Applied Microbiology (Focus on Biotechnology)

APPLIED MICROBIOLOGY VOLUME 2

FOCUS ON BIOTECHNOLOGY Volume 2

Series Editors MARCEL HOFMAN Centre for Veterinary and Agrochemical Research, Tervuren, Belgium

JOZEF ANNÉ Rega Institute, University of Leuven, Belgium

Volume Editors ALAIN DURIEUX and JEAN-PAUL SIMON Institut Meurice, Brussels, Belgium

COLOPHON Focus on Biotechnology is an open-ended series of reference volumes produced for Kluwer Academic Publishers BV in co-operation with the Branche Belge de la Société de Chimie Industrielle a.s.b.l. The initiative has been taken in conjunction with the Ninth European Congress on Biotechnology. ECB9 has been supported by the Commission of the European Communities, the General Directorate for Technology, Research and Energy of the Wallonia Region, Belgium and J. Chabert, Minister for Economy of the Brussels Capital Region.

Applied Microbiology Volume 2

Edited by

ALAIN DURIEUX and JEAN-PAUL SIMON Institut Meurice, Brussels, Belgium

KLUWER ACADEMIC PUBLISHERS NEW YORK / BOSTON / DORDRECHT / LONDON / MOSCOW

eBook ISBN: Print ISBN:

0-306-46888-3 0-792-36858-4

©2002 Kluwer Academic Publishers New York, Boston, Dordrecht, London, Moscow All rights reserved No part of this eBook may be reproduced or transmitted in any form or by any means, electronic, mechanical, recording, or otherwise, without written consent from the Publisher Created in the United States of America Visit Kluwer Online at: and Kluwer's eBookstore at:

http://www.kluweronline.com http://www.ebooks.kluweronline.com

EDITORS PREFACE This book illustrates the major trends in applied microbiology research with immediate or potential industrial applications. The papers proposed reflect the diversity of the application fields. New microbial developments have been done as well in the food and health sectors than in the environmental technology or in the fine chemical production. All the microbial genera are involved : yeast, fungi and bacteria. The development of biotechnology in parallel with the industrial microbiology has enabled the application of microbial diversity to our socio-economical world. The remarkable properties of microbes, inherent in their genetic and enzymatic material, allow a wide range of applications that can improve our every day life. Recent studies for elucidating the molecular basis of the physiological processes in micro-organisms are essential to improve and to control the metabolic pathways to overproduce metabolites or enzymes of industrial interest. The genetic engineering is of course one of the disciplines offering new horizons for the « fantastic microbial factory ». Studies of the culture parameter incidence on the physiology and the morphology are essential to control the response of the micro-organisms before its successful exploitation at the industrial scale. For this purpose, fundamental viewpoints are necessary. Development of novel approaches to characterise micro-organisms would also facilitate the understanding of the inherent metabolic diversity of the microbial world, in terms of adaptation to a wide range of biotopes and establishment of microbial consortia. Improvement of selection methods is a crucial step to initiate any research in applied microbiology. This is necessary both for traditional strains which are used as starter in the food industry and for more specific strains which are of interest in fine chemical synthesis and bioremediation. Since the two last decades, in the fermented food and beverage industry, micro-organisms have been regarded to increase shelf-life and to improve flavours and nutritional value of food. Antimicrobial activity against pathogens and spoilage micro-organisms is an important criteria for the selection of a strain as starter. Beside the development of starters, techniques of foodborne bacterial pathogens detection are in complete evolution with the introduction of molecular detection and new typing methods. The constant apparition of novel applications is the driving force which allows the constant progress of applied microbiology. Recent development in enzymes production and discovery of new metabolic pathways related to food or environmental technology are representative examples .

Editors: A. Durieux and J-P Simon Brussels March 2000

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IN MEMORY On September 25th, 1999, Professor Charles A. Masschelein died here in Belgium. To the end, Professor Masschelein remained active in the brewing and biotechnology industries and had, in fact, just recently returned from visiting a number of African breweries. That day, I lost a friend. Moreover, at his moment of passing, I understood that I had lost my mentor in the field of applied microbiology and the world had lost one of the truly great scientists of the 20th century. Twenty years before, I joined Masschelein’s department of brewing science at the Institut Meurice, CERIA. Masschelein was a leader, a dedicated spirit to R&D with an acutely inquiring mind. He completely devoted himself to the training of people and transfer of technology from the academic to the industrial world. Since 1954, C.A. Masschelein’s vision, as a Belgian scientist, was to understand the physiology of micro-organisms in their industrial environment. He taught this idea at all levels ; to large numbers of students in Europe and internationally, but also to a large number of engineers already in charge of fermentation plants around the world. Masschelein’s major topic has been the brewery. In this sector he was the defender of a strict understanding of biochemical and microbiological principles balancing this with both the reality and progression of the industrial method and the traditional quality of the final product he knew. For more than 35 years, he held a number of responsible positions in the European Brewery Convention as well as contributed greatly to brewing science worldwide. Biotransformation was always a major driving force for Masschelein. He was able to introduce this philosophy to R&D in the 1960s and apply the results to processes in order to use micro-organisms to produce industrially significant materials. For this reason too, C.A. Masschelein has always been capable of combining new developments in molecular biology on one hand to new materials and processes on the other. This challenge was such a reality for him that, when he retired as professor at CERIA, he was responsible for setting up a company to develop new continuous brewing processes. In July, 1999, during the Ninth European Congress of Biotechnology, C.A. Masschelein gave his last lecture delivering his idea concerning the future of applied microbiology in correlation with new generation of continuous fermentation processes. As with all great scientists, Masschelein’s work will be remembered and referred to for many decades in the future. J.P. Simon

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TABLE OF CONTENTS

EDITORS PREFACE ....................................................................

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IN MEMORY .................................................................................

1

TABLE OF CONTENTS ...............................................................

3

PART 1 - STARTERS ................................................................. 11 NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS .......................................................................................................................... Rolf Geisen and Paul Färber .................................................................................... Abstract ........................................................................................................... 1. Introduction ...................................................................................................... 2. Penicillium nalgiovense .................................................................................... 2.1. Taxonomic relationships at the molecular level ........................................ 2.2. Penicillin production is a common feature of p. nalgiovense .................... 2.3. Heterologous Gene Expression in P . nalgiovense .................................... 2.4. Heterologous Gene Expression in P . nalgiovense 2.4. Cloning of genes from P . Nalgiovense important for the fermentation process .......................... 3. Penicillium camemberti .................................................................................... 4. Penicillium roqueforti ................................................................................. 5. Conclusions ...................................................................................................... References ............................................................................................................

13 13 13 13 15 15 17 20 21 23 25 27 27

STARTERS FOR THE WINE INDUSTRY .............................................................. 31 Aline Lonvaud-Funel ................................................................................................ 31 Abstract ................................................................................................................ 31 1. Introduction ...................................................................................................... 31 2. Yeast starters in winemaking ........................................................................... 32 2.1 The objectives of yeast starters .................................................................. 32 2.2 Properties of yeast used as selective criteria for active dry yeast producers and winemakers ............................................................................................... 34 2.3 Evaluation of the settlement of active dry yeast during alcoholic fermentation : ................................................................................................... 37 3. Malolactic starters in winemaking .................................................................... 38 3.1 Indications for use of malolactic starter and description ............................ 39 3

3.2 The influence of lactic acid bacteria starters on wine quality and their selection ........................................................................................................... 3.3 Efficiency of malolactic starters ................................................................ 4. The future of starters for winemaking .............................................................. 5. Conclusion ........................................................................................................ References ............................................................................................................

41 42 43 45 45

PART 2 - PHYSIOLOGY, BIOSYNTHESIS AND METABOLIC ENGINEERING ........................................................................... 49 METABOLISM AND LYSINE BIOSYNTHESIS CONTROL IN BREVIBACTERIUM FLAVUM: IMPACT OF STRINGENT RESPONSE IN BACTERIAL CELLS .................................................................................................. 51 M. Ruklisha, R. Jonina, L. Paegle and G. Petrovica ...................................................... 51 Abstract ................................................................................................................ 51 1. Introduction ....................................................................................................... 51 2. Materials and Methods ..................................................................................... 52 3. Results and Discussion ..................................................................................... 52 4. Conclusions ...................................................................................................... 56 References ............................................................................................................ 57 MOLECULAR BREEDING OF ARMING YEASTS WITH HYDROLYTIC ENZYMES BY CELL SURFACE ENGINEERING ................................................ 59 Mitsuyoshi Ueda, Toshiyuki Murai, Shouji Takahashi, Motohisa Washida, and Atsuo Tanaka ....................................................................................................................... 59 Abstract ................................................................................................................ 59 1. Introduction ...................................................................................................... 60 2. Principle of Cell Surface Engineering of Yeast ................................................ 63 3. Display of Amylolytic Enzymes on the Yeast Cell Surface ............................. 65 4. Display of Cellulolytic Enzymes on the Yeast Cell Surface ............................ 67 5. Display of Lipase on the Yeast Cell Surface .................................................... 70 6. Cell Surface Engineering as a Novel Field of Biotechnology .......................... 70 References ............................................................................................................ 71 METABOLIC PATHWAY ANALYSIS OF SACCHAROMYCES CEREVISIAE ....................................................................................................................................... 75 Simon Ostergaard, Lisbeth Olsson and Jens Nielsen ................................................ 75 Abstract ................................................................................................................ 75 1. Introduction ...................................................................................................... 75 2. Metabolic pathway analysis .............................................................................. 76 2.1. Metabolic control analysis ........................................................................ 76 2.2. Metabolic flux analysis ............................................................................. 77 3. Steady-state continuous cultivation – an excellent tool for metabolic pathway analysis ................................................................................................................. 79 4. Metabolic pathway analysis applied to Saccharomyces cerevisiae .................. 80 4

4.1. Kinetic studies of the glycolysis ............................................................... 4.2. Metabolic pathway analysis of the galactose metabolism ........................ Acknowledgements .............................................................................................. References ............................................................................................................

80 81 85 85

PART 3 - STATE PARAMETERS AND CULTURE CONDITIONS ................. . ................................................................................... 87 EFFECT OF AERATION IN PROPAGATION ON SURFACE PROPERTIES BREWERS’ YEAST .................................................................................................... Andrew Robinson and Susan T. L . Harrison ............................................................. Abstract ................................................................................................................ 1. Introduction ...................................................................................................... 2. Materials and Methods ..................................................................................... 2.1 Propagation conditions .............................................................................. 2.2 Hydrophobicity .......................................................................................... 2.3 Surface charge ............................................................................................ 2.4 Flocculation ............................................................................................... 3. Results .............................................................................................................. 3.1 Yield coefficients ....................................................................................... 3.2 Cell growth rates ........................................................................................ 3.3 Hydrophobicity .......................................................................................... 3.4 Zeta potential ............................................................................................. 3.5 Flocculation ............................................................................................... 4. Discussion ......................................................................................................... 5. Conclusions ...................................................................................................... Acknowledgements .............................................................................................. References ............................................................................................................

OF 89 89 89 89 90 90 90 91 92 92 92 92 93 94 95 96 98 98 99

EFFECT OF THE MAIN CULTURE PARAMETERS ON THE GROWTH AND PRODUCTION COUPLING OF LACTIC ACID BACTERIA ............................ 101 A. Amrane and Y. Prigent ....................................................................................... 101 Abstract .............................................................................................................. 101 1. Introduction .................................................................................................... 101 2. Materials and methods .................................................................................... 102 2.1 Microorganism ......................................................................................... 102 2.2 Media ....................................................................................................... 102 2.3 Fermentors and culture conditions ........................................................... 102 2.4 Analytical methods .................................................................................. 103 3. Results and Discussion ................................................................................... 103 3.1. Preculture conditions .............................................................................. 103 3.2. Nutritional limitations ............................................................................. 105 3.3. Initial lactate additions ............................................................................ 106 4. Conclusions .................................................................................................... 107 Acknowledgements ............................................................................................ 107 5

References ..........................................................................................................

107

PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE ............................................................................................................. 109 F.F. Bauer and I.S . Pretorius ................................................................................... 109 Abstract .............................................................................................................. 109 1. Introduction .................................................................................................... 109 2. Signal transduction in Saccharomyces cerevisiae ........................................... 110 3. Molecular nature of signal transduction processes resulting in pseudohyphal differentiation ..................................................................................................... 112 3.1. Signal transduction modules .................................................................. 113 3.1.1. Nutrient availability is sensed by permeases ................................... 113 3.1.2. Transmission via receptor associated elements ............................... 114 3.1.3. Intermediate signal transduction modules ....................................... 116 3.2. Transcriptional regulators ....................................................................... 122 3.2.1. Ste12p and Tec1 .............................................................................. 123 3.2.2. Msn1p and Mss11p: Central elements in the pseudohyphal growth pathway ..................................................................................................... 123 3.2.3. Sfl1p. Sok2p and Flo8p: Factors depending on the cAMP dependent kinase ........................................................................................................ 124 3.2.4. Other factors .................................................................................... 125 3.3. Effector proteins ..................................................................................... 125 3.3.1. MUC1. a gene encoding a mucin-like protein subjected to complex transcriptional regulation .......................................................................... 126 3.3.2. Starch degrading enzymes: a direct metabolic link ......................... 127 4 . Scientific and industrial relevance .................................................................. 127 Acknowledgements ............................................................................................ 129 References ................ ................................................................................... 129 MICROBIAL PRODUCTION OF THE BIODEGRADABLE POLYESTER POLY-3-HYDROXYBUTYRATE (PHB) FROM AZOTOBACTER CHROOCOCCUM 6B: RELATION BETWEEN PHB MOLECULAR WEIGHT. THERMAL STABILITY AND TENSILE STRENGTH ........................................ 135 Quagliano Javier C. and Miyazaki Silvia S ... ......................... 135 Abstract .............................................................................................................. 135 1. Materials and methods .................................................................................... 135 1.1 Microorganism and culture media ........................................................... 135 1.2 Fermentor experiments ............................................................................ 135 1.3 Extraction and purification procedure ...................................................... 136 1.4 Analytical methods .................................................................................. 136 2. Results and discussion .................................................................................... 136 2.1 Effect of MW on PHB thermal stability .................................................... 136 2.2 Effect of aeration rate on PHB MW .......................................................... 137 2.3 PHB tensile strength (σ) at different MW ................................................. 2.4 PHB as a matrix for microencapsulation ................................................. 6

138 138

3. Conclusions .................................................................................................... References ..........................................................................................................

PART 4 - NOVEL APPROACHES TO THE STUDY OF MICROORGANISMS ................................................................

139 139

141

SHARING OF NUTRITIONAL RESOURCES IN BACTERIAL COMMUNITIES DETERMINED BY ISOTOPIC RATIO MASS SPECTROMETRY OF BIOMARKERS .......................................................................................................... 143 Wolf-Rainer Abraham, Christian Hesse, Oliver Pelz, Stefanie Hermann, Michael Tesar, Edward R. B . Moore, and Kenneth N . Timmis ............................................ 143 1. Introduction .................................................................................................... 143 2. Taxon specific biomarkers .............................................................................. 144 2.1. Polar lipids .............................................................................................. 144 2.2. Outer membrane proteins ........................................................................ 145 3. Isotopic fractionation in microorganisms ....................................................... 146 4. Carbon sharing in a pollutant degrading bacterial community ....................... 147 4.1. Origin and characteristics of the microbial consortium .......................... 147 4.2. Incorporation of [U-13C]-metabolites in microbial biomasses ................ 148 4.3. Substrate competition ............................................................................. 149 4.4. Community physiology of the microbial consortium ............................. 150 5. Outlook ........................................................................................................... 152 Acknowledgement .............................................................................................. 152 References .......................................................................................................... 152 A COMPARISON OF THE MECHANICAL PROPERTIES OF DIFFERENT BACTERIAL SPECIES ............................................................................................ 155 C . SHIU, Z . ZHANG AND C.R. THOMAS ........................................................... 155 Abstract .............................................................................................................. 155 1. Introduction .................................................................................................... 155 1.1 Relative resistance of different microorganisms to mechanical disruption ....................................................................................................................... 155 1.2 Cell wall structure .................................................................................... 156 1.3 Bacterial biomechanics ............................................................................ 157 1.4 Micromanipulation ................................................................................... 158 2. Materials and methods .................................................................................... 158 2.1 The micromanipulation system ................................................................ 158 2.2 Culture conditions .................................................................................... 159 3. Results and discussion .................................................................................... 160 4. Conclusions and future developments ............................................................ 161 References .......................................................................................................... 162

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PART 5 - NOVEL APPLICATIONS ..........................................

163

KOCURIA ROSEA AS A NEW FEATHER DEGRADING BACTERIA ........... 165 Nereida Coello and Luis Vidal ................................................................................ 165 Abstract .............................................................................................................. 165 1. Introduction .................................................................................................... 165 2.Isolation, identification and adaptation of feather-degrading microorganisms 166 2.1. Isolation and degradation of feathers by a microbial isolate ................... 166 2.2. Morphological and ultrastructural characteristics of the feather-degrading isolate ............................................................................................................. 168 3. Microbial growth and feather degradation ...................................................... 168 3.1. Effect of quantity of feathers .................................................................. 168 3.2. Effect of culture temperature on feather degradation and growth of LPB-3 ....................................................................................................................... 171 3.3. Kinetic fermentation ............................................................................... 171 4. Industrial applications ..................................................................................... 171 4.1. Fermented feather meal ........................................................................... 171 4.2. Enzymes .................................................................................................. 173 4.3. Pigments ................................................................................................. 173 Acknowledgements ............................................................................................ 174 References .......................................................................................................... 174 COMPARISON OF Pb2+ REMOVAL CHARACTERISTICS BETWEEN BIOMATERIALS AND NON-BIOMATERIALS .................................................. Dong Seog Kim and Jung Ho Suh ........................................................................... Abstract .............................................................................................................. 1. Introduction .................................................................................................... 2. Materials and methods .................................................................................... 2.1. Materials ................................................................................................. 2.2. Microorganisms and culture conditions .................................................. 2.3. Pb2+ removal experiment ........................................................................ 3. Results and discussion .................................................................................... 3.1. Pb2+ removal characteristics .................................................................... 3.2. Initial Pb2+ removal rate .......................................................................... 4. Conclusions .................................................................................................... References ..........................................................................................................

177 177 177 177 178 178 178 178 179 179 182 183 183

HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERIA . 185 Gy. Barabás, Gy. Vargha, I. Szabó, A. Penyige, J. Szöllõsi, J. Matkó, S. Damjanovich and T . Hirano .................................................................................... 185 Abstract .............................................................................................................. 185 1. Materials and methods .................................................................................... 185 1.1 Test organisms . oligocarbophylic streptomyces ...................................... 185 1.2 Biomass preparation ................................................................................ 186

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1.3 Incorporation of radioactivity from labelled n-Hexadecane into mycelia . ....................................................................................................................... 186 1.4 Fluorescence measurements ..................................................................... 186 1.5 Analysis of fatty acids .............................................................................. 187 1.6 Investigations with GTP analogues .......................................................... 187 2. Results and discussion .................................................................................... 187 3. Conclusion ...................................................................................................... 190 References .......................................................................................................... 190

PART 6 - FOOD SECURITY AND FOOD PRESERVATION ... 191 MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS: A REVIEW ...................................................................................... 193 M . Heyndrickx, N . Rijpens and L . Herman ............................................................. 193 Abstract .............................................................................................................. 193 1. Introduction .................................................................................................... 194 2. Characteristics of the foodborne bacterial pathogens ..................................... 194 3. Molecular detection and identification of foodborne bacterial pathogens ...... 198 3.1 Nucleic acid based identification methods ............................................... 198 3.2 The use of virulence genes as target for molecular identification ............ 198 3.3 The use of RRNA genes as target for molecular identification ............... 199 3.4 The use of specific sequences with a known or unknown function as target for molecular identification ........................................................................... 200 3.5 The available molecular identification systems ....................................... 201 3.6 PCR detection of bacterial pathogens in food products ........................... 203 3.6.1 Influence of food components on PCR performance ....................... 203 3.6.2 Sensitivity and contamination of PCR ............................................. 203 3.6.3 The detection of the viability of cells by DNA based technology .... 204 3.7 Evaluation and validation of DNA based methods .................................. 205 3.8 DNA amplification methods for quantification of foodborne pathogens . 207 4. Molecular typing of foodborne bacterial pathogens ....................................... 208 4.1 Terminology and general information ..................................................... 208 4.1.1 Necessity of bacterial typing of foodborne pathogens ..................... 208 4.1.2 Species-subspecies-variety-clone-strain-isolate ............................... 209 4.1.3 Molecular typing techniques used for bacterial pathogens .............. 210 4.1.4 Analysis of DNA fingerprints .......................................................... 219 4 .. 2 Prospects in molecular typing ................................................................. 220 5. Molecular typing of some specific bacterial foodborne pathogens ............... 221 5.1 Salmonella ............................................................................................... 221 5.2 Campylobacter jejuni ............................................................................... 226 5.3 Listeria monocytogenes .......................................................................... 227 5.4 Escherichia coli 0157 .............................................................................. 228 5.5 Some other foodborne bacterial pathogens .............................................. 229 References .......................................................................................................... 229

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BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION ........... 239 Cahill, S.M., Upton, M.E., and McLoughlin. A.J. .................................................. 239 Abstract .............................................................................................................. 239 1. Introduction .................................................................................................... 240 2. Meat preservation ........................................................................................... 241 2.1 Biological fermentation ........................................................................... 241 2.2 Chemical acidification ............................................................................. 243 3. The application of encapsulation technology to meat preservation ................ 243 3.1. The application of encapsulation technology to a microbial fermentation ....................................................................................................................... 243 3.1.1. Encapsulation matrices and the encapsulation process ................... 244 3.1.2. The benefits of meat starter culture encapsulation .......................... 246 3.1.3. Commercial applications ................................................................. 247 3.2. The application of encapsulation technology to chemical acidification . 248 3.2.1. Encapsulation matrices and the encapsulation process ................... 248 3.2.2. The benefits of acidulant encapsulation .......................................... 249 3.2.3 Commercial availability ................................................................... 249 4. Control of emerging pathogens ...................................................................... 250 5. The application of encapsulation technology to bacteriocin delivery ............. 251 5.1 Bacteriocins ............................................................................................. 251 5.2 Nisin ......................................................................................................... 251 5.2.1 Encapsulation of nisin ...................................................................... 252 6. Conclusions and future work .......................................................................... 261 References .......................................................................................................... 261

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

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Part 1 STARTERS

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NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS ROLF GEISEN AND PAUL FÄRBER Federal Research Centre for Nutrition Haid- und Neustr. 9 76131 Karlsruhe

Abstract The production of a variety of foods include a fermentation step by filamentous fungi. Nowadays these fermented foods are produced by selected fungal starter cultures instead of relying on the indigenous flora, which may contain spoilage or mycotoxinogenic strains. The most important fungal species for food fermentation are Penicillium nalgiovense for the production of mould fermented meat products, P. camemberti for the production of white cheeses and P. roqueforti for the production of blue veined cheeses. Before a fungal strain of these species can be used as a starter culture for human consumption it must fulfill several requirements. Not all strains isolated from the food environment and with characteristics suitable for starter cultures fit to these conditions. In addition also the currently used starter strains possess undesired properties. On the other hand the modem techniques of molecular biology or genetics offers various possibilities for screening, characterisation and for specific improvement of fungal strains. In this article examples for characterisation and improvement of fungal starter cultures by molecular techniques are described. 1. Introduction Mould fermented foods play an important role, especially in Asian countries where the production process for many foods include a fungal fermentation. Fungal species which are found in Asian type foods belong to different genera: Aspergillus oryzae, A. glaucus, Rhizopus chinensis, Neurospora intermedia or Monascus purpureus (Hesseltine, 1983). In contrast to this situation in European countries mainly three species from the genus Penicillium are used for the production of fermented foods, in particular Penicillium camemberti for white cheeses, P. roqueforti for blue cheeses and P. nalgiovense for mould fermented meat products. It has to be mentioned that P. chrysogenum also can often be isolated from fermented meat products and is sometimes used as a starter culture for meat products in some countries (Leistner, 1986). 13 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 13-29. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

ROLF GEISEN AND PAUL FARBER

Fungal starter cultures extensively contribute to the flavour and texture formation of the mould ripened food product. The most important enzymatic activities in this respect are proteases and lipases (Kinsella and Hwang, 1976). Proteases degrade proteins to flavour active peptides or amino acids, whereas lipases hydrolyse triacyl glycerins to free fatty acids and glycerol. The fatty acids can be converted into methylketones by fungal lipoxygenases that also contribute to the flavour formation. These compounds are especially important in the ripening of blue cheese (Fox and Law, 1991). But not only these enzymatic processes play a role in flavour development. It has been shown by Jacobsen and Hinrichsen (1997) that also volatile compounds produced by the fungus contribute to this process. Another important feature of the fungal starter culture is the competition against undesired microorganisms, which would otherwise cover the surface of the fermented product. These microorganisms are mainly other fungal species that could lead to a discoloration of the product or the production of undesired secondary metabolites. In addition the competition of fungal starter cultures agai nst undesired pathogenic or toxinogenic bacteria is of interest for microbiological safe production of mould fermented foods. During the fermentation process the pH of the product raises due to the metabolic activity of the fungus. Lactic acid that may be produced by accompanying lactic acid bacteria is degraded and also proteins may be hydrolysed to ammonia by the fungal starter culture. Both processes lead to an increase in pH during the fermentation which enables the growth of pathogenic or toxinogenic bacteria like Listeria monocytogenes or Staphylococcus aureus. Of course starter cultures which would suppress the growth of these undesired bacteria would be advantageous for improved food safety. Before a fungal strain can be used as a starter culture it has to fulfil several requirements (Tab. 1). Table 1. Requirements for a fungal starter culture

----------------------------------------------------

It should not produce a mycotoxin It should not produce another undesired secondary metabolite It should produce the desired flavour changes in the product It should be adapted to the food product It should compete against undesired moulds It should have antibacterial activity against pathogens ---------------------------------------------------Not all the currently used strains fulfil all these requirements. Many species of the genus Penicillium are able to produce toxic secondary metabolites, the mycotoxins. Even some species currently used, as starter cultures are able to produce mycotoxins or other undesired secondary metabolites. For this reason it is important that useful methods 14

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

exist, which enables the screening of strains for particular characteristics and that fungal strains can be optimised according particular requirements. 2. Penicillium nalgiovense 2.1. TAXONOMIC RELATIONSHIPS AT THE MOLECULAR LEVEL P. nalgiovense was first described by Laxa (1932). The type culture was isolated from a bohemian cheese. Strains of these species are mainly found on meat and meat products (Leistner et al, 1989). But they can also be isolated as spoilage organisms from different cheeses (Lund et al. 1995). P. nalgiovense is not able to produce any of the known mycotoxins, according to Frisvad (1988). Until recently P. nalgiovense was regarded as a fungal species with poor capability of producing secondary metabolites. Because of morphological similarities P. nalgiovense has long been regarded as a domesticated form of P. chrysogenum, the species which is industrially used for penicillin production (Samson and van Reenen-Hoekstra, 1988). In a recent publication Banke et al. reclassified the P. chrysogenum complex (Banke et al, 1997). According to their results, based on isozyme analysis, the P. chrysogenum complex was separated into 4 distinct species: P. chrysogenum, P. dipodomyis, P. flavigenum and P. nalgiovense. Molecular data confirmed the high relationship between P. nalgiovense and P. chrysogenum (Geisen, 1995). With several strains of P. nalgiovense a RAPD analysis was performed. This type of analysis is a molecular typing technique, which was developed by Williams et al. (1990). This technique is a modified PCR approach in which an arbitrary primer is used. It generates specific patterns if always the same reaction conditions are used. These patterns are related to the genotype of the analysed strain. Differences in the pattern reflects differences in the genotype and give information about the relatedness between different strains. Depending on the primer used the patterns can be species- (Guthrie et al., 1992), subspecies- (Hamelin et al., 1993) or even strain specific (Bidouchka et al. 1994). This approach revealed that P. nalgiovense is a homogenous species, irrespective of the origin of the strains analysed (Figure 1). The figure shows that all the patterns generated with a random primer were identical. The same result was derived with a second primer. The only difference in the pattern exhibited one strain (BFE 61). This was due with all primers used, indicating that it might be a misclassified strain To compare P. nalgiovense and P. chrysogenum at the molecular level, the same approach was carried out with both species. Figure 2 shows the results of this analysis. They clearly demonstrate, that P. nalgiovense and P. chrysogenum are two distinct species, because the RAPD patterns of both species are different, however a subsequent hybridisation analysis, in which the labelled RAPD products of P. nalgiovense were hybridised to the RAPD-PCR products of P. chrysogenum revealed, that both species has homologies at the nucleotide sequence level, which indicate the close relationship between the species.

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ROLF GEISEN AND PAUL FARBER

Figure 1: RAPD pattern from different strains of P. nalgiovense generated with primer aril. Lanes 1 and 2, P. roqueforti BFE54, P. camemberti BFE224; lanes 3 - 18, P. nalgiovense BFE71, BFE75, BFE55, BFE61, BFE57, BFE59, BFE67, BFE56, BFE66, BFE65, BFE52, BFE60, BFE69, BFE64, BFE58, BFE74; lanes 19 and 20 size marker 1 and 2. Fragment sizes are indicated in kb.

Figure 2: Comparison of the RAPD patterns of P. nalgiovense and P. chrysogenum. Lanes 1 to 5: P. nalgiovense BFE71, BFE76, P. chrysogenum DSM 844, DSM 1075, size marker. The fragment sizes are given in kb.

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NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

2.2. PENICILLIN PRODUCTION IS A COMMON FEATURE OF P.NALGIOVENSE Another aspect also indicates the close relationship between both species. P. chrysogenum is the industrial producer of penicillin and for P. nalgiovense the capability to produce also penicillin has been described independently by Färber and Geisen (1994) and Andersen and Frisvad (1994). All strains analysed were able to produce this secondary metabolite. It was demonstrated by PCR (polymerase chain reaction), that obviously the genes for penicillin production are very similar at the nucleotide level between P. chrysogenum and P. nalgiovense. With primers deduced from the sequences of the three penicillin biosynthetic genes of P. chrysogenum it was possible to amplify the respective PCR fragments from P. nalgiovense. The PCR products of both species were identical in length, indicating homology between the genes (Figure 3).

Figure 3: PCR analysis of the three penicillin biosynthetic genes of P. nalgiovense (lanes 3 to 5 and 7 to 9) and P. chrysogenum (lanes 11 to 13). Lanes 3, 7, 11 show the PCR products for the penDE genes, lanes 4, 8, 12 show the PCR products for the pcbC genes and lanes 5, 9, 13 show the PCR products of the pcbAB genes.

Subsequent sequence analyses of the pcbC gene, the gene coding for isopenicillin synthetase, revealed that both genes are 94% identical. It is known from the genes of P. chrysogenum that they are clustered and all located on chromosome 4, the largest chromosome (Fiero et al. 1993). To analyse this situation in P. nalgiovense an electrophoretic karyotyping was carried out. This analysis revealed, that like P. chrysogenum, P. nalgiovense has also 4 chromosomes of high molecular weight, but 17

ROLF GEISEN AND PAUL FARBER

compared to the chromosomes of P. chrysogenum they are smaller in size resulting in a total genome size which is considerably smaller than that of P. chrysogenum (26,5 Mb instead of 34,1 MB). These results again reflect the close relationship between both organisms, but also demonstrates the distinct differences at the molecular level. A distinct difference became also obvious when the location of the penicillin biosynthetic gene cluster have been elucidated. In the case of P. chrysogenum it is located on the largest chromosome, whereas on the smallest in case of P. nalgiovense. As mentioned above, one important requirement for a fungal strain used as a starter culture is the point that it does not produce any undesired secondary metabolite. Antibiotics like mycotoxins are secondary metabolites produced in the idiophase of the fungal growth curve. The differentiation of these secondary metabolites in antibiotics and mycotoxins depend only to the point of view. A fungal starter culture should not produce any of them. All analysed P. nalgiovense strains obviously were able to produce the secondary metabolite penicillin, also the strains that are currently used as fungal starter cultures. As these strains are well adapted to the processing conditions and have long been used for fermentation purposes. For this reason great experiences exist concerning the optimal fermentation conditions. It would be advantageous that exactly these strains would be optimised in a way that they do not produce penicillins. To achieve this goal the "gene disruption" approach was followed. A gene disruption is a gene technological method to specifically inactivate an undesired gene within an organism. In contrast to the inactivation of a gene by mutation with an chemical mutagene or irradiation only the target gene is inactivated by this approach. This characteristic is of special interest for the optimisation of strains that are already in use. With this approach no secondary mutations that may have negative consequences on the activity of a starter culture can occur. This might be the case with the conventional mutation/selection approach. The principle of the "gene disruption" approach is shown in Figure 4.

Figure 4: Scheme of the "gene disruption" approach.

18

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

There are two important points that must be fulfilled before this method can be used: 1. a transformation system for the particular organism must be established to be able to introduce DNA into the microorganism and 2. the gene to be targeted must be available. If this is the case a central fragment of that gene is cloned into a transformation vector. This plasmid is than transformed into the fungal strain. The resulting transformants are screened for activity of the undesired gene. It is highly probable, that some of the transformants are no longer active in the undesired feature. In that cases the transforming DNA had integrated into the undesired gene by homologous recombination. This integration leads to a situation in which the target gene is duplicated and separated by vector sequences. However both copies of the gene are inactive, as the first copy is truncated at the 3' end and the second copy is truncated at the 5' end. This approach was adapted to P. nalgiovense. A transformation system for that microorganism was available (Geisen, 1989). The central fragment of the penDC gene was cloned into the transforming vector and introduced into P. nalgiovense. From the resulting transformants several could be identified, which were no longer able to produce penicillin. Figure 5 shows the results of the experiment.

Figure 5: Comparison of the antagonistic activity against Micrococcus luteus of a wild type strain (right) and the gene disrupted transformant (left)

The transformed strain of P. nalgiovense is no longer able to produce penicillin. As a strain that is currently used as a starter culture has been used. The non-penicillinogenic strain for this reason is isogenic to the commonly used strain with the only difference in respect to penicillin production.

19

ROLF GEISEN AND PAUL FARBER

2.3 HETEROLOGOUS GENE EXPRESSION IN P. NALGIOVENSE As mentioned above it is necessary, for microbiological stability of a product, that fungal starter cultures are able to compete against undesired pathogenic or toxinogenic microorganisms, which can grow in mould fermented products. However this inhibition should be caused by "food grade" inhibitory principles. During fermentation unstable conditions may occur, due to the metabolic activity of the fungal starter culture. The pH may raise which enables the growth of pathogenic bacteria like Listeria monocytogenes or Staphylococcus aureus (Marth, 1987). Fungal starter cultures that would be able to suppress the growth of these organisms would improve the microbiological safety of these products. Several antagonistic proteins exist, which have GRAS status (generally recognised as safe) and could improve the antagonistic activity in a food-grade way, if they would be produced by the fungal starter culture. Examples of these proteins are the bacteriocins (Stiles, 1994), lysozyme (Bester and Lombard, 1990) or glucose oxidase (Tiina and Sandholm, 1989). The glucose oxidase (GOD) is an enzyme system that is widely used on food industry. In the presence of glucose it oxidises glucose to gluconic acid and hydrogen peroxide. For this reason it is used as a scavenger for traces of glucose or oxygen in food systems like dried egg or beverages (Reed and Underkofler, 1969). In addition to this activities, the GOD has also inhibitory activities against a range of pathogenic and toxinogenic bacteria (Tiina and Sandholm, 1989). Mainly the production of hydrogen peroxide is responsible for antibacterial activity. GOD is active against L. monocytogenes, Staphylococcus aureus, Clostridium perfringens, Samonella infantis and Bacillus cereus. This inhibitory activity is dependent on the concentration of the glucose in the medium. The god gene has been cloned from Aspergillus niger (Kriechbaum et al. 1989). A. niger also has GRAS status. P. nalgiovense has an endogenous god gene, however the expression of that gene is weak. For this reason the endogenous GOD activity of P. nalgiovense is not inhibitory for bacteria. By using the P. nalgiovense transformation system (Geisen, 1989) the god gene from A. niger was introduced into P. nalgiovense. Transformed strains could be identified, which leads to a colour change in a medium containing an pH-indicator, whereas the wild type showed no colour change under these conditions. This was an indication of increased production of gluconic acid. Subsequent Southern blotting experiments revealed that all the strains which exhibited this colour change had copies of the god gene of A. niger integrated into their genome. An analysis of the GOD enzyme activity with these strains revealed, that one of the strains had a nearly 6 time and another strain a nearly 3 time higher god activity than the wild type. With these strains an inhibitory agar diffusion assay was performed. For that purpose indicator bacteria mixed in soft agar were spread out onto an agar plate containing glucose and 5 day old colonies of P. nalgiovense strains, either transformed with the plasmid carrying the god gene or wild type. The plates were incubated over night. The result of that experiment is shown in Figure 6. The transformed strains clearly suppressed the growth of the indicator organism S. aureus. This growth inhibition was dependent on the glucose concentration in the medium. The higher the concentration, the higher the inhibitory effect. The wild type showed no antibacterial activity under these conditions indicating that the higher GOD activities from the transformed strains were obviously responsible for this effect. This

20

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

inhibition could be observed with S. aureus, L. monocytogenes und S. enterititis as indicator organisms.

Figure 6: Inhibitory activity of the transformed P. nalgiovense strains (2, 5 and 7 o'clock) compared to the wild type (10 and 12 o'clock). The plates were covered with 100 µl of an overnight culture of S. aureus mixed with 10 ml soft agar.

These results indicate that optimised strains in contrast to the wild type are able to suppress the growth of undesired bacteria, which may occur in mould fermented foods. 2.4 HETEROLOGOUS GENE EXPRESSION IN P. NALGIOVENSE 2.4. CLONING OF GENES FROM P. NALGIOVENSE IMPORTANT FOR THE FERMENTATION PROCESS It was mentioned above, that especially proteases and lipases play a role in the flavour formation of the fermented product. The change of proteolytic activity of strains either by gene amplification or gene inactivation by the gene disruption approach can lead to strains isogenic to the wild type, but with the differences in the proteolytic activity. This in term can lead to strains with different fermentation properties. It is known from P. roqueforti, that this species has different proteolytic systems. It produces extra and 21

ROLF GEISEN AND PAUL FARBER

intracellular proteases. (Stepaniak et al. 1980). The relative activity between the particular proteases can differ from strain to strain (Paquet and Gripon, 1980). P. roqueforti shows its highest proteolytic activity at acidic pH, which is in agreement with our results, as obviously the acidic protease of P. nalgiovense is the main protease of P. nalgiovense. From P. roqueforti an aspartic proteinase (Zevaco et al. 1973) and an metallo proteinase (Gripon and Hermier, 1974) have been characterised. Much less is known about the proteolytic system of P. nalgiovense. In an attempt to isolate genes for proteases a gene bank of P. nalgiovense was constructed in an expression plasmid, For this purpose the chromosomal DNA of P. nalgiovense have been isolated partially, digested with an restriction enzyme and fragments of 4 - 5 kb were cloned into an E. coli expression vector. Highly competent cells of E. coli were transformed with the plasmid gene bank of P. nalgiovense. About 20.000 independent clones of E. coli, each carrying a particular plasmid were screened for proteolytic activity by growing on medium containing skim milk. Among these clones one could be identified, which exhibited proteolytic activity. The detailed analysis of the clone revealed that it contained a chromosomal DNA fragment from P. nalgiovense of 1.4 kb, which obviously coded for a protease gene. According to Southern blot experiments the gene occurs only in a single copy in the genome of P. nalgiovense. To verify that the cloned fragment indeed codes for a protease gene from P. nalgiovense the plasmid was modified and reintroduced into the P. nalgiovense wild type strain. From the resulting transformants one could be identified with highly reduced proteolytic activity (Fig. 7)

Figure 7: Proteolytic activity of the wild type (left) compared to the transformed strain of P. nalgiovense (right).

Apparently in this strain a gene replacement has taken place. Subsequent Southern Blot analysis support this possibility (Geisen, 1995). An analysis of the pH optimum of the cloned protease, by comparing the proteolytic activity of the different strains on media 22

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

with skim milk and different pH revealed, that the cloned protease has its optimum at acidic pH. The described analysis demonstrated that a protease gene of P. nalgiovense has been cloned, and that it can be used for influencing the proteolytic activity of a given strain, either by gene replacement or by gene amplification. 3. Penicillium camemberti P. camemberti was first described by Thom (1906). Some synonyms exist for this species like P. caseicola, P. album, P. candidum or P. rogeri. P. camemberti is nearly exclusively found in cheeses and cheese factories. This species is thought to be a domesticated form of P. commune (Pitt et al. 1986). As already discussed above, one important prerequisite for a fungal starter culture is its toxicological acceptability. All strain of P. camemberti analysed so far, are however able to produce cyclopiazonic acid (CPA) according to Frisvad (1988). CPA is a secondary metabolite with toxic activity in animal experiments (Le Bars, 1979). CPA is toxic mainly against the liver, kidneys or the pancreas. CPA can also be produced by P. camemberti on cheeses, especially at higher storage temperatures. According to an assessment of the toxicity of this mycotoxin in cheese, it is not a real health problem, however strains that would not produce this toxic secondary metabolite would be advantageous. About the genetics of CPA production nothing is known, so the gene disruption approach as described above cannot be used with this species. For this reason it was tried to isolate a CPA- mutant of P. camemberti by mutation/selection. CPA consists of the amino acid tryptophane and the isoprenoid dimethylallypyrophosphate (Fig. 8).

Figure 8: Molecular structure of cyclopiazonic acid.

For mutation, spores of P. camemberti were treated with nitrite. Single spore colonies from the mutation experiment were plated out on agar plates and screened for the production of CPA. From 5000 colonies two strains could be isolated with reduced CPA production. One strain Cpa1 did not produce any detectable amounts of CPA, whereas the other strain Cpa2 produced only very small amounts of CPA compared to the wild type. The wild type was able to produce 34 µg CPA per gram mycelium (wet weight), whereas the mutant Cpa2 produced only 0,8 µg, which is only 2% of the amount of the wild type (Geisen and Leistner, 1990). However after TLC analysis Cpa2 accumulated a new metabolite, which could not be identified in the wild type (Fig. 9). 23

ROLF GEISEN AND PAUL FARBER

Figure 9: Thin layer chromatogram of the chloroform extractable secondary metabolites of different mutants of P. camembert (lane 1 to 4, Cpa2, Cpa3, Cpa4, Cpa5; different mutant strains), the wild type (lane 5) and purified CPA as standard (lane 6).

The possibility exists that this might be an intermediate of the CPA biosynthetic pathway, which accumulates just before the mutational block. To analyse this possibility the mutant strain Cpa2, as well as the wild type strain were grown in the presence of radioactively labelled 3H-tryptophan, which is a precursor of CPA. The extracted metabolites of both the wild type and the mutant were separated by twodimensional TLC and subjected to autoradiography. The CPA spot of the wild type gave a strong signal, however the new metabolite, which occurred in the mutant was not labelled. The isolated spots were counted in a scintillation counter and the incorporated radioactivity was quantified. This data show that the new metabolite has the same background activity than the control. This results indicate that obviously the mutational block must be located "before" the ligation of the isoprenoid moiety to the amino acid tryptophane. This means that the mutation should be located in the part of the pathway in which the isoprenoid moiety is produced. For that reason, the same experiment were carried out, but in the presence of 14C acetate, which is the direct precursor of the isoprenoids. The extracted secondary metabolites of the wild type and the mutant Cpa2, which were grown on the labelled medium, were also subjected to two dimensional TLC. This time a clear difference in the pattern of the separated labelled secondary metabolites could be observed (Geisen, 1992). This indicates that a mutation, which influences the production of isoprenoid precursors, is the reason for the inability of this strain to produce CPA. A physiological analysis showed that the mutated strain has the same growth behaviour than the wild type strain.

24

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

4. Penicillium roqueforti P. roqueforti was first described by Thom (1906). Until recently P. roqueforti was a heterogeneous species of blue green sporulating fungi. Because of their phenotypic differences they were first grouped to different species, but were combined into one species by Raper and Thom (1949). Recently Boysen et al. (1996) divided the species according to secondary metabolite production and to nucleotide sequence differences in the ribosomal ITS regions into three different species: P. roqueforti, P. paneum and P. carneum. P. roqueforti can produce PR toxin, marcfortines and fumigaclavine A; P. carneum patulin, penitrem A and mycophenolic acid and P. paneum patulin, botryodiploidin and other secondary metabolites. Beside its activity as starter culture P. roqueforti is an important spoilage organism for different food and feed commodities, like bread (Spicher and Isfort, 1987), cheese (Wendorf, 1993), fruits (Harwig, 1978) and silage (Häggblom, 1990). Beside other mycotoxins most strains of P. roqueforti are able to produce PR toxin (Scott et al. 1977; Wie and Liu, 1978; Lafont et al., 1979; Chang et al. 1991). PR toxin is not stable in cheese and is degraded to the less toxic PR imine (Siemens and Zawistowski, 1993). However strains which would not produce this mycotoxin would be advantageous over the producing strains. PR toxin was hepato- and nephrotoxic in studies with mice. According to Moulé et al. (1979) it inhibits the protein biosynthesis in eukaryotic systems. PR toxin is a sesquiterpene (Fig. 10). Strains that would not produce this mycotoxin would be advantageous over producing strains to be used as starter cultures.

Figure 10: Molecular structure of PR toxin

However screening for the PR toxin negative phenotype only by analysis of the PR toxin producing ability by chromatographic methods do not guarantee that the identified strains are indeed not able to produce PR toxin. The production of PR toxin is highly dependent on the environmental conditions, which means that the identified strains might be able to produce the toxin under other conditions.

25

ROLF GEISEN AND PAUL FARBER Table 2: Aristolochene synthases specific PCR reactions with different P. roqueforti strains

BFE No

PR-toxin

PCR

42

+

+

50

-

-

53

+

+

168

++

+

169

++

+

17-

++

+

172

nd

+

208

++

+

209

++

+

210

++

+

211

++

+

215

++

+

216

-

+

219

++

+

269

++++

+

270

+++

+

A better way to screen for PR toxin negative strains, is to look for the presence of certain genes coding for key enzymes in the biosynthetic pathway of the secondary metabolite. If strains can be identified, which do not have a gene coding for an enzyme in the biosynthetic pathway of PR toxin, they should not be able to produce PR toxin under any condition, if no other metabolic shunt exists. One of the genes for a key enzyme in the metabolic pathway to PR toxin has been cloned and sequenced (Proctor 26

NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS

and Hohn, 1993). It codes for the aristolochene synthase ( ari1 ). Aristolochene is a precursor in the biosynthesis of PR toxin. The knowledge of the nucleotide sequence of that gene can be used for a molecular screening method for strains that did not carry the aril gene by PCR. Strains, which do not carry the ari1 gene, should not be able to produce PR toxin. For these purpose primer sequences have been deduced from the published sequence of the ari1 gene and different P. roqueforti strains have been tested for the presence of the ari1 gene by using these specific primer in a PCR reaction. The results are shown in Table 2. As can be seen in this table one strain could be identified by this method, which apparently did not carry an ari1 gene. As it was possible that only the primer binding sites may have been mutated in that gene, which also would give rise to a negative PCR reaction, the results were confirmed by dot blot analysis. This negative strain was also not able to produce PR toxin under the conditions used. On the other hand another strain could be identified, which also did not produce PR toxin, however which gave a positive result in the PCR reaction. These results suggest, that this strain contains either a mutant gene, which is responsible for the non-producing phenotype, or the strain may produce the toxin under other conditions. In any case the strain with the negative PCR results should be preferred. 5. Conclusions Fungal strains should be carefully selected according the specific need of a fermented product. If it is not possible to find a strain that fulfils all the requirements, it can be optimised by several methods. Either molecular methods can be used to introduce additional characteristics to a strain or to inactivate specifically undesired features of a particular strain. In cases were molecular data are not available, the conventional mutation/selection approach may be used, however it has to be kept in mind that this approach is less specific and that secondary mutations with undesired phenotypic changes may occur. Screening for particular characteristics, based on molecular data is preferable over conventional phenotypic screening, as the results are more reliable. References Andersen, S.J. and Frisvad, J.C. (1994) Penicillin production by Penicillium nalgiovense.. Lett. Appl. Microbiol. 19, 486-488. Banke, S., Frisvad, J.C. and Rosendahl, S. (1997) Taxonomy of Penicillium chrysogenum and related xerophilic species, based on isozyme analysis. Mycol. Res. 101, 617-624. Bester, H. B., and S. H. Lombard. 1990. Influence of lysozyme on selected bacteria associated with gouda cheese. J. Food Prot. 53, 306-311. Bidouchka, M.J., McDonald, M.A., St. Leger, R.J. and Roberts, D.W. (1994) Differentiation of species and strains of entomopathogenic fungi by random amplification ofpolymorphic DNA (RAPD). Curr. Genet. 25, 107-113 Boysen, M., Skouboe, P., Frisvad, J.C., and Rossen, L. (1996) Reclassification of the Penicillium roqueforti group into three species on the basis of molecular genetic and biochemical profiles. Microbiology 146, 541-549. Chang, S.C., Wei, Y.H., Wei, D.L., Chen, Y.Y. and Jong, S.C. (1991) Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti. Appl. Environ. Microbiol. 57, 2581-2585. Färber, P. and Geisen, R. (1994) Antagonistic activity of the food-related filamentous fungus P. nalgiovense by the production of penicillin. Appl. Environ. Microbiol. 60, 3401-3404.

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ROLF GEISEN AND PAUL FARBER Färber, P. (1998) Biosynthese des β-Lactan Antibiotikums Penicillin durch den filamentösen Pilz Penicillium nalgiovense, einer Starterkultur zur Herstellung Schimmelpilz-gereifter Fleischwaren. Dissertation, Universität Hohenheim Fiero, F., Gutiérrez, S., Diéz, B. and Martin, J.F. (1993) Resolution of four large chromosomes in penicillinproducing filamentous fungi: the penicillin gene cluster is located on chromosome II (9.6 Mb) in Penicillium notatum and chromosome I (10.4 Mb) in Penicillium chrysogenum. Mol. Gen. Genet. 241, 573-578 Fox, P. F. and Law, J. (1991) Enzymology of cheese ripening. Food Biotechnology 5, 239-262 Frisvad, J. (1988) Fungal species and their specific production of mycotoxins, in R. A. Samson and E.S. and Reenen-Hoekstra (eds.) Introduction to food borne fungi, Publishers: Centralbureau voor Schimmelcultures, Baarn, pp. 239-249. Geisen, R. and Leistner, L. (1989) Transformation of Penicillium nalgiovense with the amdS gene of Aspergillus nidulans. Curr. Genet., 15,307-309. Geisen, R., Glenn, E. and Leistner, L. (1990) Two mutant strains of Penicillium camemberti affected in the production of cyclopiazonic acid. Appl. Environ. Microbiol. 56, 3587-3590. Geisen, R. (1992) Characterisation of a mutation in a strain of Penicillium camemberti affecting the production of cyclopiazonic acid. Fungal Genetics Newsletters 39, 20-22. Geisen, R. (1995) Expression of the Aspergillus niger glucose oxidase gene in Penicillium nalgiovense. World Journal of Biotechnology 11, 322-325. Gripon, J.C. and Hermier, J. (1972) Le systeme proteolytique de Penicillium roqueforti. 1. Conditions de production et nature du systeme proteolytique. Le lait 52, 497-514. Guthrie, P.A.I., Magill, C.W., Frederiksen, R.A. and Odvody, G.N. (1992) Random amplified polymorphic DNA Markers: A system for identifying and differentiating isolates of Colletotrichum graminicola. Phytopathology, 82,832-835. Häggblom, P. (1990) Isolation of roquefortin C from feed grain. Appl. Environ. Microbiol, 56, 2924-2926. Hamelin, R.C., Ouellete, G.B. and Bemier, L.: Identification of Gremmeniella abietina races with random amplified polymorphic DNA makers. (1993) Appl. Environ. Microbiol,, 59, 1752-1755. Harwig, J., Blanchfield, B.J. and Scott, P.M. (1978) Patulin production by Penicillium roqueforti Thom from grape. Canadian Institute of Food Science and Technology Journal 11, 149-151. Hesseltine, C.W. (1983) Microbiology of oriental fermented foods. Ann. Rev. Microbiol. 37, 575-601 Jacobsen, T. and Hinrichsen, L. (1997) Bioformation of flavour by Penicillium candidum, Penicillium nalgiovense and Geotrichum candidum on glucose, peptone, maize oil and meat extract. Food Chemistry 3, 409-416. Kriechbaum, M., H. J. Heilmann, F. J. Wientjes, M. Hahn, K. D. Jany, H. G. Gassen, F. Sharif and G. Alaeddinoglu.(1989) Cloning and DNA sequence analysis of the glucose oxidase gene from Aspergillus niger. FEBS Lett. 255, 63-66. Lafont, P., Debeaupuis, J., Gaillardin, M. and Payen, J. (1979) Production of mycophenolic acid by Penicillium roqueforti strains. Appl. Environ. Microbiol. 37, 365-368. Laxa, O. (1932) Überdie Reifung des Ellischauer Käses. Zentral.f. Bakt. 86, 160-165. Le Bars, J. (1979) Cyclopiazonic acid production by Penicillium camembert Thom and natural occurrence of this mycotoxin in cheese. Appl Environ Microbiol 38, 1052-1055. Leistner, L. (1986) Mould-ripened foods. Fleischwirtschaft 66, 1-4. Leistner, L., Geisen, R. and Fink-Gremmels, J. (1989) Mould-fermented foods of Europe: hazards and developments, in eds: S. Natori, K. Hashimoto and Y. Ueno (eds.), Mycotoxins and Phycotoxins, Publishers, Elsevier Science Publishers, Amsterdam, pp. 145-1 54 Lund, F., Filtenborg, O. and Frisvad, J.C. (1995) Associated mycoflora of cheese. Food Microbiol. 12: 173180 Marth et al (1987) Dairy Products, in L. R. Beuchat (eds.), Food and Beverage Mycology, Publishers, Van Nostrand Reinhold, New York, pp. 175- 209. Proctor, R.H. and Hohn, T.M. (1993) aristolochene Synthase: Isolation, characterisation and bacterial expression of a sequiterpenoid biosynthetic gene (Ari1) from Penicillium roqueforti. J. Biol. Chem. 268, 4543-4548. Paquet, J. and Gripon, J.C. (1980) Intracellular peptide hydrolases of Penicillium roqueforti. Milchwissenschaft, 35, 169-1 77. Pitt, J.I., Cruickshank, R.H. and Leistner, L. (1986) Penicillium commune, P. camemberti, the origin of white cheese moulds and the production of cyclopiazonic acid. Food Microbiology 3, 363-371. Raper, Raper, K. B. and Thorn, C. (1949) Manual of the Penicillia. Williams and Wilkins, Baltimore. Reed, G. & Underkofler, L.A. (1966) Enzymes in food processing, New York, Academic press.

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NEW ASPECTS OF FUNGAL STARTER CULTURES FOR FERMENTED FOODS Scott, P.M., Kennedy, P.B.C., Harwig, J. and Blanchfield, B.J (1977) Studies for conditions for production of roquefortine and other metabolites of Penicillium roqueforti. Appl. Environ. Microbiol. 33, 249-253. Siemens, K. and Zawistowski, J. (1993) Occurrence of PR imine, a metabolite of Penicillium roqueforti, in blue cheese. J. Food. Prot. 56,317-319. Spicher, G. und Isfort, G. (1987) Die Erreger der Schimmelbildung bei Bachwaren. IX Die auf vorgebackenen Brötchen, Toast und Weichbrötchen auftretenden Schimmelpilze. Deutsche Lebensmittelrundschau 83, 246-249. Stepaniak, L., Kornacki, K., Grabska, J., Rymaszewski, J. and Cichozs, G. (1980) Lipolytic and proteolytic activity of Penicillium roqueforti, Penicillium candidum and Penicillium camemberti strains. Acta Alimentaria Polonica 6, 155-164. Stiles, M. E. 1994. Potential for biological control of agents of foodborne disease. FoodRes. Int. 27,245-250, Tiina, M. and M. Sandholm (1989) Antibacterial effect of the glucose oxidase-glucose system on foodpoisoning organisms. Int. J. Food Microbiol. 8, 165-174. Thom, C. (1906) Fungi in cheese ripening: Camembert and Roquefort. US Dept. Agr. Bur. Anim. Und. Bul. 82, 1-39. Wei, R. and Liu G. (1978) PR Toxin production in different Penicillium roqueforti strains. Appl. Environ. Microbiol. 35, 797-799. Wendorf, W.L., Riha, W.E. and Muehlenkamp, E. (1993) Growth of moulds on cheese treated with heat or liquid smoke. J. Food. Prot. 56,963-966. Williams, J.K.G., Kubelik, A.R. Livak, K.J., Rafalski, J.A. and Tingey, S.V. (1990) DNA polymorphism amplified by arbitrary primers are useful as genetic markers. Nucleic Acid. Res. 18, 6531-6535. Zevaco, C., Hermier, J. and Gripon, J.C. (1973) Le systeme proteolytique de Penicillium roqueforti. II. Purification et proprietés de la protease acide. Biochemie 55, 1353-1360.

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STARTERS FOR THE WINE INDUSTRY ALINE LONVAUD-FUNEL Faculté d’œnologie - Université Victor Segalen Bordeaux 2, 351 Cours de la Libération, 33405 TALENCE Cedex

Abstract Grape must is naturally seeded with yeasts and lactic acid bacteria. Both microorganisms are needed : yeasts to carry out alcoholic fermentation, and bacteria to degrade malic acid to lactic acid during malolactic fermentation. Indigenous microflora is often sufficient. However, use of selected yeast and malolactic starters allow a better control of the process. Strains are selected for their influence on the sensorial and hygienic quality of wine. 1. Introduction Winemaking requires the successive involvement of two microorganisms. Yeasts ferment the grape must into wine, then lactic acid bacteria complete the malolactic fermentation. The later fermentation is not obligatory but recommended for optimising the quality of nearly all red wines and most white wines. Alcoholic fermentation is, of course, the main microbial event in winemaking. Pasteur was the most famous microbiologist interested in wine production. The role of yeast in alcoholic fermentation was assessed by several authors at the end of the 19th century. Their observations and experiments were so accurate and judicious that they discovered the essential proceedings, describing the primary (ethanol) and secondary products of alcoholic fermentation, the influence of several factors on the fermentation rate and on the quality of wine. As early as 1895 numerous experiments led one of the oenologists of this time to write a chapter on the use of selected yeasts for improving wine fermentation (Brunet, 1894). Modern oenology does not refute this concept. It confirms and proves that using yeast starters makes alcoholic fermentation safer and faster, thus conferring to the wine better sensorial quality. Today several dozens of yeast starters are available and are used intensively in all the wine producing areas in the world. The situation is somewhat different for lactic acid bacteria that are responsible for malolactic fermentation. Indeed, this second step of winemaking was recognised as essential in the 1940s by some oenologists in the Burgundy and Bordeaux areas. It took more than 20 years in some producing areas to consider that malolactic fermentation 31 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 31–47. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

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was not an accident but a second microbial transformation that improves wine quality. Usually lactic acid bacteria develop after the end of alcoholic fermentation, i.e. during and after the decline phase of yeasts. Thus they multiply in a medium which is not only acidic but which also contains up to 13 % ethanol. They face very harsh conditions. The addition of selected malolactic starters was first considered in the early 70s. However, the success of such preparations was much more difficult than for yeast starters, precisely because of the composition of wine. Therefore, progress has been slowed down by many drawbacks, so today the state of the art for lactic acid bacteria starters is approximately the same as it was two decades ago for yeasts. However, due to the increasing knowledge on the physiology and metabolism of such bacteria, it is expected that the gap will be soon filled. In the early 90s the first malolactic starter for direct inoculation of wine was commercialised (Nielsen et al., 1996). The demand for yeast starters is high both in the new and older wine producing countries. Yeast producers are still selecting new starters on a wide scale, since the variety of wines produced all over the world is great, and the potential ability of yeasts seems greater than as used at present. As for lactic acid bacteria , the knowledge of their influence on wine is just in its infancy and more needs to be discovered about their adaptation to the wine medium to increase the success of malolactic starters. 2. Yeast starters in winemaking After grape crushing, the must is naturally seeded with the microorganisms that are on the surface of the berries and the cellar equipment. Soon after the grapes have been sent to the fermentation tank or barrel, alcoholic fermentation normally starts with the indigenous yeasts, Several yeast species can be isolated on the grapes and thus in the grape must before fermentation: Candida sp., Kloeckera apiculata, Metschinikovia pulcherrima, Torulaspora delbruekii, Schizosaccharomyces pombe and Saccharomyces sp. K. apiculata, T delbrueckii and S. cerevisiae often predominate (Herraiz et al., 1990). However, most of them quickly disappear except for Saccharomyces cerevisiae that is the main agent of alcoholic fermentation (Fleet et al, 1984). According to the way alcoholic fermentation is carried out defects may be induced such as off-flavours, volatile acidity or even stuck fermentations. This might result in the predominance of some undesirable yeast strains. The non-Saccharomyces species are mainly involved in such problems. In addition, some Saccharomyces strains are not well–adapted to the fermentation of sugar-rich medium and cannot complete the whole process. Some also produce high levels of SO2 and acetic acid. This explains why spontaneous fermentation can be considered as a hazardous phase. Today yeast starters are exclusively selected strains of Saccharomyces cerevisiae. 2.1 THE OBJECTIVES OF YEAST STARTERS The reasons for using yeast starters were established at the end of 19th century. The experiments consisted in seeding the grape must by actively growing yeasts which were taken from fermenting musts of good quality. The experimenters observed that the quality of wine was consequently higher, and fermentation more regular and quick (Brunet, 1903). In addition, Pasteur (1876) noted that if the same grape must was 32

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inoculated by several distinct yeasts, the wines would also be distinct. The present objectives of winemakers are exactly the same. The only difference between the two centuries is that the selected strains today are individual clones while they were « mixed » starters before, since there was no isolation procedure. However, 100 years ago, wines were probably obtained by Saccharomyces sp. Now winemakers use single strain starters in order to control the process more closely. There are two reasons for using starters. One is to start the alcoholic fermentation quickly after the harvest. Indeed, in some cases, and preferably at the beginning of the winemaking the yeast population is too low (less than 104 CFU.ml-1). Multiplication up to 106 and more takes several days especially if the temperature is low. During this time, other microorganisms can develop : yeasts with oxidative metabolism and acetic acid bacteria that take advantage of the presence of oxygen to produce volatile acidity and many other defects. Thus, inoculation with starters at the concentration of 106 CFU.ml-1 prevents the growth of such microorganisms. The second reason for the winemaker to use yeast starters is to improve the final phase of alcoholic fermentation. Indeed, grape musts are so rich in sugar and sometimes so poor in essential nutrients that yeast cannot survive long enough to ferment all sugars. Stuck fermentation is one of the major problems in winemaking. The fermentative ability of the yeast cell decreases during alcoholic fermentation due to its sensitivity to increasing ethanol and fatty acids. Both compounds are toxic for yeast (Lafon-Lafourcade et al , 1984) and the target of this effect is the membrane. Oenologists and winemakers have long known that aeration is essential to improve alcoholic fermentation. This was also explained by the work of Larue and Lafon-Lafourcade (1989) who demonstrated that the survival of yeast in a fermenting must depends on the sterol composition of the membrane, which itself depends on the O2 available during growth. This also explains why yeast starters that are produced in aerobiosis can respond more efficiently to these conditions than indigenous yeasts. The addition to must of selected starters also named « active dry yeasts » has become essential in white winemaking. It has replaced the former starters that were prepared by multiplying yeasts from a spontaneous fermenting wine in highly sulfited must to prevent bacterial growth. White grape must is not easily fermentable. Indeed, after crushing it generally undergoes to several operations (decanting, clarification) to improve its sensorial quality, and these make it poorer in nutrients and in natural microflora. Therefore, it is necessary to add yeast very soon, once the must has been clarified or adjusted to the adequate turbidity by addition of light solids (Ollivier et al , 1987). Active dry yeasts are added to reach 106 CFU.ml-1, i.e. 10 to 15 g.hl-1 of the commercial preparation. They are added just after the clarification of grape juice, after a reactivation in grape must and water (v/v ; 1 :1) for 20 minutes at 40°C. Yeast starters are less used, and in fact less necessary in red winemaking. The operations that follow grape crushing do not deprive the medium of the natural microflora, since all the parts of the berries skin, pulps and seeds are poured into the vats. Moreover, the process includes several pumping over phases to extract the wine colour, and some of these are done with aeration. It is only when the temperature is too low or if rain has considerably washed the grapes that alcoholic fermentation for red wines may take several days to start. In these cases active dry yeasts are necessary.

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However, as winemakers are more and more aware of the difficulties encountered in obtaining the alcoholic fermentation, they also use starters for red wines. The second reason to use active dry yeasts is to restart uncompleted alcoholic fermentation. When several g.l-1 of sugar remain unfermented, the wine is not finished and is exposed to spoilage. Indeed during the active phase of fermentation, yeasts inhibit bacteria, but since their population decreases and may lead to stuck fermentation, lactic acid bacteria can develop. This induces one of the major accidents in winemaking called « piqûre lactique » characterised by the heterolactic fermentation of sugars. The result is a high volatile acidity. Therefore as soon as alcoholic fermentation slows down a sufficient population of active yeasts must be added. The commercial active dry yeasts need a preliminary preparation otherwise they cannot survive, since the medium is high in ethanol and fatty acids. The reactivation procedure is conducted as follows in a medium prepared with the wine itself. The alcohol content is reduced to 8-9 % with water and adjusted to 15 g.l-1 of sugar. After sulfiting at 3 g/hl-1, 20 g.hl-1 of active dry yeast are added. At 20°C growth occurs within several days and the fermentation is monitored by determination of sugar concentration or density. When the sugar has just totally disappeared, the yeasts are in the active growing phase. The starter is added at the concentration of 5-10 % in the wine to be treated. The completion of alcoholic fermentation still takes several days. The efficiency of such starters can be increased by the preliminary addition, 24-48 hours before, of yeast cell walls. Lafon-Lafourcade et al (1984) demonstrated that the preparation obtained from yeast cells by elimination of the yeast cellular components - i.e. the yeast cell walls – enhances yeast survival in fermenting must. It is now an authorised procedure to prevent or to treat stuck fermentations. This regulatory property is mainly due to the capacity of cell walls to fix and to remove yeast inhibitors such as fatty acids. 2.2 PROPERTIES OF YEAST USED AS SELECTIVE CRITERIA FOR ACTIVE DRY YEAST PRODUCERS AND WINEMAKERS All the active dry yeasts available share the same basic characters ; i.e. good multiplication rate, high yield of ethanol production, ability to complete fermentation of sugars (resistance to ethanol and other toxic compounds such as pesticide residues), adaptation to low and high temperatures, low production of volatile acidity of ethanol, sulphur dioxide, sulphur hydrogen, foam, and high glycerol production. In addition, it is preferable for them to be neutral towards the killer toxins or even producers of the toxin, and not to be sensitive since they mightbehampered by indigenous killer strains (Jacobs and van Vuuren, 1991). Finally, such selected strains must withstand the drying process and recover their properties after storage. These are the minimum requirements for selected yeast starters. However, among the dozens of preparations available, one must choose the best adapted to the special problem to be solved. They can be divided in several categories : quick starter yeasts, those for restarting stuck alcoholic fermentation, others for « prise de mousse » of sparkling wine, some for wines used to make spirits and finally yeasts aimed to develop regional or aroma typicality. The first are selected for their resistance to the hostile conditions of wine fermentation. However, more and more work is now underway to find strains able to develop aromas and to optimise phenolic compound extraction in red wine. « Primeur » red wines and white wines are mainly characterised by fruity and 34

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floral aromas. Esters, especially shorter chain fatty acid ethyl esters and acetate esters, are the main volatile components involved in the sensorial quality of wines, together with carbonyl compounds such as acetaldehyde and diacetyl. They are produced by yeast metabolisms (Henschke and Jiranek, 1993). The conditions of alcoholic fermentation, notably temperature and the presence of solids through yeast metabolism modifications change the final amount of volatile compounds. However, the strain itself has a real impact (Vasconcelos et al., 1996), e.g. if it produces two much of one of the volatile components. Some are revealed by yeast activity on the aroma precursors found in grape berries (Darriet et al , 1991).

Figure 1 : Influence of the yeast strain on the relative intensity of the Sauvignon aroma (after Darriet et al., 1991)

On the contrary, some yeasts may suppress the variety aromas ; they must be used only for the fermentation of neutral grape varieties. Among many other compounds, yeast can produce vinyl phenols from p-coumaric acid and ferulic acid in must. Above a certain threshold these give the wine pharmaceutical odours. The enzymatic activity, cinnamate decarboxylase, depends on the yeast strain (Figure 2) (Chatonnet et al, 1993). The role of yeast in the production of varietal aromas is currently intensively studied. The involvement of yeast in the release of terpenes which characterise grape varieties such as muscat, gewürztraminer, riesling, etc.... and in that of norisoprenoids that are very odorous, is not clear. More is known about the characteristic aroma of 35

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Sauvignon that appears during alcoholic fermentation. The non-odorous precursors which have been identified in grape must are cysteine complexes. The aromas are revealed by the action of a specific β-lyase. The activity varies according to the yeast strain. The S-cysteine conjugates are hydrolysed by yeasts during alcoholic fermentation (Tominaga et al, 1998). Therefore strains have been selected for their capacity to reveal the Sauvignon aroma. Other varietal aromas such as gewürztraminer and manseng are also enhanced.

Figure 2 : Influence of the yeast strain on the vinylphenol concentration of a white wine (Sauvignon variety) (Chatonnet et al., 1993)

In red wines, the type and amount of polyphenols is very important. An aim of winemaking is to obtain the highest amounts of the « good » polyphenols. It has also been shown that yeast can influence the final composition in phenolic compounds and thus the wine colour. This study led to the selection of two yeast strains that will be dried and tested in a pilot-scale study (Arioli et al., 1996). From such studies, companies producing active dry yeast for the wine industry can make new preparations. However, winemakers must remember that the wine aroma precursors are in the must. Therefore, the yeast has to be adapted to the grape must. The aim is to reveal the aromas without excess, since the overproduction of some components is undesirable.

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2.3 EVALUATION OF THE SETTLEMENT OF ACTIVE DRY YEAST DURING ALCOHOLIC FERMENTATION : Active dry yeasts are usually added to grape must containing indigenous microflora originating from the grapes and the cellar equipment. This microflora is usually low in white grape musts due to several operations aimed to eliminate solid materials from the medium. The level of microflora is normally higher in red grape musts. However, until recently it was not easy to assess the efficiency of the starter, i.e. it was difficult to ascertain whether alcoholic fermentation was done by the starters or by the indigenous yeast population. Today molecular methods are very reliable tools to monitor and to assess the role of starters. Several methods can be used to differentiate a Saccharomyces strain from another. The first method that was developed was the analysis of restriction patterns generated by hydrolysis of mitochondrial DNA (mtDNA). Oenological strains were successfully characterised by Dubourdieu et al (1987) and Hallet et al (1988). The profile of the active dry yeast used as starter is compared to that obtained from the lees sampled during or after alcoholic fermentation. If there is no extra band, this means that the starter has grown and has replaced the original yeasts. However, in spite of its reliability, this method is no longer used because it is unwieldy and time-consuming. The analysis of karyotypes needs less preparation. It consists in a separation by pulse field gel electrophoresis of the 16 yeast chromosomes. Since their size varies from 250 to 2500 kbp, this gives a profile for each strain . The preparation of samples is rapid and easy, but the electrophoresis of such large DNA molecules requires special electrophoresis equipment. Blondin and Vezhinet (1 988), then Frezier and Dubourdieu (1991) applied this method for wine yeast strain identification during winemaking. Like others, Roulland et al (1996) using this method reported that spontaneous alcoholic fermentation is carried out by one or at the most two dominant strains in wine used to make Cognac. Finally PCR (Polymerase chain reaction) using repeated sequences as primers is another very interesting method for the identification of strains during winemaking (Ness et al, 1992). It is much more rapid than the other methods and can be used directly on whole cells. The profile generated by specific amplification can distinguish most of the strains very easily. Like the mtDNA RFLP profiles and the karyotypes, the PCR patterns of the yeasts taken in lees are compared to those of the starter. They are identical if the starter has really taken part in the process. By PCR it is shown that 90 % of the population is derived from the starter when the profiles are identical. Usually the starter chosen is added as soon as possible to the grape must to inhibit and eliminate the indigenous yeasts. The chance of success is 90 % if the ratio starter/indigenous yeast is 10. Some have recommended successively adding two different starters in order to enhance wine complexity. A study was undertaken to verify the role of both starters inoculated in the same vat (Meurgues et al., 1998). Molecular typing by PCR of the yeasts taken at the end of alcoholic fermentation clearly showed that the second starter, which was added two days after the first one, had completely disappeared. As expected, addition at about 6.106 CFU.ml-1 of the second starter, i.e. three times the normal dose, could not replace the active fermenting population of the first starter which was about 108 CFU.ml-1. Thus, the addition of selected yeasts during the course of alcoholic fermentation is of little use. 37

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Analysis of the diversity of yeast strains during alcoholic fermentation has also demonstrated that a selected starter may be easily disseminated in a cellar in red wine winemaking (Frezier and Dubourdieu,1991). While only the first vat filled was inoculated, the presence of the starter was dominant even in vats filled several days after and not inoculated (Table I). Table I : Percentages of yeast strains isolated in fermenting grape musts inoculated or not with active dry yeasts (after Frezier and Dubourdieu, 1991)

Isolated Vat 1 inoculated with strain F5 strains F5 80 F10 0 522M 0 FIb4 10 FIb9 10 FIIIb8 0

Vat 2 inoculated Vat 3 inoculated Non with strain F10 with strain 522M inoculated 0 0 60 100 0 40 0 90 0 0 0 0 0 0 0 0 10 0

This means that in red winemaking mere cellar handling is sufficient to disseminate yeasts. However, the use of massive concentrations of different starters allows their settlement in several vats. In white winemaking, the conditions and the results may be similar except when must is fermented in barrels, thus making dissemination less possible. Progress in the identification of yeast strains has also led to real advances also for controlling the production of starters themselves. This is obviously very important since winemakers can now really monitor the role of the starters they have chosen according to the type of wine. 3. Malolactic starters in winemaking Lactic acid bacteria must reach the population of 106 CFU.ml-1 in order to start malolactic fermentation. Depending on the variety, the producing area and the degree of maturity, the grape must contains about 2 to 8 g.l-1 of malic acid. In most cases all malic acid must be transformed to lactic acid. The benefit is a softer taste and greater aroma complexity. However, some white wines only need the partial degradation of malic acid that results in deacidification while preserving the fruity aromas that normally disappear with total malolactic fermentation. Bacteria must grow after alcoholic fermentation in very hostile conditions due to ethanol, fatty acids, other inhibitors and acidic pH. However, in normal conditions, some days after the yeasts have completely fermented the sugars, bacteria multiply very quickly from 102-103 CFU.ml.-1 to more than 106 CFU.ml-1. The growth rate depends on the environmental conditions, the adaptation of the bacteria to the medium and on the yeast responsible for alcoholic fermentation ( Lonvaud-Funel et al, 1988). Like yeasts, the indigenous lactic acid bacteria originate from grapes and cellar equipment. In the beginning of winemaking up to eight to nine species are usually identified. However after a few days, a natural selection occurs in the fermenting must. At the end of alcoholic fermentation, the Oenococcus oeni species 38

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is predominant or exclusive, so it is the species most adaptable to wine conditions (Lonvaud-Funel et al, 1991). 3.1 INDICATIONS FOR USE OF MALOLACTIC STARTER AND DESCRIPTION The start of malolactic fermentation still remains completely uncontrolled. After alcoholic fermentation, the winemaker can only watch and wait. Temperature is the only parameter that may be controlled in order to promote malolactic fermentation. The use of special equipment to adjust it to about 20°C is very common and efficient. The wine is heated until malic acid degradation starts. However, in spite of temperature control, malolactic fermentation does not start sometimes due to a very low pH or to unknown reasons. It is not rare for it to occur several months after alcoholic fermentation, generally in May-June following the harvest. This proves that bacteria can adapt their composition and physiology to wine. This of course presents several disadvantages. Wine cannot be protected from spoilage microorganisms by sulfiting that would prevent lactic acid bacteria growth. Therefore ageing and maturation may be delayed and « young » wines cannot be sold. Moreover, as long as malolactic fermentation has not begun, the temperature must be maintained even during wintertime, thus creating additional costs. The aim of using malolactic starters is to compensate the deficient indigenous microflora by a high population of lactic acid bacteria. The first experiments with liquid cultures were for a long time unsuccessful. The bacterial cultures added to the wine really lost their activity and viability. Later when work on this subject began again, the objective was to inoculate the wine with very concentrated populations. Since it is difficult to obtain the multiplication of bacteria in wine, the idea was to add the necessary level of bacteria (more than 106 CFU.ml-1) in order to do without the growth phase. The starters were prepared with the species O. oeni. The latter is always used nowadays in spite of several attempts to use other species such as lactobacilli that can multiply more easily in industrial conditions. The concentrated cultures were first frozen, then lyophilised. However, even the addition of more than 106-107 CFU.ml-1 in wine failed and nearly no malic acid could be transformed. The first really successful use of malolactic starters was obtained by Lafon-Lafourcade et al (1983). Their research showed that the survival of lyophilised bacteria after inoculation could be greatly enhanced by a reactivation step. The lyophilised preparation was incubated for 1-2 days in a reactivation medium composed of grape juice diluted ½ in water, enriched with 5 g.l-1 yeast extract and adjusted to pH 4.8. Other reactivation media were later described; all contain yeast extract. The population during this step is around 1010 CFU.ml-1 and does not increase. Wine is inoculated with 1 vol. for 1000 vol. with this reactivated culture to reach a viable population of 107 CFU.ml-1. In these conditions the bacteria survive after inoculation and malic acid is completely degraded. Thus reactivation allows a better adaptation of bacteria to wine. Very often such starters are inoculated in small volumes of wine which are then poured into larger ones, up to the volume of the total vat which needs to be inoculated, This procedure is also successful but needs more time and care (Gerbaux et al, 1995). Obviously, in spite of their efficiency, such reactivated starters are not very convenient for winemakers. They necessitate special attention and good organisation.

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New research in the early 90s was undertaken on the resistance of O. oeni to several inhibitors such as ethanol and fatty acids. The focus was cell membrane studies since it was thought that the membrane was the primary target of the inhibitors, which kill the bacteria. Lonvaud-Funel and Desens (1990), then Garbay and Lonvaud-Funel (1 996) demonstrated that the cultivation in media added with ethanol or fatty acids or other stresses such as heat shock induced changes in the membrane composition. The most striking feature was the overproduction of some membrane proteins and a decrease in phospholipids. Such stressed cells were also more apt to survive in wine. Following these results, the first O. oeni lyophilised preparation for direct inoculation in wine became available in 1993 (Viniflora oenos, Chr. Hansen, Denmark) (Nielsen et al, 1996). The population is about 5-106 CFU.ml-1 and it grows or survives until the complete transformation of malic acid (Figure 3). A few other preparations are now available. But it is clear that the production of malolactic starters is much more difficult than yeast starters for which the winemaker can chose amongst dozens of preparations. The other problem when using a malolactic starter is the time of inoculation. While there is no alternative for yeast starters, it has often been suggested that malolactic starters can be used before, during or after alcoholic fermentation. Of course addition before alcoholic fermentation avoids the inhibition of bacteria by wine components. However, as for the indigenous bacteria, survival is readily inhibited by the active growing yeasts. Moreover, if bacteria survive during alcoholic fermentation, they can ferment sugar, in competition with yeasts. The real problem is not the loss of ethanol but the production of volatile acidity which accompanies the heterolactic fermentation of hexoses and pentoses by O. oeni. In addition, it has clearly been demonstrated that at the end of alcoholic fermentation, at a time when yeast cells are losing their activity, too many bacteria (over than 105-106 CFU.ml-1) tend to accelerate the yeast decline phase Paraskevopoulos (1988). Bacterial enzymatic activity probably contributes to the hydrolysis of the yeast cell wall. In such conditions the major problem is incomplete alcoholic fermentation. This frequently occurs naturally when the indigenous bacterial population grows too early (Ribereau-Gayon et al, 1998). At this time wine still contains some 5-10 g.l-1 of fermentable sugars, a concentration high enough to produce volatile acidity. Since the behaviour of the bacteria is completely unpredictable, it is not recommended to add malolactic starters as long as the sugar have not been completely fermented.

Figure 3A : Evolution of malic acid in a red wine inoculated or not by a malolactic starter. (Nielsen et al , 1996)

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Figure 3B : Evolution of bacterial population in a red wine inoculated or not by a malolactic starter. (Nielsen et al , 1996)

3.2 THE INFLUENCE OF LACTIC ACID BACTERIA STARTERS ON WINE QUALITY AND THEIR SELECTION Since the crucial problem of the survival of malolactic starters in wine has been at least partially solved, studies now focus on the important question of their influence on wine quality. Two aspects are now considered : hygiene and the sensory qualities. For a long time oenologists considered lactic acid bacteria only as the agents of malic acid degradation. Even if it is the principal biochemical reaction, bacteria also metabolise many other wine components. The most studied has been citric acid that is transformed into acetic acid and acetonic compounds. Only about a maximum 0.3 g/l of citric acid are metabolised but this is enough to induce sensorial changes due to the production of acetic acid (volatile acidity) and diacetyl which is very aromatic. Only a few mg/l of the latter gives the wine a buttery aroma. The threshold is dependent on the wine, and is less for white wines (= 4-5 mg/l) than for red wines (7-8 mg/l). Sensorial analyses show that some judges prefer « buttery » wines, while others consider high diacetyl concentrations as a defect. The other sensorial effects are characterised by the production of fruity and floral notes. It is not definitively established that they depend on the dominant bacterial strain. Numerous studies have compared wines inoculated by distinct bacterial strains and have shown their individual influence (Henick-Kling T., 1993). However, sensorial analyses rarely show significant differences. Moreover in such experiments, the repetition of inoculation by a given strain may often give more sensorial differences than different strains. This point remains unclear and such results

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show that malolactic fermentation has a real influence on wine aroma, but this is less strain-dependent than alcoholic fermentation. Other compounds are undesirable such as biogenic amines, which might be responsible for allergies, and ethyl carbamate that is thought to be carcinogenic. For a long time only non-malolactic bacteria (all species except O. oeni), and especially Pediococcus sp. were thought to produce histamine. However O. oeni strains may be involved. Such strains contain the hdc gene that codes for the histidine decarboxylase. Surprisingly, it has been shown that many strains have this property (Coton et al, 1998). During and mainly after malolactic fermentation, tyramine and putrescine (diaminobutane) also increase in some wines. However, until now the bacteria involved have not been completely characterised. Another observation is that some wines do not contain any biogenic amine, while others produced from year to year in the same cellar always contain them. At least for histamine, it is concluded that the indigenous bacterial flora that is established in the cellar comprises aminoacid decarboxylating strains. Ethyl carbamate that is produced from precursors such as urea, carbamylphosphate and citrulline, is also studied by food hygienists. In wine, urea is produced by yeast, and citrulline is produced by lactic acid bacteria from arginine (Liu et al., 1994). Current studies aim to study and characterise O. oeni that may utilise the arginine deiminase pathway and increase the risk of ethyl carbamate in wine. Therefore, the selection criteria for malolactic fermentation starters are based both on the capacity of the strain to be prepared at the industrial level for survival in wine and on some special metabolic pathways. Usually the strains are isolated from wines during malolactic fermentation. The only species considered is here O. oeni. After identification the first selection procedure includes sensitivity to ethanol and to pH. The latter is certainly the most important parameter. Malolactic activity is not a real selection marker since it is generally high for all strains. The ability to metabolise citric acid is also a general trait of this species and must be considered as a positive character. The other property to be considered is the inability to produce biogenic amines. Since many studies are focusing on sensorial quality and secondary metabolisms, selection will probably include more criteria in the future. 3.3 EFFICIENCY OF MALOLACTIC STARTERS Unlike yeast starters, malolactic fermentation starters have been difficult to promote because in the beginning they were very unreliable. Moreover, in numerous studies, authors have concluded that they were efficient because malolactic fermentation occurred after inoculation. Frequently, it was not clear if the malic acid is degraded by the starter or by the indigenous bacteria, especially when malolactic fermentation starts long after inoculation. This probably explains why the market for malolactic fermentation starters is still unsure. Winemakers are not convinced of the effectiveness of the starter for the above mentioned reasons. Today, it is possible to verify the settlement and the role of added bacteria with molecular methods, such as for active dry yeasts. The most relevant method is to compare the genomic fingerprints of the bacteria taken at the end of malolactic fermentation to those of the starters. Genomic studies of O. oeni have shown that each strain can be distinguished from others by the electrophoretic pattern of digested DNA. Restriction by rare cutting enzymes generates DNA fragments that are well separated by 42

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pulse field electrophoresis. Three enzymes are frequently used and are well adapted for this species. Thus, the procedure consists in the cultivation on solid medium of bacteria present in wine at the end of malolactic fermentation. The DNA of the whole biomass (or individual colonies) is analysed. If the starter has really dominated the indigenous microflora, the profile is strictly similar to that of the lyophilised bacteria. The presence of additional bands proves that other bacteria were in the wine at the same level and probably took part in the process (Daniel et al. 1993, Gindreau et al., 1997). Of course, analyses of samples during the process can indicate whether the starter undergoes a real competition with the indigenous bacteria. This advance is very new but very promising. Indeed in none of the previous studies, notably those to analyse the sensorial impact of starters, could the survival of the starter be assessed. Today, wine scientists have a suitable method to verify the ability of the starter to induce malolactic fermentation before the decision is taken to implement fastidious and difficult sensorial and fine chemical analyses. After malolactic fermentation sulphur dioxide is added to eliminate as far as possible all the microorganisms and to protect wine from oxidation. However, in many wines of relatively high pH, sulphur dioxide reduces its antimicrobial activity and the bacterial population remains relatively high. Identification to the strain level by fingerprinting has recently shown an interesting property of malolactic fermentation starters. Comparison of populations in inoculated and non-inoculated wines proved that the population remaining after sulfiting did not contain the starter strain at least at a sufficient level to be detected by DNA fingerprinting. It was composed of the indigenous lactic microflora (Millet and Lonvaud-Funel, unpublished results). Thus, if this result was confirmed, it would mean that starters are eliminated soon after malolactic fermentation. This is very positive, since it means that a given starter would be very unlikely to be perpetual in the cellar. 4. The future of starters for winemaking The interest of winemakers for yeasts and bacteria starters is motivated by the potential they offer to control the fermentation processes. Nowadays, active dry yeasts are widely used. Winemakers in USA, Canada, South Africa, Australia, New Zealand were the first users by the mid-1960s and it took about 10 years more for the European producers to start with yeast inoculation. Malolactic starters came very later and the industrial preparations for direct inoculation are very recent. Yeasts are inoculated : i) to shorten the time between the harvest and the active alcoholic fermentation which is crucial to prevent spoilage, ii) to avoid incomplete fermentation, iii) more and more, to develop specific aromas. In fact, the application of elementary oenological rules solves the second point at least. The judicious aeration of fermenting must provide enough O2 for yeast to synthesise the indispensable sterols it needs for survival (Lafon-Lafourcade, 1983). Temperature control also minimises the problems. Nevertheless, it seems that more and more active dry yeasts are widely used for safety, even if they are not really necessary. While they are indeed almost indispensable in white wine production because of all the preliminary treatments of the grape musts, this is not the case for red wines. More and more the principal objective of the producer is to develop aromas during alcoholic fermentation. Of course, it is highly 43

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preferable that they are typical aromas characterising if possible not only the grape variety but also the grape variety in its environment, i.e. marked by the « terroir » Even if there is already a considerable choice of starters , much work is still underway to improve selection. The increasing knowledge of the aroma compounds revealed during alcoholic fermentation is of course the driving force of this activity. Typicality related to wine variety is consistently influenced by the dominant strain during alcoholic fermentation. Therefore, aroma quality could be enhanced by the selection of the best-adapted strains in view of their enzymatic activities. However, typicality is also related to many other factors, and the use of a given starter will not necessarily result in the same wine in different wine areas. This justifies the increasing attention of yeast selectors for the aromas produced by candidate strains for starters. As in the early age of wine microbiology, some oenologists have in recent years proposed using yeast species other than S. cerevisiae. Indeed it is well known that the indigenous yeast microflora is composed of several genera such as Candida, Rhodotorula, Brettanomyces, Torulopsis, Harsemaspora, and Kloeckera. They are in variable proportions in the must and disappear due to their sensitivity to ethanol and O2 depuration. However, some of them may survive relatively late during the alcoholic fermentation. Their influence on wine aromas has been demonstrated (Herraiz et al., 1990). The apiculate yeasts, Kloeckera apiculata and Hanseniasporia guillermondii, were assayed associated to Saccharomyces cerevisiae (Romano et al, 1992 ). They were found to greatly influence the composition of wine and aromas. Thus there is now renewed interest in the non-Saccharomyces microflora, and active dry yeast producers are studying their potential use. Raguiel et al (1998) describe the results obtained with a T. delbruekii strain and a strain obtained by fusion of S. cerevisiae and Schizosaccharomyces pombe characterised by its capacity to degrade malic acid. While the latter was studied for deacidification, the former was studied for its property to produce strawberry aroma. The authors conclude that the use of the T. delbruekii strain mixed in suitable proportions with S. cerevisiae is interesting and justifies further experiments in various environmental conditions. Selection of yeasts as starter should also include, strains for deacidification and on others which retain or even increase the initial acidity of grape must. These mainly differ by their ability to degrade or produce more or less L-malic acid. The traditional selection of yeast strains might also be replaced in the future by genetically modified yeasts. Even if the use of such microorganisms is subjected to controversy several research teams are already preparing this approach. The first work in the early 90s concerned the transformation of S. cerevisiae with the malolactic bacterial gene. The idea was to add to the yeast the malolactic activity which would allow the simultaneous alcoholic fermentation and malic acid degradation (Ansanay et al., 1993, Denayrolles et al, 1995, Volschenk et al, 1997 ). Following this research several other functions were considered for cloning in the wine yeast : higher production of glycerol (Remize et al, 1999), of L-lactic acid (Dequin and Barre, 1994), secretion of proteolytic enzymes (Lourens and Pretorius, 1996). However, the success and the demand for such yeasts starters is very unpredictable. As regards lactic acid bacteria starters, much future work will concern the adaptability of inoculated bacteria to the stress conditions of wine. Even if great progress has been made with the starters for direct inoculation, there are still too many 44

STARTERS FOR THE WINE INDUSTRY

unaccountable cases where the best ones fail. Today, the present results on aminoacid decarboxylation and the production of ethylcarbamate can be added to the conventional traits for the selection of new starters. Therefore, much more knowledge is necessary regarding the sensorial influence of lactic acid bacteria at the strain level. If this is demonstrated and considered relevant, then malolactic starters will also be selected for these features. 5. Conclusion The production of quality wine is tightly linked not only to the quality of grapes but also to the microorganisms that completely change the composition of the medium where they develop. The fate of grape juice is to promote the growth of yeasts and bacteria. Alcoholic fermentation and malolactic fermentation spontaneously occur in most cases. However, fewer winemaking accidents occur now than in the former times by controlling yeasts and bacteria. Yeast starters are readily available and the choice gets wider each year. However, the winemaker now has a new challenge: he must choose the most suitable strain according to the grape must and the type of wine. This is not so difficult, but there are many examples of bad choice. This is especially true when strains produce too high concentrations of aromas ; this might finally lead to standardising wine. It is preferable to choose the strain which express the best the wine typicality in relation to the grape and its environment. Sometimes the best solution is to use a « neutral » strain that does not mark the wine too heavily. The present interest of researchers for non- Saccharomyces strains is very important since most reports demonstrate that wine aroma complexity is enhanced by inoculation of such yeasts. However, mixed cultures even from selected starters may be as difficult to control as the indigenous microflora. Finally, the most worrying problem is the dissemination and above all the remanence of the starters in the cellar. It has already been shown that yeast starters recur in a given cellar from year to year. Of course this does not prevent to use different strains efficiently because they are massively added. However, this will surely suppress the natural biodiversity of grape and wine microflora. At least for the moment, the danger seems, much lower for lactic acid bacteria starters which seem to be unable to survive after malolactic fermentation. References Ansanay, V., Dequin, S., Blondin, B., Barre P. (1993) Cloning sequence and expression of the gene encoding the malolactic enzyme from Lactococcus lactis. FEBS Letters 332, 74-80. Arioli, X., Colas, S., Cuimei, C., Daumas, F. and Lurton, L. (1996) Incidence de la souche de levure sur l’acidité, la couleur et la teneur en composes phénoliques des vins rouges. Application á la sélection de souches pour les vins de la Vallée du Rhône. In Œnologie 95, pp 246-250. A. Lonvaud-Funel Coordonnateur, Technique et Documentation Lavoisier (eds.) Paris. Blondin, B. and Vezinhet, F. (1988) Identification des souches de levures œnologiquespar leurs caryotypes obtenus en électrophorése en champs pulsés. Revue Française d’Oenologie 115, 7-11. Brunet, R. (1894) Les levures in Traité de vinification. pp 13-29. Masson (eds.) Paris. Chatonnet, P., Dubourdieu, D., Boidron, J.N. and Lavigne, V. (1993) Synthesis of volatile phenols by Saccharomyces cerevisiae in wines. Journal of Science Food and Agriculture. 62, 191-202.

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ALINE LONVAUD-FUNEL Coton, E., Rollan, G., Bertrand, A., Lonvaud-Funel, A. (1998) Histamine producing lactic acid bacteria in wines : early detection frequency and distribution. American Journal of Enology and Viticulture 49, 199204. Daniel, P., De Waele, E. and Hallet, J.N. (1993) Optimisation of transverse alternating field electrophoresis for strain identification of Leuconostoc oenos. Applied Microbiology Biotechnology 38, 638-641, Darriet, P., Lavigne, V., Boidron, J.N. et Dubourdieu, D (1991) Caractérisation de I’arômevarietal des vins de Sauvignon par couplage chromatographie en phase gazeuse-odométrie. Journal International des Sciences de la Vigne et du Vin 25, 167-174. Denayrolles, M., Aigle, M., Lonvaud-Funel, A. (1995) Functional expression in Saccharomyces cerevisiae of Lactobacillus lactis mleS gene encoding the malolactic enzyme. FEMS Microbiology Letters 125, 37-44. Dequin, S., Barre, P. (1994) Mixed lactic acid-alcoholic fermentation by Saccharomyces cerevisiae expression the Lactobacillus casei L(+)LDH. Biotechnology 12, 173-177. Dubourdieu, D., Sokol, A., Zucca, J., Tharlouan, P., Datte, A. and Aigle, M. (1987) Identification des souches de levures isolées de vin par analyse de leur ADN mitochondrial. Connaissance de la Vigne et du Vin 21, 267-278. Fleet, G.H., Lafon-Lafourcade, S. and Riberéau-Gayon, P. (1984) Evolution of yeasts and lactic acid bacteria during fermentation and storage of Bordeaux wines. Applied Environmental Microbiology 48, 10341038. Frézier, V. and Dubourdieu, D. (1991) Incidence du levurage sur I’écologie des souches de Saccharomyces cerevisiae au cours de la vinification dans deux crus du Bordelais. Journal International de la Vigne et du Vin 25, 63-70. Garbay, S. and Lonvaud-Funel, A. (1996) Response of Leuconostoc oenos to environmental changes. J. Appl. Bacteriol. 81, 619-625. Gerbaux, V., Cuinier, C., Barrère, C. and Berger, J.L. (1995) Bilan de cinq années d’essais d’ensemencement de vins en bactéries lactiques sélectionnées dans quatre vignobles français. Revue Française d’œnologie 154, 18-22. Gindreau, E., Joyeux, A., de Revel, G., Claisse, O., Lonvaud-Funel A. (1997) Evaluation de I’établissement de levains malolactiques au sein de la microflore bacterienne indigene. Journal International de la Vigne etdu Vin 31, 197-202. Henick-Kling, T. (1993) Malolactic fermentation in Wine Biotechnology , pp. 289-326, G.H. Fleet (eds.), Harwood Academic Publishers, Switzerland. Henschke, P.A. and Jiranek, V. (1993) Yeasts metabolism of nitrogen compounds, in Wine Biotechnology, pp. 77-1 64, G.H. Fleet (eds.), Harwood Academic Publishers, Switzerland. Herraiz, T., Reglero, G., Herraiz, M., Martin-Alvarz, P.J., Cabezudo, M.D. (1990) The influence of the yeast and type of culture on the volatile composition of wines fermented without sulphur dioxide. American Journal of Enology and Viticulture 41, 313-318. Jacobs, C.J. and Van Vuuren, H.J.J. (1991) Effects of different killer yeasts on wine fermentations. American Journal of Enology and Viticulture 42, 295-300. Lafon-Lafourcade, S. (1983) Wine and Brandy, in Biotechnology Vol. 5, H.J. Rehin and G. Reed (Eds). Springer-Verlag, Publ. London. Lafon-Lafourcade, S., Carre, E., Lonvaud-Funel, A. and Riberéau-Gayon, P. (1983) Induction de la fermentation malolactique des vins par inoculation d’une biomasse industrielle de L. oenos après reactivation, Connaissance de la Vigne et du Vin 17, 55-71, Lafon-Lafourcade, S., Geneix, C., and Ribéreau-Gayon, P. (1984) Inhibition of alcoholic fermentation of grape must by fatty acids produced by yeasts and their elimination by yeast ghosts, Applied Environmental Microbiology 47, 1246-1249. Larue, F. and Lafon-Lafourcade, S. (1989) Survival factors in wine fermentation, in Alcohol toxicity in yeasts and bacteria. N. Van Uden (eds.), pp. 193-215, Florida, CRC Press Inc. Liu, S.Q., Pritchard, G.G., Hardman, M.J. and Pilone, G.J. (1994) Citrulline production and ethyl carbamate (urethane) precursor formation from arginine degradation by wine lactic acid bacteria Leuconostoc oenos and Lactobacillus buchneri. American Journal of Enology and Viticulture 45, 235-242. Lonvaud-Funel, A. et Desens, C. (1990) Constitution en acides gras des bactéries lactiques du vin. Incidence des conditions de culture. Sciences des Aliments 10, 811-829. Lonvaud-Funel, A., Joyeux, A. and Ledoux, O. (1991) Specific enumeration of lactic acid bacteria in fermenting grape must and wine colony hybridisation with non-isotopic DNA probes. Journal of Applied Bacteriology 71, 501-508. Lonvaud-Funel, A., Masclef, J. Ph., Joyeux, A. et Paraskevopoulos, Y. (1998) Etude des interactions entre levures et bacteries lactiques dans le moût de raisin. Connaissance de la Vigne et du Vin 22(1) 11-24.

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STARTERS FOR THE WINE INDUSTRY Lourens, K. and Pretorius, S. (1996) Genetic engineering of Saccharomyces cerevisiae for the efficient synthesis and secretion of proteolytic enzyme. In Œnologie 95, pp 290-292, A. Lonvaud-Funel Coordonnateur, Technique et Documentation Lavoisier (eds.) Paris. Malepeyre, F. et Brunet, R. (1903) Emploi des levures sélectionnées in : Fabrication et emploi de la levure. pp 258-265. Encyclopédie Roret. Mulol (Eds.) Paris. Meurgues, O., Monamy, C. and Dulau, L. (1998) Etude sur le levurage fractionné. Revue des Oenologues 89, 23-24. Ness, F., Lavallee, F., Dubourdieu, D., Aigle, M. and Dulau, L. (1992) Identification ofyeast strains using the polymerase chain reaction. Journal of Science Food and Agriculture 62, 89-94. Nielsen, J.C., Prahl, C. and Lonvaud-Funel, A. (1996) Malolactic fermentation in wine by direct inoculation with freeze-dried Leuconostoc oenos cultures. American Journal of Enology and Viticulture 47, 42-48. Ollivier, Ch., Stonestreet, T., Larue, F. and Dubourdieu, D. (1987) Incidence de la composition colloïdale des moots blancs sur leurs fermentescibilité. Connaissance de la Vigne et du Vin 21, 59-70. Paraskevopoulos, Y. (1988) Utilisation des enveloppes cellulaires de levure pour la stimulation de la fermentation malolactique. Interpretation de leur mode d’action. Thése de Docteur Ingénieur, Université de Bordeaux II. Pasteur L. (1876) Etudes sur le vin. Librairie Impériale, Paris Querol, A., Barrio, E., Huerta, T. and Ramon, D. (1992) Molecular monitoring of wine fermentations conducted by active dry yeast strains. Applied Environmental Microbiology 58, 2948-2953. Raginel, F., Forsgren, K., Nonals, S. and Dulau, L. (1998) Ensemencement mixte de moût avec des levures Saccharomyces et non-Saccharomyces. Revue Française d ‘Oenologie 171, 14-19. Remize, F., Roustan, J.L., Sablayrolles, J.M., Barre, P., Dequin, S. (1999) Glycerol overproduction by engineered Saccharomyces cerevisae wine yeast strains leads to substantial changes in by-product formation and to stimulation of fermentation rate in stationary phase. Applied Environmental Microbiology 65, 143-149. Ribereau-Gayon, P., Dubourdieu, D., Donèche B. and Lonvaud-Funel A. (1998) Le developpement des battéries lactiques dans le vin. In Microbiologie du Vin. Vinifications. Traité d’œnologie I. pp. 197-224. Dunod (eds.), Paris. Romano, P. , Suzzi, G., Comi, G and Zironi, R. (1992) Higher alcohol and acetic production by apiculate wine yeasts Journal ofApplied Bacteriology 73, 126-130. Roulland, C., Versavaud, A., Galy, B., Lurton, L., Dulau, L. and Hallet, J.N. (1996) Analyse de la biodiversité des populations fermentaires et mise en oeuvre d’une stratégie de sélection de souches pour I’élaboration du Cognac. In Œnologie95, pp 163-166. A. Lonvaud-Funel Coordonateur, Technique et Documentation Lavoisier (eds.), Paris. Tominaga, T., Peyrot des Gachons, C. and Dubourdieu, D. (1998) A new type of flavour precursors in Vitis vinifera L. cv. Sauvignon blanc : S-cysteine conjugates. Journal of Agricultural Food Chemistry 46, 5215-5219. Vasconcelos, I., Moreira, N., Herdeiro, M.T., Pereira, O. and de Revel, G.(1996). Etude de l’influence de la levure d’implantation et de la composition chimique du moût sur les caractéristiques aromatiques des vins blancs issus de cinq cépages portugais. In Œnologie 95, pp 260-265. A. Lonvaud-Funel Coordonnateur, Technique et Documentation Lavoisier (eds.), Paris. Volschenk, H., Viljoen, M. Grobler, J. Petzold, B., Bauer, F., Subden, R.E., Young, R.A., Lonvaud-Funel, A., Denayrolles, M., Van Vuuren, H.J.J. (1997) Engineering pathways for malate degradation in Saccharomyces cerevisiae. Nature Biotechnology 15, 253-257

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Part 2 PHYSIOLOGY, BIOSYNTHESIS AND METABOLIC ENGINEERING

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METABOLISM AND LYSINE BIOSYNTHESIS CONTROL IN BREVIBACTERIUM FLAVUM: IMPACT OF STRINGENT RESPONSE IN BACTERIAL CELLS M. RUKLISHA*, R. JONINA, L. PAEGLE AND G. PETROVICA *Corresponding author, Institute of Microbiology and Biotechnology, University of Latvia, Kronvalda blvd.4, L V 1586, Riga, Latvia; Fax: (+371) 7 323 065; E-mail: [email protected]

Abstract Parameters affecting lysine biosynthesis by Brevibacterium flavum RC 115 cells under stringent response conditions (test guanosine 5'-diphosphate 3'-diphosphate accumulation in cells) caused by threonine limitation were investigated. Experimental results confirmed that an increase in lysine biosynthesis by this bacterium under stringent response conditions might be a result of an increase in the intracellular concentration of NADPH as well as lysine export activity. 1. Introduction Corynebacterium glutamicum and closely related Brevibacterium flavum species are widely used for lysine production on an industrial scale. An understanding of the physiology of amino acid producing bacteria is of special significance to choose the most suitable environmental conditions in which bacteria will show their maximum product synthesis rate and yield from the consumed sugars. As has been shown previously, the decreasing of bacterial growth rate and maintenance of its value below maximum for an extended period is an essential method to improve the process productivity under fed-batch cultivation conditions of C. glutamicum and B. flavum strains (Ruklisha et al., 1992). On the contrary, an increase in the bacterial growth rate in the exponential growth phase up to maximum was observed to be followed by a decrease (sometimes even irreversible) in the lysine synthesis activity of cells in the stationary phase (Ruklisha et al., 1976). An increase in lysine synthesis under growth limiting conditions was suggested to be a consequence of the stringent control mechanism mediated via guanosine 5'-diphosphate 3'-diphosphate (ppGpp) accumulation (Ruklisha et al., 1995). 51 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 51–57. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

M.RUKLISHA, R.JONINA, L.PAEGLE AND G.PETROVICA

The aim of the present studies was to investigate the effect of stringent response in B. flavum RC 115 cells on changes in the concentrations of most relevant internal metabolites, required for lysine synthesis and bacterial lysine synthesis activity as its consequence. 2. Materials and Methods The microorganism used was B. flavum RC 115 - auxotroph for threonine and methionine (Culture collection of the University of Latvia). Bacteria were cultivated under batch conditions in fermenter (MBR, Switzerland) on a glucose/corn steep liquor containing media (Ruklisha et al., 1995). Respiratory activity of the cell culture (QO2) under fermentation conditions was monitored by oxygen balance method using gasanalysing system to estimate difference between oxygen concentration in the inlet and outlet gas of fermenter (Baburin et al., 1986). In some experiments bacteria, collected from fermenter, were recultivated for one hour in flasks on a rotary shaker in slight modified mineral medium described by Kiss and Stephanopoulos (1991) with 1.5-mM threonine or without its complementation. Biomass, lysine (as lysine.HCl) and sugar concentration in the medium, intracellular concentration of ppGpp, as well as parameters of bacterial physiological activity and lysine biosynthesis (µ, h-1; qP, g lysine .g cells-1. h-1 and YP/S, g lysine. g glucose-1) were estimated as described by (Ruklisha et al., 1995). Pyruvate and NADPH were extracted from cells by the respective procedures described by Weibel et al., 1974, Matin & Gottschal, 1976 and measured by enzymatic methods. Intracellular lysine was extracted by a method proposed by Erdman et al. (1994). Concentration of amino acids in cell extracts or fermentation medium was assayed by HPLC (Shimadzu C-R4A chromatograph, Japan). The samples were treated with 10% sulphosalicylic acid in order to remove proteins, and filtered prior to analysis. The amino acids were quantified by automatic precolumn derivatisation with o-phtalaldehyde, separated in the column (Nova Pak C 18, Waters, Milford, Mass., USA) using a buffer/methanol gradient, and detected fluorometrically using Shimadzu RF-530, fluorescence HPLC monitor (Japan). 3. Results and Discussion The effect of stringent response on changes in metabolite concentrations in B. flavum RC 115 cells and lysine biosynthesis was investigated under batch cultivation conditions. Bacterial respiratory activity profile during fermentation, measured by oxygen analyser, was applied to test changes in bacterial growth rate and to establish appropriate intervals of time during the fermentation process to collect samples. Applying this test, cell culture was collected from fermenter within 10 minutes from the moment when stringent response in bacterial cells was induced (test: an increase in intracellular concentration of ppGpp). The results of the experimental work showed that the lysine biosynthesis of B. flavum RC 115 cells significantly increased under batch cultivation conditions when bacterial growth rate decreased below 0.08 h-1 (Fig.1A) as a result of threonine limitation (Fig 1B). Besides, the lysine yield from consumed sugar in the time interval 52

METABOLISM AND LYSINE BIOSYNTHESIS CONTROL IN BREVIBACTERIUM

18-22 h increased up to 0.55 ± 0.04 g lysine .g glucose-1. The increase in the lysine synthesis activity of cells was correlated with a sharp increase in ppGpp concentration in cells (Fig.2A).

Figure 1. Changes in the rate of bacterial growth (µ) and lysine biosynthesis (qP) by B. flavum RC 115 cells as well as changes in the concentration of threonine in growth medium during batch cultivation.

Hence, it was assumed that the increase of lysine biosynthesis might be a consequence of the stringent response in bacterial cells like changes in RNA synthesis and modification of metabolic pathway functioning, changes in intracellular concentrations of lysine precursors and others

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Figure 2. Changes in the bacterial respiratory activity (QO2) and internal concentrations of ppGpp, pyruvate as well as NADPH in B. flavum RC 115 cells during batch cultivation.

In further experiments, changes in the intracellular concentrations of NADPH and pyruvate as well as that of lysine biosynthesis by B flavum RC 115 cells under exponential growth and stringent response conditions were investigated. It was shown that a stringent response in bacterial cells resulted in a significant accumulation of NADPH and some increase in the pyruvate concentration in bacterial cells (Fig.2B). However, no direct correlation between induction of stringent response and changes in the internal concentration of pyruvate in cells was demonstrated since its internal concentration during exponential growth increased in parallel with the increase of bacterial growth rate. Therefore, the increase of the internal concentration of NADPH and not pyruvate might be considered as a parameter favouring lysine biosynthesis by B. flavum RC 115 cells under stringent response conditions.

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METABOLISM AND LYSINE BIOSYNTHESlS CONTROL IN BREVlBACTERIUM

The fact that with the increase of lysine synthesis concomitantly increased synthesis of other NADPH-consuming amino acids, e.g. valine (Fig.3), proline and isoleucine (data is not presented), confirmed the possible role of NADPH accumulation in cells on amino acid biosynthesis under stringent response conditions. It might indicate that the physiological meaning of the increase of biosynthesis of lysine and other NADPH-consuming amino acids under stringent response conditions would be NADP+ regeneration in cells.

Figure 3. Changes in the external concentrations of valine and lysine in the cell culture of B. flavum RC 115 during batch cultivation.

Changes in the intracellular concentration of lysine and the specific rate of its extracellular accumulation by B. flavum RC 115 under stringent response conditions differed. An internal concentration of lysine increased in parallel with the increase of bacterial growth rate but did not increase further under stringent response conditions (Fig.4). On the contrary, the extracellular accumulation of lysine under the latter conditions significantly enhanced (see: Fig. 1A). Hence, it was suggested that, under stringent conditions, induced by threonine starvation, lysine export from B. flavum RC 115 cells possibly increased. The lysine export with the help of specific excretion carriers by C. glutamicum had been proved by Erdman et al. (1994). A possible role of the stringent response on lysine export activity was investigated using cells of different growth rate values, collected in appropriate moments of batch cultivation. Cell culture of each sample was divided in two parts; cells were separated from the medium by centrifugation and transferred in two versions of fresh pre-warmed mineral media (with or without threonine). Cell culture with an initial concentration of biomass 10 ± 1 g cells.l-1 were recultivated in shake flasks for one hour. The results of short-term experiments Metabolism and Lysine Biosynthesis Control in Brevibacterium flavum showed that lysine as well as valine (the latter data are not presented) were accumulated in the medium only as a result of threonine limitation (Table 1). The higher was the

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initial rate of bacterial growth the higher increase in lysine synthesis was achieved under conditions of threonine limitation.

Figure 4. Changes in the internal concentration of lysine in B. flavum RC 115 cells during batch cultivation.

Hence the stringent response, induced in B. flavum RC 115 cells by threonine limitation, probably resulted with the increase in lysine export activity. It might be assumed that a mechanism exists, which favour increase the export of lysine from bacterial cells under stringent response conditions. Rapid induction of the export of some amino acids from bacterial cells as a consequence of stringent response was experimentally proved in other bacteria. Data obtained by Burkovski et al. (1995) clearly showed that glutamate export from Escherichia coli cells was induced under stringent response conditions and this export was rel-A gene controlled. However direct role of the stringent response on lysine export activity in corynebacteria should be further investigated. Table 1 Effect of cell incubation in the medium with or without threonine on their specific lysine synthesis activity (qP) *Cells collected at appropriate times of batch cultivation (see Fig. IA) **Data represents average means ±SE of four experiments Growth rate of initial cells* (Time of collection, h)

Incubation with .5 mM threonine

0.18 (6) 0.27 (8) 0.12 (11) 0.08 (1 8) 0.03 (20)

0 0 0 0 0

Incubation without threonine

qP ( g lysine g cells-1 h-1 )** traces 0.03 ± 0.004 0.07 ± 0.005 0.06 ± 0.005 0.05 ± 0.005

4. Conclusions The data presented in this study showed that lysine biosynthesis by B. flavum RC 115 cells increased as a consequence of stringent response, induced by threonine limitation. 56

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It was explained as a result of NADPH accumulation in bacterial cells and probably that of an increase in lysine export activity. References Baburin, L., Shvinka, J., Ruklisha, M. and Viesturs, U. (1986) Gas balance method for testing of microbial growth efficiency after carbon substrate pulse, Acta Biotechnol. 6, 2, 123 - 128. Erdman, A., Weil, B. and Kramer, R. (1994) Lysine secretion by Colynebacterium glutamicum wild type: regulation of secretion carrier activity, Appl. Microbiol. Biotechnol. 42, 604 - 610. Kiss, R.D. and Stephaanopoulos, G. (1991) Metabolic activity control of the L-Lysine fermentation by restrained growth fed batch strategies, Biotechnol Progr 7, 501-509. Matin, A. and Gottschal, J.C. (1976) Influence of dilution rates on NAD(H) and NADP(H) concentrations and ratios in a Pseudomonas sp. grown in continuous culture, J. Gen. Microbiol. 94, 333-341. Ruklisha, M., Ekabsone, M., Viesturs, U., Mezina, G. and Selga,, S. (1976) Control of growth and lysine synthesis by Brevibacterium sp.22L by variations in medium mixing intensity, Appl. Biochem. & Microbiol. 2, 4, 518-523 (in Russian). Ruklisha, M., Shvinka, J. and Viesturs, U. (1992) Biotechnology of Bacrerial Synthesis, Zinatne, Riga (in Russian); Annex: 1993 (in English). Ruklisha, M., Viesturs, U. and Labane, L. (1995) Growth control and ppGpp synthesis in Brevibacterium fravum cells at various medium mixing rates and aeration intensities, Acta Biotechnol. 15, 1, 41 - 48. Weibel, K.E., Mor, J.-R. and Fiechter, A. (1974) Rapid sampling of yeast cells and automated assay of adenylate, citrate, pyruvate and glucose-6-phosphate pools, Analyt. Biochem. 58, 208 - 216.

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MOLECULAR BREEDING OF ARMING YEASTS WITH HYDROLYTIC ENZYMES BY CELL SURFACE ENGINEERING MITSUYOSHI UEDA, TOSHIYUKI MURAI, SHOUJI TAKAHASHI, MOTOHISA WASHIDA, AND ATSUO TANAKA* Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 6068501, Japan *Corresponding author. Mailing address: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606-8501, Japan. Phone: +81-75-753-5524, FAX. +81-75-753-5534, E-mail: atsuo@sbchem. kyoto-u.ac.jp

Abstract Novel yeast cells armed with biocatalysts - glucoamylase, -amylase, CM-cellulase, βglucosidase, and lipase - were constructed by a cell surface engineering system of yeast Saccharomyces cerevisiae. These surface-engineered yeast cells were termed “Arming yeasts”. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half of yeast α-agglutinin. Glucoamylase was shown to be displayed on the cell surface of S. cerevisiae in its active form, anchored covalently to the cell wall. S. cerevisiae is unable to utilise starch, while the arming cells could grow on starch as the sole carbon source. For enhancement of the ability to directly ferment starchy materials by the arming yeast, a surface-engineered yeast cell displaying two amylolytic enzymes was constructed. The gene encoding R. oryzae glucoamylase with its own secretion signal peptide and a truncated fragment of the a-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast a-factor, respectively, were fused with the gene encoding the C-terminal half of the yeast α-agglutinin. The arming cell codisplaying glucoamylase and a-amylase could grow faster on starch as the sole carbon source than the cell displaying only glucoamylase. Furthermore, a novel celluloseutilising yeast cell displaying cellulolytic enzymes in their active forms on the cell surface of S. cerevisiae was constructed by the cell surface engineering. An arming yeast co-displaying FI-carboxymethylcellulase (CM-cellulase), one of the endo-type cellulase, and (3-glucosidase from Aspergillus aculeatus was endowed with the ability of cellooligosaccharide assimilation, suggesting the possibility that the assimilation of 59 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 59-13. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

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cellulosic materials may be carried out by S. cerevisiae expressing heterologous cellulase genes on the cell surface. Furthermore, a yeast cell armed with R. oryzae lipase was also constructed. These idea will be open to all living cells and the technique will be able to endow them with novel abilities. 1. Introduction Cell surface is crucial to the life of the cells since it is the interface between the inside and the outside of the cells. It encloses the cells and maintains essential differences between the cytosol and extracellular environment. Surface proteins are responsible for most cell surface functions, serving as cell-cell adhesion molecules, specific receptors, molecular recognising devices, catalytic enzymes, transport machinery, and so on. Many surface proteins are bound through noncovalent or covalent interaction to the cell surface structures. Cells have means of anchoring specific surface proteins and of confining surface proteins to particular domains of the cell surface structure. Recently, it has been partially clarified that how these protein molecules are targeted and localised to the cell surface. Thus, it is possible to remake the functions of cell surface by taking advantage of the known transport mechanisms of proteins to the cell surface. We have been developing "Cell Surface Engineering" of yeast based on the techniques of genetic engineering to endow the cells with novel functions by displaying target proteins on their cell surface. The cell surface display system is intended to be applied to bioindustrial processes because it is required to be safe. In practical use, the most suitable microorganism is the yeast S. cerevisiae, which has 'generally regarded as safe (GRAS)’ status and can be used for food and pharmaceutical production. S. cerevisiae is a useful organism to develop the cell surface expression system. It is also an advantageous cell as a host for genetic engineering, since it enables folding and glycosylation of eukaryotic heterologous proteins expressed and is easy to handle with ample genetic techniques. Moreover, the yeast can be cultivated to a high cell density with a cheap medium. S. cerevisiae lacks both amylolytic and cellulolytic activities and is unable to ferment starchy or cellulosic materials, although they are the most abundant and utilisable resources of plant origin (Barnett, 1976). In the conventional procedure to convert starchy materials to ethanol at a high yield, the mash should be cooked at 140180°C prior to amylolysis and the energy consumed in this process results in high production costs (Maiorella, 1985). To save energy in the cooking process, the development of a non-cooking fermentation system using the enzymes, which efficiently digest raw starch, has been led. Intensive research has been conducted to construct further improved starch-utilising systems by introducing heterologous genes encoding amylolytic enzymes into yeast cells by the genetic engineering for secretive production of the enzymes. Construction of a yeast cell secreting glucoamylase from R. oryzae has been attempted for application to fermentation of raw starch without cooking (Ashikari et al., 1989). Glucoamylase from R. oryzae was an exo-type amylolytic enzyme cleaving α-1,4-linked and α-1,6-linked glucose effectively from starch. The surface-engineered yeast cell displaying glucoamylase on the cell surface

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may be able to saccharify starch by glucoamylase on its cell wall and assimilate the released glucose to proliferate and ferment (Fig. 1).

Figure 1. A model of cell surface-engineered (arming) S. cerevisiae displaying hydrolytic enzymes. E: Hydrolytic enzymes.

On the other hand, utilisation of cellulosic materials in fermentation has not been realised successfully. Cellulose, consisting of glucose units linked together by 1,4glycosidic bonds, is the most abundant carbohydrate in the biosphere. An estimated synthesis rate of cellulose is approximately 4x107 tons per year. Cellulose is the most promising renewable carbon source that is available in a large quantity for a long-range solution to resource problems of energy. However, yeast S. cerevisiae is unable to utilise cellulosic materials in spite of its versatility in industrial fermentation. Enzymatic hydrolysis of cellulose has the potential to surmount many of the drawbacks of acid hydrolysis. The cellulase system of the anaerobic cellulolytic bacterium, Clostridium thermocellum, was shown to naturally construct a discrete multi-enzyme complex located on the cell surface (Lamed et al., 1983; Tokatlidis et al., 1991), "cellulosomes", which is considered to help C. thermocellum to obtain the source of carbon and energy efficiently by enzymatic degradation of cellulose on its cell surface. An attempt to genetically display cellulolytic enzymes in their active form on the cell surface has been tried to construct a novel cellulose-utilising yeast, S. cerevisiae (Murai et al., 1997b, 1998b). As one of the target enzymes, carboxymethylcellulase (CMcellulase) from Aspergillus aculeatus classified as endo-1,4-β-D-glucan glucohydrolase (endoglucanase) which cleaves the β-1,4-glycosidic linkage of cellulose was utilised. Since CM-cellulase is an endo-type cellulase, enzymatic degradation of cellulose to glucose requires synergistic hydrolysis by different types of cellulolytic enzymes. It is predicted that short-chain cellooligosaccharides formed by the endo-action of CMcellulase is converted quickly to glucose by β-glucosidase (1,4-β-D-glucoside 61

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glucohydrolase) in A. aculeatus. However, S. cerevisiae lacks β-glucosidease activity and consequently is unable to utilise cellobiose as the carbon source. Thus, to construct a S. cerevisiae cell which is able to utilise one of cellulosic materials, cellooligosaccharides, display of β-glucosidase, in addition to CM-cellulase, on cell surface of S. cerevisiae is also necessary. In this article, our cell surface engineering system of S. cerevisiae is summarised. Display of hydrolytic enzymes on the cell surface of S. cerevisiae has a great interest since such yeast strains are expected to contribute to the production of energy and the bioremediation of environmental pollution.

Figure 2. Structure of α-agglutinin and partial amino acid sequence of its C-terminal region containing GPI anchor attachment signal sequence (A), and the molecular design of cell surface-displayed enryme (B). A: arrows indicate the predicted cleavage site. The predicted ω site is underlined.

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2. Principle of Cell Surface Engineering of Yeast To target heterologous proteins to the outmost surface of the glycoprotein layer of the cell wall, molecular information of α-agglutinin was utilised (Murai et al., 1997a). The anchoring signal of α-agglutinin was combined with the signal of the secreted enzymes using genetic engineering techniques. Fig. 2 shows the general structure of the gene for cell surface display of an enzyme. The C-terminal half of α-agglutinin (320 amino acid residues) contains a glycosylphosphatidylinositol (GPI) anchor attachment signal at the C-terminal end, like other cell surface proteins, and is utilised as an anchoring domain for heterologous proteins since these proteins are covalently linked with glucan. The cell wall of S. cerevisiae is mainly composed of mannoproteins and β-linked glucans (Fleet, 1991), and has a bi-layered structure consisting of an internal skeletal layer of glucan, composed of β-1,3- and β-1, 6-linked glucose (Manners et al., 1973), and a fibrillar or brush-like outer layer, which is composed predominantly of mannoproteins (Horisberger and Vonlanthen, 1977). These proteins are linked to glucan through covalent bond. Two types of mannoproteins are present in the cell wall of S. cerevisiae (Klis, 1994; Cid et al., 1995). Mannoproteins loosely associated with the cell wall through non-covalent bond are extractable with sodium dodecylsulfate (SDS). The other type of mannoproteins are extractable by glucanase, which are released by p1, 3- or β-1, 6-glucanase digestion of the glucan layer of the cell wall but not by SDS extraction (Fleet and Manners, 1977). Among these glucanase-extractable mannoproteins on the cell surface of S. cerevisiae, the mating-type specific agglutinins (Lipke and Kurjan, 1992) that mediate direct cell-cell adhesion between cells of opposite mating type during mating, which are supposed to be located on the outermost surface. Mating type a and α cells express a-agglutinin and a-agglutinin, respectively (Terrance et al., 1987). α-Agglutinin is encoded by AGα1 gene (Lipke et al., 1989) and interacts with the binding subunit of the agglutinin complex of a cells (Cappellaro et al., 1991). A-agglutinin consists of a core subunit encoded by AGA1 gene (Roy et al., 199 1) that is linked through disulfide bridges to a small binding subunit encoded by a different gene AGA2 (Cappellaro et al., 1991). The structures of both a-agglutinin and the core subunit of a-agglutinin are composed of a Secretion signal region, an active region, a support region rich in serine and threonine, and a putative GPI anchor attachment signals, and presumably a heavily O-glycosylated form (Wojciechowicz et al., 1993). The structure of GPI anchors is highly conserved among molecules from various organisms (Ferguson and Williams, 1988). The core structure of the yeast GPI anchor is similar to that found in other eucaryotes (Lipke et al., 1989); ethanolamine phosphate(6) mannose(α1,2) mannose(α1,6 )mannose(α1,4)glucosamine-(α1,6)inositol phospho-lipid (Fig. 3). Many of cell surface proteins in yeast, for example, Agα1 (Lipke et al., 1989), Aga1 (Roy et al., 1991), Flo1 (Watari et al., 1994), Sed1 (Hardwick et al., 1992), Cwp1, Cwp2, Tip1,Tir1/Srp1 (Van der Vaat et al., 1995), have GPI anchors, which play important roles for surface expression of cell-surface proteins and are essential for the viability of yeasts. These glycophospholipid moieties are covalently attached to the C-termini of proteins and their primary function is to afford the stable association of proteins with the membrane. GP1-anchored proteins contain 63

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hydrophobic peptides at their C termini. After the completion of protein synthesis , the precursor protein remains anchored in the endoplasmic reticulum (ER) membrane by the hydrophobic carboxyl-terminal sequence, with the rest of the protein in the ER lumen. The hydrophobic carboxyl-terminal sequence is cleaved at the site and concomitantly replaced with GPI anchor presumably by a transamidase. The localisation of both α-agglutinin and α-agglutinin to the cell surface occurs through the secretory pathway (Tohoyama and Yanagishima, 1987) (Fig, 3). Secreted proteins are first translocated into the lumen of the ER, and then transported from the ER to the Golgi apparatus and from there to the plasma membrane in membrane-enclosed vesicles (Schekman, 1992). Fusion of the Golgi-derived secretory vesicles with the plasma membrane releases the secreted proteins to the cell exterior. Post-translational proteolytic modification of precursors of secretory peptides occurs in the late compartments of the secretory pathway (trans cisternae of the Golgi apparatus and secretory vesicles). α-Agglutinin was proposed to be further transported to the outside of the plasma membrane through the general secretory pathway in a GPI-anchored form and then released from the plasma membrane by phosphatidylinositol-specific phospholipase C (PI-PLC) and transferred to the outmost surface of the cell wall (Lu et al., 1994).

Figure 3. Transportation of a-agglutinin to cell surface (A) and the structure of GPI anchor (B), A: ER, endoplasmic reticulum: PI-PLC, phosphatidylinositol-specific phospholipase C. B: a, ethanolamine phosphate bridge; b, glycan; c, inositol. Man, Mannose; GlcNH2, glucosamine.

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3. Display of Amylolytic Enzymes on the Yeast Cell Surface To construct a novel yeast displaying amylolytic enzymes which hydrolyse starches at the cell surface, a multi-copy plasmid (pGA11) harbouring the R. oryzae glucoamylase gene was prepared for expression of glucoamylase/α-agglutinin fusion gene containing the secretion signal sequence of the glucoamylase under the control of the GAPDH promoter (Fig. 4). When cells were cultivated aerobically with 1 % soluble starch as the sole carbon source, the cells harbouring the plasmid pGA11 proliferated to reach an absorbance of about 10, which was the same level as in the case of the culture on 1% glucose, while no growth was observed with the control cells (Fig. 5). These data show that the cell surface-anchored glucoamylase reacted sufficiently for the hydrolysis of starch. Cultivation on an agar plate demonstrated that the cells harbouring the plasmid pGA11 hydrolysed starch and produced a halo strictly around the colony, while no halo formation was observed around the cells harbouring the control plasmid, revealing that the cell surface-engineered cells obtained amylolytic activity due to the expression of the glucoamylse/α-agglutinin fusion gene (Murai et al., 1997a). Immunofluorescent labelling of cells with anti-glucoamylase IgG showed that cells expressing the glucoamylase/α-agglutinin fusion gene were uniformly labelled, although the intensity was different from cell to cell, being probably due to differences in expression levels among the cells (Fig. 6). The localisation of glucoamylase/αagglutinin fusion protein on the cell wall was further confirmed by immunoelectron microscopy. Thermal stability, optimal temperature, and optimal pH of glucoamylase anchored on the cell surface were evaluated by comparing with those of the secreted free enzyme (Ueda et al., 1998). The activity of the anchored glucoamylase was stable in the temperature range of 0°C to 45°C. The optimal temperature and the optimal pH of the anchored glucoamylase was 50°C and 4.5, respectively. While no differences on the thermal stability and the optimal pH were observed between anchored and free glucoamylases, the optimal temperature of anchored glucoamylase was a little lower than that of the free enzyme. To stable express the enzyme on the yeast cell surface, a plasmid pIGA11 to be integrated into chromosome of S. cerevisiae was constructed (Fig. 4) (Ueda et al., 1998). The glucoamylase/α-agglutinin fusion gene-integrated cells exhibited the glucoamylase activity on the cell surface as well as the cells harbouring pGA11 and utilised starch as the sole source of carbon and energy. Mitotic stability of the cells harbouring pIGA11 was much higher than that of the cells harbouring pGA11. As the result of the examination whether or not the novel yeast displaying glucoamylase from R. oryzae on its cell surface can ferment starch directly, it was suggested that the fermentation efficiency would be enhanced by the addition of aamylase to the medium (Murai et al., 1998a). Furthermore, the enzyme displayed on the cell surface was regarded as a kind of a renewable catalyst depending on cultivation conditions. And, bacterial contamination was expected to be prevented, because the incorporation of glucose, which was liberated on the cell surface, was considered to be rapid.

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Figure 4. Structures ofplasmids for displaying of enzymes. pGA11 (A) and plGA1I (B), for displaying of glucoamylase; pIAA1I (C), for displaying of α-amylase; pCMC11 (D), for displaying of CM-cellulase (CMCase); pBG21 1 (E), for displaying of β-glucosidase.

In addition to display glucoamylase, to co-display α-amylase from Bacillus stearothermophilus CU21 (BSTA) (Nakajima et al., 1985) on the cell surface, the BSTA/C-terminal half of α-agglutinin fusion gene (pIAA11) constructed with the same method as in the case of glucoamylase was integrated into the chromosome of S. cerevisiae (Fig. 4). In this case, about half of the total α-amylase activity was leaked into the culture supernatant, due to the proteolytic processing of the fusion protein. The deduced amino acid sequence of BSTA contained one possible processing site for the Kex2 endopeptidase, Val-Pro-Arg, at the amino acid residues of No. 481 to No. 483. Kex2, one of the endopeptidases in the secretory process of α-mating factor and killer toxin precursors, exhibits the substrate specificity toward each carboxyl site of Lys-Arg, Arg-Arg, and Pro-Arg sequences (Mizuno et al., 1989). A Kex2-resistant BSTA/αagglutinin fusion protein was designed by eliminating 33 residues from the C-terminus of BSTA, because this domain was demonstrated not to be essential for the activity (Vihinen et al., 1994). The cell harbouring this C-terminal truncated BSTA/α-agglutinin fusion gene (pIAA∆) exhibited α-amylase activity only in the cell pellet fraction. On plate assay, a large halo was detected around the colony of the cells harbouring pIAA11, while a small halo formation was observed strictly around the cells 66

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harbouring pIAA∆ as in the case of the glucoamylase-displaying cells (Murai et al., 1999). When the arming cells with both enzymes were cultivated in the medium containing 1% soluble starch as the sole carbon source, the growth of the cells harbouring pIGA11 and pIAA∆ reached the absorbance of about 10, which was the same level as in the case of the culture on 1% glucose, although the growth on glucose of these strains was more rapid. No growth on starch was observed with the control cells and the cells harbouring only pIAA∆ (Fig. 5). The cell co-displaying glucoamylase and α-amylase could grow faster than the glucoamylase-displaying cells. α-Amylase is an endoglucanase which hydrolyses starch in a random fashion, producing oligosaccharides. Therefore, the co-operative and sequential reaction probably resulted in the increased concentration of molecules with non-reducing ends produced from starch by α-amylase, which in turn could serve as substrate molecules for glucoamylase, thereby increasing the rate of formation of free glucose. Immunofluorescent labelling of cells with anti-glucoamylase IgG and FITC-goat IgG to rabbit IgG as the second antibody and the halo formation of the cells harbouring pIGA11 and pIAA11 or pIAA∆ on a starch-containing plate showed the expression of both glucoamylase and αamylase on the cell surface of the same cell. The fermentation system using the novel cells endowed with a rapid-starch-utilising ability by displaying two sequential amylolytic enzymes on their cell surface should be further evaluated in comparison with other systems. 4. Display of Cellulolytic Enzymes on the Yeast Cell Surface As vectors for co-display of CM-cellulase and β-glucosidase on the cell surface, multi-copy plasmids, pCMC11 and pBG211 (Fig. 4), were constructed (Murai et al., 1997b; 1998b). The cells harbouring the plasmid pCMC11 and cells harbouring pCMC11 and pBG211 had the cell-associated CM-cellulase activity, and the cells harbouring the plasmid pBG2 1 1 and cells harbouring pCMC11 and pBG211 exhibited the cell-associated β-glucosidase activity. Moreover, the cells harbouring the plasmid pCMC11 and pBG211 showed both CM-cellulase and β-glucosidase activities. The parent cells exhibited neither of the activities. From these results, CM-cellulase and βglucosidase proteins were efficiently co-displayed on the cell surface in their active forms. Immunofluorescence microscopy with FITC-goat IgG to rabbit IgG as the second antibody confirmed the presence of CM-cellulase protein on the cell surface (Fig. 6). Anti-CM-cellulase IgG and anti-β-glucosidase IgG were used as the first antibodies, Control cells were not labelled with either antibodies, while cells harbouring pCMC 1 1 were labelled by fluorescence with anti-CM-cellulase IgG. Cells harbouring pBG211 were also labelled with anti-β-glucosidase IgG (Murai et al., 1998b). The cells harbouring pCMC11 and pBG2 1 1 were labelled by fluorescence with both anti-CM-cellulase IgG and anti-P-glucosidase IgG, indicating that these cells codisplayed the CM-cellulase and β-glucosidase proteins on their cell surface (Murai et al., 1998b).

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Figure 5. Aerobic cultivation of S. cerevisiae MT8-1 cells harbouring pGA11 (A) and harbouring various in tegrative glucoamylase and α-amylase/α-agglutinin fusion genes (B) in the medium containing starch as the sole source of carbon and energy, and aerobic cultivation of arming cells in the medium containing cellobiose (C) and cellooligosaccharides (D) as the sole source of carbon and energy. A, cell growth ( ); starch concentration ( ); ethanol produced ( ); glucoamylase activity in cell pellet ( ); glucoamylase activity in culture medium ( ). B, cell growth of the cells harbouring no plasmid ( ), pIGA11 ( ), pIAA ( ), and pIGA11/pIAA∆ ( ); the concentration of starch in the culture medium of the cells harbouring no plasmid ( ), pIGA11 ( ), and pIGA11/pIAA∆ ( ). C and D, symbols for cells: S. cerevisiae MT8-1 ( ); MT8-1 harbouring pCMC11 ( ); MT8-1 harbouring pBG211 ( ); MT8-1 harbouring pCMC11 ). C, Cell growth was monitored by absorbance of culture broth at 600 and pBG211 ( nm. D, Cell growth was monitored by counting colonies appeared on plates.

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Figure 6. Immunofluorescent labelling of arming cells displaying glucoamylase (A, B), CM-cellulase (C, D) on the cell surface. Nomarsky differential interference micrographs (A and C) and immunofluorescence micrographs (B and D).

The arming cells with both enzymes were cultivated aerobically in the medium containing cellobiose as the sole carbon source, and cell growth was monitored (Fig. 5). Cells harbouring pBG211 and cells harbouring pCMC11 and pBG211 could grow on cellobiose and reached the absorbance of about 2, which was a little lower than that in the case of the culture on 1% glucose, while no growth on cellobiose was observed with the control cells and cells harbouring pCMC11. The arming cells were cultivated aerobically in the medium supplemented with cellooligosaccharides (approximately 11% (W/W) cellohexaose, 29% (W/W) cellopentaose, 33% (W/W) cellotetraose, 17% (W/W) cellotriose, 4% (W/W) cellobiose, and less than 1% (W/W) glucose) as the sole carbon source and the cell growth was monitored by counting colonies appeared on YPD plates. The cells harbouring pBG211 and cells harbouring pCMC11 and pBG211 could grow on cellooligosaccharides employed, while no growth on cellooligosaccharides was again observed with control cells and cells harbouring pCMC11. Since β-glucosidase was reported to be capable of degrading cellooligosaccharides with glucose units of 2 to 6 (Sakamoto et al., 1985), the breakdown of cellooligosaccharides by β-glucosidase seemed to be sufficient to sustain the growth of the yeast displaying this enzyme. However, the difference between the growth of the cells harbouring pCMC11 and pBG211 and that of the cells harbouring pBG211 suggested that the yeast strain co-displaying CM-cellulase and β-glucosidase had the enhanced ability to degrade cellooligosaccharides. 69

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Much efforts have been devoted to utilise cellulosic materials by employing S. cerevisiae and cellulase complex from cellulolytic bacteria (Van Rensburg et al., 1996). However, these attempts failed because β-glucosidase was not expressed. Although several researches have also been made to express heterologous β-glucosidase genes in S. cerevisiae, cellobiose could not access to the enzyme remained intracellular or the enzyme was not expressed sufficiently to allow the transformant to grow on cellobiose (Adam et al., 1995; Kohchi and Toh-e, 1986; Penttila et al., 1984, Raynal and Guerineau, 1984) . After that, secretive expression of β-glucosidase gene in S. cerevisiae was reported (Cummings and Fowler, 1996), where growth on cellobiose was not examined. Thus, the results described here are the first step for the assimilation of cellulosic materials by S. cerevisiae expressing heterologous cellulase genes and also the first report for assimilation of cellooligosaccharides by yeast and for the construction of the prototype of cellulosomes (Murai et al., 1998b). 5. Display of Lipase on the Yeast Cell Surface When lipase of R. oryzae was displayed as mentioned above, little activity was observed on the cell surface. The reason of this phenomenon seemed to be derived from the fact that the active site of lipase resides in the C-terminal domain of the molecule. Fusion of the C-terminus of lipase with the 3'-half of α-agglutinin may inhibit the access of substrates to the active site. Therefore, to insure the free movement of the lipase molecule, a spacer of an appropriate length was inserted between these two molecules. This system will be further proved to be effective for the cells to express the lipase activity on the cell surface, since the length of the spacer seemed to be affected the enzyme activity. 6. Cell Surface Engineering as a Novel Field of Biotechnology Schreuder et al. (1993, 1996) reported that they had succeeded in targeting αgalactosidase from Cyamopsis tetragonoloba seeds, as a reporter enzyme, to the cell wall of S. cerevisiae. However, the engineered S. cerevisiae cells described here are the first example of yeast in which proteins were targeted to the cell surface and endowed the cells with new beneficial properties. These surface-engineered yeast cells were termed "Arming yeasts" (Anonymous, 1997). The displayed enzymes are also regarded as a kind of self-immobilised enzymes on the cell surface, this phenomenon being passed on to daughter cells as long as the genes are retained by the cells. This display system could turn or remake S. cerevisiae into a novel and attractive microorganism as a whole-cell biocatalyst by surface expression of various enzymes, especially when target substrates are not able to be taken up by the cells, and will make it possible to produce renewable self-immobilised biocatalysts. Here, the combination of the cell surface display system with endowment of an additional metabolic reaction to the yeast cells was performed by cell surface engineering. Thus, the cell surface can be regarded as a new target for giving additional characteristics of metabolic reactions. This research will open a new frontier of cellular engineering not only in yeast but also in all living cells. From the viewpoint of 70

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biotechnology, the system mentioned here is also regarded as a combination of the immobilisation of biocatalysts and the genetic engineering. In this system, the cell surface of yeast was used as a carrier for immobilisation, and the living whole cells were remade as a “cell biocatalyst”. This system is expected to have several merits; the enzyme is readily supplied only by the activation of the promoter and is provided as “naturally” immobilised on the cell surface, saving tedious purification and immobilisation processes. The cell surface engineering will be able to endow all living cells with novel abilities (Fig. 7) and open a new way in the field of biotechnology.

Figure 7. Application ofarming yeast constructed by cell surface engineering.

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MOLECULAR BREEDING OF ARMING YEASTS BY CELL SURFACE ENGINEERING Schekman, R. (1992) Genetic and biochemical analysis of vesicular traffic in yeast. Curr. Opin. Cell Biol. 4, 587-592, Schreuder, M. P., Brekelmans, S., Van den Ende, H., and Klis,F. M. (1993)Targeting of a heterologous protein to the cell wall of Saccharomyces cerevisiae. Yeast 9, 399-409. Schreuder, M. P., Mooren, A. T., Toschka, H. Y., Verrips, C. T., and Klis, F. M. (1996) Immobilising proteins on the surface of yeast cells. Trends Biotechnol. 14, 115- 120. Terrance, K., Heller, P., Wu, Y, -S, and Lipke, P. N. (1987) Identification of glycoprotein components of αagglutinin, a cell adhesion protein from Saccharomyces cerevisiae. J. Bacteriol. 169,475-482. Tohoyama, H. and Yanagishima, N. (1987) Site of pheromone action and secretion pathway of a sexual agglutination substance during its induction by pheromone a in a cells of Saccharomyces cerevisiae. Curr. Genet. 12,271-275. Tokatlidis, K., Salamitou, S., Beguin, P., Dhurjati, P., and Aubert, J. P. (1991) Interaction of the duplicated segment carried by Clostridium thermocellum. FEBS Lett. 291, 185-188. Ueda, M., Murai, T., Shibasaki, Y., Kamasawa, N., Osumi, M., and Tanaka, A. (1998) Molecular breeding of polysaccharide-utilising yeast cells by cell surface engineering. Ann. N. Y. Acad. Sci. 864, 528-537. Van der Vaart, J. M., Caro, L. H. P., Chapman, J. W., Klis, F. M., and Verrips, C. T. (1995) Identification of three mannoproteins in the cell wall ofSuccharomyces cerevisiae. J. Bacteriol. 177, 3104-3110. Van Rensburg, P., Van Zyl, W. H., and Pretorius, I. S. (1996) Co-expression of a Phanerocbaete chrysosporium cellobiohydrolase gene and a Butyrivibriofibrisolvens endo-β-1,4-glucanse gene in Saccharomyces cerevisiae. Curr. Genet. 30,246-250. Vihinen, M., Peltonen, T., Iitia, A., Suominen, I., and Mantsala, P. (1994) C-terminal truncations of a thermostable Bacillus stearothermophilus -amylase. Protein Eng. 7, 1255-1259. Watari, J., Takata, Y., Ogawa, M., Sahara, H., Koshino, M., Onnela, M. -L., Airaksinen, U., Jaatinen, R., Penttila, M., and Keranen, S. (1994) Molecular cloning and analysis of the yeast flocculation gene FLO1. Yeast 10, 211-225. Wojciechowicz, D., Lu, C. -F., Kurjan, J., and Lipke, P. N. (1993) Cell surface anchorage and ligandbinding domains of the Saccharomyces cerevisiae cell adhesion protein a-agglutinin, a member of the immunoglobulin super-family. Mol. Cell. Biol. 13:2554-2563.

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METABOLIC PATHWAY ANALYSIS OF SACCHAROMYCES CEREVISIAE SIMON OSTERGAARD, LISBETH OLSSON AND JENS NIELSEN Center for Process Biotechnology, Department of Biotechnology, Technical University of Denmark, Building 223, DK-2800 Lyngby, Denmark.

Abstract In metabolic engineering, analysis of pathways is central and it involves quantification of pathway fluxes and how these fluxes are controlled. Therefore metabolic pathway analysis rely on metabolic control analysis and metabolic flux analysis. This paper introduces the basic concepts of metabolic pathway analysis that serves as a valuable analytical tool for examination of cellular metabolism especially in connection with designing directed genetic changes to achieve specific objectives, e.g. increase flux toward a product of interest. In the paper, metabolic pathway analysis of the glycolysis and the galactose metabolism of Saccharomyces cerevisiae are used to illustrate the power of this analytical tool. 1. Introduction The multidisciplinary field of metabolic engineering aims at creating and improving microorganisms for several purposes. The tasks of metabolic engineering can be classified into the following groups: Extension of substrate range; improvement of productivity or yield; elimination of by-product formation; improvements of cellular properties; and extension of product range. When operating in either of these categories, it is of outmost importance to analyse the cellular metabolism thoroughly in order to succeed. Metabolic pathway analysis serves as an analytical tool that may help to identify the most promising targets for metabolic manipulation, and furthermore, metabolic pathway analysis can also help elucidating metabolic changes of the cell caused by certain genetic modifications. In this paper we roughly divide metabolic pathway analysis into two categories that take advantage of two different concepts: Metabolic control analysis and metabolic flux analysis. The metabolic control analysis examines perturbations in the enzymatic activities on the systemic metabolic behaviour to determine which enzyme(s) that exerts control over flux within a given pathway (Kacser and Burns, 1973; Heinrich and Rapoport, 1974). Thus, the overall aim with this 75 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 75–85. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.

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analysis is to identify the best alternatives for genetic manipulation that may lead to an increase of overall flux through a given pathway. The principles of metabolic flux analysis have proven successful as a tool for pathway analysis, since the carbon distribution within the cell may be evaluated (Stephanopoulos et al., 1998). Hence, the study of flux control targets the cellular metabolism ‘locally’, whereas the metabolic flux analysis serves as a ‘global’ cellular approach where the intracellular fluxes of a given metabolic network are estimated. In this paper the concepts of metabolic pathway analysis are described. Furthermore, examples of metabolic pathway analysis applied to the glycolysis and the galactose metabolism of Saccharomyces cerevisiae will be referred to for demonstrating the use of this analytical tool in metabolic engineering. 2. Metabolic pathway analysis 2.1. METABOLIC CONTROL ANALYSIS To elucidate which enzyme(s) that controls the flux through a given biochemical route, it is necessary to quantify the control over the overall flux exerted by the individual enzymes. The metabolic control analysis aims at calculating the flux control coefficients (FCC’s) of the individual enzymatic steps in a given pathway in order to quantify which enzyme(s) that is (are) responsible for the control over the flux. The FCC’s are normalised to one and they describe the relative change in steady-state flux (Jk) through reaction k caused by an infinitesimal change in enzymatic activity (vi) of enzyme i, as depicted in equation 1 where CiJk designates the flux control coefficient. Thus, the closer a FCC is to one, the more control over flux is exerted by the respective enzyme. (1)

The FCC’s can directly be obtained from measurements of the flux at various enzymatic activities or indirectly by calculation of the elasticity coefficients (εij) (equation 2), that describe the relative change in enzymatic activity (vi) caused by an infinitesimal change in the concentration of metabolite j. Assuming validity of the flux-control summation theorem (equation 3) and the flux-control connectivity theorem (equation 4) (Kacser and Burns, 1973), the elasticity coefficients can be applied for deduction of the FCC’s. (2)

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(3)

(4)

To determine the FCC’s indirectly via calculation of the elasticity coefficients, it is crucial to obtain detailed knowledge of the enzyme kinetics either in the form of quantitative information about the elasticity coefficients or in the form of a kinetic model for the individual enzymatic steps. Each enzymatic reaction included in the model should be described by a kinetic rate expression that correlates the concentrations of the substrate(s), the activator(s), and the inhibitor(s) of the enzyme with its specific enzymatic activity. This correlation will depend on the kinetic parameters of the given enzyme such as the maximum specific reaction rate (vmax), the affinity constant(s) (Km), inhibition constants (Ki) and in the case of a reversible reaction also equilibrium constants between certain metabolites may be included in the kinetic rate expression. When these kinetic rate expressions are processed as shown in equation 2, and the intracellular metabolite levels are measured, it is possible to calculate the elasticity coefficients, and subsequently obtain the FCC’s. 2.2. METABOLIC FLUX ANALYSIS In contrary to the metabolic control analysis that relies on the kinetics of the pathway examined, the metabolic flux analysis consists of a stoichiometric model where the metabolic pathway fluxes that span a biochemical network can be estimated by applying mass balances around the intracellular metabolites of the model i.e. metabolite balancing, or by determination of the fractional enrichment when using labelled substrate. By comparison of flux distributions, the metabolic flux analysis can serve as an analytical tool for identification of physiological changes that arise from changing the operating conditions or for comparison of the carbon distribution in different mutants. Thus, the application of the metabolic flux analysis can help investigating the cellular control of a certain biochemical branch point in order to clarify the rigidity of a certain pathway node. Furthermore, information regarding alternative pathways may also be obtained from metabolic flux analysis by including questionable pathways in the stoichiometric model and then concluding the lack or existence of the given pathway by virtue of its ability to satisfy the metabolite balances. For the performance of metabolic flux analysis based on metabolite balancing a complete stoichiometric model of the cell should be set up which includes J intracellular reactions comprising K pathway metabolites. The mass balances of the K metabolites are given in vector notation as depicted in equation 5 where Xmet designates the concentration of the K metabolites and rmet designates the vector with all the volumetric net rates of formation of the metabolites in the Jreactions:

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(5)

Due to the high turnover of the metabolite pools, the change in metabolite concentrations rapidly adjusts to a new level, and hence, pseudo steady-state may be assumed (dXmet/dt = 0). Since the metabolite levels generally are very low, the dilution of the metabolite pool due to biomass growth (described by the term is negligible. This assumption is further supported by the fact that the fluxes affecting a given metabolite pool are significant higher than the effect of dilution. Thus, rmet equals zero, and as the rmet-vector may be written as the product between the stoichiometric matrix GT comprising the stoichiometry of the J reactions organised in columns and the v-vector containing the individual rates of the J reactions, equation 6 forms the basis for metabolite balancing. (6)

This equation represents K linear algebraic balances with J unknown parameters equal to the number of pathway fluxes. This system has J-K degrees of freedom, and consequently, when J-K reaction rates are measured, it is possible to obtain estimates for all the pathway fluxes of the v-vector by solving the linear algebraic balances. To do so, all the measured rates are collected in a new vector, vm, and the residual rates that are to be calculated, are collected in another vector, vc. Furthermore, the stoichiometric matrix GT is divided into two sub-matrices GmT and GcT which contain the stoichiometry of the reactions to be measured and the stoichiometry of the residual reactions, respectively. Hence, equation 6 may be written as equation 7, and by rearrangements of this equation, the vector vc containing all the calculated reaction rates of the metabolic network can be computed as shown in equation 8. (7)

(8)

The use of metabolite balancing for estimation of intracellular fluxes may serve as a valuable tool for determination of net fluxes within a metabolic network. Nonetheless, this approach does not allow for flux determinations of reversible reactions and for the quantification of flux ratios between biosynthetic pathways leading to the same metabolite. To get information about the fluxes in these cases it is necessary to apply labelled substrates, Metabolic flux analysis using labelled substrate approaches the metabolism from an even further ‘microscopic’ view compared with metabolite 78

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balancing as the pathway fluxes are estimated from balances around each individual carbon atom in the intracellular metabolites. Hence, the method may also allow for calculation of the flux ratio between two biosynthetic routes that lead to the same metabolite, if the two biosynthetic pathways discriminate differently between the individual carbon atoms (Sonntag et al., 1993). Likewise, the method may also be used for estimation of reversible fluxes and not only net-fluxes as obtained from metabolite balancing (Marx et al., 1996; Fiaux et al., 1999). Furthermore, the use of labelled substrate may also help to elucidate compartmentation to gain information about the location of certain biosynthetic reactions within the cell (Pasternack et al., 1994). With additional biochemical knowledge regarding the cellular structure, an extended metabolic model may be established describing the cellular metabolism even more accurately. The concepts of metabolic flux analysis using labelled substrate(s) have been extensively described in Wiechert and de Graaf (1 996). 3. Steady-state continuous cultivation –an excellent tool for metabolic pathway analysis Having established the structure of a kinetic and/or a stoichiometric model for the performance of metabolic pathway analysis, it is necessary to obtain values for the model parameters. For indirectly determination of the FCC’s of metabolic control analysis, the intracellular metabolite levels of the all compounds included in the kinetic model can be used, and to estimate the flux distribution of the metabolic network established, the rates included in the vm-vector are needed. For the analysis of microbial cells it is possible to use submerged fermentation experiments for analysis of the cellular metabolism. Furthermore, through the use of continuous cultures – often referred to as chemostats – it is possible to study the cellular metabolism at a steady state. By controlling the volumetric feed rate of medium (F) fed to the bioreactor, and keeping the volume constant by having a simultaneous out-flow of medium from the bioreactor, the specific growth rate µ can be controlled at any value, since it equals the dilution rate D at steady-state. This is observed from the mass-balance of biomass (X) over the bioreactor as shown in equation 9, i.e. no accumulation of biomass ( dX/dt = 0). (9)

Furthermore, the volumetric formation rates of the extracellular metabolites (rp) which may be included in the vm-vector mentioned in section 2.2., are easily obtained when measuring the concentration of the products in the bioreactor, since the volumetric formation rates are calculated by multiplication of the dilution rate with the concentration of the extracellular metabolites at steady-state. This is can be seen from equation 10 which shows the mass balance of a given product P over the bioreactor where dP/dt equals zero at steady-state.

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(10)

Thus, from continuous operated cultivations, input parameters for the metabolic pathway analysis such as concentrations of extracellular and intracellular metabolites may be obtained in a highly reproducible fashion under preferable physiological conditions regarding the specific growth rate. The continuous operated cultivations also give the possibility to examine dynamic physiological changes of a cellular system caused by specific perturbations. These perturbations could be obtained from a pulse of the limiting component that is added to the bioreactor momentarily in order to follow the cellular response regarding substrate uptake and product formation rates. Also a step change in dilution rate may be of interest in order to study the dynamics of a culture when the specific growth rate, consequently, increases or decreases from one level to another. Furthermore, continuous cultivations may also be used for physiological studies of metabolic regulation such as glucose control (Klein et al., 1998) exerted on the metabolism of slow fermentable carbon sources. To establish repressible conditions within the medium, a high sugar concentration should be obtained which can be achieved by operation of the bioreactor under nitrogen-limited conditions. 4. Metabolic pathway analysis applied to Saccharomyces cerevisiae 4.1. KINETIC STUDIES OF THE GLYCOLYSIS The concepts described in the previous sections have been applied to Saccharomyces cerevisiae which have provided additional information to the understanding of yeast metabolism. Galazzo and Bailey (1990) established a kinetic model describing the anaerobic fermentation pathway from glucose to ethanol, glycerol, and polysaccharides in order to characterise the physiological response caused by a change in the external pH in suspended and immobilised yeast. In vivo phosphorus-31 nuclear magnetic resonance measurements for determination of the intracellular concentrations of substrates and effectors in addition to the kinetic expressions of the model were used for calculation of the FCC’s as described in section 2.1. From this study the authors found that control over flux was mainly exerted by the glucose uptake in the suspended cells regardless of the extracellular pH of 4.5 or 5.5 (FCC = 0.83 and FCC = 0.64, respectively). At the latter pH, the ATPase membrane ion pump also exhibited considerable control over ethanol production with a FCC of 0.24, since ATP is not consumed as rapidly by the ATPase at pH 5.5 compared with pH 4.5 in order to maintain the intracellular pH. In immobilised cells the glucose uptake has a lower impact, however still considerable, on the ethanol production (FCC = 0.31 and FCC = 0.30 at pH 4.5 and pH 5.5, respectively) compared with suspended cells. The control over flux in the immobilised cells was mainly exerted by the phosphofructokinase (FCC = 0.50 and FCC = 0.33 at pH 4.5 and pH 5.5, respectively), but also in these cells the ATPase exhibited considerable control over ethanol production with a FCC of 0.24 80

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when grown at an extracellular pH of 5.5. This study demonstrated the use of metabolic control analysis for explaining the physiological differences between suspended and immobilised yeast cells. Hence, metabolic control analysis may be used for identification of the best alternatives for genetic manipulation but also for description of metabolic differences in a quantitative fashion. Another strategy different from metabolic control analysis but also involving kinetic modelling, was taken for pathway analysis of the glycolysis in S. cerevisiae (Rizzi et al., 1997; Theobald et al., 1997). The kinetics of the glycolysis was examined in order to understand the regulation of the yeast metabolism during dynamic conditions. Theobald et al. (1997) established a rapid sampling technique to measure the dynamic response of the glycolytic intermediates, co-metabolites, and extracellular metabolites of S. cerevisiae caused by a physiological perturbation. In order to predict the changes of the intracellular and extracellular metabolite levels during dynamic conditions, a kinetic model of the individual enzymatic steps of the glycolysis was set up (Rizzi et al., 1997). The model comprised rate equations for the individual enzymatic steps of the glycolysis and these were included in material balances for the individual metabolites, the cometabolites, and the extracellular metabolites. The dynamic response that arose from a glucose pulse added to a glucose-limited continuous grown culture of S. cerevisiae operated at a dilution rate of 0.1 h-1, was simulated by solving the material balances for all these metabolites. To fit the experimental observations of the glucose pulse, the model was structured into a cytoplasmic and a mitochondrial compartment but with translocation of the adenine nucleotides. The model was able to simulate the steady state situation of the glucose-limited continuous cultivation and the transient response of the metabolite levels after a glucose pulse. The work of Rizzi et al. (1997) and Theobald et al. (1997) illustrates the use of continuous operated cultivations as a great tool for kinetic analysis of microorganisms, and furthermore, the studies also demonstrate the valuable use of mathematical modelling for pathway analysis in order to improve the understanding of the yeast metabolism. 4.2. METABOLIC PATHWAY ANALYSIS OF THE GALACTOSE METABOLISM The GAL genes of S. cerevisiae encoding the enzymes of the galactose utilisation pathway, serve as one of the best studied models of genetic regulation in eukaryotic systems (reviewed by Johnston and Carlson, 1992, and Melcher, 1997). The GAL genes are tightly regulated being induced by galactose and strongly repressed by glucose. The positive tramcriptional activator protein encoded by the GAL4 gene induces the expression of the structural GAL genes in the presence of galactose. The genes GAL6, GAL80, and MIG1 encode the proteins responsible for the down-regulation of the structural GAL genes that encode the enzymes responsible for uptake of extracellular galactose and subsequent conversion of intracellular galactose to glucose-6-phosphate (known as the Leloir pathway). GAL2, GAL1, GAL7, GAL10, and GAL5 constitute the structural GAL genes encoding the following enzymes: galactose permease, galactokinase, galactose- 1 -phosphate uridylyltransferase, UDP-glucose 4-epimerase, and phophoglucomutase, respectively, which are shown in Figure 1.

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Figure I The Leloir pathway containing the enzymes responsible for conversion of galactose io glucose-6-phosphate See the text for specific nomenclature of the enzymes

The galactose metabolism is interesting to approach with the tools of metabolic pathway analysis in order to investigate the remarkable physiological differences that exist between glucose and galactose metabolism. Metabolic control analysis was used for ‘locally’ examination of the galactose utilisation pathway (Østergaard et al., 1998), and metabolic flux analysis was carried out to elucidate the ‘global’ physiological effects on the entire metabolism caused by deletion of the two regulatory genes GAL80 and MIG1. To identify which enzymes control flux through the Leloir pathway, the FCC’s were determined indirectly as described in section 2.1., and hence, a kinetic model of the Leloir pathway was set up followed by calculation of the elasticity coefficients from the rate expressions of the individual enzymatic reactions. The explicit values for the elasticity coefficients were obtained from measurements of the intracellular concentrations of the intermediates of the Leloir pathway from a galactose-limited continuous cultivation operated at a dilution rate of 0.1 h-1, Thus, the FCC’s could be obtained by use of the summation and the connectivity theorem as described in section 2.1. The only unknown variable of the model was the intracellular galactose concentration, and hence, the FCC’s were computed as a function of the intracellular galactose concentration as depicted in Figure 2. According to intracellular measurements of the glucose concentration when the extracellular concentration was at or below the affinity of the transport system (1-2 mM), the intracellular glucose concentration was less than 0.1 mM (Teusink et al., 1998). Assuming the same to be the case for the galactose metabolism when the galactose concentration in the bioreactor was 1 mM, the intracellular galactose concentration was at or below 0.1 mM. Consequently, from this metabolic control analysis it was concluded that the control over flux through the Leloir pathway is mainly exerted by the galactose permease (Gal2) under the physiological conditions examined. Experimental work is in progress to examine the results of this analysis in order to gain more insight to the galactose metabolism of yeast.

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Figure 2 FCC 's calculated as a function of the intracellular galactose concentration.Gal2: Galactose permease; Gall: Galactokinase; Gal7: galactose-1 -phosphate uridylyltransferase.

The glucose and the galactose metabolism were also studied in nitrogen-limited continuous cultivations to examine the physiological differences between these two sugars. It was of interest to investigate the physiological role of the proteins encoded by the MIG1 and the GAL80 gene which are strongly involved in glucose control exerted on the galactose metabolism. Mig1 takes part of a protein complex comprising Ssn6, Tup1, and Mig1 of which the latter directs the complex to a specific consensus motif on the promoters of the target genes whereby transcription of the target genes does not occur (Keleher et al., 1992; Treitel and Carlsson, 1995). Mig1-mediated glucose control not only affects the GAL genes, but also the MAL genes responsible for maltose utilisation and the SUC genes encoding invertase that hydrolyses sucrose. In contrary, Gal80 only acts as a negative regulatory protein on the GAL genes by binding to the Cterminal end of Gal4 which prevents transcriptional activation by this protein. The specific sugar uptake rates of a mig1 gal80 double mutant strain obtained from nitrogenlimited cultivations on glucose and galactose, respectively, were compared with the corresponding specific sugar uptake rates obtained for the wild type strain (CEN.PK 113-7D). A stoichiometric matrix of the aerobic yeast metabolism was established to estimate the flux distributions of the wild type strain and the mig1 gal80 mutant strain from the data obtained by the nitrogen-limited continuous cultivations on either glucose or galactose. By measuring the uptake rates of glucose and galactose in addition to the product formation rates of pyruvate, ethanol, acetate, succinate, glycerol, ammonium, and the biomass composition, it was possible to compute the vc-vector as described in section 2.2., and hence, obtain flux distributions over the metabolic network established. The uptake rates of glucose and galactose of the wild type strain were 4.3 mmol/gDW/h and 2.7 mmol/gDW/h, respectively, and the corresponding uptake rates of the mig1 83

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gal80 mutant strain were 3.4 mmol/gDW/h and 3.8 mmol/gDW/h. Thus, the deletion of the MIG1 and the GAL80 genes had a remarkable impact on the uptake rates of these two sugars by decreasing the specific glucose uptake rate and increasing the specific galactose uptake rate. Some of the fluxes estimated from these four cultivations are shown in Figure 3.

Figure 3 Flux distributions (given in mmol/gDW/h) obtained for the wild type strain (left) and the mig1 gal80 mutant strain (right) when grown in nitrogen-limited continuous cultivations on glucose or galactose (italic). Only selected fluxes are shown for simplicity

The metabolic flux analysis provides valuable information about the cellular control of the pyruvate branch point. It is observed that the flux entering the TCA-cycle from the pyruvate node is constant for the two strains examined but it varies between glucose and galactose. The flux entering the TCA-cycle was 1.6-1.7 mmol/gDW/h when the two strains were grown on glucose, and 2.1-2.2 mmol/gDW/h for both these two strains when grown on galactose. Hence, it is concluded that not only growth on glucose results in overflow metabolism where the residual carbon above the maximum capacity entering the TCA-cycle is directed towards acetaldehyde formation, and subsequently ethanol formation, but also galactose exerts a similar response onto the metabolism. Although both glucose and galactose enter the metabolism at the glucose-6-phosphate node, the figures of the fluxes entering the TCA-cycle from the pyruvate node show that the maximum capacity entering the TCA-cycle is dependent on the specific sugar consumed. Thus, the metabolic flux analysis has demonstrated its potential by identification and quantification of metabolic changes caused by genetic manipulation, and this approach enabled us to study the control mechanism around the pyruvate node.

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Acknowledgements This work has been partly supported financially by the Danish Programme for Food Technology II (project 2409) as well as by European Commission Framework IV “Cell Factory” (contract BIO-CT95-0 107). Thanks to Kristian 0. Walløe for performance of the nitrogen-limited cultivations on glucose and galactose. References Fiaux, J., Anderson, C.I.J., Holmberg, N., Bülow, L., Kallio, P.T., Szyperski, T., Bailey, J.E., and Wüthrich, K. (1999) 13C NMR flux ratio analysis of Escherichia coli central carbon metabolism in micro-aerobic bioprocesses, J. Am. Chem. Soc. 121, 1407-1408. Galazzo, J.L., and Bailey, J.E. (1990) Fermentation pathway kinetics and metabolic flux control in suspended and immobilised Saccharomyces cerevisiae, Enzyme Microb. Technol. 12, 162-172. Heinrich, R., and Rapoport, T.A. (1974) A linear steady state treatment of enzymatic chains. General properties, control and effector strength, Eur. J. Biochem. 42, 89-95. Johnston M., and Carlson, M. (1992) Regulation of carbon and phosphate utilisation, in E.W. Jones, J.R. Pringel, and J. Broach (eds.), The molecular and cellular biology of the yeast Saccharomyces: Gene expression, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., pp. 193-281. Kaeser, H., and Burns J.A. (1973) The control offlux, Symp. Soc. Exp. Biol. 27, 65-104. Keleher, C.A., Redd, M.J., Schultz, J., Carlson, M., and Johnson, A.D. (1992) Ssn6-Tup1 is a general repressor of transcription in yeast, Cell 68,709-719. Klein, C.J.L., Olsson, L., and Nielsen, J. (1998) Glucose control in Saccharomyces cerevisiae: the role of MIG1 in metabolic functions, Microbiology 144,13-24. Marx, A., de Graaf, A.A., Wiechert, W., Eggeling, L., and Sahm, H. (1996) Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing, Biotechnol. Bioeng. 49, 111-129. Melcher, K. (1997) Galactose metabolism in Saccharomyces cerevisiae: A paradigm for eukaryotic gene regulation, in F.K. Zimmermann, K.-D. Entian (eds), Yeast sugar metabolism, Technomic Publishing Co., Inc. Lancaster, Basel, pp. 235-269. Pastemack, L.B., Laude, D.A., and Appling, D.R. (1994) 13C NMR analysis of intercompartmental flow of one-carbon unit into choline and purines in Saccharomyces cerevisiae, Biochemistry 33, 74-82. Rizzi, M., Bakes, M., Theobald, U., and Reuss, M. (1997) In vivo analysis of dynamics in Saccharomyces cerevisiae: II. Mathematical model, Biotechnol. Bioeng. 55, 592-608. Sonntag, K., Eggeling, L., de Graaf, A.A., and Sahm, H. (1993) Flux partitioning in the split pathway of lysine synthesis in Corynebacterium glutamicum, Eur. J. Biochem. 213, 1325-1331. Stephanopoulos, G., Aristodou, A., and Nielsen, J. (1998) Metabolic engineering, San Diego: Academic Press. Teusink, B., Diderich, J.A., Westerhoff, H.V., Dam, K., Walsh, M.C. (1998) Intracellular glucose concentration in derepressed yeast cells consuming glucose is high enough to reduce the glucose transport rate by 50%, J. Bacteriol. 180, 556-562. Theobald, U., Mailinger, W., Baltes, M., Rizzi, M., and Reuss, M. (1997) In vivo analysis of dynamics in Saccharomyces cerevisiae: I Experimental observations, Biotechnol. Bioeng. 55, 305-316. Treitel, M.A., and Carlson, M. (1995) Repression by SSN6-TUP1 is directed by MIG1, a repressor/activator protein, Proc. Natl. Acad. Sci. USA 92, 3132-3136 Wiechert, W., and de Graaf, A.A. (1996) In vivo stationary flux analysis by 13C labelling experiments, Adv. Biochem. Eng. Biotechnol. 54, 109-154. Østergaard, S., Olsson, L., and Nielsen, J. (1998) Metabolic control analysis of the Leloir pathway in Saccharomyces cerevisiae, in BioThermoKinetics In The Post Genomic Era. Proceedings of the 8th international meeting on Biothermokinetics, held July 2-5 1998 in Fiskebäckskil, Sweden, pp. 22-26.

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Part 3 STATE PARAMETERS AND CULTURE CONDITIONS

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EFFECT OF AERATION IN PROPAGATION ON SURFACE PROPERTIES OF BREWERS’ YEAST ANDREW ROBINSON AND SUSAN T. L. HARRISON Department of Chemical Engineering, University of Cape Town, Rondebosch, 7701, South Africa.

Abstract Traditionally, brewers’ yeast is propagated through a series of poorly aerated batch fermentation vessels. Acknowledgement of the importance of yeast quality in brewery fermentation performance and the requirement of adequate oxygen to ensure yeast quality has prompted the investigation of alternative propagation schemes. Here, the effect of aeration on yeast production, surface properties and flocculation of 4 brewers’ yeast strains (3 flocculent, 1 non-flocculent) have been investigated. The biomass growth rates and yields of all strains were improved on aeration during the propagation stage, as expected. In addition, changes were observed in the hydrophobicity and surface charge of the cell. Aeration lowers the hydrophobicity and increases the total charge on the cells while also increasing the flocculation capacity of flocculent strains of yeast. This observation is contradictory to the prediction of the DLVO theory and suggests the dominance of the lectin mechanism over classical colloidal flocculation theory. The strain classified as ‘non-flocculent’, and measured to be very weakly flocculent, showed the reduced hydrophobicity and increased charge in the presence of aeration, however its flocculence decreased in accordance with DLVO theory. This is consistent with the ‘non-flocculent’ strain lacking a lectin mechanism of flocculation. Further studies are required to determine the effect of aeration on fermentation performance and the effect of altered surface properties and flocculence on brewery fermentation. 1. Introduction Brewers’ yeast is traditionally propagated in a series of batch propagators which increases the biomass from laboratory flask size to the required quantity for full-scale brewery fermentation. These propagators are operated under oxygen limitation with low biomass yields thus requiring a number of propagation stages. This procedure increases the time required, substrate usage and risk of contamination between each 89 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 89–99. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

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transfer. Aerobic propagation offers a potential advantage over these drawbacks provided the physical properties, flocculence and fermentative ability of the yeast are not adversely affected. This paper presents a comparative study of yield coefficients, propagation rates, the surface properties of hydrophobicity and charge and the yeasts flocculence for four strains of brewers' yeast propagated under full aerobic and near anaerobic conditions. Various authors have studied surface properties of yeast to explain differences in their physical behaviour of flocculence, with contradictory findings. Amory and Rouxhet (1988) compared strains belonging to top fermenting Saccharomyces cerevisiae and bottom fermenting Saccharomyces carlsbergensis, since reclassified as S. cerevisiae. They observed that the bottom fermenting strains had more negative zeta potentials than the top fermenting strains. They also found the bottom strains to be less hydrophobic than the top cropping strains. Bowen and Cook (1989), Bowen et al. (1992) and Bowen and Ventham (1994) compared differences between bottom fermenting lager yeast, top cropping ale and bottom cropping ale yeasts. They found that yeast from top cropping ale strains had higher charge than the yeast from the bottom ale strains that in turn had more charge than the bottom cropping lager strains. Dengis et al. (1995) compared the flocculation mechanism of top and bottom cropping strains of Saccharomyces cerevisiae and found that their charge at the final pH of fermentation to be similar. 2. Materials and Methods 2.1 PROPAGATION CONDITIONS Propagation were conducted in autoclavable 2 1 glass vessels with a working volume of 1.5 1, using 16°P brewery wort (supplied by South African Breweries, Newlands brewery) at 18°C. Four commercial strains of brewers yeast were used: 3 flocculent (SAB1, SAB5 & SAB1/96) and one non-flocculent (SAB2). Agitation was provided with a Rushton impeller operated at 400 rpm. The aerobic propagation was sparged continuously with sterile air at 1.5 vvm while the near anaerobic propagation was only aerated prior to inoculation to saturate the wort with oxygen at a concentration of 9.2 mg.1-1. Foaming was controlled by the addition of 2 ml of Antifoam 289 (Sigma) prior to the start of aeration. Samples were taken to monitor cell concentration, medium density, cell hydrophobicity and surface charge. 2.2 HYDROPHOBICITY The hydrophobicity index of the cells was determined by their partitioning between a hydrocarbon and aqueous phase, based on the method of Smart et al. (1995). The cells were harvested by centrifugation, washed with distilled water, deflocculated with 2 mM EDTA and rewashed twice with deionised water. The washed cells were re-suspended to an OD660 of 0.6 – 0.75 in a phosphate-urea-MgSO4 (PUM) buffer at pH 7.1. The PUM buffer consists of 2.22% (w/v) K2HPO4.3H2O, 0.726% (w/v) KH2PO4, 0.18% (w/v) urea and 0.02% (w/v) MgSO4.7H2O. The optical density of the suspension was 90

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recorded and 2.4 ml was transferred to a wide mouth test tube. 0.2 ml xylene was added to the test tube. The mixture was vortexed for 2 minutes, and allowed to stand for 15 minutes. The aqueous phase was removed with a Pasteur pipette and its optical density determined. The hydrophobicity index was calculated as follows.

(1)

where: I = Initial optical density F = Final optical density HI = Hydrophobicity Index

2.3 SURFACE CHARGE Zeta potential of the growing yeast suspensions was measured using a Malvern ZetaSizer 4. Washed cells were suspended in 20 mM sodium acetate buffer solutions with pH values between 2.1 and 6.6 prior to zeta potential measurement. The zeta potential obtained was converted into surface charge density by using the Gouy Chapman equation (2), which describes the decay in potential around a charged particle (Hiemenz 1986).

(2)

where: n0 ε kB T Z e

= molar concentration of the electrolyte = dielectric constant of solution = Boltzman constant = temperature = valence charge = charge on the electron

For an aqueous solution at 25 °C, this reduces to equation (3).

(3)

where ψ is the surface potential of the yeast (V) and σ is the surface charge (µC.cm-2). In this study it is recommended that the charge density at the plane of shear, rather than zeta potential be used as an indicator of the surface charge of particles as this parameter removes the differences in experimental conditions used by different research groups. 91

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2.4 FLOCCULATION Samples were prepared to measure the extent of flocculation by removing a homogenous sample from the bioreactor, washing with distilled water, deflocculation with 2 mM EDTA and washing the yeast twice with deionised water. The washed yeast was resuspended in 20 mM sodium acetate buffer (pH 4.5) containing 10 mM CaCl2 to an OD660 in the range 2.3 to 2.4. The cell suspension was placed inside a glass U tube to which was fixed a square (10 mm) glass section, aligned inside a spectrophotometer. Air bubbles of approximately 0.5 cm3 were passed through the cell suspension at a rate of 60 per minute to allow an acclimatisation period and provide the reproducible mixing required to initiate flocculation. After 5 minutes, the air supply was closed and data logging of the absorbance started. The cells formed flocs and settled through the light path. Flocculation was complete within 2 to 4 minutes, after that time the plateau absorbance value was recorded. Absorbance readings were converted into biomass concentrations by calibration curves. The final extent of flocculation was reported as the wt. % of cells removed by flocculation. 3. Results 3.1 YIELD COEFFICIENTS The biomass yield coefficients were calculated at the end of each propagation and expressed as the dry mass of cells obtained per mass of total sugars used (g dry weight.g -1 These values are summarised in Table 1 for both the aerobic and near carbohydrate ). anaerobic propagation of the four strains. The increased ratio of aerobic to anaerobic yields are also presented. 3.2 CELL GROWTH RATES The rate of increase of cells during propagation was modelled using the logistic equation (Bailey and Ollis, 1986) (4). The integrated form of the equation (5) providing the cell concentration profile during propagation from the initial cell concentration, x0, has two parameters, β (ml.cell-1), the inverse of the stationary population concentration and k (h-1), the rate constant. (4)

(5)

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Since the final population size is known, the logistic equation can be solved to yield the logistic rate constant. Figure 1 illustrates the prediction of the data by the logistic equation. The rate constants obtained for each strain are summarised in Table 1. The ratio of aerobic to anaerobic rate constants is compared. Table 1. Comparison of biomass yield coefficients and cell production rates of aerobic and near anaerobic yeast propagation in high gravity brewers’ yeast.

Biomass Yield Coefficient

(gdry weigh•gcarbohydratge-1)

Strain SAB5 SAB1 SAB1/96 SAB2

Aerobic 0.139 0.134 0.095 0.117

Anaerobic 0.049 0.051 0.044 0.046

Ratio (aerobic/ anaerobic) 2.9 2.7 2.1 2.5

Logistic Cell Growth Rate (h-1) Aerobic Anearobic 0.149 0.129 0.111 0.103 0.072 0.059 0.076 0.080

Ratio (anerobic/ anearobic 1.16 1.08 1.22 0.95

Figure 1. Cell concentration during aerobic and anaerobic propagation of SAB1 with logistic equation fit to data. aerobic data, ______ aerobic logistic fit, anaerobic data --- - ---- anaerobic logistic fit)

3.3 HYDROPHOBICITY The cell growth profiles and hydrophobicity of the cell surface during propagation are shown for the four strains in Figure 2. The hydrophobicity of the cells was found to increase concomitantly with cell growth under anaerobic conditions. The aerobically grown yeast were less hydrophobic throughout propagation. Once the cells reached the stationary phase, no further change in hydrophobicity was observed. The hydrophobicity in the stationary phase of propagation is summarised in Table 2.

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3.4 ZETA POTENTIAL The zeta potential profiles of all strains measured across the pH range 2.1 to 6.6 varied with phase of growth to approach a constant charge profile towards the onset of the stationary phase. This is illustrated for the aerobic and anaerobic propagation of strain SAB2 in Figure 3a and 3b. The zeta potential of the aerobically grown strains was generally lower than the anaerobic equivalent, especially under stationary phase conditions. The iso-electric points for the stationary phase aerobic yeast were generally below the measured range of the assay, i.e. < pH 2. The charge profiles of the strains in their stationary phase of propagation are presented in Figures 4a and 4b for the aerobic and near anaerobic growth condition.

Figure 2. Increase in cell hydrophobicity with cell growth during propagation of strains under aerobic and near anaerobic conditions. ((a) SAB5, (b) SAB1, (c) SAB1/96 and (d) SAB2, aerobic hydrophobicity, _______ aerobic cell concentration, anaerobic hydrophobicity ---------- anaerobic cell concentration).

Figure 3. Zeta potential profiles for (a) aerobic and (b) anaerobic growth condition of SAB2 changing smoothly during propagation and reaching a stable profile by the end of late exponential and stationary propagation. pre-exponential, X early-exponential, phase)

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Figure 4. Zeta potential of the four strains of yeast at the end of (a) aerobic (b) and anaerobic propagation showing the different profiles observed. SAB1, SAB2, SAB1/96 and SAB5).

3.5 FLOCCULATION A typical absorbance profile for the flocculation assay is shown in Figure 5 illustrating a change in absorbance from the initial 2.44 to 0.44 after 2 minutes. These absorbencies correspond to cell concentrations of 97.6 and 3.9 million cells per ml. Based on cell concentrations the extent of flocculation is 96.4%.

Figure 5. conditions.

Flocculation assay absorbance profile for SAB5 grown under brewery

The three strains of flocculent yeast (SAB1, SAB1/96 & SAB5) were found to be more flocculent when grown under aerobic conditions as compared to propagation under anaerobic conditions. It was noticed that the yeast only became flocculent towards the end of propagation within the wort medium. This coincides with depletion of sugars, which may cause inhibition of the lectin binding required for flocculation. Samples taken during propagation were visually seen to be weakly flocculent in flocculation buffer until the late exponential phase. This coincided with the flocculation behaviour of 95

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the yeasts in situ. The extent of flocculation of yeast harvested in the stationary phase is summarised in Table 2 for both aerobic and near anaerobic growth conditions. Table 2 includes the zeta potential values at pH 4.5, the same pH as the flocculation buffer, for reference. Table 2. Summary of cell hydrophobicity, surface charge and extent of flocculation values for aerobic and anaerobic propagation of yeast.

Hydrophobicity Index Zeta Potential @ pH 4.5 Extent of Flocculation (%) (% of cells) (mV)† Strain Aerobic Anaerobic Aerobic Anaerobic Aerobic Anaerobic SAB5 2.5 12.4 -8.4(-0.39) -3.8(-0.17) 96.3 82.4 SAB1 4.5 21.0 -7.3(-0.34) -3.9(-0.18) 98.6 94.8 SAB1/96 5.5 22.8 -5.8(-0.27) -3.1(-0.14) 99.1 95.9 SAB2 1.8 25.0 -10.8(-0.50) -5.8(-0.27) 0.4 4.8 † Zeta potentials converted to surface charge values presented in brackets withunits of µC.cm-2.

4. Discussion The biomass yield coefficients of the aerobic propagation were consistently 2 to 3 fold higher than under anaerobic conditions. In addition, aerobic growth yielded higher logistic growth rate constants. The increased growth rates and higher yields illustrate that aerobic propagation is advantageous for the biomass production cycle of yeast in breweries. Before exploiting the potential advantage of aerobic propagation, the suitability of yeast produced by aerobic propagation for anaerobic brewing must be assessed. Brewing yeast is required to have good fermentative abilities as well as suitable physical characteristics necessary in the brewing environment. The physical attributes required include a suitable cell envelope structure and the yeasts’ ability to flocculate at the end of fermentation. Here the surface charge, hydrophobicity and flocculation potential of the commercial strains are assessed as a function of oxygen availability and growth phase. All flocculent strains considered in this study were more charged and yet also more flocculent when grown aerobically over those grown anaerobically. This suggests that no correlation between lower zeta potential and increased flocculence exists, contrary to the prediction of the Derjaguin-Landau-Venvey-Overbeek (DLVO) theory. Smit et al. (1992) showed that flocculation is most effective at a pH of 4.5. This is the approximate final pH attained in fermentation at which the yeast flocculates and settles out of suspension. It is therefore instructive to consider the yeasts’ surface charge at this pH when studying flocculation. From this study it is concluded that the increased surface charge associated with the aerobic propagation system does not lower flocculation in these yeast strains. Previously Amory and Rouxhet (1988), on comparing the surface charge between top and bottom cropping yeasts, showed the bottom strains to be more charged (zeta potential of -35 mV, corresponding to a surface charge of -0.12 µC.cm-2) than the top strains (zeta potential of -12 mV and charge of -0.04 µC.cm-2) at pH 4.0. In many of the early studies, zeta potentials were not measured in a defined environment. As these measurements were taken without any background electrolyte addition, even 96

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the smallest leakage of ions from the cells could alter the potential observed significantly. Bowen and Cooke (1989) determined zeta potential in wort hence surface charge can not be calculated. Dengis et al. (1995) found surface charges between -0.15 and -0.21 µC.cm-2 at pH 5.2 for top and bottom fermenting strains of Saccharomyces cerevisiae. Bowen and Ventham (1994) reported a decrease in zeta potential and surface charge during fermentation of top and bottom cropping ale yeast as well as lager yeast. The bottom ale yeast and lager yeast carried similar charge during fermentation, decreasing from -12 to -7 mV (-0.39 to -0.23 µC.cm-2) on progression from exponential to stationary phase. The top cropping ale yeast was slightly more charged throughout fermentation, decreasing from -16 mV to -12 mV (-0.53 to -0.39 µC.cm-2). These zeta potential measurements were made at the pH occurring during fermentation. Smart et al. (1995) observed a decrease in surface charge at pH 4.0 (-42.6 to -32.5 mV and -1.54 to - 1.12 µC.cm-2) when yeast was stored under conditions of nutrient starvation. From all these investigations, no clear correlation can be drawn between the flocculence or location of flocculated yeast (top vs. bottom) and the surface charge of the yeast. Van Hammersveld et al. (1994) calculated the bond strength predicted by the DLVO theory of flocculation. On comparison, they found the experimentally determined bond strength to be far higher and concluded that the additional bonding energy resulted from specific biological interactions. However, the parameters within the theory changed during fermentation in a manner that favoured flocculation predicted by DLVO. Speers et al. (1993) concurs that the DLVO theory cannot explain flocculence within flocculent yeast strains. The inability of physicochemical interactions at the surface to correlate with flocculation behaviour is further illustrated by trends in hydrophobicity determined in this study. Here a greater oxygen availability lowered the hydrophobicity but also increased the cells‘ flocculence. The hydrophobicity index of the yeast in the near anaerobic propagation increased during the exponential phase of growth while that of the aerobic propagation remained relatively low. The increase in hydrophobicity observed at the end of exponential growth, also seen by Smit et al. (1992), was found to coincide with the onset of flocculation. Smit et al. inferred that the observed hydrophobicity was attributed to flocculation proteins (lectins). This was supported by a reduction in both hydrophobicity and flocculation by protease activity. It can be concluded from these studies that the yeast lectins may contribute to the cells’ hydrophobicity, but this is not the only contributing factor and hydrophobicity alone is not responsible for flocculation. The non-flocculent strain (SAB2) showed the similar trend whereby the anaerobic propagation was less charged while at the same time being more hydrophobic than the aerobically grown yeast. This strain was observed to have a very low level of flocculence (<5%) as measured by the flocculation assay used here. For this strain, the anaerobic propagation condition caused lower charge, higher hydrophobicity and was more flocculent. This observation is consistent with the prediction of DLVO theory. Hence it is postulated that the small extent of flocculation found in this yeast strain is a result of the classical colloidal model while the flocculence observed in the flocculent strains is governed by lectin binding. The dominance of lectin-mediated flocculation is indicated.

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For all strains considered in this study, the inter-strain variation of surface charge profile and hydrophobicity was smaller than the variation generated by changes in oxygen supply or the position within the growth phase. This highlights the necessity to adequately quantify the oxygen availability during growth as well as the position in the growth cycle at which comparisons are made when comparing these properties for different strains or classes of yeast, viz. ale vs. lager or top vs. bottom fermenting yeast. Flocculation in the three flocculent strains studied here appears to be caused by the lectin/receptor mechanism described by Miki et al. (1982), as reported for other commercial strains of Saccharomyces cerevisiae. Such flocculation has been shown to require calcium ions and the sugars mannose and glucose inhibit the formation of flocs. According to the classification system of Stratford and Assinder (l991), these strains belong to the NewFlo phenotype owing to the inhibitory action of glucose. Bony et al. (1998) linked the level of flocculation observed in yeast to the amount of the flocculation lectin Flop at the surface of the cell. The protein was also detected in the endoplasmic reticulum, indicating that it was secreted along the protein excretory pathway. This pathway is known to be partially repressed under anaerobic conditions, indicating that flocculation may be repressed under anaerobic conditions as shown here. Although hydrophobicity and surface charge are not the main cause of yeast flocculation (van Hammersveld et al. 1994), these may play a role. At low levels of mixing, a sufficiently high surface charge could prevent the close interaction of individual cells to permit lectin-receptor interactions and so limit flocculation. 5. Conclusions Aerobic propagation of brewers’ yeast results in a 2 to 3 fold higher yield of biomass making it an attractive alternative to traditional oxygen limited propagation of brewers’ yeast. Aerobic propagation however does result in yeast with altered surface properties, namely lower hydrophobicity and greater surface charge. Aerobically propagated flocculent yeast also shows improved flocculation. Neither hydrophobicity nor surface charge appear to be directly responsible for this change in flocculation. Because yeast flocculence affects its fermentative ability and limited oxygen availability restricts the formation of membrane lipids and sterols, further assessment of fermentation performance is required to identify if the observed cellular changes affect brewery fermentation performance.

Acknowledgements The technical and financial support of The South African Breweries Ltd is gratefully acknowledged. Further financial support of the NRF, including sponsorship of AR through a postgraduate bursary is appreciated.

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References Amory, D.E. and Rouxhet, P.G. (1988) Surface properties of Saccharomyces cerevisiae and Saccharomyces carlsbergensis: chemical composition, electrostatic charge and hydrophobicity Biochim. Biophys. Acta 938, 61-70. Bailey, J.S. and Ollis, D.F. (1986) Biochemical Engineering Fundamentals, McGraw-Hill, Singapore. Bony, M., Barre, P. and Blondin, B. (1998) Distribution of the flocculation protein, Flop, at the cell surface during yeast growth: the availability of Flop determines the flocculation level. Yeast 14,25-35. Bowen, W.R. and Cooke, R.J. (1989) Studies of Saccharomyces cerevisiae during fermentation – an in vivo lectrokinetic investigation. Biotech. Bioeng. 33, 706-715. Bowen, W.R., Sabuni, H.A.M. and Ventham, T.J. (1992) Studies of the cell wall properties of Saccharomyces cerevisiae during fermentation. Biotech. Bioeng. 40, 1309-1318. Bowen, W.R. and Ventham, T.J. (1994) Aspects of yeast flocculation. Size distribution and zeta potential. J. Inst. Brew. 100, 167-172. Dengis, P.B., Nélissen, L.R. and Rouxhet, P.G. (1995) Mechanisms of yeast flocculation: comparison of topand bottom-fermenting strains. App. Environ. Microbiol. 61, 718-728. Hiemenz, P.C. (1986) Principles of colloid and surface chemistry. Marcel Dekker Inc., New York. Miki, B.L.A., Poon, N.H., James, A.P. and Seligy, V.L. (1982) Possible Mechanism for flocculation interactions governed by gene FLO1 in Saccharomyces cerevisiae. J. Bacteriol. 150, 878-889. Smart, K.A., Boulton, C.A., Hinchliffe, E. and Molzahn, S. (1995) Effect of physiological stress on the surface properties of brewing yeasts, J. Am. Soc. Brew. Chem. 53, 33-38. Smit, G, Straver, M.H, Lugtenberg, B.J. and Kijne, J.W. (1 992) Flocculence of Saccharomyces cerevisiae cells in induced by nutrient limitation, with cell surface hydrophobicity as a major determinant. App. Environ. Microbiol. 58, 3709-3714. Speers, R.A., Durance, T.D., Tung, M.A. and Tou, J. (1993) Colloidal properties of flocculent and nonflocculent brewing yeast suspensions. Biotechnol. Prog. 9,267-272. Stratford, M. and Assinder, S. (1991) Yeast flocculation: Flo1 and NewPlo phenotypes and receptor structure. Yeast 7, 557-574. Van Hamersveld, E.H., van Loosdrecht, M.C.M. and Luyben, K.ch.A.M. (1994) How important is the physicochemical interactions in the flocculation of yeast cells? Coll. Surf. B: Biointerfaces 2, 165-171.

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EFFECT OF THE MAIN CULTURE PARAMETERS ON THE GROWTH AND PRODUCTION COUPLING OF LACTIC ACID BACTERIA A. AMRANE* AND Y. PRIGENT Université de Rennes I, Laboratoire des Procédés de Séparation (Unité associée I.N.R.A.), Campus de Beaulieu, Bât. 10A, 263 avenue de Général Leclerc, CS 74205, 35042 Rennes Cedex, France Fax: 33 2 99 84 40 31. E-mail: [email protected]

Abstract Identification of the main culture parameters for Lactobacillus helveticus growing on whey supplemented medium was carried out from analysis of their influence on the growth and production coupling. Seed culture duration (linked to the physiological state of the inoculated cells), pH control and undissociated lactic acid concentration (the inhibitory species), as well as nitrogen limitation control this association between growth and production. 1. Introduction From the pioneer work of Luedeking and Piret (1959) lactic acid production have been recognised as partially linked to growth: (1) where the constants A and B are the coefficients for growth- and non-growth-associated production respectively. However, as previously shown (Amrane and Prigent, 1997), the best way to analyse growth and production coupling is to plot the amount of lactic acid produced (p - p0) vs. the biomass formed (x - x0), instead of the graph of the specific production rate as a function of the specific growth rate. The linear part of this plot corresponded to the growth-associated production, and its slope was the coefficient for growth-associated production (parameter A in equation 1), or the product on biomass yield YP/X. The effect of some culture parameters on growth and production coupling have been outlined in the available literature. Indeed, contradictory results concerning the effect of culture pH can be found: according to some authors the uncoupling between 101 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 101–108. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

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growth and production increase at acidic pH (Roy et al, 1987; Venkatesh et al, 1993), while Norton et al (1994) have obtained a very low value for the coefficient B for the non-growth associated production (0.01) at pH 4.3. Moreover, it has also been reported that nitrogen starvation was involved in this coupling (Turner and Thomas, 1975). However, systematic analysis of the effect of the main fermentation parameters on this linking is not at our knowledge available in the bibliography; this study will be the aim of the present work. 2. Materials and methods 2.1 MICROORGANISM Lactobacillus helveticus strain milano used throughout this work was kindly supplied by Dr A. Fur (Even Ltd, Ploudaniel, France). Stock cultures were maintained on 10% (w/v) skim milk and deep frozen at -16°C. As required, these cultures were thawed and reactivated by two transfers in 10% (w/v) skim milk (42°C, 24 h). 2.2 MEDIA Whey permeate powder (Armor-Proteines, ST Brice, France) was used as a carbon source; the powder was reconstituted at 57 g l-1, corresponding to a lactose concentration of 48 g l-1. Before use, the permeate was clarified by a heat / calcium process (Fauquant et al, 1985): it was supplemented with 3 g l-1 CaCl2, 2H2O, the pH was settled at 7.3, and the solution was pumped through two heat exchangers at 80 and 16°C respectively (mean residence time: 20 s). The solution was left to decant overnight at 4°C, and the supernatant was then supplemented with a range of yeast extract concentration (2, 5, 10, 20, 30 g l-1) or 20 g l-1 yeast extract and 5 g l-1 of both tryptic and pancreatic casein peptones (RM medium). When needed, aliquots of 1 mol l-1 lactic acid was added to RM medium in order to achieve initial concentrations p0 of 0, 2, 3, and 5 g l-1, corresponding to pH values of 5.90, 4.63, 4.34, and 4.04 respectively. During each run, the pH was maintained at its initial value by automatic addition of 10 mol l-1NaOH. The same procedure was applied to a second set of culture medium for which the initial pH was adjusted with hydrochloric acid instead of lactic acid. For another set of experiments, RM culture medium was supplemented with 1 M lactic acid at the required concentration (10,40, 50 g l-1), and then pH was settled at 5.9 by the addition of 10 M NaOH. 2.3 FERMENTORS AND CULTURE CONDITIONS Batch culture was carried out in a 2-1 fermentor (SET 2M, Inceltech, Toulouse, France), magnetically stirred (300 rpm), at 42°C. The pH was maintained at 5.9 by automatic addition of 10 M NaOH. The mass of the NaOH solution added for pH control was continuously recorded, allowing on-line calculation of the quantity of lactate anion produced at a given pH at each time point; the observed standard deviation was ±1 g l-1, 102

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Seed culture was carried out in a 0.25-1 laboratory-designed glass fermentor equipped with a sterilisable combination glass electrode (Ingold, Paris, France), cotton plug filter, magnetic stirrer, infrared lamp temperature control (set at 42°C), and an aseptic transfer line. In addition, the two fermentors were equipped with an aseptic recirculation loop (Watson-Marlow 50 1 U peristaltic pump; Volumax, Montlouis, France) incorporating a laboratory-made turbidimeter. As the turbidity was continuously recorded, the total biomass could be calculated on-line after dry weight calibration; the observed standard deviation was 10.2 g l-1. Bacteria were precultivated by inoculating sterile culture medium with 0.8 % (v/v) of the second skim milk transfer. Then, 1.6 1 of pasteurised culture medium was inoculated with 0.2 1 seed culture (11% v/v), unless specified, and the reaction proceeded. The concentration of total (p) and undissociated (HL) lactic acid was calculated using the Henderson-Hasselbach equation (pKA = 3.8), the lactate concentration (L-) and the corresponding pH value: (2)

and (3)

2.4 ANALYTICAL METHODS At the end of both the preculture and culture, the final biomass, lactose, and lactic acid concentrations were determined as previously described (Amrane and Prigent, 1994). 3. Results and Discussion 3.1. PRECULTURE CONDITIONS The effect of preculture conditions on the subsequent culture were first considered. As observed in Fig.1a, the preculture medium seemed to have no effect on the coupling between growth and production. Indeed, for both cultures carried out on RM medium and inoculated at 11 % seed level, growth and production were associated until 20-25 g l-1 lactic acid was produced, i.e. approximately half of the production; in the associated part of the curve, the same product on biomass yield (YP/X = 4.7, corresponding to the slope of the curve) was found for both batches. To examine the effect of inoculation level, another run (2) was carried out on RM medium, but inoculated at 6.3 % (v/v) 103

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instead of 11 % (additional pasteurised culture medium was fed to the fermentor to keep the total volume constant), with cells precultivated on RM medium too. No effect of the inoculation level appeared at the examination of Fig.1a: as above 20-25 g l-1 lactic acid was produced by an associated mechanism and YP/X = 4.7.

Figure 1: (a) Product on biomass yield for cells precultivated for approximately 13 h: on whey supplemented with 5 g l-1 yeast extract and inoculated at 11 % seed level, run 1 (A); on RM and inoculated at 6.3% seed level, run 2 on RM and inoculated at 11 % seed level, run 3 ; linear fitting (–). (b) Product on biomass yield for cells precultivated on RM for 13.3 h: run 3 (0); 7.5 h: run 4 4.7 h: run 5 (A) ; linear fitting for runs 3 (.....), 4 (–) and 5 (–•–•).

From Fig.1b, it appeared that the slope YP/X for the plot (p - p0) vs. (x - x0) increased in the following order for the three preculture duration tested: 7.5 h < 13.3 h < 4.7 h. Therefore, and by comparison with growth and production kinetics (Amrane and Prigent, 1998a), product on biomass yield increased when growth and production rates, as well as maximum cellular concentration, decreased.

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3.2. NUTRITIONAL LIMITATIONS As observed in Fig.2, the product on biomass yield displayed a minimum value of 3.7 for 10 g l-1 yeast extract supplementation; as previously demonstrated by derivation of growth and production parameters with respect to the YE concentration (Amrane and Prigent, 1998b and 1999), this corresponded to the best performances at minimum cost. The end of coupled production was arbitrarily defined as the point for which a 3 % deviation from the oblique asymptotic line was observed. From this, the part played by an associated mechanism in total production increased continuously with the yeast extract (YE) supplementation, from 15 % for 5 g l-1 YE to 83 % for 30 g l-1 YE, i.e. an almost total coupling between growth and production; this had to be brought together with previous result demonstrating that increasing YE supplementation resulted in a shift from a nitrogen limitation to a carbon one (Amrane and Prigent, 1998b).

Figure 2: Product on biomass yield for batch cultures of L. helveticus carried out on clarified whey supplemented with a range ofyeast extract concentrations.

Figure 3: Product on biomass yield for batch cultures of L. helveticus carried out with various initial lactic acid concentrations p0 (g l-1) : 0 –.–), 10 – –), 40 and 50 –)

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3.3. INITIAL LACTATE ADDITIONS The experiments displayed in Fig.3 were carried out on a largely nitrogen supplemented medium (RM) to avoid any nitrogen and growth factors limitation. As observed, at a given pH control (5.9), the proportion of lactic acid produced by an associated mechanism was not a function of the lactate initially added, about 60 % of the total production irrespective of the amount of lactic acid initially added; even if for 50 g 1-1 of initial lactic acid, the critical concentration (83 g l-1, Amrane and Prigent, 1998c) was achieved before a total lactose exhaustion. From Fig.3 also, increasing initial lactic acid additions resulted in increasing product on biomass yield, from 4.2 without initial addition to 15.0 for p0 = 50 g l-1.

Figure 4: Product on biomass yield for batch cultures of L. helveticus at pH 5.90, 4.63, 4.34, 4.04 initially adjusted with (a) lactic acid and (b) hydrochloric acid; (––): linear fitting.

pH control and undissociated lactic acid: As above, the experiments displayed in Fig.4 were carried out on RM medium to avoid any nutritional limitation. As observed, 106

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decreasing pH control and increasing quantity of undissociated lactic acid (the inhibitory species, Gätje and Gottschalk, 1991) resulted in decreasing amount of lactic acid produced by an associated mechanism (from 60 % for the optimal pH control, 5.9, to 17 % for a pH of 4.04 initially adjusted by addition of lactic acid), while the product on biomass yield increased (from 4.2 for pH = 5.9 to 13.5 for pH = 4.04). Moreover, by comparing Figs.4a and b, it appeared that growth and production coupling depended mainly on pH, and to a lesser extent on the undissociated lactic acid concentration. Indeed, the initial addition of lactic acid to achieve the required pH control resulted only in a slightly lower proportion of growth-associated production and also a slightly higher product on biomass yield, if compared to an initial addition of hydrochloric acid to adjust the pH. 4. Conclusions For the optimisation of the bioconversion of lactose into lactic acid, high values of the product on biomass yield YP/X were not a convenient criterion, since they correspond to a low coupling between growth and production: indeed the fermentation time was minimal, and the volumetric productivity of the bioreactor was maximal when both processes were almost totally coupled. Neither the composition of the seed culture medium nor the inoculation level had significant effect on YP/X and the percentage of associated production of the subsequent culture: among the preculture parameters, only the seed culture duration, i.e. the physiological state of cells, had such an effect. A high concentration of lactate in the culture medium (50 g l-1) resulted in a large increase of YP/X when the total lactate concentration was close to the critical one (83 g 1 1), but it had practically no effect on the coupling. Acidic culture pHs, leading to high proportions of undissociated lactic acid, resulted in a large increase of YP/X and a large drop of associated production. A similar behaviour was observed under conditions of nitrogen limitation (yeast extract concentration from 2 to 10 g l-1). Therefore it should be pointed out that concentrations of yeast extract in excess of 10 g l-1 led to a significant increase of YP/X. Acknowledgements We wish to thank Ms A. Copeland for correcting this manuscript. References Amrane, A. and Prigent, Y. (1994) Mathematical model for lactic acid production from lactose in batch culture : model development and simulation. Journal of Chemical Technology and Biotechnology 60, 241-246. Amrane, A. and Prigent, Y. (1997) Growth and lactic acid production coupling for Lactobacillus helveticus cultivated on supplemented whey: influence of peptidic nitrogen deficiency. Journal of Biotechnology 55, 1-8.

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A. AMRANE AND Y. PRIGENT Amrane A. and Prigent Y. (1998a) Identification and experimental validation of a criterion allowing prediction of cellular activity for preculture of lactic acid bacteria. Journal of Fermentation and Bioengineering 85,328-333. Amrane A. and Prigent Y. (1998b) Influence of yeast extract concentration on batch cultures of Lactobacillus helveticus: growth and production coupling. World Journal of Microbiology and Biotechnology 14, 529534. Amrane A. and Prigent Y. (1998c) Influence of an initial addition of lactic acid on growth, acid production and their coupling for batch cultures of Lactobacillus helveticus. Bioprocess Engineering 19, 307-312. Amrane A. and Prigent Y. (1999) Analysis of growth and production coupling for batch cultures of Lactobacillus helveticus with the help of an unstructured model. Process Biochemistry 34, 1-10, Fauquant, J., Viéco, A., Brulé, G. and Maubois, J.-L. (1985) Sweet whey clarification by heat-calcium aggregation of residual fat material. Lait 65, 1-20 (in French). Gätje, G. and Gottschalk, G. (1991) Limitation of growth and lactic acid production in batch and continuous cultures of Lactobacillus helveticus. Applied Microbiology and Biotechnology 34, 446-449. Luedeking R. and Piret E.L. (1959) A kinetic study of the lactic acid fermentation. Batch process at controlled pH. Journal of Biochemistry, Microbiology and Technological Engineering 1, 393-412. Norton S., Lacroix C. and Vuillemard J.C. (1994) Kinetic study of continuous whey permeate fermentation by immobilised Lactobacillus helveticus for lactic acid production. Enzyme and Microbial Technology 16, 457-466. Roy, D., Le Duy, A. and Goulef J. (1987) Kinetics of growth and lactic acid production from whey permeate by Lactobacillus helveticus. Canadian Journal of Chemical Engineering 65, 597-603. Turner, K. W. and Thomas, T. D. (1975) Uncoupling of growth and acid production in lactic Streptococci. New Zealand Journal of Dairy. Science Technology 10, 162-167. Venkatesh, K. V., Okos, M. R. and Wankat, P. C. (1993) Kinetic model of growth and lactic acid production from lactose by Lactobacillus bulgaricus. Process Biochemistry 28, 23 1-241.

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PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE Signal Transduction During Nutrient Limitation

F.F. BAUER AND I.S. PRETORIUS Institute for Wine Biotechnology, University of Stellenbosch, ZA-7600 Stellenbosch, South Africa

Abstract Cells of the yeast Saccharomyces cerevisiae can undergo profound molecular, physiological and morphological modifications in response to a limited supply of essential nutrients, in particular carbon or nitrogen sources. These include a shift in transcription patterns, the modification of the cell cycle, a change in budding pattern and strongly polarised growth. Cells having undergone these modifications do not separate after cell division is completed and form chains of elongated cells called pseudohyphae or filaments. Cells growing as filaments are able to invade agar plates and other substrates, a phenomenon referred to as invasive growth. A network of signal transduction pathways governs this switch from yeast-like growth to pseudohyphal and invasive growth. Important elements of this network have been identified, including nutrient signal-receptors, GTP-binding proteins, components of the pheromonedependent MAP kinase cascade, cAMP, and several transcription factors. In this review, we summarise our current knowledge in this rapidly progressing field. We focus particularly on the interactions between several signal transduction modules and on the different transcription factors, which are regulated by these signalling modules. 1. Introduction The line between basic and applied research is nowhere more nebulous than in yeast molecular biology and genetic engineering, and is usually traversed at will in our laboratory's research on the genetic improvement of industrial yeast strains. One of the focuses of our yeast strain development programme is to increase the efficiency of fermentation and processing. By expressing numerous genes encoding extracellular, polysaccharide-degrading hydrolases in wine, brewing and baking yeast strains, we 109 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 109–133. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

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found that Saccharomyces cerevisiae is able to change its growth pattern (pseudohyphal/invasive growth, flocculation and vellum formation) when fermentable sugars progressively become limiting in the growth medium. This observation has guided our research into fundamental aspects of cellular differentiation, Cellular adaptations to changes in extracellular parameters have been a central subject of biological investigation throughout this century. Adaptation requires the perception (sensing) of the environmental parameter, followed by the transmission of the information to the relevant compartments of the cell. This process, which will result in a specific adaptive response to a specific signal, is referred to as signal transduction. The molecular nature of signalling events hasonly become accessible during the last two decades. Signal transduction pathways are part of the most essential molecular attributes of living systems, and the ability to adapt to changing environmental conditions or the ability to co-ordinate cellular differentiation in a multi-cellular organism (which relies on the exchange of signals between cells) is essential for survival. Consequently, most signalling molecules and mechanisms have been well conserved during evolution, and signalling modules found in S. cerevisiae are very similar to those found in higher eukaryotic organisms, including mammals (reviewed in Lim et al., 1996; Widmann et al., 1999; Wilkie and Yokoyama, 1994). This situation has created new interest in S. cerevisiae as a model system, which allows investigating complex molecular interactions. Besides being traditionally the most widely used microorganism in industry, S. cerevisiae has by now probably become the scientifically best understood of all organisms, The sequence of the entire genome of S. cerevisiae has been deciphered and made public in 1996, and a large number of protein-encoding open reading frames (ORFs) has already been associated with a specific function. The availability of the entire genome sequence combined with the genetic tractability of S. cerevisiae and the existence of what is probably the widest range of molecular tools, has lead to an extraordinarily fast accumulation of new knowledge. In the not so distant future, the entire functional mapping of the genome and of the proteome of this unicellular organism will probably be achieved. This review will mainly focus on some aspects of the complex molecular interactions that occur during a specific signal transduction event. In the first part of the review, we provide some general background on S. cerevisiae and introduce the specific pathway (pseudohyphal differentiation and invasive growth) that has been the focus of our investigations. In a second part, we detail the complex molecular nature of the signal transduction events involved in this dimorphic and cell growth adaptation. Finally, in a last section, we show how the newly gained knowledge has modified our vision of signal transduction events and how this insight can be of tremendous importance for future genetic modifications of industrial strains of S. cerevisiae for biotechnological applications. 2. Signal transduction in Saccharomyces cerevisiae Like most organisms, S. cerevisiae can perceive two types of extracellular signals: signals required for intercellular communication (for example hormonal signals like the pheromones a and α), and signals emanating from the environment. Environmental 110

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signals can be either of a chemical (e.g. the concentration or availability of certain nutrients) or physical (e.g. temperature and osmotic pressure) nature, and survival requires a specific response to changes in any of these parameters. In theory, two possibilities to ensure specific responses to specific signals exist. Cells would have to either use clearly separated and different signal transduction pathways for each signal, or use the amplification, differentiation and combinatorial possibilities of a network of a limited number of cross-talking pathways. Since each signal has to be integrated to achieve a perfect co-ordination between all cellular responses to extracellular stimuli, and since the number of potentially important signals is high, specific and separated transduction pathways for each signal seem a logistical impossibility. In line with this expectation are recent data, largely obtained through research on S. cerevisiae, which suggest an increasingly complex picture of interconnected and cross-talking signalling pathways, relying on a limited amount of molecular modules (reviewed in Elion, 1995; Pawson, 1995; Levin and Errede, 1995; Madhani and Fink, 1998). One of the - at least in a research perspective - most useful phenotypic adaptations of S. cerevisiae to environmental change is pseudohyphal differentiation, which is also referred to as the dimorphic switch between yeast-form and filamentous-form (Fig. 1). This adaptation can be observed in conditions of nutritional limitation and has recently been rediscovered by molecular biologists (Gimeno et al., 1992). The phenomenon had been documented by yeast taxonomists and physiologists in the earlier half of the century, only to be forgotten when molecular biology came to the fore, probably due to the generalised use of laboratory adapted strains which were unable to implement this developmental program (reviewed in Kron, 1997). The environmental signal, which results in the dimorphic transition from yeast-form to filamentous-form, is the limitation of specific, essential nutrients. Gimeno et al. (1992) initially described the pathway as an adaptation to nitrogen limitation. However, later evidence indicates that a limitation in the carbon source or the presence of an inefficiently used carbon source like starch (Lambrechts et al., 1996a) or the presence of fusel alcohols (Dickinson, 1996) leads to similar adaptations. Phenotypically, the process refers to a number of morphogenetic processes, which underlie the transformation of round or ovoid cells into thin, elongated cells (Figure 1). Concomitantly, the budding pattern shifts from axial (most haploid strains) or bipolar (most diploid strains) to unipolar, with all buds emerging from the same pole of the elongated cells. These cells remain attached to each other and form hyphen-like structures that grow beyond the perimeter of the colony (Figure 1). Since the process is triggered by nutritional limitation, it is thought to allow yeast cells to search for nutrients. The process is not identical for haploid and diploid strains of S. cerevisiae (Gimeno et al., 1992; Roberts and Fink, 1994). Diploid cells adopt more readily an elongated, pseudohyphal growth form, and diploid filaments stretch far beyond the perimeter of the colony. Haploid filaments most frequently are limited to the area below the colony (Dickinson, 1994; Roberts and Fink, 1994). However, signal transduction events in both cell types seem to be largely identical. Cells growing in a filamentous form are able to invade solid substrates like agar, a phenomenon referred to as invasive growth. However, filamentation is not a prerequisite for invasive growth to occur, and some strains, which are unable to develop pseudohyphae, are still able to invade the 111

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substrate. The two phenotypes, filamentation and invasive growth, therefore make use of the same signal transduction pathways, but can be observed independently in some genetic backgrounds or in specific conditions. In this review, the terms pseudohyphal differentiation or filamentation will always imply invasive growth, unless otherwise stated.

Figure 1. Morphology of yeast-type and filament-type yeast cells and observed phenotypic changes.

As stated above, pseudohyphal differentiation is the result of the complex interaction of several signal transduction pathways. It requires the perfect co-ordination of essential cellular processes, including specific modifications of the cell cycle, reorganisation of the actin cytoskeleton (cell elongation), modification of the cell wall and a change in budding pattern (Figure 1). The signal transduction network governing this coordination of otherwise apparently unlinked cellular processes is one of the main foci of this review. 3. Molecular nature of signal transduction processes resulting in pseudohyphal differentiation Numerous genes have been, and are still being, identified for playing a role in - or for influencing directly or indirectly - the events associated with the dimorphic transition of S. cerevisiae cells. Several systematic genetic approaches using different phenotypic screens have been implemented to identify such genes. These approaches include the isolation of genes which (i) induce pseudohyphae formation when present in multiple copies (Gimeno and Fink; 1994), (ii) lead to elongated cell morphology (Blacketer et al., 1995) or result in the suppression of filamentous growth when mutated (Mösch and Fink, 1997), or (iii) are able to suppress such mutations (Lorenz and Heitman, 1998a). 112

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Other genes have been identified through indirect approaches, for example through their ability to induce the STA1-3 glucoamylase genes (Lambrechts et al., 1996b), which were shown to be co-regulated with invasive and pseudohyphal growth. Finally, numerous genes were linked to the process through general and systematic screening of phenotypes resulting from mutations within known genes (Radcliffe et al., 1997; Chandarlapaty and Errede, 1998; Edgington et al., 1999) or because of homologies with known developmental regulators in other species (Gavrias et al. 1996). Gene products participating in pseudohyphal differentiation can be divided into four groups: • Proteins that belong to evolutionarily conserved signal transduction modules, including plasma membrane receptors and associated heterotrimeric and small GTP binding proteins forming part of the signal sensing apparatus, as well as intermediate components of signalling cascades, for example MAP kinase cascades and second messengers like cAMP (adenosine 3', 5'-cyclic monophosphate) and their effectors. • Proteins involved in morphogenetic events. • Transcriptional regulators. • Downstream effectors of the signalling pathway. In the following section, the role of the above-mentioned signal transduction modules will be discussed in some detail. 3.1. SIGNAL TRANSDUCTION MODULES 3.1.1. Nutrient availability is sensed by permeases The reduced availability of specific and essential nutrients is the environmental signal responsible for pseudohyphal differentiation. The molecular sensors of the filamentation signal therefore have to be able to sense the presence and concentration of those nutrients. Nutrient sensing in yeast and other organisms has frequently been associated with nutrient specific transporters, permeases, or homologues thereof (reviewed in Kruckeberg et al., 1998). The suspicion that permeases could be involved in the perception of the pseudohyphal differentiation signal was confirmed by Lorenz and Heitman (1998b). The authors identified the ammonium permease Mep2p to be responsible for ammonium sensing and to provide the signal resulting in pseudohyphal differentiation (Lorenz and Heitman, 1998b). Three ammonium permeases with significant sequence homologies, Mep 1p, Mep2p and Mep3p, have been identified in S. cerevisiae (Marini et al., 1997). Of the three, Mep2p presents the highest affinity (KM=1-2 µM) for ammonium. The data of Lorenz and Heitman (1998b) suggest that Mep2p is specifically required for the sensing of ammonium, and does not play any role in the sensing of other nitrogen sources. A deletion of the MEP2 gene results in cells unable of filamentous growth in ammonium limited medium. The ∆mep2 strain, however, is perfectly able to form filaments in response to limitations in other nitrogen sources like glutamine, asparagine or proline. The deletion does not result in any growth defects on medium containing ammonium, showing that the two additional ammonium permeases Mep1p and Mep3p are efficient transporters, but play a less prominent role in signalling of ammonium availability. The data suggest that yeast cells probably require 113

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specific sensors for each of the nitrogen and carbon sources whose limitation results in pseudohyphal or invasive growth. Several other permeases can be expected to play a similar role and to specifically signal the presence or limited availability of their substrates. However, only indirect evidence has been provided so far (Lorenz and Heitman, 1998b). No receptor sensing carbon source limitation, and which would be required for pseudohyphal differentiation, has been identified, but the putative G-protein associated receptor Gpr1p is a promising candidate (Xue et al., 1998; Yun et al., 1998). Furthermore, two other putative glucose sensors have recently been identified by Ozcan et al. (1998). The proteins Snf3p and Rgt2p have homologies with hexose transporters, but apparently generate an intracellular signal without transporting glucose. As for Gpr1p, however, no direct evidence for a relation between these sensors and pseudohyphal growth has yet been presented. 3.1.2. Transmission via receptor associated elements 3.1.2.1. The role of Gpa2p. Gpa2p was initially identified because of strong homology with α-subunits of mammalian heterotrimeric guanine-nucleotide-binding proteins (Nakafuku et al., 1988). These proteins are well-studied receptor-associated signal transduction modules consisting of three subunits, α, β, and γ (reviewed in Neer, 1995). Upon activation of the receptor, the a-subunit exchanges GDP for GTP, which results in the dissociation of the heterotrimer. The signal is passed on by either the a-subunit or the β−γ-subunits, which stay attached to each other. Based on homology searches, the S. cerevisiae genome sequence revealed the presence of only two Gα subunits, Gpa1p and Gpa2p. Gpa1p is the well-studied a-subunit of the heterotrimeric G-protein associated with the pheromone receptors Ste2p and Ste3p (reviewed in Kurjan, 1992) and has not been implicated in any other signalling event so far. Gpa2p, on the other hand, has been implicated in a number of events, in general related to the control of cAMP levels in the cell (Nakafuku et al., 1988; Papasavvas et al., 1992; Lorenz and Heitman, 1997; Kübler et al., 1997; Colombo et al., 1998). Several genetic interactions with the Ras proteins, in particular the ability of overexpressed GPA2 to suppress the growth defects of temperature sensitive ras mutants, and the non-viability of a strain carrying deletions of both RAS2 and GPA2 have also been reported (Nakafuku et al., 1988; Papasavvas et al., 1992). Interestingly, whereas the β and χ subunits of Gpa1p had been easily isolated and have been well studied, no such subunits associated with Gpa2p have thus so been identified. GPA2 was more recently shown to play an essential role during pseudohyphal differentiation in diploids (Lorenz and Heitman, 1997; Kübler et al., 1997). A gpa2/gpa2 deletion strain is unable - or presents a very reduced ability - to grow in the filamentous form, whereas a strain carrying a hyperactivated allele (GPA2Gly132Val) shows enhanced pseudohyphal differentiation even on rich media. A dominant negative allele (GPA2Gly299Ala) was shown to inhibit pseudohyphal growth. Unpublished data of our laboratory indicate that Gpa2p affects invasive growth and filamentation similarly in haploid strains. Since heterotrimeric G-proteins are receptor-coupled signal transduction modules, our current knowledge strongly favours the hypothesis that Gpa2p associates with 114

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receptors like Mep2p to signal nutrient limitation. However, no direct interaction of Gpa2p with a receptor relevant for filamentation has so far been demonstrated. Genetic evidence nevertheless suggests that the ammonium transporter Mep2p requires Gpa2p for signalling (Lorenz and Heitman, l998b). In addition, glucose signalling was also shown to be dependent on Gpa2p (Colombo et al., 1998). As mentioned previously, Gpa2p has been shown to be associated with Gpr1p, a putative G protein-coupled receptor (Xue et al., 1998), and other data suggest that Gpr1p might be involved in the sensing of glucose and other fermentable sugars (Yun et al., 1998). As stated above, the relevance of this receptor for pseudohyphal differentiation remains to be established. Lorenz and Heitman (1997) and Kübler et al. (1997) present evidence that Gpa2p might transmit the signal through regulation of cAMP levels in the cell. Indeed, a strain carrying deletions of both gpa2/gpa2 and pde2/pde2, the latter gene encoding the phosphodiesterase responsible for the cleavage of cAMP, is unable to grow filamentously, but this phenotype can be suppressed by the addition of extracellular cAMP. The role of cAMP and the different signal transduction elements associated with this second messenger molecule is discussed in more detail later in this review. 3.1.2.2. Ras2p regulates filamentation via two separate pathways. The first signal transduction element to be associated with invasive and pseudohyphal growth was the small GTP-binding protein, Ras2p. Results presented by Gimeno et al. (1992) showed that the hyperactivated form of Ras2p, Ras2pVa119, stimulated both pseudohyphal and invasive growth. Initial experiments suggested that this regulation occurred through the - at this time - only recognised function of Ras2p, the control of adenylate cyclase (Cyr1p) activity and consequently the regulation of cAMP concentration within the cell. Results presented by Ward et al. (1995) strengthened the hypothesis of a cAMP dependent regulation of the process. Indeed, overexpression of the PDE2 gene (encoding, as mentioned above, a phosphodiesterase responsible for cAMP degradation) inhibits pseudohyphal and invasive growth and suppresses the pseudohyphal growth phenotype of the RAS2Va119 allele. Concomitantly, Liu et al. (1993) presented evidence for the involvement of elements of the mating specific MAP kinase cascade, in particular the MEKKK Ste20p, MEKK Ste11p and MEK Ste7p, and the transcriptional activator Ste12p (Liu et al., 1993; Roberts and Fink, 1994) in invasive and pseudohyphal growth regulation. The authors showed that other elements of the pheromone signal transduction cascade, like the pheromone receptors Ste2p and Ste3p, or the receptor-associated heterotrimeric Gprotein did not have any effect on pseudohyphal differentiation. Initially, no link between Ras2p and the MAPK cascade was established. Mösch et al. (1996) later presented evidence that the regulation by the MAPK cascade was dependent on Ras2p. The transmission of the signal via Ras2p and the MAPK cascade was shown to be cAMP independent, but dependent on the rho-like small G-protein, Cdc42p. The dominant negative CDC42 allele, CDC42Ala118, is indeed able to block the transmission of the constitutive filamentation signal created by the hyperactivated RAS2Va119 allele. Both Ras2p and Cdc42p were shown to act upstream of the MAP kinase cascade through epistasis analysis. In this case, modification of the activity of cAMP dependent kinase (PkA) through the use of bcy1 mutants or the overexpression of Tpk1p did not seem to influence filamentous growth (Mösch et al., 1996). 115

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Ras2p therefore seems to regulate pseudohyphal differentiation both via cAMP dependent and cAMP independent pathways, as represented in Figure 2 (Mösch and Fink, 1997; Lorenz and Heitman, 1997; Gagiano et al. 1999).

Figure 2. Ras2p dependent pathways leading to pseudohyphal diferentiation.

3.1.3. Intermediate signal transduction modules Ras2p and Gpa2p appear as two independent signal-transducing elements, both situated more or less directly downstream of the signal receptor. Some of the data presented in the previous paragraphs clearly suggest that the signal emanating from these two elements is further transmitted via both separated and shared signal transduction modules. 3.1.3.1. CAMP and CAMP-dependent kinase. cAMP is a key signalling molecule in most, if not all, currently existing organisms. Dependent on organism and cell type, this second messenger molecule regulates a variety of targets by controlling the activity of CAMP-dependent protein kinases (cAPK). In S. cerevisiae, cAMP is produced from ATP by the CYR1 gene encoded adenylyl cyclase, whose activity in turn is controlled by the two Ras proteins. Whereas it appears clear that basal levels of cAMP are required for normal cellular growth and cell cycle control, a specific role for cAMP during signal transduction events in S. cerevisiae has been difficult to pinpoint. Indeed, the only

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specific and significant CAMP-dependent response observed so far is linked to the sensing of glucose in some conditions (reviewed in Thevelein, 1992). The involvement of cAMP in the regulation of pseudohyphal differentiation was suspected when Ras2p was shown to regulate filamentation by Gimeno et al. (1992). Since then, a number of data have been taken as evidence that cAMP is indeed one of the major signal transduction components responsible for the establishment of invasive and pseudohyphal growth patterns, and that both Gpa2p and Ras2p are involved in the regulation of intracellular cAMP concentrations (Ward et al., 1995; Küebler et al., 1997; Lorenz and Heitman, 1997, 1998a,b; Robertson and Fink, 1998). Extracellular cAMP can enhance pseudohyphal differentiation in wild type strains, and is able to do so at low concentrations in a diploid ∆pde2/∆pde2 mutant strain. In addition, extracellular cAMP can suppress the pseudohyphal growth defect of a ∆gpa2/∆gpa2 strain in a ∆pde2 background (Lorenz and Heitman, 1997). In the same manner, the pseudohyphal growth defect of a ∆mep2/∆mep2 strain can be overcome by extracellular cAMP (Lorenz and Heitman, 1998b). As in other organisms, the main, and so far only downstream element directly regulated by cAMP in S. cerevisiae is the cAMP dependent protein kinase (cAPK), encoded by three genes, TPK1, TPK2 and TPK3 (Toda et al., 1987) These kinase isoforms have long been considered to possess largely overlapping functions, since deletion of any combination of two of the three genes encoding Tpks does not result in growth defects or reduction in cellular viability (Cannon and Tatchell, 1987; Toda et al., 1987). The role of Tpks in pseudohyphal development initially seemed doubtful, since the deletion of BCY1, which encodes the regulatory subunit of cAPK (Matsumoto et al., 1982), did not show any clear effect on pseudohyphal differentiation (Mösch et al., 1996). More recently, Robertson and Fink (1 998) were able to show that the three cAMP dependent protein kinases play different and specific roles in the control of pseudohyphal differentiation. Diploid tpk2/tpk2 mutant strains fail to grow filamentously on nitrogen limited media, whereas tpk3/tpk3 mutants or strains that overexpress TPK2 show enhanced pseudohyphal development in the same conditions. In conclusion, the authors suggest that Tpk2p is essential for pseudohyphal development, whereas Tpk1p apparently has no and Tpk3p an inhibiting effect on the process. Further data, based on 2-hybrid analysis, indicate that Tpk2p, but not Tpk1p or Tpk3p, associates specifically with the transcription factor Sf11p (discussed in more detail further on), deletion of which results in enhanced pseudohyphal growth. It is still unclear how the cAMP signal can be specifically transmitted to a specific isoform of the cAMP dependent kinase, or how different isoforms can carry out divergent signalling functions, The pronounced sequence differences in the amino terminal extension of the three Tpks might direct each kinase to a specific set of target proteins, as the direct and specific association of Tpk2p with Sf11p suggests (Robertson and Fink, 1998). 3.1.3.2. The MAP kinase cascade: α multidimensional relays station. Whereas the involvement of cAMP in a nutrient-sensing signal transduction pathway might not seem surprising, this can not be said about the contribution of several elements of the fheromone responsive MAP kinase cascade to pseudohyphal differentiation and invasive growth. (Liu et al., 1993; Roberts and Fink, 1994; Mösch et al., 1996). This 117

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cascade is represented in Figure 3. A MAP kinase signalling module consists of several protein kinases, which, upon activation, transmit the signal through a series of phosphorylation events, resulting in the activation of the MAP kinase which directly regulates various target proteins through phosphorylation (reviewed in Herskowitz, 1995; Levin and Errede, 1995; Madden and Snyder, 1998). The module is evolutionarily extremely well conserved throughout the eukaryotic kingdom (reviewed in Widmann et al., 1999).

Figure 3 Comparison between the pheromone response and the pseudohyphal growth pathways Common elements of the two pathways are in bold italics, whereas elements that impart MAPK specificity are highlighted by a grey background

The MEKKK Ste20p, MEKK Ste1 1p and MEK Ste7p were shown to be required for efficient invasive growth, as was the transcription factor Ste12p (Liu et al., 1993). Other genes which when mutated result in sterile phenotypes like STE50 were also shown to be required for the regulation of filamentous growth (Ramezani et al., 1998). Deletion of any of the above-mentioned genes results indeed in strongly reduced invasive and filamentous growth, whereas hyperactivated alleles (where available) present enhanced phenotypes for both characteristics. Epistasis analysis showed that the kinase cascade receives the filamentation signal via Ras2p and Cdc42p in a cAMP independent manner (Möschet al., 1996). Several hypotheses, recently reviewed by Madhani and Fink (1998), could explain how specific signals can result in specific responses while using, at least partially, the same elements for transmission (Figure 3). Firstly, the MEK Ste7p activates two different MAP kinases, Fus3p and Kss1p. Cook et al. (1996, 1997) and Madhani et al. 118

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(1997) showed that Kss1p is specifically required for pseudohyphal growth, whereas Fus3p is specific for mating. Both are inhibiting the activity of their specific targets when inactive (non-phosphorylated), which explains why initial genetic data based on the study of deletion mutants investigating the pheromone signal transduction suggested a partially redundant activity for the two kinases (reviewed in Herskowitz, 1995). A second mechanism to achieve MAP kinase cascade specificity relies on the combinatorial control of the transcription factor Stel 2p, which has to associate with Tec1p to activate hyphae specific genes (Gavrias et al., 1996; Madhani and Fink, 1997). A third potential mechanism to ensure MAP kinase specificity could reside in higher order association of the different kinases, which would keep particular signal transduction pathways physically separated and make cross activation impossible. This possibility is clearly demonstrated by the specific recruitment of the MEKK Stel 1p to both the mating pheromone pathway-specific scaffold protein Ste5p (Elion, 1995) and to the HOG MAPK pathway-specific scaffold Pbs2p (Posas and Saito, 1997). Indeed, besides being required for both the pheromone and pseudohyphal growth pathway, Stel1p is necessary for the osmotic activation by the osmosensor Sho1p of the HOG MAP kinase pathway (Posas and Saito, 1997). However, no pseudohyphal responsespecific scaffold protein has yet been identified. MAP kinase associated proteins might play an additional role in ensuring signalling specificity. The filamentation specific MAPK Kss1p has been shown to associate with two proteins, Dig1p and Dig2p (Cook et al., 1997; Tedford et al., 1997). The genes were identified through a 2-hybrid interaction screen and Dig1p co-immunoprecipitates with Kss1p in the nucleus. A ∆dig1/∆dig2 double mutant presents a constitutively invasive phenotype, but neither the double nor the single mutants present any defect with regard to the pheromone response. Both proteins also associate with Ste12p, the transcriptional activator regulated by both MAP kinases Fus3p and Kss1p. The data suggest that Dig2p acts as a negative regulator of invasive growth, and is directly regulated by Kss 1 p, which, as mentioned above, acts as a strong repressor of invasive growth in its non-phosphorylated state (Cook et al., 1997; Madhani et al., 1997). In addition, Bardwell et al. (1998b) show that Kss1p in its inactive form can directly bind to Ste12p, offering a further possibility for direct regulation of the protein. The different elements that might contribute to MAP kinase specificity are summarised in Figure 3. The specific contribution of the MAPK cascade to the modifications observed during pseudohyphal differentiation is unclear. However, both pheromone response and low nutrient response result in some cellular modifications of a similar nature, in particular with regard to polarised growth (see following paragraph) and changes in cell-adhesion properties. Some of these changes might be depending on the common elements in the two cascades. 3.1.3.3. Morphogenetic factors associate with the MEKKK Ste20p. The major morphogenetic event observed during filamentation is the strong elongation of the filament-forming cells. Cell elongation requires polarised growth, which in turn is dependent on the organisation of the actin cytoskeleton (reviewed in Madden and Snyder, 1998). Numerous results have been taken as evidence that the morphogenetic reorganisation of the actin cytoskeleton during pseudohyphal growth is dependent on the MEKKK Ste20p and its association with the rho-like GTPase, Cdc42p. Results 119

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show that both proteins have a more general role in the organisation of polarised growth during the cell cycle (Evangelista et al., 1997; Leberer et al., 1992, 1997; Leeuw et al., 1995, 1998; Mösch et al., 1996). Thermosensitive mutations in CDC42 result in large, round, non-budding cells, indicating the requirement for Cdc42p for polarised growth during bud emergence. Ste20p, on the other hand, directly associates with the actin cytoskeleton via an adapter protein, Bem1p (Leeuw et al., 1995; reviewed in Madden and Snyder, 1997). Ste20p also directly binds to Cdc42p, and deletion or mutation of the Cdc42p-binding site blocks pseudohyphal differentiation (Peter et al., 1996; Leberer et al., 1997). Further evidence for this more general role of Ste20p is provided by the fact that the hyperactivated allele of STE11, STE1 1-4, only partially bypasses filamentation defects of ste20 mutants, whereas mutations in STE11, STE7 or STE12 do not block cell-elongation in a RAS2Va119 strain (Mösch et al., 1996). The morphogenetic function of Ste20p and Cdc42p could explain at least in part the common requirement of the MAP kinase cascade for both mating and pseudohyphal differentiation, since in both cases a strong polarisation of the growth pattern is required, either to produce the cellular extension called shmoo during mating or to produce elongated cells during pseudohyphal differentiation. A second phenotypic change associated with both pathway is the modification of cell-adhesion properties. Other proteins that probably associate with Ste20p are the S. cerevisiae 14-3-3 proteins, Bmh1p and Bmh2p (Roberts et al., 1997). The authors provide evidence that these proteins are required specifically for the transmission of the Ras2p dependent signal to the MAP kinase cascade during pseudohyphal differentiation. However, the significance of this finding and the exact role of these proteins during signal transduction are as yet unclear. 3.1.3.4. Cell cycle dependent regulation of filamentous growth. Most responses to extracellular signals and the associated physiological adaptations can either only be carried out at specific phases of the cell cycle or result in a direct change in cell cycle control. All cellular differentiation processes could be expected to influence or be influenced by the activity of cyclin-dependent kinases (CDK). The central regulatory CDK in S. cerevisiae is Cdc28p, whose activity determines the passage of most cell division checkpoints (reviewed in Mendenhall and Hodge, 1998). A second CDK, Pho85p, has some overlapping functions, and associates with a different set of cyclins (reviewed in Andrews and Measday, 1998). Several observations indicate that CDKs are important factors for the regulation and the co-ordination of morphogenesis and polarised growth (reviewed in Madden and Snyder, 1998). Association of Cdc28 with G1 phase cyclins Cln1 and Cln2 in particular is required for polarised growth during bud formation and growth, Overexpression of these two cyclins leads to hyperpolarised growth and buds with an elongated morphology. A similar morphology can be achieved by delaying or eliminating the expression of the G2 phase specific cyclins Clb1 and Clb2. First evidence for a more direct involvement of Cdc28-CDK in pseudohyphal differentiation emerged with the finding that cells growing in filaments displayed a modified cell cycle when compared to yeast-type cells. Kron et al. (1994) reported that filamentous daughter cells start budding very soon after cytokinesis. No size control, which normally applies during GI, seems to be implemented, and daughter and mother 120

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cell bud simultaneously. The G1 delay before Start apparently is eliminated. Furthermore, pseudohyphal cells have a significantly lengthened G2 phase, during which cell elongation occurs. Further indirect evidence was provided by the observation that mutations in some genes resulting in an increased expression of Cln cyclins showed hyperactivated invasive and pseudohyphal growth (Oehlen and Cross, 1998a). An example for such a gene is WH12, a deletion of which results in overexpression of cyclins Cln1p and Cln2p and a strong pseudohyphal development (Radcliffe et al., 1997). In a more recent paper, Edgington et al. (1999) suggest a direct link between CDK alterations and specific pseudohyphal differentiation phenotypes. A strain carrying a specific mutation in CDC28, cdc28-127, which results in the replacement of cysteine residue 127 of the kinase by tyrosine, displays most filamentous-form growth characteristics constitutively. These characteristics include elongated cell shape, bud site selection and cell cycle modifications leading to simultaneous bud emergence for mother and daughter cell. Genes involved in the regulation of Cdc28p activity include ELMI, encoding a serineithreonine protein kinase which activates another protein kinase, Hs11p. ELM1 was first identified through screening of mutagenised yeast for mutations that would result in constitutive cell elongation and other filamentation specific characteristics (Blacketer et al., 1993, 1994). Deletion of ELM1 results in constitutive filamentation. A second kinase-encoding gene, which results in a similar phenotype when mutated, is HSL1. Dominant mutations within HSL1 can suppress the constitutive filamentation phenotype of elm1 mutants, placing Hs11p downstream of Elm1p. Mutant phenotypes of both ∆elm1 and ∆hsl1 strains can, on the other hand, be suppressed by inactivation of Swe1p, a third protein kinase situated in the same pathway. The deletion of ELM1 or HSP1 results in hyperactive Swe1p, which triggers the appearance of some of the filamentous growth specific phenotypes. Mutations in ELMI, HSL1, or SWE1 have no influence on the phenotype of the cdc18-127 allele, suggesting that they act upstream of the kinase. The data are consistent with the existence of a protein kinase cascade that mediates filamentation by modulating Cdc28p activity. Surprisingly, however, the effects of the protein kinase cascade on filamentation seem to be limited to haploid cells. The pathway suggested by the authors is presented in Figure 4. It is not yet clear how this cell cycle regulating pathway interacts with the various other signal transduction pathways, which seem more specifically responsible for pseudohyphal differentiation. Based on a limited amount of genetic data, Edgington et al. (1999) suggest that the proteins could be acting downstream of the MAP kinase pathway (Figure 4). CDK-dependent regulation has been shown to affect other parts of the signal transduction pathway as well. Oehlen et al. (1998b) show that Cln2p-Cdc28p kinase represses the pheromone signalling pathway at the level of Ste20p. Since Cln-cyclins are required for pseudohyphal differentiation as well, a direct influence of the Cln2Cdc28 kinase on Ste20p during this event is equally likely. Similar data were obtained for Ste11p (Wassmann and Ammerer, 1997).

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Figure 4. Cell-cycle dependent regulation of pseudohyphal differentiation as suggested by Edgington et al. (1999). This pathway seems specific to haploid cells.

3.2. TRANSCRIPTIONAL REGULATORS Besides the direct modulation of the activity of pre-existing enzymes, the main molecular response to extracellular signals resides in the activation or repression of the transcription of specific target genes. It is therefore not surprising that a large number of genes whose deletion or overexpression affects the pseudohyphal differentiation phenotype encode transcriptional regulators. There are several obvious requirements for transcription factors to be active: It has to be localised in the nucleus, bind to DNA or associate with DNA-binding proteins, and has to be able to - directly or indirectly - interact with the basal transcription apparatus, the RNA polymerase holoenzyme. Each of these requirements, i.e. nuclear localisation, DNA binding or the interaction with other proteins, can be modulated by the extracellular signal (reviewed in Hill and Treisman, 1995). In this review, we shall focus separately on each of the transcription factors that have been shown to contribute to the filamentous growth phenotype. Several of these factors had previously been identified for their role in other, sometimes related, cellular processes, underlining again the interconnected nature of all signalling processes.

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3.2.1. Ste12p and Tec1 Combinatorial control contributes to MAP kinase specificity. Fields and Herskowitz (1985) identified Ste12p as being required for the expression of cell mating-type specific genes. In the following years, Ste12p was shown to bind to DNA sequences that mediate the pheromone response, so-called pheromone response elements or PRES (Dolan et al., 1989; Errede and Ammerer, 1989; Yuan and Fields, 1991). Additional data showed Ste 12p to be phosphorylated in a pheromone-dependent manner (Song et al., 1991). Ste12p was the first transcription factor that was shown to be required for normal filamentation by S. cerevisiae (Liu et al., 1993). Deletion of STE12 results in strains with strongly reduced ability to invade the agar or to grow filamentously, whereas the overexpression from an inducible GAL1 promoter results in enhanced pseudohyphal growth. Since mating and filamentation are mutually exclusive developmental pathways, it was suggested that additional factors had to be involved to impart signal-dependent specificity. TEC1 was initially isolated by Laloux et al. (1990) for being involved in the activation of Ty1-mediated gene expression. Gavrias et al. (1996) showed that the Aspergillus nidulans transcriptional activator AbaA, a gene homologous to TEC1, was able to induce pseudohyphal differentiation in S. cerevisiae. They consequently demonstrated that a deletion of TEC1 results in strongly reduced ability of yeast strains to invade agar. Initial epistasis data either indicated that Ste12p and Tec1p act in dependent pathways or interact with one another to activate downstream functions. The latter possibility was confirmed by Madhani and Fink (1997), who showed that Ste12p was directed to enhancer-elements (FREs for filamentation and invasion response elements) that were specifically induced in response to the MAP kinase-dependent filamentation signal. The FRE consists of a Ste12p binding site, similar to the one found in pheromone-dependent genes, closely associated with a Tec1p binding site. The binding of Ste12p and Tec1p to this element occurs in a co-operative manner, and the two proteins are found associated in the same, multimeric protein complex (Baur et al., 1997; Madhani and Fink, 1997). It is therefore the combination with Tec1p that allows Ste12p to activate filamentation specific genes. Furthermore, it has been shown that the unphosphorylated form of Kss1p, which has a repressive effect on filamentation, directly binds to Ste12p (Bardwell et al., 1998a), giving a further indication on the mechanism underlying specific activation. The two proteins Dig1p and Dig2p, which also associate with Ste12p, are required for this Kss1p dependent repression (Tedford et al., 1997; Bardwell et al., 1998b). 3.2.2. Msn 1p and Mss11p: Central elements in the pseudohyphal growth pathway MSN1 has first been identified as a multi-copy suppressor of snf1 mutants (MSN1) (Estruch and Carlson, 1990). Other authors cloned the same gene as FUP1, an enhancer of iron-limited growth of S. cerevisiae (Eide and Guarente, 1992), as PHD2, a multicopy inducer of pseudohyphal growth (Gimeno and Fink, 1994) and, in our laboratory, as MSS10, a multi-copy suppressor of the repression exerted by STA10 on the STA1-3 glucoamylase-encoding genes, involved in starch metabolism (Lambrechts et al., 1996b). MSN1 has been suggested to encode a transcriptional activator since multiple copies of the gene enhance the transcription of several genes, most of which are involved in nutrient utilisation. In addition, MSN1 has been shown to activate reporter 123

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gene expression if fused to the LexA DNA-binding domain (Estruch and Carlson, 1990). MSS11, like MSN1, was identified as a suppressor of the STA10 dependent phenotype and was shown to induce STA1-3 encoded glucoamylase expression when present on a 2µ plasmid (Webber et al., 1997). The protein displays homologies with a number of transcriptional activators and suppressors, such as S. cerevisiae Snf5p, Ssn6-/Cyc8p and Drosophila NTF-1, and in particular with Flo8p, a protein initially identified for activating genes involved in flocculation (Kobayashi et al., 1996). Both MSN1 and MSS11 strongly activate the expression of MUC1, a gene required for invasive growth and pseudohyphal differentiation, when present in multiple copies (Lambrechts et al., 1996a; Webber et al., 1997). Recent data by Gagiano et al. (1999) show that Mss11p is a central factor for the establishment of pseudohyphal and invasive growth patterns. Genetic evidence indicates that Mss11p is required for both Ras2p dependent, MAP kinase dependent and independent, signal transduction pathways. Genetic evidence for Msn1p would suggest that the protein acts in a pathway downstream of Ras2p, but independent of the MAP kinase cascade. Msn1p requires the presence of Msn11p in order to induce filamentation, whereas Mss11p is able to induce the pathway in the absence of Msn1p. Mss11p therefore seems to be situated downstream of Msn1p (Gagiano et al., 1999). 3.2.3. Sf11p, Sok2p and Flo8p: Factors depending on the CAMP dependent kinase Three transcriptional regulators apparently controlled by cAPK and regulating filamentation have been identified, i.e. Sf11p, Sok2p and Flo8p. SFL1 had initially been cloned as a suppressor of flocculation (Fujita et al., 1989) since disruption of the gene leads to strong, constitutive flocculation in most strains. Robertson and Fink (1998) showed that deletion of SFL1 also results in enhanced pseudohyphal and invasive growth. Sf11p was shown to associate specifically with Tpk2p, the cAMP dependent protein kinase that specifically activates pseudohyphal growth. Interestingly, Sf11p has also been shown to interact and associate with the Srb/mediator proteins, which are part of the RNA polymerase holoenzyme and are thought to be responsible for transcriptional repression (Song and Carlson, 1998). Tpk2p therefore has to inactivate Sf11p, which in turn would relieve the repressive effects of the Srb proteins on the RNA polymerase holoenzyme and allow transcription of flocculation and pseudohyphal growth specific genes. This mechanism would provide the first evidence for a direct interaction of the cAMP dependent kinase with factors associated with the RNA polymerase holoenzyme. A second putative transcriptional repressor that regulates filamentation is encoded by the SOK2 gene, which was cloned by Ward and Garret (1 994). The gene was isolated as a dosage-dependent suppressor of conditional growth defects of temperature-sensitive cAPK mutants. Ward et al. (1995) presented evidence for the involvement of Sok2p in cAMP dependent regulation of filamentous growth by Sok2p. Diploid sok2/sok2 mutants show enhanced pseudohyphal growth, indicating a repressive activity of Sok2p. Since Sok2p presents important homologies with the helix-loop-helix DNA-binding motive of Phd1p and other fungal regulators of morphogenetic processes (Stoldt et al., 1997), it most probably directly represses the expression of filamentation related genes. Unlike Sf11 p, which directly associates with 124

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Tpk2p, no such association has yet been found for Sok2p, and its direct regulation by cAPK has yet to be demonstrated. In addition, the ∆sok2 phenotype presents defects in the regulation of genes involved in a number of other cAMP dependent metabolic regulations, including glycogen accumulation (Ward et al., 1995). It therefore behaves like a general regulator of cAMP dependent cellular responses, and is not specific for the filamentation response. A third transcriptional regulator identified as acting downstream of the cAMP signal and which induces filamentation is Flo8p. Kobayashi et al. (1996) cloned FLO8 because of its ability to induce flocculation. The gene was later shown to be required for pseudohyphal growth (Liu et al., 1996). Interestingly, the authors presented evidence that a mutation within this gene was responsible for the inability of most laboratory strains of S. cerevisiae to undergo the dimorphic switch from yeast-type to filament-type cells. Through genetic evidence, Rupp et al. (1999) showed that Flo8p positively activated pseudohyphal differentiation and that this activation was cAMP dependent. The authors showed that Flo8p activates MUC1 by directly acting on several sequences within the promoter of this gene. 3.2.4. Other factors 3.2.4.1. Ash1p. Ash1p was first identified for its role in mating type switching, where it is responsible for the daughter cell specific repression of the HO endonuclease. ASH1 mRNA is localised asymmetrically, close to the tip of large budded cells that have undergone anaphase (Long et al., 1997; Takizawa et al., 1997). This polarised localisation explains the asymmetric distribution of Ash1p between mother and daughter cell and its daughter cell specific effect on mating type switching. Ash1p contains a zinc-finger-like domain, and binds therefore probably directly to DNA to regulate gene expression. Chandarlapaty and Errede (1998) presented data suggesting that Ash1p was required for pseudohyphal growth. Cells lacking the ASH1 gene did not form filaments, whereas cells with multiple copies of the gene showed increased filamentation. Similar observations were made with regard to invasive growth in haploids. Genetic evidence places Ash1p in a pathway independent of the MAP kinase cascade but downstream of Ras2p, similar to what was observed for Msn1p (Gagiano et al., 1999). 3.2.4.2. Phd1p. This gene was identified through the screening of a genomic library for genes that, when present on a multiple copy plasmid, would enhance pseudohyphal and invasive growth (Gimeno and Fink, 1994). Phd1p showed significant homology with transcriptional regulators. However, deletion of PHD1 does not affect the ability of S. cerevisiae to form pseudohyphae or to grow invasively, and its role in pseudohyphal differentiation is unclear. 3.3, EFFECTOR PROTEINS No gene has been unequivocally shown to be directly and specifically regulated by filamentation signal transduction pathways alone. This might seem surprising, considering the large number of transcriptional regulators, which have been shown to be 125

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required for these pathways, and which have been described in the previous section. However, these regulators have mostly been cloned for specific roles in processes that only have an indirect or not yet clearly understood connection with filamentation. For example, from the above it is clear that a phenomenon like flocculation in liquid media is closely linked to the filamentation response, and some factors like Flo8p are required for both. In addition, some obvious phenotypic links between both cellular responses exist. Both processes for example require modified cell adhesion properties, and both seem to occur in media with limited amounts of nutrients. However, differences and similarities between the two events have not yet been fully investigated. The same is true for signal-dependent processes of polarised growth during both mating and filamentation. The shared elements in both pathways might at least in part be involved in aspects of cellular polarisation, but it is as yet not understood how signal dependent specificity is imparted. The data presented in this review could suggest that it is the combination of a number of different factors in a specific context that leads to the implementation of the filamentation response. If this is the case, specific effector genes and proteins of the pseudohyphal response pathway should only respond to a specific, finely balanced combination of all the pathways and factors described above. The gene coming closest to the above expectation is MUC1/FLO11. 3.3.1. MUC1, a gene encoding a mucin-like protein subjected to complex transcriptional regulation MUC1 was first identified by Lambrechts et al. (1996a) as being required for pseudohyphal differentiation and invasive growth. The gene encodes a mammalian membrane-bound mucin homologue, with strong structural similarities to dominant flocculation proteins like Flo1p. The authors showed that deletion of MUC1 results in strains unable to invade agar or to form filaments, whereas overexpression results in stronger invasion and filamentation phenotypes. These data were later confirmed by Lo and Dranginis (1998), who had previously identified the same gene, named FLO11, as being involved in flocculation (Lo and Dranginis, 1996). The structure of Muc1p suggests that it is a GPI-anchored cell wall protein, whose N-terminal section probably reaches outside of the cell wall (Lambrechts et al., 1996a; Lo and Dranginis, 1998). Particular attention has been paid to the large promoter of MUC1, the largest promoter identified in S. cerevisiae. Lambrechts et al. (1996a) initially identified MUC1 through the strong sequence homology of this promoter area with the promoters of the glucoamylase-encoding STA1, 2 and 3 genes. The promoter has been shown to be responsive to a number of the transcriptional regulators described above, including Msn1p (Lambrechts et al. 1996a,b), Ste12p/Tec1p (Lo and Dranginis, 1998; Rupp et al., 1999), Mss11p (Gagiano et al., 1999), and Flo8p (Rupp et al., 1999). MUC1 therefore is regulated by both cAMP dependent and MAPK-dependent pathways, and could be the first filamentation-specific target gene. The role of Muc1p during pseudohyphal differentiation has not yet been unequivocally established. However, based on the structure of the protein and on its cell wall bound localisation, it is highly probable that it is involved in the regulation of cell-cell attachment. An alternative hypothesis is that MUC1 would be required for

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attachment of the cell to a substrate, a step that is thought to be required for efficient invasion. Both hypotheses are not mutually exclusive. 3.3.2. Starch degrading enzymes: a direct metabolic link The promoters of the S. cerevisiae STA1-3 genes, encoding starch degrading glucoamylases, are strongly homologous to the MUC1 promoter (Pretorius et al., 1986; Lambrechts et al., 1995, 1996a; reviewed in Vivier et al., 1997), and homology extends over the entire promoter area of more than 3 kb (Gagiano et al., 1999). Experimental data suggest that the genes are indeed coregulated to a large degree, since the STA genes were shown to respond to the factors controlling pseudohyphal differentiation, including elements of the MAP kinase cascade (Gagiano et al., 1999). The coregulation of starch degradation and pseudohyphal differentiation offers some interesting evolutionary insights. The STA gene family seems to be a relatively recent evolutionary acquisition of S. cerevisiae (Lambrechts et al., 1995). Arguments in favour of this hypothesis are the subtelomeric localisation of the genes, the very limited sequence divergence between the three STA loci, and the fact that they are only found in some strains of S. cerevisiae. MUC1, on the other hand, is found in all S. cerevisiae strains investigated (Carstens et al., 1998), and has a chromosomal position far removed from the telomers. The STA genes are therefore probably the product of a recombination combining the promoter and secretion signal sequence of MUC1 with the ORF of the internal glucoamylase-encoding SGA1 gene. The evolutionary selection of a filamentation-dependent promoter might offer a real selective advantage to the yeast, since it could result in an enhanced ability of S. cerevisiae to invade nutrient-rich compartments in nature. Glucoamylases will be specifically secreted at the growing tip of the elongated cell during filamentous growth, which could allow the digestion of polysaccharide-containing barriers (cell walls of plant cells) and consequent penetration of the filament into new compartments. 4. Scientific and industrial relevance S. cerevisiae probably is the scientifically best understood of all organisms. The functional mapping of the genome and of the entire proteom of this unicellular fungus will be finalised in the not so distant future, including a physical map of protein-protein interaction. Whether the complete molecular map will provide us with new insights into the secrets of life itself, a view some will dismiss as reductionist, or whether the additional knowledge will provide us with "just" a more in-depth understanding of the molecular machinery of living organisms, remains to be seen. Already, as pointed out in this review, several new concepts have largely been based on data obtained through the study of S. cerevisiae. One of these concepts is the modular nature of signal transduction pathways. A summarised representation of the different modules and factors involved in pseudohyphal differentiation is given in Figure 5. This figure illustrates that the response to the limited availability of essential nutrients is the result of a finely balanced combination of inputs from a number of independent signalling modules. An outstanding feature of the data presented in this figure is that they combine three of the major signalling systems that, previously, had 127

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been investigated for separate and specific purposes, namely (i) the cell cycle signal emanating from CDKs, (ii) the nutrient-linked cAMP signal and (iii) the MAPK signal which might be required for the cell-cell attachment and cell growth-polarisation processes. This combination of three major pathways again demonstrates that coordination of all cellular compartments and of most molecular processes underlies cellular differentiation processes.

Figure 5. Summarised signal transduction network of the pseudohyphal growth pathway.

A different perspective can be taken by simply considering the initial phenotypes, which were used to identify most of the genes mentioned in this review. Some of the genes were initially identified for their role in the pheromone response, other for phenotypes linked to flocculation, abnormal cell cycle, abnormal stationary phase entry, starch degradation, ammonium transport, etc., again demonstrating the interconnected nature of apparently unlinked events. How does this fundamental understanding of complex processes impact on the potential use of yeast in industrial processes? Traditionally, S. cerevisiae has been used for several types of food and beverage fermentations and for the production of alcohol. For these traditional uses, industrial yeast has been optimised through centuries of mostly unaware selection by winemakers, brewers, bakers and other users. This unconscious optimisation process has lead to a number of S. cerevisiae strains specifically adapted for specific tasks, be it baking, brewing , winemaking or any other industrial use. However, from an industrial perspective, every single aspect of yeast physiology could still be optimised. Fermentation efficiency, alcohol resistance, general stress resistance, production of specific metabolites without production of additional, unwanted substances - there probably are as many potential targets for strain improvement as there are industrial users. It is in this field of strain improvement that 128

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recombinant DNA technology and metabolic engineering can provide its most important contribution. How can research of the type described in this review contribute to achieve such strain improvement targets? The answer becomes clear when looking at the results obtained with recombinant and metabolically engineered strains. These strains can frequently meet a specific target, be it a higher production of a metabolite (e.g. glycerol) or of extracellular proteins and enzymes. However, these strains have just as frequently failed to make an industrial impact. One reason for this failure can be found in the review above, and resides in the interconnection of all metabolic and signalling pathways. This interdependence makes it extremely difficult to achieve specifically improved yeast without modifying several other parameters. These secondary modifications frequently result in undesirable by-products or have a negative influence on general yeast physiology. It is therefore essential that we better understand the underlying network of metabolic and signalling connections, which would allow us to design mechanisms to avoid metabolic and physiological problems. Such integrated approaches, which will take into account not only the specific parameter to be modified, but also all potential consequences on associated or linked pathways, should result in modified yeast with much higher industrial potential. Besides this general argument for the scientific and industrial usefulness of the research outlined in this review, there are several potential industrial benefits directly associated with the study of pseudohyphal development. Some wanted -or unwantedproperties of industrial yeast strains are indeed directly linked to this pathway. The aspect retaining most attention is flocculation, which refers to the clumping of cells in liquid media and to their consequent sedimentation (reviewed in Stratford, 1992). Flocculation is due to the modification of cell-cell adhesion properties, and is of importance in several industrial processes, for example during brewing or the production of sparkling wine (reviewed in Hammond, 1995). The signal transduction pathways leading to flocculation is similar, if not identical, to the filamentation pathway, as can be seen by the number of genes shared by both (reviewed in Kron, 1997). Understanding filamentation should therefore allow us to design yeast with specific flocculation abilities. Other direct applications might reside in the development of new top-fermenting brewing and sherry yeast. Indeed, the ability of flor yeast to float on the surface of the substrate (top-ferment) is thought to be due to a modified implementation of the signal transduction pathways described in this review. Acknowledgements We thank the National Research Foundation (NRF) and the South African wine industry (Winetech) for funding of our research. References Andrews, B. and Measday, V. (1998) The cyclin family of budding yeast: abundant use of a good idea. Trends Genet. 14: 66-72.

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F.F. BAUER AND I.S. PRETORIUS Lo, W.-S. and Dranginis, A. M. (1998) The cell surface flocculin Flo11 is required for pseudohyphae formation and invasion by Saccharomyces cerevisiae. Mol. Biol. Cell. 9: 161-171. Long, R. M., Singer, R. H., Meng, X., Gonzalez, I., Nasmyth, K., and Jansen, R. P. (1997) Mating type switching in yeast controlled by asymmetric localisation of ASH1 mRNA. Science 277: 383-387. Lorenz, M. C. and Heitman, J. (1998a) Regulators of pseudohyphal differentiation in Saccharomyces cerevisiae identified through multi-copy suppressor analysis in ammonium permease mutant strains. Genetics 150: 1443-1457. Lorenz, M. C. and Heitman, J. (1998b) The MEP2 ammonium permease regulates pseudohyphal differentiation in Saccharomyces cerevisiae. EMBO J. 17: 1236-1247. Lorenz, M. C. and Heitman, J. (1997) Yeast pseudohyphal growth is regulated by GPA2, a G-protein α homologue. EMBO J. 16: 7008-7018. Madden, K. and Snyder, M. (1998) Cell polarity and morphogenesis in budding yeast. Annu. Rev. Microbiol. 52: 687-744. Madhani, H. D. and Fink, G. R. (1998) The riddle of MAP kinase signalling specificity. Trends Genet. 14: 151-155. Madhani, H. D., Styles, C. A., and Fink, G. R. (1997) MAP kinases with distinct inhibitory functions impart signalling specificity during yeast differentiation. Cell 91: 673-684. Marini, A. -M., Soussi-Boudekou, S., Vissers, S. and Andre, B. (1997) A family of ammonium transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 17: 4282-4293. Matsumoto, K., Uno, I., Oshima, Y., and Ishikawa, T. (1982) Isolation and characterisation of yeast mutants deficient in adenylate cyclase and CAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 79: 2355-2359. Mendenhall, M. D., and Hodge, A. E. (1998) Regulation of Cdc28 cyclin-dependent protein kinase activity during the cell cycle of the yeast Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 62: 1191-1243. Mösch, H. -U. and Fink, G. R. (1997) Dissection of filamentous growth by transposon mutagenesis in Saccharomyces cerevisiae. Genetics 145: 671-684. Mösch, H. -U., Roberts, R. L., and Fink, G. R. (1996) Ras2 signals via the Cdc42/Ste20/mitogen activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 93: 5352-5356. Nakafuku, M., Obara, T., Kaibuchi, K., Miyajima, I., Miyajima, A., Itoh, H., Nakamura, S., Arai, K. Matsumoto, K., and Kaziro, Y. (1988) Isolation of a second yeast Saccharomyces cerevisiae gene (GPA2) coding for guanine nucleotide-binding regulatory protein: Studies of its structure and possible functions. Proc. Natl. Acad. Sci. USA 85: 1374-1378. Neer, E. J. (1995) Heterotrimeric G proteins: organisers of transmembrane signals. Cell 80: 249-257. Oehlen, L. and Cross, F. R. (1998a) The mating factor response pathway regulates transcription of TEC1, a gene involved in pseudohyphal differentiation of Saccharomyces cerevisiae. FEBS Lett. 429: 83-88. Oehlen, L. and Cross, F. R. (1998b) Potential regulation of Ste20 function by the Cln1-Cdc28 and Cln2-Cdc28 cyclin-dependent protein kinases. J. Biol. Chem. 273: 25089-25097. Ozcan, S., Dover, J., and Johnston, M. (1998) Glucose sensing and signalling by two glucose receptors in the yeast Saccharomyces cerevisiae. EMBO J. 17: 2566-2573. Papasavvas, S., Arkinstall, S., Reid, J., and Payton, M. (1992) Yeast alpha-mating factor receptor and Gprotein-linked adenylyl cyclase inhibition requires RAS2 and GPA2 activities. Biochem. Biophys. Res. Commun. 184: 1378-85. Pawson, T. (1995) Protein modules and signalling networks. Nature 373: 573-580. Peter, M., Neiman, A. M., Park, H. O., van Lohuizen, M., and Herskowitz, I. (1996) Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast. EMBO J. 15: 7046-7059. Pretorius, I. S., Chow, T., Modena, D., and Marmur, J. (1986) Molecular cloning and characterisation of the STA2 glucoamylase gene of Saccharomyces cerevisiae. Mol. Gen. Genet. 203: 29-35. Posas, F. and Saito, H. (1997) Osmotic activation of the HOG MAPK pathway via Ste11p MAPKKK: scaffold role of Pbs2p MAPKK. Science 276: 1702-1705. Radcliffe, P. A., Binley, K. M., Trevethick, J., Hall, M., and Sudbery, P. E. (1997) Filamentous growth of the budding yeast Saccharomyces cerevisiae induced by overexpression of the WH12 gene. Microbiol. 143: 1867-1876. Ramezani, R. M., Jansen, G., Buhring, F., and Hollenberg, C. P. (1998) Ste50p is involved in regulating filamentous growth in the yeast Saccharomyces cerevisiae and associates with Ste1 1p. Mol. Gen. Genet. 259: 29-38.

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PSEUDOHYPHAL AND INVASIVE GROWTH IN SACCHAROMYCES CEREVISIAE Roberts, R. L. and Fink, G. R. (1994) Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev. 8: 2974-2985. Roberts, R. L., Mösch, H. U., and Fink, G. R. (1997) 14-3-3 proteins are essential for RAS/MAPK cascade signalling during pseudohyphal development in S. cerevisiae. Cell 89: 1055-1065. Robertson, L. S. and Fink, G. R. (1998) The three yeast A kinases have specific signalling functions in pseudohyphal growth. Proc. Natl. Acad. Sci. USA 95: 13783-13787. Rupp, S., Summers, E., Lo, H. J., Madhani, H., and Fink, G. (1999) MAP kinase and cAMP filamentation signalling pathways converge on the unusually large promoter of the yeast FLO1 1 gene. EMBO J. 18: 1257-1269. Song, D., Dolan, J. W., Yuan, Y. L., and Fields, S. (1991) Pheromone-dependent phosphorylation of the yeast Ste12 protein correlates with transcriptional activation. Genes Dev. 5: 741 -750 Song, W. and Carlson, M. (1998) Srb/mediator proteins interact functionally and physically with transcriptional repressor Sf11. EMBO J. 17: 5757-5765. Stoldt, V. R., Sonnebom, A., Leuker, C. E., and Ernst, J. F. (1997) Efg1p, an essential regulator of morphogenesis of the human pathogen Candida albicans, is a member of a conserved class of bHLH proteins regulating morphogenetic processes in fungi. EMBO J. 16: 1982-1991. Stratford, M. (1992) Yeast flocculation: a new perspective. Adv. Microb. Physiol. 33: 1-71. Takizawa, P. A., Sil, A., Swedlow, J. R., Herskowitz, I., and Vale, R. D. (1997) Actin-dependent localisation of an RNA encoding a cell-fate determinant in yeast. Nature 389: 90-93. Tedford, K., Kim, S., Sa, D., Stevens, K., and Tyers, M. (1997) Regulation of the mating pheromone and invasive growth responses in yeast by two MAP kinase substrates. Curr. Biol. 7: 228-238. Thevelein, J.-M. (1992) The RAS-adenylate cyclase pathway and cell cycle control in Saccharomyces cerevisiae. Antonie Van Leeuwenhoek 62: 109-130. Toda, T., Cameron, S., Sass, P., Zoller, M., and Wigler, M. (1987) Three different genes in S. cerevisiae encode the catalytic subunits of the CAMP-dependent protein kinase. Cell 50: 277-287. Vivier, M. A., Lambrechts, M. G., and Pretorius, I. S. (1997) Co-regulation of starch degradation and dimorphism in the yeast Saccharomyces cerevisiae. Crit. Rev. Biochem. Mol. Biol. 32: 405-435. Ward, M. P. and Garrett, S. (1994) Suppression of a yeast cyclic AMP-dependent protein kinase defect by overexpression of SOK1, a yeast gene exhibiting sequence similarity to a developmentally regulated mouse gene. Mol. Cell. Biol. 14: 5619-5627. Ward, M. P., Gimeno, C. J., Fink, G. R., and Garrett, S. (1995) SOK2 may regulate cyclic AMP-dependent protein kinase-stimulated growth and pseudohyphal development by repressing transcription. Mol. Cell, Biol. 15: 6854-6863. Wassmann, K. and Ammerer, G. (1997) Overexpression of the G1-cyclin gene CLN2 represses the mating pathway in Saccharomyces cerevisiae at the level of MEKK Ste11. J. Biol. Chem. 272: 13180-13188. Webber, A. L., Lambrechts, M. G., and Pretorius, I. S. (1997) MSS11, a novel yeast gene involved in the regulation of starch metabolism. Curr. Genet. 32: 260-266. Widmann, C., Gibson, S., Jarpe, M. B., and Johnson G. L. (1999) Mitogen-activated protein kinase: conservation of a three-kinase module from yeast to human. Physiol. Rev. 79:143-180. Wilkie, T. M. and Yokoyama, S. (1994) Evolution of the G protein alpha subunit multigene family. Soc. Gen. Physiol. Ser. 49: 249-270. Xue, Y., Batlle, M., and Hirsch, J. P. (1998) GPR1 encodes a putative G protein-coupled receptor that associates with the Gpa2p G-alpha subunit and functions in a Ras-independent pathway. EMBO J. 17: 1996-2007. Yuan, Y. L. and Fields, S. (1991) Properties of the DNA-binding domain of the Saccharomyces cerevisiae Ste12 protein. Mol. Cell. Biol. 11: 5910-5918. Yun, C.W., Tamaki, H., Nakayama, R., Yamamoto, K., and Kumagai, H. (1998) Gpr1p, a putative G-protein coupled receptor, regulates glucose-dependent cellular CAMP level in yeast Saccharomyces cerevisiae. Biochem. Biophys. Res. Commun. 252: 29-33

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MICROBIAL PRODUCTION OF THE BIODEGRADABLE POLYESTER POLY-3-HYDROXYBUTYRATE (PHB) FROM AZOTOBACTER CHROOCOCCUM 6B: RELATION BETWEEN PHB MOLECULAR WEIGHT, THERMAL STABILITY AND TENSILE STRENGTH QUAGLIANO JAVIER C. AND MIYAZAKI SILVIA S Facultad de Agronomía, Universidad de Buenos Aires. Av. San Martin 4453 (141 7) Bs.As. Argentina.E-mail:quanliano@,ifeva.edu.ar

Abstract PHB with a high molecular weight (Mw) was obtained from fermentor cultures of Azotobacter chroococcum 6B. Mw was significantly affected by aeration rate, being reduced from 2900 to 110 kDa at 0.25 and 2.5 vvm, respectively. Temperature at which maximal weight loss was obtained (decomposition temperature) markedly depended on Mw , obtaining a 40°C span between 100 and 1400 kDa. Tensile strength reached a value of 215 MPa at a Mw of 1240 kDa, being reduced to nearly 40 MPa at the lower tested Mw of 445 kDa. 1. Materials and methods 1.1 MICROORGANISM AND CULTURE MEDIA Azotobacter chroococcum 6B was isolated from rhizospheric soil samples of the Agronomy Faculty Campus, Buenos Aires, Argentina. Modified productive Burk's medium (Bm) was utilised, with glucose as carbon source (g l-1): glucose 10, MgSO4.7H2O 0.4, FeSO4.7H2O 0.012, Na2MoO4.12H2O 0.01, K2HPO4 2, NaCl 0.4 and CaCO3 0.25. (NH4)2SO4 was added at a concentration of 0.1 g l-1 and pH was adjusted to pH 7.0 with HCl 1N. Stock cultures were maintained at 4°C by periodical transfer on modified productive Burk's medium agar slants. 1.2 FERMENTOR EXPERIMENTS Batch fermentations were conducted with a 1% (v/v) inoculum of a 36 hours culture in the same medium and the working volume was 4 litres (Bioflo I, New Brunswick Sci. Co, USA; Gallemkamp Sanyo, England), both fermentors equipped with two-six blade 135 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 135–140. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

QUAGLIANO JAVIER C. AND MIYAZAKI SILVIA S.

impellers and baffles. Temperature was controlled at 30°C and initial pH was adjusted to 7.0. The air flow rate was set between 0.25 and 2.5 vvm, Initial C/N ratio (molar basis) was set between 7.3 and 138 (ammonium sulphate limitation and glucose excess) and agitation speed was 200 rpm. Batch experiments were finished at 72 h. When molasses Bm medium was utilised, glucose was replaced by sugar cane molasses (5% w/v) (Tucumán, Argentina) as carbon source. 1.3 EXTRACTION AND PURIFICATION PROCEDURE Lyophilised cells were extracted with chloroform during 24 hours in a Soxhlet apparatus. Chloroform extract (1 50-250 ml) was concentrated by simple distillation to a final volume of 25 ml. The concentrated PHB was reprecipitated in ethanol cooled in an ice bath. Reprecipitated biopolymer was filtered, washed with acetone and centrifuged (1 5 min, 5000 rpm). Purified PHB was dried at 60°C until it reached constant weight. 1.4 ANALYTICAL METHODS Cell growth was monitored by measuring the optical density at 610 nm. Cell dry weight (cdw) was measured by weighing lyophilised cells harvested from 3 ml culture broth. Residual glucose in the broth was determined by the enzymatic method using glucose oxidase/peroxidase (Wiener Lab., Rosario, Argentina). PHB content and its composition were determined by gas chromatography using 3HB standards (Braunegg et al, 1978). Mw was measured viscosimetrically and by gel permeation chromatography (GPC) using five serial Styragel columns of 102, 103, 104, 105 and 106A. Flow rate of chloroform was 0.5 ml/min. A refractive index detector was used.. Differential scanning calorimetry (DSC) studies were performed using a Mettler TA9000 calorimeter between -60 and 250°C at 10°C/min. Thermogravimetric (TG) measurements were made with a Mettler TA9900 apparatus from 25 to 800°C at 10°C/min. Tensile strength was measured on PHB probes (obtained according to ASTM 1708-84) utilising an Instron model 1122 tensiometer from slopes of strain-stress curves 2. Results and discussion 2.1 EFFECT OF MW ON PHB THERMAL STABILITY PHB samples with different Mw obtained at different fermentative conditions were analysed by measuring the temperature at which maximal weight loss is obtained when increasing sample temperature in thermogravimetric studies, called decomposition temperature, Td . This parameter is a measure of the thermal stability of PHB. Other physical parameters, such as melting temperature (Tm), glass transition temperature (Tg) and % crystallinity were also analysed. Table 1 summarises the data obtained: it was shown that an increase in Mw leads to an augmented thermal stability, as samples with a Mw around 1300-1400 kDa lost 99% of their weight at a temperature 15% higher than the sample with the lowest Mw experimented (150 kDa). These results showed that thermal stability is significantly affected by Mw. Tm was not significantly affected, as expected, as it is a physical constant independent of Mw . Tg did not showed significant 136

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differences among the analysed samples, confirming that PHB is a semicrystalline polymer, while % crystallinity was in the range of 50-90%, as generally reported for PHB (Barham et al. 1984) the differences attributed to different degrees of sample annealing. Table 1. Melting point (Tm), glass transition temperature (Tg), decomposition temperature (Td) and molecular weight (Mw) of PHB samples obtained from strain 6B.

Culture conditions

Tm (°C)

Tg (°C)

Td (ºC) (a)

%crystallin ity

Mw. 103

(kDa fixation, 0.5 vvm, 100 rpm

180.9

n.d.

291

59

1420

C/N 7.3, 0.5 vvm, 100 rpm

177.8

3.9

268

82

1330

C/N 29,0.5 vvm, 100 rpm

178.8

4.3

261.7

73

1170

molasses 5%, 0.5 vvm, 100

173.9

n.d.

286

54

1420

C/N 138,2 vvm, 400 rpm

173.0

n.d.

250

55

150

C/N 69,2.5 vvm, 100 rpm

178.2

5.7

n.d.

62

110

rpm

n.d.: not determined. Tg , Tm and % crystallinity obtained from DSC scans. % crystallinity=∆ Hm pHB (J/g°C)/∆ HmpHB100%crystalline a: Td:temperaturewheremaximal weight loss is obtained. 2.2 EFFECT OF AERATION RATE ON PHB MW PHB Mw was significantly affected by aeration rate under fermentor cultivation at a C/N ratio of 69. Figure 1 depicts the influence of aeration rate on PHB Mw: maximum Mw (2.900 kDa) was obtained at the lowest aeration rate experimented of 0.25 vvm, while when increasing aeration rate Mw diminished to 110 kDa at 2.5 vvm. This confirms that PHB is accumulated under oxygen-deficient conditions, then in such conditions 3hydroxybutyrate monomers are incorporated to the growing PHB chains, consequently increasing Mw. An increased Mw leads to an improvement of polymer performance, as lower Mw are not desirable from the point of view of plastics applications, for example, thermal stability is much lower with low Mw plastics. Also a product of low molecular mass suffer a sensitive loss of volatile degradation products as temperature is increased (Scandola et al. 1988).

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QUAGLIANO JAVIER C. AND MIYAZAKI SILVIA S.

Figure 1. PHB molecular weight (Mw) and PHB content (XPHB) under different aeration rates under fermentor culture (4 litres) with Burk’s modified medium (Bm) at a C/N ratio of 69 (mol glucose/mol ammonium sulphate) at 72 h culture. : Mw, o : XPHB (% dry weight).

2.3 PHB TENSILE STRENGTH (σ) AT DIFFERENT Mw In an attempt to know how Mw influenced PHB physical properties of mechanical resistance, tensile strength (σ) was measured on PHB samples. These results, summarised in Table 2, showed that σ increased as Mw augmented to 1240 kDa, probably as a result of the increased intermolecular forces between PHB chains on higher Mw samples. These results indicated that PHB mechanical resistance depended significantly on Mw. Table 2. PHB tensile strength

of samples with different molecular weight (Mw)

σ (MPa) 38 113 215

Mw (kDa) 445 1000 1240 2.4 PHB AS A MATRIX FOR MICROENCAPSULATION

Applications as a matrix for microencapsulating an enzyme were developed for PHB obtained from strain 6B. Cellulase was microencapsulated in PHB of three different Mw. Figure 2 showed that a Mw of 1000 kDa was the best for maintaining a higher relative cellulase activity (60%) with respect to the PHB samples of 335 and 1350 kDa., 138

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suggesting that PHB of 1000 kDa creates the most convenient pore size and microenvironment for cellulase retention and activity.

Figure 2. Relative activity of cellulase microencapsulated in PHB at different cellulase/PHB ratios. Left to right: PHB with Mw 1020, 335 and 1350 kDa.

3. Conclusions From the above results, we concluded that aeration rate should be carefully controlled under fermentor cultivation of strain 6B for PHB production, as Mw is markedly affected by dissolved oxygen tension, and consequently by aeration rate. PHB with Mw higher than 1200-1300 kDa are desirable for obtaining a bioplastic with both high tensile strength and thermal stability, in order to be successfully processed in industrial applications. References Anderson A.J. and Dawes E.A. 1990. Occurrence, metabolism, metabolic role and industrial uses of bacterial polyhydroxyalkanoates. Microbiological Reviews 54, 4: 50-472. Barham P., Keller A., Otun E. and Holmes P. 1984. Crystallisation and morphology of a bacterial thermoplastic: poly-β-hydroxybutyrate. J. Mater. Sci. 19: 2781 -2794. Braunegg G., Sonnleitner B. y Lafferty R. 1978. A rapid gas chromatographic method of the determination of Poly-6-hydroxybutyric acid in microbial biomass. European J. Appl. Microbiol. Biotechnol. 6:29-37. Quagliano J. y Miyazaki S. 1997. Effect of aeration and carbon/nitrogen ratio on the molecular mass of the biodegradable polymer poly-β-hydroxybutyrate obtained from Azotobacter chroococcum 6B. Appl. Microbiol. Biotechnol. 47: 662-664.

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QUAGLIANO JAVIER C. AND MIYAZAKI SILVIA S. Scandola M., Pizzoli H. y Ceccorulli G. 1988. Viscoelastic and thermal properties of bacterial poly-βhydroxybutyrate. Int. J. Biol. Macromol., 10: 373-377.

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SHARING OF NUTRITIONAL RESOURCES IN BACTERIAL COMMUNITIES DETERMINED BY ISOTOPIC RATIO MASS SPECTROMETRY OF BIOMARKERS WOLF-RAINER ABRAHAM, CHRISTIAN HESSE, OLIVER PELZA, STEFANIE HERMANN, MICHAEL TESARB, EDWARD R. B. MOORE, AND KENNETH N. TIMMIS GBF - National Research Centre for Biotechnology, Dept. Environmental Microbiology, Mascheroder Weg 1, D-38124 Braunschweig, Germany;a present address: ETH Zurich, Institute of Terrestrial Ecology, Schlieren, Switzerland; b present address: MorphoSys AG, Martinsried/München, Germany

1. Introduction One of the fundamental characteristics of the microbial world is its inherent metabolic diversity. An extraordinary physiological flexibility, achieved during, at least, 3.5 billion years (Doolittle et al., 1996; Feng et al., 1997; Pace, 1997), allows microorganisms not only to colonise all habitats which are inhabited by eucaryotes but also to exploit a wide spectrum of extreme biotopes (e. g., hot springs, salt and soda lakes, highly-polluted waste waters, etc.) which are inhospitable to higher organisms. The ubiquitous distribution of bacteria coincides with the observation that microorganisms, and their natural communities, are of fundamental importance for the global cycling of organic matter (Pomeroy & Wiebe, 1988). The metabolic diversity is reflected in the species diversity and, particularly, in the large evolutionary distances observed between the main phylogenetic lineages of microorganisms (Woese, 1987). It is currently estimated that probably only 1 % to 10 % of the microorganisms living in the biosphere are known (i. e., have been isolated and characterised) (Bull et al., 1992). Moreover, one should recognise that the vast majority of microorganisms present in environmental samples currently cannot be cultivated in the laboratory. An important question concerning the cultivable microorganisms arises as to their actual frequencies and significance in the ecosystem (Amann et al., 1995). The problem is further complicated due to the fact that, in nature microorganisms live in complex communities whose compositions vary depending on the environmental conditions. The activities of individual members of such communities are strongly influenced by the prevailing conditions in the environment as well as the activities of the other members (Roszak & Colwell, 1987). The structure and dynamics of natural microbial communities, i. e. the 143 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 143–154. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

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species compositions, interactions and activities over time and space, is poorly understood. This is in marked contrast to the wealth of knowledge about the community structure and dynamics of higher organisms like plants and animals. Due to the problem of non-culturability of the majority of the bacteria, traditional, cultivation-based methods of microbiology are often of very limited value (Staley, 1980; Atlas, 1984; Brock, 1987; Hugenholtz et al., 1998). Rather it is important to develop new experimental approaches, which do not need cultivation in order to clarify the relations between structure and function of microbial communities. Several approaches have been proposed to tackle the basic problems of determining microbial community structure, most of them based on the molecular analysis of ribosomal RNA or rDNA (Pace et al., 1986; Hugenholtz & Pace, 1996; Pace, 1996) and some on the analysis of polar lipids (Tunlid & White, 1992). There is an urgent need for the elucidation of the critical interactions taking place in microbial communities which determine important physiological activities, in order to be able to develop a knowledge-based strategy for the optimisation and control of biotechnological applications in the environment, and to control their safety. To achieve this long-term goal a novel approach was developed the analysis of biomarkers by isotope ratio mass spectrometry (IRMS) - for tracking the flux of carbon within microbial consortia. For this approach two components are needed: i) biomarkers which are specific for bacterial taxa, i. e. genera, families, phyla, and ii) a calibration of the degree of isotopic fractionation during the microbial degradation of the substrates and the incorporation of their carbons into the biomarkers. 2. Taxon specific biomarkers 2.1. POLAR LIPIDS Polar lipids exhibit large structural diversity, an attribute that enables them to be used as chemotaxonomic criteria. Some lipids are unique to certain taxa, whereas the relative concentrations of lipids within a taxon provides additional taxonomic resolving power. The taxonomic utility of lipids is proportional to the quality of the structural information that is obtained, Hence, in addition to conventional methods of lipid analysis (e.g. 2dimensional thin layer chromatography with selective staining and chemical cleavage, derivatisation and gas chromatographic (mass spectrometric, GC-MS) analysis of the component parts), considerable effort is currently being invested in detection/structural characterisation of lipids (e.g. Fourier transformed infrared spectrometry (FT-IR), dynamic chemical ionisation and fast atom bombardment (tandem) mass spectrometry (DCI- and FAB-(MS/)MS), two-dimensional nuclear magnetic resonance spectroscopy (1H and 13C NMR)). Because of novel technologies it is more and more possible to analyse intact lipids. Additional to the already established chromatography mainly the Tandem-MS is used for the analysis of intact lipids, During the last years we applied this unit successfully for the study of several lipid mixtures and the structure elucidation of their components. About 200 glyco-, sulfo- and phospholipids were identified for the first time as natural products and their structures characterised. Furthermore it was possible to find

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characteristic biomarkers specific to groups of bacteria and to elucidate their structures Figure 1) (Abraham et al., 1997; Niepel et al., 1997; Abraham et al., 1998b). 2.2. OUTER MEMBRANE PROTEINS The application of a newly developed technique (Tesar et al., 1996) allowed us to identify taxa specific outer membrane proteins. Polyclonal sera were developed for the identification of group-, respectively species-specific antigens against various representatives of Pseudomonades and the antisera obtained were further characterised. Here it was important to find out which sera's were active against a wide spectrum of antigens within the genus Pseudomonas. The basis were 23 different species of Pseudomonas which already have been phylogenetically characterised by their 16S rRNA sequences. With an immunological method called "Westprinting" characteristic pattern of protein bands have been generated for individual strain, which included group-specific as well as species-specific features. All tested Pseudomonas sp. revealed two dominant protein bands in the range of 8 and 18 kDa, which can be used as a genusspecific marker. In combination with the higher molecular proteins it is possible to make statements about the species, respectively subspecies. A cluster analysis based on the similarity in the protein profile revealed 5 larger arrangements (around the species P. aeruginosa, P. oleovorans, P. aureofaciens, P. syringae and P. fluorescens), which are also seen on the level of the 16S rRNA sequence analysis. To test whether these results are transferable to other strains of a species 28 different environmental isolates, which were identified as pseudomonades with the Biolog method were tested and correctly identified in agreement with other methods. Meanwhile this technique was successfully applied to other genera as well. Biomarker are not only characteristic for taxa of bacteria and can be used for their identification, the isotopic composition of the biomarkers also provide information about the substrates of these taxa. We understand the term biomarkers as chemical compounds which are characteristic for bacterial taxa. Although the chemical nature of a biomarker cannot be limited to a chemical class we focused our studies to polar lipids and outer membrane proteins and to some extent also to nucleic acids.

Figure 1: Polar lipids useful as biomarkers of bacterial taxa.

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Figure 1: Continued

3. Isotopic fractionation in microorganisms Using fatty acids this approach was calibrated in pure culture studies in order to investigate the relationship between the isotopic composition of certain fatty acids and that of the bacterial diet. In this manner representative strains of bacteria and fungi grown in a minimal medium with different carbon sources with known isotopic ratios were examined. Looking at stable carbon isotopic ratios of palmitic acid which is common in all higher organisms it was found that all microorganisms vary in their incorporation of 13C from the substrate into this fatty acid. The rates of isotopic discrimination in the various substrates and fractions of polar lipids were partially found to be very different. Especially interesting was a high depletion of 13C of all microorganisms grown on glucose and in contrast to that the high ranges of isotopic discrimination of the different organisms grown on mannose. In most cases a significant 146

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difference in the isotopic ratio between palmitic acid extracted from glycolipids and that from phospholipids was observed (Abraham et al., 1998a). The 13C of bacterial biomass is a parameter in microbial ecology gaining increasing interest (Boschker et al., 1999). It is used for the identification of substrate utilisation of bacteria, relying on the observation that bacteria grown on substrates with known isotopic values produce biomasses which differ in their isotopic ratio of only 2 λ from those of their substrates. Our studies determined isotopic differences of palmitic acid of up to 7 λ, both depletion and enrichment (Abraham et al., 1998a). We found a much larger range of isotopic fractionation for amino acids of up to 80 λ. These ranges were dependent on the biomarker and the substrate, but also on the bacterial species. From calibration studies in pure cultures we could derive some rules controlling the depletion or enrichment of carbon isotopes in the individual biomarkers. This basic new discoveries of the isotopic discrimination of individual genera of bacteria and fungi may have a great potential to study the crucial microbial catalysts of the heterotrophic dominant part of the carbon cycle in natural ecosystems. 4. Carbon sharing in a pollutant degrading bacterial community 4.1. ORIGIN AND CHARACTERISTICS OF THE MICROBIAL CONSORTIUM From Spittelwasser sediment, a stable bacterial consortium was established growing on 4-chlorosalicylate, an intermediate in the aerobic degradation of 3-chlorodibenzofurane and 2-chloronaphthalene. The small river Spittelwasser, a tributary of the Elbe river system near Bitterfeld (in the state of Sachsen-Anhalt, Germany), has served for decades as an outflow of untreated waste water from the chemical industries concentrated in this area. Until the recent shut-down of most of these factories, the river did not contain any detectable eucaryotes but a very broad spectrum of different procaryotes (Mau & Timmis, 1998). The consortium was grown in a chemostat and fed with 5 mM 4-chlorosalicylate as the sole source of carbon and energy, with a dilution rate of 0.16 d-1 and 12°C (Faude, 1995). The consortium consisted of four different bacterial strains identified by 16S rDNA sequence analysis as Pseudomonas sp. MT1, Empedobacter sp. MT2, Alcaligenes sp. MT3, and Pseudomonas sp. MT4 (Moore et al., 1996). To understand the interactions of the bacteria within the consortium it was necessary not only to identify the different bacteria but also to determine their relative abundance in the consortium and to monitor them over time. To achieve this goal rabbit antisera have been produced against all four isolates for immunochemical analysis of the microbial population and have been tested by different immunological methods. Applying these antibodies in the immunofluorescence the relative abundance's of the strains in the chemostat were determined to 84 ∀ 3 % for Pseudomonas sp. MT1, 1 % for Empedobacter sp. MT2, 8 ∀ 4 % for Alcaligenes sp. MT3 and 8 ∀ 4 % for Pseudomonas sp. MT4. Furthermore, it was found that the composition of the mixed culture remained constant over a time period of at least 3 years. The predominant strain in this consortium, Pseudomonas sp. MT1, grew on 4chlorosalicylate alone tolerating up to 1 mM, above of which cell death occurred. 147

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Incubating Pseudomonas sp. MT1 in a chemostat under conditions of the consortium chemostat led to incomplete mineralisation of 4-chlorosalicylate and the excretion of degradation metabolites into the culture medium. The accumulation of 4-chlorocatechol, 3-chloro-cis,cis-muconate, and protoanemonin was detected. Protoanemonin, first isolated from Ranunculaceae (Seegal & Holden, 1945; Didry et al., 1991) and later observed as a dead-end metabolite of 4-chlorocatechol (Blasco et al., 1995), is known for its antibiotic activity. Its formation as a metabolic intermediate represents a severe danger of poisoning for all members of the microbial consortium (Blasco et al., 1997). Increasing the dilution rate to 0.20 d-1 in the P. sp. MT1 chemostat increased, as well, the accumulation of 3-chloro- cis,cis- muconate to 4.5 ΦM and that of protoanemonin to 42.7 ΦM after 50 d. Particularly, the concentration of protoanemonin was critical and caused cell death of P. sp. MT1, expressed by rapidly decreasing cfu, optical density and substrate conversion. However, contrary to Pseudomonas sp. MT1 the consortium was able to endure much higher dilution rates, with a maximum rate of 0.80 d-1. 4.2. INCORPORATION OF [U-13C]-METABOLITES IN MICROBIAL BIOMASSES The hypothesis that protoanemonin is a key intermediate in the degradation of 4chlorosalicylate by P. sp. MT1 was tested by tracing the incorporation of the stable carbon isotope 13C of 50 nM [U-13C]protoanemonin (i. e. 1000 fold below toxicity) into biomarker fatty acids of Pseudomonas sp. MT1 . If the degradation of an intermediate is productive the fatty acids of the degrading microorganism must be enriched in the stable isotope 13C because of the incorporation of the substrat∋s carbon into the biomass. For verification of this approach in an experiment, commercially available [U-13C]4chlorocatechol was transformed first to [U-13C]protoanemonin, using a Pseudomonas sp. MT1 cell-free extract (Blasco et al., 1995), and then pulse-fed into the Pseudomonas sp. MT1 chemostat. The assimilation activity of the cells was investigated by following the flow of the tracer into fatty acids of the cellular phospholipids. Fatty acids were isolated from the biomass of Pseudomonas sp. MT1 and δ13C values were measured using a gas-chromatograph coupled via a combustion interface to an isotopic ratio mass spectrometer (GC-C-IRMS) (Abrajano et al., 1994). The phospholipid fatty acids were significantly enriched and the fatty acids C16:0 and C16:1 showed the highest 13Cenrichment found in this experiment. The substrate-utilisation of protoanemonin by Pseudomonas sp. MT1 was proven by a significant (∆δ13C+3.0∀0.5λ) enrichment of the isotope 13C in fatty acids C16:1 of Pseudomonas sp. MT1 compared to the unlabeled control. Incubation of resting cells of P. sp. MT1 with 1 mM 4-chlorocatechol in sodium phosphate buffer resulted in the formation of 25 µM protoanemonin, 17 µM cis,cis-3chloromuconate, and 16 µM cis -acetylacrylate. The detection of cis -acetylacrylate pointed to the further fate of protoanemonin because cis -acetylacrylate supported growth of Pseudomonas sp. MT1 in minimal medium and resting cells of salicylategrown MT1 cells transformed 1 mM cis-acetylacrylate within 24 h. These results indicated a conversion of protoanemonin via cis -acetylacrylate in the novel protoanemonin pathway of Pseudomonas sp. MT1. Recently, it was shown that protoanemonin can be transformed by the dienelactone hydrolase of Pseudomonas sp. B13 to cis -acetylacrylate which was further metabolised within the Krebs cycle 148

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(Brückmannet al., 1998). Since no activity of cis -dienelactone hydrolase was detected in the cell extract of Pseudomonas sp. MT1, another, unidentified hydrolase was assumed to be the responsible transforming enzyme. 4.3. SUBSTRATE COMPETITION One application of this approach was the incorporation of carbon isotopes from mixtures of substrates into the individual members of the 4-chlorosalicylate-degrading consortium. The individual strains grew on a carbon mixture of yeast extract and 4chlorocatechol, the latter of which was enriched with 13C-4-chlorocatechol to δ500λ for a better detection of incorporation levels. The isotopic ratios of two fatty acids were determined for all strains and it was found that Alcaligenes xylosoxidans MT 3 incorporated 4-chlorocatechol the best, while Flavobacterium sp. MT 2 showed almost no incorporation of this substrate. Both Pseudomonas strains showed intermediate incorporation levels. This approach will help to elucidate the functional roles of the individual members of the consortium in the mineralization of 4-chlorosalicylate.

Figure 2: Competing assimilation of 4-chlorocatechol and yeast extract by the four members of a 4-chlorosalicylate-degrading consortium. The isotopic ratios of the two fatty acids are shown (C16:1ω7 = 11-hexadecenoic acid, C18:1ω7 = 13-octadecenoic acid). Alcaligenes xylosoxidans MT 3 showed the highest and Empedobacter sp. MT 2 the lowest incorporation of 4-chlorocatechol. Note the consistent slight depletion of C18:1ω7 compared to C16:1ω7 in all strains resulting from the different biosynthetic routes of these fatty acids.

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4.4. COMMUNITY PHYSIOLOGY OF THE MICROBIAL CONSORTIUM After the discovery of the degradation of 4-chlorosalicylate via protoanemonin by Pseudomonas sp. MT1, the question remained open why the consortium could grow under much higher substrate concentrations and dilution rates than Pseudomonas sp. MT1 can tolerate or, asking the question the other way around: why are four strains continuously present in the consortium and not only one? To solve this question the approach to trace the substrate in Pseudomonas sp. MT1 was used in the study of the metabolic activity of the consortium. In order to distinguish the individual strains, immunocapture was applied, using the antibodies generated for the determination of the abundance of the individual strains. Using fatty acids C16:1 of the phospholipids as bacteria-specific biomarkers, the incorporation of 13C from metabolites of 4-chlorosalicylate into bacterial biomass of the individual strains - separated from the consortium by means of immunocapture - was determined at different times after the 13C pulse. Pulse-feeding of 0.6 ΦM [U-13C]4chlorocatechol to the consortium chemostat resulted in a 13C-enrichment in whole cells of the consortium of ∆δ13C+24λ (6 h). However, the highest 13C-enrichment in C16:1 was found for Alcaligenes sp. MT3 (2.5 h) measuring ∆δ13C+1157λ, while that of Pseudomonas sp. MT1 was only +126λ (6 h) and that of Pseudomonas sp. MT4 was ∆δ13C +24λ (6 h). The very fast and much higher 13C-enrichments in 16:1 of Alcaligenes sp. MT3, in relation to that of Pseudomonas sp. MT1 and MT3, indicated that 4-chlorocatechol, which is partially excreted by Pseudomonas sp. MT1, is predominantly used as a carbon and energy source by Alcaligenes sp. MT3. The enzyme activities found in this study were in accordance with the assumption that this strain assimilates 4-chlorocatechol via the chlorocatechol pathway. When 1.8 ΦM [U-13C]protoanemonin was pulsed into the chemostat, the 13Cenrichment in whole cells was 45.7λ (6 h). The 13C-enrichment of C16:1 of Pseudomonas sp. MT4 was ∆δ13C 17.6λ (2.5 h), whereas considerably smaller 13Cenrichments (∆δ13C 3.0-4.6λ) were determined for fatty acids of strains MT1 and MT3 at the early stages (2.5 and 6 h) after the [U-13C]protoanemonin pulse. After 24 h, the 13C-enrichment of fatty acids was ∆δ13C 14.9λ (Pseudomonas sp. MT1) and 10.8λ (MT3). The large and fast 13C-enrichment in C16:1 of Pseudomonas sp. MT4, in relation to that of Pseudomonas sp. MT1 and MT3, revealed that protoanemonin, excreted by Pseudomonas sp. MT1, is used as a carbon and energy source predominantly by Pseudomonas sp. MT4. Due to its low abundance it was not possible to determine the isotope ratios of fatty acids from immunocaptured cells of Empedobacter sp. MT2. However, previous 13Ctracer studies of the individual strains of the consortium in pure culture, using unlabeled yeast extract and 0.2 mM [U-13C]4-chlorocatechol, revealed assimilation of 4chlorocatechol into the biomasses of the strains MT1, MT3 and MT4, but not Empedobacter sp. MT2 (Pelz et al., 1997) (Figure 2). These data and the low abundance of the strain suggest that it carries out a role as consumer of cell debris with no active involvement in the degradation of 4-chlorosalicylate. Although Pseudomonas sp. MT1 is able to grow alone in a chemostat on 4chlorosalicylate, a stable microbial consortium has been isolated for the degradation of 4-chlorosalicylate. Within the stable consortium, Pseudomonas sp. MT1 is the most 150

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abundant strain, which underlines its important functional role as the primary degrader of 4-chlorosalicylate. Based on the observed high 13C-enrichments in fatty acids of strains MT3 and MT4 with the metabolites [U-13C]4-chlorocatechol and -protoanemonin the formation of a consortium can be explained by nutritional interactions of individual strains. Based on the above results, the functional roles of the consortium members were assigned (Fig 3). Using immunocapture and IRMS the sharing of substrates within the microbial community was unravelled and individual community members were shown to protect the whole consortium from critical intermediates such as 4-chlorocatechol and protoanemonin by using them as carbon sources and hence, improving the degradation of 4-chlorosalicylate by Pseudomonas sp. MT1 (Pelz et al., 1999).

Figure 3: Assignment of metabolic functions and interactions of consortium members MT1, MT3 and MT4 in the 4-chlorosalicylate degrading bacterial community.

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5. Outlook The combination of strain-specific antibodies used to immunocapture the individual strains from the community and biomarker fatty acids used to determine the flux of carbon into the biomass of the individual strains by means of IRMS proved to be a powerful tool to study microbial activities in microbial communities. Furthermore we have shown the existence of a novel pathway for the degradation of 4-chlorosalicylate via protoanemonin by means of this technique. This was possible because of the high sensitivity of the IRMS enabling the application of very small amounts of protoanemonin to the system which do not poison the strains and which do not disturb the community significantly. Future studies will be directed to the detection of microbial degraders of poorly degradable pollutants in microbial communities independent from the need to isolate them. A similar approach was recently reported for sulphate-reducing bacteria (Boschker et al., 1998). Instead of immunocapture, biomarkers like polar lipids or outer membrane proteins will be used to trace the flow of labelled substrates into the biomass of potential degraders. Since 1950 a huge number of metabolic routes within individual species have been elucidated. The challenge now is to elucidate metabolic networks in natural biological assemblages in which metabolites produced via a pathway of one cell type flow to other cells to enter new pathways. The surprises exposed in the present study are surely only a mere taste of what is in store! Exploration of metabolic networks in natural assemblages will ultimately yield an understanding of the functioning of such assemblages as biological units, of the roles of the component members and of their metabolic, physiologic and energetic benefits. Furthermore, we will more and more understand how the assemblage as a unit interacts with its abiotic environment. Such an understanding will provide the ground rules for interventions to influence environmental processes and to optimise biotechnological applications in the environment, such as in situ bioremediation. Acknowledgement We thank Dagmar Wenderoth, Tanja Jeschke, Birgit Jung, and Peter Wolff for their excellent technical assistance. The International Atomic Energy Agency, Vienna (Austria), is acknowledged for providing free reference materials for the calibration of the IRMS. This work was supported by a grant of the German Federal Ministry for Science, Education and Research (Project No. 03 19433C). References Abraham, W.-R., Meyer, H., Lindholst, S. Vancanneyt, M. and Smit, J. (1997) Phospho- and sulfolipids as biomarkers of Caulobacter, Brevundimonus and Hyphomonas, Syst. Appl. Microbiol. 20,522-539. Abraham, W.-R., Hesse, C., and Pelz, O. (1998a) Ratios of carbon isotopes in microbial lipids as indicator of substrate usage, Appl. Environ. Microbiol. 64, 4202-4209. Abraham, W.-R., Meyer, H., and Yakimov, M. M. (1998b) Novel glycine containing glucolipids from the alkane using bacterium Alcanivorax borkumensis. Biochim.Biophys. Acta 1393, 57-62. Abrajano, T. A,, Murphy, D. E., Fang, J., Comet, P., Brooks, J. M. (1994) 13C/12C ratios in individual fatty acids of marine mytilids with and without bacterial symbionts. Org. Geochem. 12,611-617.

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SHARING OF NUTRITIONAL RESOURCES IN BACTERIAL COMMUNITIES Amann, R. I., Ludwig, W., and Schleifer, K.-H. (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143-169. Atlas, R. M. (1984) Diversity of microbial communities. Adv. Microbial Ecol. 7, 1-55. Blasco, R., Wittich, R. M., Mallavarapu, M., Timmis, K. N., and Pieper, D. H. (1995) From xenobiotic to antibiotic. Formation of protoanemonin from 4-chlorocatechol by enzymes of the 3-oxoadipate pathway. J. Biol. Chem. 49,29229-29235. Blasco, R., Mallavarapu, M., Wittich, R. M., Timmis, K. N., and Pieper, D. H. (1997) Evidence that the formation of protoanemonin from metabolites of 4-chlorobiphenyl degradation negatively affects the survival of 4-chlorobiphenyl-cometabolizing microorganisms. Appl. Environ. Microbiol. 63,427-434. Boschker, H. T. S., Nold, S. C., Wellsbury, P., Bos, D., de Graaf, W., Pel, R., Parkes, R.-J., and Cappenberg, T. E. (1998) Direct linking of microbial populations to specific biogeochemical processes by 13Clabelling of biomarkers, Nature (London) 392, 801-804. Boschker, H. T. S., de Brouwer, J. F. C., and Cappenberg, T. E. (1999) The contribution of macrophytederived organic matter to microbial biomass in salt-marsh sediments: Stable carbon isotope analysis of microbial biomarkers. Limnol. Oceanogr. 44, 309-319. Brock, T. D. (1987) The study of microorganisms in situ: Progress and problems. Symp. Soc. Gen. Microbiol., 41, 1-17. Brückmann, M., Blasco, R., Timmis, K. N., and Pieper, D. H. (1998) Detoxification of protoanemonin by dienelactone hydrolase. J. Bacteriol. 180, 400-402. Bull, A. T., Goodfellow, M., and Slater, J. H. (1992) Biodiversity as a source of innovation in biotechnology. Annu. Rev. Microbiol. 46,219-252. Didry, N., Dubreuil, L., and Pinkas, M. (1991) Antibacterial activity of vapours of protoanemonin. Pharmazie 46, 546-547. Doolittle, R. F., Feng, D.-F., Tsang, S., Cho, G., and Little, E. (1996) Determining divergence times of the major kingdoms of living organisms with a protein clock. Science 271,470-477. Faude, U. C. (1995) Immunchemische Analyse mikrobieller Lebensgemeinschaften. PhD thesis, University of Leipzig, Germany. Feng, D.-F., Cho, G., and Doolittle, R. F. (1997) Determining divergence times with a protein clock: update and revaluation. Proc. Natl. Acad. Sci. USA 94, 13028-13033. Hugenholtz, P., and Pace, N. R. (1996) Identifying microbial diversity in the natural environment: a molecular phylogenetic approach. Trends Biotechnol. 14, 190-1 97. Hugenholtz, P., Goebel, B. M., and Pace, N. R. (1998) Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180,4765-4774. Mau, M, and Timmis, KT (1998) Use of subtractive hybridisation to design habitat-based oligonucleotide probes for investigation of natural bacterial communities. Appl. Environ. Microbiol. 64, 185-191, Moore, E.R.B., Mau, M., Arnscheidt, A., Böttger, E.C., Hutson, R.A., Collins, M.D., Van de Peer, Y., De Wachter, R., Timmis, K.N. (1996) The Determination and Comparison of the 16S rRNA Gene Sequences of Species of the Genus Pseudomonas (sensu stricto) and Estimation of the Natural Intrageneric Relationships. System. Appl. Microbiol. 19,478-492. Niepel, T., Meyer, H., Wray, V., and Abraham, W.-R. (1997) A new type of glycolipid, 1-[α-manno-pyra nosyl-(1α-3)-(6-O-acyl-α-mannopyranosyl)-3-O-acyl-glycerol, from Arthrobacter atrocyaneus. Tetrahedron 53, 3593-3602. Pace, N. R. (1996) New perspective on the nature microbial world: molecular microbial ecology. ASM News 62, 463-470. Pace, N. R., Stahl, D. A., Lane, D. J., and Olsen, G. J. (1986) The analysis of natural microbial populations by ribosomal RNA sequences. Adv. Microb. Ecol. 9, 1-55. Pace, N. R. (1997) A molecular view of microbial diversity and the biosphere. Science 276, 734-740. Pelz, O., Hesse, C., Tesar, M., Coffin, R. B., and Abraham, W.-R. (1997) Development of methods to measure carbon isotopic ratios of bacterial biomarkers in the environment, Isotopes Environ. Health Stud. 33, 131-144. Pelz, O., Tesar, M., Wittich, R.-M., Moore, E. R. B., Timmis, K. N., and Abraham, W.-R. (1999) Towards elucidation of microbial community metabolic pathways: Unravelling the network of carbon sharing in a pollutant degrading bacterial consortium by immunocapture and isotopic ratio mass spectrometry, Environm. Microbiol. 1, 167-174. Pomeroy, L. R., and Wiebe, W. J. (1988) Energetics of microbial food webs. Hydrobiologia 159, 7-18. Roszak, D. B., and Colwell, R. R. (1987) Survival strategies of bacteria in the natural environment. Microbiol. Rev. 51, 365-379.

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A COMPARISON OF THE MECHANICAL PROPERTIES OF DIFFERENT BACTERIAL SPECIES C. SHIU, Z. ZHANG AND C.R. THOMAS* Centre for Bioprocess Engineering, School of Chemical Engineering The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK * corresponding author.

Abstract Past attempts at direct measurement of the mechanical characteristics of cells have shown little potential for use with bacteria. However, a technique based on micromanipulation has now been used to compare the mechanical properties of two species of bacteria; Gram-negative Escherichia coli and Gram-positive Staphylococcus epidermis. Gram-negative bacteria have traditionally been considered to be weaker than their Gram-positive counterparts on the basis of them being more susceptible to disintegration by mechanical stress. This observation has been corroborated using micromanipulation, which has directly quantified the differences in cell strength on the basis of bursting force. The bursting force of rapidly growing S. epidermis was found to vary from 3 and 34 µN with an average value of 13.8 µN (standard error 0.8 µN). The corresponding value for continuously growing E. coli (specific growth rate 0.5 h-1) was 3.8 µN (standard error 0.4 µN). 1. Introduction 1.1 RELATIVE RESISTANCE MECHANICAL DISRUPTION

OF

DIFFERENT

MICROORGANISMS

TO

The physical properties of cells e.g. cell size, shape and especially wall strength, are highly species, strain and physiological state specific and will influence the relative resistance of different microorganisms to disruption, which may be used to indirectly infer the relative strength of microorganisms (Table 1, Table 2).

155 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 155–162. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

C. SHIU, Z. ZHANG AND C.R. THOMAS Table 1: Susceptibility of various kinds of cells in suspension to disintegration by various methods. A high number indicates that the cells are sensitive. Parenthesis indicates that the number is uncertain (Edebo, 1969).

Animal cells Gram - bacilli & cocci Gram + bacilli Yeast Gram + cocci Spores Mycelia a

Liquid-pressing

Freeze-pressing

Agitation

Sonication

7 6 5 4 3 2 (1)a

7 6 4 2.5 2.5 1 5

7 5 (4) 3 (2) (1) 6

7 6 5 3.5 3.5 2 1

clogging of orifice may occur

The wall thickness of yeast (> 70 nm) is typically greater than that of Gram-positive cocci (10 - 80 nm) yet the latter would appear to be the more resilient according to Table 1. This may be because size has been observed to play an important part in disruption, with larger cells showing increased susceptibility to rupture. Table 2: Protein release from microorganisms in a high-pressure homogeniser (modified from Keshavarz et al., 1987).

Microorganism

Rate constant, k 0.41 0.39 0.28 0.23 0.0085

Pseudomonas putida (Gram-negative rod) Escherichia coli (Gram-negative rod) Bacillus brevis (Gram-positive rod) Saccharomyces cerevisiae Nocardia rhodochrous

The most obvious role of the cell wall is in protecting the cell from mechanical damage, the structure and composition of which contributes greatly to the strength of individual bacteria as may' be seen from the fact that Escherichia coli (Gram-negative rod), although similar in size to Bacillus subtilis (Gram-positive rod), is disrupted more easily (Table 2). This is most likely due to the relatively thin peptidoglycan layer found in Gram-negative bacteria (see Section 0). 1.2 CELL WALL STRUCTURE Prokaryotic cells can be segregated into different groups on the basis of their cell wall structure. The Archaebacteria possess cell walls but the components and physiology are vastly different from that of eubacteria. A small fraction of eubacteria, the mollicutes, lack an outer cell wall and are bounded only by their cytoplasmic membrane (these will not be considered here). The eubacteria which do possess cell walls can be divided into two major groups: Gram-positive or Gram-negative, which refer to the manner in which eubacteria respond to the Gram stain. Under the electron microscope, the Gram-positive cell wall appears as an amorphous layer with an average thickness of 30 nm, although this value may vary from between 156

A COMPARISON OF THE MECHANICAL PROPERTIES OF BACTERIAL SPECIES

10 – 80 nm (Shockman and Barrett, 1983). Unlike Gram-negative bacteria, the periplasm is practically non-existent as the relatively large osmotic pressure, estimated at up to 25 atm (Mitchell and Moyle, 1956), found within Gram-positive bacteria forces the plasma membrane firmly against the cell wall. By comparison, the cell walls of Gram-negative bacteria are much more complex than that of Gram-positive bacteria. The cell envelope consists of essentially three layers: the cytoplasmic membrane, the murein (or peptidoglycan) layer and the outer membrane. The dual membrane system of Gram-negative bacteria gives rise to the periplasm, a space 15-20 nm thick comprising between 20-40% of the total cell volume (reference) which contains a gel-like solution of proteins and in which the murein layer is found. Peptidoglycan is less abundant in the cell walls of Gram-negative bacteria, rarely exceeding 5- 10% of the weight of the wall (Lugtenberg and van Alphan, 1983). 1.3 BACTERIAL BIOMECHANICS A number of researchers have demonstrated the flexibility of the bacterial cell wall (Mendelson and Thwaites, 1989; Baldwin, 1988; Marquis, 1968; Ou and Marquis, 1970) but perhaps the simplest and most compelling evidence was that presented by Isaac and Ware (1974) who embedded cells of different organisms in glycerol-gelatine strips and stretched them on a rack. Spirillum was found to uncoil and stretch reversibly to a rod three times the length of its original shape. The Gram-positive bacterium, B. megaterium elongated to one quarter of its original length before becoming detached from the gel matrix. E. coli showed considerable stretching and deformation - up to 100 % elongation - without detaching from the gel. With the Gram-positive coccus, S. aureus, no elongation was observed prior to detachment from the gel matrix. These results would appear to suggest the bacterial cell wall is far from being rigid and nonflexible. With Gram-positive organisms, the thicker murein leads to increased resistance to stretching while their Gram-negative counterparts show great flexibility in comparison. Without a doubt, the small size of individual bacteria has proved to be a considerable obstacle in the development of a method for directly measuring the cell mechanical properties. The approach of Thwaites and Mendelson (1985, 1989, 1991) cleverly side-stepped this difficulty by conceiving a means to produce “bacterial thread” up to 1 m long with 50 000 parallel cellular filaments, which can be subjected to similar techniques applied to textile threads. Tensile tests were performed on bacterial threads of B. subtilis mutant FJ7 to determine the cell wall properties. The length and diameter of the filaments were found to depend on the humidity and unsurprisingly so were the mechanical properties. The tensile strength and modulus of elasticity at low relative humidity were 300 MPa and 13 GPa respectively. Corresponding values when extrapolated to 100% humidity were found to be 13 MPa and 30 MPa. Such values of strength indicated that cells in vivo should theoretically be able to withstand turgor pressures of up to 2.6 MPa (Thwaites and Surana, 1991). It was found that the stress/strain curves of unwashed preparations were similar to those with the residual culture medium removed at roughly 18% higher relative humidity. The most likely reason for this was that the unwashed cells were encased by the highly hygroscopic dried medium and were therefore held in a much more humid environment relative to the bulk atmosphere. 157

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In addition, the walls of the B. subtilis mutants were shown to exhibit what was apparently viscoelastic behaviour since the load depended on the rate of strain and load relaxation also occurred in the extended state. The time-scale for decay proved to vary widely but based on the initial rate of decay, was found to occur on the order of minutes (Thwaites and Mendelson, 1985). Viscoelastic phenomena in cell walls has major implications for micromanipulation measurements; at high deformation rates, the cells would appear to be stiffer and more brittle and vice versa (Thwaites and Mendelson, 1991). Such behaviour would need to be accounted for in the development of structural models. One major drawback to the technique is that it is limited to filament forming mutants. Furthermore, the cells were not in their naturally hydrated condition when the tests were carried out and the humidity has been shown to affect the thread mechanical properties. Micromanipulation has several advantages over the latter technique in terms of simplicity, versatility and more importantly, since mechanical properties are likely to be affected by the external environment, the cells can be measured whilst suspended in the growth medium. 1.4 MICROMANIPULATION Micromanipulation is based on a relatively simple concept, which relies on squeezing a single cell, either between two optic fibre probes or between a single optic fibre and a slide, until rupture; a force transducer measures the force required. Such work has already been performed on animal cells (Zhang et al. 1991), yeast cells (Srinorakutara (1997); Mashmoushy et al., 1998) starch and latex particles. Micromanipulation studies with filamentous organisms such as fungi are slightly more involved and it has been more appropriate to determine mechanical properties by extension of the organism until breaking (Roberts, 1996). 2. Materials and methods 2.1 THE MICROMANIPULATION SYSTEM The apparatus (fig 1) was based on the rig used by Mashmoushy et al. (1998) for work with yeast. Cells are squeezed using a 50 µm optic fibre probe, one end of which is attached to a force transducer (Aurora Scientific Inc., Aurora, Canada) with paraffin wax. The end of the probe which comes into contact with the bacteria is ground down to leave a tip with a much smaller area, thus minimising alignment errors and incorrect probe-cell contacts. The transducer is attached to a stage which is driven downwards by a stepping motor, The force on the probe is measured by the transducer in terms of voltage, which is converted to give the bursting force. Lighting is provided by a slit illuminator from the side. Cells are positioned underneath the probe by moving the stage. When E. coli was being measured, the image was recorded onto video tape for later image analysis to determine the length of the cell.

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Fig 1 The micromanipulation system

2.2 CULTURE CONDITIONS Staphylococcus epidermis ATCC 5885 cells were grown on agar slopes at 30°C for 48 hours and then used to inoculate a McCartney bottle containing 10 ml of sterilised nutrient broth (Oxoid, UK). Following incubation at 30°C over 24 hours, the broth was used to inoculate a 500 ml volume shake flask containing 90 ml of sterilised nutrient broth to start the batch fermentation. The flask was maintained at 30°C and 200 rpm in an orbital shaker. The progress of the fermentation was followed by taking absorbance measurements at 6.50 nm. Samples were taken from the rapid growth phase at t = 4, 5 and 6 hours after inoculation. In preliminary fermentations, E. coli NCIMB 10214 had demonstrated a relatively short exponential growth phase. Hence, continuous as opposed to batch fermentation was used to provide physiologically identical samples. Medium of the following composition was used for E. coli fermentation: (g/l of distilled water): KH2PO4, 13.6; (NH4)2SO4, 4; MgSO4, 0.2; FeSO4 5x10-4; yeast extract, 5.0 and adjusted to pH 7.0 using 2.0 M KOH solution. Glucose solution at a final concentration of 10 g/l of medium was made up and sterilised separately and only added to the medium before inoculation. Cells of E. coli were incubated at 37°C on agar slopes for 24 hours and used to inoculate a shake flask containing 100 ml of complex medium. The culture was grown 159

C. SHIU, Z. ZHANG AND C.R. THOMAS

overnight for approximately 16 hours at 37°C and 200 rpm in an orbital shaker before being used to seed 1 1 of medium in a 2 1 fermenter. The pH was controlled using 0.5 M H2SO4 and 2.0 M ammonia solution. Once the cells had reached mid-exponential phase, the continuous fermentation was started at a dilution rate of 0.5 h-1. Samples were diluted using distilled water to a concentration of approximately 1 x 107 cells/ml. 3. Results and discussion Figure 2a and Figure 2b represent typical traces of the bursting response of individual S. epidermis and E. coli cells respectively. Both traces have a similar form; contact between the probe and the cell is made at point A after which the cell deforms until rupture (point B). Following bursting of the cell, there is a decrease in force on the probe until it reaches the slide at point D, leading to a significant increase in force.

Figure 2: Bursting traces for (a) S. epidermis and (b) E. coli

Plots of bursting force versus (equivalent spherical) diameter for S. epidermis and E. coli are shown in Figure 3a and Figure 3b respectively. In both cases, the large scatter observed in bursting force is attributed to biological variation within the culture. Three separate samples were taken (t = 4, 5 and 6 hours) and tested within one hour of sampling. Statistical testing using ANOVA indicated a strong probability that the three samples were from equivalent populations and hence they were treated as such. For cell diameters ranging from 0.5 to 1.6 µm with an average value of 0.92 µm (standard error 0.03 µm), the bursting force for S. epidermis cells was found to vary from 3 to 34 µN (standard error 0.8 µN) with an average value of 13.8 µN. Furthermore, simple linear regression appeared to suggest some relationship between bursting force and cell size, which was confirmed using an F-test with 95% confidence. 160

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Corresponding values for E. coli ranged from 1 to 9 µNwith an average value of 3.6 µN (standard error 0.4 µN) for cells with equivalent spherical diameters between 0.7 to 1.8 µm with an average value of 1.26 µm (standard error 0.05 µm). Simple linear regression showed no relation between the bursting force and equivalent spherical diameter. This was confirmed with an F-test at the 95% confidence level.

Figure 3: Plots of bursting force vs. (equivalent spherical) diameter for (a) S. epidermis and (b) E. coli

It is worth mentioning that only a small percentage of E. coli cells demonstrated bursting behaviour. In other cases, the trace either showed the deformation of the cell without bursting or nothing was observed. This might be due to rapid relaxation caused by water loss or viscoelasticity but further experiments will be required to elucidate the reasons for this phenomenon. It is significant that the Gram-negative E. coli with the thinner cell wall and lower internal pressure had a lower average bursting force than the Gram-positive S. epidermis with the thicker cell wall and higher internal pressure. Differences in cell physiology could be the underlying cause behind these differences. Although the fact that spheres are structurally stronger than cylinders could also have some bearing on this result. 4. Conclusions and future developments The bursting force measurements obtained for S. epidermis and E. coli demonstrate the usefulness of the micromanipulation technique for obtaining information on the mechanical properties of bacteria. Linked with a suitable cell model, it should be possible to estimate other mechanical properties. Biomechanics might be linked to cell physiology by carrying out micromanipulation in conjunction with experiments to determine cell wall composition, even using mutants known to lack certain cell wall components. 161

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Bacterial species do not always grow as individual cells but in a range of different morphologies. Micromanipulation techniques that have been developed for tensile testing of fungi (Roberts, 1999) and filamentous bacteria (Stocks and Thomas, 1999) might be adapted to pull apart bacteria that grown in chains of while larger probes might be used to measure the bursting forces of bacteria that grow in clumps. References Baldwin, W.W., Sheu, M.J.-T., Bankston, P.W., Woldringh, C.L. (1988) Changes in buoyant density and cell size of Escherichia coli in response to osmotic shocks. Journal of Bacteriology 170, 452-455. Edebo, L. (1969) Disintegration ofcells. In Fermentation Advances (Perlman, D., ed.). Academic Press, NY, pp.249-271. Isaac, L. and Ware, G.C. (1974) Theflexibility ofbacterial cell walls. Journal of Applied Bacteriology 37, 335-339. Keshavarz, E., Hoare, M., and Dunnill, P. (1987) Biochemical engineering aspects of cell disruption. In Verrall M.S. and Hudson M.J. (eds.), Separations for Biotechnology, pp. 62-79. Lugtenberg, B. and Van Alphan, L. (1982) Molecular architecture andfunctioning ofthe outer membrane of Escherichia coli and other Gram-negative bacteria. Biochimica and Biophysica Acta. 737,51-115. Marquis, R.E. (1968) Salt-induced contraction ofbacterial cell walls. Journal of Bacteriology 95,775-78 1, Mashmoushy, H., Zhang, Z. and Thomas, C.R. (1998) Micromanipulation measurement of the mechanical properties of baker’s yeast cells. Biotechnology Techniques, 12, 925-929. Mendelson, N.H. and Thwaites, J.J. (1988) Studies of Bacillus subtilis macrofiber twist states and bacterial thread biomechanics: assembly and material properties of cell walls. In Antibiotic Inhibition of Bacterial Cell Surface Assembly and Function (Actor, P., Daneo-Moore, L., Higgins, M.L., Salton, M.R.J. and Shockman, G.D., eds.), pp. 109-125. Mitchell, P. and Moyle, J. (1956). Osmotic function and structure in bacteria. In Bacterial Anatomy Symposium Society of General Microbiology 6, 150 -180. Ou, L.-T. and Marquis, R.E. (1970) Electromechanical interactions in cell walls of Gram-positive cocci. Journal of Bacterology 101, 92-101. Roberts, A.D. (1999). Determination of Fusarium graminearium mechanical properties by micromanipulation. PhD Thesis (submitted). The University of Birmingham. Shockman, G.D., Barrett, J.F., (1983) Structure, junction and assembly of cell walls of Gram-positive bacteria. Annual Review of Microbiology 37, 501-527. Srinorakutara, T. (1997) Mechanical Strength of Yeasts. PhD Thesis (unpublished). The University of Birmingham. Stocks, S.M. and Thomas, C.R. (1999). The tensile strength of Saccharoployspora erythraea. European Journal of Histochemistry (Proceedings of the first international conference on analysis of microbial cells at the single cell level, Como, Italy, 1999). Thwaites, J.J. and Mendelson, N.H. (1985) Biomechanics of bacterial walls: Studies of bacterial thread made from Bacillus subtilis. Proceedings of the National Academy of Sciences of the United States of America 82, 2163-2167. Thwaites, J.J. and Surana, C. (1991) Mechanicalproperties of Bacillus subtilis cell walls: Effects of removing residual culture medium. Journal of Bacteriology 173, 197-203. Thwaites, J.J. and Mendelson, N.H. (1991) Mechanical behaviour of bacterial cell walls. Advances in Microbial Physiology 32: 173-222. Zhang, Z., Ferenczi, M.A., Lush, A.C. and Thomas, C.R. (1991) A novel micromanipulation technique for measuring the bursting strength ofsingle mammalian cells. Applied Microbiology and Biotechnology 36, 208-210.

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Part 5 NOVEL APPLICATIONS

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KOCURIA ROSEA AS A NEW FEATHER DEGRADING BACTERIA NEREIDA COELLO AND LUIS VIDAL Laboratorio de Procesos BiotecnolOgicos. Instituto de Biologla Experimental. Universidad Central de Venezuela. AP 472 79 Caracas 1041-A, Venezuela.

Abstract A strain of Kocuria rosea with keratinolytic activity was isolated. This Gram-positive coccus-shaped bacterium was able to degrade whole feathers in 72 h at 40° C. In batch culture, the optimum temperature for feather degradation, bacterial growth and protease secretion was 40° C. Under these conditions, biomass and caseinolytic activity reached 3.2 g/l and 0.15 U/ml, respectively, after 36 h incubation. Extracellular protease secretion was associated with the exponential growth phase. Feather degradation reached 51 % in 72 h with a yield on biomass of 0.32 g/g. Enzymatic assay by polyacrylamide-gelatine gels showed two bands with alkaline proteolytic activities in the fermentation juice after 72 h of culture. 1. Introduction The expansion of biotechnology has produced an increasing demand for high-quality inexpensive microbial growth media. Utilisation of feathers from the poultry processing industry as a substrate for fermentation might offer an inexpensive alternative for a microbial method of metabolite production, such as enzymes and unicellular protein can be achieved provided an efficient utilisation. Feathers are a poultry by-product rich in protein (mainly keratin) and generated in very large quantities as a waste product from poultry processing industry. For many years, they have been the object of nutritional studies in order to incorporate them as nitrogen supplement in animal foodstuff. This allows poultry industry to take advantage of their usefulness and eliminates the environmental problem generated. In their native state, feathers have two important nutritional limitations: an amino acid imbalance and a poor digestibility (Xiang Lin et al. 1992). Industrially, a great part of the feather waste is cooked under high pressure and temperature producing a feather meal, which can be incorporated into poultry foodstuff as a protein supplement. This product has been evaluated extensively and the results 165 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 165–175. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

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show that it is not very digestible and the destruction of certain amino acids during the process, such as cysteine, magnifies the imbalance of essential amino acids (Bhargava & O’neil 1975; Baker et al. 1981). Thus, there is a growing interest in alternative methods for the treatment of feathers to improve the nutritional quality of the feather meal and to develop new and useful byproducts from feathers. The nutritional value of the feather meal might be improved by microbial action, this results in a modification of the structure of keratin, altering the resistance to the enzymes of the digestive tract. The literature reports that isolates of Bacillus lichenformis (Williams et al. 1991) and Streptomyces pactum (Böckle et al 1995) grown on feather media were able to modify the digestibility and profiles of the specific amino acids that were liberated. The bacterial cells incorporated into the fermented feathers may contribute to some amino acids, hence improving their balance. Also, feather as a fermentation substrate may be useful in order to obtain other microbial products (proteolytic enzymes, carotenoid pigments, etc.). We conducted a study on an isolate from the soil of a poultry-processing plant in order to determine microbial growth, possible keratinolytic activity and optimum feather degradation conditions in submerged fermentation. To our knowledge, this is the first report describing the capacity of Kocuria rosea to degrade feathers. 2.Isolation, identification and adaptation of feather-degrading microorganisms Isolating microorganisms from nature is the microbiologists first step in screening for natural microbial products. It is possible to isolate many different microorganisms by employing enrichment techniques on the appropriate culture medium. One successful approach for the discovery of new metabolites involves considering the characteristics of the desired product and process development and using ecological approaches for isolation and screening. Consideration of the ecological approaches to isolation can provide a screen with both a large number and a wide variety of microorganisms to examine for the product of interest. Even though microorganisms are highly adapted, specific microbial types are associated with different niches within a variety of ecosystems. Therefore, what is isolated from the defined ecosystem or habitat is a reflection of the isolation procedures and of the conditions found in nature. 2.1. ISOLATION AND DEGRADATION OF FEATHERS BY A MICROBIAL ISOLATE Based on the capacity of some microorganisms to grow in a medium containing feather powder as the primary organic substrate for supplying carbon and energy a featherdegrading isolate was obtained from soil of a poultry processing plant (Figure 1). It was found that a culture of the feather-degrading isolate contained microorganisms exhibiting at least two distinct colony morphologies (red and white) when streaked onto nutrient agar plates. The white colony contained only rod-shaped bacteria, whereas the red colony was a coccus-shaped bacteria, which appeared singly and in chains. Although each type displayed clearing zones when streaked onto the potter-milled feather agar plates, the red colony produced the more pronounced clearing zones. The

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isolate, designated as LPB-3, was able to degrade feathers in 72 h at 40° C when isolated cells were incubated on whole feathers suspended in saline medium. The keratinolytic activity of LPB-3 isolate was evidenced by visual observation of feather disintegration, as well as increasing turbidity of the culture due to growing cells (Figure 2). The bacterium grew aerobically and was catalase positive. Additional biochemical tests conducted on LPB-3 isolate in our laboratory were confirmed by the Pasteur Institute of France, which characterised the isolate as Kocuria rosea

Figure 1. Isolation method for feather degrading microorganisms from soil

Figure 2. Degradation of feathers by isolate. The LPB-3 strain was cultivated in assay tubes with whole feathers suspended in mineral medium (g/l): NH4CI, 0.5; NaCI, 0.5; K2HPO4, 0.3; KH2PO4, 0.4; MgCI2.6H2O, 0.1; 0.1 yeast extract; pH 7.5 at 40°Cfor 72 h. A, control; B, 24 h; C, 48 h; D, 60 h: E, 72 h.

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Only limited information regarding Kocuria rosea is currently available. This Gram positive micro-organism, which was classified in the past in the genus Micrococcus and reclassified recently in the genus Kocuria (Stakenbrandt 1999, has been studied mainly in relation to the structure and properties of the carotenoid pigments it produces (Cooney 1981; Jagamadham 1991). Until now, the feather degradation capacity of K. rosea has not been reported in the literature. Cells of the LPB-3 isolate were grown on whole-feather basal medium, in which the autoclave treatment time was progressively reduced. The isolate was able to degrade feathers which had been autoclaved for only 5 min. Attempts to maintain the growth of the isolate on non-steam-treated feathers were not successful, 2.2. MORPHOLOGICAL AND ULTRASTRUCTURAL CHARACTERISTICS OF THE FEATHER-DEGRADING ISOLATE Microscopic observations of the isolate showed a coccus (1.5 µm) which appeared singly and in chains adsorbed mostly to the feathers, although a few microorganisms swam freely (Figure 3A). The adsorption to the fermentation substrate is part of a survival strategy of microbes in certain limiting environments in order to improve feather degradation via proteolytic enzymes secretion. The ultrastructure of K. rosea was generally similar to that of other Gram-positive bacteria. The cells were separated from the surrounding medium by a microcapsule and a cell wall with a homogeneous structure, approximately 20 nm thick (Figure 3B). The cytoplasmic membrane was closely adjacent to the cell wall and the periplasmic space was poorly discernible. K. rosea was characterised by a high ribosome density in the cytoplasm and the occurrence of polyphosphate granules and glycogen accumulation was also observed. The nucleoid, consisting of DNA fibrils, was located in the centre of the cell and as cell division began, a septum was formed inside the cells. Transmission electron micrographs showed in some cells the presence of two asymmetrical septa. 3. Microbial growth and feather degradation 3.1, EFFECT OF QUANTITY OF FEATHERS Feathers promoted cell biomass production concomitant to feathers degradation, at the lowest concentration tested (10 g/l). The increase of the concentrations of free amino groups during the feather fermentation was an evidence that these bacteria produce a protease(s) capable of digesting keratin of feathers to produce short peptides and amino acids that can be used as carbon and nitrogen sources for the growth of the microorganism. The concentrations of free amino groups under these conditions are shown in Figure 4. It was observed that aerobic growth by the isolate LPB-3 on feathers throughout growth were similar for 20 and 30 g/l. After 96 h of fermentation the final concentration of free amino groups attained nearly 60 and 35 mmol for 20 and 10 g/l, respectively. Therefore for the kinetic fermentation studies, 20 g/l of feathers was used.

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KOCURlA ROSEA AS A NEW FEATHER DEGRADING BACTERIA

Figure 3. [A) Light microscopy of K. rosea cells with Gram-staining. Magnification X 10,000. Single cells and chains ofcoccus-shaped bacteria were adsorbed to the feathers. (B) Ultrastructure of K. rosea cells. Magnification X 52,000. CW, cell wall: CM, cytoplasmic membrane: AS, asymmetrical septum: CH, chromatin fibrils: PG, polyphosphate granules.

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Figure 4. Effect of quantity of feathers. LPB-3 strain was cultivated at 40°Cfor 96 h in 500ml Erlenmeyer baffled flasks, containing IO g/l 20 g/l (0) and 30 g/l scissors-cut feathers, suspended in mineral medium described earlier shaken at 75 rpm. Control non inoculated . Samples were taken and filtered through a Büchnerfunnel with Whatman paper 4. Afer removing cells by centrifugation supernatant was used for free amino groups analysis using a ninhydrin method described by Rosen (1957).

Figure 5. Growth of Kocuria rosea LPB-3 (O) and free amino groups concentration at different temperatures in feather medium (20 g/l). Samples were taken at 72 h filtered through a Büchner funnel with a Whatman paper 4. Filtrated was used to measure cell mass density by turbidity (600 nm) andfree amino groups as described earlier.

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3.2. EFFECT OF CULTURE TEMPERATURE ON FEATHER DEGRADATION AND GROWTH OF LPB-3 The optimum growth of K. rosea in scissors-cut feathers was obtained at a culturing temperature of 40° C (Figure 5). Similarly, maximum feather degradation, measured as concentration of free amino groups occurred at between 35-40° C and reached 43 mmol/l. In both cases, concentration of free amino groups and biomass decreased at culture temperatures lower than 35 C. For this reason, the optimum temperature chosen for this mesophilic strain was 40° C. Nevertheless, a psychrotrophic strain of K. rosea has been reported that growths at lower temperatures (Jagamadham 1996; Chattopadhyay 1997). 3.3. KINETIC FERMENTATION At 40° C we found a latent phase of nearly 3 h in batch fermentation, followed by an exponential growth phase in which K. rosea attained a specific growth rate of 0.17 h-1. Protease activity was detected in the culture medium throughout growth, with the maximum activity (0.15 U/ml) associated with maximum biomass (3.1 g/l) 36 h after inoculation (Figure 6). In the latter case, K. rosea caused up to 51% feather degradation in 3 days (Figure 6B). This time represents less than the reported numbers in the literature (at least 5 days) for other microorganisms with keratinolytic activity for maximum feather hydrolysis in optimum conditions: Streptomyces fradiae (Noval & Nickerson 1959), Bacillus licheniformis (Williams et al 1990) and Bacillus sp. (Atalo & Gashe 1993). Although low residual feathers was found at 96 h, the excess in incubation time was not practical because this parameter did not decrease significantly and the energy required for incubation may increase the operational costs. The increase in pH of culture medium from 7 to 8.5 after 48 h of inoculation (data non shown) was presumably a result of proteolysis and the release of ammonia following utilisation of amino acids and soluble peptides as a metabolic fuel for growth and microbial maintenance. 4. Industrial applications 4.1, FERMENTED FEATHER MEAL Industrially, feather waste is processed into hydrolysed feather meal using high temperatures and pressure. It is utilised as supplemental source of protein in poultry foodstuffs. However, the use of hydrolysed feathers meal, as a replacement protein is limited. Morris and Balloun (1973) observed that the addition of 6 % hydrolysed feather meal to diets of chickens caused a decreased body weight. Research showed that raw feathers can be fermented in anaerobiosis with a Bacillus licheniformis strain (Williams et al, 1991) to produce a fermented feather lysate that can be used as a replacement protein up to 15 % in poultry foodstuff. The feather lysate obtained with K. rosea is 171

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enriched with bacterial biomass up to 10 %, but must be studied in terms of the digestibility, amino acid profile and bioavailability in order to know more about the properties of this new product.

Figure 6. Growth -O- (A), proteolytic activity and residual feathers (B) of Kocuria rosea LPB-3 in feather medium (20 g/l) at 40° C for 96 h and 75 rpm agitation. Residual feathers are expressed as a percentage of dry weight of the residue obtained upon filtration of the culture contents through a Whatman paper No. 4. Protease activity was assayed by photometric method (280 nm) using casein as substrate based on the method of Kunitz (1947) One unit of proteolytic activity was defined as the amount of enzyme which produced an absorbance increase of 1.0 units within one min.

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4.2. ENZYMES Proteolytic enzymes are the most important industrial enzymes because of their wide applications in the detergent, dairy, pharmaceutical, leather and food industry. Many alkaline proteases are produced by bacteria and fungi and are also present in mammalian tissues. Cultures of K. rosea in feather medium stimulate the secretion of at least two alkaline proteases (Figure 7) with a broad tolerance to temperature (35-70 ° C) and high long-term storage stability. These proteases degrade keratin of feathers to produce short peptides and amino acids that can be used as carbon and nitrogen sources for the microorganism. They could be useful in the dehairing stage of leather processing and the formulation of dehairing lotion removers of cosmetic industry. Bovine and porcine pancreatic trypsin have demonstrated to be effective in reducing the requirement of lime, shortening the dehairing process time and lowering the waste treatment costs (Ward, 1985). Thus, extensive genetic and physiological studies must be carried out for a better understanding of the mechanism that controls keratinase secretion in the wild-type strain of K. rosea in order to make this protease available for industrial purposes.

Figure 7. Polyacrylamide-gelatine gels of fermentation juice of Kocuria rosea LPB-3 after 72 h of culture at different pH, A: 10; B: 9; C: 7; D: 5; E: 3.6.

4.3. PIGMENTS Many microorganisms including algae, fungi and bacteria produce carotenoid pigments. Industrially, they are used as a food colouring agents (e.g. margarine, cheese, and egg 173

NEREIDA COELLO AND LUIS VIDAL

products) and at a lesser extent as oral tanning agents in the cosmetic industry. Cantaxanthin, a xantophyll carotenoid used as feeding additive together with astaxanthin, have been used in the poultry industry to improve the colour of chicken and egg yolk and for muscle pigmentation in farmed salmonids (Elmadfa, 1998). Cantaxanthin is the major coloured carotenoid pigment biosynthesised in mesophilic K. rosea strains (Cooney, 198 1). Further research concerning qualitative and quantitative pigment content and factors influencing carotene biosynthesis in K. rosea must be done to consider the microbiological pigment processes available. Acknowledgements The authors acknowledge Lic. C. Bernal and Dr. A. Bretaña for their helpful contribution in enzyme gel assay and electron microscopy, respectively. This research was supported by grants from Interamerican Development Bank and Consejo Nacional de Ciencia y Tecnología (BTI-88), and Consejo de Desarrollo Científico y Humanístico of Central University of Venezuela (09- 12-404497). References Atalo, K. and Gashe, B. (1993) Protease production by a thermophilic Bacillus species (p-001A) which degrades various kinds of fibrous proteins. Biotechnology Letters 15, 1115-1156. Baker, D. H., Blitenthal, R. C., Boebel, K. P., Czrnecki, G. L., Southern, L.L. and Willis, G. M. (1981) Protein amino-acid evaluation of steam-processed feather meal. Poultry Science 60, 1865-1872. Bhargava, K.K. and O’Neil, J. B. (1975) Composition and utilisation of poultry by-product and hydrolysed feather meal in broiler diets. Poultry Science 54, 1511-1518. Böckle, B., Galunsky, B. and Muller, R. (1995) Characterisation of a keratinolytic serine proteinase from Streptomycespactum DSM 40530. Appl. Environ. Microbiol. 61, 3705-3710 Chattopadhyay, MK., Jagannadham, MV., Vairamani, M., and Shivaji, S. (1997) Carotenoid pigments of an Antarctic psychrotrophic bacterium Micrococcus roseus: temperature dependant. biosynthesis, structure and interaction with synthetic membranes. Biochemical and Biophysical Research Communications 239, 85-90. Cooney, J.J., and Berry, R.A. (1981) Inhibition of carotenoid synthesis in Micrococcus roseus. Canadian Journal of Microbiology 27, 421-425 Elmadfa, I. and Majchrzak D. (1998) Carotenoids and Vitamin A in fish. ZErnahrungswiss 37, 207-210 Jagannadham, M.V., Narayanan, K., Rao, C.M., and Shivaji, S. (1996) In vivo characteristics and localisation of carotenoid pigments in psychrotrophic and mesophilic Micrococcus roseus using photoacoustic spectroscopy. Biochemical and Biophysical Research Communications 227, 221-226. Jagannadham, MV., Rao, VJ., and Shivaji, S. (1991) The major carotenoid pigment of a psychrotrophic Micrococcus roseus strain: purification, structure and interaction with synthetic membranes. Journal of Bacteriology 173, 7911-7917. Kunitz, M. (1947) Crystalline soybean trypsin inhibitor II. General properties. Journal of General Physiology 30, 291-310. Noval, J. and Nickerson, W. (1959) Decomposition of native keratin by Streptomyces fradiae. Journal of Bacteriology 77, 251-263 Rosen, H. (1957). A modified Ninhydrin Colorimetric analysis for amino acids. Archives of Biochemistry and Biophysics 67, 10-15. Stackebrandt, E., Koch, C., Gvozdiak, O., and Shumann, P. (1995) Taxonomic dissection of the genus Micrococcus: Kocuria gen. nov., Nesterenkonia gen. nov., Kytococcus gen. nov., Dermacoccus gen. nov., and Micrococcus. International Journal of Systematic Bacteriology 4, 682-692. Ward, O.P (1985). Proteolytic enzymes, in Comprehensive Biotechnology Moo-Young Editor, Pergamon Press. Oxford, U.K. pp.790-817

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KOCURIA ROSEA AS A NEW FEATHER DEGRADING BACTERIA Williams, C. M., Richter, C. S., Mackenzie, J. M., Jr. and Shih, J. (1990) Isolation, identification, and characterisation of a feather-degrading bacterium. Applied and Environmental Microbiology 56, 15091515. Williams, C. M., Lee, C.G., Garlich, J.D. and Shih, J. (1991). Evaluation of a feather fermentation product, feather lysate, as a feed protein. Poultry Science 70, 85-94. Xiang Lin, Chung-Ginn Lee, Ellen S. Casale and Shih, J. (1992) Purification and Characterisation of a Keratinase from a Feather-Degrading Bacillus licheniformis Strain. Applied and Environmental Microbiology 58, 3271-3275.

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COMPARISON OF PB2+ REMOVAL CHARACTERISTICS BETWEEN BIOMATERIALS AND NON-BIOMATERIALS DONG SEOG KIM1 AND JUNG HO SUH2 1 Department of Environmental Science, Catholic University of TaeguHyosung, Kyungbuk 712-702 and 2Department of Industrial Chemistry, Ulsan College, Ulsan 680-749, Korea.

Abstract The order of Pb2+ removal capacities in chemical adsorbents were found as ion exchange resin > zeolite > granular activated carbon (GAC) > powdered activated carbon (PAC), while in biomass was Aureobasidium pullulans > Saccharomyces cerevisiae > activated sludge. Although Pb2+ removal capacity (mg Pb2+/g) of the activated sludge (30.9) was lower than those of ion exchange resin (167.7) and other pure cultures of A. pullulans (170.4) and S. cerevisiae (95.3), it was higher than those of other chemical adsorbents such as GAC (26.9), PAC (2.1), and zeolite (30.2). The initial Pb2+ removal rates in chemical adsorbents were in the order of PAC > GAC > zeolite > ion exchange resin, while in biomass was A. pullulans > activated sludge > S. cerevisiae. The initial Pb2+ removal rate of activated sludge was higher than those of GAC, zeolite, ion exchange resin and S. cerevisiae cells. 1. Introduction There are numerous reports documenting the capability of pure cultures of bacteria (Yong and Macaskie, 1997), algae (Leush et al., 1995), and fungi (Suh et al., 1998) to remove heavy metal ions from solution. According to the report of Shumate and Strandberg (1985), multi-species communities of bacteria removed silver equal to 32% of the dry cell weight, which was considerably higher than those exhibited by pure cultures of Pseudomonas maltophilia, Thiobacillus thiooxidans, and T. ferroxidans. The important point was thus made that mixed microbial cultures could be more efficient in removing heavy metal ions than pure cultures. Removal of heavy metal ions by mixed cultures of activated sludge have been studied for the last 40 years by many investigators (Ruchoft, 1949; Brown and Lester, 1982; Rudd et al., 1984) to evaluate the possibility for removing a number of heavy metal ions. 177 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 177–183. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

DONG SEOG KIM AND JUNG HO SUH

In particular, Pb2+, well recognised for its detrimental effect on the environment where it accumulates throughout the food web, is generated from mining, metal, dyestuff, electric, and petroleum industries. Pb2+ was known to be easily removed by microorganisms. Rossin et al. (1982), for example, pointed out that average heavy metal ion removal with activated sludge was as low as 1% for Ni2+ and as high as 92% for Pb2+. Equilibrium metal uptake values using freeze-dried Rhizopus arrhizus increased in the order of Sr2+ < Mn2+ < Zn2+ < Cd2+ < Cu2+ < Pb2+, and were positively correlated with the covalent index of the metal ions (Brady and Tobin, 1995). The order of adsorption for Sargassaumfluitans biomass particles (Leush et al., 1995) was in the order of Pb2+ > Cd2+ > Cu2+ > Ni2+ > Zn2+. A major goal of this research is to examine the feasibility of activated sludge on Pb2+ removal, by comparison with two pure cultures (Saccharomyces cerevisiae and Aureobasidium pullulans) and widely used chemical adsorbents (activated carbons, ion exchange resin and zeolite). 2. Materials and methods 2.1. MATERIALS Activated carbon, which is used in a filtration plant, was made with charcoal and the sizes of granular activated carbon (Sigma C2764, USA) and powdered activated carbon (Sigma C5260, USA) were 4~8 mesh and 100~400 mesh, respectively. Cation exchange resin (SK1B) with sulfonyl group was purchased from Sam Yang Co. (Korea). Zeolite (Z3125) was obtained from Sigma Co. as a powder type, of which diameter was below 10 µm. Activated sludge was prepared from the secondary return-sludge from the municipal wastewater treatment plant. 2.2. MICROORGANISMS AND CULTURE CONDITIONS Aureobasidium pullulans KFCC (Korean Foundation of Culture Collection) 110245 was aerobically cultivated with 100 ml medium containing (as g/l) 200, sucrose; 20, yeast extract; 5, K2HPO4; 2, MgSO4.7H2O; 15, NaNO3 in a rotary-shaker incubator (150 rpm) at 300 for 72 h. Saccharomyces cerevisiae KCTC (Korean Collection for Type Cultures) 1199, wasted from the brewery industry, was cultured at 30°C for 72 h in 300 ml conical flasks with 100 ml medium composed of (as g/l) 100, glucose; 8.5, yeast extract; 1.32, NH4Cl; 0.11, MgSO4; 0.06, CaCl2 in a rotary-shaker incubator with 150 rpm. Activated sludge or pure cultures were harvested by centrifugation (3,000 x g, 10 min) and then washed three times with distilled deionised water, and stored at 4 in a refrigerator until uses in the experiments. 2.3. PB2+ REMOVAL EXPERIMENT In the case of biomass, the prepared activated sludge or cell suspension was mixed with an equal volume of the initial concentration of aqueous Pb(NO3)2 solution prepared in twice the desired concentration. The pH values of the Pb2+ solution, biomass 178

PB2+ REM0VAL CHARACTERISTICS OF BIOMATERIALS AND NON-BIOMATERIALS

suspensions, and the mixture were 3.0-4.0, 5.5-5.7 and 3.5-5.5, respectively. pH adjustment was not conducted and no spontaneous metal precipitation was observed in the prepared solutions. The experiments were carried out by employing 50 ml Pb2+ solution and 50 ml activated sludge or cell suspension in 300 ml conical flasks and shake them on a rotary-shaker incubator at (150 rpm). Samples of 1.8 ml were taken at the proper time period and centrifuged immediately (10,000 x g, 10 min). The Pb2+ concentration in the supernatant was measured by atomic absorption spectrometry (Perkin Elmer 3300, USA). The dry weight of biomass was obtained after drying at 105| to a constant weight. Removed Pb2+ amounts per dry weight of adsorptive materials at equilibrium state (q) were calculated from Pb2+ mass balance yield : q (mg Pb2+/g dry weight) = (Ci - Ce)/m. Where Ci and Ce are the Pb2+ concentrations (mg Pb2+/l) in the supernatant at the initial and equilibrium state, respectively, and m is the concentration of the dried activated sludge or cells (mg dry weight/l). The initial Pb2+ removal rate (ri) was measured by calculating the slope from the plot of the removed Pb2+ amounts per dry weight (mg Pb2+/g dry weight) vs. time (min) at t=0. 3. Results and discussion 3.1. PB2+ REMOVAL CHARACTERISTICS

Fig, 1. Typical time courses of Pb2+ removal by chemical adsorbents under various initial Pb2+ concentrations: (a) granular activated carbon, (b) powdered activated carbon, (c) ion exchange resin, (d) zeolite. Initial adsorbent concentrations (g/l) in (a), 1.0; (b), 2.0; (c), 1.0; (d) 1,0,

The order of Pb2+ removal capacity in chemical adsorbents was found as ion exchange resin > zeolite > granular activated carbon (GAC) > powdered activated carbon (PAC) (Fig. 1). On the comparison between the results of GAC and PAC, the Pb2+ removal 179

DONG SEOG KIM AND JUNG HO SUH

capacity of GAC was three times higher than that of PAC. However, the time required to reach an equilibrium state with GAC was much longer than with PAC because GAC has much larger specific surface area and pore diffusion resistance than PAC. The increase in Pb2+ removal capacity according to the increase of initial Pb2+ concentration in biomass was similar to those of chemical adsorbents (Fig. 2). The Pb2+ removal capacity of the activated sludge was increased only 1.8 times (from 42 mg Pb2+/g to 74 mg Pb2+/g) even though the initial Pb2+ concentrations increased by 6-fold (from 37 mg/l to 228 mg/l).

Fig.2. Typical time courses of Pb2+ removal by biomass various initial Pb2+ concentrations: (a) activated sludge, (b) A, pullulans, (c) S. cerevisiae. Initial biomass concentrations (g/l) in (a), 1.0; (b). 0.8; (c), 0.8.

An interesting result was obtained by the two selected microorganisms. That is, in our experimental range, as the initial Pb2+ concentrations were increased accurately 5.7 times (from 49 mg/l to 278 mg/l) and 6 times (from 16 mg/l to 96 mg/l) in A. pullulans and S. cerevisiae, respectively, the Pb2+ removal capacities were increased about 5.7 times (from 53 mg Pb2+/g to 300 mg Pb2+/g) and 6 times (from 12 mg Pb2+/g to 72 mg Pb2+/g), respectively. The Pb2+ removal in pure cultures of A. pullulans and S. cerevisiae gave more favourable result than activated sludge. Considering Pb2+ removal characteristics, it was found that the time required to reach an equilibrium state in activated sludge and A. pullulans was independent on the initial Pb2+ concentrations. But, in S. cerevisiae, the time was increased in response to 180

PB2+ REMOVAL CHARACTERISTICS OF BIOMATERIALS AND NON-BIOMATERIALS

the initial Pb2+ concentration, and was much longer than those of the activated sludge and A. pullulans. This may be caused by the difference in Pb2+ removal process. In other words, most of the Pb2+ taken up by S. cerevisiae was deposited in the inner parts of the cell at equilibrium state and the Pb2+ accumulation mechanism was divided into three steps, involving metabolism-independent, -dependent and -independent (Suh et al, 1998a). On the contrary, A. pullulans used a different metabolism-independent Pb2+ accumulation process due to the existence of extracellular polymeric substances (EPS) (Suh et al., 1998b). Therefore, it was suggested that Pb2+ penetration into inner cellular regions may not occurred in the activated sludge because the trend in Pb2+ removal of the activated sludge has a resemblance to that of A. pullulans. Nevertheless, the detailed process is still in doubt because activated sludge is composed of a great number of organisms and several organic materials such as extracellular polymeric substances and cell flocs, etc. When compared the Pb2+ removal capacity of chemical adsorbents with those of biomass, the Pb2+ removal characteristics of activated sludge and A. pullulans marked similar process to that of PAC. Moreover, S. cerevisiae had a similar removal process to that of ion exchange resin. The ion exchange played an important role in the Pb2+ removal in S. cerevisiae but little occur in A. pullulans (data not shown). A similar result was observed when the initial chemical adsorbents or biomass concentrations were changed, too (data not shown). Table 1. Comparison of adsorption models and the at q10 and q200 between the chemical absorbents and biomass

Materials Granular carbon Powdered carbon

activated

Model (r2 value) Langmuir Freundlich

removed Pb2+

amounts

q10* (mg Pb2+/g)

q200 * (mg Pb2+/g)

0.95

0.84

26.0

36.5

0.96

0.90

2.1

17.3

Ion exchange resin

0.92

0.92

167.7

-

Zeolite

0.99

0.91

30.2

57.7

Activated sludge

0.93

0.98

30.9

68.8

A. pullalans

0.72

0.82

170.4

235.8

S. cerevisiae

0.97

0.94

95.3

272.7

activated

*q10 and q200 represent removal amouts of Pb2+ (mg Pb2+/g absorbent or biomass) at the equilibrium concentrations of 10 mg/l and 200 mg/l, respectively.

The Pb2+ removal capacity in chemical adsorbents, evaluating by q10, was in the order of ion exchange resin > zeolite > GAC > PAC, while that of biomass was A. pullulans > S. cerevisiae > activated sludge. A. pullulans showed remarkably higher Pb2+ removal capacity than S. cerevisiae in q10 due to the effect of EPS formation. However, the q200 value of S. cerevisiae was slightly higher than that of A. pullulans. The terms of q10 and q200 were the Pb2+ removal amounts (mg Pb2+/g) at the equilibrium concentrations of 10 mg/l and 200 mg/l, respectively. This interesting phenomenon was observed when the 181

DONG SEOG KIM AND JUNG HO SUH

initial Pb2+ concentration was very high or initial biomass concentration was extremely low. This result may be caused by the difference in Pb2+ removal mechanisms, but further study is required to reinforce this result. In general, q10 may be more reasonable than q200 from the practical points of view. The Pb2+ removal capacity of the activated sludge was relatively lower than those of other biomass (A. pullulans and S. cerevisiae) or ion exchange resin, but higher than those of other chemical adsorbents (GAC, PAC, and zeolite). This result implies the possible application of the activated sludge on Pb2+ removal process. The Pb2+ removal capacity of the activated sludge was around one to fifth and one to third as low as those of A. pullulans and S. cerevisiae, respectively. However, the utilisation of activated sludge on Pb2+ removal can be recommended, taking into consideration of economical and practical aspects and the stability of operation system. 3.2. INITIAL PB2+ REMOVAL RATE In heavy metal removal processes, not only the removal capacity but also the initial removal rates are considered to be very important factors from the practical aspects of reactor design and process optimisation. The initial Pb2+ removal rate in response to the variation of initial Pb2+ concentration is shown in Fig. 3. The initial Pb2+ removal rate was increased as initial Pb2+ concentration increased. However, it was almost independent of initial Pb2+ concentration over a critical concentration. The Pb2+ removal rates of activated sludge and A. pullulans were much higher than those of the chemical adsorbents, but S. cerevisiae was not the case. The order of initial rate of Pb2+ removal in adsorbents was found as PAC > GAC > zeolite > ion exchange resin (Fig. 3(a)). Where, the low degree of resistance in pore diffusion of PAC might cause the highest initial Pb2+ removal rate. Moreover, the ion exchange resin showed the lowest initial Pb2+ removal rate because ion exchange is here main process rather than physical adsorption.

Fig. 3. Comparison of the initial Pb2+ removal rates between (a) chemical adsorbents and (b) biomass: Symbols in (a); (0) GAC, PAC, ion exchange resin, zeolite. Symbols in (b); (0) activated sludge, A. pullulans, (A) S. cerevisiae.

182

PB2+ REMOVAL CHARACTERISTICS OF BIOMATERIALS AND NON-BIOMATERIALS

On the other hand, the initial Pb2+ removal rate in biomass was in the order of A. pullulans > activated sludge > S. cerevisiae, as shown in Fig. 3(b). The Pb2+ removal in A. pullulans was very rapid, whereas that in S. cerevisiae was very slow. This is, as mentioned earlier, because Pb2+ removal in A. pullulans was mainly achieved by adsorption onto EPS around the cell surface and that in S. cerevisiae was caused by the Pb2+ penetration into the inner cellular region. Therefore, by evaluating as initial Pb2+ removal rate, the activated sludge was placed in the middle of A. pullulans and S. cerevisiae, and it can be recommended as an useful resource for the removal process of Pb2+. 4. Conclusions The comparison of Pb2+ removal characteristics between chemical sorbents (GAC, PAC, zeolite, ion exchange resin) and biological materials (activated sludge, A. pullulans, S. cerevisiae) was conducted. The Pb2+ removal capacities of biological materials were higher than that of chemical materials. And the biological materials showed the higher initial Pb2+ removal rate than the chemical materials, except the case of ion exchange resin. Therefore, the biological materials can be applied effectively to the heavy metal removal process even though the application will be needed more research works. Especially, activated sludge that is used in municipal wastewater treatment facility may be easily applied to the continuous heavy metal removal process, in situ, compared to other biological materials References Brady, J.M. and Tobin, J.M. (1995) Binding of hard and soft metal ions to Rhizopus arrhizus biomass. Enzyme Microb. Technol. 17, 791-796. Brown, M.L. and Lester, J.N. (1982) Role of bacterial extracellular polymers in metal uptake in pure bacterial culture and activated sludge-I Effects of metal concentration, Water Res. 16, 1539-1548. Leusch, A., Holan, Z.R. and Volesky, B. (1995) Biosorption of heavy metals (Cd, Cu, Ni, Pb, Zn) by chemically-reinforced biomass of marine algae. J Chem. Tech. Biotechnol. 62, 279-288. Rossin, A.C., Sterritt, R.M. and Lester, J.N. (1982) The influence of process parameters on the removal of heavy metals in activated sludge. Water, Air, andSoil Pollution 17, 185-198. Ruchoft, C.C. (1949) The possibilities of disposal of radioactive wastes by biological treatment methods. Sewage Works J. 21, 877-883. Rudd, T., Sterritt, R.M. and Lester, J.N. (1984) Complexation of heavy metals by extracellular polymers in the activated sludge. J Water Pollut. Control Fed. 56, 1260-1268. Shumate II, S.E. and Strandberg, G.W. (1985) Accumulation of metals by microbial cells, in M. Moo-Young (ed.), Comprehensive Biotechnology, Pergamon Press, New York, vol. 4, pp. 235 - 247. Suh, J.H., Kim, D.S., Yun, J.W. and Song, S.K. (1998a) Process of Pb2+ accumulation in Saccharomyces cerevisiae. Biotechnol. Left. 20, 153-156. Suh, J.H., Yun, J.W. and Kim, D.S. (1998b) Comparison of Pb2+ accumulation characteristics between live and dead cells of Saccharomyces cerevisiae and Aureobasidium pullulans. Biotechnol. Lett. 20,247-251. Yong, P. and Macaskie, L.E. (1997) Removal of lanthanum, uranium and thorium from the citrate complexes by immobilised cells of Citrobacter sp. in a flow-through reactor: implications for the decontamination of solutions containing plutonium. Biotechnol. Lett. 19, 251-255.

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HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERIA GY. BARABÁS1, GY. VARGHA1, I. SZABÓ1, A. PENYIGE1, J. SZÖLLÖSI2, J. MATKÓ2, S. DAMJANOVICH2 AND T. HIRANO3 University Medical School, Departments of Human Genetics1 and Biophysics & Cell Biology2, H-4012 Debrecen, Hungary; E-mail: [email protected]. hu, and The Jikei University, School of Medicine3, Tokyo, Japan

Abstract Streptomyces strains from Kuwait oil field were isolated by selective inorganic media containing the oil fractions as sole carbon and energy sources. Hydrocarbon utilisation was measured by gas-liquid chromatography, and by the use of proper radioactively labelled oil fraction. Streptomyces strains rapidly used n-alkanes, and incorporated them to corresponding fatty acids of identical chain length. n-Alkane uptake was markedly increased by specific GTP-binding protein activators. Fluorescence measurements of the uptake of the hydrophobic diphenylhexatriene (DPH) showed significant difference between oil-utilising and non-utilising strains. 1. Materials and methods 1.1 TEST ORGANISMS. OLIGOCARBOPHYLIC STREPTOMYCES Strains isolated from Kuwait desert oil fields (Barabás et al., 1995) were coded as KCC (Kuwait Culture Collection). KCC26, KCC28, KCC30 and KCC42 were identified as Streptomyces plicatus, KCC25 as Streptomyces griseoflavus. Strains KCC18, KCC33, KCC36 and Khiran30 have not been taxonomically identified yet, they show, however, the typical Streptomyces morphology in light microscope. The strains, their isolation, the composition of starch-casein medium and their potential of utilising n-alkanes were previously described (Barabás et al., 1995). S. griseus 2682 was used as an oil nonutilising control (designated as ,,non-utilising”).

185 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 185–190. ©2001 KIuwer Academic Publishers. Printed in the Netherlands.

GY. BARABÁS ET AL

1.2 BIOMASS PREPARATION The biomass samples were obtained by growing the test organisms on suitable conventional media at 27°C. The Streptomyces strain was cultivated on sterile cellophane sheets covering the surface of starch-casein-agar medium: starch, 10g; casein (Difco, vitamin free), 0.3g; KNO3, 2g; NaCl, 2g; K2HPO4, 2g; MgSO4.7H2O, 0.05g; CaCO3, 0.02g; FeSO4.7H2O, 0.01g; Bacto agar, 18g; soil extract (from 1% soil suspension), 100 ml; distilled water to 1000 ml; pH 6.8. The cultures were incubated at 27°C for 5 d. The Streptomyces biomass samples were harvested by removing the cellophane sheets. For fluorescence measurement of diphenylhexatriene (DPH) uptake, n-hexadecane (C16) utilising and non-utilising strains were cultivated in filtered soybean liquid medium described previously for S. griseus 52-1 (Szabó et al., 1985). Cells were harvested after 36 h cultivation, washed twice with distilled water, resuspended in inorganic medium (IM) with the following composition in g per litre: 0.85 NaNO3; 0.56 KH2PO4; 0.86 Na2HPO4; 0.17 K2SO4; 0.37 MgSO4.7H2O; 0.007 CaCl2.6H2O; 0.004 FeIIIEDTA; 2.5 ml of a trace element solution consisting of (g per litre): 2.32 ZnSO4.7H2O; 1.78 MnSO4.4H2O; 0.56 H3BO3; 1.0 CuSO4.5H2O; 0.39 Na2MoO3.2H2O; 0.66 KI; 1.0 EDTA; 0.4 FeSO4.7H2O; 0.004 NiCl2.6H2O. Incubation was carried out at 27°C in a gyratory shaker at 300 rpm. The optical density of the cell suspension was adjusted to 0.5 at 535 nm and 2% (V/V) of n- hexadecane (C16) was added to IM. 1.3 INCORPORATION OF RADIOACTIVITY FROM LABELLED n-HEXADECANE INTO MYCELIA Strains were cultivated for 48 hours in 5 ml of inorganic medium (IM) supplemented with 20% of filtered soybean medium (Szabó et al., 1985) in 50-ml Erlenmeyer flasks. 2 µl of hexadecane-1-14C (105 dpm) was added to each culture at the inoculation. After 48 h of incubation mycelia were pelleted at 3000g. 100 mg aliquots of the mycelia were hydrolysed with 6 n HCl at 95°C for 48 h. The HCl and traces of not metabolised labelled oil was evaporated. After dissolving the dried remnant material in 200 µl water it was extracted with hexane. The radioactivities of both the water and hexane phases were measured by scintillation counting. 1.4 FLUORESCENCE MEASUREMENTS The different potential of C16 utilising and non-utilising strains to take up hydrocarbons was studied by fluorescence measurements. For this purpose, the hydrophobic compound diphenylhexatriene (DPH) was selected as the test fluorescent material. Fresh biomass samples of C16 utilising and non-utilising strains grown in filtered soybean medium were resuspended in IM than the samples were supplemented with 90 µM DPH dissolved in n-hexadecane. Their fluorescence was examined in quartz cells in a Perkin-Elmer MPF 44B spectrofluorimeter equipped with a thermostatic cell holder. The DPH fluorescence of cells was excited at 355 nm and the emission was collected at 430 nm. The cell suspensions did not show significant autofluorescence in this wavelength range. In experiments when the kinetics of DPH uptake was investigated, 186

HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERlA

small aliquots (2ml; n=3) were taken from the culture flask from time to time, washed and the DPH fluorescence (corrected for light scattering) was determined. 1.5 ANALYSIS OF FATTY ACIDS The fatty acid determinations were performed as described by Barabás et al., 1995. 1.6 INVESTIGATIONS WITH GTP ANALOGUES 0.5 g aliquots of mycelium precultivated in soy-bean medium for 36h were resuspended in 25 ml aliquots of inorganic medium containing 10 mg n-hexadecane or n-octadecane. The submerged cultures were supplemented with various amounts of GTPγS or AIF4and incubated at 30°C with a shaking frequency of 250 rpm. Biomass was harvested after 6 hours by centrifugation and the hydrocarbon uptake of the cells was determined from these samples as described in Barabás et al., 1995.

2. Results and discussion Streptomyces are soil bacteria occurring even under extreme conditions (hot desert, saltmarsh area, alpine slopes, etc.). They are capable of hydrocarbon uptake and utilisation. Uptake of oil as followed by incorporated diphenylhexatriene (DPH) from nhexadecane phase of the medium was significantly higher in the oil utilising strain than in a non-utilising one (Table 1, Radwan et al., 1998). Fluorescence measurements with DPH showed that the membrane characteristics of hydrocarbon-using strains significantly differed from that of the non-using ones (Radwan et al., 1998). Incorporation of the label from radioactive hexadecane into mycelia was also measured in several oil degrading strains and one non-degrading one (Table 2). During the 48-hour cultivation with labelled hydrocarbon the highest amount found to be incorporated (into strain KCC18) was equivalent to about 3% of the total amount of the radioactivity added to the culture. Other strains incorporated somewhat less amount, and strain Khiran30, that served as a control, could not incorporate radioactivity. Table 1 DPH fIuorescence incorporated frorn n-hexadecane phase by oil utilising (KCC 26) and non-utilising (S griseus) strains

Time (hour)

DPH fluorescence (arbitrary units) KCC 26 S. griseus 0.039 0.010 0.361 0.283 0.65 1 0.324 0.820 0.315 0.795 0.344 0.852 0.308

19 40 62 91 122 182

187

GY. BARABÁS ET AL

Table 2. Incorporation of the label from hexadecane-l-14C into mycelia of Streptomyces strains Strain

KCC42

KCC18

KCC36

KCC33

KCC25

Khiran30

Radioactivity (dpm)

1956

2985

1719

2070

393

0

Harvested mycelia were hydrolysed with 6 n HCl and the radioactivity of water-soluble components was determined. The non-utilising Khiran30 strain was used as control.

Fatty acid analysis showed that the incubation with n-alkanes resulted in an increase of the fatty acids with chain length equivalent to those of the alkane substrates (Barabás et al., 1995). The fatty acid 16:1 fraction increased from 17.4 to 29.9 in the presence of nhexadecane. Fatty acids of C18 appeared only in the presence of n-octadecane. It is known that GTP-binding proteins play essential role in mediating cellular responses to a wide variety of extracellular signals, such as hormones, growth factors, neurotransmitters, chemical signals or light (Gilman, 1987, Taylor, 1990). These proteins transmit signals via a GTP-dependent mechanism to the effector systems (enzymes or ion channels) and thereby regulate these systems that often control production of intracellular second messenger molecules. GTP-binding proteins (GBPs) act as molecular switches, their activation is catalysed by a ligand activated receptor and deactivation is established by the intrinsic GTPase activity of the GBP. According to this mechanism, the GTP-bound form is the active complex, which returns to inactive state by hydrolysing its GTP to GDP. The exchange of GDP to GTP and the rate of GTP hydrolysis is regulated by specific regulatory proteins (Gilman, 1987, Itoh et al., 1986, Taylor, 1990). GTP-binding proteins were reported to be present in S. coelicolor A(3)2 and we have shown the presence of these proteins in S. griseus and in several other Streptomyces strains (Itoh et al., 1996, Penyige et al., 1992). Our previous results suggested that in S. griseus A-factor - a γ-utyrolactone type autoregulator molecule produced by wild type S. griseus cells and required for the normal differentiation process and antibiotic production in the producer strain (Khokhlov et al., 1973) - could activate an intrinsic GTPase activity present in the cellular membrane of S. griseus NRRL B-2682 (Penyige et al., 1992). The study of their role in different cellular processes was greatly enhanced by using certain reagents, such as AIF4- or GTPγS. AlF4- mimics the effect of the γ-phosphate of GTP on the inactive GDP-bound form of the protein, GTPγS is a non-hydrolysable GTP analogue (Yatani et al., 1991).

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HYDROCARBON UTILISATION BY STREPTOMYCES SOIL BACTERIA Table 3. The effect of AlF4- on n-hexadecane and n -octadecane uptake by oil utilising micro-organisms

Microbial strains

n-alkanes

KCC25

C16 C18 C16 C18 C16 C18 C16 C18 C16

KCC 28 KCC 33 KCC 42 A. nicotiana

µM AIF40 7.2 6.3 8.0 5.2 9.2 7.2 7.6 10.3 5.9

50 10.0 ND 9.4 4.9 10.5 ND 12.3 ND 4.9

75 12.5 ND 9.8 5.3 10.9 6.2 ND 12.4 6.7

100 12.0 ND 9.7 7.8 11.8 6.6 13.7 11.2 12.9

125 11.6 ND 9.9 10.2 13.6 8.2 15.7 11.8 10.8

Data are expressed in mg alkane consumed by 1 g fresh biomass at 27°C in 6 h ND: not determined Arthrobacter nicotiana was used as control strain. In this case the incubation period was 24 h.

Table 4. The effect of GTPγS on n -hexadecane uptake by oil utilising micro-organisms

GTPγS [µg/ml] 0 10 20 50 100

KCC 25 3.2 12.3 12.8 ND 13.4

n-Hexadecane consumed Arthrobacter nicotiana 1.8 4.2 6.9 9.9 11.1

Data are expressed in mg alkane consumed by 1 g fresh biomass at 27°C in 6 h. ND: not determined Arthrobacter nicotiana was used as control strain. In this case the incubation period was 24 h.

The results of the effect of GTPγS and AIF4- – stimulators of GBPs – on the uptake of the hydrocarbon molecules (C16 and C18) are shown in Tables 3 and 4. These show that the uptake of n-hexadecane (C16) and n-octadecane (C18) from the medium by and hydrocarbon utilising micro-organisms were enhanced by the addition of AIF4- although the rate of uptake was strain specific. Moreover, the magnitude of uptake was, in most cases, directly proportional to the concentration of these effectors in the medium. These results suggest that GBPs could fulfil important physiological functions in Streptomyces strains. With electron microscopy the hydrocarbon utilising strains were found to be enriched in large less-electron dense areas as inclusions in the cytoplasm in n-alkane 189

GY. BARABÁS ET AL

containing media (Radwan et al., 1998). The oil utilising strains also eliminated hydrocarbons from soil samples artificially impregnated with hydrocarbons in laboratory experiments. Field experiments for oil bioremediation are in progress. 3. Conclusion Streptomyces strains isolated from Kuwait oil fields actively utilised oil fractions and crude oil, either in hydrocarbon containing inorganic media, or in the soil. Since these strains are typical soil bacteria tolerating extreme conditions (hot desert, alpine slopes, salt-marsh area) their practical application in bioremediation of oil pollution is promising. References Barabás, Gy., Sorkhoh, N.A., Fardoon, F. and Radwan, S.S. (1995) n-Alkane-utilisation by oligocarbophilic actinomycete strains from oil-polluted Kuwaiti desert soil. Actinomycetologica 9, 13-18. Gilman, A. G. (1987) G proteins: transducers of receptor-generated signals. Annu. Rev. Biochem. 56,615-649. Itoh, H., Kozasa, T., Nagata, S., Nakamura, S., Katada, T., Ui, M., Iwai, S., Ohtsuka, E., Kawasaki, H., Suzuki, K. and Kaziro, Y. (1986) Molecular cloning and sequence determination of cDNAs for a subunits of the guanine nucleotide-binding proteins Gs, Gi and Go from rat brain. Proc. Nail. Acad. Sci. USA 83, 3776-3780. Itoh, M., Penyige, A., Okamoto, S. and Ochi, K. (1996) Proteins that interact with GTP in Streptomyces griseus and its possible implication in morphogenesis. FEMS Microbiol. Lett. 135, 311-316. Khokhlov, A. S., Anisova, L.N., Tovarova, J.J., Kleiner, E.M., Kovalenko, O.S., Krasilnikova, O.S., Kornitskaya, E.Y. and Pliner, S.A. (1973) Effect of A-factor on growth of asporogeneous mutant of Streptomyces griseus, not producing this factor. Z Allg. Microbiol. 13,647-655. Penyige, A., Vargha, Gy., Ensign, J.C and Barabás, Gy. (1992) The possible role of ADP-ribosylation in physiological regulation in Streptomyces griseus. Gene 115, 181-185. Radwan, S.S., Barabás, Gy., Sorkhoh, N.A., Damjanovich, S., Szabó, I., Szöllôsi, J., Matkó, J., Penyige, A., Hirano, T. and Szabó, I.M. (1998) Hydrocarbon uptake by Streptomyces. FEMS Microbiol. Letters 169, 87-94 Szabó, I., Benedek, A. and Barabás, Gy. (1985) Possible role of streptomycin released from spore cell wall of Streptomyces griseus. Appl. Environ. Microbiol. 50, 438-440. Taylor, C. V. (1990) The role of G proteins in transmembrane signalling. Biochem. J. 272, 1-13. Yatani, A,, and Brown, A.M. (1991) Mechanism of fluoride activation of G protein-gated muscarinic atrial K+ channels. J. Biol. Chem. 266,22872-22877.

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PART 6 FOOD SECURITY AND FOOD PRESERVATION

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MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS: A REVIEW M. HEYNDRICKX, N. RIJPENS AND L. HERMAN Ministry of Small Enterprises, Traders and Agriculture, Centre for Agricultural Research, Department for Animal Product Quality, Brusselsesteenweg 3 70, B-9090 Melle, Belgium

Abstract In most developed countries a sharp increase in foodborne intoxications occurs since the last decade. Amongst the bacterial pathogens, the incidence rate is highest for Campylobacter jejuni and Salmonella spp. Nucleid acid based identification and detection methods have been developed for nearly all bacterial pathogens, based on probes in hybridisation assays or primers in PCR, NASBA or RT-PCR assays. As targets for molecular identification, virulence genes, the rRNA gene region or other specific sequences can be used and several commercialised systems are already available. For the (direct) detection of pathogens in food products, several problems may be encountered: PCR inhibition by food components, contamination in sensitive PCR assays, detection of living as well as dead cells. The latter problem can be solved by using mRNA as amplification target, but for routine applications the combination of a short culturing period with a less sensitive PCR is more suitable. Direct quantification of pathogens is possible with quantitative competitive PCR using an internal standard or with kinetic quantitative PCR (TaqMan or LightCycler commercial system). In bacterial typing, distinct types, strains or clones within a pathogenic bacterial species are differentiated which is important in epidemiological studies of foodborne outbreaks but also in the “from stable to table” investigation of the whole food production chain. Compared to the classical phenotypic typing techniques, molecular typing techniques have several advantages such as general applicability and a high discriminatory power. The currently available molecular techniques can be classified according to their working principle in PCR-mediated typing techniques (RAPD, repPCR), typing techniques combining PCR with restriction analysis (e.g.flaA typing of C. jejuni), typing techniques based on chromosomal restriction fragment length polymorphisms (e.g. ribotyping, pulsed field gel electrophoresis or PFGE), typing techniques combining restriction digestion with selective amplification (AFLP), and plasmid analysis. Both PFGE and AFLP are proposed as likely candidates for a uniform definite molecular typing approach using appropriate software for cluster analysis and 193 A. Durieux and J-P. Simon (eds.), Applied Microbiology, 193–238. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

M. HEYNDRICKX N. RIJPENS AND L. HERMAN

database storing of the fingerprints. For Salmonella, two typing levels can be proposed: the first important level corresponds with the serovar level and the second level can be performed by classical phage typing or molecular typing revealing clonal lineage or strain level. Several molecular techniques have a serovar dependent discriminatory power with the greatest challenge presented by the highly clonal serovar Salmonella enteritidis. 1. Introduction In the last decade a sharp increase in foodborne intoxications has been observed in most developed countries. This may sound surprising because it is generally assumed that hygiene at home during cooking and food storage and in manufacturing practices in the food industry has greatly improved and is of a sufficiently high level in this hightechnology society. This phenomenon may be attributed to several factors such as the increasing international trade in food and food and feed ingredients, international tourism, climate changes, demographic changes in the population with a higher number of elderly people, changing eating habits with a greater proportion of ready-to-eat foods and mass catering facilities. The general trend is that the zoonoses, i.e. infectious diseases caused by animal associated microorganisms such as Salmonella, are becoming a particular increasing problem which is probably associated with the current bioindustrial practice of mass production of animal products (e.g. poultry). In the food industry, food processors and regulators are asked to establish, control and monitor critical control points in the plant environment as essential part of the development and use of a HACCP concept. It is likely that this type of approach will become more important in the near future as food safety concerns increase in food processing and distribution systems. Molecular detection and typing methods are powerful tools in the whole food production chain for quality control and to unravel the prevalence and the epidemiology of the foodborne pathogens. In this paper a review is given of the molecular detection, identification and typing techniques for foodborne bacterial pathogens in the last decade. Their possibilities and eventually their associated problems, limitations and possible solutions are also discussed with reference to own observations or those of other researchers. The term “molecular” techniques is used here to denote those techniques which are “molecular biology based”, and more specific those that make use of the nucleic acids (DNA or RNA) in the bacterial cell. 2. Characteristics of the foodborne bacterial pathogens Foodborne intoxications can be caused by viruses, pathogenic bacteria, parasitic protozoa and nematodes, and by toxins of natural origin (e.g. scombrotoxin caused by bacterial histamine production in food). Most of the intoxications are of viral or bacterial nature, The most prevalent bacterial pathogens causing foodborne intoxications in the developed world are listed in Table 1. This list contains Gramnegatives, i.e. several representatives of the Enterobacteriaceae (pathogenic Escherichia coli, Salmonella, Yersinia, Shigella), Aeromonas, Campylobacter and 194

MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS

Vibrio, as well as Grampositives (Clostridium, Staphylococcus, Listeria, Bacillus, Streptococcus and Enterococcus). Other pathogenic bacterial species occurring in food can be considered as less important because their associated foodborne intoxications occur very rarely. Vibrio cholerae serogroup 01 is a major water and foodborne pathogen in underdeveloped countries because of a lack of elementary hygiene, but is almost eradicated in the western world. A lot of the minor pathogens are highly related to the bacterial species of Table 1 (e.g. Campylobacter coli, Vibrio vulnificus, V. cholerae serogroup non-O 1, enterotoxigenic, enteropathogenic and enteroinvasive E. coli, Bacillus subtilis). In addition, many bacteria can be considered as putative pathogens (e.g. Aeromonas caviae and A. sobria, marine Vibrio spp., Plesiomonas shigelloides, several enteric bacteria such as Citrobacter freundii). Some evidence has been obtained for a link between the human Crohn’s disease and Mycobacterium paratuberculosis occurring in raw and pasteurised milk (Thompson, 1994). The symptoms of acute foodborne diseases caused by bacterial pathogens (see Table 1) can roughly be divided in gastrointestinal symptoms, either of the upper gastrointestinal tract (nausea, vomiting) or of the lower gastrointestinal tract (diarrhoea, abdominal cramps), respiratory symptoms (sore throat), and neurological symptoms (visual disturbance, vertigo, paralysis). Usually not all of these acute symptoms occur in any individual with a foodborne infection and also the chronic symptoms and complications are no inevitable consequences, with the exception of botulism caused by Clostridium botulinum which in most cases leads to death if not treated with antitoxin. The course and severity of a foodborne disease is largely influenced by the virulence and the ingested dose of the bacterial strain involved, and by the fitness and immune capacity of the infected host. Infants, young children (<5 years), the elderly and debilitated persons with a compromised immune system (cancer, AIDS and transplant patients) are particular segments of the population which are more vulnerable to foodborne intoxications with a higher risk for complications, e.g. Salmonella enteritidis has a mortality rate of approximately 3.6% in hospital and nursing home outbreaks. There are large differences between the foodborne pathogens in both frequency of occurrence and in infective dose (Table 1). Salmonella and Campylobacter are by far the two pathogens, which are involved in most of the intoxications. The reported cases of Salmonella infection in Belgium have more than doubled in the period 1986 (6.092 human strains) to 1997 (14.239 human strains). This is still an underestimation because not all cases of salmonellosis, especially the less severe ones, are reported. Although not really known to the public, it is estimated that Campylobacter is even outnumbering Salmonella in this respect, but real data are not available in most countries because of the more sporadic nature of the disease. Compared to these two pathogens, the frequency of the other foodborne intoxications is mostly several orders of magnitude smaller. Nevertheless, two pathogens, Listeria monocytogenes and E. coli 0157:H7, deserve special attention because they are capable of causing severe disease of a sometimes fatal nature as has been the case in several recent, large epidemics of the latter organism in Japan, the USA and Scotland. E. coli 0157:H7, as well as Salmonella and Shigella spp. are also very infectious organisms (order of magnitude 10 cells) in comparison with others (B. cereus, C. perfringens, S. aureus, V. parahaemolyticus, enterococci) which require a much higher infective dose (>105 cells) so that an obvious spoilage of the food product should be noticeable in most cases. 195

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Table I: Foodborne bacterial pathogens, nature of the disease and associated foods

196

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Table 1: Continued

: data from the Bad Bug Book (http://vm.cfsan.fda,gov/`mow/intro.htm) most important foods incriminated are indicated in bold c: expressed per 105 inhabitants in Belgium. Data from G. Ducoffre (Surveillance van Infectieuze Aandoeningen door een Netwerk van Laboratoria voor Microbiologie, 1997, Epidemiologische Trends 1983 1996 Wetenschappelijk Instituut Volksgezondheid - Louis Pasteur, Afdeling Epidemiologie, November 1998) (http://www. iph.fgov. be/epidemio/epinl/plabnl/plabannl/index. htm). a

b

:

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3. Molecular detection and identification of foodborne bacterial pathogens 3.1 NUCLEIC ACID BASED IDENTIFICATION METHODS The conventional methodology for the identification of bacterial pathogens is based on the performance of sets of morphological and biochemical tests. Most of these methods are laborious and take several days to be completed. Nucleic acid based identification methods have a lot of advantages compared to these conventional methods. The tests are quick so that results can be obtained within the same day of the investigation. No pure culture is necessary and the results are highly reliable. The tests are as well characterised by a great specificity, offering discrimination of very closely related species and of pathogenic strains within the same species. Identification systems using nucleic acids are based on the use of unique oligonucleotide sequences either as probes in hybridisation assays or as primers for enzymatic amplification of DNA (PCR) or RNA (NASBA or RT-PCR). Probes and PCR primers, used for the identification of different foodborne bacterial pathogens, are extensively reviewed by Olsen et al. (1 995), Feng (1996), Hill (1 996) and Scheu et al. (1998). 3.2 THE USE OF VIRULENCE GENES AS TARGET FOR MOLECULAR IDENTIFICATION For molecular identification different regions of the bacterial DNA can be chosen as target. For pathogenic organisms virulence determinants are frequently used and often allow a very specific identification of a bacterial species (differentiation between C. jejuni and C. coli, Gonzalez et al., 1997) or a serotype (specific identification of S. enteritidis, Lampel et al., 1996 and of S. enteritidis and S. typhimurium, Soumet et al., 1999). Some of these PCR identification systems (Gonzalez et al., 1997, Lampel et al., 1996), however, were not sufficiently validated on field strains and have to be seen more as an indication than as a real identification (Heyndrickx & Herman, personal communication) A lot of these virulence genes are located on plasmid DNA. When possible a chromosomal localisation is preferred because of the instability of plasmids during lab manipulations, This is seen for plasmid bearing Yersinia enterocolitica, where loss of the virulence plasmid during enrichment and isolation complicates the detection of the pathogen in food products (Bhaduri & Cottrell, 1997). By PCR targeting virulence loci, pathogenic strains can be discriminated from nonpathogenic strains belonging to the same species, which is important for identification of pathogenic E. coli and Y. enterocolitica strains. Research on the use of PCR for the identification of toxin genes (LT I, LT II, ST I and ST II for enterotoxigenic E. coli; verotoxins, VT1, VT2 and virulence genes, eaeA, hly, virulence plasmid for enterohaemorrhagic E. coli) is in full expanse (Chen et al., 1998; Tortorello et al., 1998; Gilgen et al., 1998; Tsen et al., 1998; Radu et al., 1998). For the identification of pathogenic Yersinia, primers, targeting the chromosomally encoded ail (adhesioninvasion-locus) gene, can be used (Kwaga et al., 1992). Evaluation of these primers showed no signal for any non-pathogenic Y. enterocolitica strain. The American and 198

MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS

European pathogenic strains gave a positive reaction. All non-Y enterocolitica strains reacted negatively with the exception of some Y. kristensenii strains. The amplicon obtained for these strains was cloned and sequenced and it was found that the whole coding region and the 259 bp upstream region of the ail gene as present in European pathogenic Y. enterocolitica was found in these Y. kristensenii strains (Rijpens et al., 1999b). 3.3 THE USE OF IDENTIFICATION

RRNA

GENES

AS

TARGET

FOR

MOLECULAR

For many pathogens rRNA genes are targeted. The rRNA gene contains next to very conserved parts also variable regions in which specific primers for a certain species can be chosen. For some pathogens however, the variability is not sufficient for discriminating particular species and the pathogen may only be identified to the genus level. This is the case for Brucella (Herman & De Ridder, 1992; Romero et al., 1995) and Campylobacter where C. jejuni, C. coli and C. lari were be differentiated (Giesendorf et al., 1992, Linton et al., 1996). A significant advantage of using rRNA as target is the high copy number (>104/cell) allowing the use of rRNA probes for in situ hybridisation purposes (Nordentoft et al., 1997, describing a 23S rRNA probe for Salmonella; Wagner et al., 1998, describing a 16S rRNA probe for L. monocytogenes). Identification of foodborne pathogens by NASBA applies till now mostly rRNA as target molecule. In one case the mRNA from the L. monocytogenes hlyA gene was targeted (Blais et al., 1997). The RNA is amplified through the concerted action of avian myeloblastosis virus reverse transcriptase (AMV-RT), T7 RNA polymerase and RNase H (Fig 1). The reaction starts with a non-cyclic phase, in which a downstream primer containing a tail-sequence of the T7 promoter anneals to the RNA. Through the action of AMV-RT, cDNA is formed. The RNase H hydrolyses the RNA from the RNA-DNA hybrid, which results in a single strand of DNA to which the upstream primer can anneal. The AMV-RT, through its DNA polymerase activity, synthesises a second DNA strand. The T7 RNA polymerase generates then single-stranded RNA copies, which can serve as a template in a new cycle. For food pathogens, the identification of C. jejuni, C. coli and C. lari (Uyttendale et al., 1994, 1995a) and L. monocytogenes (Uyttendale et al., 199Sb) is described based on 16S rRNA probes. Also the spacer region between the 16S rRNA and 23S rRNA genes is being used as target for PCR. The spacer region is showing a considerable variation and is therefore suitable for species identification. The spacer region offers the possibility to develop ‘multipathogen’ tests allowing the simultaneous identification of different species. The line probe assay (LiPA, Innogenetics, Belgium), has been developed for the simultaneous detection of Listeria spp. and L. monocytogenes (Rijpens et al., 199.5) and recently extended to all defined Listeria species (Rijpens & Innogenetics N.V., Belgium, personal communication). The spacer region is amplified using primers targeting quasi universally conserved sequences at the 3’ end of the 16S rRNA gene and the 5’ end of the 23S rRNA gene. The amplification product is used in a reverse hybridisation assay with a strip where different specific oligonucleotide probes are immobilised as parallel

199

M. HEYNDRICKX N. RIJPENS AND L. HERMAN

lines (line probe assay or LiPA). Specific identification is achieved by hybridisation of PCR amplified spacer sequences to the different probes on the strips.

Figure 1. Schematic presentation of the NASBA method. Normal lines represent DNA, wavy lines represent RNA.

3.4 THE USE OF SPECIFIC SEQUENCES WITH A KNOWN OR UNKNOWN FUNCTION AS TARGET FOR MOLECULAR IDENTIFICATION In fewer cases a specific sequence with a known or unknown function is used as target for the molecular identification. Examples of the use of known genes for PCR are: the flaA and flaB (flagellin genes) and their spacer region for the identification of C jejuni and C. coli (Kirk & Rowe, 1994; Wegmüller et al., 1993); the IS200 element for Salmonella (Cano et al., 1993); the IS711 based multiplex-PCR system AMOS allowing the discrimination of different Brucella spp. (Bricker & Halling, 1994). DNA sequences with unknown function are used for the identification of Vibrio parahaemolyticus (Lee et al., 1995) and Salmonella (ST11-ST15 amplicon, Aabo et al., 1993; Tsen et al., 1994). The full sequence of the ST11-ST15 amplicon is recently published (Rijpens et al., 1999a). 200

MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS

3.5 THE AVAILABLE MOLECULAR IDENTIFICATION SYSTEMS For all important bacterial pathogens, DNA probes and primers are available through the international literature (see above) and/or through commercialised systems. The commercialised systems are summarised in Table 2.

Table 2: List of commercialised molecular identification and detection tests for foodborne bacterial pathogens (taken from Berben, 1998)

The first 2 systems (AccuProbe and Gene-Trak) are using a specific oligonucleotide probe with the rRNA as target molecule in a hybridisation assay. In the AccuProbe system the probe is labelled with an acridinium ester and is used in a hybridisation protection assay. When the DNA probe is hybridised to its target rRNA, the acridinium is protected from chemical hydrolysis and can react with hydrogen peroxide under basic conditions, to produce chemiluminescence. If the probe remains unbound, the ester band 201

M. HEYNDRICKX N. RIJPENS AND L. HERMAN

undergoes hydrolysis and renders the acridinium permanently non-chemiluminescent. The Gene-Trak system uses a colorimetric DNA hybridisation assay based on a sandwich hybridisation. The target RNA is hybridised with a capture probe fixed on a dipstick. The hybridisation is confirmed by a signal-generating probe hybridising to the rRNA target (Curiale & Klatt, 1990). These hybridisation systems are not using a DNA or RNA amplification step but are directed at the highly expressed 16S and 23S rRNA. They require at least 105 to 106 micro-organisms/ml (Mabilat et al., 1996).

Figure 2 Schematic presentation of TaqMan (Applied Biosystems Division of Perkin Elmer, Foster City, CA) The probe is labelled with a reporter (R) and a quencher (Q) dye

The other 4 systems (Probelia, BAX, GeneSTAR and TaqMan) are using PCR to specifically amplify the target DNA but differ in their detection system. They provide all PCR reagents in ready to use reaction mixes, including positive and negative controls. The Probelia system uses an ELISA detection protocol in a microtiter plate format. The BAX system detects the amplified product by gel electrophoresis or temperature dependent fluorescence analysis using SY BR green I (L. monocytogenes: Stewart & Gendel, 1998; Salmonella: Mrozinski et al., 1998, Bennett et al., 1998; Tige et al.,1998, E. coli O157:H7, Johnson et al., 1998, Tseng & Ghandi, 1998). In the latter case the fluorescence is monitored in the PCR tubes during an additional thermal cycle consisting of a denaturation step and a product annealing step. The GeneSTAR system uses fluorescent primers for PCR and hybridises the labelled PCR amplicon to 202

MOLECULAR DETECTION AND TYPING OF FOODBORNE BACTERIAL PATHOGENS

microtiter-bound oligonucleotide probes. The TaqMan PCR detection takes advantage of the endogenous 5’, 3’-endonuclease activity of Taq DNA polymerase to digest an internal fluorogenic probe, which is labelled with both a fluorescent reporter dye and a quencher dye (Fig. 2). A reporter dye is covalently attached to the 5’ end of the fluorogenic probe. A second fluorescent dye, which is capable of quenching the emission of the reporter dye, is covalently attached to one or more nucleotides downstream from the reporter. During the amplification of the target, the fluorogenic probe is displaced by the Taq DNA polymerase and then cleaved, releasing the reporter dye. The fluorescence emission intensity of the reporter dye increases because it is no longer quenched by the proximal quenching dye. The increase in fluorescence is quantitative for the amount of PCR product (Batt 1997, see further). 3.6 PCR DETECTION OF BACTERIAL PATHOGENS IN FOOD PRODUCTS As already mentioned, PCR opens a lot of possibilities for reliable and quick identification of bacterial pathogens. By, PCR the DNA target is enzymatically amplified and therefore could be applied to decrease the bacterial culturing time, normally applied in the conventional detection methods. It is even possible to directly detect the pathogen in the food product as is established for e.g. the detection of L. monocytogenes (Herman et al., 1995) and Brucella (Rijpens et al., 1996) in raw milk. However, when PCR is applied for the detection of pathogens in food products, some problems could be encountered. 3.6.1 Influence offood components on PCR performance Because PCR is an enzymatic reaction, it is not unexpected that a lot of food components can inhibit the amplification of the DNA target. While PCR is rarely inhibited when the technique is used for colony confirmation on agarplates, inhibition is common when PCR is applied to detect pathogens in the enrichment media. The use of a positive control is therefore indispensable when the methods are applied in routine laboratories. This positive control could be amplified in separate tubes or could be coamplified with the target DNA. In the last case one has to consider destabilisation of the system because of competition between the amplification of the positive control DNA and the target DNA. This is especially the case when the same primer pair is used to amplify both control DNA and target DNA. Although this competition could be easily stabilised to confirm colonies on agarplates, this is much more difficult when PCR is applied for detection in enrichment media (Rijpens et al., 1999a). A lot of problems with PCR inhibition could be solved by the application of a suitable sample preparation method (Lantz et al., 1994; Olsen et al., 1995; Hill 1996).

3.6.2 Sensitivity and contamination of PCR By PCR a very high sensitivity of detection can be obtained when more than 35 cycles or a two-step PCR protocol were used. Herman et al. (1995) reported a statistical determined limit between 10 and 5 cfu for the detection of L. monocytogenes in 25 mI of raw milk when a nested PCR protocol was used. The detection limit of a single 30 cycles PCR was about 1000 times higher. 203

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When highly sensitive PCR protocols are used, one has to consider the possibility of PCR contamination. A good lab hygiene protocol eventually combined with an anticarry over system can overcome most of the problems when a single 30 cycles PCR is applied. More serious contamination problems can occur when very sensitive PCR protocols are applied in routine laboratories. 3.6.3 The detection of the viability of cells by DNA based technology When no or very limited culturing of the bacterial cells occurs, it is not possible to make a distinction between the detection of living or dead cells when PCR is applied on DNA (Masters et al., 1994; Herman L., 1997). The efficiency by which dead cells are detected depends on the way they were killed. DNA could even survive a normal autoclave cycle (121°C for 15 min) in the presence of 0.5 - 2.0 M NaCl (Masters et al., 1998). Ribosomal RNA would be less stable than DNA and more closely associated with cellular viability as is shown by a decrease in NASBA signal after antibiotic exposure and an equal signal for PCR (van der Vliet et al., 1994). However, also for rRNA the way the cells are killed is important: a decrease in rRNA was observed when the bacteria were subjected to carbon starvation, heat stress and osmotic stress, no decrease was seen after cold stress, acetic acid or ethanol treatment (Tolker-Nielsen & Molin, 1996; Tolker-Nielsen et al., 1997). Ribosomal RNA would be a good indicator for viability under extreme heat inactivation and UV irradiation of cells. At moderate conditions however signals were still detected 48 h after heat exposure (McKillip et al., 1998). Many food-processing heat treatments involve moderate conditions indicating that detection of rRNA may not be associated with viability under these conditions. Uyttendaele et al. (1997) applied NASBA on the 16S rRNA in an artificial contamination experiment of poultry with heat killed (10 min at 100°C) C. jejuni cells. False-positive results were still obtained at the last measuring on 12 days after killing of the cells. To overcome the problem of the detection of dead bacterial cells the use of messenger RNA as target for the amplification reaction is investigated. Messenger RNA can be amplified by RT-PCR or by NASBA. Messenger RNA is associated with metabolic activity of the cell and is characterised by a short half life (reviewed by Pedersen et al., 1978; Higgins et al., 1988; Belasco & Higgins, 1988). More than for PCR the choice of the target is of great importance for the sensitivity and reliability of the test. Genes with an inducible expression as is the case for some virulence factors (e.g. the listeriolysin O gene of L. monocytogenes) and regulation proteins (e.g. prfA gene product as positive regulator factor of several Listeria virulence factors) seem not to be suitable for RT-PCR detection of the pathogen in food products (Herman L., 1997; Klein & Juneja, 1997). Because of the inducible expression, the extraction efficiency of their transcripts is variable. More sensitive results were obtained with RT-PCR targeting the iap gene coding for p60, a major extracellular protein of L. monocytogenes that is thought to be associated with invasion of phagocytic cells (Klein & Juneja, 1997). In this report, the specific detection of viable cells was only investigated after autoclaving conditions (15 min at 121°C). The use of housekeeping genes as the elongation factor as target for RT-PCR seems reasonable for several reasons (Vaitilingom et al., 1998). First, the elongation factor may be considered as an appropriate viability marker since inactivation is a lethal event. Second, the elongation factor gene encodes one of the 204

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most abundant proteins, allowing a considerable increase in the sensitivity level. Third, the function and the primary structure of the gene is conserved between living cells allowing the modulation of the specificity of detection. Using the elongation factor messenger as target a sensitive detection (detection level of 10 cells per ml of milk) was obtained with a specificity for viable cells within about 6 to 8 min after heat treatment (15 min at 65°C) (Vaitilingom et al., 1998). Although these results look promising, a lot of research will have to be performed before RT-PCR could be used in routine laboratory practice. The main problem is the reliable and constant destruction of spores of DNA, which could trouble the correlation with the viability of the cells. Another problem is the dependence of the sensitivity of the test on the absence of RNA degrading enzymes during the RNA extraction procedure. This is especially important because intensive DNase treatments could be necessary for sensitive detection systems (Rijpens N., personal communication). For routine application the combination of a short period of culturing (e.g. 16 h during the night) with a less sensitive PCR detection is most often used to specifically detect living cells by PCR (see protocols of all commercial PCR systems mentioned in Table 1). Using the screening/Salmonella BAX method it was estimated that 104 cfu/ml has to be present in the primary enrichment medium to be detected by PCR (Mrozinski et al., 1998; Bennett et al., 1998). For the Listeria monocytogenes BAX system the estimated number was 105- 106cfu/ml (Stewart & Gendel, 1998). Only when highly contaminated (numbers of 104 to 105cfu/g or ml) products are investigated killed cells could be detected using these combined protocols. Other advantages of inchding an enrichment step are the possibility to use less specialised sample preparation methods, the lower PCR sensitivity which decreases the problems of PCR contamination and the easier validation of the PCR method which would correlate better with the conventional method (see further).

3.7 EVALUATION AND VALIDATION OF DNA BASED METHODS As DNA based methods are used more frequently to detect bacterial pathogens in food products, there is a great need for a thorough evaluation of the numerous protocols. This is difficult because the inherent uncertainties of food analysis are coupled with new variables introduced by the so called rapid methods. Evaluation of these methods is easiest for these pathogens where the conventional method can be considered as a real standard to which the results for the rapid methods can be compared. This is the case for the detection of Salmonella. More problems are encountered when the conventional detection method lacks sensitivity as is the case for e.g. L. monocytogenes and E. coli. Artificial inoculation experiments of E. coli O157:H7 in ground beef revealed a detection rate of 96.5% with the BAX for Screening/E. coli O157:H7 (commercial PCR method) compared to 39% for the best cultural method (Johnson et al., 1998). Even the use of a combination of 4 recovery methods yields an unacceptably low recovery rate (67%). With the Probelia kit for detection of L. monocytogenes in raw milk cheeses a high sensitivity is reached due to the application of a sensitive PCR (35 cycles). On a total of 39 raw milk cheeses, 2 cheeses were found positive with the conventional method, 14 with the Probelia method (Herman L., personal communication). 205

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Because the DNA based methods can reach a higher sensitivity in comparison with the conventional method, it becomes very important to proof the validity of the positive results and to make sure that no false positives are obtained. This is not so easy because, unlike cultural methods, which almost always yield a viable pure culture isolate, the PCR methods require lysis of the bacterial cell. Therefore, viable cells needed for test confirmation can only be obtained by repeat analysis of the original enrichment medium using cultural techniques. False positive results can especially be obtained when a very sensitive PCR is used. This increases the possibility of PCR contamination and the detection of a small number of killed cells. The absence of PCR contamination is not so easy to proof. Obtaining a negative PCR control would not be sufficient to exclude the possibility of a very small contamination during sample preparation and even PCR preparation. It was experienced that even after plating of 10 ml of the enrichment culture of L. monocytogenes no strain could be isolated from some PCR positive samples (Rijpens N., personal communication). Only repeated culturing could validate the positive result in this case. For routine applications, the use of less sensitive PCR cycling programs should be encouraged. This implies, at the time being, a limitation for the use of universal protocols for detection of different food pathogens in one enrichment. Wang et al. (1997) reported the possibility to detect up to 13 species of foodborne pathogens in foods using an universal protocol of enrichment and detection. Such multi-analyte test, however, implies the use of a very sensitive PCR cycling program (40 cycles) to obtain a sufficient sensitivity. In validation experiments, the rapid method is normally directly compared to the conventional culturing method. Next to naturally contaminated samples different kinds of spiking experiments are applied. Very often pure well growing bacterial cultures are used for this purpose. Because bacterial pathogens present in foods are in a more stressed condition the sensitivities of such experiments may be overestimated. This could be overcome by an extra validation on naturally contaminated samples. For some food products (e.g. dairy products for contamination with Salmonella), however, such a validation is impossible because of the lack of possible samples. Methods could then be evaluated using artificially stressed bacterial cells. For some bacterial pathogens they are commercially available. Rijpens et al. (1999a) spiked the different dairy products with an average of 5.9 stressed S. panama or S. typhimurium cells by using the reference material prepared at the RIVM (National Institute of Public health and Environment, Bilthoven, The Netherlands; in ‘t Veld et al., 1996). The results show that, even within the group of dairy products, a differentiation of methods have to be made depending on the specific group of products tested. For ice-cream and cheeses made from pasteurised milk, PCR was applied after 16 h of preenrichment in buffered peptone water (BPW) using immunomagnetic separation (IMS, using the Dynabeads anti- Salmonella, Dynal, Oslo, Norway) and alkaline lysis as sample preparation method. For milk powder and raw milk cheeses, the 16 h preenrichment in BPW was followed by IMS and a 4 h enrichment in Rappaport-Vassiliadis broth. It is therefore clear that one have to be careful to apply simplified universal protocols without proper validation on the specific food products tested. Because of the need of validation of the DNA based methods a number of validation schemes have been developed in various countries throughout the world. In Europe 206

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there is the Association Français de Normalisation in France, the European Microbiological Method Assessment Scheme in the UK and DanVal in Denmark. However, there have been no microbiological test method assessment and validation schemes accepted by all countries of Europe. The MicroVal project has developed an European microbiological method validation and certification scheme. This validation procedure should become a CEN standard within the next 3 years (Betts and Rentenaar, 1998). In the USA the validation and evaluation scheme of the Association of Official Analytical Chemists (AOAC) is most widely accepted. Only methods that were subjected to the rigorous, multi-step, collaborative study process of the AOAC are approved as standard methods and may be used in official analysis of foods. The BAX for Screening/Salmonella was the first PCR-based product to receive the AOAC RI awarded Performance Tested Method status (Mrozinski et al., 1998). 3.8 DNA AMPLIFICATION FOODBORNE PATHOGENS

METHODS

FOR

QUANTIFICATION

OF

A lack of a simple and reliable method for quantification of the PCR products has partly hindered the use of PCR in routine food laboratories. Quantification of foodborne pathogens by PCR is possible by an indirect method based on the Most Probable Concept (MPN) and by direct quantification of the PCR product. The MPN concept was developed for estimation of the number of organisms based on the probability of getting positive and negative results in different dilutions of the bacterial culture (Cochran W., 1995). MPN-PCR has first been described for enumeration of specific micro-organisms in soil samples (Picard et al., 1992). The detection limit in these experiments was quite high, as at least 103 target cells were needed for positive results. Because of the inefficiency of a PCR of 30 cycles, this underestimation is also encountered in food samples (Herman L., personal communication). The application of a nested PCR protocol could improve the PCR efficiency to about 20 cfu/ml, allowing comparable quantitative results for MPN-PCR with plate counting methods (Mgntynen et al., 1997). For direct quantification, quantitative competitive PCR can be used based on the coamplification of the target with a known concentration of an internal standard (IS) in one reaction tube. In most cases, the IS shares the primer recognition sites with the specific template. Both template and internal standard must be amplified with the same efficiency and both end products must be analysed separately. Quantification is performed by comparing the PCR signal of the specific template with the PCR signals obtained for the known concentrations of the competitor. Wang & Hong (1999) used an ELISA-competitive-PCR method to detect and quantify L. monocytogenes in milk samples. Amplification of the target and the IS was performed in the presence of fluorescein-dUTP. The labelled PCR products were hybridised with biotinylated probes, specific for target and IS. The hybrids were bound to streptavidin-coated ELISA plates to which an alkaline phosphatase-conjugated antibody to fluorescein was added in the presence of substrate. Kinetic quantitative PCR is another direct quantification method where the amount of PCR products is followed during the PCR at several cycles. In the kinetic method, an internal standard is not mandatory and an external scale is sufficient if the reproducibility of the PCR is good. The PCR efficiency of each reaction is checked by 207

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looking at the slopes of the increase in PCR products for the experimental samples and for the external scale. If the slopes are parallel quantification is possible. Monitoring PCR kinetics has become automated by different commercially available systems, The TaqMan PCR detection system (Fig. 2, Perkin Elmer) depends on the irreversible cleavage of the probe by the polymerase exonuclease activity. Quantification was demonstrated for a pure L. monocytogenes culture and proved to be linear over a range of 50 to 5. 105 cfu of L. monocytogenes (Batt C., 1997). The Lightcycler hybridisation system (Roche Molecular Biochemicals, Mannheim, Germany) is based on the hybridisation of 2 independent probes. Resonance energy is transferred from the donor probe to the acceptor probe resulting in fluorescence emission (Fig. 3). Both amplification and hybridisation reactions are performed together and the fluorescence is automatically measured during the process.

Figure. 3. Schematic presentation of the LightCycler hybridisation system (Roche Molecular Biochemicals, Mannheim. Germany)

4. Molecular typing of foodborne bacterial pathogens 4.1 TERMINOLOGY AND GENERAL INFORMATION 4.1.1 Necessity of bacterial typing of foodborne pathogens Bacterial typing refers to the differentiation of types, strains or clones in a single species. Sometimes the term subtyping is also used for techniques, which allow the further subdivision of distinct types within a species (e.g. biotypes), which is not necessarily on the strain level. It is not surprising that medically important microorganisms have been the first candidates for molecular typing (van Belkum, 1994). Typing of bacterial pathogens is necessary in epidemiological studies where origin, transmission and persistence of pathogenic strains are investigated, and this applies to nocosomial or hospital related outbreaks as well as to foodborne outbreaks. In foodborne outbreaks, it is often necessary to distinguish the different types of the pathogen involved and to link them with the suspected foodstuffs as well as to discriminate pathogenic strains which are occurring coincidentally but independent from an epidemic caused by a single strain. This information is of major concern because it directly affects the preventive and hygienic measures to be implemented. 208

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When stable typing data are stored in a database or alternatively, the bacterial isolates themselves are collected for future typing analysis, the persistence and possible evolution of certain strains or clones can be followed, which is of importance in the investigation of the international spread of infectious agents. In the case of zoonotic pathogens, this typing approach is useful to extend the epidemiological link from the human infections to the main animal reservoirs (“from stable to table”) by thorough investigation of each step in the whole food production chain (environment, farm, slaughterhouse, distribution, retail, kitchen). In this way, the most critical points for infection as well as for control or prevention can be identified, but it is also possible to characterise the strains or clones with a changed or particular pathotype (virulence, invasion) or which have acquired (higher) resistance to certain disinfecting products (e.g. hypochlorous acid resistant Salmonella strains, Mokgatla et al., 1998) or antibiotics (e.g. quinolone resistant Campylobacter, multiresistent Salmonella typhimurium DT104). 4.1.2 Species-subspecies-variety-clone-strain-isolate The basic unit of bacterial taxonomy is the species, defined as a group of strains, including the type strain, sharing at least 70% DNA-DNA relatedness with 5°C or less ∆ Tm (with Tm the melting temperature of the hybrid). According to Vandamme et al. (1996), “the bacterial species appears to be an assemblage of isolates which originated from a common ancestor population in which a steady generation of genetic diversity resulted in clones with different degrees of recombination, characterised by a certain degree of phenotypic consistency and by a significant degree of DNA-DNA hybridisation and over 97% of 16S rDNA sequence homology”. The latter authors advocate the polyphasic taxonomic approach for bacterial classification as first step in the delineation and description of species, which takes account of all possible variation on the phenotypic and/or genotypic level. This polyphasic approach gives a higher guarantee that intra-specific reliable and stable targets (e.g. on the 16S rDNA level) for molecular identification can be found. Nevertheless, a polyphasic identification will remain necessary for atypical isolates or isolates from new niches, which may belong to unknown, related species. It must be stressed that a precise species identification is an important and necessary step before any typing can be performed. A “List of bacterial names with standing in nomenclature” is available on http://wwwsv.cict.fr/bacterio/index.html. The classification of organisms below the species level is of particular interest for epidemiology and typing. The only level with nomenclature status is the subspecies (Wayne et al., 1987), while variety has no official rank. The designation “var” (e.g. biovar, pathovar, serovar, phagovar) is commonly given to groups of strains which are distinguished by certain characters. A strain is the basic working unit in the daily laboratory practice, where the term denotes a pure culture, either fresh or stored in some way. In the Bergey’s Manual of Systematic Bacteriology, a strain is described as the “descendants of a single isolation in pure culture, and usually made up of a succession of cultures ultimately derived from an initial single colony”. A useful definition for a strain is also “an isolate or a group of isolates exhibiting phenotypic and/or genotypic traits which are distinctive from those of other isolates of the same species’’ (Struelens and members of the European Study Group on Epidemiological Markers, 1996). The 209

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meaning of the terms strain and isolate are thus synonymous in many cases. The term clone has a meaning in population biology as denoted by Ørskov and Ørskov (1983) as “bacterial cultures isolated independently from different sources, in different locations, and perhaps at different times, but showing so many identical phenotypic and genetic traits that the most likely explanation for this identity is a common origin”. For bacteria, clonality has thus a somewhat broader meaning than for eukaryotic organisms, where clones are definitely genetically identical organisms. In bacterial epidemiology, the clone concept is of an even more pragmatic nature to denote isolates obtained during clear-cut outbreaks and exhibiting the same phenotypic and/or genotypic features or to denote organisms with common features (e.g. multiple antibiotic resistance) from different geographic locations, so-called epidemic clones. The clonal relatedness between strains is usually inferred from a numerical analysis of a polyphasic typing approach combining phenotypic (e.g. multilocus enzyme electrophoresis or MEE of metabolic enzymes) and genotypic methods (comparative sequence analysis or highresolution typing with pulsed field gel electrophoresis). Many pathogenic species are of a clonal nature (e.g. Salmonella), whereas in other species genetic recombination occurs resulting in horizontal gene exchange and/or a mosaic structure of recombined segments. Because the evidence for clonality in bacteria is relative rather than absolute (unless the whole genomes of different strains could be sequenced), it may be recommended to speak only in the sense of “clonally related strains or strains of a clonal lineage” or even “clone complex” when it is to be stated that strains have probably the same origin as indicated by polyphasic typing. 4.1.3 Molecular typing techniques used for bacterial pathogens Most if not all of the currently used molecular typing techniques are DNAfingerprinting techniques, i.e. techniques that reveal DNA sequence polymorphisms within a species by the generation of fingerprints. The advent of molecular techniques and especially of the PCR technique has revolutionised the approach and possibilities of bacterial typing. Before that, typing was performed by the classical phenotypic techniques such as antibiogram typing, biotyping, serotyping, phage typing and multilocus enzyme electrophoresis (MEE). Many of these classical typing methods, especially serotyping and phage typing, are still highly used in clinical and public health laboratories as well as veterinary laboratories to gather timely information which can be used for epidemiological investigations of foodborne and zoonotic pathogens. Information of the antibiotic resistance of isolates of some pathogens such as Salmonella, Campylobacter and enterococci will also be of increasing importance. The classical typing techniques remain however laborious, require the holding of large sets of antisera and phages and are therefore only performed in some reference laboratories per country. Some strains (e.g. auto-agglutinating strains) are not typable with these techniques, Moreover, it is sometimes difficult to correlate the serotyping and phage typing data coming from different laboratories (veterinary vs. clinical) to each other for a certain pathogen such as Salmonella. Molecular typing offers important advantages such as general applicability (i.e. typability of all isolates) and the possibility of a higher discriminatory capacity or resolution which is needed to complement the classical sero- and phage typing when the above mentioned tasks (4.1.1 & 4.1.2) have to be performed. The fingerprints can also 210

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be used for numerical cluster analysis, which can be useful to delineate clonal relatedness. In several cases, these molecular techniques are characterised by their simplicity of performance, their reproducibility or their high resolution enabling the differentiation between individual strains of a species. However, it must be said that not all these interesting characteristics are automatically combined in one single technique. In Table 3, a simple and pragmatic classification of the several molecular typing techniques is given based on their basic working principles. Some indication is also given of their complexity or simplicity, universal applicability (i.e. typability of all bacterial species), reproducibility and resolution, which is a somewhat subjective matter and for the latter sometimes pathogen-dependent. Table 3: Currently used molecular typing techniques for foodborne pathogens and some of their characteristics

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Table 3: Continued

Basic principle

Molecular typing techniques (abbreviation)

Further subdivision

Characteristicsa Complex. a

and PCR plasmid analysis

plasmid

profiling

plasmid restriction (plasmid REA) a

(PP) analysis

L

N M

Resolut Reproduc ion ibility

Universal application

V N

V

M-H M-H

: H, high; L, low; M, medium; V, variable; Y, yes; N, No.; Complext., complexity

A more objective indication and comparison of the resolution between different techniques when applied to a certain pathogen can be obtained with the so-called discriminatory index (DI) of Hunter and Gaston (1988). As can be deduced from Table 3, the techniques which are characterised by their high reproducibility and resolution, are mostly also the most complex and laborious ones (PFGE and AFLP). In the choice for a certain technique, other factors will also play an important role, such as the pathogen or case involved, the time and apparatus available in the laboratory, the kind of information needed and the aim of the typing study (shortor long-term study). Nevertheless, it is generally recommended that several molecular techniques should be combined. The techniques are named here by their most commonly used names, which are not confusing or erroneous. The latter is not always fulfilled since the same names are sometimes used for different techniques (e.g. ribosomal nucleid acid gene restriction analysis) or the same techniques bear several names (e.g. arbitrarily primed PCR), which makes a simple retrieve of the literature sometimes impossible. An extensive effort to classify and name the whole array of DNA-fingerprinting techniques has been done by Vaneechoutte (1 996) and we can only strongly recommend the molecular epidemiologists to name their typing techniques in a consequent manner, for example based on the guidelines by the latter author or based on a pragmatic consensus. The several molecular typing techniques most relevant to foodborne bacterial pathogens are now described in more detail, using their commonly used abbreviation if available (see Table 3). 4.1.3.1 PCR-mediated typing techniques. The PCR has without any doubt revolutionised the molecular typing approach of bacteria and has also brought the possibility of molecular typing within the reach of routine laboratories. A large variety of PCR mediated typing techniques exists and some of them are listed in Table 3. The most widely known and used techniques in this category are AP-PCR and RAPD, developed independently by Welsh and McClelland (1990) and by Williams et al (1990), respectively. Since there are no fundamental differences between both techniques and the name RAPD is more frequently used, it is recommended that the latter name is used throughout. RAPD is based on the random amplification of genome 212

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fragments lying between arbitrary sequences, which are used as targets for priming, Usually, only one primer of 10 up to approximately 20 bases long is used in the PCR working at a low annealing temperature (around 35ºC, but higher temperatures are also used) and with a high MgCl2 concentration (up to 4 mM). As a consequence, these reaction conditions will permit specific as well as non-specific primed amplification of fragments. Another consequence is the generally low degree of reproducibility of this kind of molecular typing. A technical review on RAPD typing in microbiology has been done by Power (1996). The several experimental factors affecting the amplification reaction and the reproducibility of RAPD have been reviewed by Tyler et al. (1997). One of the important factors affecting the complexity of the banding pattern is the primer selection. As a matter of fact, for every organism studied a suitable primer has to be selected empirically. The most critical factor for reproducibility seems to be the primer concentration to template DNA concentration. Tyler et al. (1997) have formulated several recommendations for using arbitrary PCR: • quantify DNA for each organism and each extraction method, and the use of whole-cell extracts is to be avoided. • primers should be screened for priming ability and reproducibility. • quantify primers for every synthesis reaction. • titrate DNA against primer concentration to reach an ideal primer/template ratio. • standardise the use of Taq DNA polymerase by maintaining the same supplier, • titrate the Taq DNA polymerase against the primer/template ratio. • standardise the MgCI2 concentration. • use one thermocycler with a standard set of cycling conditions. • run the appropriate control blanks to account for background. It is now possible to perform RAPD in a commercialised pre-mixed, pre-dispensed reaction format (Ready-To-GoR RAPD Analysis Beads, Amersham Pharmacia Biotech) which requires only the addition of template DNA and a primer. This format will help to fulfil the above recommendations and to increase the inter- and intra-laboratory reproducibility. Bacterial genomes contain multiple interspersed repetitive DNA elements (usually <500 bp) occupying intergenic regions at sites dispersed throughout the whole genome. These repetitive elements may provide the genomic code necessary for proper chromosomal structure and function in vivo. Several classes of repetitive elements have been found in different microbial genomes: polynucleotide sequences, tandem repeats, short interspersed repetitive sequences (<50 bp) such as the repetitive extragenic palindromic (REP) element, large interspersed repetitive elements (>50 bp) such as the enterobacterial repetitive intergenic consensus (ERIC) element, and mosaic repetitive elements such as the BOX-element. Tandem repeats are polynucleotides occurring in tandem in a variable number according to the strain. A PCR based on this variable number of tandem repeats (VNTR) is very useful for typing of unicellular eucaryotes, but has also typing potential in some bacteria (Frénay et al., 1994). For bacteria, the REP-, ERIC- and BOX- elements are of greater importance. Genuine REP and ERIC elements are present in high numbers (e.g. >100 for REP) in the genomes of the Enterobacteriaceae and REP- and ERIC-like elements have also been demonstrated 213

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throughout the eubacterial kingdom. They contain a conserved inverted repeat to which complementary consensus primers have been designed for PCR-typing (Versalovic et al., 1991). The modular BOX-element is the first repetitive element found in a Grampositive organism (Streptococcus pneumoniae) consisting of the subunits boxA, boxB and boxC (Martin et al., 1992). Based on the widespread conservation of the boxA subunit among diverse bacteria, a similar PCR-typing method has been designed (Koeuth et al., 1995). Repetitive element primed PCR or rep-PCR is a general term to collect at present the REP-PCR, ERIC-PCR and BOX-PCR typing methods, based on the respective repetitive elements. In rep-PCR, amplicons are generated which contain unique sequence chromosomal segments lying between the repetitive sequences. Unlike RAPD, rep-PCR is considered not to be arbitrary because known conserved sequences are used as priming targets at higher annealing temperatures (40°C for REP-PCR and 52°C for ERIC- and BOX-PCR) (Tyler et al., 1997). However, some authors (e.g. Giesendorf et al., 1993) use only one of the repetitive element targeting primers at low annealing temperatures, which resembles more a RAPD typing. We suggest that the term rep-PCR typing and its different subdivisions should be reserved for the techniques relying on the original procedures. Recently, some discordant notes have been observed within the promising proposition of rep-PCR as a stringent PCR-typing approach with reduced experimental variation and PCR artefacts and with the potential of storing the patterns in databases for strain and species identification. Gillings and Holley (1997) concluded that ERIC-PCR, using the standard annealing temperature of 52°C, does not necessarily direct amplification from genuine ERIC sequences, and that this typing technique applied to non-enterobacterial organisms must be regarded as a highly reproducible variant of RAPD. A similar observation was made by us (Herman and Heyndrickx, in press) with REP-PCR applied on the Grampositive UHT-resistant Bacillus sporothermodurans. For Grampositives, this is not surprising since they do not contain genuine REP- and ERIC- elements and it is suggested that the BOX-elements are the preferable targets for these organisms (Tyler et al., 1997). With Salmonella, we observed small differences (band intensities) in both REP- and ERIC-PCR patterns between independent PCR-runs, but a considerable variation when primers were used from 2 different sources (Heyndrickx M., unpublished results). For all rep-PCR techniques, we recommend to adhere to the same recommendations as for RAPD and to analyse strains with the same batches of primers and, if possible, within the same PCR experiment. For a detailed protocol inclusive of the primer sequences for the different rep-PCR techniques, we refer to http://www.msu.edu/~debruijn/loadr.html. A major advantage of rep-PCR over RAPD however is the fact that a single primer set targeting a known conserved and repetitive sequence can be used for both Gramnegative and Grampositive organisms, whereas in RAPD a suitable primer with enough discriminatory power has to be selected for any individual organism. Finally, some PCR techniques aiming at other sequences occurring at multiple sites in the bacterial genome may also be effective for typing. The 2 most important targets are the tRNA sequences and the ribosomal spacer regions. tRNA sequences occur in multiple copies dispersed throughout the genome and contain shared sequence motifs from which outwardly directed primers can be derived. tRNA-PCR has more potential for species identification, but in certain cases subgroups corresponding to the variety level can be delineated (Seal et al., 1992). In many bacterial species multiple copies or alleles 214

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of the ribosomal operon are present (e.g. up to 10 copies in Bacillus). The spacer region between the 16S and the 23S rRNA genes may vary in length between species, between the rRNA alleles of a species and even between strains of a species as has been demonstrated convincingly for Clostridium difficile (Gürtler, 1993). This variation in length is in part due to the number and type of tRNA genes present in the spacer. A universal bacterial identification and typing method is based on the PCR amplification of the variable length 16S-23S rDNA spacer regions (Gürtler and Stanisich, 1996; Jensen et al., 1993). This typing method has also been called PCR-ribotyping (Kostman et al., 1992). The typing resolution of the ribosomal spacer based PCR technology can be enhanced by additional restriction analysis to detect also sequence differences between the spacers. 4.1.3.2 Typing techniques combining PCR with restriction analysis. After PCR amplification of certain genes or gene fragments, the amplicons can be digested with restriction enzymes and the resulting restriction fragments separated by agarose gel electrophoresis to reveal sequence polymorphisms. This restriction analysis is necessary because the targeted genes differ only in sequence and (usually) not in length (unless insertions or deletions have occurred in the gene) between strains of a species. Protein encoding genes showing intraspecific variability are used for this typing approach. A good example is the flagellin gene typing of C. jejuni based on the restriction analysis of the amplified flaA gene (Nachamkin et al., 1993). Since the amplicons are generally up to 1500 bp long, tetracutter restriction enzymes (i.e. enzymes with a 4-base recognition sequence) which cut theoretically after each 256 bp, are the best choice to reveal enough restriction fragments and thus to reveal sequence polymorphisms. Also enzymes with a longer recognition sequence, which contains only 4 defined bases and for the rest undetermined bases (e.g. DdeI with the recognition sequence C^TNAG, with N indicating an undetermined base), are suitable for this purpose. Information concerning all currently known restriction enzymes is available at the daily updated “Rebase” (http://rebase.neb,com/rebase/rebase.html). It must be noted that amplified ribosomal DNA restriction analysis (ARDRA) is frequently categorised under the typing techniques with potential of subspecies/strain differentiation. This is erroneous since this technique relies on the 16S rRNA gene which is conserved amongst bacterial species and is thus ideally suited for taxonomic and phylogenetic studies, but not for molecular typing (Heyndrickx et al., 1996). 4.1.3.3 Typing techniques based on chromosomal restriction fragment length polymorphisms. Before the advent of the PCR methodology, molecular typing was mainly performed by restriction enzyme digestion of the bacterial chromosome and subsequent gel electrophoretic separation of the fragments to detect restriction fragment length polymorphisms (RFLP) by comparing the number and size of restriction fragments. This technique is also simply called restriction enzyme analysis or REA. There are 3 main sources for the generation of RFLP’s: base substitution within the restriction recognition sequence, deletions and insertions. Base substitutions affect only the restriction enzyme(s), which cleaves within the original or mutated recognition sequence and result in the gain or loss of a restriction fragment(s). If a restriction site is lost, a new restriction fragment, equal to the sum of the 2 fragments flanking the previous restriction site is generated; if a restriction site is gained, 2 new restriction 215

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fragments which together equal the size of the lost fragment, are generated. Deletions and insertions affect the RFLP patterns obtained with all restriction enzymes because of the change in size of a particular restriction fragment corresponding to the size of the deletion or insertion. For RFLP analysis, hexacutter restriction enzymes (i.e. with a 6base recognition sequence) are normally used, but this still may result in >1000 bands for a normal bacterial genome of 4x106 bp, which makes separation and interpretation of the complex banding patterns very difficult. Several approaches (high-size or highfrequency RFLP using tetracutters and low-size RFLP using hexacutters and polyacrylamide gel electrophoresis, selective restriction fragment hybridisation, pulsed field gel electrophoresis) have been developed to overcome this problem by reducing the number of (visible) bands. In the selective restriction fragment hybridisation (SRFH), the separated chromosomal restriction fragments are transferred by Southern blotting on nitrocellulose or on (more durable) nylon filters or membranes by capillary action or by vacuum. The denatured membrane-bound DNA fragments are hybridised to a probe, which can be either 32P-radioactive labelled or non-radioactive labelled with a chemically modified nucleotide containing a hapten (e.g. digoxigenin- or DIG-dUTP of Boehringer Mannheim, biotin-dATP). Only the fragments hybridising with the probe are then revealed by autoradiography or with an anti-DIG or -biotin alkaline phosphatase conjugate combined with a chemiluminescent or chromogenic substrate. This hybridisation procedure results in simple and easy interpretable SRFH patterns. Several factors influence the specificity of the hybridisation reaction such as the stringency of the hybridisation temperature and several critical parameters (e.g. ionic strength of the hybridisation solution, probe length and % G+C) affecting the Tm of the hybrids formed. An essential element in SRFH analysis is the type of the probe used, which can be either a single-stranded DNA or a RNA sequence. SRFH probes can be categorised in random, directed and reiterated sequence probes (reviewed by Demezas, 1998), with the latter 2 being the most important ones. Reiterated sequence probes include transposons, insertion sequences (IS) and duplicated genes. SRFH with virulence/toxin probes, phage probes or IS-probes are very useful in the molecular typing of certain foodborne pathogens. It must be noted that most of these SRFH applications are named as RFLP techniques preceded by the name of the probe used, e.g. IS200-RFLP and The most widely used SRFH application is based on probes directed at the conserved ribosomal RNA genes and is named ribotyping (Grimont and Grimont, 1986) or (erroneously) also restriction analysis of rRNA genes. Ribotyping is universal applicable but its resolution is dependent on the number and the spatial distribution of the rRNA copies on the bacterial chromosome. In the first place, ribotyping is a useful taxonomic and identification technique and also a potential typing technique for microorganisms containing several ribosomal operons (multiplicity of rRNA operons in prokaryotes reviewed by Schmidt, 1998). The ribotyping technique has been automated with the RiboPrinter™system (Qualicon Inc., Wilmington, DE, USA). Starting from a pure colony, this automated system analyses 32 samples per day in 4 batches of 8 samples, with the results from the first batch available in 8 hours. The second approach to reduce the amount of fragments in RFLP analysis is macrorestriction analysis (MRA) by pulsed field gel electrophoresis (PFGE). This technique is based on the in situ restriction digestion of the intact unsheared bacterial 216

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chromosomes in lysed agarose-embedded cells with so-called “rare-cutting’’restriction enzymes, i.e. hexacutters or octacutters that cut the genome infrequently. This results in (usually 10 to 30) large chromosomal fragments ranging in size from several kbases up to several Megabases, which can only be separated by the specialised electrophoresis technique PFGE. In PFGE, the electric field is not linear but switched with a certain field angle and shape and pulse time. For an optimum separation of fragments over a certain molecular weight range, an appropriate ramp of the pulse time intervals has to be selected for every case. The number and size range of fragments is dependent on the restriction enzyme used, the % G+C content and the degree of methylation of the bacterial genome. In practice, it seems that appropriate restriction enzymes have to be selected for PFGE analysis of each bacterial species and that a combination of different enzymes is necessary for optimal strain discrimination within the species. An overview of rare cutting enzymes useful for PFGE analysis of several bacteria is given by McClelland et al. (1998). The standard protocol for PFGE is time-consuming (up to 6 days), but more rapid procedures for specific organisms (e.g. one day procedure for E. coli O157:H7, Gautom 1997) or in general (less than 3 days procedure of Matushek et al., 1996) have been designed without consequences for the PFGE patterns. Guidelines to interpret macrorestriction patterns have been formulated by Tenover et al. (1995). Under the condition that isolates obtained from outbreaks spanning relatively short periods are analysed by PFGE using only one restriction enzyme because of time considerations, the following categories of isolates can be distinguished on the basis of the PFGE patterns: • indistinguishable isolates showing no fragment differences and hence no genetic differences. These isolates all represent the same strain. • closely related isolates showing 2-3 fragment differences consistent with a single genetic event (point mutation, deletion or insertion). Such variation has been observed in strains of some species on repeated subculturing or multiple isolation from the same source. • possibly related isolates showing 4-6 fragment differences consistent with 2 independent genetic events. Such variation has been observed between isolates collected over longer periods or taken from extended outbreaks, and can mostly be confirmed by other typing techniques. • unrelated isolates showing ≥ 7 fragment differences consistent with ≥ 3 genetic events. Although these guidelines are in the first place intended to know the epidemiological relationship of isolates to the outbreak strain, they may be useful in a more general context when using PFGE as molecular typing tool. It is also advised not to take into account of the fragments of <40 kb because they are not reproducible comparable. As a comment on the above guidelines, we believe that the inherent genetic homogeneity of the bacterial pathogen investigated should also be taken into consideration, which is of course not facilitating the interpretation of molecular typing patterns. Indeed, there seems to be a wide span of degree of homogeneity between different microorganisms with S. enteritidis at the one edge as a very homogeneous, clonal species, S. typhimurium as a more heterogeneous species, and Helicobacter pylori and campylobacters in general at the other edge as very heterogeneous species.

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4.1.3.4 Typing techniques combining restriction digestion with selective amplification A patented high-resolution technique, which deserves a separate classification, is AFLP™(hereafter referred to as AFLP). Although AFLP should not be used as an acronym, the name is often used as an abbreviation of amplified fragment length polymorphism. AFLP has been adapted for bacteria independently by Janssen et al. (1 996) and Lin et al. (1 996) as follows: • double digestion of the chromosomal DNA with 2 restriction enzymes, a hexacutter (H), mostly ApaI, EcoRI or HindlII, and a tetracutter (T), mostly Taq1 or MseI. A correct choice of the enzymes is crucial as this will predetermine the size and number of restriction fragments and depends on the % G+C content of the genome. The enzyme combinations EcoRI/MseI and ApaI/TaqI are the most suited for bacterial genomes with a low and a high % G+C content, respectively. HindIII is the most suited to digest genomes with a 40-50 mol %G+C. • ligation of restriction halfsite-specific double stranded oligonucleotide linkers or adapters to both ends of the restriction fragments. The adapters are designed in such a way that the original restriction sites are not restored after ligation to the restriction fragments, so that the ligation can be performed in the presence of the restriction enzymes to prevent fragment-to-fragment ligation. • pre-selective amplification of all H-T fragments with primers complementary to the sequences of the H- and T-adapters, respectively. In some procedures, this step is not used and a selective amplification is performed directly. • selective amplification of a subset of the H-T fragments with primers complementary to the H- and T-adapters and with additionally one or more selective bases at their 3’-end. Under stringent PCR conditions, only the primers of which the selective base(s) is complementary to the base(s) flanking the restriction site, will match perfectly and will result in amplification. Theoretically, this means that per selective base only 1 on the 4 restriction fragments will be amplified, and thus a selective amplification of restriction fragments is achieved. The complexity of the AFLP patterns largely depends on the length and the base composition of the selective bases. Usually one selective base for both primers is sufficient. The choice of the composition of the selective base again depends on the % G+C of the bacterial genome, but also on the complexity and band distribution of the AFLP patterns obtained with the restriction enzyme combination. In some cases, it may be necessary to use 1 selective base for one of the primers only or to use 2 selective nucleotides for one of the primers to achieve an adequate number of evenly distributed bands. Only the H-complementary primer is labelled, either radioactive or with a fluorescent dye, to reveal the AFLP patterns by autoradiography or on an automated sequencing apparatus with laser fluorescent detection, respectively. AFLP is applicable in both bacterial taxonomy and typing as demonstrated by its superior resolution towards the differentiation of highly related strains (Janssen et al., 1996). Other advantages are the high reproducibility because of the stringent PCR conditions, and the large possibilities of experimental variation (choice of restriction enzymes and selective bases) so that optimal settings for each microorganism can be 218

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searched. The AFLP technique is now available in a commercial format (AFLP™ Microbial Fingerprinting kit of PE Applied Biosystems, Foster City, Ca., USA). 4.1.3.5 Typing techniques based on plasmid analysis. Plasmids were the first targets for molecular typing starting already in the mid-seventies and they are still very useful for certain pathogenic bacteria such as Salmonella. Plasmid analysis starts with the specific isolation of the plasmid DNA which is mostly accomplished by a modified procedure of the alkaline lysis method, originally described by Kado and Liu (1981). In most rapid plasmid preparations, chromosomal contamination is difficult to eliminate, but this should not present a problem for interpretation since the chromosomal DNA appears as a diffuse single band of the same length for all strains. The most simple technique is plasmid profiling (PP) in which the size and number of the plasmids present in the strains is investigated by agarose gel electrophoresis. An E. coli reference strain harbouring several large plasmids of known size is very useful for exact molecular weight determination in PP analysis. In some plasmid preparations, the same plasmid may be present in multiple forms (the native supercoiled form, the open circular form resulting from a cut in one DNA strand, and the linearised form resulting from a cut in both DNA strands), each with its own electrophoretic mobility. Especially with smaller plasmids, this may result in multiple bands for the same plasmid, which results in an overestimation of the number of plasmids present. Another problem is the instability of plasmids resulting in the possibility that a certain strain loses one or more of its plasmids. On the other hand, plasmid DNA may undergo more rapid evolution than chromosomal DNA because plasmid borne genes do not encode for essential cell functions. It may thus be expected that plasmid DNA is less subjected to evolutionary pressure and is more prone to sequence variations. Sequence polymorphisms between identical sized plasmids are revealed by restriction analysis (plasmid REA). 4.1.4 Analysis of DNA fingerprints. The DNA fingerprints generated by the various molecular typing techniques can be visually analysed to distinguish strains with identical patterns, related patterns and clearly different patterns. This analysis by eye is only possible for strains run on the same gel, which still may contain several tens of strains. When patterns on different gels have to be compared, the interpretation becomes more complicated, not only because of the restrictions in human visual memory, but also because of differences in electrophoretic mobility between equally sized bands. These differences are sufficient to make the use of specialised pattern analysis software highly recommendable (e.g. the state-of the art package GelCompar, Applied Maths, Belgium). All packages allow the same essential functions: input of the strain information, gel normalisation, cluster analysis and database generation (Seward et al. 1997). One of the most critical elements in this computerised analysis is the normalisation step, in which bands of equal size in the patterns are physically aligned to compensate for inter- and intra-gel inconsistencies and variations. By normalisation, all gels become compatible with each other which makes the generation of databases and the cluster analysis of patterns from different gels possible. By cluster analysis, patterns are grouped in a dendrogram according to their similarity to each other, which is calculated by the program for every pair of patterns based on a similarity coefficient. This coefficient can be either based on the 219

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presence/absence of bands (Dice and Jaccard coefficient) or on the whole densitometric curves (Pearson product-moment coefficient). The latter coefficient is more suited for complex patterns with large differences in band intensities (e.g. RAPD and rep-PCR), but the Dice coefficient is generally regarded as representing more reliably genetic relationships between strains on the basis of DNA fingerprints (Nei and Li, 1979) and hence clonal relationships between strains. 4.2 PROSPECTS IN MOLECULAR TYPING Molecular typing is a relatively young discipline but has already encountered many changes and variations on the same theme, in which the PCR technology has played a major role. It is the prospect that molecular typing of foodborne bacteria will increase constantly in importance and will generate data, which can be exchanged between different types of laboratories (veterinary, industrial, and clinical/public health laboratories) and between countries. On the International Meeting on Bacterial Epidemiological Markers in 1997 (IMBEM IV, Elsinore, Denmark), PFGE and AFLP were proposed as the likely candidates for a high-resolution, standardised, definitive typing approach in which patterns can be reproducibly generated, stored in databases and exchanged via networks. In the USA, PulseNet (http://www.cdc.gov/ncidod/dbmd/puIsenet/pulsenet.htm) is launched as a National Molecular Subtyping Network for Foodborne Disease Surveillance, permitting rapid comparison of PFGE fingerprints through an electronic database at the Centers for Disease Control and Prevention (CDC). Cost and speed are also important parameters for the implementation of a typing system, which can benefit from new developments or applications. Another fragment separation technique based on fluorescent labelling and capillary electrophoresis of DNA fragments (e.g. with the ABI Prism 310 Genetic Analyzer, PE Applied Biosystems) enables a higher level of automation, flexibility and throughput of samples. It is also possible to directly reveal sequence polymorphisms (down to the level of point mutations) with specialised electrophoresis techniques in which a denaturing gradient is applied in the gel, either chemically in denaturing gradient gel electrophoresis (DGGE), or in temperature gradient gel electrophoresis (TGGE) or alternatively in temporal temperature gradient gel electrophoresis (TTGE). Equally sized fragments or amplicons will stop migrating at different positions and hence will be separated in the denaturing gradient depending on the melting temperature Tm of their sequence. PCR-TGGE and DGGE analysis is currently used in molecular microbial ecology (reviewed by Muyzer and Smalla, 1998), but has potential for rapid molecular typing as well. A further prospect is the deviation of molecular typing by DNA fingerprinting towards sequencing data and hybridisation pattern data on DNA-chips. Sequence data are still the preferred type of information because of their absolute nature and it is the challenge of future molecular typing to identify suitable nucleic acid targets, which show enough intraspecific variation within a single short sequencing run. DNA chips are high-density oligonucleotide arrays which are used in fluorescent hybridisation analysis to screen sequence variation in large sets of (amplified) DNA samples in a high throughput manner. Simultaneous genotyping and species identification using hybridisation pattern recognition analysis on DNA-chips has been demonstrated for mycobacteria (Gingeras

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et al., 1998). It is expected that whole genome sequencing data will provide new targets (e.g. DNA repeats or genes) for molecular typing. 5.Molecular typing of some specific bacterial foodborne pathogens 5.1 SALMONELLA From Table 4, it can be deduced that Salmonella has been the subject of numerous molecular typing studies. It is important to clarify first the rather confusing Salmonella taxonomy. The genus Salmonella forms a single DNA homology group divided in seven sub-groups. The seventh group is given the species rank as Salmonella bongori, while the other 6 groups represent 6 subspecies of one species, which should carry the name Salmonella choleraesuis (Skerman et al., 1980). Because of confusion with the name of the serovar Choleraesuis, Le Minor and Popoff (1987) proposed the species name Salmonella enterica instead, which is now used by almost every author, but has no standing in nomenclature because not officially accepted by the Judicial Commission of the International Committee on Systematic Bacteriology. Therefore, Euzéby (I 999) has recently proposed to reject the name Salmonella choleraesuis and to designate Salmonella enterica as neotype species. The official situation will thus be that Salmonella contains 2 species, S. bongori and S. enterica, the latter being divided in 6 subspecies (Christensen et al., 1998). S. enterica subsp. enterica is the most important subspecies containing most of the >2300 serovars. Three serovars of high clinical importance have been given species rank: S. enteritidis, S. typhimurium and S. typhi. Other serovars should not be considered as species and should be designated as in the example: Salmonella enterica subsp. enterica ser. Hadar or (more practically) Salmonella Hadar. Two typing levels can be proposed for Salmonella. The serovar level is (and will remain if only for historical reasons) a very important first typing level for daily practice. Several molecular typing techniques (see Table 4) such as PCR-ribotyping (Lagatolla et al., 1996), PCR-RFLP of the ribosomal operon (Shah and Romick, 1997), ERIC-PCR (Van Lith and Aarts), REP-PCR (Heyndrickx M., unpublished results) and AFLP (Aarts et al., 1998) seem to provide the possibility of serovar identification and thus to replace the classical serotyping. However, it should be noted that some serovars seem to be polyphyletic because of recombinations in the genes encoding for the O- and H-antigens, which could be reflected in different fingerprint patterns for some strains of these serovars (Hilton and Penn, 1998).

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Table 4: Molecular typing techniques used for foodborne and animal associated Salmonella serovars and some important literature references for each of them. References are classified according to the sole typing technique involved or according to the most studied or (if possible) the most discriminatory typing technique when several techniques are involved. In the latter case, relevant information on the other typing techniques is given as well. For more complete information on a specific technique or serovar, we refer to further studies cited in the references.

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Table 4: Continued

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Table 4: Continued

Molecular typing technique

Reference

Specific comment

Threlfall et al., 1998

association of S. Anatum outbreak with formula-dried milk by plasmid profile typing and PFGE S. enteritidis isolates of human gastro-enteritis linked with eggproducing poultry flocks by plasmid profiles and REA plasmid REA of Australian S. enteritidis strains combination of plasmid analysis and PFGE most discriminatory for S. Choleraesuis strains; see also ribotyping and ISZOO-typing use of plasmid profiles and IS200-typing for distinction between epidemic and non-epidemic S. Hadar isolates discrimination of S. enteritidis poultry isolates with plasmid profiling, discrimination of S. typhimurium poultry isolates with ribotyping and IS200-typing; see also ribotyping and IS200-typing detection of molecular variation in the serotype-specific plasmid of S. enterifidis by plasmid REA using PstI and SmaI little or no differentiation amongst S. enteritidis isolates; see also PFGE subdivision of same S. enteritidis PFGE type by plasmid profile, and conversely, same plasmid profile by PFGE; see also PFGE 5 different plasmid profiles amongst S. Dublin field isolates; see also PFGE

Dorn et al., 1993 Mills et al., 1995 Weide-Botjes et al., 1996 Fantasiaet al., 1997 Millemann et al., 1995 Rankin et al., 1995 Liebisch & Schwarz, 1996a Suzuki et al., 1995 Liebisch & Schwarz, 1996b PFGE

Echeita & Usera, 1998 Liebisch & Schwarz, 1996a Powell et al., 1995 Thong et al., 1998 Murase et al., 1995 Murase et al., 1996 Wegener & Baggesen, 1996 Buchrieser et al., 1997 On & Baggesen, 1997

detection of chromosomal rearrangements in S. typhi by PFGE with I-CeuI; also ribotyping superior discriminatory value of PFGE using XbaI, SpeI and NotI for epidemiologically unrelated S. enteritidis isolates; see also plasmid profile, ribotyping and 6200-typing derivation of S. enteritidis PT9a and 7 strains from a PT4 strain revealed by PFGE and ribotyping; also ribotyping investigation of S. enteritidis gastro-enteritis outbreak by PFGE (Xbal, AvrII and SpeI) evaluation of PFGE (BlnI, XbaI) for epidemiological analysis of Salmonella outbreaks (S. typhimurium, S. Thompson, S. enteritidis) variations in PFGE patterns of S. enteritidis isolates from a food poisoning tracing back of aS. Infantis outbreak to a single pig slaughterhouse and its supplier pig herds by PFGE (XBaI) subdivision of S. enteritidis PT4 and 8 strains by PFGE using NotI and XbaI, supporting the hypothesis of a single clone pandemic determination of 2 principal clonal lines of S. typhimurium in Denmark by PFGE using XbaI and ribotyping; also ribotyping

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Table 4: Continued

Molecular typing technique

Reference

Specific comment

Boonmar et al., 1998

epidemiological analysis of S. enteritidis isolates from humans and broilers by PFGE (BlnI and XbaI) and phage typing, indicating the spread of a genetically identical clone subdivision of S. Indiana by PFGE using XbaI derivation of ampicillin-resistant S. enteritidis PT6a from PT4 revealed by PFGE (XbaI) and plasmid analysis subdivision of S. enteritidis outbreak isolates by PFGE (XbaI and NotI), plasmid profile and phage type; see also plasmid analysis PFGE using XbaI, SpeI and Avr II slightly less discriminatory than ribotyping for S. enteritidis isolates; see also ribotyping most discrimination between S. Dublin field isolates by PFGE using XbaI and SpeI; see also plasmid analysis, ribotyping, IS200typing combination of PFGE (XbaI), RAPD and phage typing for discrimination of S. enteritidis strains from different origins

Punia et al., 1998 Ridley et al., 1996 Suzuki et al., 1995 Thong et al., 1995 Liebisch & Schwatz, 1996b Laconcha et al., 1998 PCRribotyping

Lagatolla et al., 1996 Shah & Romick, 1997

rep-PCR

Van Lith & Aarts, 1994 Millemann et al., 1996 Lopez-Molina et al., 1998

Beyer et al., 1998 AFLP

Aarts et al., 1998

specific PCR profile for 7 Salmonella serovars (a.o. S. enteritidis), spacer region polymorphic for 3 serovars (a.o. S. typhimurium) restriction analysis (HinfI) of ribosomal spacer regions for Salmonella subspecies differentiation typing of Salmonella up to the serotype level with ERIC-PCR 1 and 2 fingerprints obtained respectively for S. enteritidis and S. typhimurium strains with ERIC-PCR; see also RAPD ERIC-PCR with 1 primer useful for differentiation between Salmonella serotypes, not for S. enteritidis phage types; see also RAPD REP-PCR and ERIC-PCR (with 1 primer) discriminates epidemic S. Saintpaul strain from other strains, see also RAPD Salmonella serotype and phage type identification and discrimination of strains, previously identified as identical by other methods

The classical second level is phage typing, but this method has often a too low discriminatory power for detailed outbreak investigations, a phage typing scheme is only available for some serovars, and some isolates may be untypable. Being a phenotypic method, phage typing can not be used for delineating phylogenetic relationships. Another problem is phage conversion in some serovars, which may disturb the study of epidemiological relationships (Rankin and Platt, 1995). Several molecular typing techniques have the potential of second level typing, which 225

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corresponds with clonal lineage or strain level (see Table 4). For some techniques, the discriminatory power or applicability is serovar dependent: e.g. IS200 typing gives the highest discrimination in S. typhimurium (Olsen et al., 1997), but is not applicable for S. Hadar (Weide-Botjes et al., 1998b). The demonstrated discriminatory power may also depend on the set of isolates included: e.g. ribotyping has been reported as giving no or little differentiation (Liebisch and Schwarz, 1996a) and as being slightly higher discriminatory than PFGE (Thong et al., 1995) for S. enteritidis. One of the greatest challenges is the molecular typing of S. enteritidis which is a highly clonal organism, particularly within certain phage types (e.g. PT4 and 8). Currently, it seems that RAPD gives the best discriminatory power for S. enteritidis isolates belonging to different or even the same phage type (e.g. Fadl et al. 1995), provided that a suitable primer is identified (e.g. Lin et al., 1996). Also PFGE using the enzyme combination XbaI - NotI - SpeI (Liebisch and Schwarz, 1996a) and plasmid analysis (e.g. Millemann et al., 1995) have been shown to be useful for this serovar. A combination of different molecular typing techniques used under optimal conditions and eventually complemented by phage typing, is highly recommended to trace isolates in epidemiological investigations (Weide-Botjes et al., 1998a). S. typhimurium isolates usually show more genetic variation and can be differentiated with several techniques (see Table 4). AFLP using an EcoRI primer with 2 selective bases enabled phage type identification and differentiation of strains, which were indistinguishable by other methods (Aarts et al., 1998). 5.2 CAMPYLOBACTER JEJUNI C. jejuni is one of the most common causes of sporadic gastro-enteritis with poultry products being the most important vehicles for infection. C. jejuni is divided in the subspecies jejuni and doylei, but the former subspecies is the most important one. Classical typing schemes are the Penner heat-stable and Lior heat-labile serotyping schemes and biotyping. Several molecular typing techniques have been developed or evaluated for a higher and universal applicable discrimination of isolates. A PCR-RFLP technique is based on the sequence heterogeneity of the flagellin gene flaA and is referred to as flaA typing or profiling (Nachamkin et al., 1993) using mostly the restriction enzymes DdeI and HinfI (Santesteban et al., 1996). Both the flaA and fla B genes, occurring in tandem, can also be used as combined target for restriction analysis (Ayling et al., 1996). Alternatively, a short (150 bp) variable region of theflaA gene can be used as target in direct sequence analysis for epidemiologic investigations (Meinersmann et al., 1997). Recently, evidence for intergenomic recombination betweenflaA genes of different C. jejuni strains as well as intragenomic recombination between the flaA and flaB genes within a strain has been deduced from mosaic flaA gene structures, which has as important consequence that flagellin gene typing cannot be used for long-term epidemiological monitoring and determination of clonal relationships within Campylobacter populations (Harrington et al., 1997). This problem could be overcome by combining the polymorphisms determined by PCR-RFLP of several genetic loci as demonstrated by a multiplex PCR gene fingerprinting method based on the variable gyrA and pflA genes (Ragimbeau et al., 1998). It seems that flaA types are conserved across different serotypes and that flaA typing is less discriminatory than PFGE and thus cannot be used as sole basis for grouping 226

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strains (Santesteban et al., 1996). PFGE was also shown to be the most discriminatory of 3 typing methods (including ribotyping and phage typing) for C. jejuni strains of different Penner serotypes and within a single heat stable serotype (Gibson et al., 1995), and the most discriminatory of 4 typing methods (including fatty acid profile typing, biotyping and serotyping) for C. jejuni and C. coli isolates from abattoirs (Steele et al. 1998). For the DNAse-positive strains of Lior biotype II, formaldehyde fixation of the cells is necessary to perform PFGE (Gibson et al., 1994). Definition of clonal lineages within C. jejuni with PFGE must be based on the patterns obtained with at least 2 restriction enzymes (e.g. SmaI and KpnI) (Gibson et al., 1997). PFGE typing of field isolates from Finnish patients, chicken faecal samples and meat samples (Hänninen et al., 1998), from Canadian meat processing plants (Steele et al., 1998) and from sporadic cases of diarrhoeal disease in England (Owen et al., 1997) have all shown a high degree of genomic diversity within C. jejuni. It is not unlikely that the observed large genetic variation may at least partly be attributed to genomic instability within single strains as the result of intragenomic recombinations or natural transformation by non-homologous DNA and driven by environmental pressures (Wassenaar et al., 1998). In vitro genotypic variation of C. coli induced by repeated subculturing has already been observed by PFGE (On, 1998). PFGE patterns must therefore be carefully interpreted and preferably combined with other (single locus) molecular typing techniques and/or with numerical analysis in order to evaluate relationships between Campylobacter strains. The same caution probably also applies to other whole-genome typing techniques such as RAPD, which has been shown to have an excellent discrimination ability within different Campylobacter species and Penner serotypes (Hernandez et al., 1995; Madden et al., 1996). By RAPD typing, possible transmission routes (e.g. environment, farmer’s footwear) of Campylobacter infection in well-defined settings (successive broiler flocks) have been demonstrated (van de Giessen et al., 1998; Payne et al., 1999). Because Campylobacter does not seem to be of a clonal nature, but rather consists of genomic mosaics, it may be difficult or impossible to define epidemiological links by molecular typing in less defined settings (e.g. different farms) as shown by Weijtens et al. (1997). A SRFH technique with a probe based on the highly conserved domain (HCD) of the tlpA gene, encoding for a methyl-accepting chemotaxis-like protein from C. coli, has also been proposed as a molecular typing scheme which is not likely to be subject to an extensive degree of genetic instability (Gonzalez et al., 1998). 5.3 LISTERIA MONOCYTOGENES L. monocytogenes causes a rare but severe disease in humans, which is in most if not all cases caused by industrially processed food with an international distribution (e.g. cheese). Serotyping is of limited epidemiological value as only 3 serovars (1/2a, 1/2b and 4b) are causing most of the infections. Because of the large socio-economic impact, the World Health Organisation (WHO) food safety unit in Geneva mandated in 1990 a multicenter study on L. monocytogenes subtyping methods. The results of the first phase are published in a special issue “Molecular typing of Listeria” of the International Journal of Food Microbiology (Vol. 32, 1996). The conclusions of this study can be summarised as follows (Bille and Rocourt, 1996):

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•

amongst the phenotypic typing methods, serotyping is a useful first step method with a good reproducibility for certain serotypes (4b and 1/2a), and phage typing is most valuable for mass screening, but both methods need standardization. MEE is reasonably reproducible, but too laborious for large sets of strains. • ribotyping has a limited discriminatory power, particularly for the serotype 4b which is responsible for most of the major recent outbreaks, but has the advantage of an automation of the procedure (Riboprinter) (Swaminathan et al., 1996). • high-frequency restriction endonuclease typing (REA), which is a variant of RFLP using high-frequency cutting enzymes (EcoRI, HaeIII, HhaI), shows a good reproducibility and high discriminatory power, but needs further standardization and computer analysis for objective interpretation of the patterns (Gerner-Smidt et al., 1996). • PFGE (using ApaI and SmaI) is highly discriminatory and reproducible and looks very promising if a computerised analysis of the patterns in association with a strict standardization of the protocol can be used (Brosch et al., 1996). • RAPD also looks very promising, but several problems of intra- and interlaboratory reproducibility are encountered (Wemars et al., 1996). It is expected that this Listeria typing study will serve as a model for other pathogens. Other studies have also shown the superior discriminatory power of RAPD/AP-PCR and PFGE for L. monocytogenes strains belonging to several serovars (Louie et al., 1996; Destro et al., 1996). The importance of silage as a source of listeriosis outbreaks in ruminants was demonstrated by ribotyping performed with the Riboprinter (Wiedmann et al., 1996). These authors acknowledge that ribotyping is less discriminatory than other typing methods such as RAPD, but argue that this is not necessarily a disadvantage since too sensitive a typing system might detect epidemiologically irrelevant strain differences based on point mutations and/or DNA rearrangements. Rep-PCR (REP- and ERIC-PCR) generates L. monocytogenes serotype specific patterns and within the heterogeneous serotype 1/2a REP-PCR shows a similar strain discrimination as RAPD (Jersek et al., 1996). It was also observed by rep-PCR that there is no or only little similarity between L. monocytogenes isolates from humans and animals and from food, suggesting that only a minor proportion of food strains are pathogenic (JerSek et al., 1999). 5.4 ESCHERICHIA COLI 0157 The serotype E. coli O157:H7 (motile) and O157:H- (non-motile variant), belonging to the enterohaemorrhagic E. coli (EHEC) which are a subset of the verocytotoxic E. coli (VTEC), stands as the type example of the so-called new emerging infectious diseases.. The diversity of strains in cattle and sheep, the major reservoirs of potential pathogenic VTEC and of E. coli 0157 strains, has been investigated by PFGE (Kudva et al., 1997; Beutin et al., 1997; Heuvelink et al., 1998). PFGE (using Xba I) has the highest discrimination for E. coli 0157 (giving the best discrimination) (Grif et al., 1998) and is therefore highly used in the epidemiological investigation of foodborne outbreaks, 228

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which have been linked in many cases to undercooked hamburgers (Barrett et al., 1994). Ribotyping, on the contrary, is not able to discriminate between E. coli 0157 isolates (Martin et al., 1996). In the USA, the PulseNet uses a standard PFGE procedure and an electronic pattern database for E. coli 0157 (Anonymous, 1996). However, in the interpretation of molecular typing results of E. coli 0157, 2 important phenomena must be accounted for. Firstly, for the phylogenetically highly related E. coli 0157 isolates from human infections which seem to belong to a single clone complex, PFGE has its limitations because epidemiologically unrelated strains may differ in only a few fragment bands making an unequivocal differentiation between outbreak and nonoutbreak related strains sometimes difficult (Böhm and Karch, 1992). Combination with another typing method such as RAPD (Birch et al., 1996) or phage typing is therefore highly advised (Grif et al., 1998). Secondly, E. coli 0157 can undergo rapid genotype alteration in the course of infection, so-called clonal turnover, which may be caused by the chromosomal integration or loss of verocytotoxin gene carrying bacteriophages (Datz et al., 1996), and may result in the appearance of new PFGE patterns. SRFH techniques with the use of Shiga-like (SLT) or verocytotoxins probes (SLT-RFLP) (Samadpour, 1995) and bacteriophage λ probe (λ-RFLP) (Grimm et al., 1995) have been shown to be very sensitive methods for interstrain differentiation. A PCR based typing method based on the E. coli repetitive element IS3 has been developed as a rapid screening method for the identification of unrelated E. coli O157:H7 isolates (Thompson et al., 1998). 5.5 SOME OTHER FOODBORNE BACTERIAL PATHOGENS PFGE (using Sma I) (Liu et al., 1997) and RAPD methods (Nilsson et al., 1998) have been developed for the discrimination of strains of the sporeformer Bacillus cereus. The AFLP technique has been extensively evaluated for the genus Aeromonas (Huys et al., 1996). The combination of different ribotyping procedures (classical ribotyping and PCR-ribotyping) has been shown useful for strain discrimination in Yersinia enterocolitica (Lobato et al., 1998).

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BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION CAHILL, S.M., UPTON, M.E., AND MCLOUGHLIN, A.J. Department of Industrial Microbiology, Ardmore House, University College Dublin, National University ofIreland, Belfield, Dublin 4, Ireland

Abstract The fermentation process has long been used as a method of meat preservation. In order to eliminate batch to batch variation the fermentation process must be standardised. This, combined with problems associated with emerging pathogens such as enterohaemorrhagic Escherichia coli has led to a re-examination of the process of fermented meat production in order to ensure the production of a consistently high quality and safe product. Encapsulation technology can be applied to meat fermentations with the objectives of enhancing the existing methods of preservation and in developing novel methods to combat the problem of emerging and re-emerging pathogens. Encapsulation technology has been shown to be beneficial in the production of fermented meats by both direct and indirect acidification. Indirect acidification occurs after the addition of a starter culture to the meat and encapsulation technology has been observed to enhance its activity upon its addition to meat. The success of encapsulation would appear to be based on some form of spatial organisation involving a) protection and b) controlled release. The creation of a microenvironment, which provides the desired conditions or populations and the physical regulatory systems, minimises the effect of fluctuations in the macroenvironment and protects the cells from competition, predation and lysis. Controlled delivery from the microenvironment assist the cells to adapt to the new macroenvironmental conditions and then release the adapted cells under regulated conditions. Encapsulated acidulants are already in use in the United States and their use is on the increase in Europe. Problems such as discoloration and lack of binding associated with direct acidification can be overcome by encapsulation, which allows the time and rate of acid release to be controlled. Emerging pathogens are now challenging the antimicrobial hurdles present in a fermented meat product. This has led to investigations into enhancing the safety of existing manufacturing processes for fermented meats. Bacteriocins are antimicrobial agents, which are naturally produced by lactic acid bacteria. However, their ineffectiveness towards Gram-negative bacteria and their reduced activity in meat products has excluded their use until now. Combining bacteriocins, for example nisin, with other stresses enhances their activity towards Gram-negative pathogens. 239 A. Durieux and J-P. Simon (eds.). Applied Microbiology, 239–266. ©2001 Kluwer Academic Publishers. Printed in the Netherlands.

CAHILL, S.M., UPTON, M.E., AND MCLOUGHLIN, A.J.

Encapsulation in polymer gels facilitates the optimisation of conditions for in situ nisin production and permits the controlled release of bacteriocins into the macroenvironment. Therefore, this technology has the potential to facilitate the use of bacteriocins produced by lactic acid bacteria in meat products as a means of overcoming potential problems associated with emerging pathogens. 1. Introduction The deterioration of food products is an inevitable process. However the rate at which this process occurs is dependent on the food type, composition, formulation, packaging and storage conditions (Gould, 1995). For centuries meat has been considered as a highly valued and nutritious food (Shay and Egan, 1992). However, it is due to this nutritious nature that meat is also a highly perishable product and when stored in air spoils rapidly as a result of the growth of Gram-negative bacteria (Egan, 1983). Apart from microbiological spoilage numerous chemical and biochemical changes occur during the storage of meat, which limit its shelf life. While many foodstuffs, including meat can now be preserved by refrigeration and freezing, the preservation of food has long preceded the technical advances, which permit such methods of shelf-life extension. Examples of the more traditional methods of preservation include heating, drying, curing, smoking and fermentation. The high sensory and nutritious quality of the resulting products has meant that, even in the face of modern technology, many of these methods of preservation have not only survived but also retained a high level of popularity. For example, pepperoni, a fermented meat product, has an annual consumption rate of 370 million pounds in weight in the United States (Hinkens et al., 1996). While many methods of meat preservation exist, the focus of this article will be the enhancement, using bioencapsulation technology, of meat preservation techniques based on fermentation and acidification. Although meat preservation by microbial fermentation is among the oldest forms of food preservation (Dillon and Cook, 1994), dating back to about 1000 BC (Nychas and Arkoudelos, 1990), and also one of the most successful means of prolonging the shelf-life, new challenges and research opportunities continually arise. Recent food poisoning outbreaks associated with enterohaemorrhagic Escherichia coli in fermented meat products (Anonymous, 1995b; Tilden et al., 1996) have prompted many researchers to further examine the safety of these products and to search for novel means of enhancing their safety. Also, as in any process, there is a continual search for technical innovations, which will enhance quality and production efficiency (Prochaska et al., 1998). Encapsulation, which has been described as an “old yet new” technology (Pszczola, 1998) is one means by which advancement can be made in the area of meat preservation. Encapsulation technology has been used in the food industry for more than sixty years with the encapsulation of flavourings by spray drying in the 1930’s being considered the first application of this process (Reineccius, 1995). However, it has only been adapted slowly and so can still be regarded as a developing technology within the food sector. This is probably due to the requirement for low cost food grade encapsulation materials by the food industry. Encapsulation technology in the food sector is currently growing at a level of 30% annually in the US and one of the leading 240

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producers of encapsulated food ingredients reported a doubling of sales in Europe in 1997 (Personal Communication, 1998). Encapsulation can be defined as the technology of packaging a substrate (solids, liquids, gases) within another material (Risch, 1995). In the case of bioencapsulation the material which is packaged is a biologically active agent. The resulting miniature capsules or packages can range from sub-micron to several millimetres in diameter and are ideally spherical in shape (Jackson and Lee, 1991). However this shape will depend on the physical structure of the material being encapsulated. In the encapsulate the active agent which has been entrapped is termed the core material or the internal phase while the encapsulating material is referred to as the coating, shell wall material or the carrier. A large variety of materials can be used as encapsulating agent including proteins, fats and carbohydrates (Jackson and Lee, 1991). The nature of the material used is generally dependent on the subsequent use of the encapsulate as well as the encapsulation and release properties required. Both encapsulation technology and fermentation technology are each in themselves quite complex processes. Thus the merging of two such technologies has the potential to result in interesting developments in the area of meat preservation. 2. Meat preservation The preservation of meat by acidification or the lowering of pH is currently carried out using one of two processes: • Biological acidification - the reduction of pH by microbial fermentation. This is achieved by the addition of lactic acid producing starter cultures to the meat. The reduction in pH may also be achieved by the activity of the natural lactic acid producing microflora present in the meat or inoculating the meat with the product of a successful fermentation. • Chemical acidification - the reduction of the pH of the meat to a level which will inhibit spoilage and pathogenic bacteria by the addition of a chemical acidulant such as glucono-delta-lactone. This method of acidification has developed as a means of overcoming the uncertainties, which are sometimes associated with a microbial fermentation. 2.1 BIOLOGICAL FERMENTATION Fermented meat products are defined as meats that are deliberately inoculated to ensure sufficient acidification and controlled microbial activity to alter the product characteristics (Bacus, 1986). Although this technology has been extensively researched and reviewed (Leistner and Lucke, 1988; Hammes et al., 1990; Shay and Egan, 1992; Campbell-Platt and Cook, 1995; Ricke and Keton, 1997) there continue to exist areas for improvement. The general steps in a meat fermentation process are outlined in Figure 1. Examination of the steps in the production of a fermented meat product allows the identification of potential problems in this process (Table 1). While many quality and safety problems can be overcome by the implementation of a HACCP system, this does not solve all problems, particularly those associated with the activity of the starter 241

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culture. Process control must combine optimisation of the conditions for acid production with those for drying, flavour and texture development etc. and achieving this may necessitate a compromise of the conditions. In relation to the starter culture activity, it is not be possible to control important factors such as strain degeneration, shelf-life stability and ecological competence by controlling the conditions in the fermentation process. Encapsulation has been described as a ‘technology which can solve certain problems which cannot be solved otherwise’ (Pszczola, 1998) and therefore its potential in meat preservation, particularly in the area of starter culture activity deserves some examination.

Figure 1: Outline of the main steps in a meat fermentation process

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BIOENCAPSULATION TECHNOLOGY IN MEAT PRESERVATION Table 1: Potential problems associated with the meat fermentation process

Potential problems Microbial load -presence of pathogens Environmental contamination, growth of pathogens -viability and activity - competition from natural microflora - microbial load Uniform distribution of additives in meat Environmental contamination, Process control Process control - temperature, humidity Process control - temperature, humidity

Step Meat Chopping and mixing Additives - Starter culture - Spices Mixing of meat and additives Filling the casing Fermentation Maturation and drying

2.2 CHEMICAL ACIDIFICATION While the direct addition of organic acids such as lactic acid would appear to be a faster and more reliable means of preservation than microbial fermentation, their use has been limited due to their instantaneous reaction with the meat which causes undesirable colour and texture changes (Shay and Egan, 1992). A more successful alternative has been the addition of glucono-delta-lactone (GDL) to lower the pH. After addition to the meat the GDL is hydrolysed to gluconic acid bringing about a reduction in pH (Shay and Egan, 1992). As the use of GDL does not involve the direct addition of an acid, the problems associated with discoloration and textural changes are reduced. However, the extent to which this reduction in undesirable characteristics occurs depends on the rate at which the GDL is hydrolysed. Furthermore, once hydrolysed to gluconic acid, it can be further metabolised to acetic acid and carbon dioxide which can add a bitter taste and gas pockets to the meat (Kneissler et al., 1986). On the other hand, the application of encapsulation technology to food acids permits the controlled or slow release of these ingredients thus increasing their usefulness in meat preservation (Dziezak, 1988). 3. The application of encapsulation technology to meat preservation 3.1. THE APPLICATION OF MICROBIAL FERMENTATION

ENCAPSULATION

TECHNOLOGY

TO

A

While the natural microflora in a meat system will, under suitable conditions, result in a fermentation process (Cooke et al., 1987; Gibbs, 1987; Grombas, 1989), the unreliability of this process has long been recognised. The practice of backslopping, which involves initiating a new fermentation by the addition of some of the product of a successful fermentation, was developed as a means of overcoming the uncertainty (Gibbs, 1987; Grombas, 1989; Nout and Rombouts, 1992). Today, the production or purchase of specially selected and prepared starter cultures is probably one of the most important and also expensive parts of the fermentation process since a successful fermentation is dependent on the addition of a viable and active inoculate. Therefore, 243

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the development of the technology to control the ecological competence of starter cultures in fermented meats is vital. In order to be viable and active it is necessary for the starter culture to survive the stresses associated with its preservation, storage and reactivation. Encapsulation or immobilisation of starter cultures has been studied quite extensively as a means of providing benefits such as cell retention in continuous culture systems (Prevost and Davies, 1987, 1992), protection from phage (Steenson et al., 1987; Passos et al., 1994) and improved storage stability (Kim et al., 1988; Sheu and Marshell, 1993; Sheu et al., 1993; Champagne et al., 1994). Encapsulation in polymer gels has been examined as a means of ensuring the ecological competence of meat starter cultures, although studies on the encapsulation of meat starter cultures have been quite limited (Kearney, 1990; Kearney et al., 1990a, b; McLoughlin and Champagne, 1994). However, immobilisation of meat starter cultures has recently been identified as one of the areas in meat fermentation which deserves further investigation (Ricke and Keeton, 1997; Prochaska et al., 1998). With the aid of electron microscopy studies on fermented meat products, it has been shown that the natural microflora, and also the starter culture bacteria, grow in cavities or nests within the meat (Katsaras and Leistner, 1991). Encapsulation of the starter culture provides a means of controlling the conditions and the microflora within these nests. 3.1.1. Encapsulation matrices and the encapsulation process. The encapsulation process involves the ‘trapping’ of cells or molecules within the lattices of a polymer gel. The entrapment matrix may be naturally occurring polymers such as alginate, carrageenan or chitosan or a synthetic polymer such as polyacrylamide (Woodward, 1988). Naturally occurring polymers are widely used in immobilisation, with algal polysaccharides being the most popular matrices (Willaert and Baron, 1996). Alginate, which is a polysaccharide, is most commonly extracted from the brown seaweed Laminaria hyperborea. Alginate consists of unbranched copolymers of α(1→4)-L-guluronic acid (G) and β-(1→4)-D-mannuronic acid (M) and their relative proportions (G/M ratio) determines the functional properties of the alginate (Murata et al., 1993). Gelation of alginate occurs as a result of the binding of divalent cations to the alginate and this is also accompanied by a conformational change. These cations bind preferentially to the α-(1→4)-L-guluronic acid; therefore, a high content of Gblocks results in a strong gel (Willaert and Baron, 1996). Calcium alginate beads can be produced by adding droplets of a sodium alginate solution into a calcium chloride bath where droplets form gel spheres which maintain their shape as a result of cation exchange between Na+ and Ca2+. During gelation these gel spheres entrap materials or cells which were formerly dispersed in the alginate solution within an “egg-box” type structure. Immobilisation within alginate beads has been successful because it is a mild process, which can be carried out quickly, simply and cheaply. The mild process (Figure 2) is also an important factor in that it means that encapsulation does not add additional stress to the bacteria before they undergo the preservation process. However, this method also has disadvantages, the most significant of which is the sensitivity of calcium alginate beads to chelating agents such as phosphate, citrate, lactate and EDTA (Willaert and Baron, 1996). Nevertheless, alginate in particular, has been focussed upon as an encapsulation matrix due to the mild nature of the entrapment process and also the

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status of alginate as a food grade additive (sodium alginate -E401, calcium alginate E404) (Anonymous, 1995a).

Figure 2: The process of cell encapsulation in polymer gel beads

Carrageenans are polysaccharides, which are extracted from red seaweed of which Chondrus crispus is the main source. There are three main types of carrageenan, kappa (κ), lambda (λ) and iota (i) with κ-carrageenan the most widely used for cell immobilisation (Willaert and Baron, 1996). Carrageenans are sulphated linear polysaccharides of D-galactose and 3-6 anhydro-D-galactose with the various carrageenans differing from one another in their content of 3-6 anhydro-D-galactose and the number and position of ester groups (Trius and Sebranek, 1996). Gelation can be achieved by cooling, due to the thermal properties of the gel, or by contact with a solution of gel inducing reagents such as K+, NH4+, Ca2+, Cu2+, Mg2+, amines or water miscible organic solvents (Chibata et al., 1987). This is a mild and simple process. Carrageenan gel networks are formed during cooling by a series of polymer chain associations to give rise to a 3-D helix framework. This framework is stabilised in the presence of cations by the formation of additional bonds (Trius and Sebranek, 1996). carrageenan gelation is dependant on cations but unlike alginate beads, carrageenan beads are thermally reversible (Willaert and Baron, 1996). Chitosan is another polysaccharide which is used for immobilisation but it is not as widely employed a matrix as alginate or carrageenan. Chitosan is a partially deacetylated chitin, which is formed by reacting chitin with a concentrated alkali (Vorlop and Klein, 1987). It is a high molecular weight linear polymer consisting of 1→4 linked glucosamine and has a high nitrogen content (>7% w/w) (Vorlop and Klein, 1987; Willaert and Baron, 1996). Chitosan is soluble in organic acids and to a more limited extent in mineral acids e.g. HCl. Gelation of chitosan occurs as a result of 245

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binding with multivalent anions. When chitosan is added dropwise to a cross-linking solution having a pH < 6 the NH2 groups of chitosan are protonated and an ionic crosslinking occurs. In order to form a true ionotrophic gel the pH of the cross-linking solution must be less than 6. Bead formation will also occur at a pH greater than 7.5 but this is merely a precipitation of chitosan as at this pH the chitosan is totally deprotonated and is water insoluble (Vorlop and Klein, 1987). Chitosan can be crosslinked with low molecular weight counterions such as polyphosphates and also high molecular weight counterions such as alginate. Cross-linking of chitosan with low molecular weight counterions results in globules in which cells or molecules are entrapped in a real network while cross-linking with high molecular weight counterions results in capsules (Vorlop and Klein, 1987). Also, unlike calcium alginate beads, ionotrophic chitosan beads are stable to chelators such as phosphate (Willaert and Baron, 1996). Entrapping cells in a polymer gel permits greater control of the cell microenvironment such as the juxtapositioning of cells and nutrients and controlling the rate of cell release (McLoughlin and Champagne, 1994). Also, gels have the properties of both liquids and solids, for example a 1% alginate gel contains 99% water and yet it shows solid characteristics such as a defined shape and resistance to stress (McLoughlin, 1994). Controlling the level of polymer added permits control over the characteristics of the encapsulate and so allows the properties of the encapsulate to be tailored to a certain extent. 3.1.2. The benefits of meat starter culture encapsulation. The advantages of cell encapsulation can be divided into two main groups: • a) Protection - the provision of optimal conditions for activity and the protection of the cells from a fluctuating and dynamic macroenvironment • b) Controlled release - controlling the rate of release allows the cells to adapt to their new environment (McLoughlin, 1994). With regard to meat starter cultures, the advantages can be defined more specifically as increased stability, enhanced activity or an increased rate of acidification and protection from competition and bacteriophage. 3.1.2.1. Enhanced stability of encapsulated starter cultures. The preservation of starter cultures is necessary to facilitate their storage and transport. The most commonly used methods of preservation are lyophilisation and freezing. The preservation process followed by the rehydration or reactivation process subjects the cells to stress, which can affect their subsequent activity. However, it has been demonstrated that the encapsulation of the meat starter cultures Lactobacillus plantarum and Pediococcus pentosaceous in calcium alginate beads, in the presence of cryoprotectants, prior to freezing or lyophilisation, resulted in a higher level of survival than that observed for free cells (Kearney, 1990). This increased survival appears to be due to the rate at which the cells are rehydrated or reactivated. With encapsulated cells rehydration is not instantaneous and is controlled by the diffusion properties and the actual volume of the encapsulate thus avoiding the osmotic shock associated with instantaneous hydration (Prochaska et al., 1998). Thus, the use of encapsulation to create a microenvironment,

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whose properties are influenced by the presence of polyols and skim milk, can result in increased survival rates of meat starter cultures (Kearney, 1990). 3.1.2.2. Enhanced activity of encapsulated starter cultures, Encapsulation of meat starter cultures has also been shown to increase fermentation rates compared to free cells (Kearney et al., 1990b). This is a consequence of the increased stability provided by immobilisation (section 3.1.2.1), which subsequently decreases the lag phase of growth and acidification. The encapsulation matrix also offers protection from other stresses in the meat macroenvironment such as salts (Kearney et al., 1990a). This is an important observation particularly in light of the fact that the search for techniques to control emerging food pathogens may involve the inclusion of additional stresses in the meat. 3.1.2.3. Protection from bacteriophage. Encapsulation technology has been shown to protect starter cultures from bacteriophage by means of exclusion of the bacteriophage from the microenvironment of the encapsulate (Steenson et al., 1987). While bacteriophage are recognised as a major problem within the dairy industry their role in meat fermentations appears to be of less consequence (Nes and Sarkeinn, 1984). However, the fact that entrapment within a matrix protects cells from bacteriophage also means that the cells are protected from the competitive effects of the natural microflora present in the meat. 3.1.2.4. Genetic stability. The utilisation of biotechnological approaches as a means of producing more efficient starter cultures is becoming increasingly important. The usual approach to gene cloning of the lactic acid bacteria is the development of vectors based on either cryptic plasmids or heterogeneous plasmids which are resistant to a wide range of antibiotics (Gasson, 1993). While the development of genetically engineered starter cultures for use in meat fermentations on a large scale has so far been elusive, work in this area is ongoing. However, once developed the stability of these strains will be of great importance. Encapsulation in calcium alginate beads has been shown to increase the stability of plasmid containing bacteria (O’Donnell, 1997). Therefore, encapsulation technology has the potential to enhance the biotechnological developments made in the area of starter culture technology. 3.1.3. Commercial applications. As yet the advantages of encapsulation of meat starter cultures in alginate beads has not been exploited at a commercial level. This is probably due in part to the fact that the technology for the large-scale production of polymer gel encapsulated bacteria is only evolving, The common entrapment technique, which involves adding the cell containing alginate solution into a bath of calcium ions in a dropwise manner, is difficult to perform on a large-scale. However, one engineering company has recently developed the equipment to carry out this process on a large-scale (Anonymous, 1999). This may facilitate the large-scale production of polymer gel encapsulated starter cultures and so enable the commercial exploitation of the technology.

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Methods of internal gelation, which involve the addition of the calcium source directly into the alginate solution and controlling the availability of the calcium ions through the use of calcium chelators (Monshipouri and Rice, 1995) or pH (Poncelet et al., 1992, 1995; Quong et al., 1998) have permitted the development of emulsification or dispersion methods of encapsulation. This involves the addition of the alginate/calcium mixture into a bath of oil and applying a shear in order to disperse the gel mixture throughout the oil, thus producing spherical encapsulates. The use of a calciumchelating agent slows down the gelation process sufficiently to permit the dispersion of the gel in the oil. The pH method involves the external addition of an acid to reduce the pH and cause the release of calcium ions from the calcium source present. The acid is added after the emulsification or dispersion step. While these techniques are suitable for the large-scale production of alginate beads, an improvement of the encapsulation technique is still required. The harvesting of the encapsulates from the oil has been reported to be problematic (Friel, 1998). Also, a broad size distribution of the encapsulates produced by this method has been observed (Poncelet et al., 1992; 1995). 3.2. THE APPLICATION OF ENCAPSULATION TECHNOLOGY TO CHEMICAL ACIDIFICATION In contrast to meat starter cultures, the encapsulation of acidulants has been extensively studied and commercialised (Marchot, 1993). The application of encapsulation technology to food acids has been spurred on by the fact that acidification is a valuable means of food preservation (Davidson, 1997), even though the direct addition of acids to many foods results in undesirable flavour and colour changes (Shay and Egan, 1992). Also, the application of acids to a wide range of food products including meat, dairy products, bakery products, desserts and canned fruits and vegetables (Dziezak, 1988; Janovsky, 1993; Marchot, 1993) would appear to assure the commercial viability of encapsulated acids. 3.2.1. Encapsulation matrices and the encapsulation process. A large variety of materials can be used as the coating or encapsulation matrix and the selection of the matrix will depend to a certain extent on the properties required by the encapsulate. Lipids, for example waxes, paraffin, steric acid and oils such as hydrogenated soya oil, palm oil and cottonseed oil are probably among the most commonly used encapsulation materials, although there have also been reports on the entrapment of acids in alginate (Cordray and Huffman, 1985; Ensor et al., 1990; Siragusa and Dickson, 1992). While the immobilisation of acids in alginate involves, like free cells, the crosslinking of the alginate with calcium ions, the process of entrapment within a lipid matrix is quite different. Some of the processes which have been utilised for the entrapment of acids in lipids include spray cooling, spray chilling, air suspension coating and centrifugal-suspension coating (Jackson and Lee, 199 1), techniques which allow the effective utilisation of "hot melt" technology. Encapsulation protects the acid from premature reaction with the meat and provides for controlled release when the necessary conditions are met (Janvsky, 1993). Release of the lipid-encapsulated acids is

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related to temperature but other mechanisms of release such as that caused by the gradual intrusion of water are also being investigated. Spray cooling and spray chilling involve the use of cool air to solidify the encapsulate (Lamb, 1987). These two processes differ in the encapsulating material used in that lipids with low melting points (32 – 42°C) are used in spray chilling while spray cooling utilises lipids with a higher melting point (45 – 122°C) (Dziezak, 1988). The acid/lipid mixture is extruded through heated nozzles into a low temperature chamber where the lipid solidifies, entrapping the acid. While spray chilling and cooling are suitable for liquid acids, air suspension coating is used to coat or encapsulate a solid core material. The process involves suspending the solid core material in a fluidised bed of air and spraying with the coating material (Dziezak, 1988; Kondo, 1989). The thickness of the coating material can be controlled by the length of time for which the coating is applied. Centrifugal-suspension coating involves suspending the core particles in the liquid coating and then introducing them onto a rotating disk (Sparks and Mason, 1990). This results in the coating material forming a film around the core material. Another process which has been described for acidulant encapsulation involved plating the particulate acidulant onto calcium lactate and then coating with a molten edible lipid (Percel and Perkins, 1985). The product of this process would have controlled release at a higher temperature. 3.2.2. The benefits of acidulant encapsulation. The encapsulation of acidulants permits control over the rate and time of acid release. These are important factors in meat preservation by acidification. During sausage production the ingredients must be allowed to 'bind' after mixing. This process is prevented if the pH is reduced too quickly resulting in a product with a crumbly texture, which is undesirable in a sausage. By encapsulating the acid so that the time of release is controlled, for example delaying acid release until the meat reaches smokehouse temperature, means that the use of an organic acid need not compromise sausage texture. The second problem with direct acidification is its adverse effect on colour development. The pH of preserved meats must be carefully controlled in order to allow the formation of nitroso-hematin pigments, which are responsible for the typical pinkred colour of these meat products (Jackson and Lee, 1990). Encapsulated acids can be released slowly thus allowing the desired colour pigments to form. Encapsulated acids have also been reported as a means of overcoming the batch to batch variation and long processing time which are inherent in the microbial fermentation process (DeZarn, 1995). Using encapsulated acids it is possible to develop a formulation or recipe with which a very reproducible pH can be achieved. Also, the processing time can be drastically reduced compared to a microbial fermentation thus allowing a higher level of production. 3.2.3 Commercial availability. The commercial viability of encapsulation is reflected in the number of patent applications in this area (Pszczola, 1998). Recently Doskocil Food Service Co. in the United Stated have patented a process for producing dry and semi-dry sausage products 249

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using an encapsulated acid (Anonymous, 1998). The process involves the use of an acidulant, which has been encapsulated in a material with a melting temperature of at least 90°F. Encapsulated acids are available under brand names such as CAP-SHURE® and DURKOTE® with some companies also offering the facility whereby they will tailor-make encapsulates to meet the customers requirements. It must also be noted that the cost of encapsulates compared to the free form is quite expensive, up to three times that of the free acid. For example encapsulated lactic acid costs IR£7.00/kg compared to IR£1.50/kg to IR£2.00/kg for the free form. Therefore, these encapsulates must offer some unique functionality over the free acid in order to achieve and retain economic viability. The application of encapsulation to meat products is not confined to encapsulated acids. Encapsulated salt is also used in meats (DeZarn, 1995). The use of encapsulated salt permits the addition of extra salt to meat products without adversely affecting the shelf life or the texture of the meat. 4, Control of emerging pathogens Recent food poisoning outbreaks associated with enterohaemorrhagic Escherichia coli in fermented meat products have raised questions regarding the safety of these foodstuffs. In the United Stated this has led to regulatory action requiring manufacturers of fermented meat products to demonstrate that their process incurs a five log reduction in E. coli O157:H7 numbers. It has been reported that the traditional fermentation process is not sufficient to achieve this and currently the only means of doing so is to incorporate a heat step into the manufacturing process. While incorporating a heating step will ensure the production of a safe product, taste and texture may be compromised. As a result, alternative methods of eliminating foodborne pathogens and ensuring a safe product are being sought. It is likely that any technique of eliminating foodborne pathogens will also be detrimental to the starter culture bacteria used in microbial meat fermentation. One way of overcoming this may be in the use of bacteriocins, antimicrobial agents produced by lactic acid bacteria. At present only one such agent, nisin, has GUS status (Anonymous, 1969). Nisin has been applied in the meat industry, but with limited success. This is probably due to the low solubility of nisin, uneven distribution, heat sensitivity at neutral pH values, possible binding to meat proteins and antagonising effects within the meat (De Vuyst and Vandamme, 1994). However there have been some reports where nisin was found to be effective at reducing numbers of bacteria attached to meat. Chung et al. (1989) found that nisin had an inhibitory effect on Grampositive bacteria attached to meat. Nisin has also been reported to be successful in controlling the growth of food spoilage organisms and food pathogens on cooked pork when used in combination with a modified atmosphere packaging system (Fang and Lin, 1994). The use of a nisin-producing Streptococcus lactis in reducing bacterial growth in frankfurters has also been reported (Wang et al., 1986). The application of nisin as a possible alternative or adjunct to nitrites and nitrates in the preservation of meats has been suggested (Rayman et al., 1981). This may be an important application as consumer concerns regarding carcinogenic nitrosamines in cured meats increase.

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While the successful application of nisin to meat preservation has so far been elusive, as in the case of direct acidification, encapsulation technology may provide a means of overcoming this problem. 5. The application of encapsulation technology to bacteriocin delivery 5.1 BACTERIOCINS Bacteriocins are proteinaceous antimicrobial compounds produced by a wide range of bacteria but not lethal to the producer organism. The lactic acid bacteria, probably the most important group of starter culture bacteria, produce a wide range of bacteriocins and bacteriocin-like substances (De Vuyst and Vandamme, 1994). The demand from consumers to reduce or remove chemical preservatives from foods has initiated much interest in the use of naturally occurring metabolites to inhibit the growth of undesirable microorganisms (De Vuyst and Vandamme, 1994). Bacteriocins produced by lactic acid bacteria are potential agents for use as natural food preservatives. For example, the use of bacteriocin producing starter cultures for in situ bacteriocin production may be a useful means of food preservation and thus the potential exists to use bacteriocins as natural preservatives in fermented products. While the use of bacteriocins as natural preservatives appears to be an attractive option there are a number of factors to be considered relating to their use in fermented products. Firstly, bacteriocins are generally most effective against bacteria closely related to the producer organism and therefore their effect on the starter culture must be considered before using them in fermented products (De Vuyst and Vandamme, 1994). One means by which this can be overcome is using bacteriocin producing starter cultures, but this may require the use of genetically manipulated microorganisms. Secondly, bacteriocins are generally ineffective towards Gram-negative bacteria such as E. coli and therefore their use can be limited unless they are used in conjunction with another agent such as a chelator, in order to enhance their activity towards Gramnegatives (Blackburn et al., 1989; Stevens et al., 1991, 1992; Cutter and Siragusa, 1995a, b; Shefet, et al., 1995). Thus, when applying bacteriocins in food preservation they should be delivered in such a way as to minimise their effect on the naturally occurring beneficial microflora in both the foods to be fermented and in the consumer while also having a negative effect on specific pathogens. The complex nature of the system highlights the need for a very directed delivery system. Encapsulation of the bacteriocin nisin was studied as a means of modelling a directed delivery system. While it is recognised that nisin may not be the ideal bacteriocin for application in meat fermentations, its position as the only bacteriocin with GRAS status (Anonymous, 1988) means that currently it is the only bacteriocin which can be added directly to foods. 5.2 NISIN As it is a naturally occurring preservative work to find new applications for nisin is ongoing. One area of research involves the transfer of the genetic material encoding 251

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nisin production and nisin immunity to industrial starter cultures (Hugenholtz and de Veer, 1991). This would mean that many fermented food products could be protected by nisin produced in situ without the fermentation being adversely affected. Extending the spectrum of nisin activity to Gram-negative bacteria is also being examined. The application of nisin in combination with food grade chelating agents is reported to increase the inhibitory activity and inhibitory spectrum of nisin (Blackburn et al., 1989; Stevens et al., 1991, 1992; Cutter and Siragusa, 1995a, b; Shefet et al., 1995). The use of nisin in combination with other bacteriocins such as pediocin AcH has been reported to demonstrate greater antibacterial activity against a greater number of Gram-positive bacteria (Hanlin et al., 1993). Nisin in combination with lactate has been found to reduce the numbers of Salmonella typhimurium attached to beef and nisin in combination with EDTA has been reported to reduce the numbers of Escherichia coli O157:H7 attached to beef (Cutter and Siragusa, 1995b). 5.2.1 Encapsulation of nisin The immobilisation process can be used to confine molecules to a certain region in space in such a way as to exhibit hydrodynamic characteristics which differ from those of the surrounding environment (Willaert and Baron, 1996). This can be achieved by attachment or adsorption onto a solid support or entrapment or encapsulation within a matrix. Each of these methods has been reported for the immobilisation of nisin. Encapsulation or immobilisation of the antimicrobial peptide nisin is a concept which has only recently been reported (Daeschel et al., 1992; Lante et al., 1994; Bower et al., 1995a, b; Fang and Lin, 1995; Cutter and Siragusa, 1996, 1997, 1998; Wan et al, 1997). There have been several reports on the immobilisation of nisin by adsorption onto silica surfaces which have suggested that nisin may be adsorbed onto food contact surfaces in order to prevent colonisation of food pathogens thus leading to a safer food product (Daeschel et al., 1992; Bower et al., 1995a, b). Nisin has also been immobilised on organic and inorganic matrices but will only display antimicrobial activity when desorbed from these supports (Lante et al., 1994). Encapsulation of nisin within a polymer gel has also been reported. Nisin immobilised within calcium alginate resulted in a greater reduction in bacterial numbers and also nisin activity was sustained for up to seven days under refrigeration conditions (Cutter and Siragusa, 1996, 1997). The stability of nisin on cooked pork was also improved by immobilisation of nisin in 1-% calcium alginate (Fang and Lin, 1995). Incorporation of nisin within calcium alginate micro-particles has been shown to protect the bacteriocin from degradation by proteolytic enzymes (Wan et al, 1997). This suggests that encapsulation of nisin within a polymer gel may be an effective delivery system for its protection and controlled release to meat matrices. Two methods of nisin delivery, using encapsulation in polymer gels, have been investigated. • Immobilisation of the producer organism, Lactococcus lactis, in calcium alginate beads • Encapsulation of nisin in capsules prepared using a combination of polymers. 5.2.1.1. Formulation of an optimal encapsulation system for nisin production by L. lactis. Immobilisation of cells in calcium alginate beads allows incorporation of a 252

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combination of nutrients and protective agents into the matrix, which enhances inoculum survival (Heijnen et al., 1992). Formulation of a delivery system for a microorganism by incorporating nutrient adjuncts into the bead microenvironment has been previously reported. Including maize cob grits and wheat gluten in alginate beads containing Aspergillus flavus spores enhanced the activity of the mould after field release (Daigle and Cotty, 1995). The incorporation of a combination of skim milk and bentonite resulted in the highest survival and colonisation rates of Pseudomonas fluorescens inoculated into soil (van Elsas et al., 1992). Similarly with immobilised L. lactis, co-entrapping a combination of nutrients and microenvironmental modifying agents increased the activity of the immobilised cells. Thus, one of the obvious benefits of immobilised cell technology is in the control and exploitation of the unique microenvironment associated with gel entrapment and especially using this microenvironment in the stabilisation of microbial cultures (Karel et al., 1985).

Figure 3: The effect of a combination of eo-immobilisation treatments on nisin activity by cells immobilised within calcium alginate beads.

Treatment A; Cells only Treatment B; Cells +1% glucose Treatment C; Cells + 1% tricalcium phosphate Treatment D; Cells + 3% skim milk Treatment E; Cells + 1% Tween 80

Treatment F; Cells + 1% tricalcium phosphate + 3% skim milk Treatment G; Cells + 3% skim milk +1% glucose Treatment H; Cells + 1% tricalcium phosphate +1% glucose TreatmentI; Cells + 1% tricalcium phosphate + 3% skim milk +1% Tween 80 Treatment J; Cells + 1% tricalcium phosphate + 3% skim milk + 1% glucose

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Through manipulation of the bead microenvironmental conditions, nisin production within a polymer gel can be optimised. The inclusion of nutrient and non-nutrient adjuncts with L. lactis cells entrapped in calcium alginate beads has been observed to have an advantageous effect on the activity of immobilised cells. An optimum delivery system for L. lactis was formulated by co-entrapping a combination of adjuncts and cells within the confines of the alginate bead (Figure 3). Locating both the nutrients and the cells within the confines of the encapsulation matrix resulted in an increase in the level of nisin produced by the entrapped cells. The requirement to deliver microorganisms to dynamic environments such as meat and the development of immobilisation technologies has resulted in the evolution of inoculum delivery systems in a number of application areas which improve the resistance of the culture to stress and regulate the release of cells from a protective microniche (McLoughlin, 1994). The application of immobilisation in enhancing the survival of starter cultures during freezing, lyophilisation and subsequent storage has been mentioned previously and is well documented (Kim et al., 1988; Kearney et al., 1990a; Sheu et al., 1993; Sheu and Marshall, 1993; Champagne et al., 1994). However this is only one type of stress from which immobilisation has been shown to offer protection While the limited availability of nutrients has the ability to increase bacterial resistance to stress (Gilbert and Brown, 1995), the inclusion of excess nutrients within the bead microenvironment also increased the survival of cells exposed to acidic and osmotic stresses. The activity of these cells was also enhanced. This means that the incorporation of adjuncts within the bead can be used as a means of manipulating the bead microenvironment to control bacteriocin activity (Figure 4). This is of particular interest in light of the fact that lactic acid bacteria may react to physiological inducers and so manipulation of the cellular environment may stimulate bacteriocin production (De Vuyst et al., 1996). This stimulation of bacteriocin production may be of significant importance when bacteriocin producing lactic acid bacteria are added to foods as starter or protective cultures (De Vuyst et al., 1996). However, the specific environment of the food may limit the capacity of the lactic acid bacteria to produce these antimicrobials (De Vuyst et al., 1996). This was observed to be the case in the presence of an acid and a salt stress (Figure 4). Therefore, designing the bead microenvironment so that it provided a source of nutrients and a means of acid control, as well as providing a physical barrier between the entrapped cells and the macroenvironment, allowed enhanced bacteriocin production under adverse conditions (Figure 4). This was significant in that it demonstrated the potential benefits of entrapment in a polymer gel in the development of inocula for in situ bacteriocin production.

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Figure 4: The effect of o-immobilisation treatment on a) biomass development and b) nisin production by immobilised cells grown in broth at pH 5 and 1% NaCl. and c) biomass development and d) nisin production by immobilised cells grown in broth at pH 7 and 4% NaCl. Treatment A; Cells only Treatment B; + 3% skim milk Treatment C; + 1 % tri-calcium phosphate Treatment D; + 3% skim milk + 1% tri-calciumphosphate Treatment E; + 3% skim milk +I% glucose Treatment F; + 1% tricalciumphosphate + 3% skim milk + 1% glucose

5.2.1.2 The application ofpolymer gels in the controlled release of nisin. One of the disadvantages of nisin production in an immobilised cell system is retention of the bacteriocin within the bead. Microenvironmental manipulation was shown to increase nisin yield, however, this was also accompanied by an increase in nisin retention within the encapsulation matrix. Therefore, the release of nisin from polymer gels and how the release could be controlled and manipulated were investigated. There are numerous advantages to be gained by the controlled release of nisin to the macroenvironment such as enhanced stability and protection from proteinase degradation thus, prolonging activity.

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Figure 5: The release of nisin from polymer gel capsules. The release of nisin into liquid medium DMI was followed over a one week period at 4°C and 200 rpm.

For many bioactive agents it is recognised that there is a need to prolong the duration of activity and develop a means of more efficient utilisation of the bioactive agent (Schacht et al., 1993). Within the area of pharmaceuticals, sustained release formulations are now a relatively common method of delivering low molecular weight drugs (Langer, 1990). The encapsulation of proteins and the development of slow release formulations are viewed as enhancing the stability and thus the applications of proteins in therapeutics (Putney and Burke, 1998). Natural polymers can serve as depot systems in which the agent can be incorporated and from which it is subsequently released over an extended and controllable period of time (Schacht et al., 1993). Although the application of immobilisation or encapsulation technology in the delivery of bacteriocins is a relatively new development (Degnan and Luchansky, 1992; Kirby, 1993; Fang and Lin, 1995; Cutter and Siragusa, 1996, 1997, 1998; Wan et al., 256

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1997), the encapsulation of nisin in calcium alginate has been achieved with some success. It has been observed to prolong the activity of the nisin and protect it from degradation by proteolytic enzymes (Fang and Lin, 1995; Cutter and Siragusa, 1996, 1997; Wan et al., 1997). However, manipulation of the gel matrix in order to control the release of nisin to the macroenvironment has not been reported. This, however, is an important factor in the development of delivery systems for other bioactive agents, such as therapeutic agents (Aydin and Akbuga, 1996; Berthold et al., 1996). The influence of polymers on the release of nisin from the immobilisation matrix has been examined with a view to developing a delivery system, which offered a controlled mode of nisin release. Through exploitation of the polymer composition and charge, immobilisation matrices were designed with the aim of producing encapsulates which exhibited controlled release properties for the bacteriocin nisin. The preparation of encapsulates from alginate and chitosan has been demonstrated as a means of controlling the rate of nisin release, at least into a liquid macroenvironment (Figure 5). The structure of these encapsulates (Figure 6) as well as the chemical properties of the encapsulation matrix and the core material have been identified as factors with a role in the mechanism of controlled release.

Figure 6: The internal structures of capsules preparedfrom a combination of chitosan and alginate gels. (a + b) ACC capsules (alginate core– chitosan coated capsules stabilised with calcium chloride)

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Figure 6: The internal structures of capsules prepared from a combination of chitosan and alginate gels. c) CCA capsules (chitosan core– alginate coated capsules stabilised with calcium chloride) d) CAT capsules (chitosan core– a lginate coated capsules stabilised with tripolyphosphate)

Figure 6: The internal structures of capsules prepared from a combination of chitosan and alginate gels. (e) ATCC capsules (alginate core– chitosan coated capsules stabilised with tripolyphosphate)

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The modification of polymer beads by coating with a polymer of the opposite charge has been previously reported (Huguet et al., 1996; Quong and Neufeld, 1998). It has also been reported that better slow release properties can be achieved when chitosan or polylysine are used to change the permeability of the alginate membrane (Nuissinovitch et al., 1996). Chitosan is also acceptable for oral administration as well as being a good matrix for sustained release properties (Chandy and Sharma, 1992, 1993). Alginate chitosan capsules can be produced as a result of complexing between two oppositely charged polymers, alginate being polyanionic and chitosan being a polycationic polymer (Huguet et al., 1996). Capsules have been prepared with an alginate core and a chitosan coat and vice versa, and the effect of each polymer on the release properties of the other evaluated. Low molecular weight counterions were included in the preparation of these capsules (Knorr and Daly, 1988; Pandya and Knorr, 1991 ; Polk et al., 1994; Chang et al., 1996; Hari et al., 1996; Nussinovitch et al., 1996) as this stabilised the capsule structure. Although capsules can be produced solely from alginate and chitosan (Vorlop and Klein, 1987) these were observed to be weak due to narrow width of the capsule membrane compared to the overall diameter of the beads. This may be overcome by producing capsules of very small diameter. The release pattern from capsules was dependent on a number of factors. According to Huguet and Dellacherie (1996), the release of materials encapsulated in alginate beads coated with chitosan depends on 1) the molecular weight of the material, 2) the chemical composition and 3) the conformation of the molecules. These factors can vary depending on the composition of their environment. Coating alginate beads with a polycation has been reported to drastically reduce diffusion from the beads. Nussinovitch et al. (1996) reported that after 6 days only 50% of the entrapped bovine albumin and 10% of entrapped gamma globulin was released from alginate polylysine capsules. At the surface of the membrane a chemical reaction occurs between the polycation and the polyanion and the strength of this membrane can be controlled by the polycation used thus making it possible to tailor liquid-core beads to specific aims (Nussinovitch et al., 1996). Chitosan is insoluble at a pH above 5.4 and so, while it is unsuitable for biomolecules unstable at low pH values (Huguet at al., 1996), this makes it a suitable gel for nisin encapsulation due to the high stability of nisin at acidic pH values. The molecular weight of nisin is 3,353 Da (Jung, 1991a, b), although, it often occurs as stable dimers with a molecular mass of about 7,000 or tetramers of 14,000 Da (Cheeseman and Berridge, 1957, 1959; Jarvis et al, 1968). While the rate of diffusion decreases with increasing molecular weight (Martinsen et al., 1989), this should not affect the diffusion of nisin due to its low molecular weight. Proteins with a molecular weight of between 2,500 and 66,000 Da have been reported to diffuse readily from a matrix (Nussinovitch et al., 1996). The chemical nature of the environment, such as pH or ionic charge, is an important factor in relation to nisin release as this determines how the bacteriocin reacts under particular conditions. For example, the effect of pH on the solubility of nisin is well known (Liu and Hansen, 1990). The pH is also an important factor in relation to the physical structure of the encapsulating matrix. At high pH values chitosan forms an insoluble precipitate while at acidic pH values an ionotrophic gel is formed (Vorlop and Klein, 1987). The pH at which these capsules are prepared is an important factor in 259

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relation to entrapment and release. For example, if alginate - chitosan encapsulates are prepared at a pH of about 5.5, the surface of the beads formed is highly negative and the positively charged chitosan can establish a strong ionic interaction with the alginate (Huguet et al., 1996). On the other hand, if the capsules are prepared at a very low pH (~ 2) the alginate will have a very low content of negative charges and so cannot interact strongly with the chitosan (Huguet et al., 1996). In the preparation of alginate core capsules, the pH of the alginate was 5.48, which would optimise ionic interactions with the chitosan. In the preparation of chitosan core capsules, the pH of the alginate gel was 7.17 and so it was highly negatively charged and so there was an ionic interaction between the two gels. While normally such a high pH would cause precipitation of the chitosan (Vorlop and Klein, 1987), the high molecular weight of the alginate would prevent its diffusion through the chitosan. Therefore, the chitosan would not be completely exposed to the high pH. The membrane produced as a result of the ionic interaction between the alginate and the chitosan appeared to be the important factor in relation to nisin release. The absence of any visible interaction between these two polymers in chitosan-alginate-tripolyphosphate (CAT) and alginate-tripolyphosphatechitosan-calcium chloride (ATCC) capsules and the observed differences in release from these capsules compared to chitosan-calcium chloride-alginate (CCA) and alginate-chitosan-calcium chloride (ACC) capsules highlights this fact. The inclusion of tripolyphosphate in the alginate increased its pH to approximately 8.5. Exposing chitosan to such an alkaline pH causes it to precipitate (Vorlop and Klein, 1987) and in the case of ATCC and CAT capsules this is likely to have prevented any interaction between the chitosan and the alginate. CCA capsules exhibited a very low level of nisin release and structural differences were observed between the chitosan core in these capsules and the chitosan coat in ACC capsules. This was likely to be due to the pH factor, The chitosan core of CCA capsules contained calcium chloride, which diffused outwards to stabilise the alginate coating. There is no influx of ions into the bead and so the pH of the core remained low (< 5). This is a stable environment for nisin (Liu and Hansen, 1990) and as the alginate coat will be of a higher pH than the nisin containing core the nisin was probably retained in this part of the capsule resulting in a low level of release. The solubility, stability and biological activity of nisin are highly dependent on pH. They drop sharply and continuously as the pH is increased and nisin is almost insoluble at neutral and alkaline conditions (Liu and Hansen, 1990). How the pH affects the charge of a protein will affect its release (Huguet and Dellacherie, 1996). Proteins which are stored under pH conditions lower than their isoelectric point present a positive charge and their release remains low (Huguet and Dellacherie, 1996). In this study, the low level of nisin release from CCA capsules, which have a low internal pH, reflected this. Investigating the application of polymer gels in the development of a delivery system for nisin has shown that by entrapment of the nisin-producing organism, L. lactis, in a polymer matrix, conditions for cell activity can be optimised. This permitted nisin production to be enhanced under both stress and non-stress macroenvironmental conditions. Manipulating the composition of the entrapment matrix and modifying the entrapment/encapsulation process, enabled the production of nisin encapsulates, from which the release of the bacteriocin to the external environment could be controlled. 260

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Thus, it can be concluded that naturally occurring polymer gels can be successfully applied to the development of a delivery system for nisin. The encapsulates which have been developed for the delivery of L. lactis and nisin would now need to be evaluated in a meat product. This would determine the full extent of the success of encapsulation in polymer gels to nisin delivery. 6. Conclusions and future work The production of safe food is a difficult task and while there will always be the risk of foodborne disease there is an ongoing endeavour to control or minimise the risks. Risk managers are continually looking for new options to minimise the risks associated with foods and this in turn encourages researchers to focus on developing these options. Encapsulation of food ingredients is one of these options and the technology offers great potential in the area of food preservation. The pharmaceutical/medical industry has extensively developed and used this technology for the purposes of time-release, flavour masking and improved stability of pharmaceutical formulations (DeZarn, 1995) and continues to do so to great benefit, such as limiting the side effects of drugs to the development of techniques for in situ insulin production for diabetics (Badwan et al., 1985; Langer, 1990; Duncan, 1993; Willaert and Baron, 1996). The encapsulation of food ingredients was once regarded as too expensive and customised for use in the food industry. However, the development of the technology, although relatively unsophisticated when compared to other fields, means that the use of encapsulated products has increased. Encapsulated ingredients are being designed to meet the needs of a specific application. In order to do this a variety of factors must be considered such as the functional properties of the finished product, the type of encapsulation material, the processing conditions and the desired release properties (Pszczola, 1998). It is widely recognised that much remains to be done in the area of encapsulation of food ingredients or additives. The necessity to use safe and edible materials and the associated cost continue to impede the evolution of the technology. However the potential for using naturally occurring polymer gels such as alginate, carrageenan and chitosan as encapsulation matrices has been demonstrated here and they may provide a means of overcoming such impediments. Within the area of meat preservation, emerging and re-emerging pathogens are challenging the existing methods of preservation. This means that it is necessary to seek out new solutions to these new problems. Encapsulation may provide us with a means of finding solutions and adapting and enhancing existing methods of preservation. The amount of basic laboratory research being carried out in this area is quite extensive and perhaps the greatest challenge, which faces the application of encapsulation technology to particular areas in the food sector such as meat preservation, is transposing the techniques from the laboratory to the industry. References Anonymous (1969) Specifications for identity and purity of some antibiotics. FAO/WHO Expert Committee on Food Additives. Twelfth report. WHO Technical Report Series No. 430.

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INDEX Acetate ............................................................................. 24, 35, 83, 91, 92 Acidification.................. .239, 240, 241, 243, 246, 247, 248, 249, 251, 265 Activated sludge ............................................................................. 178, 181 Active dry yeast........................................ 33, 34, 36, 37, 38, 42, 43, 44, 47 Aerobic propagation ......................................................................... 90, 98 Aeromonas ............................................................................ 194, 196, 229 Aeromonas hydrophila ........................................................................... 196 AFLP ................. 193, 211, 212, 218, 220, 221, 225, 226, 229, 232, 233 Alcaligenes sp ................................................................................ 147, 150 Alcoholic fermentation ... 31, 32, 33, 34, 36, 37, 38, 39, 40, 42, 43, 44, 45, 46 Alginate.................................................................................. 244, 259, 264 Amplified ribosomal DNA restriction analysis ............................. 215, 232 Antagonistic activity ......................................................................... 19, 20 Antibacterial activity ........................................................ 14, 20, 252, 263 Antibiotics .............................................................................................. 18 Antimicrobial ..................................................................................... 1, 263 AP-PCR .................................................................. 211, 212, 222, 223, 228 ARDRA .......................................................................................... 215, 232 Aristolochene synthase ............................................................................ 27 Arming yeasts ..................................................................................... 59, 70 Arthrobacter nicotiana .......................................................................... 189 Aspergillus aculeatus .................................................................. 59, 61, 72 Aspergillus niger ......................................................................... 20, 28, 72 Aspergillus oryzae ................................................................................... 13 Aureobasidium pullulans ....................................................... 177, I78, 183 Azotobacter chroococcum .............................................................. 135, 139 Bacilli ..................................................................................................... 156 Bacillus cereus ................................................................. 20, 196, 233, 234 Bacillus stearothermophilus ........................................................ 59, 66, 73 Bacillus subtilis............................................................. 156, 162, 195, 265 Bacteria.............................................................................. 38, 52, 103, 263 261

Bacterial biomechanics .......................................................................... 157 Bacterial pathogens 1, 193, 194, 195, 196, 198, 201, 203, 205, 206, 208, 210, 212, 229, 235, 23 7 Bacterial typing ..................................................................... 193, 208, 210 Bacteriocins............................................. 20, 239, 250, 251, 252, 256, 262 Biogenic amines ....................................................................................... 42 Biomarker .............................................................................................. 145 Biomaterials ........................................................................................... 262 Biomechanics ................................................................................. 161, 162 Biosorption ............................................................................................ 183 Blue cheese .................................................................................. 13, 14, 29 Botryodiploidin ........................................................................................ 25 Brevibacterium flavum ................................................................ 51, 55, 57 Brucella.................................................................. 199, 200, 203, 230, 235 Campylobacter 193, 194, 195, 196, 199, 201, 209, 210, 226, 227, 231, 232, 233, 234, 235, 236, 237, 238 Campylobacter jejuni 193, 196, 226, 231, 232, 233, 234, 235, 236, 237, 238 Carbon sharing ...................................................................................... 147 Carrageenan .......................................................................................... 245 Cell growth rates ..................................................................................... 92 Cell wall ........................................................................................... 72, 156 Cellulase ................................................................................................ 138 Chitosan ......................................................................... 245, 259, 262, 263 Citric .................................................................................................. 41, 42 Classical typing ..................................................................................... 210 Cloning .............................................................. 44, 73, 131, 132, 190, 247 Cloning of genes ...................................................................................... 21 Clostridium ............................................ 20, 61, 72, 73, 195, 196, 201, 215 Clostridium perfringens .................................................................. 20, 196 Community physiology........................................................................... 150 Competition......................................... 14, 40, 43, 203, 239, 243, 246, 263 Contamination ........................... 65, 89, 193, 203, 204, 205, 206, 219, 243 Cyclopiazonic acid., .......................................................................... .23, 28 Detection1, 46, 144, 148, 149, 152, 153, 193, 194, 198, 199, 201, 202, 203, 204, 205, 206, 207, 218, 223, 224, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238 DGGE ............................................................................................ 220, 234 Diacetyl............................................................................................. 35, 41 268

DNA amplification ................................................................................. 207 DNA chips. ............................................................................................. 220 DNA fingerprints ................................................................................... 219 DPH....................................................................................... 185, 186, 187 Effect of aeration ........................................................................... 137, 139 Effector protein ...................................................................................... 125 Effector proteins .................................................................................... 125 Electrophoretic karyotyping .................................................................... 17 Empedobacter sp.................................................................... 14 7, 149, 150 Encapsulation239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 254, 255, 256, 257, 259, 260, 261, 262, 263, 265 Encapsulation matrices ................................................................ 244, 248 Enterococcus................................................................................. 195, 197 Escherichia coli 0157 ................................... 196, 229, 235, 237, 252, 263 Ethylcarbamate ........................................................................................ 45 Fattyacids 14, 33, 34, 38, 40, 46, 146, 148, 149, 150, 151, 152, 185, 187, 188 FCC............................................................................ 76, 77, 79, 80, 82, 83 Feather ................................................................................................... 165 Fermentation 2, 13, 14, 18, 20, 21, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 43, 44, 46, 47, 52, 57, 60, 61, 65, 67, 71, 72, 79, 80, 89, 90, 96, 98, 99, 102, 107, 108, 109, 159, 160, 165, 166, 168, 171, 173, 175, 239, 240, 241, 242, 243, 244, 247, 249, 250, 252, 262, 263, 264, 265 Fermented feather meal ......................................................................... 171 Filamentous growth ...................................................................... 131, 132 flaA typing ...................................................................................... 193, 226 Flavor ............................................................ 14, 21, 28, 47, 242, 248, 261 Flocculation .......................................................... 92, 95, 96, 98, 129, 130 Fluorescence measurement., ................................................. 185, 186, 187 Food 27, 28, 29, 45, 47, 85, 227, 230, 231, 232, 233, 234, 235, 236, 237, 238, 249, 261, 262, 263, 264, 265, 266 Food components ........................................................................... 193, 203 Fumigaclavine ......................................................................................... 25 GAC ....................................................................... 177, 179, 181, 182, 183 Galactose .......................................................................................... 83, 85 Galactose metabolism., ............................................................................ 85 Gene disruption ........................................................................... 18, 21, 23 Genetically modified yeasts ..................................................................... 44 Genomic instability ................................................................................ 227 269

Glucoamylase..................................................................................... 59, 60 Glucose oxidase .................................................................. 20, 28, 29, 136 GRAS status ..................................................................... 20, 250, 251, 262 GTP analogues ...................................................................................... 187 GTP-binding proteins ................................................................... 109, 188 HACCP ......................................................................................... 194, 241 Histamine ......................................................................................... 42, 194 Hydrocarbon utilisation ......................................................................... 185 Hydrolytic enzyme .................................................................................... 61 Hydrolytic enzymes .................................................................................. 61 Hydrophobicity ...................................................................... 90, 91, 93, 96 Identification 37, 38, 42, 46, 77, 81, 84, 145, 147, 153, 154, 166, 175, 193, 194, 198, 199, 200, 203, 209, 214, 216, 220, 221, 225, 226, 229, 231, 232, 233, 234, 236, 237, 241 Idiophase .................................................................................................. 18 Immunomagnetic separation ........................................................ 206, 235 IMS......................................................................................................... 206 Infection ......................................... 195, 209, 223, 226, 227, 229, 265, 266 Inhibitory activity............................................................................ 20, 252 Insertion sequences....................................................................... 216, 236 Ion exchange resin ................................................................................. 181 IS200 typing ................................................................... 222, 223, 226, 235 Isoprenoid ......................................................................................... 23, 24 Isotopic fractionation. ............................................................................ 146 ITS regions ............................................................................................... 25 Kinetic fermentation .............................................................................. 171 Kinetic studies .......................................................................................... 80 Kocuria rosea ........................................ 165, 166, 167, 168, 170, 172, 173 Lactic acid ......................................................................... 14, 38, 262, 263 Lactic acid bacteria 14, 31, 32, 34, 38, 39, 41, 42, 44, 45, 46, 108, 239, 247, 250, 251, 254, 263, 264 Lactobacillus helveticus ................................................. 101, 102, 107, 108 Lactococcus lactis .................................................... 45, 252, 262, 263, 264 Leloir pathway. ........................................................................... 81, 82, 85 Line probe assay .................................................................................... 199 LiPA ....................................................................................................... 199 Lipase ....................................................................................................... 70 Lipases ............................................................................................... 14, 21 270

Listeria monocytogenes 14, 20, 195, 196, 201, 205, 227, 230, 231, 232, 233, 234, 235, 236, 237, 238, 262, 263, 264, 266 LPB-3 .................................................................... 167, 168, 170, 172, 173 Luedeking and Piret ............................................................................... 101 Lysine ................................................................................................. 55, 57 Malic .................................................................... 31, 38, 39, 40, 41, 42, 44 Malolactic fermentation ............................................. 31, 38, 39, 42, 43, 45 Malolactic starters ............................................................................. 38, 43 MAP kinase cascade 109, 113, 115, 117, 119, 120, 124, 125, 127, 131, 133 Marcfortines ............................................................................................ 25 Meat....................................................................... 241, 243, 262, 264, 265 Meatpreservation.................................................................. 239, 240, 241 Mechanical properties ........................................................................... 162 Metabolic control analysis .................................................... 75, 76, 82, 85 Metabolic engineering. ............................................................................ 85 Metabolic flux .................................................................................... 77, 78 Metabolicflux analysis .................................................... 75, 77, 79, 82, 84 Metabolic pathway analysis .................................................. 75, 76, 80, 81 Methylketones .......................................................................................... 14 Microencapsulation ...................................................... 262, 263, 264, 265 Micromanipulation ....................................................................... 158, 162 Molecular breeding .................................................................................. 73 Molecular detection ........................................................................... 1, 194 Molecular identification ....................................... 193, 198, 200, 201, 209 Molecular typing 15, 193, 208, 210, 211, 212, 215, 216, 217, 219, 220, 221, 225, 226, 227, 229, 230, 231, 232, 233, 238 Molecular weight..... 17, 135, 137, 138, 217, 219, 245, 256, 259, 260, 262 Mould fermented foods ...................................................................... 14, 21 Mould fermented meat products .............................................................. 13 MUC1 ............................................................................ 124, 125, 126, 127 Mutation........................... 18, 23, 24, 27, 28, 120, 121, 125, 130, 131, 217 Mycophenolic acid. ........................................................................... 25, 28 Mycotoxin............................................................................... 14, 23, 25, 28 NADPH accumulation ...................................................................... 55, 57 n-Alkane ................................................................................................. 185 NASBA ............................................................... 193, 198, 199, 200, 204, 237 NASBA method ....................................................................................... 200 n-Hexadecane ........................................................................................ 189 271

Nisin............................................... 250, 251, 252, 262, 263, 264, 265, 266 Nucleic acid based identification methods ............................................ 198 0157 h7 ....................................................... 195, 201, 202, 205, 217, 228, 250 Oenococcus oeni ...................................................................................... 38 Oil bioremediation ................................................................................. 190 Outbreak ........ 21 7, 222, 223, 224, 225, 229, 231, 234, 235, 236, 237, 238 Outer membrane proteins ...................................................................... 145 PAC........................................................................ 177, 179, 181, 182, 183 Pathogenic E. Coli ................................................................................. 198 Patulin., .................................................................................................... 25 Pb2+ removal........................................ 177, 178, 179, 180, 181, 182, 183 PCBC gene ............................................................................................... 17 PCR15, 17, 26, 27, 37, 193, 198, 199, 200, 202, 203, 204, 205, 206, 207, 210, 211, 212, 213, 214, 215, 218, 220, 222, 225, 226, 229, 230, 231, 232, 233, 235, 236, 237, 238 PCR detection ................................................................ 203, 205, 208, 238 PCR ribotyping ...................................................................................... 233 Penicillin.................................................................... 15, 17, 18, 19, 27, 28 Penicillin biosynthetic genes ................................................................... 17 Penicillin production ......................................................................... 17, 27 Penicillium camemberti ......................................................... 13, 23, 28, 29 Penicillium chrysogenum.................................................................. 27, 28 Penicillium commune ............................................................................... 28 Penicillium nalgiovense.. ...................................................... 13, 15, 27, 28 Penicillium roqueforti........................................................... 25, 27, 28, 29 Penitrem A ............................................................................................... 25 Peptidoglycan ........................................................................................ 157 PFGE193, 211, 212, 216, 217, 220, 222, 223, 224, 225, 226, 228, 229, 230, 231, 234, 235, 236 Phage typing., ........................................................................................ 233 PHB........................................................................ 135, 136, 137, 138, 139 Pigments ................................................................................................ 173 Plasmid.......................................................................... 219, 230, 234, 235 Plasmidanalysis .................... 193, 212, 219, 222, 223, 224, 225, 226, 234 Polar lipids ................................................................................... 144, 146 PR imine............................................................................................. 25, 29 PR toxin ....................................................................................... 25, 26, 27 272

Primers 15, 17, 29, 37, 193, 198, 199, 201, 202, 213, 214, 218, 222, 232, 236, 238 Probes 46, 136, 153, 158, 162, 193, 198, 199, 201, 203, 207, 208, 216, 229. 231. 233. 235. 236 Propagation ............................................................................................. 90 Propagation conditions ........................................................................... 90 Protease ................................................................................. 171, 172, 174 Proteases .................................................................................... 14, 21, 173 Pseudomonas sp ....................................................... 57, 145, 147, 148, 150 Pulsedfield gel electrophoresis .................................... .193, 210, 216, 238 Quantification...................................................... 75, 78, 84, 193, 207, 234 rAPD15, 16, 27, 193, 211, 212, 213, 214, 220, 222, 223, 225, 226, 227, 228, 229, 232, 234, 235, 236, 238 rAPD analysis .......................................................................................... 15 rDNA...................................................................... 144, 147, 209, 215, 232 Reactivation ................................................................. 33, 34, 39, 244, 246 Relative resistance ................................................................................. 155 Repetitive elements ................................................................................ 213 REP-PCR............................... 193, 211, 214, 220, 222, 223, 225, 228, 231 Respiratory activity .................................................................................. 52 Restriction analysis........................ 193, 212, 215, 216, 219, 222, 225, 226 Restriction fragment length polymorphism ... 193, 211, 215, 230, 234, 236 RFLP................................................................ 37, 211, 215, 216, 228, 236 Rhizopus oryzae ....................................................................................... 59 Ribosomal RNA ...................................................................................... 204 Ribotyping 193, 211, 216, 222, 223, 224, 225, 226, 227, 228, 229, 231, 232, 233, 234, 235, 236, 23 7 rRNA .............. 145, 153, 193, 199, 201, 204, 215, 216, 230, 234, 235, 237 rRNA genes., .......................................................................................... 199 RT-PCR.................................................................................. 193, 198, 204 Saccharomyces cerevisiae. 32, 47, 59, 71, 72, 73, 97, 98, 99, 130, 131, 132, 133, 183 Salmonella 193, 194, 195, 196, 199, 200, 201, 202, 205, 206, 207, 209, 210, 214, 219, 221, 222, 223, 224, 225, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 252, 262, 266 Salmonella sp ......................................................................................... 236 Salmonella typhimurium ................ 209, 234, 235, 236, 237, 238, 252, 266 Secondary metabolite ..................................... 14, 15, 17, 18, 23, 24, 25, 26 Sensitivity ............................................................................................... 203 273

Serotyping ............................................................................................. 227 Serovars ........................................................ 221, 222, 223, 225, 227, 228 Shigella ........................................................................... 194, 195, 197, 201 Signal transduction .......................................................................... 110, 113 Signal transduction modules .............................................................. 113 Spacer region ......................................................... 199, 200, 214, 225, 232 Spirillum ............................................................................................... 157 SRFH ............................................................................... 211, 216, 227, 229 Staphylococcus aureus ................................ 14, 20, 197, 201, 231, 234 Staphylococcus epidermis ............................................................. 155, 159 Starch degrading enzymes ............................................................... 127 Starter ................................................................................................... 243 Starter culture 13, 14, 18, 19, 20, 23, 25, 239, 241, 242, 243, 244, 246, 247, 248, 250, 251, 252, 254, 264, 265 Starters..................................................................................................... 11 Steady-state continuous cultivation ......................................................... 79 Streptococcus pyogenes ........................................................................ 197 Streptomyces......................... 166, 171, 174, 185, 186, 187, 188, 189, 190 Streptomyces griseoflavus ...................................................................... 185 Streptomyces plicatus ............................................................................ 185 Stuckfermentation ............................................................................ 32, 34 Substrate competition ............................................................................ 149 Surface charge ........................................................................................ 91 Surface properties ............................................................................... 99 Target for molecular identification .................................... 198, 199, 200 Target genes ................................................................................ 83, 122 Taxon specific biomarkers ............................................................. 144 Taxonomic relationships .................................................................... 15 Taxonomy ........................................................................ 123, 131, 132 TEC1 ...................................................................................... 123, 131, 132 Tensile strength.............................................. 135, 136, 138, 139, 157, 162 Texture ..................................................................... 14, 242, 243, 249, 250 TGGE............................................................................................. 220, 234 Thermal stubility .............................................................. 65, 136, 137, I39 TLC analysis ............................................................................................ 23 Toxin ........................................................................................................ 29 Transcriptional regulators ............................................................. 113, 122 Transformation., ............................................. 19, 20, 32, 40, 44, 111, 227 Transformation vector ............................................................................ 19 274

tRNA-PCR ........................................................................................ 211, 214 Tryptophane ...................................................................................... 23, 24 Two dimensional TLC .............................................................................. 24 Typing1, 37, 193, 194, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 238 Typing techniques .................................................................. 215, 218, 219 U-13 C...................................................................................... 148, 150, 151 Validation .................................................................................... 108, 205, 206 Viability of cells. .................................................................................... 204 Vibrio............................................................................. 195, 197, 200, 233 Volatile compounds .......................................................................... 14, 35 Whey....................................................................................................... 102 White cheese ..................................................................................... 13, 28 Wine .............................................................................................. 46, 109 Yeast starters .................................................................................... 33, 45 Yersinia.......................................... 194, 197, 198, 201, 229, 230, 233, 235 Yield coefficient ....................................................................................... 92 Zeolite ............................................................................................ I 78, 181 Zeta potential ......................................................................... 91, 94, 95, 96

275