on increasing amounts of glyphosate. This cell line overproduces EPSP synthase 15- to 20-fold as a result of amplification of the corresponding gene. A chimeric EPSP synthase gene, designed to overproduce EPSP synthase, has been introduced into petunia cells by means of Agrobacterium-mediated transformation. Transformation conferred a tolerance to glyphosate in cell culture as well as in regenerated transgenic plants. The transgenic plants were sprayed with Roundup herbicide (formulated glyphosate with surfactant) at two to four times the dose required to kill 100% of the wild-type plants and survived. The EPSP synthase enzyme activity is localized in chloroplasts which represent a major site of aromatic acid biosynthesis, showing a targeted approach to herbicide tolerance in plants. F. SPECIAL CONSIDERATIONSFOR DISCOVERING (AND DEVELOPING) PRODUCTS OF DIFFERENT INDUSTRIAL SECTORS

Some special limitations will be discussed as related to product categories according to industrial sectors listed in Table XV. Health care products and pharmaceuticals are receiving special attention because of their human applications. The considerations that are to be particularly addressed are mode of product delivery, proprietary position, and cost of regulatory approval. Product delivery features two observations: (1)an injectable route is not considered acceptable outside of hospital (beyond chronic routine administration), and (2) oral administration of peptide hormones is currently not feasible due to enzymatic destruction in and poor absorption from the gastrointestinal tract. This may have a negative impact on potential marketability of the products resulting from recombinant DNA technology. Consideration of a proprietary position involves three major patent protection approaches (Fig. 11):(1)“Use” patents are possible, when featuring novelty of application; (2) “Process” patents are difficult to enforce; and (3)“Composition of Matter” (COM) patents are difficult to obtain for naturally derived drugs (not if they are new). Patent protection is highly desirable although not absolutely necessary for a successful commercial product.

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I

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FIG.11. Types of patent coverage.

The cost of carrying out product discovery and of gaining approval is very high. About $100 to $150 million is required to carry a new product through the approval process to obtain a New Drug Application (NDA) approval by the FDA (Burrill and Lee, 1992). This cost is due to the fact that it takes 6 to 10 years (sometimes 15) between initial discovery of product and final FDA approval. Not all capital invested is recovered in all instances. Pharmaceutical research involves highrisk investments that lead to infrequent commercializable results. The commercialization of agricultural products involves less stringent limitation and therefore lower costs, compared to pharmaceuticals. Important factors in product discovery (and development) in the agricultural area are as follows: 1. Efficacy (effectiveness of product candidates in the control of targeted pest; effective dosage per acre)

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2 . Toxicity (human safety issues, e.g., carcinogenicity and teratogenecity in field applications) 3. Product delivery (controlled slow delivery under field conditions) and sufficient shelf-life of biological products 4. Environmental risk (biodegradation potential in the field; risks of ground water contamination by product candidate and by metabolites resulting from microbial biotransformation reactions. 5 . Patent coverage (on the average it takes about 7 years to obtain an approval through regulatory agencies and this significantly reduces the effective time of patent coverage).

Table XX presents an example of breakdown of product quantity requirements to reach the pharmaceutical or agricultural markets. The total cost is somewhat correlated with the overall effort needed to produce the required quantity. For microbially derived products, a typical concentration of undeveloped product in the fermentation broth is 1 mg/liter. Thus, to produce the necessary quantities, the required broth volumes, obtained in a dedicated fermentation capacity, are (1985 data): therapeutic agent, 600 x lo6liters; agrochemical agent, 55 x lo6liters. In the chemical environmental, and service segments, specifically in specialty and commodity chemicals, economy (profit) plays a decisive TABLE XX PRODUCT REQUIREMENTS TO DEVELOP THERAPEUTIC AND

AGROCHEMICAL AGENTS Quantity required (kg) Testing phase

Therapeuticagent ~~~

Discovery Toxicity testing Animal tests Initial field tests Acute toxicity Teratogenicity Mutagenicity Carcinogenicity Subchronickhronic testing Clinical trials Field trials Total

Agrochemical agent

~

0.001

-

0.001 0.01

0.01-0.05

-

5 35

-

0.09 0.5

-

0.04

50

-

500

-

-

-600

3.5

50 -55

Note. Reprinted with permission from Ritchie (1985), Table 1, p. 403. Copyright 1985 by Society of Chemical Industry.

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I

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role in the choice of the manufacturing route (biological vs chemical, or a hybrid). In the environmental area, the service is segmented along discipline lines: 1. Civil and environmental engineering 2. Hydraulics, soil body mechanics (and nutrient delivery and stabili-

zation) 3. Microbiology and sanitary aspects 4. Regulatory and environmental compliance. Only rarely is all required expertise available in one organization. Some services are typically subcontracted (e.g., construction at the remedial site). Typical segmentation would be along the lines: environmental assessment-process-design-contract-compliance-operation and monitoring (some being lumped together).

G. APPROACHES TO DISCOVERY 1. Organismic Approach to Product Discovery (Selective Isolation and Random Screening Method )

In summary, this approach breaks down into: selective isolation of the desired characteristic using selective conditions (selective pressure) to promote growth or early detection of a mutant (e.g., using antibiotic resistance, antimetabolite resistance, analog resistance, allosteric inhibition, or constitutive product formation); and random isolation and screen (e.g., of survivors from a mutagenized population for production of antibiotics, growth factors, amino acids, nucleotides, or pharmacologically active compounds). The screening capacity determines the speed of progress. As these approaches are well established and have been in use for several decades in the pharmaceutical industry, the reader is referred to standard textbooks (Stanburry and Whitaker, 1984; Crueger and Crueger, 1990). 2. Genetic Approach to Product Discovery (Strain Development)

As natural isolates usually produce commercially important products in very low concentrations, an effort must be made to increase the productivity of the chosen organism. Environmental manipulation (optimization of growth and production) will be limited to the maximum ability of the organism. Genetic (classical) manipulation can lead to an increase of the potential yield. For industrial microbial organisms the approaches differ according to the class of product produced: (1) pri-

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mary metabolites or (2) secondary metabolites. For primary metabolites, only biosynthetic pathway modification aspects are listed. 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11.

Concerted or multivalent feedback control Cooperative feedback control Cumulative feedback control Sequential feedback control Isoenzyme control Modifying permeability Avoiding feedback inhibition or repression Autotrophic mutation Avoiding inhibitor or repressor toxicity Constitutive mutation Screening for revertant mutations, etc.

For secondary metabolites, the following are established methods. 1. Isolation of auxotrophic mutants 2. Isolation of resistant mutants 3. Isolation of revertant mutants 4.

Use of recombination

5. Use of protoplast fusion 6. In vitro genetic manipulation 7. In vitro genetic recombination (recombinant DNA technology)

The above and other methods leading to modification of properties other than the yield of product (resistance to phage infection) are covered in Stanburry and Whitaker (1984). Cruegers’ book (Crueger and Crueger, 1990) covers the classical mutagenesis and recombinant methods in detail. Both organismic and genetic approaches in product discovery still represent a core activity within a cooperate environment, but they are rapidly being replaced by more modern approaches (covered below). Most mutagenesis, selection, recombination, and genetic engineering of organismic and strain development methods can be also applied to other organisms. 3. Molecular Recognition Approach in Product Discovery

(Receptor Model) The explosion of knowledge in biological sciences is resulting in new approaches to drug discovery. The central concept that is relevant to this process is the relationship between the structure and function of the cell surfaces, namely of receptors (Keirns and Farina, 1989). Receptors are proteins, often glycoproteins, consisting of subunits of high

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I

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molecular weight, typically functioning in a lipid environment of cell membranes. Their intimate molecular structure is often in correlation with normal or pathological state of cells. Receptors then serve as molecular targets for hormones, antibodies, and drugs. Targets for antibodies are denoted as antigens. In addition, synthetic structural analogs of these molecules, such as hormone analogs, subunits, or modified antibodies, can interact with receptors or modify them. In some instances, analogs have been isolated from nature. Analogs of hormones are of two categories: agonists, mimicking hormone function (binding to receptor) and causing the normal response, albeit in a less potent way; and antagonists, which bind to the receptor but do not activate the hormone-induced functions. The bound antagonist competes with the binding of the natural hormone (or agonist) and blocks its physiological activity. Hormones, antibodies, and other ligands feature variable affinity to their molecular targets and form chemical complexes. Together, they are denoted as a molecular recognition system. The interaction between ligands and targets is becoming an important basis for rational drug design and development. The aim of such drug design is to screen potential therapeutic agents (natural or synthetic) against convenient molecular target models of normal and abnormal metabolic states of humans or of other organisms. With further developments in biological sciences, the above view of ligands (and their targets) can be further expanded. The following list demonstrates other recognition systems with the potential for drug discovery and the development of screens. 1. Natural receptors, antibodies, and substrates (interaction with enzymes to form E-S complex) 2. Monoclonal antibodies (or their subunits) resulting from hybridoma or recombinant DNA technologies 3. Human hybridoma antibodies 4. Catalytic antibodies 5. Enzymes (e.g., reverse transcriptase for screening anti-AIDS therapy 6. Segments of DNA or RNA molecules 7. Macromolecules and metabolites involved in cellular signal transduction mechanisms (cyclic AMP, protein kinases, phosphatidylinositol, ion channels, second messengers) 8 . Carbohydrates (or glycoproteins) involved in cell-to-cell signaling, and in the extracellular cell matrix (also lectins-plant proteins having an affinity for carbohydrates)

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9. Semisynthetic proteins, sequences (segments of natural proteins) of recognition (binding) sites of complexes: ligand-receptor, antibody-antigen, enzyme-substrate

It is obvious that recombinant DNA technology (and other methods, such as site-directed mutagenesis) will play an important role in obtaining, generating, or modifying some ligands or targets. The actual screening can be carried out in several ways. 1. In solution (both ligand and target molecules) 2. Target molecule can be covalently attached to a matrix

An in vitro whole cell (cell culture) assay also can be used. 3. Cell suspension with appropriately expressed surface targets 4. Cells as above, but attached to an immobilization matrix

A special solid-phase (or solution) peptide synthesis reaction system has been proposed as a model system for generating vast chemical diversity in peptide structure. An automated synthesis of randomized peptides is then screened against a convenient target receptor, resulting in optimized ligand with the best affinity (peptide epitope library). All of the above screening leads should then be subjected to an in vivo animal model, as binding of a ligand to a target does not guarantee the therapeutic value of the tested drug. The practical advantages of ligandtarget screening are: (1)sensitivity of assay, (2) specificity, and (3)considerable reduction in number of in vivo screens to be performed. In addition, the screen can embody a search for a suitable therapeutic index. This index is a ratio of toxic to effective doses, measuring the drug safety. The greater this index, the safer the agent is. Certain aspects of drug intolerance can be tested using a convenient receptor which is known to represent drug toxicity. Table XXI lists some examples of receptors as targets for drug discovery. It should be stressed that in many instances a given receptor may serve multiple functions and thus may lead to different classes of drugs. Additional potential targets for drug discovery may involve cell surface receptors for lymphokines, growth factors, attachment factors, viruses, etc. The molecular recognition system as a means of drug discovery occupies the top level of the modern drug eifort, using a proper balance of in vitro and in vivo assays (Fig. 12).

Chemical Approach in Product Discovery (Structure and Function Model) a. Structure-Activity Relationship. The prediction of biological activity from chemical structure of biocatalysts is largely impossible. 4.

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TABLE XXI SOME KNOWN RECEPTORS AS

Receptor

DRUGTARGETS

Ligand

Functional target ~

Acetylcholine receptor

Acetylcholine Nicotine (agonist) Atropine (antagonist)

GABA receptor Somatostatin receptor

y-Aminobutyric acid Somatostatin

~

~

~~~

Cardiac, smooth muscle, insulin release Neuromuscular Excitation of CNS, local anaesthetic Convulsions, locomotion, etc. Release of human growth hormone

Some progress, however, has been made by comparing similar compounds and relating them through the structure-activity relationship (SAR)(Martin, 1989). Compounds that exhibit similar biological activities are usually similar in some chemical and physical properties: (1)molecular shape (stereochemistry and steric repulsion), (2) valence distribution (dispersion interactions, hydrogen bonds, hydrophobic interactions), and (3) electrostatic interactions (charge distribution). Such compounds are called (bio)isosteric (Burger, 1990). Two examples of

\

Molecular recognition systems

\ \

/

Intact cells

\

/

Isolated organ

\

Intact animal

/

FIG. 12. Balanced drug discovery and screening program. An intact animal with induced pathological disfunction and transgenic animal model of a disease can be added at the bottom of inverted pyramid.

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isosteric guidelines useful in drug design are presented below. First, replacement of a carbonyl group for a closely related compound will generate a pair of compounds exhibiting similar biochemical activity. Indeed, p-aminobenzoic acid (PABA, H,NC,H,COOH) and sulfanilamide (SA, H,NC6H,S02NH,) both act on an enzyme needed in the biosynthesis of tetrahydrofolic acid, an important vitamin and growth factor. PABA is a substrate of this enzyme, while SA is an antagonist blocking the biosynthesis. In this case both interact with the enzymeactive site. In other cases, carboxylic acids and structurally similar sulfonamides act in a similar rather than antagonistic manner. Second, a partition coefficient between water and oil (usually 1-octanol) phase evaluates a drug’s structural proportions between electrostatic and lipophilic groups, as well as its potential ability to penetrate cellular and subcellular membranes. However, this test may fail as it does not always predict the above properties. Note the mosaic nature of cell membranes, consisting of layers of lipids and amphoteric proteins. The simple measurement of this physical property helps to assess the product’s biological activity. Besides SAR, an advanced method called quantitative SAR (QSAR) attempts to transform chemical structure into a list of physical properties, thus examining not the structure-activity but rather the physical property-activity relationship. Several quantitative descriptors of physical properties have been proposed. 1. Effect of remote substituents on a molecule on electrostatic, dispersion, or hydrophobic interactions 2. Effect of substituent size 3. Effect of all-or-non type (presence or absence of specific group) 4. Biological potency

Details and examples are given by Hansch (1990). Chemically based drug discovery methods, employing the SAR and/ or QSAR models, can be divided as follows. 1. Screening of natural chemical motifs for new products and applica-

tions 2. Screening of chemically generated diversity (molecular modification) 3. Screening of mimetics of complex biomolecules

b. Discovering Products in Nature. Nature provides a vast resource of chemical diversity. From the viewpoint of the SAR, the search goal should be based on activity rather than on chemical structure in this

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case. A good example is the mechanism-based screen for p-lactam antibiotics, mentioned above, emphasizing the importance of enzymes in bacterial cell-wall biosynthesis. The example below represents a discovery of product application for a known biochemical entity (Gregory and Preston, 1977). Extracts from urine of pregnant women exhibited an inhibitory activity of gastric acid secretion in an assay system. The active fraction was shown to contain two polypeptides, /3 and y (y is lacking the C-terminal arginine). P-Urogastrone was then isolated from human urine, purified, and enzymatically degraded, and the amino acid sequence was established. The sequence was very similar to that of known epidermal growth factor (hEGF).The product was cloned and expressed in Escherichia coli, produced and purified from a fusion protein. Chemical synthesis was too complicated, as the polypeptide has 53 amino acids. The product has two potential new medical applications: as an antiulcer compound and as a wound-healing agent. The second activity, shown in a burn-treatment model, is related to the hEGF activity. However, the product would not compete with an oral active drug. Thus the market forces decide on drug value. c. Generating Molecular Diversity. A systematic molecular modification of natural products may have the following benefits: enhancement of potency and specificity, and lowering of unwanted side effects and toxicity. Molecular modification has become the most widely used method in the initial phase of drug discovery. This approach is based on intimate understanding of three-dimensional structures involved in the molecular recognition systems (ligand-target complex) and on modification of either component involved (Martin, 1989). The unifying methodology is that of SAR (QSAR).It consists of the following issues (not all are necessarily used at one time). 1. Three-dimensional mapping of binding site, identification of the key ligand-target structure, and confirmation by means of different physicochemical and computing methods (single-crystal X-ray diffraction, nuclear magnetic resonance spectroscopy, potential energy mapping, mechanical models, molecular graphics and modeling) 2. Design and/or modification of ligand (analogs, variants) and target molecules by means of mechanical models, molecular graphics, chemical synthesis, and site-directed mutagenesis (in case of proteinaceous molecules)

The central concept of binding-site mapping is that any small molecule that binds to a target must physically fit into the binding site and

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must have no chemical properties that prevent binding. When a tight binding is observed, it is expected that the regional chemical properties of the ligand are complementary to those of the binding site on the target molecule. Binding site mapping will help to design new molecules with greater specificity, greater affinity, and higher potency. An excellent example of a successful search for antiparasitic agents is given in Ring et a]. (1993). An entirely new approach in exploiting molecular diversity is selection of combinatorial libraries. The combinatorial selection consists of repetitious use of the following steps. 1. Use of existing or generation of randomized combinations of building blocks (molecular diversity) via chemical or biological synthesis, consisting of sequences of different molecules or via nonspecific scission of macromolecules, giving rise to random pool of fragments 2. Selection against a target molecule of a ligand from the above library of sequences and fragments (denoted as a shape library) 3. Amplification of the selected ligands, followed by a repetition of the above cycle.

This method makes use of specificity in intermolecular interactions between elements of the shape library and a ligand or isolation of sequence of interest (subset of the library) on the basis of their catalytic properties, or any other parameter that allows for enrichment of the desired molecular sequence. In principle, the isolation of a single molecule with specified properties from an initial pool is possible from an extremely large number of sequences. Subsequently, a very small amount of enriched material can be replicated in vitro (e.g., by means of the PCR method). The power of the combinatorial library method lies in its ability to isolate extremely rare sequences with precisely specified properties from a very large pool of random sequences (Szostak, 1992; Kenan et al., 1994) and in finding conceptual leads for designing high-affinity ligands and effector molecules with the potential for drug discovery. Combinatorial libraries may constitute synthetic peptides, phage-expressed antibody libraries, oligonucleotide libraries, and small-molecule libraries. As target molecules, the following can be used: (1) DNA with protein binding sites, (2) RNA with protein binding sites, (3) catalytic RNAs exhibiting catalytic activity, (4) RNA with (other) ligand binding sites, and (5)other synthetic polymer mimics. Resulting ligands would serve as structural leads for synthesizing small molecules with desired activities. Combinatorial libraries will likely play an important role in drug design and discovery (Clackson and Wells, 1994).

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d. The Synthesis of Analogs and Variants Draws from the Pharmacophore Concept. The minimum structure required for a compound to possess its particular biological activity is called the pharmacophore. It results from trimming away extraneous portions of the molecule, leaving behind skeletal structures and retaining key functional groups, and from further modification of those (PABA vs SA as mentioned above). Extensive discussion and examples of molecular modification are given by Martin (1989)and Austel (1989). Site-directed mutagenesis and other modern techniques can be used to modify products for specificity. By means of recombinant DNA technology one can change a specific portion of the DNA code for a protein in order to change specific amino acids of the protein. Consequently, a hypothesis on the role of specific amino acids in the function of proteins can be tested by systematic variation of the amino acids. Thus, new or improved proteins (variants) can be made by increasing the thermal stability or catalytic efficiency or by changing the pH profile or substrate specificity. Chimeric proteins, constructed by means of the combination of several gene regions, can be made. Protein engineering then uses thorough knowledge of protein structure and proposes changes in the structure that might result in useful functional change. A comparative analysis of proteins reveals many empirical rules that can be used as a guide for replacement of amino acids in protein engineering. A good example of molecular modification on the microbial biotechnology side is the possibility of generating p-lactam analogs using biosynthetic chemistry (Hollander et al., 1984). A complex chemistry is involved in the cyclization reaction of a tripeptide (6-~-aaminoadipy1)L-Cys-D-Val)by Cephalosporium acremonium into the plactam nucleus. The p-lactam nucleus can be considered here as a pharmacophore. The clue to discovery of this cyclase enzyme (also called isopenicillin N-synthetase) was from observations on the 3methyl hydroxylation enzyme. Because the synthetase specificity is not absolute for the natural tripeptide, the enzyme could be used to produce p-lactam analogs. The scenario could be as follows: cloning of the enzyme into a bacterium] testing for activity with a new tripeptide substrate] and characterizing the enzyme activity/specificity. e. Product Derived from Mimetics. A simple chemical replacement of complex biomolecules featuring biological activity is termed biomimetics (or a ligand that resembles the shape of another molecule and competes with it for binding with a target molecule). Much effort has

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been concentrated on modification of peptides. These include variation of the backbone (exchange of L- for D-amino acids, isosteric replacement of the peptide bond, and reversion of the peptide bond) or replacement of the peptidic by nonpeptide structures. Peptide nucleic acids are novel DNA mimetics in which the sugar-phosphate backbone has been replaced with a backbone based on amino acids.

H. ROLEOF SERENDIPITY IN PRODUCT DISCOVERY Modern rational screening for new drugs is a combination of good science and chance observation (serendipity)(Sneader,1986).Serendipity is a phenomeonon in the course of problem solving resulting in unanticipated findings and the discovery of new and unexpected phenomena. The combination of chance with creativity produces many opportunities. The artificial sweetener aspartame (Asp-Phe-Met) was discovered as the last amino acids of gastrin, a polypeptide hormone. Another way of discovering drugs is an accidental observation of biological effects on tested drugs or side effects that bear no relation to the originally planned program (e.g.,the hypotensive agent minoxidil). Until recently, traditional drug discovery depended largely on accidental observations during the drug screening. The current trends use most innovative rational methods combined with serendipity. Many drugs discovered serendipitously or by nonrational screening procedures over the last three decades have subsequently been shown to act by interfering with a particular mechanism (aspirin-an anti-inflammatory drug-inhibits prostaglandin synthetase). As the discovery cannot be planned, scientists are allowed to explore unusual ideas, to challenge a dogma, and to think beyond the ordinary experiment. A solution that they may arrive at could have eluded others for some time. This kind of thinking is denoted as “lateral thinking” in discovery (Spilker, 1989). While looking at Fig. 3, we can identify it as a combination of the T3 and T4 types.

I. HOW THE TOP 20 DRUGSWERE DISCOVERED Table XXII lists the top 20 US. drugs and their major route of discovery (their total sales approached $6 billion in 1988; Miller and Brewer, 1992). The mimetics route is not listed, as it is a path of the future. Some routes feature minor involvement of outer routes in addition to a major one, e.g., the use of chemical modification or receptor-ligand

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TABLE XXII

ROUTE OF DISCOVERYOF LEADCOMPOUNDS FOR THE BEST-SELLING U.S. DRUGS Pharmacological assay (total occurrence: code number in parentheses) ~~

~_ _ _ _ _ _ _

~

Route of lead compound discovery

In vivo (animal) screen

In vitro screen

Random screen of microorganisms and chemicals

4: (3), (lo), (4), (5)

Screen for analogs

3: (11, (21, (16)

2: (61, (9)

Unexpected clinical or biochemical observations

3: (12), (111,(18)

2: (81, (17)

6: (71, (191,(201, (131, (14). (15)

Note. The following list of the top 20 brand dollar name prescription products for 1988 in the U.S. in the order of dollar value (fmm $808 to $162 million) has been used to compile the above table. Compound names are listed first,followed by brand names. (1)ramitidine (Zantac),(2) cimetidine (Tagamet), (3)diltiazem (Cardisem), (4)naproxen (Naprosyn), (5)alpazolam (Zamax), (6)captopril (Capoten). (7)cefaclor (Ceclor), (8)atenanol (Tenormin), (9)enalapril (Vasotec), (10)nifedipine (Procardia), (11)piroxicam (Feldene), (12)paracetamol (Tylenol),(13)lovastin (Mevacor), (14)terfenadine (Seldane), (15)hydrochlorothiazine triamterene (Duaside), (16)Sulindac (Clinoril), (17)Metaprolol (Lopressor), (18)estrogens (Premarin), (19)celbriaxone (Roceptrin),(20)cefoxitin (Mefoxin).

interactions. Using these combined numbers (not shown in the table) the rational approach comprises about 75% of the overall approach. Some additional data on these drugs and their discovery are given by Bindra and Lednicer (1982), Sneader (1986), and Lednicer (1993). VI. Process Discovery

A. APPROACHES TO PROCESS DISCOVERY It is often impossible to separate process discovery from product discovery, as in many instances product discovery will simultaneously determine a way of manufacturing. The methodology used in product discovery is often shared with the process discovery. Bioprocess discovery is carried out by means of one or more of the following methods: mechanistic approach, general systems approach, and statistical approach. The mechanistic approach combines a chemical structure search and a metabolic pathway search (organism-product pathway of process discovery). The chemical structure search has been discussed in the above section, and the metabolic pathway search could be equated with some elements of Strain Development. The result is an organism with

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a suitable metabolism, generating a desired product. This step can also include genetic improvement of the organism. A modern rational version of this route is denoted as metabolic engineering. The organism-product route is thus the most common route for process discovery. An example will be presented below. Metabolic engineering attempts to reprogram metabolic activities of cells via amplification of existing protein activities of cells, activation or inhibition of existing activities, and addition of new activities, all through employment of recombinant DNA technology. An enzyme (or several enzymes of a metabolic pathway) is encoded into an organism of choice from a different source. A theoretical framework is available in terms of a computer algorithm that allows testing of qualitative and stoichiometric influences of such metabolic manipulations; that is, it allows examination of an addition or deletion of certain enzymes on pathway availability (Seressiotis and Bailey, 1986). The general systems approach at process discovery makes use of all kinds of information concerning product chemistry, enzymology, biochemistry, and microbiology, including other fields of science and engineering (e.g., bioprocess technology), and relates them in a search for patterns leading to process discovery. At the same time a great deal of serendipity is involved, making use of unexpected findings of seemingly unrelated observations, linking categories of phenomena which have previously appeared unrelated. It is obvious that there are no rational rules or steps available in such searches, requiring contribution of specialists from different disciplines. This approach may be useful for an initial decision on the type of organism-product combination. The statistical approach uses “brute force” in process discovery, searching in a multidimensional space of variables including media components. Brute force means the employment of a great number of experiments aimed at screening of variables (and arriving at important ones, systemic variables), undergoing stochastic, random changes in their scalelsize. the statistical approach has been used for optimization (of culture conditions and in media formulation).According to Haaland (1989),statistical screening involves identification of process variables, identification of important (systemic) variables, and optimization. The end result is reduction of process variables and more precise knowledge of processing conditions. The statistical experimental design will provide the most information from the smallest number of experiments and will guide a researcher in executing the experiments. The effective strategy should combine experiments with model building and numerical optimization.

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B. BIOTRANSFORMATIONS The concept of biotransformation was originally introduced from the microbial viewpoint. An expanded view covers the capabilities of all organisms, including mammals. The advantages of biotransformations relative to chemical synthesis are numerous. The specific and selective reaction aspect of biotransformations makes it a special subapproach of the mechanistic approach mentioned above. Types of microbial biotransformations are listed in Table XXIII. Two microbial-substrate views are encountered: metabolized substrate and cometabolized substrate. In the case of metabolized substrate (or xenobiotic), a product precursor or a xenobiotic serves as a substrate for microbial growth and can be completely broken down in some instances, as is the case of some pesticides. The herbicide dalapon, a chlorinated fatty acid, is converted by Arthrobacter sp. into pyruvate after oxidative dehalogenation (Crueger and Crueger, 1984). In the case of cometabolism, the organism does not obtain energy for growth. There are numerous examples from chemicals representing hazardous wastes. Xenobiotics are chemical species that do not normally occur in nature and are man-made (e.g., dalapon, DDT). Reaction products, resulting from specific biotransformation enzyme/ cell (microbial) systems, then serve for process discovery. Examples include carrier-bound immobilized glucose isomerase and penicillin acylase, and whole-cell dehydrogenation of sorbitol to sorbose (a precursor of ascorbic acid; see below) by Gluconobacter sp. For nonmicrobial biotransformations (e.g., mammals, insects, plant cells), defined as metabolic conversions of chemical substances of nonbiological origin (xenobiotics),a distinction is made between two phases TABLE XXIII

TYPICAL BIOTRANSFORMATION REACTIONS Acylation Amination Amidation Cleavage of C-C bond Condensation Decarboxylation Dehydration Dehydrogenation Deamination Demethylation Epimerization Epoxydation Esterification

Halogenation Hydration Hydrolysis Hydroxylation Isomerization Methylation Oxidation Phosphorylation Racemization Reduction Resolution of racemates Sulfoxidation Transglycosidation

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of biotransformation (Bozler and Schmid, 1989). Phase I reactions include hydrolytic, oxidative, and reductive processes. In Phase I1 reactions, the product of these processes or the parent drug itself is conjugated with hydrophilic endogenous substances. The resulting conjugates are often considerably more water-soluble and more easily eliminated from the body than the parent drug or its Phase I metabolites. Metabolically active enzymes have evolved to cope with certain requirements, to respond to toxic compounds, and to supply the organism with energy and physiologically important compounds. The Phase I enzymes involved in biotransformations are: 1. Cytochrome P-450-dependent monooxidases (mixed function oxidases), oxidizing aliphatic and aromatic hydrocarbons, alcohols, amines, amides, ethers, sulfides, steroids, and fatty acids 2. Cytochrome P-450-independent oxidative and reductive enzymes, such as monoamine oxidase, leading to deamination of primary, secondary, and tertiary amines, alcohol dehydrogenase oxidizing primary, secondary and aromatic alcohols, xanthine oxidase oxidizing purines, and aldehyde oxidase oxidizing aldehydes 3. Hydrolases, breaking down esters, amides, and epoxide bonds.

In specific chemical terms, such reactions include: 1. Oxidation of carbon atoms of aliphatic chains 2. Hydroxylation of carbon atoms of saturated rings 3. Oxidation of aromatic compounds 4. Dealkylation of short-chain alkyl ethers

5. Dealkylation and deamination of amines 6. 7. 8.

N-Oxidation of amines Oxidation of thioethers Reductions.

Phase I1 reactions increase hydrophilicity even more drastically. The metabolic conjugation reactions involve the activation of the endogenous substrate, a step requiring energy, followed by coupling reactions. Reaction types are glucuronidation, sulfation, glucose conjugation, methylation, acetylation, glutathione conjugation, and amino acid conjugation. The conjugation reactions can also result from microbial activity, although this occurs less frequently. Linkage of a pesticide or its product with naturally occurring compounds, such as amino acids or carbohydrates, results in a temporary detoxification. The transformation of a fungicide proceeds via an amino acid conjugate (Crueger and Crueger, 1990). In mammals, the liver is the most important organ for biotransformation. Other organs (skin, blood, lung, kidney, brain, and gut) perform this activity as well. In the latter, gastrointestinal microflora

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is a major player. Bozler and Schmid (1989) present many examples of nonmicrobial-based biotransformations. Such biotransformations can be used to generate important products and processes. Growing, resting, or immobilized cells and organelles, or cell-free enzymes or immobilized enzymes, are therefore a viable bioprocess alternative for process discovery.

c. EXAMPLESOF BIOPROCESSDISCOVERY A brief history of product and process discovery of two vitamins is presented below. The history of vitamin C (ascorbic acid) is an example of a mechanistic approach, the result of a search for a convenient chemical structure-organism combination (Florent, 1986). The origin of this product discovery goes back to disorders due to lack of fruit in the diet. The revelation of the chemical structure of ascorbic acid came from Haworth in 1932, following its isolation from lemons by King and Waugh in the same year. Previous studies by SzentGyorgyi in 1928 on biological cell oxidation lead to the isolation of a substance later found to be identical to that from the lemon, and, in turn, this lead to the understanding of the biological function of vitamin C. Most commercial processes for vitamin C start with glucose and lead to 2-keto-~-gulonicacid (KGA), an intermediate to vitamin C. KGA is then converted to ascorbic acid by a two-step chemical synthesis. They all represent a combination of chemical and biological reaction steps, to a different degree. 1. Reichstein-Grussner’s (1934) five-step process from glucose to KGA is a chemically dominant (80%) semisynthetic process with four chemical steps and a second step involving dehydrogenation of sorbitol to sorbose by a biotransformation reaction.

D-glucose --* D-sorbitol + L-sorbose + diacetonesorbose --* diacetone2-ketogulonic acid + 2-keto-~-gulonicacid 2. The L-idonic acid process is a semisynthetic, three-step process (1960 to 1970), largely based on microbial activity (67Y0),the second step, dehydrogenation of 5-keto-~-gluconicacid to L-idonic acid, involving chemical catalysis, 3. Shionogi’s 2,5-diketo-~-gluconicacid process, developed in the late 19703, is a two-step fermentation process (Erwinia and Corynebacterium), the second step being too slow. 4. Genentech’s genetically engineered microbial pathway process in the mid-1980’s is a single-step fermentation using Erwinia herbicola,

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improving the limiting step above. This was achieved through cloning the Corynebacteriumgene encoding the 2.5-ketogluconicacid reductase into the Erwinia from the Shionogi process.

Market Forecasting: Industry status Finn competitive position: Market Technology Market size potential Market growth potential Timing Forecast by region/ territory and product

4-D

Strategic Planning: Market changes Market niches (product line) Revenue forecast Strategic investment Investment intangibles (satisfaction)

Market Analysis (Distribution Requirements): Consumer perceptions (demographics) Consumer preferences (psychographics) Purchasing behavior (shopping patterns) Product benefits and attributes wanted Perceived problems in present products Siuational factors surrounding product usage I

I

I

Consumer

Product positioning: Perceptions (characteristics) Preferences (benefits)

Firms

Products

Product development

FIG.13. Market discovery and development.

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Detailed chemistries of the above processes are explained in Florent (1986).The first method, the first commercial process, still remains a dominant manufacturing process; the others may represent improvement in individual chemistries but remain uncompetitive. The last process, being a single-step fermentation process, looks attractive, but the fermentation is rather slow and is not being explored commercially. The history of vitamin B2 (riboflavin) involves a product discovery milestone (chemical synthesis by Karrer in 1935),three different process discoveries, and an innovative process development, all being commercially explored (Florent, 1986): 1. Pure chemical process 2. Yeast fermentation process (initially, in 1935 Eremothecium ashbyii was a winner, but genetic variation prevented its widespread use-this is an example of bioprocess discovery within a chemically dominated process); Ashbya gossypii proved in the 1980’sto be more reliable (Merck)-this is an example of bioprocess discovery within a purely bioprocess route Market discovery and strategic planning

kid Market potential

Product

Market development

I

Pilot market study

t Market development

Area market test

Market in place

Product in marketplace

Consume; check 4

life-cycle

FIG.14. Market discovery and development and consumer check.

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3. A semisynthetic five-step process, the first step involving glucose fermentation to ribose using Bacillus pumilus (Takeda, 1974),followed by four chemical synthetic steps; this process has captured more than 50% of the market.

A recombinant bioprocess discovery for vitamin B2 has also been reported, although it is not competitive enough. VII. Market Discovery

Market discovery is not a term used in the business literature. It is used in the context of this article to parallel it to other discovery terms. Market discovery features three main components (Fig. 13): market forecasting, strategic planning of market, and market analysis. They are all important for a new product prior to its commercial release. Once a product is on the market, consumers, and their perceptions and preferences (benefits), will determine its viability. Based on this information a company may proceed with market (product) development (Fig. 14). The consumer position is of critical importance for this process.

External analysis Market size Competition Patent and technology position Economic environment Legislative and regulatory environment Political and social conditions

Financial analysis Revenue analysis Cost analysis Profitability analysis

I Internal analysis Purpose or mission of a company Corporate objectives Resources (physical and human)

I

I

FIG.15. Business environment of a company.

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CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I

Market is only a small part of the whole business environment, which includes three components (Fig. 15): external analysis, financial analysis, and internal analysis.

VIII. Interactions of Discovery Aspects within the D&D Cycle A. PRODUCTDISCOVERY vs PRODUCTDEVELOPMENT Often, as was the case with product/process discovery, boundaries between product discovery and development are blurred. Product development is achieved in several ways; some of them were already presented in the product discovery section. The actual route depends on the product type. 1. New class of compounds 2. Analogs for nonprotein products (e.g., penicillins)

/

/

Product development

\

P

Anal( non-proteins

\c/

New classes of compounds via screens

\

FIG. 16. Product discovery vs product development: enlargement in scope and expenses.

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3. Variants for protein products (e.g., via a site-directed mutagenesis within a protein gene) 4. Improved product form (e.g., for better drug delivery) 5. Products resulting from mimicking of biological activity (mimetics).

A limited view of product discovery would encompass only lead compounds, the rest being attributed to the product development. The relationship between product discovery and development is illustrated in Fig. 16. Although product discovery may represent considerable effort, product development is more expanded in scope and expense. B. PRODUCT DISCOVERY vs PROCESS DISCOVERY~DEVELOPMENT

Figure 17 shows a growth/innovation matrix. Product discovery, reinforced with patents, is thus an important tool in an innovation process. Figure 17 also lists some relevant examples.

PRODUCT Existing ~~

New

~

Process innovation (Process development) e.g., penicillin Existing

Product discovery (surprised new use) "Use" or "COM" patent, e.g., aspartame as sweetener or microbial transformation of steroids (product analog)

PROCESS

New

Process discovery "Process" patent, e.g., insulin via genetic engineering (*)

Product discovery/Process discovery (double discovery) "Use"/"Process" patent or "Process"/"Product" patent, e.g., EPO via genetic engineering(*) or EPO variant (*)

FIG.17. Processlproduct growthlinnovation matrix. * May include "COM" patent (e.g., amino acid sequence of proteins].

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C. MARKET DISCOVERY vs DEVELOPMENT Figure 18 presents a product/market growth/innovation matrix, showing how to expand the existing products/markets and to develop new products/markets. In a market penetration strategic option, there is an advantage of both product and market knowledge; however, the product life-cycle may quickly mature and then decline. In a product development strategy, market knowledge is in place; however, product knowledge is limited. It aims to introduce new products to the existing markets. Market discovery and development entails introduction of existing products to new markets through new geographical markets (foreign countries) or new market segments. Product knowledge is already in place. Diversification strategies can take various forms, all typical in biotechnology: 1. “Down” the channel-expanding internally or through acquisitions (taking over wholesale or retailing functions) 2. “Up” the channel (growth via ownership of supplier of raw materials) 3. Horizontal (acquisition of competition).

I Existing

Existing

I

PRODUCT

Market penetration (product life-cycle is limited)

New

Product development (knowledge of produdmarket is limited)

~

New

Market discovery and development (product knowledge in place)

Diversification: (1) “down’ the channel (wholesale, retailing) (2) ‘up” the channel (supplier) (3) horizontal (acquisition of competition)

FIG.18. Market/product growth/innovation matrix (loosely adapted from Stevens and Shenvood (1987),Fig. 2-2, p. 20).

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Other diversification strategies may involve adding products which are technology related. Each of the growth strategies provides alternatives that can be pursued at the same time. REFERENCES Affymax, N. V. (1989).Private placement memorandum (internal report). AN, Palo Alto, CA. Austel, V. (1989). The medicinal chemist’s approach. In “Modern Drug Research. Path to Better and Safer Drugs” (Y. C. Martin, E. Kutter, and V. Austel, eds.), pp. 243-307. Dekker, New York Bindra, J. S . , and Lednicer, D., eds. (1982).“Chronicles of Drug Discovery,” Vol. 2.Wiley, New York. Bozler, G., and Schmid, J. (1989).Priniciples of pharmacokinetics and drug metabolism. In “Modern Drug Research. Path to Better and Safer Drugs” (Y.C. Martin, E. Kutter, and V. Austel, eds.), pp. 77-160. Dekker, New York. Burger, A. (1990).Drug design. In “Drug Development” (C. E. Hamner, ed.), 2nd ed., pp. 39-49. CRC Press, Boca Raton, FL. Burrill, G. S . , and Lee K. B., Jr. (1992).“Biotech 93.Accelerating Commercialization. An Industry Annual Report.” Ernst 8r Young, San Francisco. Campbell, W. C., Fisher, M. H., Stapley, F. O., Alberts-Schonberg, G., and Jacob, T. A. (1983).Ivermectin: A potent new antiparasitic agent. Science 221,823-828. Clackson, T., and Wells, J. A. (1994).In vitro selection from protein and peptide libraries. Trends Biotechnol. 12, 173-184. Crueger, W., and Crueger, A. (1990).“Biotechnology: A Textbook of Industrial Microbiology,” 2nd ed., Chapters 2 and 3. Sinauer Assoc., Sunderland, MA. Cushman, D. W., Ondetti, M. A., Cheung, H. S., Sabo, E. F., Antonaccio, M. J., and Rubin, B. (1980).Angiotensin-converting enzyme inhibitors. In “Enzyme Inhibitors as Drugs” (M. Sadler, ed.), pp. 231-247. Macmillan, London. deStevens, G. (1990).Lead structure discovery and development. In “Comprehensive Medicinal Chemistry. The Rational Design, Mechanistic Study and Therapeutic Application of Chemical Compounds” (C. Hansch, ed.), Vol. 1,pp. 261-278. Pergamon, Oxford. Finn, R. K. (1988).Preface. In “Biotechnology Focus” (R. K. Finn et al., eds.), p. v. Hanser, Munich. Fleming, I. D., Nisbet, L. J., and Brewer, S. J. (1982).Target directed antimicrobial screens. In “Bioactive Microbial Products: Search and Discovery “J, D. Bu’Lock, L. J. Nisbet, and D. J. Winstanley, eds.), pp. 107-130. Academic Press, London. Florent, J. (1986).Vitamins. In “Biotechnology” (H.J. Rehm and G. Reed, eds.), pp. 115-158. Verlag Chemie, Weinheim. Gilbreath, R. D. (1987).“Forward Thinking: A Pragmatist’s Guide to Today’s Business Trends.” McGraw-Hill, Boston. Gregory, H., and Preston, B. M. (1977).Primary structure of hormone urogastrone. Int. 1. Pep. Protein Aes. 9,107-118. Haaland, P. D. (1989).“Experimental Design in Biotechnology.” Dekker, New York. Hansch, C., ed. (1990).“Comprehensive Medicinal Chemistry. The Rational Design, Mechanistic Study and Therapeutic Applications of Chemical Compounds,” Vol. 4.Pergamon, Oxford. Hartmeier, W. (1986).“Immobilized Biocatalysts. An Introduction.” Springer, Berlin.

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Ho, C. S., and Wang, D. I. C., eds. (1991). “Animal Cell Bioreactors.” ButterworthHeinemann, Boston. Hollander, I. J., Shen, Y.-Q., Heim, J., and Demain, A. L. (1984). A pure enzyme catalyzing penicillin biosynthesis. Science 224, 610-612. Kahn, M., Wilke, S., and Chen, B. (1988). Nonpeptide mimetics of p-turns: A facile oxidative intramolecular cycloaddition of an azodicarbonyl system. 7. Am. Chem. SOC.110, 1638-1639. Keirns, J. J., and Farina, P. R. (1989). Drug action and receptor theory. In “Modern Drug Research. Path to Better and Safer Drugs” (Y. C. Martin, E. Kutter, and V. Austel, eds.), pp. 1-34. Dekker, New York. Kenan, D. J., Tsai, D. E., and Keene, J. D. (1994). Exploring molecular diversity with combinatorial shape libraries. Trends Biochem. Sci. 19, 57-64. Lednicer, D., ed. (1933). “Chronicles in Drug Discovery,” Vol. 3. American Chemical Society, Washington, D.C. Lefton, R. E., Buzzotta, V. R., and Sherberg, M. (1980). “Improving Productivity Through People Skills: Dimensional Management Strategies,” pp. 436-462. Ballinger, Cambridge, VK. Liddell, J. E., and Cryer, A. (1991). “A Practical Guide to Monoclonal Antibodies.” Chichester, New York. Martin, Y.C. (1989). Theoretical basis of medicinal chemistry: Structure-activity relationship and three-dimensional structures of small and macromolecules. In “Modern Drug Research. Path to Better and Safer Dugs” (Y. C. Martin, E. Kutter, and V. Austel, eds.), pp. 161-216. Dekker, New York. Miller, J. S., and Brewer, S. J. (1992). The discovery of medicines and forest conservation. In “Conservation of Plant Genes: DNA Banking and In Vitro Biotechnology” (R. P. Adams and J. E. Adams, eds.), pp. 119-134. Academic Press, San Diego, CA. Moody, P. C. E, and Wilkinson, A. J. (1990). “Protein Engineering.” IRL Press at Oxford University Press, Oxford. Nakanishi, H., Ramurthy, S., Raktabutr, A., Shen, R., and Kahn, M. (1993). Peptidomimetics of the immunoglobulin supergene family-a review. Gene 137, 51-56. Navia, M. A., and Murcko, M. A. (1992). Use of structural information in drug design. Curr. Opin. Struct. Biol. 2, 202-210. Office of Technology Assessment (1984). “Commercial Biotechnology. An Interdisciplinary Analysis,” OTA-BA-218, p.3. U.S. Congress, Washington, D.C. Ondetti, M. A., Cushman, D. W., and Rubin, B. (1993). Captopril. In “Chronicles of Drug Discovery” (J. S. Bindra and D. Lednicer, eds.), Vol. 1,pp. 1-32. Wiley, New York. Primrose, S.B. (1987). “Modern Biotechnology.” Blackwell, Oxford. Prokop, A. (1982). Systems analysis and synthesis in biology and biotechnology. Int. I. Gen. Sys. 8, 7-31. Ring, C. S., Sun, E., McKerrow, J. H., Lee, G. K., Rosenthal, P. J., Kuntz, I. D., and Cohen, F. E. (1993). Structure-based inhibitor design by using protein models for the development of antiparasitic agents. Proc. Natl. Acad. Sci. U.S.A. 90,3583-3587. Ritchie, G. (1985). Biologically active secondary metabolites. Chem. Ind. (London), June 17, pp. 403-407. Seressiotis, A., and Bailey, J. E. (1986). HPM-an algorithm and data-base for metabolic pathway synthesis. Biotechnol. Lett. 8, 837-842. Shah, D. M., Horsch, R. B., Klee, H. J., Kishore, G. M., Winter, J. A,, Tumer, N. E., Hironake, C. M., Sanders, P. R., Gasser, C. S., Aykent, S., Siegel, N. R., Rogers, S. G., and Fraley, R. T. (1986). Engineering herbicide tolerance in transgenic plants. Science 233,478-481.

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Sneader, W. (1986).“Drug Discovery. The Evolution of Modern Medicines.” Wiley, London. Spilker, B. (1989).“Multinational Drug Companies: Issues in Drug Discovery and Development,” pp. 28,57.Raven Press, New York. Stafford, A., and Warren, G., eds. (1991).“Plant Cell and Tissue Culture.” Milton Keynes at Open University Press. UK. Stanburry, P. F., and Whitaker, A. (1984).“Principles of Fermentation Technology,” Chapter 3. Pergamon, Oxford. Stevens, R. E., and Sherwood, P. K. (1987).“Market Opportunity Analysis.” Lexington Books, Lexington, MA. Szostak, J. W. (1992).In vitro genetics. Trends Biochem. Sci. 17, 89-93. Takeda Chemical Industries, Ltd. (1974).French patent 2, 189,513. Weinberg, G. M. (1975).“An Introduction to General Systems Thinking.” Wiley, New York. Weinberg, G. M. (1982).“Rethinking Systems Analysis and Design.” Little, Brown, Boston. Wheelwright, S. M. (1991).“Protein Purification. Design and Scale up of Downstream Processing,” p. 1. Hanser, Munich.

Challenges in Commercial Biotechnology. Part 11. Product, Process, and Market Development ALESPROKOP Department of Chemical Engineering Vanderbilt University Nashville, Tennessee 37235

I. Introduction 11. Product Development

A. Industrial Rationale: Strategies and Constraints B. Resources Statistics: Pipeline, Funnel, and Triangle Concepts C. Product Formulation and Delivery Concepts D. Microbial Biotechnology Route in Product Development E. Examples of Product Development 111. Process Development and Scale-Up A. Stages in Process Development B. Process Analysis vs Process Synthesis C. Alternatives in Process Development D. Process Scale-Up E. Process Control and Optimization F. Process and Plant Design G. Technology Transfer IV. Market Development A. Holistic Approach B. Current Status of the Biotechnology Market C. Market Development Concepts V. Outlook References

I. Introduction

Once product discovery is complete, the development process begins. This is a very lengthy, expensive, and tightly regulated process. Pharmaceutical companies spend much of the product development conducting clinical trials required to pass the safety, efficacy, and quality requirements of the drug, followed by the FDA review and approval. Product development also includes pharmaceutical development of dosage forms and formulations. In parallel with product development, process development is carried out. In microbial process biotechnology, it may start at the time of product discovery, when a new microbial product is available and when an appreciable amount of product is needed for chemical characterization. The technical development is expanded when a compound is more advanced and enters more extensive biologi155 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 40 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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cal and preclinical test. Further process improvements and scale-up are needed for clinical tests and as the production decision is near. Market development begins with the cost/benefit analysis, grows with product innovation, and peaks with capturing a substantial share of market. II. Product Development

A.

INDUSTRIAL RATIONALE:

STRATEGIES AND CONSTRAINTS

Product development is necessary for capturing market. Five different product discovery/development strategies have been briefly presented in Part I (AAM, vol. 40, pages 95-154) of this series. New Class of Compounds, originated from screening (discovery);example: cephalosporins Product analogs (analog compound) for nonprotein products: Improved potency/efficacy, reduced daily, dose and extended duration of action; example: protamine-insulin Reduced side effect: improved specificity of biological effect, broader spectrum (e.g., antimicrobial), reduction in selective toxicity between species (microbe vs host); examples: ampicillin vs penicillin G, porcine insulin Product variants (variant molecules) of active proteins that are: Closer in similarity to the host (human or animal);examples: human insulin (humulin), growth hormone Farther in similarity to the natural molecule with modified specificity; example: humulin via recombinant DNA technology Improved product formulation: Higher product purity (overall quality upgrade); example: lower content of pyrogens in insulin Mimetics, resulting from mimicking of biological activity; example: cyclodextrins (having an enzymatic activity) Patent coverage provides another means for securing market. The categories of patents, as delineated in Fig. 11 of Part I, assume the following meaning in terms of product and process development. 1. Composition of Matter (“COM”)patent is convenient for product analogs and variants 2. “Use” patents can cover alternate product use (another indication for a pharmaceutical) 3. “Process” patent is useful for new manufacturing process

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The patent life, that is, the number of years of effective patent protection, is of particular importance to biotechnology products. In contrast to products from other, nonregulated industrial segments, the average patent life for a biotechnology product when it reaches the market is significantly less than the original 17-year patent term. A considerable loss of patent life is due to time expended in the lengthy development and regulatory procedures required for a new drug (see Fig. 3). The drug Price Competition and Patent Restoration Act of 1984 enables the restoration of up to 5 years of the patent term. In the best case, a maximum of 14 years of patent protection from the time of regulatory approval can be obtained. Since the introduction of this act many new human and animal biotechnology products have been granted considerable restoration periods on their patent life. In addition, new legislation is also pending to extend the patent term from 1 7 years to 20. A patent would become effective from the time that the application is filed, rather than from the date the patent is issued. Product development requires time, financial resources, and regulatory expertise. Most new drugs, generated by new biotechnology companies, are being jointly developed with established pharmaceutical companies, having resources to develop products. Recombinant insulin is a good example: initially cloned and developed by Genentech scientists, it was later manufactured and marketed by Eli Lilly (approved by the FDA in 1982). After successful completion of preclinical and the three phases of clinical testing (an example of timing is in Figure l), the sponsoring company submits a New Drug Application (NDA), or a Product License Application (PLA) in case of biologics, to the FDA. The average NDA approval time runs 2 to 3 years. For new drugs available for life-threatening and severely debilitating diseases a new regulatory route [Treatment Investigational New Drug (IND)] has been available since 1989, the goal being to reduce approval time. In addition to its major role in product development (clinial trials evaluation), the FDA regulates R&D, testing, manufacturing, quality control, labeling, marketing, and postmarketing studies of drugs. The FDA has made its intent to regulate the product, not the process, clear and has stated that it sees no need to institute new procedures or requirements for new biotechnology products, such as generated via recombinant DNA technology or hybridoma-derived technology. The same position has been stated for the use of new biotechnology products in food products. Other useful strategies for product development involve the use of the concept of Biochemical Unity (diversity) of Nature (BUON). This

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ALES PROKOP Pre-clinical: Acute 1 month

6 month

Long term Carcinogenicity Mutagenicity Teratology PerVpostnatal Fertility Clinical Submit IND Phase I Phase II Phase 111 Prepare NDA Submit NDA Post-market studies

1

2

3

4

5

6

7

8

9

10

Years

FIG.1. Clinical bar diagram (Gantt chart). Possible timing of preclinical and clinical studies.

concept provides guidelines and rules which are applicable to wholecell systems. As such they are not generally applicable (exceptions exist to such rules). One can assume the similarity of an unknown and this can reduce the need for extensive experimentation. As such they are only a first approximation or order of magnitude estimate as further experiments are needed to verify the validity of assumptions used. The BUON concept assumes similarity in composition, biochemical pathways, and physiological responses between certain classes of organisms. For example, elementary analyses of organisms reveal, surprisingly, that within certain boundaries, the composition of living organisms does not vary greatly between different groups (Atkinson and

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Mavituna, 1983). Similarly, this is true for the role of essential elements in various groups of organisms: many have been shown to be essential for several species, all for most of them; many are interchangeable with others in their function. Another good example of the BUON rule is the universal presence of the Krebs cycle in aerobic organisms. On the contrary, while looking at many species, Biochemical Diversity of Nature (BDON) is commonplace. Thus, the nucleic acid content of different organisms varies considerably. A variation in the nucleic acid base content and of their ratio is, as matter of fact, used for taxonomic purposes. Molecular genetics thus provides a precise, quantitative basis for defining biological relatedness. How can one use BUON and BDON concepts at product development? (They are also being used at the discovery stage; however, no examples were introduced in Part I of this series).The BUON rule can be applied as an inductive tool to minimize experiments and it can provide reasonable conclusions to solve bioprocess development problems. However, assuming uniformity (say,in a microbial biomass composition) means that one is liable to extrapolate from limited data into potential erroneous conclusions. The BDON rule assumes diversity, i.e., the use of unique features of nature for product discovery/development. A good example is the use of unique features of Actinomycetes to come up with a diverse range of antibiotics or the use of unique low DNA and high RNA content in yeasts for RNA-derived products (flavor enhancers). Assuming diversity, however, means that we need to design additional experiments to obtain specific data on a system in question. Other examples of the use of BUON/BDON rules are listed below. It should be mentioned in this context that a restatement of these two concepts is contained in Perlman’s rules of thumb of an applied microbiologist’s understanding of microbial systems (Perlman, 1980). One of them (rephrased by the author of this article) states that “the microorganisms can do and will do (almost) anything”-a BDON rule telling us that there is a tremendous resource in nature in terms of microbes’ (or orther organisms’) capability (source of metabolites, enzymes, etc.). B. RESOURCES STATISTICS: PIPELINE, FUNNEL, AND TRIANGLE CONCEPTS 1. Pipeline Concept

Major steps involved in product development (and product discovery, process and market development) are depicted in the Pipeline Concept diagram (Fig. 2). It is an expanded view of product discovery presented in Fig. 6 (Part I). The lead compound will successively assume several

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Testing of new chemicals (natural or synthetic) via differe t screens

f

Lead compound

t

t

Product discovery

Candidath comDound In-depth analysis in animal studies

t

Project,compound

1

Product development

proiT 1

Market

Marketed drug

t

I

I I I

drug Clinical studies 1-111 regulatory approval

Post-market surveillance, new clinical indications, new dosage forms

I Process development

I I I I I I I I I I

Chemical modification and animal studies

Technical development, toxicology, metabolism, animal studies, pre-clinicaltests on humans

AI

7

FIG.2. Pipeline concept of drug discoveryldevelopment (adaptedfrom Spilker (1989a), Fig. 1.2, p. 18).

different names until it reaches market. Process and market development will be discussed below. 2. Funnel Diagram

The funnel approach (Fig. 3) was originally used as a visual model of drug development (Spilker, 1989b). The diameter of the funnel may be used as a measure of the total number of compounds tested over time. It is a convenient way of demonstrating how resources are funnelled from research to market in terms of cost, time, activity, and statistics. Figure 3 presents the latter three measures. Statistical data on product discovery/development are those of Vagelos (1991). An industry (pharmaceutical and biotech companies) average for total cost to develop a drug is $231 million and 1 2 years (1991 data). An average for the development costs of therapeutic drugs for biotech companies is lower, $125 million, because of their more aggressive approach (Bur-

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'Creativitycircle" Research 2-5

Project development Bam" )evelopment analogs, variants)

ars 20 substances for animal studies and modification

stage 10 substancesfor clinical trials

0

77

INDstage "Qualitycircle"

U

8-10 years

0 I substance for FDA approval

1

n

Manufacturing

0 1 compound on market

Market

FIG.3. Funnel diagram of pharmaceutical R&D statistics (adapted from Spilker (1989b), Fig. 8.4, p. 185).

rill and Lee, 1993). In estimating the cost of drug development, an attempt is made to include expenses for products and projects that are not successful and never reach the market. Other forms of the development funnel diagram have been suggested (Clark and Wheelwright (1993), exhibits 5-1, 5-4, and 5-6). 3. Inverted Triangle Concept

Another way of presenting industry statistics is the Inverted Triangle (Pyramid) concept. Figure 4 presents data of Xenova Ltd., UK (Anonymous, 1991). When compared with Fig. 3, the success rate (probability of survival) is much lower. Obviously, creativity and serendipity in an industrial environment play an important role. Figure 4 presents data in a narrower target area, as is typically sought by the new biotechnology industry. Concluding from the above, pharmaceutical R&D is very risky and companies are not guaranteed any return for several years, if at all. There is no assurance that any project will lead to a marketable product.

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characterized

clinical evaluation

FIG.4. Inverted triangle for new biotechnology statistics (numerical data from Anonymous (1991),p. 373).

Only 1 drug of 10 that enter clinical trials will make it to market, and only 30% of marketed drugs recover their R&D costs. As the developmental cost represents the bulk of expenditures, it is useful to compare the performance of different biotechnology sectors in terms of R&D expenses (Table I). The average fiscal year 1993 R&D expenditure, as a percentage of sales, for leading health care companies (reporting sales of $75 million or more and R&D expenses of at least $1 million) was 10.6% (13.0% for pharmaceutical companies), the highest among industries (Anonymous, 1994). R&D expenses as a percentage of profits were the highest in health care companies with computer companies/services trailing behind. Selected data for new and established biotechnology and pharmaceutical companies demonstrate that new biotechnology companies strive to catch up and secure a lead within this industrial sector. Both R&D scores signify the importance of R&D in pharmaceutical product development.

c. PRODUCT FORMULATION AND DELIVERY CONCEPTS A classification of modes of formulation of pharmaceutical products follows the different physical state of the drugs (Florence and Halbert, 1990): 1. Liquid (solutions, suspensions, emulsions.) 2. Semisolid (creams, ointments, gels.) 3. Solid (tablets, capsules, molded products).

163

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1 TABLE I

R&D SCOREBOARD: BIOTECHNOLOGY vs OTHERINDUSTRIALSEC~ORS Industrial segment (composite)

R&D expenses as %

Food Fuel Chemicals Electricallelectronics Computer companies/services Health care Drugs Products/services Selected new biotechnology companies Immunex Genetics Institute Chiron Biogen Genentech Genzyme Selected established leading pharmaceutical companies Bristol-Myers Squibb Eli Lilly Merck Upjohn

of sales 0.8 0.8 4.1 5.5 8.0 10.6 13.0

6.8 340.9 97.6 58.4 58.1 48.5 36.0

9.9 14.8 11.2 17.6

R&D expenses as % of profits (pretax income) 10.7 12.0 66.3 59.4 15,739.5

72.7 77.3 61.6

NEGo NEG 606.5 229.1 501.1

NEG

43.9 136.0 37.2 130.9

Note. Adapted from Anonymous (1994). NEG,negative earning.

Depending on the chemical characteristics of a drug and its formulation as well as on the type of body barrier, different conventional administration routes can be used (Johnson and Lewis, 1990): Oral Parental Rectal Vaginahterine 5. Nasal 6. Buccal/sublingual 7. Transdermal 8. Pulmonary 9. Ocular

1. 2. 3. 4.

The most desirable approach is oral delivery, in which the drug is intended to be absorbed from the gastrointestinal (GI) tract. The in-

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jectable route (including implants) and transdermal devices have a limited place in current therapy. The delivery of therapeutic peptides and proteins, resulting from new biotechnology, via the GI tract will be difficult because of their inherent instability and the poor permeability of the epithelial membranes to high-molecular-weight substances. A universal delivery system for peptides and proteins is not possible because of the diverse range of physical and chemical characteristics and different sites and modes of action at their sites, including different spatial and temporal requirements. To circumvent these problems, the industry has invested in drug delivery research. In order to use future generations of therapeutic proteins clinically, their physicochemical properties, route, and pattern of administration will need to be such as to ensure that their delivery to their sites of action is relevant. Structural changes may be brought about by (Tomlinson, 1990): 2. Deletion mutation, in order to improve stability, and specificity (e.g., second-generation tissue plasminogen activator with increased potency due to an increase in the effective concentration in the proximity of the fibrin clot) 2. Heterologous hybridization, in order to provide effector functions, protection, and recognition (e.g., interleukin-2 bound to diphtheria toxin) 3. Change in the glycosylation pattern, via expression host selection or enzymatic modification (e.g., use of mammalian cells to secure ycarboxylation of glutamyl residues) 4. Chemical conjugation to hydrophilic polymers to alter biological disposition (e.g., L-asparaginase-dextran complex to lower antigen reactivity and extend clearance).

These will result in changes in protein stability, site recognition and binding, and disposition. The administration will have to be carried out via the correct route and at an appropriate amount, rate, frequency, and duration, as well as with proper mediators (enhancers). In addition, advances in materials science (and biotechnology) are permitting the development of new physical and chemical methods of drug delivery. They include matrix devices, gastric retentive devices, bioadhesives, absorption enhancers, coated dosage forms, liposomes, microcapsules, controlled release systems, degradable polymers for delivery, nasal delivery permeation enhancers (surfactants), and peptide backbone modification (mimetics). A special subsection of this activity is denoted as drug “targeting” to specific cells, featuring a linkage of a bioactive agent to a suitable recognition system (e.g., monoclonal antibody).

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D. MICROBIAL BIOTECHNOLOGY ROUTEIN PRODUCT DEVELOPMENT There is a common misconception among academic and industrial researchers that a product must be manufactured in the same mode by which it was discovered and developed. For example, when a product is discovered through biological assays or screens, it is often assumed that a biological synthesis, such as fermentation, is the logical mode of production. In reality, however, the best route of manufacture for a biologically derived product may be chemical synthesis or a combination of chemical and enzymatic processes. The biological route of manufacturing (ROM), via fermentation, is often restricted to more complex molecules. There are several reasons for choosing a chemical synthesis route of manufacture: 1. Large economic advantages of chemical synthesis over microbial (biological) synthesis 2. A chemical analog, derived from a molecule discovered on the basis of a biological assay (route of discovery, ROD), is more active or more stable (e.g., in the GI tract) 3. A chemically derived product is of higher quality/purity with fewer of the by-products often generated by a biological route.

Nevertheless, microbial biotechnology has been instrumental in securing many product leads not only at the discovery stage, but also at the product development stage, both directly and indirectly. In the following, several noted examples will be presented. 1. Direct Microbial Route (ROD equals ROM)

a. Nonprotein Product Analogs. Examples of such products are analogs of antibiotics, fungicide analogs, and microbial polysaccharide analogs [this example can be denoted as “from kelp alginates to microbial gum” (kelp alginates are derived from seaweed Macrocystis pyrifera): The first step involved Pseudomonas aeruginosa (an example of the use of BUON in the process discovery), the second step a move from microbial alginate to xanthan gum of Xanthomonas campestris in the 1960~1. b. Protein Variants. Examples include production of bovine somatotropin, porcine somatrotropin, and human growth hormone in genetically engineered (recombinant) bacteria. 2. Indirect Microbial Route (ROD not equal to ROM)

The following examples show how the initial microbial impact materialized in a nonmicrobial manufacturing route, with product develop-

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ment playing a “pro-drug” role. A pro-drug is defined as a substance that is converted inside the body into an active drug, usually via a metabolic process. A pro-drug may be inactive on its own or it may share some part of a biological profile of the active drug. Examples include: variant proteins serving as a model for the chemist to biomimic their structure and function via understanding of protein folding patterns; and analog leads, providing a basis for semisynthetic drugs (e.g., semisynthetic antibiotics).

E. EXAMPLESOF PRODUCT DEVELOPMENT To illustrate the role of microbial biotechnology in product development in the pharmaceutical and environmental industries, three examples are presented below. One more excellent example of R&D history is that of lovastatin, a drug that reduces blood cholesterol levels. It was discovered on the basis of an enzyme inhibition study and isolated from Aspergillus terreus, and is being produced microbially. Both discovery and development aspects of this drug have been reported (Vagelos, 1991). 1. From Penicillin to a New Class of Antibiotics

The product development matrix in Fig. 5 demonstrates how new products evolved: 1. Pencillin V, an analog of penicillin G, with different delivery mode 2. Ampicillin, a broader-spectrum antibiotic

3. Cephalosporins, a new class of antibiotics, belonging, with penicillins, to the same family of p-lactam antibiotics 4. Streptomycin, an entirely new class of antibiotics, not related to penicillins (an example of the BUON rule in product development).

It should be emphasized that the product development in these cases involved the use of chemical or microbiological approaches. Ampicillin is an example of a broad class of semisynthetic antibiotics, originating from the “penicillin nucleus,” 6-amino penicillanic acid (6-APA), which is also made microbially. Different side-chains attached to 6APA result in a variety of biological activities meeting, to different degrees, the different requirements for an ideal antimicrobial agent. At the same time, as has been illustrated in the history of penicillin product development, a new class of compounds (Fig. 5, C3) with broader antimicrobial spectrum and more efficacious activity, or an entirely new class (different from penicillin; Fig. 5,C4), originated from microbially based screens and from further product development. The

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

Return to A (Discovery)

c Product Development cycle

w A Product and Process Discovery

cycle for pniclllln 0

Product

Discovery

Development

A1 Fleming (1928) A3 UK patienl (1941)

C

-

B Procesa Development cycle for penlclllin 0

Chain/Florey

A4 Peoria (1 940's)

Physiology

Return t o A (Discovery)

167

I 1 83 Complex media

B4 Defined media

:

FIG.5. Mind map of penicillin G product/process discovery and development.

example of penicillin G shows, in addition, the open-ended nature of R&D activity. The whole pathway of product/process discovery and development in its entirety (it is possible to separate developmental aspects completely) will be presented retrospectively to:

1. Present historical highlights of penicillin G product discovery and development and process discovery and development activities (Elder, 1970;Sneader, 1985;Calam, 1987) 2. List relevant critical methodology and concepts for each important step in penicillin G R&D

This topic is explained by means of matrix charts, followed by appropriate comments. Figure 5 presents three matrix charts.

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A. Historical productlprocess discovery (and development) cycle (2 x 2 morphological matrix)

B. Process development cycle (genetics, physiology, and engineering matrix) C. Product development cycle (chemistry and microbiology matrix).

In the following, only salient points are presented. Product and Process Discovery Cycle Product discovery Alexander Fleming observed in 1928 a growth inhibition zone on a plate with a Gram-positive bacterium around a colony of Penicillium Fleming’s discovery is part intuition, part serendipity Nature favors those who are prepared Process (purification) discovery Chain and Florey (UK) in the late 1930s developed a large-scale process to purify the active penicillin secreted by fungi by extraction with solvents (amylacetate from acidic solutions, followed by the back-extraction to alkaline aqueous solution) Product (application) discovery Therapeutic effect of penicillin proven in 1941 with successful treatment of a UK policeman Process (industrial production) discovery Labor-intensive “tray” process is transferred from UK to Peoria, Illinois (USDA) Introduction of submerged culture vessel in the 1940s Process Development Cycle Process engineering (submerged culture) development USDA Peoria and pharmaceutical companies (Squibb, Merck, Pfizer, E. Lilly) develop an industrial submerged culture fermentation in the late 1940s (a precursor of the present-day stirred tank bioreactor) Strictly batch processing Turbine-agitated vessel design and air introduction by blowingin for mixing and oxygen transfer Air “sterilized” by heating Higher productive process developed via screening of fungi Different sources of fungi from nature used Parent strain mutation and selection of survivors Process improvement via formulation of complex media using Corn-steep liquor Antifoams

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY, 11

169

Higher productive process via better understanding of the physiology of the production strain (defined media) Improved nutrient and precursor feeding strategy Secondary metabolite control Further improvement via process engineering using chemical engineering principles Continuous media sterilization and sterilizer design Glass wool for air filter design Sterilizable pH probes and other control instrumentation Reactor scale-up Fed-batch processing Environmental control and monitoring Introduction of strain improvement via genetic engineering Single-gene cloning Multigene cloning Product Development Cycle Screening of microbial strains for new antibiotics (Waksman’sstreptomycin in 1943) Screening of microbial strains for analog of penicillin G (penicillin V) Chemical modification of penicillin (semisynthetic penicillins in 1958,e.g., ampicillin with Gram+ and Gram- activity) New class of therapeutic agents (in 1949 and later, e.g., Cephalosporins) When both B and C (development) are accomplished the whole cycle is typically restarted (return to A). Table I1 lists concepts covered in this example. It is important to extract this type of information to realize all possible tools that are available. 2. Historical Perspective of Insulin’s Discovery and Development

This example will briefly cover the product discovery phase and then concentrate on product development [most factual data are from Sneader (1985)and Kehoe (1990)].In addition, it will demonstrate the usefulness of the rules of thumb, BUON, and BDON, as well as how nonmicrobial ROD will finally lead to microbial ROM. Product and process discovery (the dog used as a model for humans represents the BUON rule): 1. Demonstration in 1889 that the removal of the pancreas from a dog makes it diabetic, leading to medical research on injection of pancreatic extracts to diabetics 2. Rumanian Paulesco in 1920 isolated antidiabetic hormone

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ALES PROKOP TABLE I1 CONCEPTS RELEVANT TO PENICILLIN PRODUCr/PROCESSDISCOVERY AND DEVELOPMENT EXAMPLE

~~

Cycle

Conceptlrule

A

Product discovery as an intuitive process, role of serendipity Process discovery by means of alteration of physicochemical properties at purification Product (application) discovery Process discovery-chemical reactor concept applied to an industrial submerged culture

B

Process engineering development via introduction of batch processing Chemical reactor agitation and aeration Screening of biocatalysts (BDON rule] Screening of complex media sources (trial-and-error, statistically bound) Design of defined media (use of mass balance, concept of secondary metabolites) Chemical engineering principles Continuous processing Air filter design Aseptic technology Scale-up rules and concepts Temperature and nutrient profiling

C

Microbial screening for new antibiotics (BDON rule) Chemical analog screening for other biological activity (BDON rule] Chemical and functional screening for broader biological activity Screening for new classes of antibiotics (BDON rule]

3. Banting and Best in 1921 in Canada developed (process discovery!) insulin isolation by means of alcoholic extraction, avoiding high pH (instability) and preventing action of the protein-digesting enzyme trypsin 4. Collins in Cleveland in 1922 succeeded in further process optimization by precipitating insulin from aqueous alcoholic extracts of dog’s pancreas by pure alcohol and improving product purity 5. Sanger in Cambridge in 1955 elucidated the amino acid sequence of insulin.

Product development (recognition that BDON rule can lead to a source of side-effect problems): 1. Abel at Johns Hopkins in 1926 obtained purer crystalline insulin (zinc precipitation), replacing amorphous insulin, leading to reduced local irritation

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

171

2. At Nordisk’s Laboratory (later Novo) in 1936 a long-lasting product form was obtained by adding a small protein-protamine-obtained from fish sperm (note that this is an exception to the BDON rule; see Fig. 6 for a drop in product purity). Protamine’s function is lower insulin solubility for slow dissolution in body fluids. 3. Novo’s further product formulation improvement in the 1950s resulted from replacement of phosphate buffer (removing zinc) by acetate buffer and from removing protamine, resulting in longer-action insulin 4. Novo’s application of chromatographic purification in the 1970s lead to higher purity product 5. Eli Lilly’s “discovery” of the BDON concept in the 1970s emphasized structural differences between animal insulins (porcine, ox,cow) and human insulin. Lilly, being a major microbial fermentation company, decided to use a genetic engineering option (license from Genentech) for production of human insulin in Escherichia coli. This lead to microbial ROM of human insulin. In fact, Lilly initially investigated the “chain process,” based on separate production of two chains (A and B) as fusion proteins, followed by reconstitution of the proinsulin Human insulin protein “BDON’ rule

Product purity Chromatographic purification

Nonhuman insulin protein ‘BUON“ rule

Acetate buffer

Reduced Zn Zn crystalline insulin Protamine impurity (long-lasting)

1890

1926

1936 1938

19601970 1980s

Time

FIG.6. Historical perspective on insulin product development.

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molecule through disulfide bridges. Later, the one-step “pro-insulin” route was applied industrially, based on expression of multiple joined proinsulin genes in E. coli. The essential step of this process is a folding of inclusion bodies of isolate proinsulin to gain the proper molecular configuration. 6. Novo’s alternate product/process discovery in 1970s features the use of enzyme engineering technology, substituting one of the amino acids in pig insulin to convert it into human insulin.

A summary of historical perspective on insulin in product development is presented in Fig. 6. From. the 1890s to the 1960s,BUON was the concept assumed by researchers involved in insulin production. During that time nonhuman insulin product development consisted of either improving protein purification or new product formulation (buffer). From the 1970s,with the advent of genetic engineering, the paradigm of animal insulin for human use was altered; product development specialists started to recognize the importance of BDON. The introduction of human insulin through a product development effort represented a change in composition of matter and a novel process to manufacture. 3. Environmental Biotechnology/Services

This activity will be described in the context of a waste minimization program applicable to the chemical-, food-, and pulp-processing industries. The role of biotechnology in this attempt will be outlined and the potential for generation of new products and services will be reviewed. Waste minimization programs are typically implemented as a result of audits trying to identify sources of wastes and their quantities and nature. The goal of waste minimization as applied to processing sites is ultimately to seek a “zero-discharge” criterion via: “Point-source” reduction consisting of: Preventive maintenance to reduce leaks Prevention of accidental spills Lowering of by-product generation Using nontoxic process chemicals By-product utilization consisting of: Solvent recovery Evaporative concentration “End-of-pipe” treatment involving physical, chemical, thermal, and biological treatment technologies. Such an approach involves quite limited application of environmental biotechnology in dealing with biotechnology wastes at “nonactive”

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

173

dumps or spill sites. A broader perspective for biotechnology involvement in the waste minimization would require identification of locations where such application is possible, and identification of biocatalyst type. The identification of biotechnology-based solutions to environmental problems should involve: Process plants Reduction of biocorrosion and biofouling Microbe reduction in cooling waters Enzyme applications in food-processing streams Utilization of hemicellulases in pulp processing streams By-product utilization, in addition to existing utilization (canelbeet molasses or cheese whey for protein and ethanol production, corn steep liquor for antibiotic production) Conversion to methane, ethanol, acetone, etc. Use of enzymes and biotransformation to produce more active isomers of agricultural chemicals “End-of-pipe” treatment Use of the upper zone of the soil (microbe)-plant system in land applications Use of in situ bioremediation of soil/water Use of microbes to produce fuels and chemicals Use of biofilters for dilute organic waste gas cleaning Aerobic and anaerobic water treatment (high salt and organic nitrogen can be problematic). Among prospective applications are those in bold type above. The biofilters are compost or soil beds intended for microbial oxidation of volatile organic compounds. They can handle gas flow rates greater than 5000 m3/h (Bohn, 1992). The identification of catalyst type is a critical step. Hierarchies of biocatalyst systems as applicable to bioenvironmental applications are shown in Fig. 7. In summary, environmental biotechnology should be an integral part of waste minimization programs for several industrial sectors, particularly because there is a minimum of by-products.

Ill. Process Development and Scale-Up

This part will initially discuss generic issues of process development and then concentrate on scale-up of organismic-based processes, with an emphasis on microbial process biotechnology. Most of the material has been drawn from Tong and Inloes (1990).

174

ALES PROKOP Destruction

By-product use:

I

I

technology:

' PlanVsoil 13microbes

Land application

Plant

Vegetative sink for gaseous pollutants

____)

-

Organics for fuel and chemicals

More active agricultural chemicals

Microbes

Enzymes

Air biofilter

Water aerobic anaerobic

FIG.7. Hierarchy of biocatalyst systems with potential in bioenvironmental area.

A. STAGES IN PROCESS DEVELOPMENT Process development and scale-up include qualitative and quantitative determination of all key factors which permit the successful conversion of a laboratory procedure into an industrial scale. It is generally not possible to use conditions that have worked in a laboratory and blindly apply them to industrial scale equipment. However, certain economic and performance limits must be satisfied during the process development. Development should be done in a timely manner Development should be economical Process must be stable and performance predictable Product quality must be guaranteed 5. Production rate should be reasonable

1. 2. 3. 4.

Biocatalyst and process development consists of several steps: 1. Biocatalyst development (usually carried out in conjunction with product discovery; note that inoculum development applies only to organismic systems-strain development is described in Part I) 2. Product recovery and purification development (note that this step will not be covered in this review) 3. Process scale-up (sometimes referred to as equipment sizing)

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CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

4. Process control and optimization (note that this step will not be covered in this review) 5. Process and plant design, including rather specialized engineering and design functions.

The overall picture of process development and scale-up within the whole R&D activity is depicted in Fig. 8 (simplified Fig. 1 of Part I). The inoculum, product recovery, and process design, as well as process scale-up, are lumped together inthe central box (bold). A short overview on inoculum (seed) development is presented below. A general course of inoculum development is as follows: inoculum preservation + inoculum build-up + prefermentor culture * production fermentor. Potential problems involve (adapted from Reisman, 1993): 1. Age of inoculum (optimal transfer time) 2. Inoculum hold time (criteria for hold before use or discard) 3. Cross-inoculation (use of a production run to inoculate instead of

“lost” inoculum) 4. Inoculum transfer time (set limits for transfer at a scale-up process) 5. Number of transfers (minimize number of inoculum growth cycles and transfers before reaching a production volume)

_ _ - - - -- 1 I l 8

Discovery

21 I I

--

6

v-

-

I 4a Product Development +

-

7 Product in Market Market Development

7a

A--

!- -

I

J

7b

4b

- - - _Information transfer Product transfer

FIG.8. Process development and scale-up in perspective. Numbers represent approximate sequence of steps.

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ALES PROKOP

6. Draw-fill technique (check on the possibility of use of a small fraction of completed batch as a seed for new cycle.

Other problems related to inocula development are given in Chapter 6 of Stanburry and Whitaker (1984).

B. PROCESSANALYSIS vs PROCESSSYNTHESIS While discussing process development and scale-up steps in detail below we will not forget to employ the main theme of these articles, process analysis vs synthesis. By employing analysis, we will attempt to understand how a problem is broken into elements and how they work. Via synthesis, we will combine individual observations into a coherent picture. This methodology will be particularly important in the area of process scale-up. The traditional approach tends to view scale-up as a closed-ended, static problem that can be solved using well-defined equations (quantitative rules of thumb). In addition, the traditional view often limits its focus to the bioreactor itself, neglecting other scale-up problems. The holistic (general systems) view will thus enrich this approach and provide many solutions to problems. IN PROCESS DEVELOPMENT c . ALTERNATIVES

Variables and parameters involved in individual steps of process development and the result of such investigation (or type of analysis necessary) are listed in Table 111. A rather different group of professionals solve individual steps of process development within a company’s R&D departments (Table IV). However, successful completion of this cycle is only possible if criteria are set by marketing and manufacturing divisions (Table V). Note that technical criteria for optimization and control are identical to those of the biocatalysthnoculum development group. The process design function is fulfilled by a specialized group. A successful scale-up, by definition (Reisman, 1993), means that a process has been developed and designed, giving a predictable increase in production capacity, while the product potency is maintained and no unexpected chemical entities are found in the product. Such a process can be validated at the production scale; clinical quality and quantity (in case of pharmaceuticals and biologics) are achieved and appropriate documentation is available for regulatory purposes. Scale-up becomes a problem when large-scale production exhibits one or more of the following characteristics when compared with the known performance at small scale:

177

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1 TABLE I11 DESIGNPARAMETERS OF PROCESS DEVELOPMENT Step Biocatalyst/ inoculum d eve1op m en t

Variable or parameter Catalyst storage and operative stability Organism stability Organism preservation Nutritional requirements Growth rate/productivity Temperature cardinal points Morphology and viscosity Response to oxygen, carbon dioxide Product and by-product formation

Product recovery and purification development

Degree of product recovery

Result or analysis Storage and operational conditions Handling, degeneration rate Maintenance methods Optima for growth and product formation Design of process Heat load, sterilization Power input Agitation-aeration needs Recovery method cost and effluents Number of recovery/purification steps Number of steps Recovery methods, regulatory issues

Degree of product purity Nature of impurities (pyrogens) Product stability (pH, temperature) Recovery options Product inventory

Type of processing, hold-up times Regulatory issues Refrigeration needs, segregation

Process scale-up

Unit operations Scale-up criteria Design alternatives Equipment sizing Material of construction Batch cycle time Utilities consumption Mixing/aeration needs

Scaleability Size dependency Ease of scale-up Cost and design Stresslsize dependency Scale dependency Proportionality to scale Scale dependency

Process control and optimization

Response to variables Key variables to control Nutrient feeding Control schemes

Process dynamics Control schemes Monitoring and control Computerization

Process and plant design

Raw material cost and selection Uniformity of raw material Contaminants

Contractual arrangements Process response Process response (continued )

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Variable or parameter

Result or analysis

Ease of sterilization, method of sterilization Water quality Sequence of steps Power requirement Cooling requirement Air requirement Multiple vessel Cycle times Nutrient feeding Cleanliness Recovery options Utility needs Equipment maintenance Process flow diagram Plant start-up Design constraints

Batch vs continuous Water purification Optimal configuration Reactor and agitator selection Cooling method Compressor design Vessel assignment Plant layout Batch or continuous operation Sterility, clean-in-place systems Economic design Costs, co-generation Cost, spare parts Balances and control schemes Equipment specification/operation Safety and containment procedures

Note. Adapted from Reisman (1988). Table 15.

1. Product concentration at harvest or formation rate is significantly lower (20% or more) 2. Co-product or by-product distribution differs significantly in terms of concentration or formation rate TABLE IV INDUSTRIAL PROFESSIONAL GROWSINVOLVED IN PROCESSDEVELOPMENT Step

Professionals

Biocatalyst/inoculum development

Microbial physiologists Biochemists Geneticists

Product recovery and purification development

Biochemists Chemical engineers

Process scale-up

Chemical engineers

Process control and automation

Chemical engineers Electrical/control engineers

Process and plant design

Chemical engineers Mechanical engineers

179

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1 TABLE V

INDUSTRIAL DEFINITION OF CRITERIA FOR SUCCESSFUL PROCESS DEVELOPMENT AND SCALE-UP

Technical criteria determined by process and scale-up groups

Commercial criteria defined by marketing/manufacturing divisions Development time

Product quality

Production volume

Product cost

~_________

Biocatalyst/inoculum development and process control 81 optimization Product yield Product concentration Product stability Productivity , Product recovery and purification Product recovery Product purity Nature of impurities Process scale-up Equipment sizing Construction material Process and plant design Utilities cost Energy requirements Operational mode Recovery options

J J J J

J

J J J

J J

J

J

J

J

J

J J

J

J J J J

J

J J

J

J

J

J J J J

3. Harvested fermentation broth properties differ significantly, resulting in significantly reduced product recovery yields or lower product quality.

Before discussing individual scale-up methodologies, a quick look will show what happens when reactor size is increased. In addition, it will also be shown that different alternatives for scale-up are affected by product amount needs, by its timely supply, and by availability of reactor options. 1. Geometric Scale-Up

The following three examples illustrate how different scale and dimensions affect a vessel’s performance (loosely adapted from Oldshue, 1983b).Figure 9 shows a scale-up of a cube. If two cubes are compared, one with edges three times as long as the other, the surface area of the

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ALES PROKOP

FIG.9. Relationshipbetween length, area, and volume.

larger cube is 9 times greater, and its volume is 27 times greater. A disproportionate decrease in the areaholume ratio with scale (1/1 to 9/27) might have a negative consequence when designing jacketmediated cooling. A two-dimensional scale-up of a mixing vessel maintains many individual geometric and mixing ratios constant (Fig. 10). Such scale-up has been suggested for large-scale bioreactors for biomass production. A three-dimensional scale-up, in a geometrically similar manner, points to likely differences in important mixing parameters (Fig. 11).Table VI illustrates an independence of scale-up parameters for geometrically similar bioreactors. The scale-up criteria require some comments. The energy input Pois an ungassed power input and pumping rate is a measure of circulation in a vessel. Some of the criteria have traditionally been used for scale-up (POW,Q/V, NDizplp). That means that a given chosen criterion is intended to be maintained at both scales. From Table VI it appears that at scale-up there is no way to fix all geometric and scale-up criteria at a larger scale; only one can be maintained at a given time, while the rest undergo substantial change. For example, by fixing an energy input per volume at larger scale, other parameters differ widely from unity, showing that many quantitative mixing indices have not been maintained. Specifically, homogeneity of such a reactor will suffer, as the pumping rate per volume is down to 0.34. On the other hand, the maximal shear rate increases to 1.7, perhaps a damaging situation to shear-sensitive cells. From the above example it is clear that geometric scale-up will not be successful.

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

181

FIG.10. Bioreactor scale-up keeping every individual mixing criterion constant.

FIG.11. Geometrical scale-up without maintaining important mixing parameters. Reprinted with permission from Oldshue (1983a), “Fluid Mixing Technology,” (19831,Fig. 9-2, p. 193. Copyright 1983 McGraw-Hill, Inc.

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182

TABLE VI INTERDEPENLlENCE OF SCALE-UP PARAMETERS FOR SIMILAR BIOREACTORS

Scale-up criterion Energy input Energy inputlvolume Impeller rotation number Impeller diameter Pump rate of impeller Pump rate of impeller/ volume Maximum shear rate Reynolds number

Designation

Po PJV N Di

Q QlV N/D, ND;p/p

19-liter bioreactor 1.0 1.0 1.0 1.0 1.0 1.o 1.0

1.0

2370-liter bioreactor 125 3125 1.0 25 0.34 1.0 5.0 5.0 42.5 125 0.34 1.0

25 0.2 0.2 5.0 25 0.2

5.0 25.0

1.0

1.7 0.5

5.0

0.2 0.0016

0.04 5.0 5.0 0.04 0.2 1.0

Note. Reprinted with permission from Oldshue (1983a), Table 9-5, p. 197. Copyright 1983 by McGraw-Hill.Note that all criteria have been set to 1.00 at 19-liter scale with the objective of seeing how they change numericallywith scale-up. At a larger scale, PJV, QIV, NID,, and Reynolds number are each successively held constant.

2. Scale-Up Alternatives for New Products

Criteria for successful resolution of industrial scale-up problems depend on the types of scale-up situations encountered. The relative importance of process development, product development, and market development may change the chosen pathway. Thus, multiple pathways exist for the resolution of these open-ended problems. Efficient and effective resolution requires the use of different specialists, from microbiologists to engineers, to meet the multidisciplinary nature of these problems. For example, successful scale-up in microbial biotechnology is often achieved by different R&D groups using the same microbial strain but different types of agitator design, aeration conditions, and fermentation media. The relative importance of product supply versus process, product, and market development needs breakdown into the four types of scaleup challenges (Table VII): 1. Fast track testing-during the screening for a new biotechnology product or a new product candidate, product supply needs are larger than available process development information; here, high priority is given to meeting product supply needs while lower priority is given to defining process scale-up needs. 2. When the efficacy of a compound and the likelihood of the biotechnology (e.g., microbial) route as the preferred route for manufacturing have been established, the scale-up situation for the new product under-

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

183

TABLE VII SCALE-UP

ALTERNATIVES FOR NEWPRODUCTS ~

Scale-up type

Scale-up activity loopa

Activity ratio

I. Fast-track testing of new product candidate

1-2-3a-3b-4a-4b-3a-3b

Product supply %product development

11. Simultaneous product and process development

3b-4a-4b-3a-3b1-2-3 < 5-8-9-5

Product supply = process development

111. Manufacturing cost reduction of existing or new product

5-8-9-5

Process development P product supply

IV. Market-derived product modification

7b-4b-3a-5

Process development P product SUDDlV

See Fig. 8 for details.

goes simultaneous product and process development; new product supply needs are comparable to process development information. 3. Manufacturing cost reduction of the existing or new product-where the process development information exceeds product supply needs; an example is the introduction of a superior production culture or lower cost media in the manufacture of existing antibiotics. 4. When the product is modified as a result of new market development, where the market development information exceeds the product supply needs. The resource allocation to scale-up is controlled by priorities and types of challenges as the relative importance of completely resolving scale-up challenges depends on the stage of process/product development (Fig. 12): 1. For early stage (I) of process/product development (the most frequent scale-up situation), priority resources are allocated to new product supply as much as possible within the minimum development time, and acceptable performance ratio can be significantly less than 1.0 2. For later stages (11) of process/product development, the competitive production cost targets shift resources to process development and the performance ratio should more closely approach 1.0.

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184 1 .o-

Resource allocate to scale-up (relative)

Scale-up performance ratio (large/small scale)

0.7Product supply emphasis

ProcesdProduct development emphasis

FIG.12. Industrial objectives as related to type of the scale-up challenge.

The scale-up performance ratio is measured in terms of productivity, raw material consumption efficiency (product yield), and/or product quality (large to small scale). Ideally, the performance on a large scale is identical or superior to the best performance on a small scale. The above two stages can be used to illustrate how criteria for determining scale-up success may change. For example, an appropriate criterion during stage I is the performance ratio measured in terms of the minimum development time. It may include the time required for using multiple batches at small scale. At the same time, this is an example of performance ratio including factors other than the traditional use of product concentration ratio at harvest. Such a scenario would, obviously, include the use of the existing hardware. During stage I1 it is often necessary to simultaneously minimize both production cost and development time. This requires effective integration of the fermentation group’s objectives with those of the upstream group (which is concerned with host-vector and mutant selection) and the downstream group (which deals with product recovery and purification). To produce a microbial product, one needs to properly select the organism (host, gene, vector in case of recombinant products) to maximize productivity. Selection of optimum medium is determined by the need to minimize product recovery capital and operating costs and to maximize productivity.

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

185

3. Reactor Hardware vs Software Alternative

The relationship between the production scale (capacity) and product market value serves as a suitable basis for a classification of biotechnology products (Table VIII). The bioreactor scale is thus the most important economic indicator for potential success. A detailed plot of productivity vs market value is in Fig. 33.The above product classification is then useful for selection of bioreactor hardware at scale-up. The following hardware options are considered. 1. Grass-root installation, a brand new plant construction focused on a perfect match between design and process requirements 2. Retrofit installation, featuring a major modification of the existing bioreactor/equipment hardware 3. Process adaptation to existing hardware

Table IX lists characteristics of the three classes of biotechnology products which influence the selection of bioreactor hardware options, a software option making use of charged culture conditions. Figure 13 distinguishes hardware and software options.

TABLE VIII SCALE-UP-BASED CLASSIFICATION OF BIOTECHNOLCGYPRODUCTS

Product class A. High-volume, low-value product

B. Medium-volume, medium-value product C. Low-volume, high-value product

Production capacity (t/year)

Bioreactor Unit market scale value ($/kg)

100-10,OOO

100-1000

m31

1-100

10-100

0.001-1

1-10

1-100

Typical example Organic acids Amino acids

1~0-~0,000Antibiotics

10,000-

Interferons

10,000,000 Insulin

Recombinant proteins Monoclonal antibodies

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186

TABLE IX HARDWARE OPTIONS AS INFLUENCED BY CLASSOF BIOTECHNOLOGY PRODUCTS Hardware option

Hardware characteristics

Grass-root installation (Class A products)

Economy of scale Existing capacity insufficient Optimize capital utilization Process specific bioreactor design required

Retrofit installation (Class B products]

Moderate economy of scale Market incentives to lower production costs Minimize capital expenditures Flexibility

Process adaptation to existing hardware (Class C products)

Marginal economy of scale Low production costs relative to selling price Market uncertainties Flexibility

Examples: Hardware option: Alter biorector and accessory hardware

to meet specific

1. Grass-root installation 2. Major redesign of existing

hardware

Software option: microbial cultures or producing organisms

requirements of the culture

2. Screen alternative

fermentation control strategies (e.g.,substrate feed)

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

187

D.PROCESSSCALE-UP Microbial scale-up methodologies can be classified into two categories (Tong and Inloes, 1990). 1. Qualitative methods, identifying useful ways to search for the sources of scale-up problems. These methods organize information obtained from various multidisciplinary members of a scale-up team to determine, what is known, what is not known, and operational conditions at the scale-up site. 2. Quantitative methods, describing the alternative experimental strategies that enable us to obtain data to identify exactly the actual sources of the problem. These methods provide answers to questions developed from the qualitative methodologies and, identify specific sensitive scale-up factors and range of acceptable operational conditions.

The process of identifying scale-up problems occurs in two sequential steps that use both methodologies (Fig. 14),details of which are given below.

6 Engineering

Rules of

Rules of thumb

Inconsistency

Regime analysis

Heterogeneity

Scale-down

L Substitution

J Pattern

Open ended solutions

FIG.14. Mind map of microbial scale-up methods.

Specific

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1. Qualitative Methods

The qualitative search for potential sources of scale-up problems is an important first step that requires an exhaustive and comprehensive examination of all potential sources of problems. One must adopt a holistic (general systems) view of the physical origins of scale-up problems. Besides the often-assumed origin of problems in the production bioreactor itself, a more comprehensive outlook considers (Fig. 15): 1. Bioreactor auxiliaries (e.g., seed bioreactors, nutrient, addition, vessels, media sterilizers, transfer lines) 2. Site-related issues (e.g., process/cooling water supply, raw material sources, process steam supply) 3. Technology transfer steps.

The application of individual items of two sets of methodologies to the four common origins of scale-up, as listed above, is summarized in Table X. The reader is urged to revisit this table after completing the comprehensive discussion below.

FIG.15. Holistic view for identifying the four potential sources of scale-up problems.

189

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1 TABLE X APPLICATION OF SCALE-UPMETHODOLOGIES TO THE FOURCOMMON ORIGINS OF SCALE-UP PROBLEMS Four potential sources of scale-up problems Scale-up methodologies Quantitative Checklists/ROT Morphological analysis/ROT Quantitative Statistical analysis Pattern analysis Specific rate analysis Engineering process analysis

Bioreactor auxiliaries

Bioreactor

Site-relted issues

Technology transfer

Yes

Yes

Yes

Yes

Yes

Yes

No

Yes

Yes Yes

Yes Yes

Yes Yes

No No

Yes

No

No

No

Yes

Yes

No

No

Note. Reprinted with permission from Tong and Inloes (1990),Table 3, p. 560, copyright 1990 by American Chemical Society).

a. Rules of Thumb. The three following rules provide a useful conceptual framework for developing the two remaining qualitative scale-up methodologies: use of checklists and morphological analysis. The rules of thumb, used to “hunt” for sources of scale-up problems, are as follows: Rule 1. Substitutions, deletions or additions of process or unit operations or new materials are automatic suspect sources of scale-up problems. Engineering design is often simplified during the initial construction of fermentation plants to achieve capital or operating cost savings. Therefore, additions, substitutions, or deletions of unit operations are facts of life in industrial fermentation scale-up. For example, substitution of batch sterilization with continuous sterilization in fermentations using complex media is a common source of scale-up problems. Scaleup of batch media sterilization also can result in changes in chemical composition of bioavailable nutrients, leading to differences in productivity (Singh et al., 1989).(See discussion below to resolve this type of scale-up problem.) Another example deals with the lower cost of raw materials for large-scale fermentation. Thus, the use of “city water” having a relatively high level of iron has been known to cause scaleup problems in fermentations that are sensitive to iron content (vitamin B2).

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Rule 2. Heterogeneity of the chemical and physical environment or of the microbial and genetic properties at commercial scale are potential sources of scale-up problems. For highly viscous fermentation broths, the macromixing change on scale-up is likely to cause temperature, pH, and nutrient gradients at various positions within the bioreactor. This heterogeneity of the physicochemical environment, in turn, will result in relatively greater performance variability on scale-up because smallscale fermentations normally operate under better mixing conditions. Heterogeneity in the microbial and genetic properties can influence scale-up performance. 1. Infungal antibiotic scale-up, seeding of media with a high-spore inoculum (vs heterogeneous spore-vegetative cells) leads to more consistent fungal fermentation performance (Calam, 1987) 2. Secondary metabolite fermentations require a high proportion of mutant cells (low degree of mutant reversion to wild, nonproductive cells) to ensure stable scale-up (Vanek and Hostalek, 1986).A similar situation exists in recombinant fermentations (high degree of plasmid retention). Rule 3. Inconsistencies either of physical and chemical or of microbiological and genetic processes can result in performance variability or failure at scale-up. These inconsistencies are often caused by unavoidable differences in operational procedures between the laboratory, pilot plant, and commercial-scale departments (organizations). Often, these differences are due to physical or staffing constraints. Examples of inconsistencies include the following.

1. Differences in the extent or type of chemicals used to clean bioreactors and accessory equipment, which can leave residues that influence the biological response differently 2. Differences in the operational acceptability as to whether frozen or fresh inocula are suitable 3. In processes for sterilization of media, the zero-hour (fresh media sterilization) chemical composition at large scale can differ from that at small scale (Boeck et a]., 1988).The extended cool-down period for large vessels may considerably damage heat-labile nutrients 4. Differences in the response time and oscillatory behavior of the process control systems (e.g., temperature, pH) at different scales 5 . Differences in analytical approaches at small vs large scale (e.g., use of g/liter vs kg/t) by laboratory and technology transfer groups

Not all inconsistencies result in differences in the physical, chemical, or microbial processes at commercial scale. Rule 3 is merely suggested

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

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as a reminder to the scale-up staff to ensure that seemingly innocuous inconsistencies are at least considered as possible causes of scale-up problems.

b. Checklists. The use of checklists is considered a deliberate, conscious effort of the entire scale-up team to obtain a comprehensive list of potential issues by applying Rules 1-3 to a particular biotechnology process: 1. What are the known substitutions of processes, unit operations, and/or raw materials at the industrial scale relative to those practiced at the laboratory or pilot scale? 2. What are the known inconsistencies or differences in operational functions (standard operating procedures) between the industrial and laboratory pilot scales?

A typical example of a scale-up checklist is shown in Table XI. The entire scale-up team is defined to include plant personnel, the design engineers whose experience in describing the designed parameters for the existing equipment is invaluable, and the technical R&D and pilot plant technical staff acquainted with the process. Another example of a checklist is given in Reisman (1993). Checklists are a valuable, comprehensive scale-up tool because they require involvement of all personnel who are knowledgeable with all four sources of scale-up problems. Table X shows that this method is the only one that covers all four sources of scale-up problems. The checklist approach integrates the use of other methods and resolves issues identified by the entire team, c. Morphological Analysis. This analysis (and quantitative methods

described below) tends to be the domain of the bioprocess engineers or research scientists. As such, they tend to exclude valuable input, such as that of plant operators. The morphological analysis examines the major factors influencing the bioprocess performance, called “parameters of importance.” In microbial biotechnology, there are four major factors of importance (Table XII). The morphological analysis focuses thinking of the four specialist groups to scale-up, who are asked to answer the following questions. 1. Can the known substitutions of processes, unit operations, or raw materials at the commercial scale relative to those practiced at the laboratory/pilot scale influence the biological performances on scale-up?

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ALES PROKOP TABLE XI

SAMPLE OF CHECKLIST FOR SCALE-UP IDENTIFICATION IN MICROBIAL BIOTECHNOLOGY Source

Problem

Production bioreactor

Foaming Reproducibility of productivity Oxygen and heat transfer capabilities Localized effects-glucose entry point and mixing pattern Potential iron limitation Ability to maintain dissolved oxygen profile Response time of process controllers Back pressure control

Auxiliaries to production bioreactor

Continuous sterilizer performance characteristics Precipitate formation and trace metal bioavailability Accuracy of flow rate measurements Seed bioreactor-inoculum stability, pattern, response time of process controllers

Site-related issues

Water supply-composition Air supply-relative humidity Cleaning agents-residues Process steam-contaminants Raw material preparation-mixing

Technology transfer

time

Defining and identifying differences in terminology Defining differences in assay methods

Note. (Reprinted with permission from Tong and Inloes (1990). Table 2, p. 559. Copyright 1990 by American Chemical Society).

2. Will the known heterogeneity of the environment or in the properties cause a difference in biological response on scale-up? 3. Can the known inconsistencies or differences in operational practices between the commercial and laboratory/pilot scales affect the biological performance at scale-up?

The objective of the multidisciplinary group is to discover potential physical, chemical, and microbial-genetic interactions that result in differences in performance at different scales of operation (e.g., effect of raising bioreactor pressure to increase oxygen transfer-an example of physical/microbial interaction). The ability to accurately extrapolate (that a potential scale-up factor will or will not have a significant effect) from limited laboratory data is a key for success. This is necessary for proper prioritization (or truncation) of the checklist of possible experiments or actions.

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TABLE XI1 FACTORSOF IMPORTANCE IN SCALE-UP FOR MICROBIAL BIOTECHNOLOGY Factor

Potential source of problem

Tools used for identification

Microbial

Source of culture (slant, frozen vial) Culture stability (mutant, plasmid) Inoculum conditions at seed transfer Microbial growth rate

Protocol based on shake flask results Shake flask controls, plasmid marker assay Transfer time (age), density, pH, shake flask control Growth time profile, oxygen and carbon dioxide rate Flask-to-flask and week-to-week variability Microscopic examination, streak plate pH, streak plate Microscopic examination

Mixed culture Contamination Cell lysis/phage attack Cell morphology Genetic

Host-plasmid stability Reversion of high productivity mutant to wild parent strain

Convenient plasmid selection Environmental pressure

Chemical

Nutrient concentration in initial medium Media preparation/precipitate formation lonic content of water

Sugar, nitrogen, phosphate, sulfate, total solids Total solids, color of precipitate, phosphate, Mg Fe, Ca, Mg, material of construction Shake flask controls

Seed/production media, raw material lots Mode of media sterilization pH of media Inoculum transfer Defoamer addition, time of addition Carbon balance Physical

Temperature Backpressure/hydrostatic pressure Evaporation loss Foaming Mixing

Color of broth, pH, phosphate, total solids

PH

-

By-product distribution, mass transfer Sugar, organic acids, RQ Thermistor, thermometer Dissolved oxygen and carbon dioxide, pH Relative humidity, volume/ weight balance Volume balance, capacitance probe Impeller Reynolds number, mixing time (continued)

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ALES PROKOP TABLE XI1 (Continued)

Factor

Potential source of problem Location of nutrient, acid, and base additions Shear rate Viscosity Oxygen transfer rate Liquid-to-solid transfer rate Heat transfer rate

Tools used for identification

Flow and mixing profiles Impeller tip seed, agitator speed, mixing time Viscometer, power required Dissolved oxygen, off-gas analysis, transfer coeff. Check its value Heat balance

Note. Adapted from Tong and Inloes (1990).

2. Quantitative Methods

The quantitative scale-up methodologies lead to alternative experimental strategies to answer the questions developed in the checklists or morphological analyses. The use of these four methodologies (below) identifies the specific sensitive scale-up factor(s) applicable to the fermentation of interest and determines the range of acceptable operating conditions for the industrial scale-up. Executing the resulting experimental program ensures the highest probability of scale-up success in the minimum time. a. Multivariable Screening. Statistical multivariable screening has been used traditionally in the fermentation industry to screen for new organisms and products and to empirically develop optimum culture conditions (Stark et al., 1976).The approach is characterized by using multiple bioreactors of identical size. Each bioreactor operates under a different set of conditions, identified through the use of either checklists or morphological analysis as a potential source of problems. The analysis searches for those significant variables that influence fermentation performance at large scale (Fig. 16). b. Statistical Analysis. This is usually required to reduce the large amount of data generated in the multiple bioreactors and to elucidate those factors contributing in a significant way to the problem. Experimental factors can be varied singly with the remaining factors held constant, but this approach suffers from possible failure to detect and estimate possible interactions between critical factors and from the requirements for a large number of runs. A more efficient approach that reduces the number of required runs and determines potential interactions involves the use of a factorial design experimental plan

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1 Input variables (x)

Multiple Bioreactor units

195

Final Product concentration

Type of analysis: y = 0 (x) search for significant input variables which influence productivity Type screens: Culture genetics:

1. Natural selection 2. Mutate and select for product formation

Culture environment: 1. Empirical selection of process conditions to guarantee high productivity

FIG.16.

Statistical multivariable screening method.

(Box et al., 1978). The statistical approach is discussed briefly in Part I (Section VI, Approaches to Process Discovery). c. Pattern Analysis (Heuristic). This approach is most applicable in scale-up involving complex media. The basic premise used in this analysis is that of Young (1979), which states that “comparing chemical concentration profiles (e.g.,pH, ammonia, trace metals, chloride) as well as metabolite concentration profiles (e.g., acetic acid, carbon dioxide, product) between different scales (laboratory vs. pilot or pilot vs. plant) will lead to identification of major sources of scale-up problems.” Differences in the pattern of the acetic acid the profile (Fig. 17), for instance, can lead to examination of strategies for glucose control and dissolved oxygen control in the pilot and plant fermentation (this is of particular significance for a high-density bacterial culture). Similarly, differences

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196

Small scale Concentration

Large scale

or Productivity

Time

FIG. 17. Example of pattern analysis at different scales.

in media makeup or sterilization can be determined by comparing preand poststerilized media from pilot and plant samples. Pattern analysis is characterized by the use of single bioreactor hardware and by multiple and detailed chemical/biological assays carried on time samples (Fig. 18)at two different fermentation scales. Examples of profiles are: 1. Microbial factors-growth rate, CO, release rate 2. Chemical factors-glucose, ammonia, pH, chloride, trace metals,

product 3. Physical factors-temperature, dissolved oxygen. Pattern analysis can likewise be applied to areas beyond the bioreactor. One can compare seasonal variations in the chemical composition of the water supply and of media sources that can cause seasonal scaleup problems. Differences in process controller responses (e.g., to temperature, pH, pressure) for bioreactor auxiliary equipment such as continuous sterilizers and nutrient supply tanks can lead to significant physiochemical effects that are unique to only one scale of operation. d. Specific Rate Analysis. This analysis is applicable only to examining metabolic patterns within the bioreactor, whereas the pattern analysis also applies to elements external to the bioreactor. Specific rate analysis employs a single bioreactor in conjunction with multiple chemical and biochemical assays to characterize the process. Unlike pattern analysis, which uses only productivities and concentrations, specific rate analysis evaluates and compares the metabolic patterns and wholecell physiology of average cells at the two different scales of operation. The measures of whole-cell physiology are provided by specific rates, namely of growth, nutrient uptake, and product formation. The analysis

197

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1 ,Tpx X’,

XNX

Glucose feed1 - 1

3 1 4 1 15+1

45g/l/h-)+I x’

Nitrogen feed I t 18mg/l/hx/+I

f’

0

3 80

7

-C-CA-AA-A&-.~-&~-~-~-~ JAmmonlo

/x

1

Oi

20

I

40

60

80 Cycle time ( h 1

1

I

100

120

J 140

FIG.18. Pattern analysis of penicillin fermentation with Penicillium chrysogenum (reprinted with permission from Queener and Swartz (1979), Fig. 5, p. 52, copyright 1979 by Academic Press).

searches for those specific performance parameters that correlate with productivity and then identifies those fermentation parameters that significantly affect the specific parameters (Fig. 19). In a fed-batch bioreactor producing penicillin, the average-cell physiology is represented by specific growth and product rates; a critical specific growth rate is a key input parameter affected by a specific substrate feed input rate (see Fig. 4.36 of Ryu and Hospodka, 1980). This leads to accurate identification of the optimum feed regime at both scales.

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Single bioreactor

Input variables (x)

I

I I

Performance parameters

Final product concentration

(4

I I I I I I

I I I I I I I I

Type of analysis: L = F (x)

Correlate input variables with specific performance parameters

y = f (2)

Search for significant performance parameters which correlate with productivity

Types of approaches: Culture genetics:

1. Mutate/select for specific biochemical deletion or derepression

control 2. Gene construction Culture environment: 1. Control of biochemical pathways 2. Qualitative whole-cell physiology 3. Process design which manages cell growth separately from product formation

FIG.19. Specific rate analysis scale-up method.

The engineering process analysis often uses the following quantitative tools: rules of thumb, regime analysis, and scale-down approach.

e . Rules of Thumb (ROT). Based on maintaining constant powerhnit volume at mixing of a vessel (POW),constant oxygen transfer (k,a), tip speed, or dissolved oxygen level (Table VI) on scale-up, rules of thumb have been used with some success in situations where oxygen transfer or shear stress limits performance (Fig. 20). In general, rules of thumb in engineering process analysis are not suitable as the sole scale-up

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

Yield

100

-

10s

+

(% of maximum)

0

0

75

-

I I

I

0

Selected value

P0

0

50

0

O I

/

-+

199

I I I I I

-

10

20

30

40

k a (l/min) L

FIG.20. Nystatin titers as related to oxygen transfer (k,a) during scale-up (reprinted with permission from Jarai,(1972),Fig. 13, p. 102, copyright by the Society for Fermentation and Bioengineering, Japan).

procedure for growth and production processes. It is frequently impossible to achieve the same conditions at prediction scale as those used at pilot scale (e.g., powerhnit volume). Furthermore, several factors responsible for controlling performance at production scale are not necessarily affected by these engineering parameters (criteria of scaleup). In such cases other factors responsible for controlling the culture behavior on scale-up must be identified.

f. Regime Analysis. This approach is potentially useful as an adjunct to other engineering approaches which attempt to identify and focus on only those mechanisms that are rate-limiting. Characteristic times, which are measures of the rates of different mechanisms, can be estimated either from theoretical considerations or by experimentation. These times are used to determine the most probable rate-limiting step on scale-up. By establishing the most likely operating regimen, it is possible to simulate the rate-limiting mechanisms (and thus conduct scale-up studies) at laboratory scale. The following are excerpts or adaptations taken from Prokop (1982), explaining how to use the regime analysis. The dynamic hierarchy of time characteristics, i.e., the relaxation times of individual subsystems involved, is useful. The relaxation time (characteristic time, time constants, half-time, etc.) is a measure of the rate of a mechanism: time needed by that mechanism to smooth out a change to a certain fraction

200

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of response. A low value for a characteristic time means a fast mechanism; a high value means a slow mechanism (the last one is of importance to us). Other than the characteristic time of biological subsystems, one can use the cell generation time, rate of substrate consumption, and rate of oxygen uptake (rate terms can be appropriately converted to time values) on a population level. Also, engineering characteristic times as a mixing time (a degree of homogenization of a vessel) can be included. Figure 2 1 presents two sets of characteristic times for both biological and reactor levels, as applicable for a mammalian cell culture (Prokop, 1991). Some methodology on their estimation can be found in Sweere et a ] . (1987). While comparing values of characteristic times it is possible to delineate the “state” of cells: in steady-state, in a dynamic state, or in a “frozen” state. When environmental changes are too slow (long characCHARACTERISTIC TIME 10-6 (s) }

10.~

1oo

10.2 I

I

Mass action law

I I

‘

’

synthesis

Enzyme reactions Protein diflusion in cytoplasm

H

4

I

2’

mRNA

I2

12

stability

I

13

I

108 I (s)

‘’

Cell division

stability

Lateral diflusion of proteins

BIOLOGICAL MECHANISMS

o6

Cell multiplication

1 mRNA

-P-

41

1

I

I

Subtances translocation into cells

WWsis

41

1o4

102

I I

8

in membrane Cell attachment -/-:5 on microcarriers Ligand to elicit response Receptor

Protein flip/flop between membranes

’3

42 Receptor

6 1 , y c l e 6

deformation

Oxygen consumption

Substrate consumption’“

------------_------------Oxygen transfer Microcarrier

PHYSICAL MECHANISMS

I

’ CELL ‘FROZEN

settllng’b Mixing

Circulation time Laminar stretching

I

Ditf?sion 11

’

H

i

time

‘lo

’

Heat production Heat transler

la

111

1

Turbulent erosion

111

CELL IN A DYNAMIC STATE

CELL IN STEADY-STATE

FIG.21. Characteristic times of biological and physical mechanisms (mammalian cell culture). Data sources (numbers) are in the original reference (Prokop (1991),Fig. 2-14, p. 52).

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

201

teristic time), the organism will be at steady-state in relation to the particular environment. In case the time needed for oxygen transfer to the liquid phase is much larger than that of oxygen consumption, oxygen depletion will occur in the bioreactor. Long characteristic times of liquid circulation, as well as short ones of heat transfer and oxygen transfer, could be related to this, resulting in temperature and dissolved oxygen gradients (synthesis of stress proteins may occur!) The other extreme is that of small characteristic times of the environment (change is too fast) compared to that of the organism. This means that the organism cannot adapt to such a change and is frozen in the initial state. For example, mammalian cells will not attach to microcarriers (they will be frozen in the initial suspension state and will die) when turbulent erosion times are too short compared to the cell attachment. The turbulent erosion represents the Reynolds stresses in the impeller region. For the overlapping time domains between biological and environmental mechanism, the organism will dynamically respond to a change. For example, as long as the characteristic times of the turbulent erosion and the laminar stretching on one side and that of mammalian cell deformation (e.g., hybridomas) are comparable, no damage to cells occurs since the cell can recover from a deformation. The laminar stretching represents viscous stress in the bulk liquid. The knowledge originating from the characteristic time analysis can be used for identifying rate-limiting (bottleneck) mechanisms and will allow or maintain the same regime at different levels for a reactor scaleup. A major advantage associated with the characteristic time-based scale-up (using a scale-down reactor) is saving time and resources, since production scale experiments are always expensive. g. Scale-Down Analysis. Because design and operating limitations often exist in production-scale bioreactors, use of scale-down technique to stimulate at small scale the conditions existing at large scale can be quite useful (Fig. 22). Another scale-down approach relies on the preparation of media or culture broth at large scale to a certain point in the fermentation cycle, followed by transfer of the material into smallscale bioreactors for further evaluation and optimization (Fig. 23). This can be used to substantiate the existence of sensitive scale-up factors and to find appropriate process conditioning. Among its potential uses are evaluating the impact of media sterilization conditions at large scale (e.g., batch vs continuous) and identifying optimum culture conditions following induction of a recombinant protein. Simulated scale-down methodology has been applied successfully to a 500-liter pilot bioreactor to simulate improper dissolved oxygen con-

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202

Cell mass concentration (mg/ml) Oxygen uptake rate (mmoVVhr)

Chlottetracycline(unildl)

12 10

ReDlica

10

8

6

4

2

0 0

20

40

60

80

100

120

140

Time (hr)

FIG.22. Example of scale-down of chlortetracyclinefermentation (reprinted with permission from Biotechnology and Bioengineering, 8, Jensen et a ] . (1966), Fig. 5, p. 531, copyright 1966 by John Wiley, reprinted by permission of John Wiley & Sons).

trol or inadequate oxygen transfer in avermectin production. Potential scale-up problems caused by the introduction of new media at production scale were averted (Buckland et al., 1985). h. Other Approaches. Other approaches to engineering process analysis are somewhat limited. Dimensional analysis, for example, always results in geometrical similarity (e.g., dimensionless ratio of characteristic lengths, impeller diameter to vessel diameter). It neglects biological components of the system. Mechanistic models are seldom available due to insufficient biochemical and physiological information. Empirical models usually are incapable of prediction and add little to the

CHALLENGES IN COMMERCIAL

BIOTECHNOLOGY. I1

203

Large-scale batch or continuous sterilization

Media transfer

Small-scale Small-scale Small-scale bioreactor #2 biorector #n bioreactor #I FIG.23. Application of scale-down analysis at media sterilization. Small-scale fermentations can be performed either under identical conditions (replica) or under different conditions (optimization).

fundamental understanding of microbial dynamics. However, they can all be used together (Fig. 24). The methodologies discussed above, although illustrated by examples from microbial biotechnology, are generally adaptable to other biological reactor scale-up problems such as those involving tissue culture, mixed culture, immobilized whole cell, and enzyme systems. The classification of scale-up methods according to types of cognitive thinking is shown in Table XIII. It also includes degree of risk vs cost trade-offs. Methods of thinking are those explained in Part I, Table VI, Tl-T4. Table XI11 demonstrates a need, in addition to a multidisciplinary team of specialists, for different-thinking-oriented people for successful handling of all scale-up challenges. E. PROCESS CONTROL AND OPTIMIZATION

Newer biotechnology plants are run under some type of control; some are computerized. It is important to divide control variables to those which require monitoring, monitoring plus control, and computer control. Present development facilities are highly instrumented, some with on-line analysis of important parameters, allowing for rapid process development and optimization. In addition to direct control algorithms, some parameters are often calculated from mass and energy balances. The employment of computers then allows more operative actions to

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204

Mechanistic analysis

Dimensional analysis

Empirical models

Scale-down analysis

analysis

Experimental design

Rules of thumb

Experience

be taken for process improvement. Further discussion of this topic is beyond the scope of this article.

F. PROCESS AND PLANT DESIGN The goal of process design is a set of specifications allowing a selection of equipment, controls, and services to effectively carry out the developmental activity. Common tools used include mass and compound balancing, energy balances, flow rates, specifications, installation drawings, and operating parameters. Process design is an iterative process requiring the cooperation of many technical personnel (Fig. 25). Plant design criteria must also be adapted in terms of expected manufacturing flexibility or rigidity. Advantages and disadvantages of a fixed single production train vs a multiproduct production plant are listed in Fig. 26. A major result of the process design is a process flow diagram, a pictorial representation of unit operations and processes involved. Figure 27 is a flow chart for citric acid production (fermentation and recov-

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

205

TABLE XI11 CLASSIFICATION OF SCALE-UPMETHODSACCORDING TO “METHODS OF THINKING’ AND RISK/COST ANALYSIS Scale-up methods (“methods of thinking”)

Scale-up challenge”

Degree of risk

Time cost and dollar cost

TI. Speculative thinking Rules of thumb of engineering analysis Clues of biochemists, microbiologists, geneticists and technicians

Type 1

High

Low time Low cost

T2. Personalized thinking Origins of scale-up problems Pilot plant personnel experience with facilities characteristics Microbiologist’s knowledge of culture characteristics Chemist’s knowledge of product/coproduct stability

Type I&II

Medium

Medium time Low cost

T3. Data bound thinking Statistical and pattern analysis with heavy reliance on analytical assays and access to many bioreactors

Type III&IV

Low

High time High cost for assay development and interpretation

T4. Relational thinking Specific rate analysis, regime and scale-down analysis, morphological analysis, requiring training in biochemical and pathway mechanisms and an ability to interrelate multidisciplinary knowledge

Type III&IV

Low

High time High cost but maximize value of data use beyond scale-up, e.g., at optimization

Scale-up challenges I-IV are listed in Table VII.

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206

I

I

I

General (multifunctional) batch multi-product train

Dedicated continuous single train 1

I

I

Known utility and manpower needs Amenable to computer control Lower insurance and taxes

Adaptable in both scale and type of product Applicable to nonstandardor low volume Subcontractingor leasing is possible (custom manuf.)

Disadvantages Higher initial cost potential overdesign Greater maintenance complexity Higher inventory or spare parts Work-in-processbuild-up likely Long training periods/ complex instructions Process planning may be more complex Complex instrumentation Utility and waste treatments loads may vary Greater need for warehousing Gross contamination potential exists

Inflexibility Difficult changeover Expansions or major revision needed to accommodate volume change Difficult to respond to change in demand or experimentation

I

ery /purification). Such a sheet should comprise overall material balances and chemical and physical data for each stream, along with energy inputs and removals. Engineering flow sheets are considered an expansion of flow diagrams. Some greater detail might be included, but detailed piping and electrical drawings are not normally available at this point. Piping and instrument diagram (PID) results from later stages of process and plant design. A PID for a bioreactor will show pipe sizing, valve type and sizing, pressure relief devices, instrumentation including indicators] final control elements, leads into and out of the drawing (to associated equipment drawings), special notes for construction details and materials of construction, pressure and vacuum ratings, agitator

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

207

Selection of design options with the use of "universal multidisciplinary approach"

t

User team fRB ), Operations

. Conceptual, holistic brainstorming

+ by multidisciplinary team; ask right questions End-result: identifv obiectives and issues of project

..

DesigdFinance Session #2a Quantified constraint list

.Budget

.Time . Space . "Acts of God"

Design Team

Session #2b - Need list

.Detailed user's needs identification ..List of: Process needs Production needs Future RBD, Market needs

Session #3 Prioritized need list

.Needs classified via constraints list: wish list want list must list

Session #1b Alternatives list

. List of design

options (subsystems) . List of overall plant design alternatives

-r

t Session #4 Prioritized alternative list

.Alternatives ranked and classified

according to must, want & wish list

Selected design (optimum)

FIG.26. Approaches to design of a natural products process plant.

and filter ratings, and alarms (Reisman, 1988). An example of a typical bioreactor PID is also given in Reisman (1988). The same source discusses special tasks involved in actual start-up or process introduction. It is a nonroutine operation without guidelines, requiring careful planning. An additional constraint on the process design side could be in case of the use of recombinant organisms: biocontainment.

DEFOAMER

FERMENTOR SEmlNG (sterile operation) TANK

I

AIR>=

SEED

,

NUTRIENTS PRECIPITATION

ML RECYCLE

MYCELIUM REMOVAL

LIME

FILTRATE (waste)

VACUUM FILTER

"2"4 ACIDULATOR

CaS04

TO

L

c

EVAPORATOR

BY PRODUCT

-

FILTER

VACUUM

FIG. 27. Flow chart for citric acid production (courtesy of Reisman (19881,Fig. 4, p. 45). Reprinted by permission of CRC Press, Boca Raton, FL.

-

TREATMENT and I or ION EXCHANGE

-0 CARBON

pH CONTROL

LABORATORY INOCULUM

MEDIUM

SUGAWMOLASSES

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

209

G. TECHNOLOGY TRANSFER This section focuses on how biotech companies can most effectively transfer information for product development both in-house and externally (Wheelwright, 1994). Given the unique nature of biotech product development, there are many barriers that must be overcome. In addition, because of the different environments of the R&D organization and the manufacturer, different goals and objectives emerge (understanding of principles vs production goals). According to Wheelwright, there are three different ways to transfer technology: 1, Documentation transfer (“over-the wall transfer”). The information is passed from one group to another without the benefit of direct involvement. 2. Cross-training of personnel, providing a direct interaction between the transferring and receiving group. This may include the training of a factory worker by a member of the development group. 3. Moving personnel, including and on-site exchange of people and information between the groups (e.g., the transfer of a manufacturing person into development during the course of new process design or transfer of an R&D person into manufacturing during the start-up phase of new process). From an organizational viewpoint, a sequential information transfer is germane to all categories of technology transfer as above. The parallel development model, however, promotes better transfer between individuals in different groups as all groups work on the project at the same time, but with staggered, overlapping times. This allows for considerable feedbacks between the R&D groups and manufacturing and for a better learning environment. In the end, the parties involved see a wider “systems” view of problems, combining the overall goal with the specifics. The technology transfer ceases to exist and the successful endpoint is reached only when the acceptable product is delivered to market. However, this is only possible via market development. IV. Market Development

A. HOLISTIC APPROACH Marketing and market development are extremely expensive activities for a biotechnology/pharmaceutical company. These companies tend to make the bulk of their profits from only a few products, This is primarily due to the high risk of R&D activity, forcing companies to spread investment into many areas and products with expectations that only a few will bring profit.

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This section will follow (with one exception) the outline of a product/ market development presented in Table XIV. Some of the entries in this table will not be discussed, as they pertain to general marketing problems. Only those which bear a relevance to specifics of biotechnology and pharmaceutical industries will be covered at length (in bold). Table XIV merely shows the logic involved in market development.

B.

CURRENT STATUS OF THE BIOTECHNOLOGY MARKET

1. The World Biotechnology Market Estimates of the potential annual market of biotechnology products is about $15 billion for 1995 and $56 to $69 billion for the year 2000 (Table XV). Unfortunately, there is no more updated estimate available. 2. U.S.Biotechnology Market

The share of individual market segments is shown in Tables XVI and XVII. Note that the majority (82%) of the public companies (the breakdown is not given) specialize in human health care. In Table XVI, this table does not reflect the market value of products. Table XVII presents the most recent data (1992and 1993)and attempts to make a forecast over the next decade. Viewing this table, one would conclude that human therapeutics and human diagnostics will generate most of the sales. On the other hand, the fastest growing segments will be in the rest of the industry, particularly in environmental, agricultural, and specialty chemicals applications. Its market share is forecast to grow rapidly. Within each market segment the identification of a suitable niche is the goal of market research, following the strategic activity. New biotechnology seeks to replace traditional niches (e.g., by recombinant insulin) or introduce entirely new products (e.g,, G-CSF). The pace of introduction of biotechnology products into the market has been spectacular over the last few years. Table 1-2of the OTA report (Office of Technology Assessment, 1991) lists approved biotechnology products as of 1990.The list has been expanding since and many products await approval. Since 1981,14 biopharmaceutical products have been approved for a variety of indications and 143 biotechnology products are currently in the regulatory pipeline, many being investigated for more than one indication (Shame1 and Keough, 1994). It is convenient to review here the structure of the U.S. biotechnology industry in terms of different capabilities. Table XVIII presents a breakdown of the U.S. main players in the pharmaceutical/biotechnology industry. It is apparent that not every company can afford to cover all necessary functions.

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. 11 TABLE XIV PRODUCUMARKET DEVELOPMENT Strategic planning Business Environment Resources Product strategy techniques Life-cycle Experience curve Strategic business unit Strategic concepts Follower strategy Importance of fit Risk management New product strategy Product matrix Categories of product Strength required for success Choosing suitable field of activity Specific markets Key factors Selecting product for market entry Market forecasting World vs U.S. market Biotechnology and pharmaceutical market Total vs available market Market share New Product development New product evolution (idea-selection-development-testing-market] Generating new idea Encouraging environment for innovation Product selection and screening Pre-screening Product-fit desirability matrix New product desirability matrix Business attractiveness-company fit matrix New product profile chart Business analysis New product development and testing Setting technical objectives Marketing and research interface Concept testing Key customers Specifications Technical support Applied research Process development Commercialization (continued)

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212

TABLE XIV [Continued) Product introduction strategy Marketing plan Product pricing Selective distribution channels Advertizing and promotion Market development techniques Product introduction Field work Selection of timing Management and control ~~

Note. Adapted from Pacifico and Polacek (1981) and Pacifico and Witroer (1981).

C. MARKETDEVELOPMENT CONCEPTS 1. Product Cycle

From an economic standpoint the cycle concept is normally viewed on sales vs time coordinates. The product life-cycle in the market context is also denoted an “S-shaped curve of innovation.” It relates the incremental progress in technology (performance, an increase in profit) to incremental amounts spent on R&D to promote that technology (Fig. 28). It is a characteristic feature of the biotechnology industry that there is a persistent move toward a periodic replacement of top-selling

TABLE XV ESTIMATED WORLD MARKET FOR BIOTECHNOLOGY PRODUCTS (MILLIONS OF 1985 DOLLARS; INFLATION IS CONSIDERED AT 6 TO 8%

PER YEAR)

Industrial segment Pharmaceuticals Human therapeutics Human diagnostics Ag-bio Specialty chemicals and food Commodity chemicals Environmental applications Industrial suppliers Total

1990

3,500 1,500 4,000 2,100

N/A

N/A

2000

20,000-30.000 5,000 13,000-14,000 6,500-8,500 1,000

N/A

4,075

10,500

15,175

56,000-69,000

Note. Adapted from Anonymous (1988),Table 3.1.

CHALLENGES IN COMMERCIAL B I O T E C H N O L O G Y . I1

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TABLE XVI U.S. COMMERCIAL ACTIVITYAS DISTRIBUTED IN SPECIFIC SECTORS (TOTAL BIOTECHNOLOGY INDUSTRY SALES)

Percentage

Industrial segment

1992

1993

Pharmaceuticals Human diagnostics Human therapeutics Ag-bio I n d u s t r i a l supplier C h e m i c a l , environmental, and services

95 30 65 2

68 26 42 8 15 9

NIA 3

Note. Adapted from Glaser (1992) and Lee and Burrill (1994). Note that the 1993 data should be considered more reliable as all the sectors are accounted for.

T A B L E XVII U.S. BIOTECHNOLOGY

Industrial sector

Pharmaceuticals Human therapeutics Human diagnosostics Subtotal Ag-bio Specialty chemicals and food Commodity chemicals and energy Environmental applications Bioremediation Environment monitoring Subtotal Industrial suppliersb

SALES FORECAST: 1992-2002

(MILLIONS OF 1992 DOLLARS)

Base year sale

%of total

Forecast growth

(1992)

(1992)

1997

2002

Ippa)"

2250 1050

4600 1700

9,200 2,500

70

48 22 70 1.5

375

1,400

15 9 13.1 35

95

2.0

400

1,300

30

NIA

-

N/A

1,000

N/A

65

1.5

300(1995) [820(1997)]

10

0.02 1.5

1000

940(1991)

25

1992/2002

250

1993/2003 (ppa)

15

38

2740(1996) [1165(1992)] [3400(1997)]

Note. Adapted from Shame1 and Keough (1994) and Szoka (1992). Numbers in brackets have been added (calculated) for comparable statistics. ppa, growth in percentage per annum. Bioinstrumentation: DNA sequencerslsynthesizers,PCR, separations instruments, bioreactors, monitoring instruments.

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TABLE XVIII U.S. PLAYERS IN PHARMACELJTICALIBIOTECHNOLOGY INDUSTRY Four essential components for succe88 ~~

Innovative

R&D Type of company

technology

Competent production in market

I. Fully integrated 100% pharmaceutical (e.g., Merck. Lilly)

Chemicel/pharmaceutical (e.g.. Pfizar, Squibb. Hoffman-LaRoche) Recent pharmaceutical/ chemical [e.g., Monsanto-Searle, DuPont. Kodek) U. Highly research-oriented pharmaceutical Genetic engineering (e.g.. Genentech. Cetus. Amgen) Product formulation oriented (e.g.. ALZA)

***

“t.

t*.

..t

*.

...

...

**

No

No

Entrepreneurship

Product development and formulation

...

**

..t

f.

*.

**

No

.*

Note.***,Good command; *+, limited resources; +,minimal resources; No, no resources.

product(s) by new entries, resulting from new R&D effort. Such replacement is driven by advancement in new drug discovery by means of molecular, biochemical, and recombinant DNA technology tools. However, the spending on new technology may be less productive than incremental spending on the old technology. In many instances a considerable discontinuity exists between two S-curves, representing early excessive investments with little measurable advancement (typically seen in new biotechnology). The competitive innovation and new product entry is necessary for survival and has important implications for the performance of the biotechnology industry. 2. Strategic Concepts

a. Follower Strategy. The market follower strategy intends to find a niche (i.e.,a market segment) and requires proper timing and an intimate knowledge of the environment. In the pharmaceutical industry, such a strategy results in “me-too” drugs, usually developed by means of standard methods with a low level of innovation (note, relative to the existing or applied technology). The me-too drugs offer little therapeutic advantage over the existing drug products on the market. They have little justification, therefore, unless they show clear clinical superiority to existing competitors. An example of the latter is that of variants of

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

215

New use

Performance I / I

T New technology

/

Old technology

Original use

Effort

FIG.28. S-curve of innovation (product replacement). Note that product’s life can be extended by discovering of new product use.

captopril. Many variants of this ACE inhibitor are in clinical trials or have entered the U.S. market, It is doubtful that they will make much of an impact unless they are significantly more effective (e.g., enalapril). Captopril has captured a significant share of the antihypertensive market but enalapril is also expanding and promises to be a widely used drug.

b. RisklBenefitAnalysis. The factors involved in risklbenefit analysis and management include technological, commercial, legal, and financial considerations. Companies conduct cost and benefit analysis along with R&D to justify the expense of product development and to determine the potential for return on investment. Legal and regulatory risks present a special category for the biotechnology industry. Complex proprietary situations (uncertainties in interpretation of patent protection, patent infringement litigation) and particularly regulatory risks may present significant obstacles and delays in commercializing many biotechnology products.

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3. New Product Strategy

a. Product Matrix. The simplified product matrix has already been presented (Fig. 18 of Part I). An expanded version is shown in Fig. 29 (as technology/market matrix), with some examples. An intermediate row and column has been inserted into Fig. 18 (of Part I) to represent expanded market or improved technology. Figure 30 attempts to show the complexity of the product-process-market innovation system without stressing binary interactions. Six different scenarios for innovation presented in the upper part of this figure are redrawn in the lower part. It shows many possibilities of reaching market. Dotted arrow No. 7 is not a very frequent pathway in the industry. It is rarely used in biotechnology. It can work in the case of total replacement of an existing chemical productlprocess by a biotechnology-related process (e.g., semisynthetic penicillins). The term “technology” used in the upper part of Fig. 30 can be equated with process. Box 1 represents improvements and extension to present product and process to make them more attractive to present customers (market). Box 2 requires some input into a product, Box 3 into a market. Thus Boxes 1-3 fall into the traditional processing technology.

No technological improvement

Expanded market

Improved technology

New technology

Product reformulation (e.g., improved quality of insulin)

Product replacement (e.g., recombinant insulin in place of animal)

Sales development (e.g.9 expanded market)

Product development improved product via RBD and marketing

Product development product line extension (e.g., additional indication for a drug)

Market development new use of product

Market development market extension

Commercial development: diversification

FIG. 29. Expanded technology (product)/market matrix (adapted from Pacific0 and Polacek, 1981).

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

1 Present product Present technology Present market

2 Newproduct Present technology Present market

3 Present product Present technology New market

4 Newproduct Present technology New market

5 Newproduct New technology Present market

6 Newproduct New technology New market

1&2

183

Present Product

New Product

Present

b

~

6

-

217

+

Process

b

Present Market

New Market

5&6

FIG.30. The innovative system (PMT matrix).Note that route 6 is typically denoted as new business (venture)activity, often set as an independent operating company (reprinted with permission from Collier (1975), Fig. 1, p. 91; copyright 1975 American Chemical Society).

Box 4, however, requires inputs into the product and market. To manufacture such a product the existing technology is used, but the market is new. Developments in Box 5 require both a new product and new manufacturing technologies, while the existing sales department can be used. Most developments falling into Box 6 represent a completely new business for a company. To handle such developments, a new business organization, often largely separated from the parent organization and called a venture company, needs to be set up, nurtured by the R&D division. The venture companies typically are a spin-off from a large corporation (e.g., the former Invitron from Monsanto). This is not to be confused with venture capital. It is clear that the product strategy is indispensable for a successful innovation program. A higher level of innovation results from an interaction between the market and technology (product/process). b. MarketlProcess Matrix. The commercial value of a product or market segment relative to others can be represented in the technology/ market matrix. One such matrix may compare the estimated technologi-

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cal availability (success or technical maturity) and estimated market need (commercial value), perhaps via brainstorming or other approaches. Such a comparison can be carried at a specific product level (e.g., the probability of submitting NDA vs cost for product development) or at market segment (or subsegment) levels (Fig. 31).Each circle in Fig. 31 refers to a particular market segment, and the size of a circle is used as a third parameter (1990 market size). Such a matrix can determine driving forces and/or commercialization hurdles to help predict the relative rate and direction of product/market development. In addition, such a matrix can be used for presentation of forecasted data.

Technical availability Low

Medium

High

Contaminant monitoring

High

0

:hemicals

0

0 Therapeutics

Medium

Agriculture Oilrecovery 0 Low

0 Energy

Pollution control

0 Biochip

Market need FIG.31. Technological availability/marketneed profile matrix. Circle size indicates 1990 market size [reprinted with permission from Shame1 [1991), Fig. 19-3 p. 351, copyright 1991 by Butterworth-Heinemann).

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

219

c. ProductlMarket Strategy. From the above, one can conclude that there are three prime factors contributing to product strategy and to its success (PMT matrix). 1. Uniquenedsuperiority of product (P) is the most important, particularly in the health care market 2. Strategy for marketplace (M), including market potential, customer’s purchase decision, and competitive situation in the marketplace 3. Technical (T) component, i.e., engineering, design, and manufacturing skills of the firm

The PMT factors represent conditions that make a new product successful. In addition, several rules of thumb can be proposed for new products: stay away from me-too products, markets where new-product introductions are frequent, and projects new to the firm (unless you have resources to enter expensive R&D in a new direction). 3. Product Categories and Strategies

This part describes the primary product areas (market segments] on which the industry is currently based, focuses on the major business and marketing concerns facing each market segment, and offers product strategy for each. Most of the information is drawn from Price (1991). The market segmentation is that already adopted in Table 15 of (Part 1). In the therapeutic segment over 150 new therapeutics are at various stages of regulatory approval and several dozen are expected to reach the market in the early 1990s. This segment is characterized by: Enormous costs involved in developing a product Significant overlap among developing activities of most pharmaceutical/biotechnology firms, resulting in highly competitive and innovative activity Diversified market development Employment of existing sales forces and distribution channels Development of own distribution for new products Alliances between established pharmaceutical companies, providing product development, market capabilities and sales forces, and new biotechnology companies (not possessing capital to do so] Product application is limited to U.S.A., Europe, and Japan New dosage forms to be developed for proteidpeptide human applications Extensive purification needed for bacterial-derived proteins/products High product liability in U.S.A. (human vaccines).

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Strategies for this segment include: 1. Extension of the survival period for new biotechnology firms to the point of revenue generation from the first product and that by raising additional capital, establishing a joint venture with a manufacturer and shortening the regulatory approval process. The highest single expenditure is connected with developing, constructing, and validating a largescale manufacturing facility. 2. Carry out clinical tests in other geographic areas with low product liability 3. Knowledge of competitors position and technologies, including a survey of the firms involved and, possibly, obtaining new technologies via an acquisition or merger 4. Market penetration, which includes proper product positioning on the market, establishment of local distribution and foreign channels, and convenient product pricing.

The diagnostic segment has been growing rapidly in recent years and new products are continuously being introduced. It features: 1. Well-established market 2. Lower R&D costs (compared to pharmaceuticals) 3. Smaller regulatory burden 4. Importance of first to market rather then proprietary technology 5. Diagnostic products are perceived as “to small” and “too diffused”

market for effective management Strategies for this segment include: 1. Expand academic contracts in order to tap know-how developed there and quickly move it to market 2. Create niche markets and avoid competition in the large markets (valid for small biotechnology companies) 3. Aggressively penetrate foreign markets (Europe, Pacific, rim) 4. Cross-license technologies and products in Japan 5 . Involve new and small biotechnology companies, featuring more entrepreneurship.

The agricultural biotechnology segment will see an explosion in commercialization of many agricultural products (e.g., genetically engineered crops with improved yield and resistance to microbial and insect pests, primarily via replacement for products that already exist; e.g., Calgene’s Flavr Savr tomato). This program has been hampered by: 1. Small knowledge base in plant molecular biology (compared to animal and human area)

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

221

2. Challenges of developing gene transfer technologies 3. Highest R&D costs in comparison with other segments (lengthy

development times and time-consuming regulatory process). On the other hand, plant breeding, cell culture, and microbial selection allows for focusing product development on precise isolation of genetic traits and their transfer to crops or an organism of choice. At present there are few genetically engineered products on the market, but several are now in advanced stages of development and are undergoing field trials. The employment of genetic engineering will dramatically reduce the time necessary for breeding. 4. Public concern over the release of genetically altered plants, organisms, and microorganisms into the environment. The benefactors (market) of these products will be farmers, distributors, food processors, retailers, and the general consumer. Strategies for this segment include: 1. Broadening of alliances of small companies with large agricultural companies in order to reduce developmental costs 2, Establishing and broadening governmental and public relations functions in order to cut down on regulatory approval time and to increase public awareness 3. Formation of R&D and marketing alliances with European agricultural chemical firms to penetrate the European pesticide market.

Suppliers to the industry segment includes instrumentation, equipment, suppliers for cell culture and its cultivation, purification, and monitoring equipment and services. It features: 1. In-house marketing and sales forces and services 2. Specialized services for quality assurance testing and clinical trial

development 3. Lowest R&D costs 4. Very fragmented market. Strategies for this segment include: I. Positioning of products properly in the market via establishing firms, image, and product awareness 2. Establishing client service function to solve the customer’s problems 3. Creating new product/service niches.

Specialty chemicals and food segment is a broadly defined category. As most current biotechnologies do not provide distinct advantages for specialty chemicals, more R&Dis required before such products become

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competitive with chemically derived products. Strategies for this segment include the use of codbenefit models to identify potential replacements of chemicals by a biotechnology route, and targeting environmentally problematic chemical processing for replacement by bioprocessing (chemical vs enzymatic biopulping). The environmental application segment is the fastest growing and most dynamic part of biotechnology. Some problems include: 1. Lack of fundamental understanding of behavior of soilwater-contaminant-microbe complex 2. Reluctance to accept biotreatment technologies by government, industry, and service sectors (majorityof actions rely on waste immobilization rather than on removal and treatment) 3. Public concerns over environmental consequences of applications of biotechnology 4. Mismanagement of federal public funds (lack of direct use, excessive legal expenses).

Strategies for this segment include educating the public on risk/benefit of biotreatment technologies, and creating new product/service niches (e.g., general consumer check for presence of contaminants, environmental diagnostics). 4. Selecting Market Segment

Because of lack of funds, biotechnology companies focus their developmental activity in a limited number of areas. Only some of them will lead to tangible results at one time. Choosing suitable segments and depth of their coverage and proportions of R&D will determine the overall success of a company’s portfolio. In some cases clinical studies will be carried out on drugs that will never be marketed. The testing will be done only at an experimental or research basis to answer some specific questions. Such side development may turn into a new drug development. The development of new markets for existing products prolongs the growth phase of that product. 5. Selecting Product and Screening

The product selection process has three stages (Fig. 32):prescreening, screening for degree of fit, and detailed business analysis. As is the case of product discovery, product screening for market can be represented by an inverse triangle. In this case the statistics are more favorable, and the success rate is rather high. In the example in Fig. 32,60new product ideas entered the screening process and 5 were taken for further development and testing. The goal is to identify unique products, with the

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

223

New product ideas

i 60

Pre-screening

30

Screening for degree of fit

12

Detailed business analysis

5

FIG.32.

To development & testing Product selection process.

highest chance on the market. The business analysis is beyond the scope of this review article. New Market Development Industry and research interface: The probability of commercial success is dependent on the distance between the technical and marketing points of view. Two extreme situations are encountered in the biotechnology industry: technology-driven development, and market-driven development. For a mature industry, a proper balance between the two extremes should be struck, with proper communication between marketing and research and adequate effort to understand each other. Technology-driven development occurs typically for new technology companies pursuing a less defined market opportunity. In the human therapeutics field, neither the market nor the drug’s mechanism of action is well defined; that means that the ultimate marketplace success is unpredictable. Commonly, potential uses are being tested as well as additional indications for approved drugs (e.g., interferons, interleukins, TNF). Commercial biotechnology in the late 1970s was driven exclusively by the science. The R&D pace was so fast that it made new products obsolete in a few years. In the 199Os, the commercial environment is substantially different. Financing is harder to come by than previously. The market needs are and will be the driving forces of the industry during the 1990s and beyond. Thus, the majority of present day development is market-driven, with a defined and expectant market (e.g., erythropoietin, human growth hormone, insulin, tissue plasminogen activator, recombinant hepatitis B vaccine). The products originating from such development are much needed new drugs. The prerequisite for the market-driven development is, however, new technology as well. 6.

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7. Commercialization

a. Marketing Plan. The four P’s of the marketing mix necessary for success are (McCarthy, 1981)product (especially its technical level and uniqueness), price, place (distribution), and promotion.

b. Product Pricing. As far as the relative potentials for biotechnology manufactured products are concerned, two plots are useful (Figs. 33 and 34). Figure 33 shows the effect of productivity and Fig. 34 of the world production volume on price. The former figure allows estimates as to the likely selling price depending on bioreactor productivity and

Bulk selling price, $/t 2~ 107 Vitamin 812 2x 106

2x10~

2 x lo4

2x1~3

2X1O2

t

EthanoI

2 x 10 1i4

10

-2

10

-1

1

lo2

103

10

4

Productivity,Vm31a

FIG.33. Productivities vs prices of some biotechnological products in 1983 (reprinted with permission from Lewis and Kristiansen (1985), Fig. 1, p. 572, copyright 1985 by Society of Chemical Industry).

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY, 11

225

Price, M

loll

-

Human factor Vlll Human growth hormone

lo9

-

10’

Vitamin812

lo5

-

lo3

-

0

Benzyl penicillin

o

Beer Bakeh yeast

-6 10

-4 10

-2 10

10

2 10

4 10

6 10

8 10

10 10

World production, t/a

FIG.34. World production and prices of some biotechnological products (reprinted with permission from Lewis and Kristiansen (1985), Fig. 2, p. 572, copyright 1985 by Society of Chemical Industry).

suggests whether it would be competitive with similar products produced through other routes. For example, pullulan polysaccharide with low productivity of 2 t/m3 is liable to be expensive at around $10,000/ t when placed on the graph. Figure 34 shows an inverse relationship between production volume and price, representing the supply-demand interface. From this figure it can be concluded that the price tends to be low when world production is high. In the case of new therapeutic drugs, initially for a leading unique product, a company will attempt to set an exceptionally high price. As the number of manufacturers increases, the price will drop. Few drugs

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maintain a large market share for extended time periods. Product competition results in continuous price reduction on older products. The general downward trend is consistent with the existence of vigorous, dynamic competition in the pharmaceutical industry. Although the patent privilege provides a monopoly over individual drugs, the appearance of similar drugs introduces competition before the patent expiration. The temporary preeminence, the relatively rapid displacement of the leading products, and the rise of similar products and substitutes emerges as the general pattern. Replacement or competitors result from parallel development of the same product by many biotechnology companies, as seen in case of interferons, and from continued product development or from me-too efforts. In the pharmaceutical industry, entry into particular therapeutic markets generally requires new patented products if the products are to command prices comparable to those of the leading established products. A second vehicle of entry into therapeutic markets is based on generic products (without patent protection). Me-too drugs by definition do not represent true significant new therapeutic agents. However, their introduction causes the prices of older drugs to decline. The product imitations and substitutions result in high elasticity of demand over broad groups of drugs within the given product class. Thus, the supply-demand curve (Fig. 33) can be partially relaxed (market elasticity). In some countries, governments attempt to control prices and provide an environment for cost-cutting measures and cost-effective therapies. This is achieved: 1. By imposing some price controls through partial exclusion of costly drugs from reimbursement 2. By allowing higher prices for innovative drugs 3. By allowing higher prices in return for increased R&D 4. By price freezes and limits on profit.

Generic drugs are making a dramatic impact on the pharmaceutical industry in the U.S., especially in the managed care environment. As many product patents expire, pharmaceutical products are losing ground to generics. To maintain their piece of the market, pharmaceutical companies are focusing their own generic businesses, licensing out generic conversions of their drug, or acquiring generic companies (Lee and Burrill, 1994).

c. Selection of Timing. To be successful, the introduction of a drug into the marketplace must be timely. If a product is unique, the need and demand will be great and its success is assured. Usually, such a product will set in motion a great flurry of activity in other firms to

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

227

find a derivative with improved activity or adverse effects. This was the case with captopril. It is doubtful whether many variants will make much of an impact (except enalapril). Several other examples are given in deStevens (1990). Evaluating the commercial potential of new biotechnology products is an inexact science that is subject to many changes in the market as well as changes in product performance. Size of market (e.g., number of patients to be treated) is difficult to estimate. When a product is already in the market, it is easier to extrapolate its value more accurately. V. Outlook

The two parts of this series attempted to combine technical and business aspects of biotechnology in a unique way. As such, they represent only few steps in the overall commercialization pathway (Table XIX). From this table it is obvious that the other steps are under a low degree of control within a biotechnology company but cannot be neglected. The greatest impact on the ultimate success or failure of a product once it is past the initial product development effort hinges on its performance in the clinical setting and on the ability of the organization to produce the material at an acceptable cost. A hierarchy of commercial decisions is shown in Table XX. The following expands the last item in Table XIX (business impact of regulatory and commercialization pathway). For the first-generation biotechnology products, manufacturing costs and price were not of strategic importance because of lack of competition (as was the case of t-PA). As competition and production requirements increase in the second-generation products low production cost (and high capacity) is of prime importance. In this respect, the impact of biocatalyst and process selection is obvious (Bisbee, 1993).Selection between a bacterial, mammalian, or baculoviral (in insect cells) production host includes issues such as degree of expression, product accumulation, processing volumes, media requirements, complexity of separation/purification, contamination (viruses and oncogens), and native vs refolded product (protein) issue. The choices are difficult and complex. For example, media cost may be excessive for mammalian cell culture; on the other hand, for a bacterial system, the downstream process represents the majority of cost due to the expensive refolding step. The fidelity of product glycosylation may dictate a final decision. The preservation of biological activity and assurance of purity is a central issue in the GMP reinforcement. The present approach (FDA) is based on a case-by-case scenario as any validation activity depends on the nature of impurities, on their risk to patients, on a method of their removal,

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TABLE XIX BIOTECHNOLOGY PRODUCT COMMERCIALIZATION PATHWAY ~~

Issue Regulatory aspects Health, safety, and environment (FDA, USDA, EPA, environmental release of recombinant organisms, safety, and health) Industrial regulations [Good lab practice (GLP),good manufacturing practice-(GMP), quality control, validation, total quality (IS0 9000)] Legal aspects Patent law Trade secrets Copyright and trademarks Plant variety protection Ownership of tissues Corporate law and tort liability International legal instruments Joint ventures and licensing Social aspects National security concerns Antitrust law Technology transfer University-industry relationship International transfer and export control Public perception and ethical issues Public opinion and confidence in biotechnology Genetic screening and counseling Genetic therapy and organ implantation Animal rights Biological warfare Business impact of regulatory and commercialization pathway Impact of biocatalyst selection (product discovery) Impact of processing mode (reactor, recovery/purification) GMP reinforcement Clinical demonstration of product safety and efficacy Regulatory climate

Control Complete control over the selection process

Mostly under control; negotiable

Only some control; largely under a societal (public) scrutiny and control; sometimes negotiable

Mostly under control; business and management choices available; some societal and governmental constraints: regulatory and costcontainment climate

(continued)

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1

229

TABLE XIX (Continued) Issue

Control

Cost effectiveness Processlpurification cost Regulatory cost Cost-containmentclimate Total production management

and on validation of their absence (Sofer, 1994).Elaborate spiking experiments, using nucleic acids, viruses, and protein impurities, coupled with appropriate detection methods are set up to follow the “clearance factor.” Endotoxin removal presents a separate challenge. GMP and IS0 9000 standards serve as convenient vehicles for implementing a quality assurance system, although both do not provide automatic guarantees. A coupling of GMP with a clinical demonstration of product safety and efficacy is at the center of attention. For most biopharmaceuticals and biologics the bulk production cost and clinical performance results are affected by downstream (purification) processing. Further product development then depends on an expedited review by regulatory agencies. In spite of the FDA’s accelerated approval policy, changing standards and unexpected requests may delay product commercialization. The Orphan Drug Act is under review and will likely result in a significant decrease in the period of market exclusivity-from 7 to 4 years. The general consensus is that the U.S.governmental regulatory process is unnecessarily stringent in terms of seeking maximal safety (as compared to other industries and countries). Such regulatory slow-downs and uncertainty surrounding clinical and regulatory developments will result in lower industry success and in lower U.S. competitiveness (Burrill, 1994). Related to the above are the cost-cutting measures and the health care reform attempted by the present administration. The health care reform is focusing on capping costs for different elements TABLE XX

HIERARCHY OF COMMERCIAL DECISIONS Activity

Main output

Technology inputs Regulatory aspects Legal aspects Social inputs and checks Business inDuts

Efficacy Safety Novelty Acceptance (market) cost

TABLE XXI ?fIE CHANGING INDUSTRY MODEL

Vertically integrated companies

Virtually integrated companies

Change ~~

h)

0 W

~~

Impact ~

Product development

Inward focus on all aspects

Outward focus toward partners and resources

Ideas went to big companies for development; now, ideas originate everywhere and are shared

Accelerated development of technology; more efficient process; resources are shared to maximize intellectual capital

Technology

Closely guarded to maintain control and competitive edge

Technology sharing among partners is required for success

Intellectual capital is fluid; business culture must be built around people

Payoff is faster, product development enhanced; trust between partners is required

Manufacturing

Central to enterprise; growth focuses on adding manufacturing infrastructure across value chain

Greater flexibility in speed and capacity can be achieved; whoever is best, manufactures

Excess capacity is leveraged, wherever it resides; captive assets are irrelevant

Efficiency, flexibility, and and reduced capital requirements

Marketing and sales

Large sales forces calling on everbroadening customer base

Cooperative marketing; use the selling assets closed to the customer

Captive sales and marketing organizations could become liabilities

Quicker market penetration; lower-cost distribution

Management

Highly structured; hierarchical decision process

Fluid decision-making: collaborative process

Loss of top-down control; manager retraining may be required

Organizations are interdependent; managements are equal, less parochial

Communication

Largely closed and held within the organization

Open and spread throughout the enterprise

Transcedence of parochial culture; information and ideas are shared

Issues and problems are addressed in real time

Capital spending

Internally driven; capital needs met through financing or operations

Shared among partners; Capital assets housed in most strategic locations

Assets are obtained by leastcost means, by whichever partner is most appropriate

Lower costs; ability to access broader resources than otherwise

Organizational structure

Organizational structures are fixed; control is inward and downward

Organizations are fluid, changing in response to common needs

Each enterprise defines the appropriate structure and culture for accomplishing objectives

Participants are empowered; organization supports the shared focus

Note. Reprinted with permission from Burrill and Lee (1993). Fig. 11, p. 22. Copyright 1993. Ernst & Young.

232

ALES PROKOP

in the health care industry and as a result major (voluntary) changes are already occurring in the marketplace, including changes in pharmaceutical companies-and this is before any reform has been enacted [pharmacoeconomics, Thompson and Burrill, 1994). This is certainly a positive development. On the other side, there are concerns that unnecessary governmental interventions would inject further uncertainty into the financial, scientific, and technical communities, greatly hindering further development of biotech companies. Reduced capital flow would mean that many promising technologies will never make it to market (Whitman, 1994). On a more positive side, cost-cutting measures lead to streamlining the R&D process and create unexpected opportunities for the biotech industry (Burrill, 1994). As the economy will play a greater role in current and future activity, new business forms are being adapted, resulting in the emergence of a “virtually integrated” model of the biotech industry. The current concept of “vertically integrated” company features, within an independent entity, a full command over their R&D, manufacturing, marketing, and sales, including regulatory activity (Burrill, 1994). One single biotech company cannot afford to have all components in place anymore. Thus the expensive trial-and-error screening of new compounds may be eliminated and replaced by an acquisition/alliance with a new biotech company, having the expertise to do so. Fundamental restructuring trends are evidenced by “increased partnering between companies, increased acquistion or partnering activity with suppliers, customers, generic drug and diagnostic businesses, and biotech companies . . . More and more biotechnology companies are forming many varieties of mutually dependent strategic relationships. They are prepared to restructure arrangements and operating processes when circumstances change. By these means, companies are building long-term values without incurring onerous long-term risks and costs, and they are overcoming shortterm hurdles without risking long-term sustainability (reprinted with permission from Burrill, 1994; Copyright 1994 BioPharm). Tables XXI and XXII present basic features of a virtually integrated company as contrasted to a vertically integrated one. In conjunction with these changes in management, changes are apparent in the ways that financing is provided through new “creative” financing vehicles (see Fig. 31 of Burrill and Lee, 1993). to summarize the present business and commercial climate in the pharmaceutical and biotech industry, the following features are noteworthy: 1. High cost of product purification 2. GMP reinforcement 3. Clinical demonstration of safety/efficacy

CHALLENGES IN COMMERCIAL BIOTECHNOLOGY. I1 IALtLE

233

XXII

WHY VIRTUAL INTEGRATION? Pharmaceutical industry drivers

Biotechnology industry drivers

Need to deliver new products with real impact to a rapidly changing market

Need to access regulatory, marketing, distribution capabilities

Pricing and earnings pressures

Need to support net burn rates while companies develop their long-term value

Inefficient infrastructure

Lean infrastructure and efficient development process

Intellectual capital from academic institutions is “draining” to the biotech world

Talent-the core asset in a marketplace that places a premium on innovation

Need to become move adaptable

Need to prove that it’s here to stay

Note. (Reprinted with permission from Burrill and Lee (1993),Fig. 9, p. 19. Copyright 1993, Ernst8rYoung).

4. Cost-containment climate 5. Industry restructuring and new financing vehicles.

The most recent U.S. biotech business characteristics is depicted in the latest Ernst & Young report (Lee and Burrill, 1994). ACKNOWLEDGMENTS Most of this material was generated over several semesters in conjunction with the teaching of a graduate course on commercial aspects of biotechnology at Washington University, St. Louis. Besides my participation, many persons from the local industrial environment got involved. Among them, particularly Dr. Godfred E. Tong and Douglas S. Inloes (both at that time at Monsanto) contributed in a considerable manner. I express my gratitude to them for providing valuable materials and a regret that they were not available for writing these articles. Particularly, Figs. 1, 11, and 16 of Part I, Figs. 5-8, 12,14-16, 19,23,and 25 of Part 11, Tables VI, VII, and XXII of Part I, and Table V, VII-IX, XIII, and XVIII of Part I1 were adapted from their sources.

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Atkinson, B., and Mavituna, F. (1983).“Biochemical Engineering and Biotechnology Handbook.” Nature Press, New York. Bisbee, C. A. (1993).Current perspective on manufacturing and scaleup of biopharmaceuticals. Gene. Eng. News 13(4),8-9. Boeck, L. D., Wetzel, R. W., Burt, S. C., Huber, F. N., Fowler, G. L., and Alford, J. S., Jr. (1988).Sterilization of bioreactor medium on the basis of computer-calculated thermal input designated as F,. J. Ind. Microbiol. 3, 305-310. Bohn, H. (1992).Consider biofiltration for decontaminating gases. Chem. Eng. Prog. 88, 34-40.

Box, D. E. P., Hunter, W. G., and Hunter, J. S. (1978).“Statistics for Experimenters,” pp. 291-432. Wiley, New York. Buckland, B., Brix, T., Fastert, H., Gbewoyo, K., Hunt, G., and Jain, D. (1985).Fermentation exhaust has analysis using mass spectroscopy. BiolTechoIogy 3, 982-988. Burrill, G. S. (1994).Emerging trends: 1994 first quarter update. BioPharm 7(5),22-26. Burrill, G. S., and Lee, K. B., Jr. (1993).“Biotech 94.Long-term Value Short Term Hurdles. The Industry Annual Report.” Ernst & Young, San Francisco. Calam, C. T. (1987).“Process Development in Antibiotic Fermentations.” Cambridge University Press, Cambridge, UK. Clark, K. M., and Wheelwright, S. C. (1993).“Managing New Product and Process Development. Text and Cases,” pp. 294,301,and 306. Free Press, New York. Collier, D. W. (1975).The creative link between market and technology. CHEMTECH, February, pp. 90-93. destevens, G. (1990).Lead structure discovery and development. In “Comprehensive Medicinal Chemistry. The Rational Design, Mechanistic Study and Therapeutic Applications of Chemical Compounds” (C. Hansch, ed.), Vol. 1,pp. 261-278. Pergamon, Oxford. Elder, A. L. (1970).The history of penicillin production. AIChE Symp. Ser. 66(100), 97 PP. Florence, A. Y., and Halbert, G. W. (1990)Formulation. In “Comprehensive Medicinal Chemistry. The Rational Design, Mechanistic Study and Therapeutic Applications of Chemical Compounds” (C. Hansch, ed.), Vol. 5, pp. 593-613. Pergamon, Oxford. Glaser, V. (1992).Strong growth in biotechnology market sectors predicted for 1992-2002, Gen. Eng. News 12(3),6-7. Jarai, M. (1972).Oxygen transfer in the fermentations of primary and secondary metabolites. Ferment. Technol. Today, Proc. Int. Ferment. Symp., 4th, 1972,pp. 97-103. Jensen, A. L., Schultz, J. S., and Shu, P. (1966).Scale-up of antibiotic fermentations by control of oxygen utilization. Biotechnol. Bioeng. 8,525-537. Johnson, M. C. R., and Lewis, S. J. (1990).Routes of administration. In “Comprehensive Medicinal; Chemistry. The Rational Design, Mechanistic Study and Therapeutic Applications of Chemical Compounds” (C. Hansch, ed.), Vol. 5,pp. 593-613. Pergamon, Oxford. Kehoe, J. A. (1990).The study of biosynthetic human insulin. In “Frontiers in Bioprocessing” (S. K. Sikdar, M. Bier, and P. Todd, eds.), pp. 45-49. CRC Press, Boca Raton, FL. Lee, K. B., Jr., and Burrill, G. S . (1994).“Biotech 95: Reform, Restructure, Renewal. The Industry Annual Report.” Ernst & Young, San Francisco. Lewis, C., and Kristiansen, B. (1985).Chemicals manufacture in biotechnology-The prospects for western Europe. Chem. Ind. (London), (No. 171,pp. 571-576. Office of Technology Assessment (1991).“Biotechnology in a Global Economy,” OTABA-494,p. 9.US.Congress, Washington, DC.

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Oldshue, J. Y. (1983a). “Fluid Mixing Technology.” McGraw-Hill, New York. Oldshue, J. Y. (1983b). Mixing in fermentation process. In “Annual Reports on Fermentation Processes” (G. T. Tsao, ed.), Vol. 6, pp. 75-99. Academic Press, New York. Pacifico, C., and Polacek, R. (1981). “New Product Development. ACS Audio Course.” American Chemical Society, Washington, DC. Pacifico, C. R., and Witroer, D. B. (1981). “Practical Industrial Management.” Wiley, New York. Perlman, D. (1980). Some problems on the new horizonx of applied microbiology. Dev. Ind. Microbio. 21, xv-xxiii. Price, B. J. (1991). In the trenches: The marketing and selling of biotechnology. In “The Business of Biotechnology” (R. D. Ono, ed.), pp. 225-248. Butterworth-Heinemann, Boston. Prokop, A. (1982). Systems analysis and synthesis in biology and biotechnology. Int. 1. Gen. Syst. 8, 7-31. Prokop, A. (1991). Implications of cell biology in animal cell biotechnology. In “Animal Cell Bioreactors” (C. S. Ho and D. I. C. Wang, eds.), pp. 21-58. ButterworthHeinemann, Boston. Queener, S . W., and Swartz, R. W. (1979). Penicillins: Biosynthetic and semisynthetic. In “Economic Microbiology” (A. H. Rose, ed.), Vol. 3, pp. 35-122. Academic Press, New York. Reisman, H. B. (1988). “Economic Analysis of Fermentation Processes.” CRC Press, Boca Raton, FL. Reisman, H. B. (1993). Problems in scale-up of biotechnology production processes. CRC Crit. Rev. Biotechnol. 13(3), 195-253. Ryu, D. D. Y., and Hospodka, J. (1980). Quantitative physiology of Penicillium chrysogenum in penicillin fermentation. Biotechnol. Bioeng. 22, 289-298. Shamel, R. E. (1991). Biotechnology megatrends: Lessons from the past visions of the future. In “The Business of Biotechnology” (R. D. Ono, ed.), pp. 345-463. Butterworth-Heinemann, Boston. Shamel, R. E., and Keough, M. (1994). Trends in biopharmaceutical development and commercialization. Gene. Eng. News 14(1), 6, 8. Singh, V., Hensler, W., and Fuchs, R. (1989). Optimization of batch fermentor sterilization. Biotechnol. Bioeng. 33, 584-591. h e a d e r , W. (1985). “Drug Discovery. The Evolution of Modern Medicine.” Wiley, Chichester. Sofer, G. (1994). Biotechnology product validation, Part 4. Clearance of impurities from protein and peptide biotherapeutics. BioPharm 7(4), 46-50. Spilker, B. (1989a). “Multinational Drug Companies: Issues in Drug Discovery and Development,” pp. 10,18. Raven Press, New York. Spilker, B. (1989b). “Multinational Drug Companies: Issues in Drug Discovery and Development,” p. 185. Raven Press, New York. Stanburry, P. F., and Whitaker, A. (1984). “Principles of Fermentation Technology.” Pergamon, Oxford. Stark, W. M., Know, W. G., Wilgus, R., and Dubus, R. (1976). Nebramycin fermentation. Culture and fermentation development. Dev. Ind. Microbiol. 17, 61-77. Sweere, A. P. J., Luyben, K. C. A. M., and Kossen, N. W. F. (1987). Regime analysis and scale-down: Tools to investigate the performance of bioreactors. Enzyme Microb. Technol. 9, 386-398. Szoka, P. (1992). Instrumentation markets prosper with biotechnology product development. Gene. Eng. News 12(3), 8, 35.

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Thompson, K., and Bunill, G. S. (1994)Pharmacoeconomics and health care reform. BioPharm 7(1), 50-52. Tomlinson, E. (1990).Selective delivery and targeting of therapeutic proteins. In “Protein Production by Biotechnology” (T. J. R. Harris, ed.), pp. 207-225. Elsevier, London. Tong, G. E., and Inloes, D. S. (1990).Making more. CHEMTECH 20(9),556-573. Vagelos, P. R. (1991).Are prescription drug prices high? Science 252, 1080-1084. Vanek, Z., and Hostalek, Z., eds. (1986).“Overproduction of Microbial Metabolites.” Butterworth-Heinemann, Boston. Wheelwright, S. M. (1994).Commercializing biotech products. Bio/Technology 12, 877-880.

Whitman, C. T. (1994).Keep politics out of biomedical innovation. Gene. Eng. News 14(3),4-5.

Young, T. B. (1979).Fermentation scale up: Industrial experience with a total environmental approach. Ann. N. Y.Acad. Sci. 326,165-180.

Effects of Genetically Engineered Microorganisms on Microbial Populations and Processes in Natural Habitats JACK

D. DOYLE,* GUENTHER STOTZKY,+ GWENDOLYN MCCLUNG,* AND CHARLES W. HENDRICKS~ * ManTech Environmental Technology, lnc. Corvallis, Oregon 97333 f

Laboratory of Microbial Ecology, Department of Biology New York University New York, New York NY 10003 $

United States Environmental Protection Agency Washington, DC 20460

United States Environmental Protection Agency Environmental Research Laboratory-Corvallis Corvallis, Oregon 97333

I. Introduction 11. Aquatic Environments 111. Activated Sludge IV. Soil

V. Plants VI. Discussion References

I. Introduction

There has been a relatively limited number of studies on the survival of (e.g., Table I) and gene transfer by (e.g., Table 11) genetically engineered microorganisms (GEMs) in natural environments (e.g., Armstrong et a]., 1987, 1989; Bentjen et a]., 1989; Levy and Miller, 1989; Stotzky, 1992; Stotzky and Babich, 1984, 1986; Stotzky et a]., 1991; Trevors et al., 1987; Walter et al., 1987,1991;Wellington and van Elsas, 1992). Even fewer studies have been conducted on the effects on the environment of the introduction of GEMs (e.g., Table 111) (e.g., Casper and Landsmann, 1992; Fredrickson and Hagedorn, 1992; Fuxa, 1989; Levin et al., 1992; MacKenzie and Henry, 1990; Stotzky, 1990, 1991, 1992). Furthermore, few studies have been conducted on the effects of nonengineered microorganisms introduced to the environment [e.g., biocontrol agents (Flexner et al., 1986; Genthner and Middaugh, 1992; 237 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 40 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

TABLE I SURVIVAL OF GENE~CALLY ENGINEERED MICROORGANISMS boCULATED INTO VARIOUS HABRATS

CFUlg or ml Habitat Aquatic

Activated sludge Soil

Organism Erwinia carotovom L-864 (GEM) L-863 (parent] Pseudomonas sp. B13FRl(pFRC20P) Pseudomonas putida UWCl Pseudomonas sp. B13FRl(pFRC20P) Pseudomonas putida KT2440(pWWO-EB62) Streptomyces lividans Spores Mycelium Erwinia carotovora L-864 (GEM) L-863 (parent) Escherichia coli (various strains with and without plasmids) Pseudomonas putida PP0301(pRO103) (GEM] PP0301 (parent)

Initial

Final

Duration (days)

103-109

10' 10'

32

105-106

28

lo6- 1o8 104-105

56 6

Reference Scanferlato et al. 1989)

103-109 107

106-107 106-107

104-105 104-105

32

6

106-1 08

104-107 102-105

33 33

1o8

104

108 105

104 102-103

55 55

107 107-108

105 105

105-108

Wagner-DObler et al. (1992); Pipke et aJ. (1992) McClure et al. (1991a) Niisslein et ol. (1992) Nusslein et al. (1992) Wang et al. (1989)

Orvos et al. (1990) 27

Devanas and Stotzky (1988) Doyle et al. (1991)

52 52

GENETICALLY ENGINEERED MICROORGANISMS

239

TABLE I1 EXAMPLES OF GENETRANSFER BY GENETICALLY ENGINEERED MICROORGANISMS IN SOIL‘ Frequency Conjugation Chromosomal genes: to Plasmid-borne genes: to Transduction: to lo-’ Transformation: to Transfer of chromosomal genes by conjugation Enhanced by montmorillonite but not by kaolinite Greatest at neutral pH Plasmid transfer by conjugation Enhanced by montmorillonite, nutrients, and optimal water content Intra- and intergeneric with broad host-range plasmid Narrow host-range plasmid transferred only in sterile soil Affected by residence time in soil (e.g., donor E. coli less functional than recipient P. aeruginosa) Transduction Unaffected by clay minerals Enhanced somewhat by nutrients Transduction frequencies higher with phage lysates than with lysogenic bacteria Inactivation of transducing phages similar to that of other viruses Survival of transducing phages greater in presence of montmorillonite than of kaolinite Transformation Higher in sterile than in nonsterile soil Adsorption and binding of DNA on clay minerals and other particles is pH-dependent (greater below PI of DNA] Transformation reduced by presence of clay minerals and other particles DNA bound on clay and other particles is protected against degradation by DNase and retains its transforming ability DNA bound on clays can be amplified by the polymerase chain reaction DNA binds primarily on edges of clay and does not intercalate clay General comments Reduced by presence of indigenous microbiota No transfer of novel genes to indigenous bacteria detected (others have reported such transfer) Not clear whether gene transfer occurs continuously or only after introduction of donor and recipient Possibility of the persistence of “cryptic genes’’ (especially with transduction and transformation] Compiled from Devanas and Stotzky (1986, 1988), Devanas et al. (1986), Gallori et al. (1994), Khanna and Stotzky (1992),Krasovskyand Stotzky (1987).Leeand Stotzky (19901,Lorenz and Wackernagel (19941,Stotzky (1989,1992). Stotzky and Krasovsky (1981).Weinberg and Stotzky (1972).Zeph and Stotzky (1989), and Zeph et 01. (1988, 1991).

TABLE 111 SOME EFFECTS OF &“ICALLY

Habitat Aquatic

ENGINEERED MICROORGANISMS ON MICROBIAL POPULATIONS AND PROCESSES IN NATURALHABITATS

Organism Alcaligenes sp. BR60

Pseudomonas sp. Activated sludge

N $P

0

Soil

Pseudomonas putida UWCl Pseudomonas sp. BlJFRl(pFRC2OP) Pseudomonas putida KT2440(pWWO-EB62) Pseudomonas aeruginosa Pseudomonas putida (PBS~) Streptomyces lividans (TK23-3651(pSE5) Pseudomonas sp. RCl

Pseudomonas putida PP0301(pR0103)

Effects and comments

Reference

Degraded 3-chlorobenzoate, 4-chloroaniline, 3chlorobiphenyl, and 2,4-dichlorophenoxyacetate; transfer of pBRC6O to indigenous bacteria was observed Degraded 3-chlorobenzoate, 4-chlorobenzoate, and 4methylbenzaate via the ortho-cleavage pathway

Fulthorpe and Wyndham (1989, 1991, 1992)

Degraded 3-chlarobenzoate; indigenous population was more effective in degrading 3-chlorobenzoate Degraded 3-chlorobenzoate and 4-methylbenzoate

Strains containing appropriate plasrnid-borne genes Degraded 3-chlorobenzoate; transfer of degradative genes to indigenous Pseudomonas sp. was observed Transiently increased CO, evolution when lignocellulose was added (pSE5 codes for enhanced production of extracellular lignin peroxidase and H,O,) Mutagenesis with Tn5 resulted in a reduction in the colonization of wheat rhizoplane by indigenous fluorescent pseudomonads, which apparently suppress take-all disease of wheat by the fungus, Gaumannomyces gmminis var. triciti Degraded 2,4-dichlorophenaxyacetate(2,4-D) to 2chloromaleylacetate; the accumulation of the first degradative product, 2,4-dichlorophenol (2,4-DCP), reduced CO, evolution for 35 days and decreased the

Pipke et al. (1992); Wagner-Dobler et al. (1992) McClure et al. (1991a) Niisslein et al. (1992)

Pertsova et al. (1984) Wang et aJ. (1989)

Fredrickson et al. (1989)

Doyle et al. (1991); Short et al. (1991)

Pseudomonas cepacia ACllOO Bmdyrhizobium japonicum

Ahizobium meliloti RM 6963 Bradyrhizobium sp. IC3554(pSM4) Bacillus thuringinesis

Plants

Pseudomonas cepacia (R388::Tnl721), Enterobacter cloaceae

number of fungal propagules to undetectable levels after 10 days; dehydrogenase activity was enhanced after most of the 2,4-D was converted to 2,4-DCP; the diversity of other groups of the microbiota and the activity of other enzymes studied were affected only transiently caused changes Degraded 2,4,5-trichlorophenoxyacetate; in the taxonomic diversity of the indigenous microbiota, possibly as the result of the metabolic activity of the genetically engineered bacterium Roots of soybean were nodulated less by nonmotile mutants (generated by mutagenesis with Tn7) than by the motile wild-type;no changes were observed in other physiological responses studied Mutagenesis with Tn5 resulted in a transient decrease in fixation of N,, smaller plants of alfalfa, and some changes in the morphology and physiology of the bacterium Insertion of the genes from Bacillus thuringiensis subsp. ismelensis that code for the production of antidipteran toxins partially protected the nodules of pigeon pea from being eaten by R. angulata Insecticidal toxins bind on clay minerals, which reduces the susceptibility of the toxins to microbial degradation but does not eliminate their insecticidal activity

Spraying of these bacteria onto leaves of bean and radish plants grown in microcosms containing cutworms (Peridroma saucia) resulted in no detectable impacts

Bej et al. (1991)

Liu et al. (1989)

Lagares et al. (1992)

Nambiar et al. (1990)

Venkateswerlu and Stotzky (1990, 1992); Tapp et al. (1994);Tapp and Stotzky (1994); Koskella and Stotzky (1994) Armstrong et al. (1989); Armstrong (1992) (continued )

TABLE III (Continued) Habitat

Organism

Effects and comments ~ _ _ _ _ _ _

~~

(pBR322), Klebsiella planticola (pBR322) , Elwinia herbicola (pBR322) Pseudomonas syringae (iceminus)

_____

Reference

___

on the variables examined; transfer of the plasmids in the insect was indicated No detectable effect of this genetically engineered bacterium, in which the genes for ice nucleation were deleted, on the areas surrounding the spray zones of strawberries and potato; only the geographic distribution of the organism was studied

Lindow and Panopoulus (1988);Seidler and Hern (1988); Stetzenbach et al. (1992); Lindow et al.

Both genetically engineered bacteria (one that chromosomally encoded resistance to erythromycin and one that contained a plasmid-borne cellulase gene from Clostridiumthermocellum)and the parental strain equally reduced the pH and became the dominant microbiota in silos containing primarily ryegrass (Lolium perrene)

Sharp et al. (1992)

(1992)

hctobacillus plantarum NO01193

GENETICALLY ENGINEERED MICROORGANISMS

243

Gilbert et al., 1993; James et al., 1993; Laird et al., 1990; Linderman et al., 1991; Snarski, 1990), rhizobia for dinitrogen fixation (Thies et al., 1991,1992),pure cultures and consortia for the degradation of xenobiotics and other aspects of bioremediation, coal scrubbing, biomass conversion, fuel production, ore mining, mineral leaching, oil recovery, and waste treatment], even though the introduction of nonengineered microorganisms to do a specific job has a long history. This is surprising, as a fundamental concern in the release to the environment of microorganisms, whether genetically altered or not, should be the potential ecological effects of these releases (i.e., effects other than those for which the microorganisms were released). A number of questions need to be addressed when assessing the effects of releasing of GEMs to the environment (e.g., Table IV). However, even if an introduced microorganism survives and/or transfers its genetic information to indigenous or other introduced microorganisms, there should be little cause for concern unless the expression of the novel genes causes an unacceptable change in the environment (Stotzky et al., 1991). The introduction of foreign genes to “wild-type” microorganisms, via traditional or recombinant techniques, results not only in alterations of the genotype of the organisms, but also can produce unanticipated changes in the phenotypic expression of the recipient microorganisms, which, in turn, could result in unanticipated environmental impacts (e.g., Austin et al., 1990; Bentley et al., 1990; Doyle et al., 1991; Kozyrovskaya et al., 1984; Lagares et al., 1992; Morisaki et al., 1992; Nambiar et al., 1990; Shoham et al., 1991; Short et al., 1991; Tapp et al., 1994; Venkateswerlu and Stotzky, 1992; Williamson and Hartel, 1991). The effects that GEMs may have on the structure and function of an ecosystem could also be affected by their mode of introduction, their spatial and temporal distribution, and the physicochemical and biological characteristics of the environment to which they are released (Table V) (e.g., Doyle, 1993; Doyle and Hendricks, 1993; Seidler and Hern, 1988; Stotzky, 1989, 1990, 1991, 1992; Stotzky et al., 1991, 1993; Trevors et al., 1990). Among the reasons why the potential ecological impacts of introduced organisms have not been studied more extensively are: the inability to predict how potential impacts will be expressed; the lack of ecological understanding needed to decide at what environmental level (e.g., micro- vs macroecology) the effects should be sought; the scarcity of information on which techniques are most appropriate for detecting impacts in different circumstances (e.g., Pickup et al., 1991; Smit et al., 1992; Stotzky et al., 1993); and the question of how the results should be interpreted (e.g., Babich et al., 1983; Stotzky et al., 1993) (Tables VI

244

JACK D. DOYLE ET AL. TABLE IV IN ASSESSING THE RISKS INVOLVED QUESTIONS THATMUSTBE ANSWERED IN RELEASINGGENETICALLY ENGINEERED MICROORGANISMS (GEMs) TO THE ENVIRONMENT‘

Survival of the GEMs Establishment (colonization) Multiplication Dispersal of the GEMs to other sites and environments Transfer of the novel genetic material to indigenous microbes Survival Establishment Multiplication Potential ecological and health impacts of the novel genetic material Probability of containment, decontamination, and mitigation Compiled from Stotzky (1989,1990,1991,1992).

and VII). A brief review and evaluation of the few studies that have been conducted on the ecological effects of the release of GEMs to aquatic and terrestrial environments are presented, to illustrate the current, and inadequate, state of knowledge on the potential impacts of GEMs on the environment (e.g.,aquatic, activated sludge, soil, and plants). TABLE V ECOLOGY. FACTORS THATAFFECTTHE ACTIVITY, POPULATION DYNAMICS OF MICROORGANISMS IN NATURAL HABITATS’

AND

Carbon and energy sources Mineral nutrients Growth factors Ionic composition Available water Temperature Pressure Atmospheric composition Electromagnetic radiation PH Oxidation-reduction potential Surfaces Spatial relationships Genetics of the microorganisms Interactions between microorganisms ~~

“Compiled from Stotzky (1974)and Stotzky et al. (1991).

GENETICALLY ENGINEERED MICROORGANISMS

245

TABLE VI POSSIBLE REASONSWHYPOTENTIAL ECOLOGICAL IMPACTSOF INTRODUCED MICROORGANISMS MOREEXTENSIVELY HAVENOTBEENSTUDIED Inability to predict how potential impacts will be expressed. At what environmental level should ecological impacts be sought (micro-vs. macroecology)? What techniques are most appropriate to detect impacts? How should results be interpreted (e.g., statistical vs ecological significance)?

II. Aquatic Environments

Scanferlato et al. (1989) studied the survival of a genetically engineered Erwinia carotovora subsp. carotovora L-864 and the effects of this GEM on the numbers of total, proteolytic, pectolytic, and amylolytic bacteria in the water column and sediments of bubble-aerated, freshwater-sediment microcosms (glass jars containing 300 ml of sediment and 550 ml of water incubated at 20°C with a 12-hr light/dark cycle). The GEM was constructed from E. carotovora L-543 (wild-type) by mutational deletion of a pectate lyase gene from a plasmid, insertion of a 1.4-kilobase (kb) DNA fragment from Tn903 encoding kanamycin resistance into the plasmid, and insertion of part of this engineered plasmid in a cis relation to the wild-type pectate lyase gene in the chromosome. Spontaneous rifampin-resistant mutants of E. carotovora L-833 (the engineered L-543, designated L-864) and L-543 (the wildtype control, designated L-863) were used in the microcosm studies. The numbers of the GEM and control strains declined at the same rate in both the water column and the sediment, and they were below TABLE VII DESIRABLE CHARACTERISTICS OF METHODSFOR ASSESSING ECOLOGICALEFFECTSOF GENETICALLY ENGINEERED MICROORGANISMS' Relevance Representative of community Sensitivity Reproducibility Ease (facility, rapidity) Cost effectiveness Interlaboratory validation Predictiveness (transferability, modeling) Ecological vs statistical significance From Stotzky et al. (1993).

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detection (about 10' CFU/ml) 32 days after inoculation, regardless of the numbers inoculated (lo3,lo6,or lo9CFU/ml).Although the numbers of total, proteolytic, and pectolytic bacteria were higher in microcosms inoculated with log CFU/ml of the GEM or the wild-type strain than in uninoculated microcosms, there were no statistically significant differences between the effects of these strains. The numbers of indigenous bacteria decreased with time in parallel with the decrease in the numbers of inoculated organisms. Inoculation of the microcosms did not affect the numbers of amylolytic bacteria, as neither the GEM nor the wild-type strain was apparently able to degrade starch. The results of these studies indicated that the genetically engineered strain of E. carotovora had no effect on the indigenous bacterial populations investigated. However, a strain of E. carotovora, engineered to produce a 30% reduction in the weight of macerated plant tissue as compared with the wild-type strain, persisted significantly longer (35 days) in thermally perturbed microcosms than in nonperturbed microcosms (5 days) (Scanferlato et al., 1990). The GEM, constructed from E. carotovora subsp. carotovora EC14 (Roberts et al., 1986) as described by Scanferlato et al. (1989) for E. carotovora L-543, persisted longer in the perturbed microcosms apparently as a result of decreased pressure from competitors and predators and increased nutrient availability. After 10 days of incubation and for the remainder of the study, the perturbed microcosms inoculated with the GEM had greater numbers of total and cellulolytic bacteria than the unperturbed microcosms inoculated with the GEM. Competitive exclusion by the GEM was postulated as a reason for the failure of the indigenous community to recover in unperturbed microcosms inoculated with the GEM. Although the numbers of total and cellulolytic bacteria in the uninoculated, perturbed microcosms were initially low, they increased after 10 days to levels similar to those in perturbed microcosms inoculated with the GEM. The activity of the electron transport system (ETS) [measured by the reduction of 2-( p-iodophenyl)-3-( p-nitrophenyl)-5-phenyl tetrazolium chloride] was also initially lower in the uninoculated perturbed microcosms than in the perturbed microcosms inoculated with the GEM. The levels of ETS activity in perturbed microcosms inoculated with the GEM decreased, in concert with the numbers of the GEM, to the level of ETS activity in the uninoculated, perturbed microcosms. Consequently, the presence of the GEM appeared to be the sole factor responsible for the changes in community dynamics and processes in the thermally disturbed microcosms. Furthermore, the effects of the GEM appeared to be influenced by the environment into which it was intro-

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duced, as differential effects were observed between perturbed and unperturbed microcosms inoculated with the GEM. The results of this study support the conclusions of others (e.g., Babich and Stotzky, 1983; Babich et al., 1983; Doyle and Hendricks, 1993; Doyle and Stotzky, 1993; Doyle et al., 1991; Gustafsson and Jansson, 1993; Kawar and Sherlock; 1989; Short et al., 1991; Stotzky, 1989, 1990, 1991, 1992; Stotzky et al., 1993; U.S. General Accounting Office, 1988) that the assessment of the potential ecological effects of GEMS released to the environment should be conducted on a case-by-case basis. Wagner-Dobler et al. (1992) studied the effect of Pseudomonas sp. B13FRl(pFRC20P), genetically engineered to degrade mixtures of chloro- and methylaromatics through an ortho-cleavage pathway, on a variety of environmental parameters in microcosms containing water and sediment from the Rhine River. The GEM was constructed from Pseudomonas sp. strain B13 by expanding its ortho-pathway for the degradation of 3-chlorobenzoate (3BC) to include enzymes encoded by the TOL plasmid (i.e., toluate dehydrogenase and dihydrocyclohexadiene carboxylate dehydrogenase). This modification enabled the recombinant strain, B13FR1, to metabolize 4-chlorobenzoate (4CB) and to transform 4-methylbenzoate (4MB), via methylcatechol, to 4-methyl-2-enelactone. The gene encoding the enzyme, 4-methyl-2enelactone isomerase, was recruited from Alcaligenes eutrophus JMP134 and enabled the conversion of 4-methyl-2-enelactone to 3methyl-2-enelactone, thereby allowing the complete degradation of 4MB (Pipke et al., 1992). When the GEM was added to the water phase of the microcosm at 5 x lo6 CFU/ml, with or without a mixture of 3CB and 4MB (25 pM each), it survived in the surficial sediment (top 5 mm) at densities of 5 x lo4 to 5 x lo5 CFU/g of sediment (dry weight) for 4 weeks and degraded the aromatics. The GEM did not signficantly affect the oxygen flux in the water, photosynthesis (measured by the content of chlorophyll a), the rate of incorporation of 3H-thymidine,or the community structure of heterotrophic bacteria (total, starch- and cellulose-utilizers, enterobacteria, pseudomonads and fluorescent bacteria, and nitrate reducers) in the sediment. Similar results were obtained when these microcosms were used to study the degradation of a mixture of 3CB and 4MB (25 p M each) by the GEM in sediments and water derived from an unpolluted lake (Lake Plussee) and the Rhine River (Pipke et al., 1992). The GEM survived for 26 days in both sediments at densities 5 x lo4 to 5 x lo5 CFU/g of sediment (dry weight). The removal of 3CB, but not of 4MB, from the water column and the degradation of both 3CB and 4MB in the sediments was enhanced, indicating that the GEM expressed the engineered

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catabolic pathway in situ. The addition of neither the GEM nor the chlorobenzoates affected the total number of bacteria detected in the sediments. The degradation of 3CB, 4-chloroaniline (PCA), 3-chlorobiphenyl (3CBP), and 2,4-dichlorophenoxyacetate(2,4-D) by Alcaligenes sp. strain BR60 was studied in sediment and flowthrough microcosms containing lake water (Fulthorpe and Wyndham, 1989,1991,1992).Alcaligenes sp. BR60 contains genes for the catabolism of 3CB in a 17-kb transposon (Tn5271) integrated into an 85-kb broad host-range plasmid, pBRC6O. Populations of bacteria carrying Tn5271 (determined by a Most-Probable-Number-DNAhybridization method; Frederickson et al., 1988) were significantly higher in microcosms treated with 3CB, PCA, and 3CBP than in the untreated microcosms, indicating that these chemicals exerted a selection pressure for this genotype. The rates of uptake of 14C-PCAand the subsequent evolution of 14C0, were correlated with the size of the population carrying Tn5271. Transfer of pBRC6O from Alcaligenes sp. BR60 to indigenous bacteria occurred in microcosms treated with 3CB and 3CBP. Three phenotypic clusters of chlorobenzoate-degraders were enriched, but only one of these clusters was closely related to Alcaligenes sp. BR60. No selective effect on populations containing pBRC6O or Tn5271 was observed in the presence of 2,4-D. However, the indigenous recipients of the novel genes had a lower affinity for 3CB and/or greater losses of the 3CB+ phenotype than did the GEM. These studies demonstrated that horizontal gene transfer from an introduced GEM to the indigenous microbiota can occur during the adaptation of bacteria to some chlorinated aromatic compounds in a freshwater microcosm. Ill. Activated Sludge

Pseudomonas putida UWCl(pDlO), engineered to degrade 3CB and introduced at 4 x lo6 to 3 x lo8 CFU/ml, survived for more than 8 weeks at lo4 to lo5 CFU/ml in laboratory-scale, activated-sludge units without 3CB, and plasmid pDlO remained stable during the 8-week incubation (McClure et al., 1991a,b). However, the addition of 3CB to the units reduced the survival of the GEM, presumably by enriching for indigenous microorganisms capable of degrading 3CB, and the GEM did not enhance the degradation of 3CB. The introduction of 7 x lo7 CFU/ml of P. putida ASR2.8, a transconjugant derived from activatedsludge and which contained the recombinant plasmid, pD10, to the activated-sludge units resulted in the degradation of 32% of the added 3CB, suggesting that the genetic engineering of isolates derived from the site of intended release is more likely to produce strains that are

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capable of performing the desired functions in that environment. Neither of these GEMs appeared to affect other biological components in the units, as the units consistently produced high-quality effluent with low biochemical oxygen demand and low content of suspended solids. Moreover, the numbers of total (10' to lolo celldml, measured by epifluorescence) and heterotrophic bacteria ( lo7 to 10' CFU/ml, measured by dilution plating) remained relatively constant, and the numbers of protozoa, primarily peritrichs and crawling ciliates, remained high (lo5 protozoa/ml), indicating that the activated-sludge systems were functioning properly. The numbers of Pseudomonas sp. B13FRl(pFRC20P) and P. putida KT2440(pWWO-EB62) added at concentrations of lo6 to lo7 CFU/ml to activated-sludge microcosms declined to stable levels of lo4 to lo5 CFU/ml within 6 days (Niisslein et al., 1992). B13FRl(pFRC20P) degraded combinations of 3CB and 4MB (1mM each) after 3 days in the microcosms, whereas the indigenous microbiota required 8 days before 4MB was degraded, and 3CB was degraded only after the concentration of 4MB had been reduced. The numbers of indigenous heterotrophic bacteria declined (from i07-i08 to
Orvos et al. (1990) obtained results in soil inoculated with E. carotovora subsp. carotovora L-864 or the wild-type strain L-863 that were similar to the results observed by Scanferlato et al. (1990) with the same

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strains in an aquatic system. The survival of the GEM, L-864, was the same as that of the wild-type strain, L-863, during a %-day incubation in a nonsterile loam soil (Orvos et al., 1990).The numbers of both the GEM and the wild-type strain decreased from approximately 10' to lo4 CFU/g of soil, but the GEM decreased more rapidly than the wildtype. The numbers of total bacteria, pseudomonads, actinomycetes, and salt-tolerant bacteria (including Staphylococcus spp.) remained essentially constant, after an initial decrease of about one order of magnitude, in soil inoculated with either the GEM or the wild-type, indicating that neither of these strains had any lasting effect on the indigenous microbiota. Strains of wild-type (Streptomyces viridosporus T7A), mutant ( S . viridosporus T7A-81, obtained by ultraviolet irradiation of T7A and having an enhanced lignin-solubilizing ability), and recombinant species of Streptomyces [Streptomyces Iividans TK150(pIJ702), TK23(pSP10.5),TK23(pSEl), and TK23-3651(pSE5)]survived in a silt loam soil for 33 days (the duration of the study), whether introduced as spores or mycelia (Wang et a]., 1989). The survival of spores (inoculated at lo5 to 10" CFU/g of nonsterile soil and declined to lo4 to 10' CFU/g) was greater than that of mycelia (inoculated at lo6 to 10' CFU/g of nonsterile soil and declined to 10' to lo5CFU/g),and survival of both types of propagules was greater in sterile than in nonsterile soil. When lignocellulose was added to the soil, only the recombinant species, S . lividans TK23-3651(pSE5)and TK23(pSEl), had a statistically significant, albeit short-term, effect on the rate of carbon mineralization (as measured by the evolution of CO,), Neither the wild-type, the mutant, nor the other recombinant strains affected the rate of CO, evolution from the soil. Streptomyces lividans TK23-3651(pSE5),containing plasmid-borne genes coding for the enhanced production of extracellular lignin peroxidase (3- to 4-fold) and H,O, (20-fold), produced a significant, but transient (about 1 2 days), increase in CO, evolution from soil amended with lignocellulose, as well as from soil not amended with lignocellulose. The total amount of CO, evolved over 36 days was significantly greater from soil inoculated with the GEM than from the control soil or from soil inoculated with the wild-type, mutant, or other recombinant strains. Inasmuch as the GEM did not increase CO, evolution from unamended sterile soil, the enhanced evolution of CO, was thought to be the result of the indigenous microbiota mineralizing lignin-associated carbon, in both the amended and the unamended soil, that was made more available by the enzymes produced by the GEM. However, the initial enhanced production of lignin peroxidase and H,O,, which ap-

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parently resulted in the transient increase in the evolution of CO,, may have been the result of the protoplasting/regeneration procedures used to construct this GEM. Consequently, the apparent effect of this GEM on the enhanced degradation of soil organic matter could not be definitively attributed to the novel genes. Streptomyces lividans TK23(pSEl), which contained a 5.5-kb insert of DNA (that presumably codes for an endoglucanase) from the chromosome of S. viridosporus in plasmid pSPl0 (Eaton, 1988), significantly, but temporarily, reduced CO, evolution for 1 2 days after inoculation into unamended soil. This reduction in the metabolic activity of the indigenous microbiota in the presence of the GEM was postulated to be the result of the production of a transitory metabolic inhibitor by the GEM. Inoculation of nonsterile soil with another recombinant species, S . lividans TK23.l(pIJ702.LP) [engineered to produce lignin peroxidase constitutively at an enhanced rate by the transformation of plasmid pIJ702.LP into S. lividans TK23 (Wang et a]., 1990)], resulted in a statistically significant, albeit short-term, increase in CO, production compared with soil inoculated with the wild-type strain, TK23 (Wang et al., 1991). However, no statistically significant difference was observed in the rate of CO, evolution from sterile soil inoculated with either TK23.l(pIJ702.LP) or TK23. Consequently, the enhanced rate of CO, production in nonsterile soil inoculated with the GEM probably resulted from synergistic activities between the GEM and the indigenous microbiota. The enhanced degradation of native lignin (no exogenous lignocellulose was added in this study), as the result of lignin peroxidase produced constitutively by the GEM, presumably increased the availability of lignified organics to the indigenous microbiota. Inasmuch as TK23.l(pIJ702.LP) survived in nonsterile soil at lo5 to lo7 CFU/g of soil for 30 days and plasmid pIJ702.LP remained stable in the GEM for at least 29 days, the introduction of this or similar GEMS might affect the cycling of carbon in soil. Consequently, the effects of TK23.l(pIJ702.LP) on humification, the cycling of carbon, nitrogen, phosphorous, and sulfur, soil enzyme activities, and functional groups of microorganisms in soil were studied (Crawford et al., 1993). Two different methods, in two different laboratories (for interlaboratory validation; e.g., Table VII), were used to measure CO, evolution from the same batch of soil: (1)“respiration cabinets” (Wang et al., 1989), into which were placed 250-ml flasks containing 5 g of soil inoculated with TK23 or TK23.l(pIJ702.LP)or not inoculated and amended with lignocellulose labeled as either l4 C-lignin or l4 C-cellulose or not amended (Department of Bacteriology and Biochemistry, University of Idaho,

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Moscow, ID); and (2) “master” jars (Stotzky, 1965; Stotzky et al., 1993), into which were placed vials, each containing 50 g of soil inoculated with TK23 or TK23.1(pIJ702.LP)or not inoculated and amended with unlabeled lignocellulose or not amended (U.S. Environmental Protection Agency, Corvallis Environmental Research Laboratory, Corvallis, OR). Both methods used potentiometric titration for the detection of evolved CO, after trapping in NaOH. In studies conducted in the respiration cabinets, significantly more CO, was evolved during the first 3 days of the incubation from the soil amended with lignocellulose and inoculated with the GEM than from the amended soil inoculated with the parental strain. A similar increase in CO, evolution was observed in the unamended soil inoculated with the GEM compared to the parent strain. In studies conducted in the master jars, there were no statistically significant differences in the rates of CO, evolution between the soil inoculated with the GEM, the parent, or not inoculated, regardless of whether or not the soil was amended with lignocellulose. The studies comparing the production of CO, in respiration cabinets and master jars were repeated several times, and similar results were obtained with each replicated experiment. Consequently, different methods (e.g., respiration cabinets versus master jars) can produce different results when determining the effects of GEMs on carbon mineralization (as measured by CO, evolution) in soil. This indicates that when comparing results from different laboratories, the methodologies used for assessing the potential ecological effects of GEMs should be considered in the evaluation of the results. In addition to affecting the rate of carbon mineralization in soil (as measured by CO, evolution in the respiration-cabinet method), S. lividans TK23.l(pIJ702.LP)also transiently affected the incorporation of lignocellulose-derived carbon into humus fractions and the numbers of cellulose-utilizing bacteria. The amount of 14Cincorporated into humic acids was greater in soil amended with 14C-lignin-lignocellulose and inoculated with the GEM than in soil inoculated with the parental strain after 30 days but not after 60 days of incubation. In contrast, the incorporation of 14C from ‘‘C-lignin-lignocellulose into fulvic acids was greater after both 30 and 60 days of incubation in soil inoculated with the GEM than with the parental strain. In soil amended with 14Ccellulose-lignocellulose, the incorporation of 14Cinto the humic- and fulvic-acid fractions was significantly greater in soil inoculated with the GEM than with the parental strain after 30 days, but these differences were not detectable after 60 days of incubation. The numbers of cellulose-utilizing bacteria were significantly greater in unamended soil inoculated with the GEM than with the parental strain on Days 7 and 14

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of the incubation. No significant differences in the numbers of celluloseutilizing bacteria were observed in soil amended with lignocellulose, regardless of the type of inoculum and whether inoculated or not. The incorporation of lignocellulose-carbon into soil humus and the increase in the numbers of cellulose-utilizing bacteria in the presence of the GEM support the hypothesis that the enhanced production of lignin peroxidase by the GEM increased the availability of lignocellulose to the indigenous microbiota. These studies also show that without the various tests used, the effects of S. lividans TK23.1(pIJ702.LP)on carbon cycling and on the indigenous microbiota in soil would not have been detected, further emphasizing the importance of using a battery of tests to assess the potential ecological effects of GEMs that are to be introduced to the environment (e.g., Doyle and Stotzky, 1993; Doyle et al., 1991; Short et al., 1991; Stotzky et al., 1993). As the GEM S. lividans TK23.l(pIJ702.LP)is a constitutive overproducer of lignin peroxidase, it could also have unanticipated effects on the microbial ecology of soil other than that predicted, i.e., the degradation of lignin. Lignin peroxidase, as well as other peroxidases, has been shown to be involved in the degradation of a number of polycyclic compounds, including several of environmental concern (e.g., Aitken et al., 1989; Fernando et al., 1990; Harvey and Palmer, 1990; Lamar et al., 1990; Ramachandra et al., 1988). Moreover, the activity of lignin peroxidase is generally assayed by monitoring the oxidation of 2,4-dichlorophenol, a secondary metabolite in the degradation of the herbicide 2,4-D (Adhi et al., 1989; Don and Pemberton, 1985; Foster and McKercher, 1973; Perkins et al., 1987; Ramachandra et al., 1988). Consequently, although the lignin peroxidase produced by TK23.l(pIJ702.LP)degrades lignin, it may also degrade priority pollutants, possibly resulting in metabolites that could cause undesirable impacts on the environment (e.g., Doyle et al., 1991; Lamar et al., 1990; Short et al., 1991). As metabolites produced by GEMs may have an effect(s) beyond that for which the GEMs were constructed, they need to be examined for their potential to affect adversely the environment to which the GEMs are to be introduced. The effects of genetically engineered strains of Pseudomonas solanacearum on the numbers of protozoa in soil were studied by Austin et al. (1990). Five strains of bacteria were evaluated: (1)a Pseudomonas sp. routinely used in vitro as a food source for protozoa; (2) a mucoid variant of the P. solanacearum wild-type strain, designated AW; (3) a nonmucoid variant of AW, designated AR; (4) a strain of AW containing Tn5, designated PS-6; and (5) a strain of AW that harbored a recombinant

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plasmid containing a cosmid vector, pLAFR3 (20 kb), which incorporated a 30-kb fragment from P. solanacearum that contained a gene encoding for /3-1,4-endoglucanase,designated AW(pHE-3) (Roberts et al., 1988). When the Pseudomonas sp. or the P. solanacearum variant AR was added, the numbers of all the types of protozoa evaluated increased from approximately lo2to 104/gof soil during the 5-day study. When either strain AW or PS-6 was added, the numbers of flagellated protozoa increased to approximately 105/gof soil, whereas the numbers of ciliates and amoebae remained at the level present in uninoculated soil (about 102/gof soil). The addition of strain AW(pHE-3) resulted in a significant decrease in the numbers of flagellated protozoa after 2 days compared with the numbers of flagellated protozoa in soil inoculated with the parental strain, AW. These results showed that the numbers of the different protozoa evaluated were affected by differences in the genotype of their food souce and that if a GEM, AW (pHE-3), is not consumed by predators, it may persist longer in soil than the wild-type strain. Consequently, these results demonstrate that: (1)changes in the genetic composition of bacteria can result in phenotypic changes that may influence the community structure of higher organisms (e.g., Bamforth, 1988; Elliott et al., 1984; Foissner, 1994; Hunt et al., 1984; 1987; Ingham et al., 1986a; Moore and de Ruiter, 1991; Pussard et al., 1994; Zwart et a]., 1994),emphasizing that the effects of GEMs on ecosystems need to be assessed on numerous trophic levels in the environments into which they may be released; and (2) the persistence of GEMs in soil may depend, in part, on their palatability as prey (Gurijala and Alexander, 1990). In a subsequent study, the effects of gene alterations on the growth and virulence of P. solanacearum (a causal agent of bacterial wilt) were studied in bulk soil and in the rhizospheres of host and nonhost plants (Williamson and Hartel, 1991). The amounts of endopolygalacturonase A or of endopolygalacturonase A plus endoglucanase produced by P. solanacearum were either reduced by marker-exchange mutagenesis and/or transposon mutagenesis with Tn5 (P. solanacearum strains PG-3 and 209B) or enhanced by increasing the copy number of the genes, pgl+ and egl+, with a recombinant plasmid [P. solanacearum strains AW(pDR340)and AW(pK593)I.Regardless of the plant type that was inoculated [Lycopersicon esculentum Mill. cv. Marion (tomato-host plant), Portulaca oleracea L. (common purslane-nonhost plant), or Pennisetum glaucum (L.) R. Br. (millet-nonhost plant)],none of the introduced strains increased their numbers in either nonsterile bulk or rhizosphere soil. Therefore, neither the reduction nor the enhancement of enzyme activity increased the competitive fitness of P.

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solanacearum. Furthermore, tomato plants did not wilt significantly faster in the presence of the GEM strains PG-3 and 209B than in the presence of the wild-type strain, i.e., the GEM strains with reduced enzyme activity (PG-3 and 209B) were no less virulent than the wildtype strain. This lack of difference in virulence was attributed to the reduced population densities of these strains that resulted from an impaired ability to grow and, hence, to compete with the indigenous microbiota. In contrast to the GEM strains with reduced enzyme activity, the GEM strains AW (pDR340) and AW(pK593) did not cause any wilting despite their elevated production of endopolygalacturonase and endoglucanase. Changing the phenoiypic expression of the bacterium by genetic engineering (i.e., increasing the expression of specific enzymes by increasing the copy of number of genes coding for enzymes) resulted in changes that were mirrored by deviations from the normal plant-microbe interaction, i.e., the induction of wilting. These studies also indicated that ecological effects induced by introduced GEMS can be detected by monitoring the condition of organisms at higher tropic levels, e.g., plant health. In studies with intact soil-core microcosms (Bentjen et al., 1989; Bolton et al., 1991a,b; Fredrickson et al., 1989, 1990), the addition of engineered (Tn5 mutant) or nonengineered rhizobacteria, Azospirillum lipoferum (about lo8 CFU/seed), and a spontaneous, rifampin-resistant mutant of the root-growth inhibiting rhizobacterium, Pseudomonas sp. RC1 (about lo8 CFU/g of soil) (Fredrickson and Elliott, 1985), did not produce any significant differences in the level of soluble nutrients in soil leachates, plant (corn and wheat) uptake of nutrients, soil dehydrogenase activity, plant biomass, or populations of heterotrophic, symbiotic, and nonsymbiotic nitrogen-fixing, and nitrifying bacteria. However, the addition of Pseudomonas sp. RC1 resulted in a reduction in the colonization of the rhizoplane of wheat by fluorescent pseudomonads indigenous to the soil, although the total numbers of pseudomonads increased. A similar reduction in the numbers of indigenous, fluorescent pseudomonads was not observed in the rhizoplanes of wheat or corn inoculated with either the engineered on nonengineered strains of A. lipoferum. As fluorescent pseudomonads apparently suppress take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici, their exclusion as a result of competition or niche displacement by an introduced microorganism could increase the vulnerability of wheat crops to root disease (Hamdan et al., 1991). When Pseudomonas cepacia ACllOO, genetically engineered to degrade the herbicide 2,4,5-trichlorophenoxyacetate(2,4,5-T),was added (about lo5 cells/g of soil) to soil amended or not amended with

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2,4,5-T and planted with radish seeds (Raphanus sativus), both the phenotypic and the genotypic diversity [as determined by the reassociation kinetics (Cot values) of melted DNA; Kornberg, 19801 of the soil microbial community increased slightly and transiently over that observed in uninoculated soil amended with 2,4,5-T and in unamended and uninoculated soil (Bej et al., 1991).After 6 weeks, both the phenotypic and the genetic diversity of soil inoculated with the GEM and either amended or not amended with 2,4,5-T approximated the level of diversity in the uninoculated, unamended soil. The addition of 2,4,5T alone caused a decrease in phenotypic diversity over the length of the study, as measured by the Shannon diversity index (Kaneko et al., 1977; Shannon and Weaver, 1963). As the DNA used in the genetic diversity assays was total DNA extracted from the soil, the contribution of DNA from the introduced GEM to the apparent genetic diversity of the soil microbiota could not be determined. The transient increase in the diversity of soil inoculated with the GEM and amended or not amended with 2,4,5-T was thought to have resulted from changes in the indigenous microbiota caused by substrates produced by the metabolic activities of the GEM and/or genetic interactions between the introduced GEM and the indigenous populations. The results of this study, although not entirely clear, suggested that: (1)the introduction of a GEM can affect the indigenous microbiota, albeit transiently; and (2) the effects of GEMS on the indigenous microbiota may be influenced by the presence of the substrate upon which the products of the novel genes act. The introduction of Pseudomonas fluorescens C5t, encapsulated in alginate, to a nonsterile loam soil affected soil respiration, as measured by CO, evolution from and 0, consumption by the soil [Trevors, 1991). Pseudomonas fluorescens C5t was constructed by inserting a &endotoxin gene from Bacillus thuringiensis subsp. israelensis into transposon Tn5 contained in the suicide vector pSUPlOl, producing Tn5::tox, which was then inserted into the chromosome of P. fluorescens R2f by conjugation with the mobilizing donor strain Escherichia coli S17.1 (Trevors et al., 1990). Over the 14-day period of the experiment, the numbers of cells of P. fluorescens encapsulated in alginate or suspended in water decreased from about 10' to lo6 CFU/g in nonsterile soil and from about 10' to CFU/g in sterile soil. The evolution of CO, from nonsterile soil inoculated with the encapsulated P. fluorescens C5t was greater and the consumption of 0, was lower than that from uninoculated soil and soil inoculated with P. fluorescens C5t suspended in distilled water during the first 7 days. The reasons that nonsterile soil inoculated with the encapsulated GEM had lower 0, consumption

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than nonsterile soil inoculated with the GEM suspended in distilled water or not inoculated are not known. However, the lower amont of 0, consumed by nonsterile soil inoculated with the encapsulated GEM was thought not to be the result of limited 0, diffusion into or CO, diffusion from the beads, especially as CO, evolution from soil inoculated with the encapsulated GEM was greater than that from soil inoculated with the suspended GEM. Both 0, consumption and CO, evolution were lower in nonsterile soil than in autoclaved soil inoculated with either encapsulated or water-suspended bacteria. The results of this investigation, although not entirely clear, suggest that the ecological effects of GEMS introduced to the environment may be influenced by how they are introduced to that environment (e.g., the introduction of a GEM encapsulated in alginate versus the same GEM suspended in distilled water). The addition of the GEMS E. coli W3110(R702), E. coli J53(RP4), Enterobacter cloacae 108(R388::Tn1721),P. putida PPO2OO(pWWO), and P. putida PP0301(pR0103) or of their homologous, plasmidless, parental strains individually to nonsterile soils from New York and Oregon (at lo6 to 10’ CFU/g of soil) produced no statistically significant differences in the overall metabolic activity (i.e., CO, evolution) of the soils, regardless of whether they were unamended or amended initially and then again after 2 weeks with 1% glucose (Doyle and Stotzky, 1993; Stotzky et al., 1993). There were also no consistent, statistically significant effects on species diversity or the activities of soil enzymes. These studies were repeated several times, and the statistically significant differences observed in the numbers of a specific functional group of the indigenous microbial community (e.g., in ammonia oxidizers or denitrifiers) or in the activities of some soil enzymes (e.g.,acid phosphatases) between soil inoculated with these GEMs, their homologous parents, or not inoculated were transient. There were also no consistent, statistically significant differences in nitrification and in nonsymbiotic nitrogen-fixation among soil inoculated with most of these GEMs, their homologous parents, and those not inoculated (Jones et al., 1991). However, the amounts of nitrogen fixed were so small that any differences between microbial treatments were probably not detectable. Statistically significant differences in nitrification were observed only between the parental E. cloacae and its plasmid/transposon-containing counterpart; nitrification was reduced in the presence of the GEM. The reasons for and significance of this observation are not known. Most of the differences in the effects of these GEMSand their homologous parents did not appear to be ecologically significant. However,

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when the differences were statistically significant, the effects appeared to be dependent on the type of GEM, the type of soil [some studies were conducted with the New York soil amended with different concentrations of montmorillonite or kaolinite (e.g.,Jones etal., 1991; Stotzky, 1986)], and the microbiological parameter evaluated (e.g., Doyle, 1993; Stotzky et a]., 1993) (Table 111). In addition to these studies with soil amended or not amended with glucose and inoculated with GEMScontaining novel genes that imparted resistance to antibiotics and heavy metals, studies were also conducted in soil amended with the specific substrate on which the catabolic products of novel genes in a GEM function (Doyle et al., 1991; Short etal., 1991). Pseudomonas putida PP0301(pR0103) was added to soil amended with 2,443. Plasmid pR0103 contains constitutively expressed genes that code for enzymes that convert 2,443 first to 2,4dichlorophenol(2,4-DCP) and then eventually to 2-chloromaleylacetate (2-CMA). A xeric soil from central Oregon was used in these studies, as the microbiota of this soil did not detectably metabolize 2,4-D. The soil was either not amended or amended with 1% glucose, 500 ppm 2,4-D, or 1%glucose plus 500 ppm 2,4-D and then either inoculated (about lo7 CFU/g of soil) with P. putida PP0301, the parental strain, or PP0301 (pR0103) or not inoculated. Soil inoculated with PP0301(pR0103) and amended with glucose plus 2,4-D evolved significantly less C02 during the first 35 days of the 52-day experiment than did inoculated soil amended with only glucose. No significant differences in the rate of C02 evolution were detected between soil amended with glucose or with glucose plus 2,4-D when the soil was inoculated with the parental strain and soil that was not inoculated. The concentration of 2,4-D in soil inoculated with the GEM decreased rapidly to less than 80 ppm, and 2,4-DCP accumulated to about 90 ppm by Day 10 and then began to decrease by Day 38, as determined by high-performance liquid chromatography and gas chromatography-mass spectrometry. In soil inoculated with the parental strain or not inoculated, there was no decrease in the concentration of 2,4-D and no accumulation of 2,4-DCP. Accumulation of 2,4-DCP in soil inoculated with the GEM was apparently the result of: (1)the tfdB gene, which codes for the synthesis of 2,4-DCP hydroxylase, being regulated by a different operon, in contrast to the other constitutive genes involved in the conversion of 2,4-D to 2-CMA (Kaphammer et al., 1990): and (2) the xeric soil not having indigenous bacteria capable of mineralizing 2,4-D (Short et al., 1991). Short et a]. (1990) did not observe the accumulation of 2,4-DCP in an agricultural soil containing indigenous bacteria capable of mineralizing 2,4-D.

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The numbers of fungal propagules in soil amended with 2,4-D and inoculated with the GEM decreased to undetectable levels by Day 10. Comparable decreases were not observed in soil that received the other combinations of amendments and inocula, except that the numbers of fungal propagules also decreased by Day 40 in unamended soil inoculated with the GEM. In in vitro studies, 50 ppm of 2,4-DCP (the lowest concentration evaluated) completely inhibited the growth of representative fungi isolated from the unamended, uninoculated soil used in this study, whereas 200 ppm of 2,4-D (the highest concentration evaluated) had essentially no effect on fungal growth (Short et al., 1991). In soil amended with 500 ppm 2,4-D, dehydrogenase activity was inhibited in both the presence and the absence of glucose (an inhibition also reported by other investigators; see Doyle et al., 1991; Short et al., 1991). However, in soil inoculated with the GEM, but not in soil inoculated with the parental strain, this inhibition of dehydrogenase activity was relieved after the conversion of most of the 2,443 to 2,4-DCP (Doyle et al., 1991). In contrast, the addition of 2,4-D without the GEM did not inhibit the overall metabolic activity of the soil microbiota or the reproduction of fungi. There were no consistent effects of 2,4-D and the GEM, either separately or together, on the numbers of total, spore-forming, and chitin-utilizing bacteria or on the activities of arylsulfatases and acid and alkaline phosphatases (the other parameters evaluated). Moreover, the survival of the GEM and its parental strain was similar in all treated soils, indicating that the ability to catabolize 2,4-D did not provide the GEM with a selective advantage. The breakdown of 2,4-D to 2-CMA provides only two carbons for anabolism (Short et al., 1991). An important conclusion derived from these studies and those of Bej et al. (1991) is that when evaluating the potential effects of GEMs on microbial populations and processes in soil or other natural habitats, the substrate on which the products of the novel genes function or the specific inhibitor to which the genes confer resistance should be provided to detect any potential effects (Stotzky, 1990). For example, P. putida PP0301(pRO103)appeared to be a good candidate for eventual release to soil: it is genetically engineered to degrade an herbicide, 2,4D, to an environmentally harmless intermediate, 2-CMA, that is readily mineralized by the indigenous soil microbiota; and as the GEM derives essentially no energy from the transformation of 2,4-D to 2-CMA, it has no competitive advantage in soil. However, the unanticipated accumulation of 2,4-DCP and its inhibitory effects emphasize that, with the current state of knowledge, the potential ecological effects of GEMs should be evaluated on a case-by-case basis (e.g., Babich and Stotzky, 1983; Babich et al., 1983; Doyle and Hendricks, 1993; Doyle and Stotzky,

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1993; Doyle et al., 1991;Gustafsson and Jansson, 1993; Kawar and Sherlock, 1989; Short et a]., 1991; Stotzky, 1989, 1990, 1991, 1992; Stotzky et al., 1993;US.General Accounting Office, 1988) (Table 111). Another aspect that needs to be considered in the assessment of the potential ecological effects of recombinant organisms is the environmental release of GEMS and transgenic plants containing novel genes that do not code for catabolic functions but whose nonenzymatic products may accumulate in soil and other natural habitats and may result in ecological perturbations. For example, genes from various subspecies of B. thuringiensis that code for insecticidal proteins have been genetically engineered into plants and bacteria that are indigenous or adapted to a specific habitat(s) in which they can persist, proliferate, and synthesize the toxins. This is in contrast to the use of commercial preparations of B. thuringiensis that usually consist of mixtures of cells, spores, and insecticidal parasporal crystals. These commercial preparations have been used as insecticides for more than 30 years with no apparent toxicities, probably because B. thuringiensis does not survive or grow well in natural habitats, such as soil, and the concentration of the toxins are rapidly decreased by ingestion and biodegradation (e.g., Tapp et al., 1994;Venkateswerlu and Stotzky, 1992). However, if production exceeds consumption by insect larvae, inactivation, and/or degradation, the toxic proteins could accumulate to concentrations that could constitute a hazard for nontarget organisms (e.g., beneficial insects and other animal classes) (e.g., Flexner et al., 1986;James et al., 1993)and could result in the selection and enrichment of target insects that are resistant to the toxins. This potential hazard is exacerbated by modifications of the inserted toxin genes to code only for the synthesis of the toxins or a portion of the toxins rather than for the synthesis of the nontoxic crystalline protoxins. Consequently, organisms ingesting the toxins will not need to have a high pH (ca. pH 10.5)and specific proteolytic enzymes in their gut for the solubilization and cleavage, respectively, of the protoxins into toxic subunits. Nontarget insects, earthworms, and other organisms in higher trophic levels could, therefore, be susceptible to the toxins. Although specific receptors for the toxic proteins on the epithelium of the gut appear to be necessary, and these are apparently present in larger numbers in susceptible larvae, their absence in nontarget organisms has not bee definitively established. Moreover, the accumulation of toxins would be enhanced if they are bound on particles (e.g., clay minerals] in the environment, and thereby are rendered less accessible to microbial degradation but still retain their toxic activity, as has been shown for numerous other proteins (e.g., Stotzky, 1986).

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The equilibrium adsorption and binding of the protoxins (M, = 132 kDa) and toxins (M, = 66 kDa) from B. thuringiensis subsp. kurs-

taki, which have antilepidopteran activity, were greater on montmorillonite than on kaolinite, and two- to three-fold more toxin than protoxin was adsorbed on both clays (Venkateswerlu and Stotzky, 1992). Maximum adsorption occurred within 30 min (the shortest time interval measured), indicating that toxins released from transgenic plant or microbial biomass would be free and available for microbial degradation in soil for only a short time. Adsorption on clay was greater at low pH and decreased as the pH increased, and it was not significantly affected by tempertures between 7 and 50°C. Similar results were obtained with the toxins (M, = 68 kDa) from B. thuringiensis subsp. tenebrionis, which have anticoleopteran activity (Tapp et al., 1994), and with clays extracted from natural soils. The M, of the proteins appeared to be unaltered after adsorption on the clays, and no significant modifications occurred in their structure as the result of binding on the clays. The proteins did not significantly intercalate the clays. Furthermore, the clay-protein complexes retained their toxicity to lepidopteran and coleopteran larvae (Tapp and Stotzky, 1995), and the binding of the proteins on the clays reduced their susceptibility to biodegradation (Koskella and Stotzky, 1994). The results of these studies indicate that because these toxins bind on clay minerals, become resistant to biodegradation, and retain their insecticidal activity, they have the potential to accumulate in soil and, therefore, could affect the environment. Studies such as these and those of A u s t i n z a l . (1990), Doyle et al. (1991), and Short et al. (1991) demonstrate that: (1) the expression of novel genes may lead to unanticipated effects on microbial populations and processes that are not apparent from their construct; (2) products formed as a result of novel gene expression may affect a variety of trophic levels; (3) the ecological effects of metabolites produced by GEMs may develop so gradually as to be well established before becoming apparent; and (4) the ecological effects of GEMs can vary depending on the type of GEMs and the environment into which they may be released. V. Plants

The field release of an ice-minus strain of Pseudomonas syringae had no apparent deleterious impact on the environment (Lindow and Panopoulos, 1988). However, these studies demonstrated the effects of the speed and direction of the wind on the dispersion of GEMs applied to plants as aerosols (Lighthart and Kim, 1989; Lindow and Panopoulos, 1988). The numbers of released bacteria decreased rapidly from the

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source, as measured by sentinel plants [bush bean (Phaseolus vulgaris L.) and oat plants (Avena sativa cv. Cayuse)]and a variety of air samplers [gravity plates, AGI-30s (all-glass impingers), Andersen impactor samplers, and Reynier samplers] (e.g., Stetzenbach et al., 1992). When the GEM was sprayed onto strawberry plants in Brentwood, California, 99.99% of the cells settled on the target plants and soil within the test site, and 0.0001% was dispersed beyond a 15-m-wideuncultivated buffer zone surrounding the site (Seidlerand Hern, 1988).In a subsequent spray of the GEM onto potato plants in Tulelake, California, viable cells were detected beyond a 30-m-wide buffer zone in a southerly to easterly direction. Nozzle size and wind speed apparently influenced the extent of the drift of the sprayed GEM and the numbers of cells present in each aerosol droplet. As the objective of the study was only to determine the distribution of the GEM on and off the test site, the ecological effects of the release of this GEM were not studied. However, if GEMs spread beyond the sites of aerosol application, as in these studies, to other habitats, which may contain different and susceptible biological communities, the GEMs could induce unanticipated ecological effects. Spraying the leaves of bean (P. vulgaris cv. Bush Blue Lake) or radish plants (R. sativus cv. Cherry Belle) growing in nonsterile soil in microcosms containing cutworms (Peridroma saucia) with lo6 to lo* CFU/ ml of P. cepacia (R388 : :Tnl721), E. cloacae(pBR322),Klebsiella planticola(pBR322),or Erwinia herbicola(pBR322)resulted in no detectable impacts on the cutworms (Armstrong, 1992; Armstrong et al., 1989). However, the cutworms transported the introduced bacteria, which also grew in the frass of the insects after passage through the gut, and gene transfer occurred within the guts of the cutworms (Armstrong et al., 1989, 1990). The spread of GEMs and their novel genes by insects, coupled with their survival and growth in the frass of insects, could enable the GEMs to establish competitively in new environments, wherein the expression products of the novel genes could have unexpected ecological effects. When two genetically engineered strains of Lactobacillus plantarum NCDO 1193-one containing pSA3 (a plasmid encoding resistance to erythromycin) integrated into the genome, and the other containing a derivative of pSA3 (designated pM25) into which a cellulase gene from Clostridium thermocellum was inserted and maintained as a plasmid-were inoculated into silo microcosms containing perennial rye grass (Lolium perenne) at lo6 CFU/g of grass, both the GEMs and the parent strain proliferated, dominated the epiphytic microbiota, and shortened the time required to decrease the pH (Sharp et al., 1992). Apparently, the presence of the extra genetic material was not a disadvantage to the GEMs.

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To determine whether the presence of a genetically engineered endophytic bacterium alters the chemical properties of the host plant and the decomposition of colonized residues of the host plant, 18-day-old plants of corn (Zea mays L.) were inoculated with a endophytic bacterium, Clavibacter xyli subsp. Cynodontis(MDE1)(designated the “wildtype”), a genetically engineered construct, C. xyli subsp. cynodontis(MDR1.3) (which contained a gene encoding for the production of the &endotoxin from B. thuringiensis subsp. kurstaki), or sterile buffer (Tester, 1992). Leaves and stalks were harvested at maturity and, after various chemical and physical properties were analyzed, were incorporated into soil to determine the extent of their decomposition in a 63day laboratory incubation study. Leaf residues from plants inoculated with the GEM retained significantly more water than those from plants inoculated with the wild-type strain or buffer. Furthermore, the leaves from plants inoculated with the GEM contained significantly more nitrogen than leaves from plants inoculated with the wild-type strain or sterile buffer. Generally, the leaves of plants inoculated with the wildtype strain contained more nitrogen than plants inoculated with sterile buffer, but this difference was not always significant. During the 63-day incubation, about 33% of the corn residue was decomposed. Leaves from corn plants inoculated with only buffer were decomposed significantly more than those from plants inoculated with the GEM or the wild-type strain. In contrast, corn stalks inoculated with the GEM were decomposed significantly more than those inoculated with the wild-type strain or buffer. The types of organic substances present in the stalks of plants inoculated with the GEM (e.g., a trend toward more soluble protein but significantly less soluble carbohydrates than in the stalks of plants inoculated with the wild-type strain or not inoculated) were suggested to be responsible for these differences in the rate of decomposition. However, the differences in the rate and extent of decomposition were so small, even though they were statistically significant, that they may not be detectable in the field where large fluctuations in environmental conditions can occur (e.g.,Brubaker et al., 1993; Wiens, 1977),and hence, they may not be ecologically significant. These results suggest that GEMs can affect the physiology of plants and the rate of turnover of their biomass. However, in the absence of field studies, the potential ecological effects of endophytic GEMs remain unknown, particularly as environmental conditions in the field are highly variable. Neither the GEM Pseudomonas aureofaciens Ps373RNLll nor its parental strain, P. aureofaciens Ps3732RN, affected: (1)the growth of bean ( P . vulgaris L.) in vermiculite or in loam or sandy loam soil; (2) the number of nodules resulting from the symbiotic relation between

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Rhizobium phaseoli and the bean roots in vermiculite; and (3) the evolution of CO, or consumption of 0, by loam or sandy loam soil (England et al., 1993).To take advantage of the inability of most fluorescent pseudomonads to metabolize lactose, the lac operon genes, lacZ and lacy, from E. coli were inserted into the chromosome of P. auerofaciens Ps3732RN to create P.auerofaciens Ps3732FWLll (Drahos et al., 1986; Kluepfel et al., 1991). Nursery containers (6 liters) filled with either sterile or nonsterile vermiculite, loam, or sandy loam soil and planted with either Whitebean mutant No. 103 (produced by mutagenesis with ethyl methane sulfonate and selected for positive nodulation in the presence of fixed N,) or ExRico 23 1991 Breeder bean were inoculated with 1 ml of 1 x 10’ to 1 x lo9 cells/ml of Ps3732RN or Ps3732RNL11 and 0.5 g of pellets of R. phaseoli-peat (The Nitragin Co., Milwaukee, WI) and incubated at 22°C at 60 to 71% relative humidity for 25 days (vermiculite-containing microcosms) or 46 days (microcosms containing either loam or sandy loam soil). The numbers of P. auerofaciens Ps3732RNLll [6.51 (loam) and 6.75 (sandy loam) log CFU/g dry weight of soil] recovered from the soil microcosms after 2 8 days were similar to those of P. auerofaciens Ps3732Rn [6.26 (loam) and 6.72 (sandy loam) log CFU/g dry weight of soil]. Although soil type did not affect the survival of either strain of pseudomonas, the recovery of both the GEM and the parental strain was about 2 orders of magnitude greater from sterile than from nonsterile soils. No statistically significant differences in the final dry weights of bean biomass or in plant height were observed between soils or vermiculite inoculated with the GEM (Ps3732RNLll) or the parental strain (Ps3732RN),suggesting that the GEM did not affect plant growth. Furthermore, the number of nodules on plants grown in the presence of R. phaseoli in vermiculite inoculated with the GEM were not statistically different from the number of nodules on plants grown in vermiculite inoculated with the parental strain and R. phaseoli. The presence of the GEM also did not affect the evolution of CO, or consumption of 0, by the soils, as no statistically significant difference was found in the rates of respiration for soil inoculated with the GEM or parental strain, regardless of soil type. In growth-chamber studies with corn, wheat, and soybean grown in nonsterile farm soil, no significant differences were noted in the rates of survival of P. auerofaciens Ps3732, the strain from which, P. aureofaciens Ps3732RN was derived, and engineered constructs of Ps3732 containing chromosomal inserts of the &endotoxin gene from B. thuringiensis subsp. kurstaki (Watrud et al., 1985).In field studies with corn,

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the survival of Ps3732 was comparable to that observed in greenhouse studies. Nodulation of the roots of soybean (Glycine max cv. Hodgson) by a nonmotile mutant of Bradyrhizobium japonicum, generated by mutagenesis with Tn7, was significantly less than that by the wild-type strain (Liu et al., 1989). Bradyrhizobium japonicum Webster 48 (the wildtype strain, designated Web 48) served as the recipient in plate matings in a 1: 4 ratio with the donor strain E. coli AB2463(Rp4-ColEl: : Tn7) to obtain the motility-minus mutant, B. japonicum Web 48 : :Tn7. Both the mutant and the wild-type strains exhibited the same: (1)growth rate in pure culture, although the mutant had a longer lag phase; (2) ability to bind soybean lectin; (3) flagellar morphology; and (4) nodulating capacity. As the mutant and wild-type strains were equally prevalent in the rhizosphere of the soybean seedlings at about the time of the initiation of the nodules, motility was not considered to be an important factor in the colonization of the rhizosphere. The longer lag phase of the Tn7 mutant was thought to contribute to its inability to nodulate plant roots at the smae rate as the wild-type strain. The results of this study, as those of Austin et al. (1990)and Williamson and Hartel (1991)with protozoa, indicate that the creation of novel genotypic traits in bacteria, when expressed, may affect the structure and/or function of organisms at higher trophic levels, e.g., plant nodule formation in this study. Transposon mutagenesis also affected the ability of Rhizobium meliloti to nodulate alfalfa (Medicago sativa) (Lagares et al., 1992). After mutagenesis with Tn5, R. meliloti Rm2011 (designated RM6963) had a rough colony morphology, contained lipopolysaccharide components of lower molecular weight than the wild-type strain, lost its ability to grow in Luria-Bertani medium containing sodium deoxycholate or sodium dodecyl sulfate, showed decreased survival in tryptone-yeast extract medium supplemented with high concentrations of calcium, and had a delayed and reduced ability to nodulate the primary root of alfalfa. Plants nodulated by the mutant Rm6963 were smaller 2 to 3 weeks after inoculation than control plants inoculated with wild-type R. meliloti. This effect on plant size was correlated with a transient decrease in the fixation of N,. One month after inoculation, most plants nodulated with Rm6963 recovered and expressed a full nitrogen-fixing phenotype, but the plants developed an abnormally large number of small nodules on their lateral roots. Changes in the genotype of this bacterium, therefore, not only resulted in changes in the phenotype of the modified bacterium, but also affected the symbiotes of the bacterium.

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Species of Bradyrhizobium also nodulate the roots of pigeon pea (Cajanus cajan), an important source of protein in tropical regions, especially in the absence of nitrogen fertilization. These nodules are eaten by larvae of the dipteran, RiveJlia angulata, thereby reducing the yield of an important food crop. In an attempt to control these larvae biologically, a fragment of DNA from plasmid pSY368 that encodes for one of the antidipteran toxins produced by B. thuringiensis subsp. israelensis was inserted into plasmid pSM4, derived from a broad hostrange group Inca plasmid, pKT230, and transferred by conjugation into Bradyrhizobium sp. IC3554 (Nambiar et aJ., 1990). The root nodules subsequently formed by strain IC3554(pSM4) were partially protected against insect damage (86% of the nodules containing the wild-type strain IC3554 were damaged and only 46% of the nodules containing the GEM were damaged), and the amount of nitrogen in the leaves was increased (1.4% in the control plants and 2.9% in the GEM-containing plants). The GEM expressed a protein with a M, of 45 kDa, which was in reasonable agreement with the M, of one of the antidipteran toxins produced by B. thuringiensis subsp. israelensis. These investigations were done in nonsterile soil, and not all of the nodules formed in soil inoculated with the GEM contained strain IC3554(pSM4). Consequently, some of the nodules were susceptible to being eaten by larvae of R. angulata. However, in the presence of the GEM, only 46% of the nodules were damaged by the RiveJJia larvae, whereas 90% were damaged in the uninoculated soil. The GEM, therefore, may have provided limited protection to nodules formed by nonengineered, indigenous species of Bradyrhizobium present in the nonsterile soil. The ecological of effects of this GEM on the populations and processes of the indigenous microbiota of the soil are not known, as no studies on these aspects were conducted. However, these studies did demonstrate that an expression of an effect induced by a GEM (Lea,protection of nodules) may be reflected in a secondary manner, e.g., the increase in the nitrogen content of leaves. Pea (Pisum sativum cv. Dark Skinned Perfection) inoculated with the GEM Rhizobium leguminosarum bv. viceae 1045rf(pPBS8),and white clover (TrifoJium repens L. cv. Menna) inoculated with the GEM R. Jeguminosarum bv trifolii RCR46sp(pPBS8) suffered less root and nodule damage when exposed to the clover weevil Sitona Jepidus than the corresponding control roots inoculated with the parental strains, R. leguminosarium bv. viciae 1045rf(pKT230)or R. leguminosarium bv. trifolii RCR46sp(pKT230) (Skert et al., 1990). The engineered strains of Rhizobium were constructed by digesting plasmid DNA containing the toxin gene from B. thuringiensis subsp. tenebrionis (Btt) with BamHl and ligating the resulting fragments (between 5.5 and 6.3 kbp) to a

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BamH1-digested pUC12 vector. These fragments were then cloned in E. coli JM83 that had been transformed with the pUC12 vector, taking a 3.0-kbp toxin-gene fragment from the E. coli JM83 clones and subcloning it into the Hind111 site of pKT230, which was transformed into E. coli S17-1, and conjugating E. coli S17-1 with the Rhizobium sp. by crossings on membrane filters. Cell extracts of the recombinant Rhizobium and E. coli cultures were shown to contain a 6-endotoxin from Btt by immunodetection using a primary rabbit antiserum raised against solubilized crystal protein isolated from Btt. Nodules of white clover or leaf discs of dock plants (Rumex sp.) painted with the purified Sendotoxin from Btt and protein extracts from the recombinant Rhizobium and E. coli cultures were lethal to larvae of S . lepidus and Gasterophysa viridula Deg. Furthermore, the inoculation of pea and white clover with the recombinant rhizobia provided some protection against damage of roots and nodules by larvae of S . lepidus. However, the authors suggest that this protection may have resulted from the larvae consuming recombinant rhizobia colonizing the soil and the surfaces of root nodules, as the expression of the toxin by recombinant rhizobia within root nodules has not been demonstrated. The results of this and the previous study suggest that recombinant strains of rhizobia may be useful in the biological control of insect pests of leguminous plants.

VI. Discussion

Despite a paucity of studies on the ecological effects of microorganisms introduced to the environment, whether genetically engineered or not, the available data, as summarized in this review, indicate that the introduction of GEMs has the potential to affect the structure and function of ecosystems. Numerous ecological effects resulting from the introduction of GEMs to aquatic and terrestrial environments have been reported. For example, GEMs have been shown to: (1)compete successfully with the indigenous microbiota of disturbed ecosystems; (2) transfer their novel genes in situ with the expression of these genes in a new host; (3) affect both gross and specific metabolic activities and the rate of biomass turnover; (4) influence the community structure and function of the indigenous microbiota in various habitats; (5) affect interactions between symbiotes and organisms at different trophic levels; and (6) produce metabolites that may have unanticipated impacts on the environment (Table 111).However, these effects of GEMs are, in most cases, based on laboratory studies and, therefore, may not reflect effects in the field where large fluctuations in environmental conditions can occur (Brubaker et al., 1993; Wiens, 1977).

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The assessment of the potential ecological effects of GEMs before their release to the environment is usually performed in laboratories and greenhouses. Whether data obtained in the laboratory/greenhouse are relevant to the field remains unclear, as data from attempts to correlate field and laboratory results are generally inconclusive (Schuster, 1991). For example, Bolton et al. (1991a) found that populations of Pseudomonas sp. strain RC1 inoculated into soil-core microcosms (incubated in growth chambers with temperature fluctuations that mimicked average field temperatures), into field lysimeters, and into field plots (all systems were planted with winter wheat) had similar survival rates and colonized the rhizoplane of wheat in a similar manner in the microcosms, field lysimeters, and field plots. However, the effects of RC1 on the numbers of fluorescent pseudomonads and heterotrophic bacteria, dehydrogenase activity, plant biomass production, and 15Nfertilizer uptake by wheat were different in each of these systems (Bolton et al., 1991b).Kroer and Coffin (1992)reported differences in the trophic interactions of microbes in aquatic microcosms and a salt-water pond: although the microcosm:pond ratios for primary productivity, bacterial production, and nannoflagellate grazing of bacteria were qualitatively similar, the rates of total primary production, bacterial production, and nannoflagellate grazing were 25 to 40% lower in the microcosms than in the pond. Consequently, there is a need to verify assessments of ecological effects induced by GEMs in the laboratory through field trials (Committee on Scientific Evaluation of the Introduction of Genetically Modified Microorganisms and Plants into the Environment, 1989; Fox, 1988; Fredrickson and Seidler, 1989). This need is an integral part of current proposals for tier-testing schemes for assessing the potential risks of GEMs before their release to the environment (e.g., Committee on Scientific Evaluation of the Introduction of Genetically Modified Microorganisms and Plants into the Environment, 1989; Dean-Ross, 1986; Debabov, 1991; Doyle and Hendricks, 1993; Gustafsson and Jansson, 1993; Levin et a]., 1987). Surrogate microorganisms (parental or wild-type strains) have been used to: (1) evaluate methods for monitoring GEMs in the field; (2) investigate the survival, dispersal, and potential for gene exchange of GEMs introduced to the field; and (3) validate microcosm and mesocosm studies with GEMs (Bott and Kaplan, 1991, 1993; Donegan et al., 1991; Fisher and Briggs, 1988; Omenn, 1986; Seidler, 1992; Walter et al., 1990). Although the use of surrogate organisms may provide some information in survival and dispersal studies, surrogate organisms are not appropriate for validation in the field of ecological effects induced by GEMs in the laboratory/greenhouse. Surrogates neither contain the

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novel genes of the GEMs nor produce the metabolic products that may be responsible for the environmental effects observed with GEMs in the laboratory (Alexander, 1986). Although mesocosms and greenhouses probably simulate field conditions better than laboratory microcosms, mesocosms are also limited in their usefulness in validating laboratory studies, as they are also an environmentally regulated, contained, and anthropogenic construct of the natural world. In contrast, limited releases of GEMs to the field, under stringent conditions of containment and with adequate contingency procedures for decontamination and mitigation (e.g., Donegan et al., 1992; Ingham et al., 1986a; Lindow et al., 1992; Vidaver and Stotzky, 1992) would enable the collection of considerable information necessary to validate laboratory results. Lindow and Panopoulos (1988) and Kluepfel et al. (1991) demonstrated the applicability of using limited field releases to verify laboratory studies on the fate and survival of different GEMs. Although introductions of GEMS to microcosms have resulted in some changes of the structure and function of the indigenous microbiota that were statistically significant (e.g., Doyle et al., 1991),these effects were often of short duration and were highly variable. This transiency of statistically significant differences emphasizes the need to be able to distinguish between the “statistical” and “ecological” significance of measurement endpoints (Sutter, 1990) when evaluating the effects of GEMs to be released to natural habitats. The relationship between the statistical significance of highly variable cause-and-effect data and their ecological significance is being debated actively within the scientific community (Murdoch, 1994; Nyholm et al., 1992). Because of the transient nature of the responses of the indigenous microbiota to the presence of GEMs, the use of inferential statistics [usually based on some form of an analysis of variance and comparisons of means (e.g., least significant difference, Tukey’s analysis, Duncan’s multiple-range test) (e.g., Steel and Torrie, 1980)] for determining the ecological significance of observed effects is often equivocal (Denny and Gaines, 1990). A variety of statistical methodologies are being adapted to aid in the understanding of the effects of stressors on indigenous populations in the environment: e.g., analysis of the dynamics of metapopulations, which involves an analysis of the dynamics of indigenous populations that have, as a group, a patchy distribution within a particular habitat and which attempts to evaluate how this group of populations interacts and changes as it is influenced by the various genetic and ecological processes that are occurring in that habitat (Schemske et al., 1994). This approach takes into consideration an array of discrete microhabitats that may differ in the size, quality, number, size-

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distribution, spatial arrangement, and genetic heterogeneity of the populations. The most effective use of the meta-population model has been in understanding invasion, persistence, and spread of infectious diseases in animals and plants (Anderson and May, 1986;Bailey, 1975). However, this approach may also be useful for studying the fate and effects of GEMS introduced to natural habitats. The regulation of populations is a fundamental process in the ecology of microorganisms, but whether this regulation results from the presence of local stabilizing factors, either abiotic or biotic, or from the dynamics of meta-populations is unresolved (Murdoch, 1994). For a GEM to affect the stability or expression of a population, it must disrupt these stabilizing factors. Consequently, GEMs may affect a particular measurement endpoint or some component of the regulatory processes. If a particular assemblage of populations is affected by the presence of a GEM, other indigenous organisms or ecological processes may respond to the stress in a manner entirely different from that predicted, and, thus, something other than a mathematical expression based on normal distributions may be required to describe the behavior of the system (Parsons, 1991).Gaines and Denny (1993)showed that when indigenous populations are lost (i.e., extinction occurs), the extremes of a parameter (e.g., the highest population number necessary to affect change, the lowest temperature, the greatest change in population structure or function when an individual is killed or damaged) are not adequately described by biostatistics that emphasize normal distributions. However, normal distributions may be useful in discerning effects that are transient (Gumbel, 1958). The transient nature of statistically significant observations of ecological effects induced by GEMs also suggests that the adequacy of the systems (e.g., microcosms) used to assess the effects of GEMs must be addressed. The relative merits of an artificial system used for investigating the potential ecological effects of GEMs depends not only on the scientific question(s) asked (Fredrickson et al., 1990), but, more importantly, also on whether the test system can be used to model the behavior of GEMs in environments to which they may be introduced. The concept of “environmental realism,” first introduced by Blanch and Gustafsson (1978),emphasizes that stressors must behave in test systems in a manner similar to how they would behave in situ. The demonstration of environmental realism when assessing the potential for GEMs to induce ecological effects requires the use of causeand-effectdata acquired by evaluating ecological endpoints that reflect realistic changes in environmental parameters (Sutter, 1990). Babich and Stotzky (1983)emphasized that laboratory analyses of ecological

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endpoints based on only one species are not suitable indicators of environmental risk, because they fail to integrate or model the expression of stressors in the environment. Consequently, they proposed the concept of the “ecological dose” as one approach with which to evaluate the ecological significance of changes in measurement endpoints that result from the introduction of abiotic pollutants to the environment. Present microbiological and statistical methods lack the ability to assess effectively the ecological significance of the effects of GEMs on the structure and function of indigenous microbial communities (Nannipieri et al., 1990). Approaches have been developed, however, that strengthen measurements for ecological significance by integrating information on effects over time or across several different measurement endpoints. Among these approaches, long-term ecological testing (Magnuson, 1990; Schindler, 1989), the application of bio- and environmental probes [i.e., a system that includes a bioindicator, such as cells, enzyme systems, etc., and a reporter system that can function in situ (Peichl and Reiml, 1990; Parsons, 1991)], and multispecies analysis using diversity measures (Landis et al., 1993) appear to be most promising. Furthermore, the use of a broad-based battery of physicochemical and biological endpoints (e.g., Doyle and Stotzky, 1993; Holmes and Ingham, 1993; Stotzky, 1974; Stotzky et al., 1993) still has the best potential for the assessment of the effects of GEMs. Unfortunately, microbial ecology, as a scientific discipline, lacks adequate hypotheses and methodologies with which to determine the ecological significance of changes in the structure and function of microbes in natural habitats (Doyle and Stotzky, 1993). Consequently, the determination of ecological significance continues to be based on the intuitive judgement of the investigator (Babich and Stotzky, 1983; Babich et al., 1983; Stotzky et al., 1993). Nevertheless, the occurrence of some ecological perturbations induced by some GEMs indicates that until methodologies and theories for establishing ecological significance are developed and verified, the potential ecological impacts resulting from the release of GEMs to the environment should be evaluated on a caseby-case basis (e.g., Babich and Stotzky, 1983; Babich et al., 1983; Doyle et al,, 1991; Doyle and Hendricks, 1993; Doyle and Stotzky, 1993; Gustafsson and Jansson, 1993; Kawar and Sherlock, 1989; Short et al., 1991; Stotzky, 1989, 1990, 1991, 1992; Stotzky et al., 1993, U S . General Accounting Office, 1988). A number of studies support the suggestion that the assessment of the risks of introducing GEMs to the environment should be made on a case-by-case basis. For example, Doyle (1993) presented evidence that the effects of GEMs on the populations and processes of microbes

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indigenous to nonsterile soil are influenced by both the GEM and the soil type. Doyle and Hendricks (1992, 1993) observed differences in microbial populations and processes in soils from Oregon and Idaho that were similarly amended and inoculated with the GEM S. lividans TK23.l(pIJ702.LP).Doyle et al. (1991) and Short et al. (1991) showed that the substrate on which the products of the novel genes function must be provided to detect any potential effects resulting from the introduced GEM. The accumulation of 2,4-DCP and the subsequent reduction in CO, evolution and of fungal propagules occurred only in soil inoculated with P. putida PP0301(pR0103) and amended with 2,4D but not in unamended soil. Scanferlato et al. (1990)demonstrated that the introduction of a GEM to two similar sediment-water microcosms produced differential impacts when one microcosm was thermally perturbed. Jones et al. (1991) observed statistically significant differences in nitrification in a New York soil amended with different concentrations of montmorillonite or kaolinite and inoculated with various GEMs. The introduction of the GEM E. coli W3110(R702)to a xeric sandy loam soil from Oregon (Doyle and Stotzky, 1993) or to a sandy loam soil from New York (Stotzky et al., 1993) induced transient changes in the indigenous microbial community that were probably not ecologically significant. However, the introduced GEM did not survive as well in the Oregon soil as it did in the New York soil. Furthermore, numbers of the indigenous microbiota in these two soils fluctuated differentially through time. In addition to assessing the potential effects of GEMs on a case-bycase basis, the effects should be assessed: (1)with a battery of tests to determine the effects of GEMs on various indicators of ecosystem function (e.g., gross metabolism, physiological diversity, enzyme activities) and structure (e.g., genotypic and phenotypic diversity) (Stotzky, 1974); (2) with standard methodologies and conditions; (3) in the presence of the specific substrates on which the products of their novel genes function; (4) at different trophic levels; and (5) over extended periods of examination (Magnuson, 1990). A variety of tests is needed to ensure that additive, synergistic, and potentially unanticipated effects are detected (Stotzky et al., 1993). Additionally, the use of a variety of tests provides quality assurance for the results obtained. For example, the kinetics of CO, evolution can be related to dehydrogenase activity and changes in pH. As several studies have shown that experimental conditions and methodologies can affect the results of tests used to investigate the effects of GEMs on the microbial ecology of soil (e.g., the rate of CO, evolution; Crawford et al., 1993;Doyle, 1993; Doyle and Hendricks, 1993), tests need to be conducted using standardized methods and

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conditions in the presence of the specific substrate(s) upon which the expression products of the novel genes function (Stotzky, 1990). The potential effects of GEMs should be assessed not only at the microbial level, but also at higher trophic levels, as organisms at higher trophic levels may influence or be influenced by the potential effects of GEMs (e.g., Bamforth, 1988; Elliott et al., 1984; Hunt et al., 1984, 1987; Ingham et al., 1986a,b; Moore and de Ruiter, 1991). For example, insects have been shown to transport GEMs over plant and soil surfaces, to serve as an incubator in which the transfer of novel genes can occur, and to provide a nutrient base (frass) that may promote the survival and adaptation of the introduced GEM in a new habitat (Armstrong et al., 1989,1990).Furthermore, studies with protozoa (Austin et al., 1990; Williamson and Hartel, 1991) and with plants (Liu et al., 1989) have demonstrated that the expression of novel genetic traits carried by a GEM can result in phenotypic changes in the GEM that may influence the community structure and physiology of organisms at higher trophic levels. As not all ecological processes are manifested over short periods of time, the effects of GEMs must be assessed over extended periods (Magnuson, 1990). For example, Doyle et al. (1991) would not have detected the reduction in the numbers of fungal propagules in unamended soil inoculated with the GEM P. putida PP0301(pR0103) if the soil microcosms had not been incubated for longer than 30 days. However, the results obtained in studies with extended periods of incubation must be carefully interpreted. For example, although s. lividans TK23.l(pIJ702.LP)constitutively expresses an enhanced production of lignin peroxidase (Wang et al., 1991),Doyle and Hendricks (1992) only observed a significant increase in the rate of CO, evolution from soil amended with lignocellulose and inoculated with this GEM after 96 days of incubation at 25°C.These results suggest that depletion of easily degraded endogenous carbon substrates by the indigenous microbiota was necessary before the effects of this GEM could be observed. As the depletion of endogenous carbon sources is not likely to occur in the natural environment, the observed increase in the rate of CO, evolution from soil inoculated with the GEM and amended with lignocellulose is probably an artifact of the experimental design. Although the studies reported in this review indicate that GEMs may have the potential to affect the structure and function of ecosystems, these data must be interpreted and applied cautiously. There is still insufficient basic information about the ecological consequences of introducing organisms to natural habitats to enable the establishment of far-reaching and long-lasting policies and criteria. Although there is no

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reason to question the veracity or quality of the current information on the effect of GEMs, the establishment of policies and criteria based on assumptions and on incomplete and possibly imprecise interpretation of data could restrict progress in the assessment of the risks of the products of biotechnology. The current federal policy for regulating biotechnology and, hence, the release of GEMs to the environment was published in 1986 by the United States Office of Science and Technology Policy (OSTP) and is referred to as the “Coordinated Framework for Regulation of Biotechnology Products’’ (Office of Science and Technology Policy, 1986). The OSTP decided that products of recombinant DNA technology will not differ fundamentally from unmodified organisms or from conventional products, and, therefore, existing federal statutes are adequate for regulating organisms and products developed by biotechnology. The Coordinated Framework specified the jurisdiction of various Federal Agencies, such as the Food and Drug Administration (FDA), the United States Department of Agriculture (USDA),and the United States Environmental Protection Agency (USEPA),with respect to review of GEMs. The FDA is responsible for reviewing genetically engineered microorganisms that are used for food, pharmaceutical, or cosmetic purposes under the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 United States Code [U.S.C.] 301 et seq.; Office of the Law Revision Counsel of the House of Representatives, 1989c), the Public Health Service Act (PHSA) (42 U.S.C. 262; Office of the Law Revision Counsel of the House of Representatives, 1989d), and the National Environmental Policy Act (NEPA)of 1969 (42 U.S.C. 4321-4347; Office of the Law Revision Counsel of the House of Representatives, 1989d) (Arbuckle et a]., 1991). Under FFDCA, the FDA has the authority to prohibit the introduction into interstate commerce of adulterated human food and animal feed [other than meat and poultry products, which are regulated by the USDA/Food Safety Inspection Service (FSIS) under the Federal Meat Inspection Act (FMIA) (21 U.S.C. 601 et seq.; Office of the Law Revision Counsel of the House of Representatives, 1989c) and Poultry Products Inspection Act (PPIA) (21 U.S.C. 451 et seq.; Office of the Law Revision Counsel of the House of Representatives, 1989c),respectively]. A food is considered adulterated if, among other things, it carries or intrinsically contains an added poisonous or deleterious substance at a level that may render harm or if it contains an unapproved food additive. Microorganisms, including GEMs, used as sources or components of foods or food additives are, thus, under the jurisdiction of the FDA. Under the requirements of the NEPA, the FDA also considers the potential effects that the manufacture and use of a food or food additive might have

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on the environment. Such effects could include those resulting from commercial-scale fermentation and the use of microorganisms for the manufacture of foods, food products, or food additives. Under the FFDCA, new drugs for use in humans and animals may not be marketed until they are licensed by the FDA. Such licenses, issued only for a particular product of a manufacturer, are given only upon demonstration that the product is safe and effective for its intended use. Biological drugs for use in humans are also subject to the PHSA. Under the PHSA, both the product and the manufacturing establishment are licensed, and licenses are granted only if the product and the manufacturing establishment meet specific standards designed to ensure continued safety, purity, potency, and effectiveness. Licensure for drugs derived from microorganisms includes a review of all aspects of the microorganism and its use relevant to the safety, efficacy, and purity of the final product. As with foods, food products, and food additives, the FDA considers, under the requirements of the NEPA, any potential environmental effects of the use or manufacture of the drug that could result from granting licensure, such as those resulting from the commercial-scale fermentation and use of the microorganism as a source of the drug. The USDA reviews GEMS under its broad regulatory authority to prevent the introduction and dissemination of plant pests, to protect agriculture from threats to animal health, and to protect against the adulteration of food products made from livestock and poultry. The Animal and Plant Health Inspection Service (APHIS)of the USDA regulates these first two areas, and the FSIS is responsible for the third. Under the Plant Quarantine Act (PQA) (7 U.S.C. 151-164a, 166-167; Office of the Law Revision Counsel of the House of Representatives, 1989a) and the Federal Plant Pest Act (FPPA) (7 U.S.C. 150aa et seq.; Office of the Law Revision Counsel of the House of Representatives, 1989a),the USDA has the authority to prevent the introduction, spread, or establishment of plant pests that are new or that are not widely prevalent in the United States. By statute, plant pests are broadly defined to include organisms that can directly or indirectly injure or cause disease or damage in any plants or parts thereof, or to any processed, manufactured, or other parts of plants. Regulations provide for a permit system for field testing of certain organisms produced using biotechnology. The USDA/APHIS prepares analyses of the impacts of proposed field tests under the requirements of the NEPA, and field tests are approved only if the proposed trial poses no risk of creating a plant pest and the introduction is not likely to produce a significant impact on the environment. ~~~

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Under the Virus-Serum-Toxin Act (VSTA) (21 U.S.C. 151 et seq.; Office of the Law Revision Counsel of the House of Representatives, 1989c), the USDA/APHIS has the authority to regulate all veterinary biologicals that are imported into the United States, shipped or delivered for shipment interstate or intrastate, and exported. GEMSand naturally occurring organisms and products are reviewed in the same manner. The USDA/APHIS reviews applications for field testing of any veterinary microbial product under the VSTA and NEPA. Licensure is granted after completion of all requirements to ensure purity, safety, potency, and efficacy. The FMIA and PPIA allow the USDA/APHIS to regulate livestock and poultry used for research and to prevent the adulteration of food products made from livestock and poultry. Several animal quarantine statutes (21 U.S.C. 111, 114, and 134; Office of the Law Revision Counsel of the House of Representatives, 1989c) also give the USDA/APHIS the authority to prevent the introduction and dissemination of communicable diseases of livestock and poultry. Regulation of GEMSby the USEPA falls under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136 et seq.; Office of the Law Revision Counsel of the House of Representatives, 1989a), the FFDCA (21 U.S.C. 301 et seq.; Office of the Law Revision Counsel of the House of Representatives, 1989c),and the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601 et seq.; Office of the Law Revision Counsel of the House of Representatives, 1989b). Under FIFRA, the Office of Pesticide Programs (OPP) regulates the development, sale, distribution, use, storage, and disposal of all pesticides. The USEPA also regulates pesticide residue levels in foods and feed that enter commerce under FFDCA. Microbial pest control agents (MPCAs),whether GEMS or naturally occurring microorganisms, are subject to regulation under FIFRA. Registration of a pesticide occurs only after the USEPA determines that the pesticide does not cause unreasonable adverse effects. The data requirements for registration of MPCAs are found in the Code of Federal Regulations (CFR) (40 CFR 158; Office of the Federal Register National Archives and Records Administration, 1993).Guidance to producers for testing protocols to obtain these data is found in the Pesticide Assessment Guidelines, Subdivision M [US.Environmental Protection Agency (USEPA), 19891. MPCAs are tested not only for mammalian toxicity/pathogenicity to address human health effects, but also for ecological effects on nontarget organisms in the ecosystems of application based on a tier-testing scheme, the use of a maximum-hazard approach, and the selection of appropriate test species. The tier-testing scheme consists of four tiers, whereby adverse effects observed at lower tiers trigger further testing

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at higher tiers. The tiered scheme is, however, flexible, depending on the effects observed. Tier-I consists of acute pathogenicity/toxicity testing using a single maximum-hazard dose of the MPCAs (i,e., the maximum amount of active ingredient expected to be available to the target host x a safety factor, as microorganisms may be capable of reproducing in the environment) for the screening of terrestrial wildlife (e.g., birds, rabbits), aquatic animals (e.g., fish, invertebrates), nontarget plants, and beneficial insects. If adverse effects are observed at Tier-I, Tier-I1 testing, which assesses the survival and fate of the MPCAs in the environment, may be conducted. Tier-I11 is a more thorough investigation of the effects observed in Tier-I, and testing may involve the effects of chronic exposure on avian species, on a greater number of species of aquatic invertebrates, on fish during a complete life cycle, on aquatic ecosystem processes or functions, or on additional plant species. Tier-IV involves simulated or actual field studies. The USEPA Office of Pollution Prevention and Toxics (OPPT) regulates, under the TSCA, the manufacturing, importation, distribution, use, and disposal of chemicals in commerce that are not specifically covered under other statutes. Products specifically excluded from jurisdiction under the TSCA include: (1)pesticides (but not pesticidal intermediates); (2)foods and food additives; (3) drugs, (4) cosmetics and their intermediates; (5) tobacco and tobacco products; and (6) nuclear materials. The TSCA serves as a “catch-all” statute encompassing a wide variety of products that are not covered under other statutes. Although the USEPA considers microorganisms as chemical substances, only some microorganisms are regulated under TSCA. Only those microorganisms that are “new” microorganisms, defined as those that are (1)intergeneric (containing DNA from different genera), and (2)not listed on the TSCA Inventory of Chemical Substances, are subject to premanufacture review under TSCA Section 5. Naturally occurring microorganisms are implicitly listed in the Inventory. The OPPT reviews commercial use of new microorganisms under the TSCA, Section 5, through premanufacture notices (PMNs) that are required to be submitted to the Agency at least 90 days before manufacture or import is to commence. The process of risk assessment within the USEPA is conducted using the paradigm of Risk = Hazard x Exposure. Consequently, if either the hazard or the exposure is low, then the overall risk is low. In the OPPT, various documents are written in support of the risk assessment process for a PMN: confirmation of microbial taxonomy, an analysis of the construct of the GEM, a human health hazard assessment, an ecological

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hazard assessment, an engineering report (worker exposure and releases to environmental media), and an exposure assessment (environmental exposure and fate). For environmental release applications, field protocols are also reviewed. These reports are integrated into a final risk assessment and a risk characterization of the effects on human health and the environment is made before the manufacture or importation of a GEM commences. A marketing analysis is also incorporated into the decision-making process, as the TSCA is a risk/benefit statute, and the benefits to society are weighed against the risks of use of the product. In contrast to the FIFRA, there are no formalized data requirements under the TSCA that commercial manufacturers must meet with submission of a PMN to the Agency. However, commercial manufacturers should submit sufficient information to enable the Agency to conduct a risk assessment. The OPPT has a guidance document that lists the types of data that may be necessary to conduct such a risk assessment. This document, sent to potential submitters, is “Points to Consider in the Preparation and Submission of TSCA Premanufacture Notices (PMNs) for Microorganisms” (USEPA, 1990). The document requests: (1)a description of the recipient and donor microorganisms; (2) the construct of the PMN microorganism; (3) the characteristics of the microorganism; (4) a description of the production process; (5) predicted worker and consumer exposure; (6) information on the environmental behavior of the new strain; and (7) the environmental release protocols. Accurate identification of the recipient and donor microorganisms involved in the construction of the GEM is essential so that information from the existing scientific literature can be obtained about any hazardous characteristics or traits that the microorganisms may possess. Familiarity with the recipient microorganism and knowledge of its behavior in the environment may help mitigate some of the uncertainty or concern associated with the release of recombinant microorganisms. Indications of pathogenicity or toxicity (by virtue of toxin production) of the donor or recipient microorganism to either plants or animals can, for the most part, be obtained from the literature. An examination of the genetic modifications made to the microorganism is also essential, to ensure that only the traits of interest were inserted into the recipient microorganism and that there are no unknown, uncharacterized pieces of DNA large enough (i.e., open reading frames) to code for unknown gene products. Furthermore, an examination of the genetic construct is needed, to establish that there are no traits engineered into the microorganism that would increase its potential for pathogenicity, toxicity, or other environmental hazards relative to the unmodified parental strains.

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The mobility of the introduced DNA, judged by factors such as its location in the genome and the presence of transposons or conjugative plasmids, is also of concern, especially if there is any potential hazard associated with the inserted DNA. The assessment of the more subtle ecological effects that could potentially result from the use of GEMs is the most difficult. In January, 1994, a workshop (entitled Development of Ecological Tier-Testing Schemes for Microbial Biotechnology Applications), jointly sponsored by the USEPA [the OPPT and Office of Research and Development (ORD)]and Environment Canada (Commercial Chemical Branch of Conservation and Protection), was held to develop ecological tier-testing schemes for GEMs that may be subject to regulation under the TSCA. Included were GEMs used in a variety of applications, such as bioremediation, biomining, desulfurization of fossil fuels, coal transformation, oil recovery, nitrogen fixation, fuel production, biomass conversion, waste treatment, and closed-system fermentations. Experts from academia, industry, and government were initially divided into workgroups to: (1)identify ecologically significant endpoints of effects and fate; (2) identify hazards and exposure scenarios associated with the specific technologies; (3) discuss the pathogenicity and toxicity of GEMs to be used in these technologies; and (4) develop a tier-testing, strawman scheme, which was to be refined later in the workshop. The experts were then divided into three groups, depending on the level of containment of the technologies: (1)closed systems (e.g., bioreactors, fermentations, desulfurization of petroleum); (2) semicontained (e.g., biomining, waste treatment, some bioremediation processes); and (3) open, intentional releases (e.g., bioremediation of oil spills, nitrogen fixation). Some of the recommendations made by the workgroup on endpoints of ecologically significant effects included the following. Sustainability of an ecosystem was thought to be most important, and in some cases, generic endpoints were thought to be appropriate, whereas other endpoints may have to be site-specific. Recovery of an ecosystem and delayed effects were also identified as warranting more investigation. Endpoints, such as primary productivity, nutrient cycling, community structure, and predation, were thought to be of ecological significance. Details of the effects and fate endpoints thought to be of significance, along with the tiered schemes that rank the testing and information needed to review GEMs, will be published by the USEPA in the workshop proceedings. From a regulatory viewpoint, insufficient data on the ecological effects of GEMs released to the environment exist in the literature. Furthermore, most of the GEMs evaluated to date have had a limited effect on the basic ecology of the environments to which they have been

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introduced, and as a result, thorough testing of the risk assessment paradigm to predict environmental change, as defined by the USEPA, has not been possible (Orvos, 1993). More research is needed to aid regulators in predicting how potential impacts will be expressed, deciding at what environmental level (e.g., micro vs macro) the effects should be sought, identifying the most appropriate techniques for detecting impacts, and interpreting the results of ecological effects studies with GEMs (Table VI). Moreover, further work is needed to develop assessment and statistical techniques for evaluating the potential of introduced microorganisms to affect the ecological status of the environment, to understand how the environment may modify or interact with the expression of the novel genes in a GEM, and to develop risk assessment protocols that permit the early detection of high-risk organisms before they are released to the environment. Assessment of the potential ecological effects resulting from the use of GEMs is a formidable process, as the science of microbial ecology is in its infancy. Without a basic understanding of the structure and function of the microbiota indigenous to the environment and some insight into what constitutes an ecologically significant change in that structure and function, the assessment of ecological effects, other than pathogenicity or toxicity, that could potentially result from the release of GEMs will remain difficult.

ACKNOWLEDGMENTS The preparation of this article and some of the research described has been funded, in part, by the U.S. Environmental Protection Agency. This article has been subjected to the Agency’s peer and administrative review and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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Detection, Isolation, and Stability of MegaplasmidEncoded Chloroaromatic Herbicide-Degrading Genes within Pseudomonas Species DOUGLASJ. CORK AND AMJADKHALIL Department of Chemical and Biological Sciences Biology Division Illinois Institute of Technology Chicago, Illinois 60616 I. Introduction 11. Selected Model Haloaromatic and Aromatic Compounds of Agricultural and Industrial Importance A. Dicamba, A Model of Chlorinated Aromatic Metabolism B. Other Chlorinated and Nonchlorinated Aromatic Compounds Subject to Environmental Biotransformations 111. Detection and Isolation of Large Plasmids from Pseudomonas spp. A. Wheatcraft Method B. Casse Method C. Kado and Liu Method D. Allen Method IV. Effect of Alternative Carbon Sources on the Stability and Curing of a Large Molecular Weight Plasmid, pDKl A. Nutritionally Induced Instability of Dicamba Degradation B. The Effect of Dicamba, Salicylate, and Succinate on Batch Growth and Dicamba Degradation C. The Effect of Succinate Concentration on Batch Growth and Dicamba Degradation D. Succinate-Dependent Curing of pDK1 V. Summary References

I. Introduction Dicamba (3,6-dichloro-2-methoxybenzoic acid), a model chloroaromatic herbicide, may be used as a model for the in situ bioremediation of many recalcitrant chloroaromatic compounds. Dicamba is a popular herbicide, and can be used as the sole carbon source for the growth of Pseudomonas sp. strain Pseudomonas-Xanthomonas maltophilia (PXM). The dicamba-degrading ability of PXM is attributable to the presence of a large plasmid. This large plasmid was purified by the methods of Casse et al. (1979), Wheatcraft and Williams (1981), Kado and Liu (1981),and L. Allen (personal communication, 1994). Each of these methods has been compared and contrasted with respect to typical 289 ADVANCES IN APPLIED MICROBIOLOGY, VOLUME 40 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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plasmid profiles obtained during standard agarose gel electrophoresis trials. This large plasmid consists of approximately 250 kb, and loses part of the plasmid (the dicamba-degrading locus) when the microorganism is grown on alternative sources of constitutive carbon metabolites, such as succinate. When the organism is grown on succinate-limited medium, Pseudomonas sp. strain PXM, hereafter also referred to as PXM, loses the dicamba-degrading activity after about 20 cell doublings. When this microorganism enters into late stationary phase, part of plasmid begins to disappear and these cells begin to lose their viability when transferred to medium containing dicamba as the sole carbon source. Salicylate appears to maintain the stability of the large plasmid. The effect of the sole carbon source on the stability of a strain’s aromaticor chloroaromatic-degrading ability has been previously noted by many investigators (Saint and Venables, 1990; Saint et al., 1990; Duetz and van Andel, 1991; Duetz et al., 1991; Lloyd-Jones et al., 1994). Changing carbon sources is often accompanied by loss of part or all of a catabolic plasmid in the strains. For example, it has been suggested that “benzoate curing” of part of a large plasmid occurs in other Pseudomonoaceaeharboring unstable plasmids encoding for biphenyl-degrading ability (Lloyd-Joneset al., 1994).The ability to utilize dicamba as a sole carbon source is especially remeniscent of the studies by Saint and Venables (1990; Saint et al., 1990) on the loss of Tdn catabolic genes in Pseudomonas putida. In their strains, the ability to degrade aromatic amines and m-toluate was lost after subculture in benzoate, succinate, acetate, and glucose minimal medium. This alteration in nutritional requirements occurs concomitant with the recombinational deletion or complete loss of the entire pTDNl plasmid (Saint and Venable, 1990; Saint et al., 1990). Because of the instability of the dicamba-degrading phenotype, the DNA content of cultures of Pseudomonas sp. strain PXM grown on different carbon sources provides another example of this phenomenon. This is a review of techniques used in the detection, isolation, and stability of pDK1, a large plasmid which encodes for dicamba-degrading proteins. It is hoped that this information can further enhance the development of optimal growth policies for stabilizing other large plasmidmediated chloroaromatic herbicide biotransformations important to applied microbiologists interested in bioremediation. II. Selected Model Haloaromatic and Aromatic Compounds of Agricultural and Industrial Importance

The use of herbicides has greatly increased the world’s supply of food and fiber. The continued use of herbicides over the last three

CHLOROAROMATIC HERBICIDE-DEGRADING GENES

291

decades has led to the accumulation of toxic chemicals in the environment. Soil, water, air, and biological systems are sinks for herbicides and pesticides and their hydrophilic and hydrophobic metabolites. However, microorganisms have developed the ability to degrade a large number of these recalcitrant compounds. Fundamental investigations of pesticide and herbicide biotransformations have expanded our knowledge of the genetic and metabolic regulation that is so important to the application of these microorganisms in the environment. Chlorinated aromatic compounds have been utilized in agriculture and industry for many years as solvents, lubricants, plasticizers, and insulators, as well as for use as pesticides and herbicides. As environmental pollutants, chloroaromatics must be detoxified after their initial benefit to agricultural or industry has been achieved. Microorganisms can bioremediate these haloaromatic compounds as a function of the number of attached halogens, the stability of the genes encoding the detoxication pathway, and the mass transfer properties of the contaminant, the contaminant-degrading organism, and the contaminated soil or water (Tursman and Cork, 1992).The degradation of haloaromatics may occur through their utilization as a sole source of carbon and energy by various bacteria. Aromatic compounds such as catechol, salicylate, and benzoate are often involved in the control and regulation of haloaromatic metabolism. As bacteria evolve new pathways for haloaromatic detoxication, it is important to determine whether the genotype is chromosomal or plasmid-borne and what factors control the regulation of gene expression and metabolism of the detoxifying system. These factors must be understood before developing an optimal growth policy for in situ soil bioremediation by these microorganisms. Pseudomonadaceae are well known for their detoxification of many haloaromatic compounds. The biological dissimilation of many dichloroaromatic compounds has been examined in many excellent reviews. This review focuses on a well-studied dichloroaromatic benzoic acid herbicide, dicamba, and the stability of a large plasmid, pDK1, which encodes for this herbicide’s catabolism. A. DICAMBA, A MODEL OF CHLORINATED AROMATIC METABOLISM

Dicamba (Banvel) is a herbicide used to control broadleaf weeds in field and silage corn, grain sorghum, small grains, grass seed crops, and noncropland areas such as fence rows, roadways, golf courses, and wastelands (Ofiara, 1990). Unfortunately, dicamba affects the growth of important commercial crops, such as soybean and peas. As a soluble

292

DOUGLAS J. CORK AND AMJAD KHALIL

anionic herbicide (see Table I), dicamba can be persistent in soil, especially under dry field conditions. Using 14C-labeleddicamba, Krueger demonstrated the ability of three enriched Pseudomonas strains to completely mineralize dicamba to 14C02(Krueger et al., 1989, 1991a,b). With thin-layer chromatography and high-performance liquid chromatography, Cork and Krueger (1991) proposed a pathway for microbe-mediated dicamba degradation (Fig. 2). One of these strains, Pseudomonas sp. strain IIT 7,was characterized by Fogarty and Tuovinen (1995). Pseudomonas sp. strain PXM is another strain which shows ability to utilize dicamba, 3,6-dichlorosalicylate (DCSA), or salicylate as a sole carbon and energy source. Dicamba at 2000 pg/ml was found to be optimal for the growth of PXM in chemically defined liquid and solid media. Some other characteristics of this strain are now described. 1. Microbial Identification and Purity

Identification of PXM as a dicamba-degrading pure culture is based on a number of qualitative and quantitative determinations. After transfering the microorganism from dicamba minimal medium, the API 20ER TABLE I CHEMICAL AND PHYSICAL PROPERTIES OF DICAMBA Empirical formula Molecular weight Physical state Color Odor Melting point Boiling point Specific gravity Vapor pressure (25°C) Dissociation constant KO PKO Octanol/water coefficient Flash point Hydrolysis Oxidation Strong acid Strong base Solubility of dicamba acid (g/liter H,O 25°C) Solubility of dicamba Na salt (g/liter HzO 25°C) Solubility of dicamba K salt (g/liter H,O 25OC) Source: Taken from Cork and Krueger, 1991.

C*H&,ZO, 221.04

Crystalline solid White Odorless 114-1 16°C Decomposes at >2OO"C 1.57

mm Hg

3.41 x 1.16 X 1.94 0.1

lo-'

150°C Stable Stable Resistant Resistant 6.5 360 480

CHLOROAROMATIC HERBICIDE-DEGRADING GENES

293

aCl 0cdc, + COOH

COOH

COOH

I

i

COOH

COOH

COOH

I

\

FIG.1. Ring cleavage of chlorinated benzoic acid by Pseudomonas sp. (Hartmann et al., 1979).

system reveals that this dicamba-degrading strain is Pseudomonas maltophilia. The results for these tests are shown in Table 11. The results of gas chromatographic fatty acid analysis appear in Table 111, revealing that this dicamba-degrading strain is Xanthomonas maltophilia (Reba, 1992). The literature reveals a current conflict in the systematics of X. maltophilia (Swing et a]., 1983; Van Zyl et a]., 1992; Vautern et a]., 1992). This is because there are several differences between X. maltophilia, P. ma1tophilia, and Stenotrophomonas maltophilia. Therefore, we decided to use the strain abbreviation PXM to designate this Pseudomonas sp. PXM culture purity is maintained through dicamba-selective pressure in liquid and solid media and by following this selection protocol:

294

DOUGLAS J. CORK AND AMJAD KHALIL TABLE I1 API IDENTIFICATIONOF A DICAMBA-DEGRADING ORGANISM API reactions ONPG(f ]" Citrate Acetone Inositol Oxidase Arginine H2S Gelatin Sorbitol Manitol

-b

+ -

-

-

Lysine Urea Glucose Sucrose Rhamnose Ornithine Tryptophan Melibiose Amygdalin Arabinose

Gram strain: Gram negative (f). Fermentation. +, positive reaction; -, negative reaction.

1. Smooth, round, large, brownish-yellow colonies typically appear on a solid Gel-rite plate with dicamba (2000 pg/ml) as the sole source of carbon for growth. After 2 to 3 days of growth, they are about 1 mm in diameter. All API and fatty acid analyses were performed on samples TABLE I11 GASCHROMATOGRAPHIC FATTY ACEI ANALYSIS FOR THE DICAMBADEGRADING XAhTHOMONAS MALTOPHILIA Fatty acid

1o:o 11:o IS0 11:O IS0 3 0 H 13:O IS0 12:O 3 0 H 14:O IS0 14:O 13:O IS0 3 0 H 13:O 2 0 H 15:O IS0 15:O ANTIS0 16:O IS0 16:l B 17:O IS0 18:l CIS 9 19:o IS0

Percentage at 28°C

Percentage at 35°C

0.57 3.30 1.58 0.49 2.50 0.78 3.46 2.91 0.35 36.68 11.76 1.31 3.09 3.09 1.25 0.35

0.52 3.20 1.89 0.47 2.54 0.93 2.61 2.84 0.34 36.44 11.62 2.18 1.88 4.68 0.99 0.45

Source: Taken from Reba, 1992.

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295

taken from these plates. Switching to alternative carbon sources designated for these analyses results in species changes in these microorganisms. 2. When these colonies are transferred to liquid dicamba medium containing 2000 pg/ml dicamba, they consistently produce a pinkish color. 3. When serial liquid dilutions of this culture are made and samples are transferred back to solid medium containing Luria broth (LB), the colonies on LB plates appear large and spherical and have a whiteyellowish color. Unfortunately, these are cured of the pDKl and genus and species changes are induced (Reba, 1992; Cantrell, 1993). 2. Antibiotic and Nutritional Profile

Table IV illustrates the antibiotic sensitivity profile of PXM while growing on 2000 pg/ml dicamba or LB in either liquid or solid medium. Table V lists several sole carbon sources in a chemically defined minimal medium (Cork and Krueger, 1991), and whether Pseudomonas sp. strain PXM can grow on these compounds. B. OTHER CHLORINATED ANDNONCHLORINATED AROMATIC COMPOUNDS BIOTRANSFORMATIONS SUBJECT To ENVIRONMENTAL There have been many studies of the ability of soil and aquatic microorganisms to dissimilate chlorinated aromatic hydrocarbons such as chlorotoluene (Pierce et al., 1981, 1983, 1984), chorobenzoates (Chatterjee et al., 1981; Chatterjee and Chakrabarty, 1982), methoxychlorobenzoates (Cork and Krueger, 1991), chlorophenols (Apajalahti and Salkinja-Salonen, 1986), chloroacetamide (Saxena et al., 1987), and TABLE IV THE EFFECTOF ANTIBIOTICS ON THE GROWTHOF PXM IN

DICAMBAAND LB LIQUIDMEDIUM Antibiotics (50 CLg/ml)

Growth on dicamba liquid

Streptomycin Kanamycin Tetracycline Ampicillin Chloroamphenicol Erythromycin Chlorotetracyline

+a

+ , Resistant; - , sensitive.

-

-

-

Growth on LB liquid

296

DOUGLAS J. CORK AND AMJAD KHALIL TABLE V

GROWTHOF PSEUDOMONAS SP.PXM ON SEVERAL CARBON SOURCES Source

PXM

Cured Pseudomonas sp. PXM (pregrown on succinate)

Dicamba DCSA Catechol Gentisal 2FB 3FB Benzene Succinate Salicylate POHB Anthrinilate Quinate DNSA Tolulene 3,5-Dichlorobenzoate

+ + + + + + + + + + + + + +

+ + + + + + + + + + +

-

4-chlorophenylacetate (Klages et al., 1981).In this review we focus on a special class of plasmid alteration, “deletion curing,” which results in the partial or complete loss of haloaromatic-degrading ability. It is suggested that dicamba-degrading plasmid pDKl exemplifies deletion curing. Before examining the dicamba-degrading genotype and phenotype, we first examine the stable expression of other aromatic- and chloroaromatic-degrading pathways. 1. Induction and Regulation of Biphenyl Hydrocarbon Catabolism

a. Metabolism of Substituent Biphenyls. The isolation of bacteria capable of utilizing biphenyl was initially described by Lunt and Evans (1970). Furukawa et al. (1983, 1989; Furukawa and Miyazaki, 1986) have studied the effect of alternative aromatic hydrocarbons on the induction and regulation of biphenyl catabolism by Pseudomonas paucimobilis strain Ql. This microorganism can grow on biphenyl, xylene, toluene, both xylene and toluene, salicylate, or octane. Evidence has been presented that the catabolism of biphenyl, xylene, toluene, and salicylate is regulated by a common operon encoding a common metabolic pathway. Incubating strain Q1 cells with biphenyl or 2,3-dihydroxybiphenyl leads to the production of a yellow meta-cleavage compound with a A,, at 434 nm. The meta-cleavage compound is converted to benzoic acid. The same cell converts catechol to 2-hydroxymuconic 375 nm); similarly, some other biphenyl derivatives semialdehyde (A,

297

CHLOROAROMATIC HERBICIDE-DEGRADING GENES

are metabolized to the corresponding substituted benzoates through the same pathway.

b. Induction of Oxidizing Activities and Enzyme Levels. A comparative description of the respiratory metabolism of a typical microbe growing on biphenyl, xylene/toluene, and salicylate is presented in Table VI. Biphenyl-grown cells oxidized all of the biphenyl, 2,3-dihydroxybenzoate (2,30HBP), benzoate, toluene, m-xylene, m-toluate, salicylate, and catechol substrates with high oxygen consumption. Similarly, cells grown on m-xylene and its metabolic intermediates, such as methylbenzyl alcohol, m-tolualdehyde, and m-toluate, could oxidize biphenyl, and m-xylene/toluene, as well as salicylate itself. Benzoate, in contrast to m-toluate and salicylate, was as poor an inducer as succinate was for the catabolism of biphenyl, m-xylene/toluene, and salicylate. However, the benzoate-grown cells rapidly oxidized benzoate and catechol. These result demonstrate that biphenyl, m-xylene/toluene, and salicylate can be common inducers for the oxidation of the same compounds. Benzoate, however, cannot induce the enzymes of these three different compounds. Enzyme levels in cell extracts of biphenyl- and m-toluate-grown strain Q1reveal that biphenyl and m-toluate can induce 2,30HBP oxygenase (meta-cleavage enzyme of 2,30HBP) and C230 (meta-cleavage enzyme of catechol and its methyl derivatives), but not ClzO (ortho-cleavage enzyme of catechol). TABLE VI RATE OF OXYGEN UPTAKE FOR BIPHENYL, XYLENEITOLUENE, SALICYLATE AND THE RELATED BY STRAIN0 1 GROWNON VARIOUS SUBSTRATES COMPOUNDS

Growth substrate

BP

2,30HB

B

T

m-X

m-T

Sal

Ca

Succinate BP Benzoate m-Xylene m-MBA m-TD m-Toluate Salicylic Acid

1.2 28.5 1.3 34.9 13.0 27.3 24.6 28.0

4.1 19.4 3.1 20.6 7.1 10.5 16.3 15.9

0.8 17.0 33.2 18.6 2.4 5.1 10.5 6.0

0.1 20.1 0.1 20.0 7.6 16.2 18.0 10.8

0.5 16.7 0.1 15.7 7.5 21.4 16.4 19.0

4.5 16.7 9.0 26.4 2.7 6.9 16.4 19.0

2.0 22.1 1.7 23.7 6.3 7.9 19.4 21.5

14.0 29.1 56.6 64.5 16.5 29.6 34.3 51.4

Abbreviations: BP, biphenyl; m-MBA,m-Methylbenzyl alcohol; -TD. m-Tolualdehyde;B. Benzoate; T, Toluene; m-X,m-xylene; m-T, m-Toluate; Sal, Salicylate; Ca, Catechol; 2,30HB, 2,3-Dihydroxybenzoate. * Numbers illustrate 0, uptake in (nmollml per min). From Furukawa et al., 1983.

298

DOUGLAS J. CORK AND AMJAD KHALIL

As described above, the catabolism of biphenyl, xylene/toluene, and salicylate is interrelated, since benzoate and toluene are common metabolic intermediates of biphenyl and xylene/toluene, and salicylate is produced from 2-hydroxybiphenyl. All enzymes of the biphenyl, xylene/toluene, and salicylate degradative pathways are induced when cells are grown on biphenyl, xylene/toluene, or salicylate (Furukawa et al., 1983). Growth of P. paucimobilis Q1cells with benzoate as a sole carbon source allows the induction of only the ortho pathway enzymes, suggesting that biphenyl, xylene/toluene, or salicylate specifically induces the meta pathway enzymes for the oxidative degradation of these compounds. 2. Biochemical Mechanisms of Selected Haloaromatic

Transformations in Pseudomonas sp. Since the degradation of benzoic acid is well known, and not an unusual event for Pseudomonas, it has been used as a model for suggesting intermediates in new pathways involving chloroaromatic metabolism. The aromatic ring is cleaved through ortho or meta pathways and metabolic breakdown products are utilized by the TCA cycle as shown in Fig. 2. Addition of hydroxy group(s) to the aromatic ring before the ring cleavage has also been observed with other bacterial strains (Johnson and Stanier, 1971). The proposed pathways for ring cleavage of chlorinated benzoic acids by Pseudomonas sp. are shown in Fig. 1. Even though it is the added chlorine atom(s) that contribute to the chlorinated aromatic compounds’ greater resistance to chemical and biological degradation, some bacterial strains have attained the abilities to degrade and utilize these xenobiotics. Degradation of 3-chlorobenzoic acid by a strain of P. putida has been reported (Chatterjee et al., 1981). The chlorine atom is released after the ring cleavage. The addition of hydroxy groups to the benzene ring also occurs prior to the ring cleavage in these pathways. Degradation of 4-methoxybenzoate by a strain of P. putida was previously studied (Bernhardt et al., 1975).The first step in this biodegradation is demethylation and the reaction products are 4-hydroxybenzoate and formaldehyde. An NADH-dependent reductase and an ironcontaining and acid-labile sulfur-containing monooxygenase are involved in this reaction. The introduction of hydroxyl groups to the aromatic ring, either by addition of an hydroxy group or by demethylation of the methoxy group, is a common event for most cases of chloroaromatic microbial degradation. The proposed pathway for dicamba degradation is presented in Fig. 2 (Cork and Krueger, 1991).

CHLOROAROMATIC HERBICIDE-DEGRADING GENES

299

Coon

&l+@ c-

Dlcamba

/

3,6-dichlorosaiiclylic

' & - W T C A

cycle intermediate

<m 3,6-dichloro

2,5-dihydroxysalicylic

acid

acid

I

O02

FIG.2. Proposed pathway of dicamba degradation (Cork and Krueger, 1991).

Salicylate is a common aromatic compound involved in the metabolism of some aromatic and haloaromatic compounds. Salicylate hydroxylase is a flavoenzyme present in some soil Pseudomonads. It catalyzes the decarboxylative hydroxylation of salicylate to form catechol. Currently, there are at least two distinct salicylate hydroxylase enzymes reported in the literature. One is the P. putida salicylate hydroxylase first described by Yamamoto et al. (1965) and Takemori et al. (1974). This enzyme is a flavoprotein containing 1mol of flavin adenine dinucleotide (FAD), and is a monomer with an approximate molecular weight of 54 kDa. The other is the Pseudomonas cepecia salicylate hydroxylase. It contains 2 mol of FADS and two identical subunits with a total molecular weight of 91 kDa (Kamin et al., 1976; Tu et al., 1981). A

300

DOUGLAS J. CORK AND AMJAD KHALIL

structural determination is essential for further detailed understanding of the function of salicylate hydroxylases of Pseudomonas species. You et al. (1990)purified and partially characterized salicylate hydroxylase from P. putida salicylate hydroxylase (Yamamoto et al., 1965),the P. putida PpG7 salicylate hydroxylase appears to be unstable. Significant loss in specific activity is observed during the storage of enzyme preparations during the time required for column purification of the protein. The purified enzyme preparation is slightly yellow in color, thus providing preliminary supporting evidence that it is a flavoenzyme. Table VII shows the amino acid analysis of salicylate hydroxylase (You et al., 1990).

TABLE VII THEAMINOACIDCOMPOSITION OF SALICYLATE HYDROXYLASE FROM P. PUTIDA P P G COMPARED ~ TO THE AMINO ACIDCOMPOSITION OF THE KNOWN P. PUTIDA SALICYLATE HYDROXYLASE

P. putida (Takemori Amino acid Ala Arg Asn ASP CYS Gln Glu GlY His Ile Leu LYs Met Phe Pro Ser Thr TrP TYr Val

MW

P. putida (PpG7) 53,48 26,31

et al., 1974) 58 20

-

38,36 6-7

36 5

46,43 60,61 11,13 17,16 54.56 7,6 58 14,14 20,17 19,19 14,16 12,11 31.32

37 37 14 19 41 17 7 12 21 21 21 8 12 32

45 kDa

54 kDa

-

Source: Taken from You et al., 1990.

CHLOROAROMATIC HERBICIDE-DEGRADING GENES

301

111. DETECTIONAND ISOLATION OF LARGEPLASMIDS FROM PSEUDOMONAS SPP. When microorganisms encounter a new organic chemical in their environment, they may obtain the new catabolic genes needed for degradation of that compound from other microorganisms through conjugational or transformational events, or they may modify existing genes through mutational processes (Chaudhry and Chapalamadugu, 1991). Even though the chlorinated hydrocarbons have appeared only during the past few decades, microorganisms have evolved catabolic pathways for these compounds. The genes for the degradation of these compounds are often plasmid associated, and are found within naturally occurring microogansims (Tables VIII and Table IX). Many Pseudomonas and related species carry large biodegradative plasmids (Chakrabarty, et al. 1976; Don and Pemberton, 1981; Franklin et al., 1981; Pierce et al., 1981; Fujimoto, 1991).Because these plasmids are so large (20to 300 MDa; Chakrabarty, 1976),they have necessitated the development of several methods of isolation and purification TABLE VIII PLASMID-ENCODED DEGRADATION OF CHLORINATED HYDROCARBONS P1asm id puu204 pKFl *a

pSS50 pAC21 pGS1 pJP4 pAC27 pAC31 pRC10 pEML159 *

* pwWlO0 NAH7 pDK1

Compound 2-Monochloropropionic acid 4-Chlorobiphenyl 4-Chlorobiphenyl 4-Chlorobiphenyl 4-Chlorobiphenyl 1,4-Dichlorobiphenyl DNT 3CBA, 2,4-D, and MCPA 3CBA 3,5DCBA 3CBA, 2,4-D, and MCPA 2,4-D 2,4-D PCP biphenyl Naphthalene Dicamba, DCSA, salicvlate

Molecular size 53 kb 82 kb 16 and 72 kb 50 MDa 53 kb 65 MDa 150 kb 80 kb 110 kb 105 kb 45 kb ?b

50-150 MDa 80-100 kb 200 kb 80 kb 250 kb

*, No designation. ?, Size unknown. Source: Taken from (Chaudhry and Chapalamadugu, 1991;Cork et al., 1992).

a

DOUGLAS J. CORK AND AMJAD KHALIL

302

TABLE IX CAPABLE OF SELECTED CHLOIUNATED AROMATIC COMPOUNDS AND MICROORGANISMS DEGRADING THEM Comuound

Microorganisms

P1asm id

~

1,ZDCB 1,3DCB 1,4DCB 1,4DCB 2,6-Dichlorotoluene 2CBA 3CBA 3CBA 3CBA 3CBA 4CBA 4CBA 3,5DCBA 3,5DCBA 2,4DCBA 4-Chloro-2-nitrophenol 4-Chloro-2-nitrophenol 2,3,4,-Chlor and 2,4DCP PCP PCP PCP PCP 3-, 4-, and 5-Chlorosalicylate

Pseudomonas sp. Alcaligenes sp. Alcaligenes sp. P. putida P. cepacia HCV P. cepacia A. eutrophus Pseudomonas sp. B13 P. putido Flavobacterium sp. Arthrobocter sp. Pseudomonas sp CBS3 Pseudomonas sp. Pseudomonas sp. Cornybacterium sp. Pseudomonas sp. Alcaligenes Alcaligenes sp. Aerobes P. cepacia ACllOO Anaerobes Flavobacterium sp. Pseudomonas sp.

+ + pJP4 pAC27 pAC27 (110 kb) pRCl0 (45 kb) pAC31 From pJP4

+

-

Source: Taken from Chaudry and Chapalamadugu, 1991.

(Wheatcraft and Williams, 1981; Casse et al., 1979; Kado and Liu, 1981; L. Allen et al., personal communication). Previous procedures developed for Escherichia coli plasmids typically rely on a differential precipitation step in which high molecular weight chromosomal DNA is precipitated and the relatively small plasmids (1-10 MDa) remain in the supernatant (Birnboim and Doly, 1979).Table X compares and contrasts several methodologies involved in the isolation of large plasmids. The following section compares and contrasts the above techniques as applied to the problem of detecting and purifying plasmid pDKl from Pseudomonas spp.

METHOD A. WHEATCRAFT Methodologies for detection and isolation of the large plasmid pDKl from PXM all fail when the boiling method (Holmes and Quigley, 1981),

CHLOROAROMATIC HERBICIDE-DEGRADING GENES

303

TABLE X COMPARISON OF THE FOURPROTOCOLS FOR PURIFICATION OF THE LARGEPLASMID PDKl ~

Protocol used

Lysis buffer used

Wheatcraft and Williams (1981)

1 N NaOH with

Kado and Liu (1981)

3% SDS in 50 mM

Casse et al.

1%SDS in TE

(1979)

Allen (1994, personal communication)

Extraction by phenol-chloroform

Precipitation of plasmid DNA

Not used

SDS at room temp Used

Phenol only

buffer, pH 12.4

0.05 M glucose,

0.009 M EDTA, 0.024 M Tris, sucrose,

3 M Na acetate, 95%

ethanol at -20°C for 30 min

Tris, pH 12.6, at 65°C for 1 hr

0.3 M Na acetate,

95% ethanol at -20°C overnight Not used

Isopropanol at room temp for 15 min

the clear lysate method (Birnboin and Doly, 1979), and precipitation by polyethylene glycol (Maniatas et al., 1989) are used. A direct method (Wheatcraft and Williams, 1981) was successfully used. A 1.5-ml sample of PXM culture was harvested and centrifuged. The cell pellet was resuspended with 100 p1 of TE buffer (50 mM Tris, 50 mM EDTA, pH 8.0) and 25 pl of sodium dodecyl sulfate (SDS) solution (saturated in 1 N NaOH) was added to the suspension. The tube was inverted 20 times in 1 min to lyse the cells. A very viscous lysate was obtained. The lysate was sheared by full speed Vortex for 3 min. The viscosity of the sample decreased rapidly during this treatment. The sample was then directly subjected to agarose gel electrophoresis. TBE buffer (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA) was used for the 0.7% agarose gel electrophoresis of the DNA samples. The electrophoresis was run at 40 mA for 3-5 hr at 4°C. The gel was stained with ethidium bromide and was observed under UV light. The following bands were observed from the top to the bottom of the gel in Fig. 3: (A)open circular plasmid DNA, and probably chromosomal DNA; (B) supercoiled closed circular plasmid DNA; (C) linear and nicked plasmid DNA: and (D) chromosomal DNA. This typical distribution of the DNA bands was first suggested by Meyer et al. (1976).Table XI suggests a correlation between the presence of the plasmid and the dicamba-degrading phenotype. Cells were not

DOUGLAS J. CORK AND AMJAD KHALIL

3 04

1 2 3 4 5 6 7 8 9 A B C

D

FIG.3. PI.asmi.d disappearance during incubation of PXM on 20010 pg/ml diccamba medium. The 1method of Wheatcraft (Wheatcraft and Williams, 1981) was used. A, B, and C refer to open circular, supercoiled, and linear, respectively. D is the chromosomal DNA. (1)Lambda marker; (2) 18-hr cells; (3) 20-hr cells; (4) 22-hr cells; (5) 24-hr cells; (6) 26-hr cells; (7) 28-hr cells; (8) 46-hr cells; (9) 96-hr cells.

able to grow when they were transferred back to dicamba-containing solid medium after 49 hr of growth on liquid dicamba medium. Figure 3 illustrates initial curing of the plasmid after 49 hr. Since partial curing of the plasmid was observed at the same time as loss of dicambadegrading ability (after 96 hr of growth), it was suggested that the large plasmid might contain genes coding for dicamba metabolism. TABLE XI CORRELATION OF CELLGROWTH WITH THE: PRESENCE OF THE LARGE PLASMID PDKl Batch growth time (hr)

Absorbance at 600 nm

18 20

0.31 0.40 0.43 0.47

22 24 26

Growth on dicamba solid medium

0.50

28

0.50

49 96

0.48 0.48

Plasmid starts to disappear. D, Detected; PD, partially detected.

Plasmid detection

D D D D D D D' PD

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305

B. CASE METHOD The method of Casse et al. (1979) was used to detect and isolate the covalently closed circular DNA (CCC-DNA)from PXM. In this method, cell lysis was carried out in alkaline buffer at a pH used for denaturation (pH 12.45). Lysis in highly alkaline buffer provides a protein denaturation effect which served to reduce the enzymatic degradation of plasmid DNA. A 50-ml sample of PXM culture was harvested and centrifuged before the end of the exponential phase. The pellet was mixed with 1M NaCl and shaken vigorously for 30 min. Bacteria were washed twice with TE buffer (0.05 M Tris, 0.02 M EDTA, pH 8.0) and the final pellet was weighed and resuspended in TE buffer (100 mg bacteria to 0.5 ml buffer for better reproducibility). The lysing buffer consisted of TE buffer containing 1%(w/v) SDS, adjusted to pH 12.4. To 0.5-ml bacterial suspension in a 50-ml beaker, 9.5 ml of the lysing buffer was added and the mixture was placed on a magnetic stirrer at 100 rpm for 90 sec before incubation at 34°C for 20-25 min. The pH was then lowered to 8.5 to 8.9 by adding 0.6 ml of 2 M Tris buffer (pH 7.0) while stirring the mixture at 100 rpm for 2 min. The lysate was made to 3% (w/v) NaCl and after 30 min at room temperature, 10 ml of phenol [previously saturated with a solution of 3% (w/v) NaCl in water] was added. The two phases were mixed by stirring at 300 rpm for 2 min. The mixture was then centrifuged at 5000g for 10 min and the clear aqueous upper phase was transferred to a new centrifuge tube. It was brought to 0.3 M sodium acetate 2 vol cold ( - 20°C) 95% ethanol was added to precipitate the DNA. The tube was kept at -20°C overnight. The precipitated DNA was recovered by centrifugation at 12,000 g at 4°C for 20 min. The ethanol was removed from the tube and the DNA pellet was dissolved in 300 pl TES buffer, pH 8.0 (0.05 M Tris, 0.05 M EDTA, 0.05 M NaC1). The DNA samples were analyzed immediately by agarose gel electrophoresis or stored at - 20°C until ready for use. Unfortunately, the CCC-DNA recovery was low. Agarose gel electrophoresis of the large plasmid by the Casse Method (Fig. 4, lanes 2 and 3) again revealed a distribution of the plasmid into the three regions as described by Meyer et al. (1976).

c. KADO AND LIU METHOD Approximately 5-10 ml of PXM culture was harvested and centrifuged before the end of the exponential phase, A 3OO-pl sample of lysing solution was added to the cell pellet and mixed gently to resuspend the cells in the lysing buffer. The lysate was incubated at 60-65°C for

DOUGLAS 1. CORK AND AMJAD KHALIL

306

1

2

3

23 kb

FIG.4. Isolation of the large plasmid by the method of Casse et al. (1979). (1) Lambda HindIII; (2) PXM cells from midlog phase; (3) PXM cells from midlog phase.

1hr. The lysate was extracted twice with a 1: 1(v/v)phenol-chloroform mixture. The upper aqueous phase which contained the DNA was transferred to a new tube and then extracted with ether (1 vol) in order to remove excess phenol. The DNA was precipitated by adding 100 pl of 3 M sodium acetate and 800 p1 cold (-20°C) 95% ethanol. The tube was kept at -20°C overnight. The precipitated DNA was recovered by centrifugation at 12,000 g at 4°C for 20 min. The ethanol was removed from the tube and the DNA pellet was dissolved in 100 pl of autoclaved water. The samples were then applied directly to agarose gel for electrophoresis. A low salt buffer system composed of 40 mM Tris-acetate and 2 mM sodium EDTA was used to run the gel. The concentration of the agarose was 0.7%. The electrophoresis was run at 20-25 mA for 4-5 hr. The gel was stained with ethidium bromide (1pg/ml) and DNA was observed under a UV light box. A modification of the method of Kado and Liu for isolation of pDKl was attempted. In this protocol the same steps were used as in the above method (Kado and Liu 1981) except for the following modifications:

CHLOROAROMATIC HERBICIDE-DEGRADING GENES

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1. Cells were lysed at 75°C for 15 min. 2. After the phenol-chloroform extraction step, no ether extraction

was performed. 3. The plasmid DNA was precipitated with isopropanol at room temperature instead of using ethanol at -20°C. These modifications resulted in less chromosomal DNA and more of an enrichment of the large plasmid (Fig. 5). Compared to the Casse Method, the method of Kado and Liu was easier to perform and resulted in reproducible, clear detection of the plasmid. By using highly alkaline SDS lysing buffer (pH 12.5)and high temperature (65"C), the CCC-DNA was released after denaturing the chromosomal DNA with a highly alkaline buffer. Proteins and cell debris were removed by extraction with phenol-chloroform. The clarified extract was used directly for the electrophoretic analysis seen in Fig. 5.

D. ALLENMETHOD Midexponentialphase cells were harvested from one liter of PXM grown on 2000 ppm dicamba in batch culture. The cells were resuspended in 13.5 ml lysis buffer (for 100 ml, 0.9 g glucose, 0.35 g EDTA, 0.3 g Trizma 1

2

3

A

kb 23.1

9.41 6.68

4.36

2.32 2.02

FIG. 5. Isolation of large plasmid by the modified method of Kado and Liu (1981). PXM cells from midlog phase, grown on 2000 pg/ml dicamba. (1) Lambda marker HindIII; (2-5)

308

DOUGLAS J. CORK AND AMJAD KHALIL

base, 25 g sucrose, pH 8.0). Lysozyme was added at 1 mg/ml. The suspension was incubated at 37°C for 15 min. Ten milliliters of alkaline SDS (0.8 g NaOH and 1 g SDS in 100 ml water) was added to the mixture, mixed gently, and kept on ice for 15 min. Then 13 ml of 7.5 M ammonium acetate was added to the mixture and it was kept on ice for 15 min. The mixture was centrifuged at 5000 rpm for 10 min. The supernatant was filtered through cheese cloth into a new sterile centrifuge tube. A i5-ml volume of isopropanol was added to the filtered solution, mixed and kept at room temperature for 15 min, and centrifuged at 5000 rpm for 10 min. The pellet was resuspended in 2 ml 2 M ammonium acetate and kept on ice for 15 min. The supernatant was saved and 2 ml of isopropanol was added; it was kept at room temperature for 10 min and centrifuged at 5000 rpm for 15 min. The pellet was drained for 15 min at room temperature and resuspended in 250 p1 TE buffer. Samples were subjected to 0.7%agarose gel electrophoresis. This method is based on a previously published protocol (Lee and Rasheed, 1990), as well as suggestions from Dr. Larry Allen (ThermoGene Inc., Chicago, IL). Figure 6 (left and right) is a typical agarose gel electrophoresis of the sample prepared by the method of Allen (1994) with very litle chromosomal DNA contamination. In this method, lysis of the cells was performed by both SDS and lysozyme. No highly alkaline pH and no high temperature was used. To eliminate the possibility of shearing of the large plasmid, no phenol-chloroform extraction was performed as phenol may shear the large plasmid. Precipitation of the plasmid DNA was performed with isopropanol at room temperature. The restriction profiles of this plasmid with four different restriction enzymes are shown in Fig. 6, indicating an estimated molecular size of about 250 kb. This estimation was taken from the sum of the fragment sizes after digestion with Sphl (Fig. 6, lane 6), and EcoRI (Fig. 6, lane 3). A standard curve for A DNA digested with BstGII and another curve for A DNA digested with Hind111 were used. IV. Effect of Alternative Carbon Sources on the Stability and Curing of a Large Molecular Weight Plasmid, pDK1

A. NUTRITIONALLY INDUCED INSTABILITY

OF DICAMBA DEGRADATION

PXM was first grown on 2000 pg/ml liquid dicamba medium until the cells reached midexponential phase. These cells were subsequently transferred to gel-rite plates containing dicamba as the sole source of carbon (2000 pg/ml). A transfer was then made to various plates con-

CHLOROAROMATIC HERBICIDE-DEGRADING GENES 1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

0.70% Agarose Gel

1% Agarose Gel

309

kb 8.4

7.2 6.3 5.6 4.8 4.3

3.6

FIG.6. isolation of the large plasmid from PXM by the method of Allen. The left gel is run with 0.7% agarose, while the right gel is 1% agarose. The lanes for both gels are the same. 11) Lambda DNA cleaved with BstGII; (2) pDKl large plasmid purified by method of Allen; (3) pDK1 digested with EcoRI; (4) pDKl digested with BamHI; (5)pDKl digested with HindIIi; (6) pDK1 digested with SphI; (7) pDKl digested with BglII; ( 8 ) pDKl digested with ApaI.

taining one of the several alternative carbon sources listed in Table XII. The cells were subsequently transferred back to medium containing dicamba as the sole source of carbon. The results indicate that the dicamba-degrading phenotype was lost after growth on succinate and benzoate. B. THEEFFECTOF DICAMBA,SALICYLATE, AND SUCCINATE ON BATCH GROWTHAND DICAMBADEGRADATION A 2000 pg/ml dicamba concentration was found to be optimal for batch growth of the organism and a 5% (v/v) inoculum was found to be optimal for successive transfers to liquid medium. When dicamba is limited to 1000 pg/ml, it is completely degraded with a corresponding release of approximately 8.1 mM free chloride and accumulation of biomass (Fig. 7). Typical growth curves of PXM on 2000 ppm dicamba are shown in Figs. 8 and 9.

DOUGLAS J. CORK AND AMJAD KHALIL

310

TABLE XI1 THE EFFECTOF A PREVIOUSLY USED CARBON SOURCE ON

SUBSEQUENT DICAMBA-DEGRADING ABILITY Transfer from a specific carbon source to second sole carbon source

Ability to grow

+ + + + + +

Dicamba to DCSA DCSA to dicamba Dicamba to succinate Succinate to dicamba DCSA to succinate Succinate to DCSA Dicamba to salicylate Salicylate to dicamba Dicamba to benzoate Benzoate to dicamba DCSA to benzoate Benzoate to DCSA

+

-

+ -

In order to correlate the presence of different limiting carbon sources with the presence of the large plasmid, several growth experiments were performed. For example, batch growth of PXM was analyzed in flasks containing 2000 pg/ml dicamba, 500 pglml salicylate, or 1000 pg/ml succinate as the growth-limiting substrate. Cell growth was measured by optical density at 600 nm, viable plate counting, and direct

O.O

c .OO

0

0

-0.60

In

>.

t v)

z

I,. -0.40

1

-d

0.20

TIME (hr)

* OICAMEA

* CHLORIDE

-

OPT.

DENS.

FIG.7. Conversion of dicamba into free chloride and biomass.

u

t0

CHLOROAROMATIC HERBICIDE-DEGRADING GENES

311

1 0.9

0.8 E C

8

ID

5 w

$

0.7 0.6

0.5

a 0.4

32 0.3 0.2 0.1

0

5

0

15

10

25 30 TIME (hr)

35

40

45

FIG.8. Growth curve of PXM on 2000 pg/ml Dicamba.

hemocytometer counting. Typical growth curves for PXM on 500 pgl ml salicylate and 1000 pg/ml succinate are shown in Figs. 10 and 11, respectively. As a function of time of growth on these sources of carbon, the cells were lysed and examined for the presence of the large plasmid. The data, summarized in Table XIII, suggest a succinate-dependent plasmid curing time. This curing time was quantitated in terms of 40

35

30 v)

Aw -g25 0

UI?

2

.20 El5

z

10 5 0

0

5

10

15

25

30

35

40

45

TIME (hr)

-+Vioble Cell Count

Direct Cen Count

+

FIG.9. Normal cell growth of PXM on 2000 pglml dicamba.

DOUGLAS J. CORK AND AMJAD KHALIL

312 a""

800 700

9

58 2

600

?so0 g400

9

300 200

100 0

0

5

3

7

9

11

13

15

17

TYAE (hr)

I -m-

VioMe

CSII

count

+Direct

cet~count

I

FIG.10. Typical growth curve of PXM on 500 pg/ml salicylate.

number of cell doublings which occurred during exponential growth on the designated carbon source. Table XI11 shows the effect of the sole carbon source on the specific growth rate (p),the corresponding generation time (tgen),and the detection of the large-plasmid pDKl. In general, specific growth rate and plasmid curing were enhanced by

1

0.9 0.8

E 8 W t4

0.7

0.6

w

Y 0.5 sa 0.4

8 0.3 9

0.2 0.1

0 D

3

5

7

10

TIME (hr)

FIG.11. Growth curve of PXM on 1000 pg/ml succinate as a sole carbon source.

313

CHLOROAROMATIC HERBICIDE-DEGRADING GENES TABLE XI11

THEEFFECTOF LIMITING SUBSTRATE ON SPECIFIC GROWTH RATE. GENERATION TIME,AND DETECTION OF THE LARGE PLASMID P

t,,

Substrate (mM)

[Specific growth rate

(hr-91

[Generation time (hr)]

Dicamba (9 mM) Salicylic acid (3.6 mM) Succinate (9 mM)

0.091 0.207 0.425

7.62 3.35 1.63

Detection of the plasmid

+ + -a

Note. Data obtained from Figs. 8-10, After 20 doublings in succinate-limited batch medium, the plasmid begins to disappear.

succinate. Growth rate is enhanced and the plasmid is stabilized by salicylic acid.

c. THEEFFECTOF SUCCINATE CONCENTRATION ON BATCHGROWTHAND DICAMBA DEGRADATION Dicamba (2000 pg/ml) minimal medium was prepared in the presence of 4.5 mM succinate. Figure 1 2 shows a 1.5-fold enhancement in cell growth rate and complete elimination of the dicamba. These preliminary data suggest a relationship between succinate utilization and dicamba 2400 1

P2000 P

(2.4 2.0

t\

Y

0

1.6

1600-

g

2

v

4

8

p t

1200-

1.2

000-

0.8

400-

0.4

O

0

Y

1

5

10

15

20

25

- --

30

#

2 i 8

0.0 35

FIG.12. Cell growth and dicamba depletion in dicamba medium containing 4.5 mM succinate.

314

DOUGLAS J. CORK AND AMJAD KHALIL

degradation. The addition of either 9 mM succinate (Fig. 13) or 18 mM succinate (Fig. 14)reveals a succinate-dependent repression of dicamba degradation. It is suggested that this is indirectly due to succinate curing of pDKl. Dicamba degradation is completely repressed when 18 mM succinate is present in the medium. These data suggest the following: 1. Preferred utilization of succinate over dicamba by PXM at high succinate concentration. 2. Initial enhancement of dicamba degradation at low succinate concentrations. 3. Complete suppression of dicamba degradation at higher succinate concentration.

D. SUCCINATE-DEPENDENT CURING OF pDKl Washed PXM cells were grown on 1000 pglml succinate through six successive batch experiments. This is approximately equivalent to 20 cell doublings. The plasmid profile of these cells is shown in lane 4 of Fig. 15. Compared to DCSA-, salicylate-, and dicarnba-grown cells, it is evident that curing of pDKl occurs after such extended subculturing on succinate-limited medium. This curing is observed as a significant deletion or loss of the pDKl as seen in Fig. 15, lane 4, compared to the plasmid profile in lanes 3, 5 , and 6, respectively. Lane 4 in Fig. 15 shows one lower band which is chromosomal DNA, confirmed by the appearance of smearing when cut with restriction enzymes.

I

2400 3

(2.4

2.0 1.6

0

2

v

0.8

i

0.4

E8

le2

2

350.0

4

MCAMBA (ug/d) +OPTICAL DENSITY

FIG.13. Cell growth and dicamba depletion in dicamba medium containing 1000 pgl ml succinate.

315 2400 1 -

(2.4 ,2.4

P

-2.0

-1.6

E s 0

W

t

-1.2

-0.8 4

-0.4

0

5

4

10

15

20

25

30

b

g 2

4

F8

A35 o 70.0 .0 35

DICAMBA (ug/ml) +OPTICAL DENSITY

FIG.14. Cell growth and dicamba depletion in dicamba medium containing 2000 pg/ ml succinate.

FIG. 15. The effect of succinate-limited growth on curing of the large plasmid pDKl from PXM. (A) Open circular, (B) linear, (C) supercoiled, (D) chromosomal. (1) Lambda DNA, uncut; (2) lambda DNA cleaved with HindIII; (3) complete pDKl from PXM grown on 2000 pg/ml dicamba; (4) complete pDKl from PXM grown on 1000 pg/ml succinate; (5) complete pDKl from PXM grown on 500 pgIm1 salicylate; (6) complete pDKl from PXM grown on 500 pg/ml DCSA.

316

DOUGLAS J. CORK AND AMJAD KHALIL

Figure 16 shows that, unlike succinate, there is no dose-dependent curing of large-plasmid pDKl by salicylate. When PXM is grown on 2000 pg/ml dicamba in the presence of variable concentration of salicylate (300 or 600 pg/ml), pDKl is detected (lanes 2, 3, 4, 5, 7, 8, 9, 11, 12,13). When the culture reached stationary phase, the plasmid began to disappear (lanes 6, 10, 11, 15, 16, 17). Figure 17 shows the SDS-PAGE protein profile for PXM grown on different carbon sources including dicamba (lane 2), salicylate (lane 3), or succinate (lane 4). These differences indicate a significant change 1

3

?

d

5

6

7

8

9

10

11

12 13

14

15 16

17

18

FIG.16. The effect of 2000 pg/ml dicamba with salicylate on the detection of pDKl of PXM. The method of Wheatcraft (Wheatcraft and Williams, 1981) was followed. (A) Plasmid DNA (pDKl), (B) chromosomal DNA. (1)Lambda Hind111 marker; (2) PXM cells on 2000 pg/ml Dic. at 18 hr; (3) PXM cells on 2000 pglml Dic. at 22 hr; (4) PXM cells on 2000 pg/ml Dic. at 26 hr; (5)PXM cells on 2000 pg/ml Dic. at 30 hr; (6) PXM cells on 2000 pg/ml Dic. at 34 hr; (7) PXM cells on 2000 pg/ml Dic.+300 pg/ml Sa1./22 hr; (8) PXM cells on 2000 pg/ml Dic.+300 pg/ml Sa1.126 hr; (9) PXM cells on 2000 pg/ ml Dic.+300 pg/ml Sa1./30 hr; (10) PXM cells on 2000 pg/ml Dic.+300 pg/ml Sal.1 34 hr; (11) PXM cells on 2000 pg/ml Dic.+300 pg/ml Sa1./38 hr; (121 PXM cells on 2000 pg/ml Dic.+6OO pg/ml Sa1./22 hr; (13) PXM cells on 2000 pg/ml Dic.+6OO pg/ml Sa1./26 hr; (14) PXM cells on 2000 pg/ml Dic.+6OO pg/ml Sa1./30 hr; (15) PXM cells on 2000 pg/ml Dic.+6OO pg/ml Sa1.134 hr; (16) PXM cells on 2000 pg/ml Dic.+600 pg/ml Sa1./38 hr; (17)PXM cells on 2000 pg/ml Dic. at stationary phase; (18) PXM cells on 1000 pg/ml succinate at 26 hr.

CHLOROAROMATIC HERBICIDE-DEGRADING GENES 1

2

3

317

4

kd 66

45

29

14.3

FIG. 17. SDS-PAGE profile from PXM grown on dicamba, salicylate, or succinate. (1) Protein size markers (bovine albumin, 66 kDa; egg albumin, 45 kDa; trypsinogen, 29 kDa; P-lactoglobin, 18.4 kDa; lysozyme, 14.3 m a ) ; (2) sample from PXM grown on 2000 pglml dicamba; (3) sample from PXM grown on 500 pg/ml salicylate; (4) sample from PXM grown on 1000 pg/ml succinate.

in the protein expression when these microorganisms switched from dicamba to salicylate or succinate as carbon sources. Some of these differences may be due to proteins encoded by pDKl. V. Summary

Dicamba is used as a model system for microbial degradation of chloroaromatic benzoic acids. The detection, isolation, and stability of a megaplasmid within a Pseudomonas sp. is described as the first step in optimizing the growth of this microorganism and other microorganisms similar to it. A large plasmid, pDK1, consisting of approximately 250 kb, was purified from dicamba-degrading Pseudomonas sp. PXM. This plasmid was purified by the method of Allen (personal communication, 1994), which is a modified version of several that have been attempted for the isolation of large plasmids (Lee and Rasheed, 1990).

318

DOUGLAS J. CORK AND AMJAD KHALIL

The restriction analysis of this plasmid (pDK1) from PXM. revealed many distinctive bands on agarose gel electrophoresis. Based on the preliminary restriction enzyme analysis, the estimated size of this plasmid is 250 kb, which could make it one of the largest procaryotic plasmids encoding for chloroaromatic degrading enzymes. Allen’s methodology results in very high purity and reproduciblity compared to the other methods used in this study. As described in this work, the method of Kado and Liu (1981) is easier to perform and results in a more reproducible plasmid preparation than the method of Casse et al. (1979).Casse’s protocol requires the use of a highly alkaline SDS solution (pH 12.45) in order to eliminate the chromosomal DNA. However, only incomplete removal of the chromosomal DNA results. Compared to the Casse et al. protocol, the Kado and Liu protocol requires the use of a highly alkaline solution (pH 12.6)and a high temperature (55-65°C) to eliminate the chromosomal DNA. This results in a nearly complete removal of the chromosomal DNA. The high temperature treatment also quickly eliminates the RNA. Another advantage of the protocol of Kado and Liu over the protocol of Casse et al. is that the former uses phenol-chloroform extraction while the latter uses only phenol extraction. The phenol-chloroform extraction step denatures the DNA along with the proteins. In addition to this, the phenol-chloroform mixture minimizes the formation of a brown oxidation pigment that usually occurs with phenol extraction alone. Finally, the time needed to complete the Kado and Liu protocol is much shorter (2 hr) than the time needed to complete the Casse protocol (8 hr). As described previously, a highly purified plasmid preparation with minimal chromosomal DNA was prepared by following the suggestions of L. Allen. Compared to the other protocols, it is suggested that this protocol yields an extremely pure plasmid DNA preparation for the following reasons: 1. The lysis solution includes sucrose and lysozyme which gently break down the cell wall. This is followed by use of the alkaline SDS solution to lyse the cells. The other protocols require the use of alkaline SDS without lysozyme. 2. The use of 7.5 M ammonium acetate neutralizes the pH of the cell lysate and facilitates the interaction of proteins to the SDS. 3. No phenol-chloroform or phenol extraction is used in this protocol. Use of this step sometimes results in resistance to restriction enzyme digestion. 4. The plasmid DNA pellet can dissolve faster in the TE buffer or sterile double-deionized water after the plasmid DNA is precipitated with isopropanol at room temperature. 5. The total time for DNA purification is about 3 hr.

CHLOROAROMATIC HERBICIDE-DEGRADING GENES

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Curing and instability of this plasmid occur when PXM is grown on succinate or other rich alternative sources of reduced carbon including complex media such as LB or TSA. This curing results in the inability of PXM to grow back on dicamba (loss of dicamba phenotype). Curing also occurs when cells growing on dicamba reach stationary phase. Once PXM reaches late stationary phase, the plasmid is cured and subsequent subculturing on dicamba minimal medium is usually unsuccessful. These observations suggest that the pDKl is essential for the dicamba-degrading ability of PXM. Growth of PXM on salicylate or succinate and the effect of these compounds on dicamba degradation and the stability of pDKl were investigated. Growth of PXM on 500 pg/ml of salicylate stabilizes the plasmid and dicamba-degrading ability, while growth on succinate results in curing and instability of pDK1. Once the specific genes for the dicamba metabolism have been identified and isolated, it will be feasible to consider the cloning of these genes into a well understood, stable organism that could be used for soil bioremediation and treatment. This would allow for the quick and efficient breakdown of dicamba and protection of dicamba-susceptible species such as soybeans. The cloning of these genes into dicambaresistant plants is an area of ongoing, intensive investigation. Partial deletion versus lethal deletion of pDKl in PXM as a function of alternative, growth-limiting nutrients is also an area of ongoing investigation. Lethal deletion of the pDKl results in the inability of PXM to grow back on dicamba. This study suggests that this plasmid may be one of the largest chloroaromatic-degrading plasmids yet detected, and may be stabilized by vigorous environmental control and genetic manipulation.

ACKNOWLEDGMENTS The authors would like to acknowledge the laboratory assistance of Dr. Ben Stark,

IIT;Dr. George Gaines 111, Isogenetics Corp.; and Dr. Larry Allen, Thermogene Corp. REFERENCES Apajalahti, J. H. A., and Salkinja-Salonen, M. S. (1986).Appl. Microbiol. Biotechnol. 25, 62-67.

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Chakrabarty, A. M. (1976).Annu. Rev. Genet. 10, 7-30. Chatterjee, D. K., and Chakrabarty, A. M. (1982).Mol. Gen. Genet. 188, 279-285. Chatterjee, D. K.,Kellogg, S. T., Hamada, S., and Chakrabarty, A.M. (1981).J. Bacteriol. 146,639-646.

Chaudhry, G. R., and Chapalamadugu, S. (1991).Microbiol. Rev. 55, 59-79. Cork, D. J., and Krueger, J. P. (1991).Adv. Appl. Microbiol. 36, 1-66. Cork, D.J., Khalil, A., and Ofiara, K. R. (1992).Gas, Oil, and Environmental Biotechnology. “Proceedings of the Institute of Gas Technology Symposium, Chicago,” (Akin, C., Smith, J., and Markuszewski, L., eds.), Vol. V (Sept.),pp. 15-30. IGT Press, Chicago. Don, R. H., and Pemberton, J.M. (1981).J. Bacteriol. 991, 681-686. Duetz, W. A., and van Andel, J. G. (1991).J. Gen. Microbiol. 137, 1369-1374. Duetz, W. A,, Winson, M. K., van Andel, J, G., and Williams, P. A. (1991).J. Gen. Microbiol. 137,1363-1368.

Fogarty, A. M., and Tuovinen, 0. H. (1995).J. Ind. Microbiol. (in press). Franklin, F. C. H., and Bagdasarion, M., and Timmis, K. N. (1981).“Microbial Degradation of Xenobiotics and Recalcitrant Compounds,” pp. 107-130.Academic Press, London. Fujimoto, S. (1991).M. S. Thesis, Illinois Institute of Technology, Chicago. Furukawa, K., Simon, J. R., and Chakrabarty, A. M. (1983).J. Boct. 154,1356-1362. Furukawa, K., and Miyazaki, T. (1986).J. Bacteriol. 166, 392-398. Furukawa, K., Hayase, N., Taira, K., and Tomizawa, N. (1989). J. Bacteriol. 171, 5467-5472.

Hartmann, J., Reinke, W., and Knackmuss, H. J. (1979).Appl. Environ. Microbiol. 37, 421-428.

Holmes, D. S., and Quigley, M. (1981).Anal. Biochem. 114,193. Johnson, B. F., and Stanier, R. Y. (1971).J. Bacteriol. 107,468-475. Kado, C. I., and Liu, S. T. (1981).J. Bacteriol. 145, 1365-1373. Kamin, H., White-Stevens, R. H., and Presswood, R. P. (1976).In “Methods in Enzymology,” (Fleishers and Packer, L.), Vol. 53. Academic Press, New York. 527-543. Klages, U., Markus, A., and Lingens, F. (1981).J. Bacteriol. 146, 64-68. Krueger,J. P., Butz, R. G., Atallah, Y. H., and Cork, D. J. (1989).Isolation and Identification of Microorganisms for The Degradation of Dicamba. J. Agric. Food Chem. 37,534-538. Krueger, J. P., Butz, R. G. and Cork, D. J. (1990).Specificity of a Flavobacterium in The Metabolisms of Substituted Chlorobenzoates. J. Ind. Microbiol. 5, 147-152. Krueger, J. P., Butz, R. G., and Cork, D. J. (1991a).J. Agric. Food Chem. 39, 995-999. Krueger, J. P., Butz, R. G., and Cork, D. J. (1991b).J. Agric. Food Chem. 39, 1000-1003. Lee, S. Y., and Rasheed, S. (1990).BioTechniques 9, 676-679. Lloyd-Jones, G.,DeJong, C., Ogden, R. C., Duetz, W. A., and Williams, P. A. (1994).Appl. Environ. Microbiol. 60,691-696. Lunt, D., and Evans, W. C. (1970).The microbial metabolism of biphenyl 54p. Biochem. J.118,54-55. Maniatis, R. T., Fritsch, E. F., and Sambrook, J. (1989).“Molecular Cloning: A Laboratory Manual, Second Edition,” Laboratory Press, Cold Spring Harbor, N.Y. Meyer, J. A., Sanches, D., Elwell, L. P., and Falkau, S. (1976).J. Bacteriol. 127,1529-1537. Ofiara, K. R. 1990.M. S. Thesis, Illinosis Institute of Technology, Chicago. Pierce, G. E., Facklam, T. J., and Rice, M. J. (1981).Dev. Ind. Microbiol. 22,401-408. Pierce, G. E., Robinson, J. B., and Colaruotolo, J. R. (1983).Dev. Ind. Microbiol. 24, 499-507.

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INDEX

A

Acetovibrio cellulolyticus, cellulolytic enzyme source, 3,5-6 Activated sludge, genetically engineered microorganism effects, 248-249 Agglutinin, in milk, 53-56 Alcaligenes eutrophus, genetically engineered, in aquatic environments, 247-248

Allen plasmid isolation method, from Pseudomonos, 307-308 Antibiotics, product development, 166-169

Aquatic environments, genetically engineered microorganism effects, 245-248

Aromatic compounds, see Chloroaromatic herbicide-degrading genes Aspergillus, cellulolytic enzyme source, 3-5,15

Avena sativo, genetically engineered microorganism effects, 262

B Bacillus cellulolytic enzyme source, 3, 5-6, 13 growth inhibition in milk, 70-78 Bacteria, see also specific bacteria cehlolytic enzyme source, 3,5-6 growth in milk, 45-83 antimicrobial systems applications, 81-82

cow ration effects, 69-71 early studies, 47-48 inhibition bacilli, 78-79 effect on milk quality, 71-72 enterobacteria, 77-78 lactic acid bacteria, 53-69 leptospiras, 80

mycoplasmas, 80-81 propionibacteria, 79 staphylococci, 79-80 lactenin studies, 48-53 lactic acid bacteria agglutinin effects, 53-56 autoinhibition by hydrogen peroxide, 60-61 culture manipulation, 67-68 heated growth medium, 65-67 lactoferrin role, 62 lactoperoxidase system, 56-60 other inhibitors, 62-63 seasonal activity variation, 63-65 starter cultures, 68-69 stimulators, 72-76 thiocyanate role, 61 stimulators, 72-76 commercial substances, 76 complex substances, 74-76 components, 72-73 minerals, 73 Biocatalysts, discovery sources in biotechnology, 108-113 classification, 108-110 enzyme-based, 110-1 12 organismic, 110 processing, 112-113 reactors, 112-113 Biotechnolopy, commercial, 95-233; see also Genetically engineered microorganisms biocatalysts, 108-113 classification, 108-110 enzyme-based, 110-1 12 organismic, 110 processing, 112-113 reactors, 112-1 13 discovery and development cycle commercialization pathway, 114-115 internal interactions, 149-152 market discovery versus development, 151-152

323

324

INDEX

product discovery versus process discovery, 150 product discovery versus product development, 149-1 50 markets, 148-149 process, 141-148 approaches, 141-143 biotransformations, 143-145 examples, 145-148 products, 115-141 approaches, 131-140 classification, 115-120 discovery versus manufacturing routes, 121-125 industrial sector products, 128-131

new chemical class discovery, 125-127

plant product discovery, 127-128 screens, 115-121 serendipity, 140 top 20 drugs, 140-141 industrial aspects, 97-99 interdisciplinary nature, 96-97 market classification, 113-114 market development, 209-227 concepts commercialization, 224-227 follower strategy, 214-215 marketlprocess matrix, 217-218 market segment selection, 222 new market development, 223 product categories, 219-222 product cycle, 212-214 product/market strategy, 219 product matrix, 216-217 product screening, 222-223 product selection, 222-223 product strategies, 219-222 risk/benefit analysis, 215 current status, 210-212 holistic approach, 209-210 market discovery, 148-149 outlook, 227-233 overview, 95-107 industrial aspects, 97-99 interdisciplinary nature, 96-97 process systems, 95-96 systems approach, 99-107

process development, 173-209 alternatives, 176-186 geometric scale-up, 179-181 new product scale-up, 182-184 reactor hardware versus software alternative, 185-186 analysis versus synthesis, 176 control, 203-204 optimization, 203-204 plant design, 204-208 scale-up geometric, 179-181 multivariable screening, 194 new products, 182-184 pattern analysis, 195-196 qualitative methods, 188-193 quantitative methods, 194-203 regime analysis, 199-201 scale-down analysis, 201-203 specific rate analysis, 196-198 statistical analysis, 194 stages, 174-176 technology transfer, 209 process discovery, 141-148 approaches, 141-143 biotransformations, 143-145 examples, 145-148 process systems approach, 99-107 overview, 95-96 product development, 156-173 constraints, 156-1 59 delivery concepts, 162-164 examples, 166-173 environmental services, 172-173 insulin discovery and development, 169-172 from penicillin to new class of antibiotics, 166-169 microbial route development, 165-166

product formulation, 162-164 resource statistics, 159-162 funnel diagram, 160-161 inverted triangle concept, 161-162 pipeline concept, 159-160 strategies, 156-159 product discovery, 115-141 approaches, 131-140

325

INDEX classification, 115-120 discovery versus manufacturing routes, 121-125 new chemical class discovery, 125-127 other industrial sector products, 128-131 plant product discovery, 127-128 screens, 115-1 2 1 serendipity, 140 top 20 drugs, 140-141 regulation of genetically engineered microorganisms, 2 74-280 Biotransformation environmental, 289-300 dicamba, 291-295 other chlorinated and nonchlorinated compounds, 295-300 process discovery, 143-145 Biphenyl hydrocarbons, in catabolic regulation, 296-297

C

Casse plasmid isolation method, from Pseudornonas, 305 Cellobiohydrolases molecular properties, 7-10 synergistic properties, 20-24 Cellulases, microbial, 1-35 biosynthesis, 26-34 localization, 30-31 in recombinant cells, 31-34 regulation, 26-30 catalytic properties, 15-26 catalytic mechanisms, 24-26 glycosylation, 19-20 structural organization, 15-19 synergistic mechanisms, 20-24 enzyme sources, 2-6 bacterial, 5-6 fungal, 3-5 future prospects, 34-35 molecular properties, 6-15 cellobiohydrolases, 7-10 endoglucanases, 10-13 p-glucosidases, 13-15

Cellulornonas fimi in cellulolytic enzyme analysis, 12, 18, 20 cellulolytic enzyme source, 3, 5-6 Cellvibrio gilvus, cellulolytic enzyme source, 3, 5-6, 15 Chemicals, commodities, see Biotechnology, commercial Chemical services, see Biotechnology, com m ercia1 Chloroaromatic herbicide-degrading genes, in Pseudomonas species, 289-319 alternative carbon source effects, 308-317 dicamba, 308-317 salicylate, 309-313 succinate, 309-317 large plasmid isolation, 301-308 Allen method, 307-308 Casse method, 305 Kado and Liu method, 305-307 Wheatcraft method, 302-304 models, 290-300 dicamba metabolism, 291-295 environmental biotransformations, 295-300 overview, 289-290 Clostridiurn thermocellum in cehlolytic enzyme analysis, 15, 18, 25-26 cellulolytic enzyme source, 3, 5-6 Commercial biotechnology, see Biotechnology, commercial Commodity chemicals, see Biotechnology, commercial

D Dicamba, in Pseudomonas chlorinated aromatic metabolism model, 289-295 degradation nutritionally induced instability, 308-309 salicylate effects on batch growth, 309-313 succinate effects on batch growth, 309-314

326

INDEX

Discovery, see Biotechnology, commercial, discovery and development cycle Drug research, see Biotechnology, commercial E Endoglucanases catalytic mechanisms, 26 molecular properties, 6,10-13 synergistic properties, 20-24 Enterobacteria, growth inhibition in milk, 77-78

Environmental biotransformation, see Biotransformation, environmental Environmental services, see Biotechnology, commercial Enzymes bacterial, 5-6 biocatalysts, 110-112 cellulolytic, see Cellulases, microbial fungal, 3-5 Erwinia carotovora, genetically engineered, in aquatic environments, 245-247

F Follower strategy, in biotechnology market development, 214-215 Fungi, cellulolytic enzyme source, 3-5 Funnel diagram, product development model, 160-161 Fusarium solani in cellulolytic enzyme analysis, 20-24 cellulolytic enzyme source, 3-5 G

Genetically engineered microorganisms, see also Biotechnology, commercial in activated sludge, 248-249 in aquatic environments, 245-248 overview, 237-245, 267-280 in plants, 261-267 regulation, 274-280

in soil, 249-261 strain development, product discovery approach, 131-132 Geometric scale-up, in biotechnology process development, 179-181 /3-Glucosidases catalytic mechanisms, 24-25 m o h u l a r properties, 13-15 synergistic properties, 20-24 Glycine max, genetically engineered microorganism effects, 265 Glycosylation, by microbial cellulases, 19-20

Growth inhibitors, bacteria in milk bacilli, 78-79 effect on milk quality, 71-72 enterobacteria, 77-78 hydrogen peroxide, 60-61 lactic acid bacteria, 53-69 leptospiras, 80 mycoplasmas, 80-81 propionibacteria, 79 staphylococci, 79-80 thiocyanate, 61 Growth stimulators, bacteria in milk, 72-76

commercial substances, 76 complex substances, 74-76 components, 72-73 minerals, 73 pancreas extract, 74-75

H Haloaromatic compounds, see Chloroaromatic herbicide-degrading genes Herbicide-degrading genes, see Chloroaromatic herbicide-degrading genes Hybridoma technologies, see Biotechnology, commercial Hydrogen peroxide, bacterial growth inhibition in milk, 60-61

I

Industry, see Biotechnology, commercial Inhibitors, see Growth inhibitors

INDEX Insulin, discovery and development, 169-172

Inverted triangle concept, product development model, 161-162

K Kado and Liu plasmid isolation method, from Pseudomonas, 305-307

L

327

inhibition bacilli, 78-79 effects on milk quality, 71-72 enterobacteria, 77-78 lactic acid bacteria, 53-69 leptospiras, 80 mycoplasmas, 80-81 propionibacteria, 79 staphylococci, 79-80 lactenin studies, 48-53 lactic acid bacteria, 53-69 agglutinin effects, 53-56 autoinhibition by hydrogen peroxide, 60-61

Lactenin, bacterial growth studies in milk, 48-53 Lactic acid bacteria, growth in milk agglutinin effects, 53-56 autoinhibition by hydrogen peroxide, 60-61

culture manipulation, 67-68 heated growth medium, 65-67 lactoferrin role, 62 lactoperoxidase system, 56-60 other inhibitors, 62-63 seasonal activity variation, 63-65 starter cultures, 68-69 stimulators, 72-76 thiocyanate role, 61 Lactoferrin, bacterial growth inhibition in milk, 62 Lactoperoxidase system, bacterial growth inhibition in milk, 56-60 Leptospiras, growth inhibition in milk, 80 Loliurn perenne, genetically engineered microorganism effects, 262

M Marketing, see Biotechnology, commercial, market development Medicago sativa, genetically engineered microorganism effects, 265 Milk, bacterial growth, 45-83 antimicrobial systems applications, 81-82

cow ration effects, 69-71 early studies, 47-48

culture manipulation, 67-68 heated growth medium, 65-67 lactoferrin role, 62 lactoperoxidase system, 56-60 other inhibitors, 62-63 seasonal activity variation, 63-65 starter cultures, 68-69 stimulators, 72-76 thiocyanate role, 61 stimulators, 72-76 commercial substances, 76 complex substances, 74-76 components, 72-73 minerals, 73 Minerals, bacterial growth stimulation in milk, 73 Molecular recognition, product discovery, 132-134 Multivariable screening, in biotechnology process development, 194 Mycoplasmas, growth inhibition in milk, 80-81

P Pancreas extract, bacterial growth stimulation in milk, 74-75 Pattern analysis, in biotechnology process development, 195-196 Penicillium funiculosum in cellulolytic enzyme analysis, 20-24 cellulolytic enzyme source, 3-5 Pharmaceutical research, see Biotechnology, commercial

328

INDEX

Phaseolus vulgaris, genetically engineered microorganism effects, 262-263

Pipeline concept, in product development, 159-160 Pisum sativum, genetically engineered microorganism effects, 266-267 Plant design, in commercial biotechnolo n , 204-208 Plants, genetically engineered microorganism effects, 261-267 Plasmids isolation from Pseudomonas, 301-308 Allen method, 307-308 Casse method, 305 Kado and Liu method, 305-307 Wheatcraft method, 302-304 pDK1 nutritionally induced instability of dicamba degradation, 308-309 salicylate effects on batch growth, 309-313

succinate effects on batch growth, 309-314

Problem-solving techniques, in biotechnology research and development, 102-107

Propionibacteria, growth inhibition in milk, 79 Pseudomonas cellulalytic enzyme source, 3, 5-6, 12 chloroaromatic herbicide-degrading genes, 289-319 alternative carbon source effects, 308-317

batch growth with dicamba, 309-313

instability of dicamba, 308-314 salicylate, 309-313 succinate, 309-317 large plasmid isolation, 301-308 Allen method, 307-308 Casse method, 305 Kado and Liu method, 305-307 Wheatcraft method, 302-304 models, 290-300 dicamba metabolism, 291-295 environmental biotransformations, 295-300

overview, 289-290

genetically engineered species in activated sludge, 248-249 in aquatic environments, 247-248 on plants, 261, 263 in soil, 253-259, 268, 272-273

R Regime analysis, in biotechnology process development, 199-201 Risk/benefit analysis, in biotechnology market development, 215

S

Salicylate, haloaromatic transformations in Pseudomonas biochemical mechanisms, 299-300 effects on batch growth, 309-313 Scale-up, in biotechnology process development geometric, 179-181 multivariable screening, 194 new products, 182-184 pattern analysis, 195-196 qualitative methods, 188-193 quantitative methods, 194-203 regime analysis, 199-201 scale-down analysis, 201-203 Screening multivariable, 194 new products, 222-223 in product discovery, 115-121 random, 131 Selective isolation, product discovery, 131

Sludge, genetically engineered microorganism effects, 248-249 Soil, genetically engineered microorganism effects, 249-261 Staphylococci, growth inhibition in milk, 79-80

Stimulators, see Growth stimulators Streptomyces lividans, genetically engineered, in soil, 250-253 Streptomyces viridosporus, genetically engineered, in soil, 250-253

329

INDEX Succinate, haloaromatic transformations in Pseudomonas effects on batch growth, 309-314 pDK1 curing, 314-317

T Thinking methods, in biotechnology research and development, 103-105 Thiocyanate, bacterial growth inhibition in milk, 61 Trichoderma reesei cellulase biosynthesis, 27-35 cellulolytic enzymes analysis, 13-19, 25-26 sources, 3-5, 8

V Vitamins, bacterial growth stimulation in milk, 75-76

W Wheatcraft plasmid isolation method, from Pseudomonas. 302-304

2 Zea mays, genetically engineered microorganism effects, 263

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CONTENTS OF PREVIOUS VOLUMES

Volume 30

Interactions of Bacteriophages with Lactic Streptococci Todd B. Klaenhammer Microbial Metabolism of Polycyclic Aromatic-Hydrocarbons Carl E. Cerniglia Microbiology of Potable Water Betty H. Olson and h s l o A. Nagy Applied and Theoretical Aspects of Virus Adsorption to Surfaces Charles P. Gerba Computer Applications in Applied Genetic Engineering Joseph L. Modelevsky Reduction of Fading of Fluorescent Reaction Product for Microphotometric Quantitation G. L. Picciolo and D. S . Kaplan INDEX

Apparatus and Methodology for Microcarrier Cell Culture S. Reuveny and R. W. Thoma Naturally Occurring Monobactams William L. Parker, Joseph O'SulIivan, and Richard B. Sykes New Frontiers in Applied Sediment Microbiology Douglas Gunnison Ecology and Metabolism of Thermomatrix thiopara Daniel K. Bmnnan and Douglas E. Caldwell Enzyme-Linked Immunoassays for the Detection of Microbial Antigens and Their Antibodies John E. Herrmann The Identification of Gram-Negative, Nonfermentative Bacteria from Water: Problems and Alternative Approaches to Identification N. Robert Ward, Roy L. Wolfe, Carol A. Justice, and Betty H. Olson INDEX

Volume 31 Genetics and Biochemistry of Clostridium Relevant to Development of Fermentation processes Palmer Rogers

Volume 32

Microbial Corrosion of Metals Warren P. Iverson

The Acetone Butanol Fermentation B. McNeiJ and B. Kristjansen

Economics of the Bioconversion of Biomass to Methane and Other Vendable Products Rudy J. Wodzinski, Robert N. Gennaro, and Michael H. Scholla

Survival of, and Genetic Transfer by, Genetically Engineered Bacteria in Natural Environments G. Stotzky and H. Babich 331

332

CONTENTS OF PREVIOUS VOLUMES

The Microbial Production of 2.3Butanediol Robert J. Magee and Nain Kosoric

Recovery of Bioproducts in China: A General View Xiong Zhenping

Microbial Sucrose Phosphorylase: Fermentation Process, Properties, and Biotechnical Applications Erick J. Vandamme, Jan Van Loo, Lieve Machtelinckx, and Andre De Laports

INDEX

Antitumor Anthracyclines Produced by Streptomyces peucetius A. Grein INDEX

Volume 33

The Cellulosome of Clostridium thermocellum Raphael Lamed and Edward A. Bayer Clonal Populations with Special Reference to Bacillus sphaericus Samuel Singer Molecular Mechanisms of Viral Inactivation by Water Disinfectants A. B. Thurman and C. P. Gerba Microbial Ecology of the Terrestrial Subsurface William C. Ghiorse and John T. Wilson Foam Control in Submerged Fermentation: State of the Art N. P. Ghildyal, B. K. Lonsane, and N. G. Karanth Applications and Mode of Action of Formaldehyde Condensate Biocides H. W. Rossmoore and M. Sondossi Occurrence and Mechanisms of Microbial Oxidation of Manganese Kenneth H. Nealson, Bradley M. Tebo, and Reinhardt A. Rosson

Volume 34

What’s in a Name?-Microbial Secondary Metabolism J. W. Bennett and Ronald Bentley Microbial Production of Gibberellins: State of the Art P. K. R. Kumar and B. K. Lonsane Microbial Dehydrogenations of Monosaccharides MiloS Kulhdnek Antitumor and Antiviral Substances from Fungi Shung-Chang Jong and Richard Donovick Biotechnology-The Golden Age V. S. Malik INDEX

Volume 35

Production of Bacterial Thermostable aAmylase by Solid-state Fermentation: A Potential Tool for Achieving Economy in Enzyme Production and Starch Hydrolysis B. K. Lonsane and M. V. Ramesh Methods for Studying Bacterial Gene Transfer in Soil by Conjugation and Transduction G. Stotzky, Monica A. Devanas, and Lawrence R. Zeph Microbial Levan Youn W. Han

CONTENTS OF PREVIOUS VOLUMES

333

Review and Evaluation of the Effects of Xenobiotic Chemicals on Microorganisms in Soil R. J. Hicks, G. Stotzky, and P. Van Voris

An Evaluation of Bacterial Standards and Disinfection Practices Used for the Assessment and Treatment of Stormwater Marie L. O’Shea and Richard Field

Disclosure Requirements for Biological Materials in Patent Law Shung-Chang Jong and Jeannette M. Birmingham

Haloperoxidases: Their Properties and Their Use in Organic Synthesis M. C. R. Franssen and H. C. van der Plas

INDEX

Medicinal Benefits of the Mushroom Ganoderma S. C. Jong and J. M. Birmingham

Volume 36

Microbial Degradation of Biphenyl and Its Derivatives Frank K. Higson

Microbial Transformations of Herbicides and Pesticides Douglas J. Cork and James P. Krueger An Environmental Assessment of Biotechnological Processes M. S. Thakur, M. J. Kennedy, and N. G. Karanth Fate of Recombinant Escherichia coli K12 Strains in the Environment Gregg Bogosian and James F. Kane

The Sensitivities of Biocatalysts to Hydrodynamic Shear Stress Ales Prokop and Rakesh K. Bojpai Biopotentialities of the Basidiomacromycetes Somasundarom Rajarathnam, Mysore Nanjarajurs Shashirekha, and Zakia Bano INDEX

Microbial Cytochromes P-450 and Xenobiotic Metabolism F. Sima Sariaslani Foodborne Yeasts T. Dedk High-Resolution Electrophoretic Purification and Structural Microanalysis of Peptides and Proteins Erik P.Lillehoj and Vedpal S. Malik INDEX

Volume 37

Microbial Degradation of the Nitroaromatic Compounds Frank K. Higson

Volume 38

Selected Methods for the Detection and Assessment of Ecological Effects Resulting from the Release of Genetically Engineered Microorganisms to the Terrestrial Environment G. Stotzky, M. W. Broder, J. D. Doyle, and R. A. Jones Biochemical Engineering Aspects of Solid-state Fermentation M. V. Romana Murthy, N. G. Karanth, and K. S. M. S. Raghava Rao The New Antibody Technologies Erik P. Lillehoj and Vedpal S. Malik

334

CONTENTS OF PREVIOUS VOLUMES

Anoxygenic Phototrophic Bacteria: Physiology and Advances in Hydrogen Production Technology K. Sasikala, Ch. V. Romano, P. Raghuveer Rao, and K. L. Kovacs

Microbial Pentose Utilization Pmshant Mishra and Ajay Singh Medicinal and Therapeutic Value of the Shiitake Mushroom S. C. Jong and J. M. Birmingham

INDEX

Yeast Lipid Biotechnology Z. Jacob

Volume 39

Asepsis in Bioreactors M. C. Sharma and A. K. Gurtu Lipids of n-Alkane-Utilizing Microorganisms and Their Application Potential Samir S. Radwan and Naser A. Sorkhoh

Pectin, Pectinase, and Protopectinase: Production, Properties, and Applications Takuo Sakai, Tatsuji Sakamoto, Johan Hallaert, and Erick J. Vandamme Physicochemical and Biological Treatments for EnzymaticMicrobial Conversion of Lignocellulosic Biomass Purnendu Ghosh and Ajay Singh INDEX

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