Practical Guide to Microbial Polyhydroxyalkanoates

Practical Guide to Microbial Polyhydroxyalkanoates

Kumar Sudesh

Hideki Abe

FeJSMITHEKS Smithers ismithers - A Smithers Group Company

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reface

P

Polyhydroxyalkanoates (PHA) are plastic-like polymers produced naturally by many types of bacteria. PHA are among the most promising next-generation plastics because they are bio-based and biodegradable. The book begins with a few brief chapters on the biology of PHA focusing particularly on their synthesis by microorganisms. Unlike chemical polymerisation processes, microbial syntheses involve multiple biological catalysts that convert renewable carbon sources into monomers, which in turn are polymerised into high-molecular-weight polymers in an aqueous environment at ambient conditions. This complicated process is described by giving specific and well-established examples, along with new and recent findings on the catalytic capabilities of the key enzyme PHA synthase. With this brief background on the biology of PHA, readers will be exposed to the main focus of this book: PHA properties. Readers are provided with important information on the crystalline and solid-state structures of various types of PHA, as well as their physical and mechanical properties, which are governed by the monomer types, their composition, and molecular weight. The final objective of this book is to provide detailed information on the currently understood mechanism of PHA biodegradation because this is one of the most important factors determining the final applications of PHA. We are extremely grateful to Dr Kesaven Bhubalan, Mr Aaron Ooi Wei Yang, Dr Tang Hui Ying and Ms Rathi Devi Nair Gunasegaven for their invaluable help, particularly during the final stages of the preparation of this book. Last but not least, we thank ismithers for publishing this book. The exemplary support and guidance provided by ismithers staff, in particular Mrs Eleanor Garmson, expedited completion of the book. Besides serving as a reference material, we hope that this book will encourage more people to get involved and contribute to the development of these interesting and promising microbial polyesters. Kumar Sudesh Hideki Abe

...

111

ontents

C 1.

Background ...................................................................

1

2.

Polyhydroxyalkanoates (PHA) Types ............................

5

3.

2.1

Naturally Occurring PHA ...................................................

5

2.2

Unnatural PHA ...................................................................

6

Microbiology of Polyhydroxyalkanoate (PHA) Synthesis .....................................................................

13

4.

Production of Polyhydroxyalkanoates (PHA).............. 17

5.

Extraction and Purification of Polyhydroxyalkanoates (PHA) from Microbial Cells ........................................ 21

6.

Crystalline and Solid-state Structures of Polyhydroxyalkanoates (PHA)....................................

25

6.1

Poly[(R)-3-hydroxybutyrate] (P(3HB)) .............................

25

6.2

Poly[(R)-3-hydroxyvalerate](P(3HV))..............................

29

6.3

Medium-chain-length Poly [(R)-3-hydroxyalkanoate]s (mcl-P(3HA)).................................................................... 30

6.4

Poly(4-hydroxybutyrate) (P(4HB))....................................

33

6.5

Poly(3-hydroxypropionate)(P(3HP))................................

36

6.6

Poly[(R)-3-hydroxybutyrate]-basedCopolymers ...............38

V

Practical Guide to Microbial Polyhydroxyalkanoates

7.

Physical Properties of Polyhydroxyalkanoates (PHA).. 51 Crystallisation Kinetics .....................................................

51

7.2 Thermal Properties ...........................................................

55

7.1

7.3

7.2.1

Melting Temperature ....................................................

55

7.2.2

Glass Transition Temperature (T, ) ................................

59

7.2.3

Thermal Degradation Temperature ...............................61

Mechanical Properties ...................................................... 7.3.1

Films and Fibres of P(3HB) Homopolymers .................67

7.3.2

Films and Fibres of P(3HB)-based Copolymers .............74

7.3.3

Mechanical Properties of mcl-P(3HA) and their Modification Techniques ..............................................

77

P(3HB)-based Polymer Blends ......................................

77

7.3.4

8.

Intracellular degradation (mobilisation)of Polyhydroxyalkanoates (PHA).................................... 8.1 8.2 8.3 8.4

9.

85

Endogeneous Degradation of PHA ...................................

85

Intracellular P(3HB)Depolymerases and Degradation Systems.............................................................................

87

Intracellular 3HB Oligomer Hydrolases ............................ Intracellular mcl-PHA Depolymerases ..............................

Extracellular Degradation of Polyhydroxyalkanoates (PHA).................................... 9.1

67

91 91

97

Effect of Environmental Conditions on the Degradation of PHA ............................................................................. 97

9.2 Structure and Properties of PHA-degrading Enzymes........98

9.3

vi

9.2.1

Short-chain-length PHA Depolymerase ........................

9.2.2

Medium-chain-length PHA Depolymerase ..................110

Effect of Chemical Structures on Enzymatic Degradability ..................................................................

99

110

Contents

9.3.1

Enantiomer Selectivity of PHA Depolymerase ............ 110

9.3.2

Substrate Specificity of the Catalytic Domain .............111

9.3.3

Binding Specificity of the Substrate-binding Domain ..114

9.4

Effect of Solid-state Structures on Enzymatic Degradability ....................................................

9.5

Molecular Mechanisms of the Enzymatic Degradation of PHA ...........................................................................

123

Conclusion and Future Perspectives ................................

126

9.6

..............116

Abbreviations.. .................................................................. 137 Index.. ............................................................................... 143

vii

1

Background

The ability of some microorganisms to synthesise an intriguing plastic-like material has received much attention from academia and industry over the past 30 years [1-91. Polyhydroxyalkanoates (PHA)are high-molecular-weight biological polyesters synthesised by many types of bacteria. PHA serve as carbon- and energy-storage compound for the bacteria. Figure 1.1 shows the general process of PHA accumulation by a bacterial cell.

Carbon and Energ

Figure 1.1 PHA accumulation process by a bacterial cell (schematic). If R is CH,, the PHA is known as poly(3-hydroxybutyrate) [P(3HB)]or PHB. P(3HB) was the first type of PHA to be discovered. It is also the commonest type of PHA synthesised by most microorganisms

1

Practical Guide to Microbial Polyhydroxyalkanoates In conditions that are unfavorable for growth, many bacteria are equipped with intricate metabolic pathways that convert excess carbon sources into 3-, 4- and 5-hydroxyalkanoyl-CoA [ 10, 111. An enzyme called PHA synthase polymerises, via a condensation process, these hydroxyalkanoyl-CoA compounds into linear polyester molecules having number-average molecular masses in the range 0.05-20 MDa. The resulting amorphous PHA molecules are simultaneously packed into the form of waterinsoluble granules and kept in the cell cytoplasm. The accumulated PHA granules are usually large enough to be readily viewed under phase-contrast light microscopy. The entire process from monomer synthesis to polymerisation takes place in an aqueous environment in the bacterial cell under ambient conditions. Under controlled cultivation conditions in a fermenter, PHA content in the bacterial cells can increase to as much as 90 wt% of the dry weight of the cell within 48 hours. In contrast to the chemical polymerisation process that requires relatively extreme conditions, biological processes take place in the relatively mild conditions of the cell cytoplasm. To date, approximately 150 types of monomers have been identified as substrates for PHA synthase, which is the key enzyme for PHA biosynthesis. This shows that PHA synthases are a class of enzymes that have broad substrate specificity. PHA synthases are highly stereospecific. They specifically polymerise only the ( R ) enantiomer of the hydroxyalkanoate monomers. Therefore, microbial PHA are a source of enantiomerically pure compounds. By hydrolyzing high-molecular-weight PHA, it is possible to obtain the monomers, which can be used to synthesise useful compounds.

References 1.

A.J. Anderson and E.A. Dawes, Microbiological Reviews, 1990,54,4,450.

2.

Y. Doi, Microbial polyesters, VCH, New York, US, 1990.

3.

S.Y. Lee, Biotechnology and Bioengineering, 1996,49,1, 1.

4. L.L. Madison and G.W. Huisrnan, Microbiology and Molecular Biology Reviews, 1999,63, 1,21. 5.

Y. Poirier, D.E. Dennis, K. Klomparens and C. Somerville, Science, 1992, 256, 520.

6.

A. Steinbuchel in Biomaterials, Ed., D. Byrom, MacMillan Publishers, Basingstoke, Hampshire, UK, 1991, 125.

2

Background 7.

K. Sudesh, H. Abe and Y. Doi, Progress in Polymer Science, 2000,25, 10, 1503.

8.

H.E. Valentin, D.L. Broyles, L.A. Casagrande, S.M. Colburn, W.L. Creely, P.A. DeLaquil, H.M. Felton, K.A. Gonzalez, K.L. Houmiel, K. Lutke, D.A. Mahadeo, T.A. Mitsky, S.R. Padgette, S.E. Reiser, S. Slater, D.M. Stark, R.T. Stock, D.A. Stone, N.B. Taylor, G.M. Thorne, M. Tran and K.J. Gruys, International Journal of Biological Macromolecules, 1999,25, 1-3, 303.

9.

M. Zinn, B. Witholt and T. Egli, Advanced Drug Delivery Reviews, 2001, 53, 1,5.

10. A. Steinbuchel and H.E. Valentin, FEMS Microbiology Letters, 1995, 128, 3, 219.

11. H.E. Valentin, A. Schonebaum and A. Steinbuchel, Applied Microbiology and Biotechnology, 1996,46,261.

3

2

Poly hyd roxyaIkanoates (PHA) Types

Studies have shown that there are many types of monomer constituents of PHA. The various types of PHA monomers can be classified into ‘natural’ and ‘unnatural’. The former can be found naturally accumulated by microorganisms in the environment. Unnatural PHA are produced by microorganisms in the laboratory if they are fed specific chemicals. These chemicals are usually not the normal substrates used by the microorganisms for growth and survival.

2.1 Naturally Occurring PHA Poly(3-hydroxybutyrate) or P(3HB)is the commonest among the various types of PHA that have been reported. The biosynthesis pathway of P(3HB) is shown in Figure 2.1. P(3HB) was the first type of PHA to be studied and characterised [l,21; it was initially thought that P(3HB)was the only type of bacterial polyester that existed naturally [3]. This is why, in most early literatures, this bacterial storage polyester was referred to as ‘P(3HB)’or ‘PHB’, which refers to a polymer of 3-hydroxybutyric acid. It was not until much later that researchers realised the existence of several types of monomers that can be incorporated into the storage polyester [4-61. Therefore, the general name ‘PHA’ is more appropriate to refer to this microbial polymeric material. About 150 types of monomers have been identified as PHA constituents. This variety of monomers reflects the broad substrate specificity of PHA synthases. Although the known PHA synthases can polymerise various monomers, bacterial cells cannot synthesise most of the monomers. Most bacteria can naturally synthesise only a few of these monomers. Most of the other monomers have to be supplied to the bacteria in the form of precursor carbon sources for the bacteria to polymerise them.

5

Practical Guide to Microbial Polyhydroxyalkanoates

Acetyl-CoA

Acetoacetyl-CoA

H3C

SCOA

H3C~

S

C

O

A

HaCuSCOA

reductase 3-hyd roxybutyry I-CoA

H3C

Poly-(R)-3-hydroxybutyrate

Figure 2.1 Schematic pathway for the biosynthesis of P(3HB) in bacteria. This pathway has been studied in great detail using Cupriavidus necator H16, which is the model microorganism for studies on PHA biosynthesis. The genes encoding all the three enzymes have been cloned from various microorganisms and characterised

2.2 Unnatural PHA The quest for novel PHA with unique material properties has resulted in the identification of unnatural PHA. Most of the diverse range of PHA is unnatural. These ‘exotic’ PHA are synthesised only if structurally related carbon sources or precursor compounds are provided to the bacterial cells under specific cultivation conditions. Cells are usually devoid of suitable metabolic pathways to enable them to use such precursor compounds as sources of carbon andlor energy. Many bacterial cells can take up a wide range of precursor compounds from the culture medium and polymerise them into PHA. The key factor that determines the type of PHA that is produced by a microorganism is the substrate specificity of the polymerising enzyme PHA synthase [7, 81. Among the types of unnatural PHA monomers produced only by microorganisms in the laboratory are 4-hydroxybutyrate (four-carbon compound) [9-181, 4-hydroxyvalerate (five-carbon compound) [ 191, 4-hydroxyhexanoate

6

Po lyhy d r oxyal ka noates ( PHA) Types (six-carbon compound) [20], 5-hydroxyhexanoate (six-carbon compound) [21], 4-hydroxyheptanoate (seven-carbon compound) [211and 4-hydroxyoctanoate (eightcarbon compound) [21]. Regardless of the types of PHA, all of them are accumulated in the form of waterinsoluble granules in microbial cells. Models on the formation of PHA granules have been proposed. Two well-known models for the in vivo formation of PHA granules are the ‘micelle model’ [22,23] and ‘budding model’ [24], which both consider the defined location of the PHA synthase (Figure 2.2). In the micelle model, a micelle-like structure is formed due to the aggregation of P(3HB)-linked synthase. In this model, synthase proteins residing on micelle surface acquire the 3-hydroxybutyrate-CoA substrate from the cytosol, leading to the growing insoluble PHA chain within the micelle. On the other hand, in the budding model the synthase proteins adhere to the inner face of the plasma membrane. As the PHA chain elongates and phasin proteins (specific proteins that are associated with the PHA granules) are produced, budding of a vesicle with a phospholipid layer could ultimately lead to granule formation. This model of granule formation was supported in recent works by two research groups [25-271. Aside from these two models of formation of PHA granules, Tian and co-workers noticed that emerging granules arose from the cell centre of wild-type Cupriavidus necator, which were localised at unknown mediation elements [28,29]. They proposed that these mediation elements may act as nucleation sites for initiation of P(3HB) granules. An increasing number of new monomers have been found to be active as substrate for the PHA synthase. Recently, it has been demonstrated that the PHA synthase can polymerise even non-PHA polymers such as polythioesters [30-321 and polylactic acids (PLA) [33-361. In the latter case of lactic acid, a mutant PHA synthase was used to produce the unnatural copolymer of PHA and PLA. These new findings further confirm that PHA synthases are interesting enzymes whose potential has not been fully tapped. However, attempts to crystallise this enzyme and obtain its crystal structure have proved to be extremely challenging. Therefore, not much is known about its catalytic mechanism. What is known is that this key enzyme in the biosynthesis of PHA can polymerise high-molecular-weight water-insoluble polyesters in an aqueous environment at room temperature [23,24,37- 431. One can imagine its potential applications if the enzyme mechanism can be understood and exploited for chemical syntheses.

7

Practical Guide to Microbial Polyhydroxyalkanoates ~~~

A.

~

s

Amphipathic polymer synthase

4 4

.... ,.., ..,,_: : '.....,....." ..1

Polymer inclusion

$/'

9

... .. .. .. . . . .......... ..... ,... ......

I \

'

\ \

........

Polymer particle Bacterial cell /

B.

/

Y

--f

/

' 8 I !-.A,

Y

/

/

I I I

/'

I

I I I I I I

I I I Cytoplasmic I membrane I

,

J

.....

. ~, . ...... i

J

'

a

.,.-. .. ,.,

I

4

J)

r)

....... . .... . . . ............. . :. ... :~ ...,.'.

, .

.I

,

Bacterial cell

Figure 2.2 Models of formation of PHA granules in bacteria. The process of self-assembly of granules occurs randomly in the cytosol for (a) the micelle model whereas it occurs near the cytoplasmic membrane for (b) the budding model. The irregular lines in the diagram represent the elongation of polymer chains Regardless of whether PHA are natural or unnatural, they are classified into two categories according to the number of carbon atoms in their monomers. Those consisting of fewer than six carbon atoms are referred to as 'short chain-length' (scl)

8

,

Polyhydroxyalkanoates (PHA) Types monomers, whereas those that contain 6-14 carbon atoms are referred to as ‘medium chain-length’ (mcl)monomers. PHA that consist of scl monomers are called ‘scl-PHA’ whereas those that consist of mcl monomers are called ‘mcl-PHA’. This type of classification was favoured because all the naturally occurring bacteria that were initially studied were found to synthesise scl-PHA or mcl-PHA. Initial studies did not detect bacteria with the ability to produce PHA that contain scl monomers and mcl monomers. It is now known that this apparent preference for scl monomers or mcl monomers is because of the substrate specificity of the PHA synthase. The PHA synthase of Cupriavidus necator H16 (formerly known as Alcaligenes eutroghtis H16), which is the most studied bacterium for PHA biosynthesis, can efficiently produce PHA that contain monomers such as 3-hydroxybutyrate (3HB)and 3-hydroxyvalerate (3HV). The central metabolic pathways of C. necator naturally generate 3HB from sugars, fatty acids and other carbon sources. However, 3HV generation has to be initiated by the addition of structurally related precursor carbon sources such as propionic acid and valeric acid. Several other bacteria which have the natural ability to accumulate PHA containing 3HV from structurally unrelated carbon sources such as glucose have been identified. Rhodococcus sp. NCIMB 40136 is one such naturally occurring bacterium which can synthesise PHA containing primarily 3HV from sugars [44]. Nocardia corallina is another strain of bacterium known for its ability to synthesise PHA containing 3HV from most carbon sources. It was reported that most of the 3HV monomers produced by N. corallina are synthesised via the methylmalonyl-CoA pathway [45].

References 1.

M. Lemoigne, Bulletin de la Societe de Chimie Biologique, 1926, 8, 770.

2.

M. Lemoigne, Annales de Virologie, 1927,41, 148.

3.

E.A. Dawes and P.J. Senior, Advances in Microbial Physiology, 1973,10, 135.

4.

M.J. De Smet, G. Eggink, B. Witholt, J. Kingma and H. Wynberg, Journal of Bacteriology, 1983, 154,2, 870.

5.

R.H. Findlay and D.C. White, Applied and Environmental Microbiology, 1983,45, 1,71.

6.

L.L. Wallen and W.K. Rohwedder, Environmental Science & Technology, 1974, 8, 6,576.

9

Practical Guide to Microbial Polyhydroxyalkanoates 7.

B.H.A. Rehm, Biotechnology Letters, 2006,28,4,207.

8.

B.H.A. Rehm and A. Steinbiichel, International Journal of Biological Macromolecules, 1999,25, 1-3, 3.

9.

Y. Doi, M. Kunioka, Y. Nakamura and K. Soga, Macromolecules, 1988,21, 9,2722.

10. Y. Doi, A. Segawa and M. Kunioka, lnternationalJourna1 of Biological Macromolecules, 1990,12,2, 106. 11. S. Hein, B. Sohling, G. Gottschalk and A. Steinbiichel, FEMS Microbiology Letters, 1997, 153, 2,411. 12. M. Kunioka, Y. Kawaguchi and Y. Doi, Applied Microbiology and Biotechnology, 1989,30,569. 13. M. Kunioka, Y. Nakamura and Y. Doi, Polymer Communications, 1988,29, 174. 14. S. Nakamura, Y. Doi and M. Scandola, Macromolecules, 1992,25, 17,4237. 15. K. Sudesh, T. Fukui, K. Taguchi, T. Iwata and Y. Doi, International Journal of Biological Macromolecules, 1999, 25, 1-3, 79. 16. H.E. Valentin and D. Dennis,Journal of Biotechnology, 1997, 58, 1, 33. 17. H.E. Valentin, S. Reiser and K.J. Gruys, Biotechnology and Bioengineering, 2000,67,3,291. 18. H.E. Valentin, G. Zwingmann, A. Schonebaum and A. Steinbiichel, European Journal of Biochemistry, 1995,227, 1,43. 19. H.E. Valentin, A. Schonebaum and A. Steinbuchel, Applied Microbiology and Biotechnology, 1992, 36, 507. 20. H.E. Valentin, E.Y. Lee, C.Y. Choi and A. Steinbiichel, Applied Microbiology and Biotechnology, 1994,40, 710. 21. H. E. Valentin, A. Schonebaum and A. Steinbuchel, Applied Microbiology and Biotechnology, 1996,46,261. 22. D. Ellar, D.G. Lundgren, K. Okamura and R.H. Marchessault, Journal of Molecular Biology, 1968,35,3,489.

10

Polyhydroxyalkanoates (PHA) Types 23. T.U. Gerngross, K.D. Snell, O.P. Peoples, A.J. Sinskey, E. Csuhai, S. Masamune and J. Stubbe, Biochemistry, 1994,33, 31,9311. 24. J. Stubbe and J. Tian, Natural Product Reports, 2003,20, 5,445 25. D. Jendrossek, Biomacromolecules, 2005,6, 2, 598. 26. V. Peters and B.H.A. Rehm, FEMS Microbiology Letters, 2005,248, 1, 93. 27. D. Schultheiss, R. Handrick, D. Jendrossek, M. Hanzlik and D. Schuler, Journal of Bacteriology, 2005, 187, 7,2416. 28. J.M. Tian, A.M. He, A.G. Lawrence, P.H. Liu, N. Watson, A.J. Sinskey and J. Stubbe,Journal of Bacteriology, 2005, 187, 11, 3825. 29. J.M. Tian, A.J. Sinskey and J. Stubbe,]ournal of Bacteriology, 2005, 187, 11, 3814. 30. T. Liitke-Eversloh, K. Bergander, H. Luftmann and A. Steinbuchel, Microbiology-UK, 2001,147, 11.

31. T. Liitke-Eversloh, K. Bergander, H. Luftmann and A. Steinbiichel, Biomacromolecules, 2001,2,3, 1061. 32. T. Liitke-Eversloh, A. Fischer, U. Remminghorst, J. Kawada, R.H. Marchessault, A. Bogershausen, M. Kalwei, H. Eckert, R. Reichelt, S.J. Liu and A. Steinbuchel, Nature Materials, 2002, 1,4, 236. 33. S.H. Park, S.H. Lee, E.J. Lee, H.O. Kang, T.W. Kim, T.H. Yang and S.Y. Lee, inventors; LG Chemical Ltd., Korea Institute of Science and Technology, assignees; W O 2008062995,2008. 34. S.H. Park, S.H. Lee, E.J. Lee, H.O. Kang, T.W. Kim, T.H. Yang and S.Y. Lee, inventors; LG Chemical Ltd., Korea Institute of Science and Technology, assignees; WO 2008062996,2008. 35. S.H. Park, S.H. Lee, E.J. Lee, H.O. Kang, T.W. Kim, T.H. Yang and S.Y. Lee, inventors; LG Chemical Ltd., Korea Institute of Science and Technology, assignees; WO 2008062999,2008. 36. S. Taguchi, M. Yamada, K. Matsumoto, K. Tajima, Y. Satoh, M. Munekata, K. Ohno, K. Kohda, T. Shimamura, H. Kambe and S. Obata in the Proceedings of The National Academy of Sciences of The United States of America, 2008, 105,45, 17323.

11

Practical Guide to Microbial Polyhydroxyalkanoates 37. T.U. Gerngross and D.P. Martin in the Proceedings of The National Academy of Sciences of The United States of America, 1995,92, 14, 6279. 38. T.U. Gerngross, P. Reilly, J. Stubbe, A.J. Sinskey and O.P. Peoples, Journal of Bacteriology, 1993, 175, 16, 5289. 39. Y. Jia, T.J. Kappock, T. Frick, A.J. Sinskey and J. Stubbe, Biochemistry, 2000, 39, 14, 3927. 40. Y. Jia, W. Yuan, J. Wodzinska, C. Park, A.J. Sinskey and J. Stubbe, Biochemistry, 2001,40,4, 1011. 41. U. Miih, A.J. Sinskey, D.P. Kirby, W.S. Lane and J.A. Stubbe, Biochemistry, 1999, 38,2, 826.

42. S.J. Sim, K.D. Snell, S.A. Hogan, J. Stubbe, C.K. Rha and A.J. Sinskey, Nature Biotechnology, 1997, 15, 1,63. 43. W. Yuan, Y. Jia, J.M. Tian, K.D. Snell, U. Miih, A.J. Sinskey, R.H. Lambalot, C.T. Walsh and J. Stubbe, Archives of Biochemistry and Biophysics, 2001, 394, 1, 87. 44. G.W. Haywood, A.J. Anderson, G.A. Williams, E.A. Dawes and D.F. Ewing, lnternational Journal of Biological Macromolecules, 1991, 13,2, 83. 45. H.E. Valentin and D. Dennis, Applied and Environmental Microbiology, 1996,62,2,372.

12

3

Microbiology of Polyhydroxyalkanoate (PHA) Synthesis

The microorganism plays the key part in the biological synthesis of PHA. The successful syntheses of PHA is dependent upon not only on the type of microorganism used but also its cultivation conditions. PHA-producing microorganisms can be readily found in almost any environmental sample (e.g., soil, water). The best place to look for interesting PHA producers is samples from the activated sludge of wastewater treatment plants because of the availability of rich organic compounds. Various methods have been used to isolate PHA-producing microorganisms from environmental samples [ 1- 41. The simplest method is by streaking environmental samples on nutrient agar plates until pure colonies of bacteria are isolated. Pure colonies are then streaked on minimal medium agar plates that contain certain carbon sources such as glucose. If the isolated bacterium can use glucose as the carbon source, cells will multiply on agar plates. PHA accumulation usually starts if certain elements such as nitrogen source become limited while the carbon source is available. Various types of carbon sources besides glucose can be added. However, bacteria that do not possess the PHA synthase enzyme will not accumulate PHA. PHA accumulation in the bacterial cell can be determined quite easily. PHA are accumulated in the form of discrete water-insoluble granules, so an optical microscope operated in phase-contrast mode can be used to detect such granules in the cell cytoplasm. Phase-contrast optical microscopy allows direct visualisation of PHA granules in the microbial cell without the need for staining. Compositional and density differences between PHA granules and the cell cytoplasm make the granules appear as light refractive inclusions. Figures 3.l(a) and (b) show the differences between cells with and without PHA granules. Cells without PHA granules have a homogeneous cytoplasm, and therefore do not show contrast and appear dark. It is usually necessary to use an oil immersion objective lens at a magnification of x100. Some microorganisms accumulate other compounds that may look like PHA granules. Spores also appear as highly light-refractive inclusions in the cell cytoplasm and may be mistaken for PHA by the untrained eye (Figure 3.1(c)). If in doubt, specific staining methods can be employed to identify PHA granules. The oxazine dye Nile blue A is widely used for this purpose, and results in a bright orange fluorescence of PHA granules if observed under an ultraviolet (UV) light microscope.

13

Practical Guide to Microbial Polyhydroxyalkanoates

Figure 3.1 Observation of cells without PHA granules (a), with PHA granules (b) and cells with PHA granules and spores; and (c) under phase-contrast light microscopy

14

Microbiology of Polyhydroxyalkanoate (PHA) Synthesis Electron microscopic techniques can be used to obtain further ultrastructural details of PHA granules. Transmission electron microscopy (TEM) of thin sections of cells reveals PHA granules as white spherical areas with a distinct boundary region in the cell cytoplasm. By using a freeze-fracture replica technique coupled with TEM, the properties of PHA granules can be elucidated [5].The ability of PHA granules to show plastic-like deformation even at temperatures far below their glass transition temperature (T,)can be seen in Figure 3.2. The freeze-fracturing process was carried out at -160 "C and the type of PHA contained by the cells was poly(3-hydroxybutyrate) or P(3HB) homopolymer with a T, of about 4 "C.

Figure 3.2 Freeze-fracture electron micrograph showing spherical PHA granules embedded in the cytoplasm of bacterial cells. The cell on the left reveals at least five PHA granules that have been stretched during the freeze-fracture process. The cell on the right reveals six PHA granules that have been completely 'scooped out' from the cell cytoplasm, and one PHA granule that has been slightly stretched. The scooped out granules leave crater-like holes in the frozen cell cytoplasm. The cells contain approximately 50 wt% PHA of the dry cell weight based on gas chromatographic analyses. The white arrow shows the shadowing direction. The shadow appears white

15

Practical Guide to Microbial Polyhydroxyalkanoates

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A.A. Amirul, A. Yahya, K. Sudesh, M. Azizan and M. Majid, World Journal of Microbiology and Biotechnology, 2009,25, 1199.

2.

A.A. Amirul, S.N. Syairah, A.R.M. Yahya, M.N.M. Azizan and M.I.A. Majid, World Journal of Microbiology and Biotechnology, 2008,24, 1327.

3.

M. Berlanga, M.T. Montero, J. Fernindez-Borrell and R. Guerrero, International Microbiology, 2006, 9,2, 95.

4.

S.S. Kung, Y.C. Chuang, C.H. Chen and C.C. Chien, Letters in Applied Microbiology, 2007, 44,4, 364.

5.

K. Sudesh, T. Fukui, T. Iwata and Y.Doi, Canadian Journal of Microbiology, 2000,46,4, 304.

16

4

Production of Polyhydroxyalkanoates (PHA)

Large bioreactors are needed for the cultivation of bacterial cells under controlled conditions to produce PHA on a commercial scale. In general, the PHA biosynthesis medium is prepared from a mixture of phosphate salts and by limiting certain nutrients (usually nitrogen) in the presence of a source of excess carbon. The depletion of this selected nutrient triggers the metabolic shift in microorganisms to accumulate PHA as a reserve of carbon and energy. Two approaches have been developed in batch cultivation: one-stage cultivation and two-stage cultivation. In the former, cell growth and PHA accumulation occur simultaneously. Two-stage cultivation consists of a cell growth phase which is carried out in a separate nutrient-enriched medium. Cells are then transferred into a nutrientlimited medium for the PHA accumulation phase. The method of cultivation differs according to bacterial strains, and one-stage cultivation is usually preferred for largescale production of PHA. The cultivation period for PHA biosynthesis is usually 24-96 hours. During the period of cultivation in the one-stage approach, PHA production is initiated at the exponential phase of cell growth and continues until the late stationary phase. In a fed-batch culture, cells are continuously fed with a selected carbon source after it has entered the late exponential phase. Large-scale or industrial scale production systems usually employ the fed-batch cultivation mode [ 1,2]. In general, the fed-batch method yields high cell densities which consequently reduces the overall production cost [3-51. Other modes of cultivation have also been evaluated, such as: pH-stat-based cultivation whereby the carbon source is fed based on the fluctuation in p H [6]and the chemo-stat method whereby the culture medium is continuously exchanged with sterile growth medium [7]. Selection of the suitable carbon source is one of the key factors that can reduce the cost of PHA production. Studies estimated that the contribution of substrate cost was approximately 28-50% compared with the total production cost [ 5 , 8 , 9 ] . Thus, suitable and relatively cheap carbon feedstocks had to be identified to maximise cost-effective production of PHA. Among some of the suitable carbon sources are sugars [lo], whey [ l l , 121, molasses 13, 141, triacylglycerols 151, and starch [16].

17

Practical Guide to Microbial Polyhydroxyalkanoates The type of PHA synthesised depends on the carbon substrates, monomer-supplying pathways, and the specificity of PHA synthases. Sugars such as glucose and sucrose are the commonest carbon sources for large-scale production of PHA. Plant oils [2, 17-20] and fatty-acid derivatives [211have also been investigated for PHA production. Compared with sugars, they are cheaper, renewable, and produce higher yields of polymer. Studies have shown that poly(3-hydroxybutyrate) or P(3HB) production from plant oils was almost twofold higher (0.6-0.8 g/g) as that of glucose (0.3-0.4 g/g) [22]. This is mainly due to the higher number of carbon atoms per gram of oil compared with sugar. Recently, palm oil by-products and used cooking oil have been investigated for P(3HB) production in laboratory-scale experiments [23]. This was done to generate value-added green material from palm oil waste.

References 1.

G.Q. Chen, G. Zhang, S.J. Park and S.Y. Lee, Applied Microbiology and Biotechnology, 2001,57, 50.

2.

P. Kahar, T. Tsuge, K. Taguchi and Y. Doi, Polymer Degradation and Stability, 2004,83,1,79.

3.

M.B. Kellerhals, W. Hazenberg and B. Witholt, Enzyme and Microbial Technology, 1999,24, 1-2, 111.

4.

M.B. Kellerhals, B. Kessler and B. Witholt, Biotechnology and Bioengineering, 1999,65,3,306.

5.

S.Y. Lee and J.I. Choi, Polymer Degradation and Stability, 1998, 59, 1-3, 387.

6.

J.I. Choi and S.Y. Lee, Applied and Environmental Microbiology, 1999,65, 10,4363.

7.

Q. Ren, K. Ruth, L. Thony-Meyer and M. Zinn, Macromolecular Rapid Communications, 2007,28,22,2131.

8.

G. Braunegg, R. Bona and M. Koller, Polymer - Plastics Technology and Engineering, 2004,43,4, 1779.

9.

L.R. Lynd, C.E. Wyman and T.U. Gerngross, Biotechnology Progress, 1999, 15,5,777.

10. D. Byrom, FEMS Microbiology Letters, 1992, 103,2,247.

18

Production of Polyhydroxyalkanoates (PHA) 11. W.S. Ahn, S.J. Park and S.Y. Lee, Biotechnology Letters, 2001,23,3,235. 12. S.Y. Lee, J. Choi and H.H. Wong, lnternationalJourna1of Biological Macromolecules, 1999,25, 1-3, 31. 13. F. Liu, W. Li, D. Ridgway, T. Gu and Z. Shen, Biotechnology Letters, 1998, 20,4,345. 14. D. Solaiman, R. Ashby, T. Foglia and W. Marmer, Applied Microbiology and Biotechnology, 2006, 71, 783. 15. D.K. Solaiman, R.D. Ashby and T.A. Foglia, Applied Microbiology and Biotechnology, 2001, 56, 664. 16. J. Yu,Journal of Biotechnology, 2001, 86,2, 105. 17. M.S.M. Annuar, I.K.P. Tan, S. Ibrahim and K.B. Ramachandran, Food and Bioproducts Processing, 2007, 85,2, 104.

18. K. Bhubalan, W.H. Lee, C.Y. Loo, T. Yamamoto, T. Tsuge, Y. Doi and K. Sudesh, Polymer Degradation and Stability, 2008,93, 1, 17. 19. W.H. Lee, C.Y. Loo, C.T. Nomura and K. Sudesh, Bioresource Technology, 2008,99, 15, 6844. 20. C.Y. Loo, W.H. Lee, T. Tsuge, Y. Doi and K. Sudesh, Biotechnology Letters, 2005,27,18, 1405. 21. Y. Doi, S. Kitamura and H. Abe, Macromolecules, 1995,28, 14,4822. 22. M. Akiyama, T. Tsuge and Y. Doi, Polymer Degradation and Stability, 2003, 80,1,183. 23. Y.K. Kek, W.H. Lee and K. Sudesh, CanadianJournal of Chemistry, 2008,86, 6,533

19

5

Extraction and Purification of Polyhydroxyalkanoates (PHA) from Microbial Cells

PHA are intracellular products, so the methods employed to recover them focus on their solubilisation or on the solubilisation of non-PHA biomass. PHA polymers are extracted using chlorinated solvents (e.g., chloroform) and isoamilic alcohols (e.g., methanol) [l, 2-41. In general, this solvent extraction method has been adapted for recovery of PHA on the laboratory scale because a large quantity of solvent is normally required. Chloroform extraction results in a high level of polymer purity without polymer degradation [4].However, usage of large quantities of volatile solvents is expensive and hazardous to the environment. Therefore, solubilisation of the non-PHA biomass is the preferred method for PHA extraction at the industrial scale. Sodium hypochlorite is a well-known cell solubiliser which has been used for extraction of poly(3-hydroxybutyrate)or P(3HB) [2,5]. Cells are initially treated with this solution and the PHA granules separated by centrifugation. Although this method is simple and effective, it was found to cause severe degradation of P(3HB), resulting in a reduction of up to 50% in weight-average molecular weight (MJ compared with that of intact cellular P(3HB). This is due to the strong oxidation effects of sodium hypochlorite. A modified method of recovery using a dispersion solution of sodium hypochlorite and chloroform was used by Hahn and co-workers. [ 6 ] .The dispersion enabled them to take advantage of differential digestion by hypochlorite and solvent extraction by chloroform. Up to 91% of P(3HB)was recovered with a purity >97%, but a reduction of -20% in the M w of the polymer recovered was observed. Besides sodium hypochlorite, other chemicals have also been tested for the solubilisation of non-PHA biomass, including acids (HCI and H2S04),alkalis (NaOH, KOH, N H 4 0 H )and surfactants (sodium bis(2-ethylhexyl)sulfosuccinate(AOT), cetyl trimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), Triton X-100, Tween 20).Among these chemicals, SDS, NaOH and KOH were more efficient in recovering P(3HB) from recombinant Escherichiu coli [7]. The purity of P(3HB) recovered in this manner is 84-98%, and the total polymer that could be recovered is -90-94% of its initial content. There was a slight reduction in the Mwofthe polymer recovered by alkali digestion (-9-13%).These values are much lower compared with the recovery methods using sodium hypochlorite as the solubilising agent. Much attention has also been given for the enzymatic recovery and purification of PHA. This recovery process usually involves heat shock treatment of the culture broth for

21

Practical Guide to Microbial Polyhydroxyalkanoates

a short period of time before enzymatic treatment. Heat shock ruptures the cells and denatures proteins, which subsequently prevents a detrimental increase in medium viscosity. Various enzymes capable of solubilising non-PHA biomass were evaluated for efficient recovery while maintaining a high level of purity [8-111. Proteases, cellulose and lysozymes are examples of commonly used enzymes. In general, enzymatic digestion is employed with chemicals (eg., the use of anionic detergents such as SDS aids further decomposition of protein and lipids; extraction and purification of PHA using ethylenediamine tetra-acetic acid (EDTA) aids in the degradation of the lipopolysaccharride layer enveloping the membrane of the cell wall). It is also possible to recover and purify PHA by enzymatic digestion without using chemicals [lo]. The advantage of using enzymes is dependent upon their specificity and on the mild operational conditions required. These operational conditions result in less damage to the product and efficient recovery of polymers. It has been reported that purity and polymer recovery of >90% could be obtained using enzymatic digestion methods. Molecular mass analysis of polymers recovered by enzymatic digestion showed no significant signs of polymer degradation [lo]. The high efficiency and low operational cost makes enzymatic recovery a promising approach for large-scale applications.

References 1.

Y. Doi, Microbial polyesters, VCH, New York, NY, USA, 1990.

2.

E. Berger, B.A. Ramsay, J.A. Ramsay, C. Chavarie and G. Braunegg, Biotechnology Techniques, 1989,3,4,227.

3.

R.V. Nonato, P.E. Mantelatto and C.E. Rossel, Applied Microbiology and Biotechnology, 2001, 57, 1-2, 1.

4. J.A. Ramsay, E. Berger, R. Voyer, C. Chavarie and B.A. Ramsay, Biotechnology Techniques, 1994,8,8,589.

5.

J.A. Ramsay, E. Berger, B.A. Ramsay and C. Chavarie, Biotechnology Techniques, 1990,4, 4,221.

6.

S.K. Hahn, Y.K. Chang, B.S. Kim, K.M. Lee and H.N. Chang, Biotechnology Techniques, 1993,7, 3,209.

7.

J.I. Choi and S.Y. Lee, Biotechnology and Bioengineering, 1999,62,5, 546.

22

Extraction and Purification of Polyhydroxyalkanoates (PHA)from Microbial Cells 8.

G.J.M. de Koning and B. Witholt, Bioprocess and Biosystems Engineering, 1997, 17, 7.

9.

P.A. Holmes and G.B. Lim, inventors; Imperial Chemical Industries PLC, assignee; US 4910145, 1990.

10. EM. Kapritchkoff, A.P. Viotti, R.C.P. AIL, M. Zuccolo, J.G.C. Pradella, A.E. Maiorano, E.A. Miranda and A. Bonomi, Journal of Biotechnology, 2006, 122,4,453. 11. K. Yasotha, M.K. Aroua, K.B. Ramachandran and I.K.P. Tan, Biochemical Engineering Journal, 2006, 30, 3,260.

23

6

Crystalline and Solid-state Structures of Polyhydroxyalkanoates (PHA)

6.1 Poly[(R)-3-hydroxybutyrate] (P(3HB)) P(3HB)molecules within the native granules are present in an amorphous form [l-41. P(3HB)has a perfectly isotactic structure with only the (R)-configuration, so isolated P(3HB) reveals 55-80% crystallinity [5]. The densities of crystalline and amorphous P(3HB) are 1.28 g/cm3 and 1.18 g/cm3, respectively [6]. The crystal structure of P(3HB) has been determined by X-ray diffraction analysis of P(3HB) oriented fibre and intramolecular potential energy calculation by two research groups [7-91 (Figure 6.1(a)). The estimated crystallographic parameters are in close agreement. The unit cell is orthorhombic with dimensions a = 0.576 nm, b = 1.320 nm and c (fibre axis) = 0.596 nm, and space group of P2,2,2,. Two anti-parallel chains are packed in a unit cell, and the molecule has a left-handed 2,-helix conformation (Figure 6.1(b)).Bruckner and co-workers [lo] refined the crystalline structure of P(3HB) using the Rietveld fitting method applied for powder X-ray diffractograms. When the geometrical parameters obtained from powder diffractograms were compared with those from oriented fibres, disagreement factors were observed. However, all structure analyses converge to models whose crystalline conformation and crystallographic characteristics are similar. P(3HB) forms a lamellar crystalline structure if prepared as single crystals from dilute solution or if prepared as spherulites from the melt. Single crystals of P(3HB) have been prepared from a dilute solution of polymer in many kinds of organic solvent 16, 12-23]. The surface morphology and crystal structure have been investigated using microscopic analyses. Typically, P(3HB) forms lath-shape crystals with dimensions of -0.3-2 pm along the short and of -5-10 pm along the long axes (Figure 6.2).The thickness of single crystals is in the range 4-10 nm, and is dependent upon the molecular weight, solvent, and crystallisation temperature.

25

Practical Guide to Microbial Polyhydroxyalkanoates

f

8

n

2E

a-form (2, helix)

p-form (planer zigzag)

Figure 6.1 Crystal structure of bacterial P(3HB) (a) Projection of the crystal structure of P(3HB) in the ab and bc planes, and (b) molecular conformations for two types of crystalline form: 2, helix conformation (a-form) and planar zigzag conformation (p-form) [ 8 , 9 , 111

Based on the electron diffractogram of P(3HB) single crystal, the polymer chains align perpendicular to the lamellar base of the crystal, and the long axis of P(3HB) single crystals is the crystallographic a-axis. Barham and co-workers [6] showed that the single crystal of P(3HB) was split into small crystal fragments along the long-axis direction by stretching perpendicular to its long axis. When the single crystal was stretched parallel to its long axis, periodic cracks yielded intersections at the long axis. In addition, the polyethylene decoration on the surface of P(3HB) single crystals had also been carried out. The decorated polyethylene appeared as crystals, and had its long axis perpendicular to the long axis of the P(3HB) single crystal [12, 131. These results suggested that the predominant chain folding in P(3HB) crystals is along the long axis of the single crystal, i.e. along the (100) direction with existing successive folds in the (110) and (1-10) directions (see Figure 6.2).

26

LZ

"lUU

5

(a)

Practical Guide to Microbial Polyhydroxyalkanoates Due to the twisting of P(3HB) lamellar crystals, P(3HB) spherulites typically show a banded texture (Figure 6.3). Periodicity and regularity of the banding texture depend on the crystallisation temperature and molecular weight.

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

I

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

CH2

Poly [(R)-3-hydroxybulyrate] P(3HB)

Banded texture of P(3HB) spherulite (crystallized at 85 "Cfrom melt)

5- 10 nm

(Lame11ae)

r

f

(Spherulite) Figure 6.3 Schematic representation of multi-oriented lamellar aggregates for P(3HB)

P(3HB) from biological sources can be of very high purity, resulting in P(3HB) undergoing a homogeneous nucleation from the pure melt. The nucleation density for P(3HB) is excessively low compared with common polymers. The low nucleation rate leads to development of extremely large spherulites within P(3HB) materials.

28

Crystalline and Solid-state Structures of Polyhydroxyalkanoates (PHA) The sizes of P(3HB) spherulites developed from the pure melt vary from several micrometres to a few millimetres depending on the crystallisation temperature and molecular weight [6]. The spherulites of P(3HB) usually contain cracks radially or along the circumference of the spherulite [26,27]. Radial cracks occur even on spherulites crystallised at room temperature. Circumferential cracks appear if films crystallised at high temperatures are cooled to room temperature. The cracking pattern may be due to the difference between the radial thermal expansion coefficient and circumferential thermal expansion coefficient of the spherulite. For example, if the radial thermal expansion coefficient is much greater than the circumferential coefficient, a large tensile stress in the radial direction results on cooling of the spherulites from its crystallisation temperature. Such stress could be released by circumferential cracking. In general, P(3HB) is crystallised in an orthorhombic unit cell with a 2, helix conformation. X-ray fibre diagrams of P(3HB)with highly oriented films and fibres indicated two types of molecular conformation: the 2, helix conformation (a-form) and the planar zigzag conformation (p-form) [9,11,28-311. The p-form of P(3HB) was first suggested by Yokouchi and co-workers [9] from the X-ray diffraction measurement of a further drawn, uniaxially stretched P(3HB) film. The X-ray diffraction pattern of the sample revealed a sharp reflection on the equator and streaks on the layer lines. This suggested formation of a twisted planar zigzag conformation with a fibre period of 0.47 nm. The p-form structure was confirmed, and a detailed structure of this form analysed by Orts and co-workers [ 111 using a cold-drawn film of P(3HB-co-21 mol% 3HV). From the calculated value of fibre repeat length for fully extended P(3HB) (fibre repeat of 0.474 nm), the p-form has a near completely extended chain conformation (see Figure 6.1(b)). The p-form is introduced by the orientation of free chains in the amorphous regions between a-form lamellar crystals [9, 11, 291. The equatorial reflection derived from the p-form disappeared in the X-ray diagrams when the film was annealed at temperatures >160 "C, but the reflections from the a-form remained. Therefore, the shrinkage and reorganisation of the molecular chains occurred at a high annealing temperature, and the a-form is more stable than the p-form [28].

6.2 Poly[(R)-3-hydroxyvalerate] (P(3HV)) The crystal and molecular structure of P(3HV)was first studied using X-ray diffraction of isotactic polymers chemosynthesised from the racemic monomer [32]. Chain conformation and packing, derived from the chemosynthetic P(3HV) fibre diagram, are similar to those of P(3HB) except for a decrease of nearly 10% in the c-axis of the unit cell. Subsequently, the crystalline structure of bacterially synthesised P(3HV)

29

Practical Guide to Microbial Polyhydroxyalkanoates was examined using the polymer of (R)-3HV containing approximately 20 mol% of the (R)-3HBunit [33]. Furthermore, through recent electron diffraction study on single crystals of bacterial P(3HV) homopolymers, it was shown that the two structures from bacterial P(3HV) and synthetic P(3HV) coincided [34, 351. The diffraction analysis combined with X-ray and electron diffraction diagrams for pure P(3HV) homopolymers indicated that P(3HV) crystallises in the P212,21orthorhombic space group with lattice parameters a = 0.950 nm, 6 = 1.010 nm, and t (fibre axis) = 0.556 nm, and with a twofold screw left-handed symmetry along the molecular axis (Figure 6.4). Solution-grown lamellar single crystals of P(3HV) appear to be square-shaped with a lateral size of 2-4 pm and a thickness of 5-6 nm (Figure 6.5) (34, 351. The electron diffraction diagram indicates that the polymer chains align perpendicular to the base of the crystal, and that the growth planes of crystals are (110) [20]. From the electron micrographs of single crystals decorated with polyethylene and lateral force microscopic observations, the average direction of chain foldings is parallel to the growth planes.

6.3 Medium-chain-length Poly[(R)-3-hydroxyaIkanoate]s (mclP(3 HA)) PHAs of medium-chain-length (R)-3-hydroxyalkanoates (mcl-(R)-3HA) with 6-14 carbon atoms, mcl-P(3HA), can be produced by several prokaryotes, including many fluorescent pseudomonads. The metabolism of these bacteria can add or cleave-off two carbon atoms from the fed carbon-source molecule, so the produced mcl-P(3HA) is usually a random copolymer rather than a homopolymer [36]. For example, Pseudomonas oleovorans grown on sodium octanoate, will produce polyesters consisting of 86 mol% of (R)-3-hydroxyoctanoate ((R)-3HO),10 mol% of (R)-3-hydroxyhexanoate ((R)-3HHx),and 4 mol% of (R)-3-hydroxydecanoate ((R)-3HD)[37]. For reasons of simplicity, mcl-P(3HA)s are named after their major component. The repeating units in mcl-P(3HA) molecules also have complete (R)-configuration, so the polymer is also crystallisable. McLP(3HA) crystallises as a 2, helix in an orthorhombic lattice with two molecules per unit cell [38]. The side chains were proposed to form ordered sheets with extended conformation in the crystalline structure. For example, the unit-cell dimensions of P(3HO)are a = 0.512 nm, b = 3.605 nm, and c (fibre axis) = 0.455 nm [38]. Compared with the unit-cell dimensions of P(3HB) homopolymer, the compressed fibre repeat is attributed to the intramolecular interactions of the extended alkyl side chains.

30

Crystalline and Solid-state Structures of Polyhydroxyalkanoates (PHA)

c

4

I

Figure 6.4 Crystal structure of P(3HV) [32]

31

Practical Guide to Microbial Polyhydroxyalkanoates

Figure 6.5 Transmission electron micrograph of solution-grown P(3HV) single crystals [34]

The parameters of the crystals with 2, helix conformations for P(3HA) are summarised in Table 6.1. The dimensions of the unit cell (fibre repeat, unit-cell dimension of the a b plane, and volume of the unit cell) are plotted as a function of the length of the side chain in Figure 6.6. The volume of the unit cell of P(3HA) expands from 0.453 nm3 for P(3HB) up to 0.938 nm3 for P(3HD).The unit-cell dimension of the a b plane also expands from 0.760 nm2to 2.06 nm2in the same range. These expansions are a result of the increase in the volume of the (R)-3HA monomer. The fibre period decreases from 0.596 nm to 0.455 nm as the length of the side chain increases. The decrease in the fibre repeat suggests that the crystallisation is driven by side-chain packing influences. Longer side chains can form a stable ordered sheet with an extended alltrans conformation. As for P(3HN) and P(3HD), further increase in the length of the side chain has no influence on the fibre period of mcLP(3HA). Therefore, 0.45 nm appears to be a limit of compression for the 2, helix of P(3HA) [45], and further compression may require new helix symmetry [38]. Modelling and intramolecular energy calculations have led to two models being developed for the crystalline helix of mcLP(3HA). In the ‘comb-like’ model, the trans-zigzag side chains extend in the directions perpendicular to the chain axis. In the ‘herringbone’ model, the side chains extend diagonally from the main chain.

32

Crystalline and Solid-state Structures o f Polyhydroxyulkanoutes (PHA)

Table 6.1 Crystallographic parameters of

P H A crystals

“91, h[34, 381, “381, d[39],“401, “411, g[42, 431, h[44]

6.4 Poly(4-hyd roxybutyrate) (P(4HB)) Chemosynthetic techniques such as polycondensation of 4-hydroxybutyrate (4HB) and ring-opening polymerisation of y-butyrolactone (y-BL) have not yielded a homopolymer of 4HB (P(4HB))with high molecular weight because the 4HB readily forms a ring ester structure by dehydration and such five-membered ring esters (y-BL) are relatively stable. A random copolymer of (R)-3HB and 4HB, termed P(3HB-co4HB), with a wide range of compositions was produced by Cupriuvidus necutor, Alcaligenes lutus and Delftiu acidovoruns from various carbon sources [46-501. When 4HB or lP-butanediol was used as sole carbon source for D. acidovoruns, P(4HB) homopolymer could be produced [49, SO]. Currently, P(4HB) homopolymer is commercially produced by Tepha Incorporated, USA, in a large-scale fermentation of geneticany engineered Escherichia coli [51].Therefore, P(4HB) homopolymer is one of the characteristic molecules in PHAs.

33

Practical Guide to Microbial Potyhydroxyalkanoates

2.5

2.0

I.5

1.o

0.5

0 0

I

2

3

4

5

6

7

Side-chain length (carbon number) Figure 6.6 Dimensions of P(3HA) crystals as a function of the length of the side chain The crystal structure of P(4HB) homopolymer was analysed from the X-ray fibre diagram. Mitomo and co-workers [52] reported the X-ray fibre diagram obtained from the necking-stretched and annealed film of P(4HB).The unit cell of P(4HB) is orthorhombic with space group P2,2,2,, and parameters a = 0.775 nm, b = 0.479 nm, and c (fibre axis) = 1.194 nm. In one unit cell, two chains exist in an anti-parallel arrangement. Pazur and co-workers [53] carried out energy calculations on a single chain of P(4HB) and found five minimum energy conformations. Among them, one conformer possessing a pitch of 1.19 nm was in good agreement with the observed fibre repeat distance. The calculated value of fibre repeat length for P(4HB) with the all-trans planar zigzag conformation is 1.240 nm, so they claimed that P(4HB) molecules in crystals have an inherent regular torsion along the molecular backbone. Su and co-workers [54] reported almost identical unit-cell dimensions of P(4HB) determined by a X-ray fibre diagram of a stretched-annealed film combined with electron diffraction patterns of single crystals. The same research group reported the

34

Crystalline and Solid-state Structures of Polyhydroxyalkanoates (PHA) helical conformation (which is a slightly distorted all-trans conformation) and the molecular packing structure in detail based on diffraction data and energy calculations (Figure 6.7) [44].

f a

b

Figure 6.7 Crystal structure of P(4HB)and transmission electron micrograph of solution-grown P(4HB)single crystals [44]

35

Practical Guide to Microbial Polyhydroxyalkanoates Lamellar single crystals of P(4HB) grown from a dilute solution of ethanol showed a ‘lozenge shape’ with a ratio between the two diagonal axes of -3/5, and crystal thickness was 7-8 nm (Figure 6.7)[54]. Based on the electron diffraction diagram, it has been confirmed that the growth planes of P(4HB) single crystals correspond to the crystallographic (110) planes. From the electron micrographs of single crystals decorated with polyethylene, chain foldings occurred on the surface of P(4HB) single crystals along the (110) growth planes.

6.5 Poly(3-hydroxypropionate) (P(3HP)) In general, P(3HP) can be obtained as poly(P-propiolactone) by a chemosynthetic method as a ring-opening polymerisation of P-propiolactone (p-PL). A random copolymer of (R)-3HB and 3-hydroxypropionate (3HP), termed P(3HB-co-3HP), with a wide range of compositions was produced by Ralstonia Eutropha [55] or A. latus [56, 571, when 3HP was used as the carbon source, whereas the pure P(3HP) homopolymer has yet to be synthesised by bacteria. However, using a similar strategy for the P(4HB)homopolymer, the homopolymer of 3HP can be produced by microbial synthesis. Therefore, the crystalline structure of P(3HP) has been introduced. There are some different chain conformations and crystalline structures of P(3HP) depending on the sample preparation conditions [39-42, 581. Based on X-ray diffraction and electron diffraction studies, it has been reported that P(3HP) has at least three crystal forms: a, p, and y. The a-form crystal is obtained by hot-drawing and annealing procedures from solution-cast film. The molecular chain of the a-form has a 2, helix conformation with a fibre period of 0.702 nm, but there is no other report regarding the structure of the a-form [39]. The p-form crystal is generated by cold-drawing and annealing procedures from film. The p-form crystal shows a characteristic X-ray fibre diagram, and the diffraction patterns have discrete reflections on the equatorial line and diffuse continuous scatterings on the layer lines. From the observed fibre distance of 0.482 nm, the p-form has a planar zigzag chain structure for the molecular conformation [39]. The crystal structure of the p-form was further investigated using single crystals prepared from an irradiated bulk polymerisation of p-PL. The unit cell of the p-structure is orthorhombic, with parameters a = 0.773 nm, b = 0.448 nm and c (fibre axis) = 0.477 nm [40]. The crystals in the p-form were reported to be in a paracrystalline state, like a liquid crystalline nematic. The y-form originated from solution-grown lamellar single crystals [41]. The y-form consists of a two-chain, C-faced, orthorhombic unit cell with parameters a = 0.700 nm, b = 0.490 nm and c (fibre axis) = 0.493 nm (Figure 6.8).Although p-form and y-form molecular chains crystallise in an all-trans conformation, the molecular arrangement from the projection to the ab-plane differs. Besides the three crystal forms described above, the helical conformation structure with a fibre distance of 0.430 nm is generated in 36

Crystalline and Solid-state Structures of Polyhydroxyalkanoates (PHA)

P(3HP) polymer prepared by irradiated bulk polymerisation of p-PL [42]. Furuhashi and co-workers IS91 examined the crystalline structure formed in the solution-cast film of P(3HP)with high molecular weight weight-average molecular weight (Mw)= 3.7x1OS,Mwlnumber-averagemolecular weight (M,) = 2.2).

f

0.492 nm

Figure 6.8 Crystal structure of P(3HP) (y-form) and transmission electron micrograph of solution-grown P(3HP)single crystals [41]

37

Practical Guide to Microbial Polyhydroxyalkanoates The X-ray pattern of solution-cast film was almost identical to that of the y-form sedimented mat, whereas other reflection peaks which could not be indexed in terms of the y-form unit cell (including the reflection with a d-spacing value of 0.429 nm) were present in the X-ray diagram of the solution-cast film. Therefore, the authors concluded that the solution-cast film of P(3HP) with high molecular weight crystallised mainly in a y-form. More recently, Zhu and co-workers [43, 60, 611 studied the polymorphic crystallisation of P(3HP) dependent upon molecular weight and crystallisation conditions. They reported that a new crystal form (&form) was formed in the solution-cast film of P(3HP)together with the y-form crystal, and that the low-molecular-weight fraction of P(3HP) was preferentially organised in the &form. The &form crystal was successfully prepared by solution-casting and optimal ~. melt-crystallising for the P(3HP) with molecular weight below Mn = 5 . 6 ~ 1 0The characteristic X-ray reflection peak corresponding to the &form crystal was observed at a d-spacing value of 0.429 nm. Based on the Fourier-transform infrared (FTIR) analysis and conformation prediction, they proposed that P(3HP) molecules in the &form take on the 2, helix conformation rather than the all-trans conformation adopted by the y-form. However, the detailed structural analysis of the &form has not been carried out. Lamellar single crystals of P(3HP) with y-form molecular chains, which are grown from cyclohexanone solution and are isothermally crystallised, exhibit a lath-like morphology with dimensions of -0.5-1.5 pm in width, -5-8 pm in length, and 5 nm in thickness (Figure 6.8) [59]. The general fold direction is along the a-axis (long axis of the crystal), and the chain folds successively in the (110) and (1-10) planes, similar to P(3HB) lamellar crystals.

6.6 Poly[(R)-3-hydroxybutyrate]-based Copolymers The structure and properties of random copolymers of (R)-3HBand (R)-3HV have been extensively investigated. X-ray diffraction patterns of the P(3HB-co-3HV) films showed only two crystalline forms; the crystalline lattice found in the copolymers with (R)-3HVcompositions up to approximately 40 mol% was the same as that of pure P(3HB), whereas P(3HB-co-3HV) containing more (R)-3HV units had the P(3HV) crystalline lattice [62, 631. One of the interesting properties of the P(3HBco-3HV) copolymers is that relatively high crystallinity (up to 60%) is preserved even at intermediate comonomer composition [64] (Figure 6.9). This observation is in keeping with the hypothesis of isodimorphism, i.e., (R)-3HV units are partially included into the P(3HB) crystalline lattice, and vice versa [62].

38

Crystalline and Solid-state Structures of Polyhydroxyalkanoates (PHA)

0

0

A

20

-

01 0

H.AAO

0

A

A

0

0

0

I

I

I

I

20

40

60

80

100

Second monomer fraction (mol?”) Figure 6.9 Relationship between crystallinity and the fraction of second monomer units for random copolymers of (R)-3HBwith different HA units. ( 0 ) :P(3HBC O - ~ H V()A, ): P(~ H B - c o - ~ H H x( )W, ): P(3HB-co-mcl-3HA), ( 0 ) :P(3HB-co-3HP), and (A): P(3HB-co-4HB)

The second monomer composition in the crystalline phase is not the same as the whole copolymer composition even in an isomorphous copolymer. The extent of co-crystallisation has been measured using high-resolution solid-state 13C-NMR spectroscopy [65-671. Kamiya and co-workers [65, 661 and VanderHart and coworkers [67] concluded that some fraction of the second comonomer units was included in the crystalline phase of the crystalline lattices of P(3HB)and the P(3HV), thereby confirming the hypothesis of P(3HB-co-3HV) isomorphism. In the P(3HB) lattice, the (R)-3HB units crystallise more readily than the (R)-3HV units, and vice versa in the P(3HV) lattice. Thus, the content of the second monomer is lower than the whole composition. The extent of co-crystallisation in the P(3HB) lattice has also been determined by measurements of density and lattice strain [27, 681. The results of these studies supported the hypothesis of isomorphism, although both studies and NMR studies 39

Practical Guide to Microbial Polyhydroxyalkanoates failed to reach an agreement on the amount of (R)-3HV units in the P(3HB) lattice. This amount varies from 30% to 100% of the total (R)-3HVcontent in the copolymer [65,66,68]. When the extent of co-crystallisation in the P(3HB) lattice was compared with that in the P(3HV) lattice, it was reported that more (R)-3HB units crystallise in the P(3HV) lattice than (R)-3HVunits in the P(3HB) lattice [65, 661. Wide-angle X-ray diffraction studies showed that, for the crystals in the P(3HB) lattice, the d-spacing of ( 1 10)reflection extends as the (R)-3HVcontent of the overall copolymer increases [62]. Expansion of d-spacing was not observed for the P(3HV) lattice [63]. Thus, the less bulky minor component probably co-crystallises more readily than the more bulky one. The effect of crystallisation temperature on the extent of co-crystallisation has also been investigated [69,70]. The (R)-3HVcontent in the P(3HB)lattice and the (R)-3HB content in the P(3HV)lattice decrease as the crystallisation temperature increases, and the degree of co-crystallisat ion is lowered as the crystallisation temperature increases. Barham and co-workers [71] pointed out that the mechanism for the inclusion of comonomer units into the crystals is by kinetic, rather than by equilibrium, methods. Crystals formed under kinetic conditions contain more co-monomer units than crystals formed under thermodynamic conditions. The kinetic model also predicts that the extent of co-crystallisation decreases with increase in crystallisation temperature because more rearrangement of the chain can take place during crystallisation at higher temperature, providing the opportunity to approach the equilibrium state. Single crystals of P(3HB-co-3HV) have been reported by Mitomo and co-workers [19], Marchessault and co-workers [17], Nobes and co-workers [72], and Iwata and co-workers [ 13, 731. Mitomo and co-workers obtained P(3HB-co-3HV) single crystals with six different (R)-3HVcontents of <30 mol% by isothermal crystallisation in propylene carbonate [ 191. They reported that the morphological characteristics of single crystals up to 10 mol% of (R)-3HVcontent were very similar to P(3HB), whereas single crystals of 17 and 30 mol% (R)-3HVcontents yielded morphological irregularity with bumpy surfaces [19]. They also described a slight increase in the crystallographic a parameter from 0.576 nm to 0.581 nm (10 mol% (R)-3HV)[19]. Single crystals of P(3HB-co-3HV) with increasing (R)-3HVcontent (up to 21 mol%) were subsequently grown from ethylene glycol by Marchessault and co-workers, who reported that single crystals exhibited the electron diffraction diagrams of the P(3HB) lattice but with a slight increase in the a and b parameters [ 171. Iwata and co-workers prepared single crystals of P(3HB-co-8 mol% 3HV) from chlorofordethanol, and reported that there is no essential difference in the d-spacings of electron diffraction diagrams between the P(3HB) homopolymer and P(3HB-co-8 mol% 3HV) single crystals [13,73]. Therefore, the (R)-3HVunits are excluded from crystals and mainly

40

Crystalline and Solid-state Structures of Polyhydroxyalkanoates (PHA) exist on the crystal surfaces during the slow crystallisation from the dilute solution. In the case of random copolymers of (R)-3HB with (R)-3HHx having a propyl side chain, only one crystalline form of the P(3HB) lattice is observed for the X-ray diffraction patterns of P(3HB-co-3HHx) copolymers with compositions of 0-25 mol% (R)-3HHx. In contrast to P(3HB-co-3HV) copolymers, the expansion of lattice spacing of the (110)reflection is not detected in the X-ray diffractograms of P(3HB-co-3HHx) copolymers, and the crystallographic parameters of the P(3HB-co-3HHx) copolymers are only slightly influenced by the (R)-3HHx unit. The X-ray crystallinities decrease from 60% to 18% as the (R)-3HHxfraction is increased from 0 mol% to 25 mol% (Figure 6.9) [74,75]. These results suggest that the (R)-3HHxunits are excluded from the P(3HB)crystalline phase. Due to their sterically bulky side chain, (R)-3HHxunits act as defects of P(3HB) crystals, and co-crystallisation does not occur in P(3HB-co3HHx) copolymers at low compositions up to 25 mol% of (R)-3HHx. In similar situation in P(3HB-co-3HHx) copolymers, the random copolymers of (R)-3HB with mcl-(R)-3HA form the P(3HB) lattice, and the mcl-(R)-3HA units are excluded from the P(3HB) crystalline phase in P(3HB-co-mcl-3HA) copolymers. The X-ray crystallinities decrease to 18% as the mcl-(R)-3HA fraction increases to 18 mol%. The crystal structures and degrees of crystallinity for P(3HB-co-3HP) copolymers have been determined from the X-ray diffractions of solution-cast films with a wide range of compositions from 0 mol% to 100 mol% 3HP [57,76-781. Only one crystalline form of the P(3HB) lattice is observed for the copolymers with 3HP composition <38-43 mol%, whereas the copolymers with 3HP content >78 mol% crystallise in the P(3HP) lattice. The crystallographic parameters of the copolymers with compositions up to 38-43 mol% represent almost the same values as the P(3HB) crystal, whereas the d-spacing values corresponding to the P(3HP) crystalline lattice remained constant regardless of 3HP composition for the copolymers with >78 mol% 3HP. X-ray crystallinities steeply decrease from 6O-62% to 16% as the 3HP fraction is increased from 0 mol% to 38-43 mol% [57, 771. Copolymers with -60-67 mol% of 3HP are found to be in an amorphous form at room temperature. Then, the crystallinities increase from 13-21% to 37-62'3'0 with an increase in 3HP fraction from 71-78 mol% to 100 mol% [57,77]. The presence of minor co-monomer units significantly suppresses the crystallisation of the copolymers, suggesting that the co-monomer units are excluded from the crystalline phase of the copolymers. From the measurement of I3C spin-lattice relaxation time using solid-state NMR spectra, it has been confirmed that the co-crystallisation of (R)-3HBand 3HP units in the same crystal lattice does not occur in P(3HB-co-3HP) copolymer [79, SO]. Although the 3HP co-monomer unit has the same carbon number of the main chain with the (R)-3HB unit and no side chain, the 3HP units are excluded from the P(3HB) crystalline phase in P(3HB-co-3HP)

41

Practical Guide to Microbial Polyhydroxyalkanoates copolymers at low 3HP compositions. It is predicted that the trans arrangement will be energetically more stable as the conformation about the CH,-CH, bond of 3HP unit rather than the gauche arrangement due to the absence of a chiral centre in the main chain. The P(3HP) homopolymer with high molecular weight predominantly forms a y-form in solution-cast film [59], and the molecular chain has an all-trans conformation in the y-form. Such differences in the conformational features of main chains between (R)-3HBand 3HP units results in the exclusion of co-monomer units from the crystalline phase of the copolymers. The crystallinities of P(3HB-co-4HB) decrease from 60% to 1 4 % as the 4HB content increases from 0 mol% to 49 mol% [49, 501. Only one crystalline form of the P(3HB) lattice is observed for the X-ray diffraction patterns of P(3HB-co-4HB) copolymers with compositions of 0-29 mol% 4HB. In contrast, only the P(4HB) lattice is observed for the P(3HB-co-4HB) copolymers with compositions of 78-1 00 mol% 4HB. Thus, the P(3HB-co-4HB) copolymer system also does not display cocrystallisation because the crystalline con formational features of 4HB repeating units are different from those of (R)-3HB. Iwata and co-workers prepared single crystals of three types of P( 3HB)-based copolymers with (R)-3HHx,4HB, and 6-hydroxyhexanoate (6HHx; E-caprolactone) units as the second monomer [24, 731. Each single crystal of copolyester showed a lath-shaped morphology, and yielded a sharp electron diffraction pattern similar to that of P(3HB) homopolymer. In their study, there was no essential difference in the d-spacings of electron diffraction diagrams between P(3HB) homopolymer and copolymer single crystals, suggesting that all of the second monomers used were excluded from crystals and existed mainly on the crystal surfaces. P(3HB)-basedcopolymers also form spherulites if crystallised from the melt in bulk materials. The sizes of spherulites vary depending on copolymer composition and crystallisation temperature. The primary nucleation rate and spherulite growth rate of copolymers are reduced by the introduction of second monomer units. As a result, larger spherulites are organised in the P(3HB)-based copolymers with longer time spent, whereas the overall crystallinity is smaller than for the P(3HB) homopolymer. The lamellar thicknesses in spherulites vary depending on the chemical structure of each second monomer, copolymer composition, and crystallisation temperature. The long period distance (Lp)and lamellar core thickness (1J values of P(3HB-co-3HV) with (R)-3HV content of <21 mol% are 7-24 nm and 4-17 nm, respectively, and increase in the crystallisation temperature causes increments in Lpand lc values [64, 811. In addition, at a given crystallisation temperature, both values tend to increase if (R)-3HV content increases. P(3HB)-based copolymers with second monomer content of <10 mol%, except for P(3HB-co-3HV), reveal the changes in Lp and 1, values from 5.3 nm to 9.9 nm and from 1.6 to 3.5 nm, respectively, depending on

42

Crystalline and Solid-state Structures of Polyhydroxyalkanoates (PHA) the crystallisation temperature [81, 821. The I, values (1.6-3.5 nm) of copolymer samples except for P(3HB-co-3HV) are apparently smaller than those of P(3HB) homopolymer, whereas the Lp values (5.3-9.9 nm) are almost consistent with those of P(3HB), indicating that the interlamellar amorphous region is thickened by the introduction of second monomers. The 1, data of copolymer samples support the notion that a portion of (R)-3HV units is included in the P(3HB) crystalline lattice, whereas the other second monomer units are excluded from the P(3HB) crystalline lattice. In addition, 1, values for copolymers decreased with an increment of side-chain length of the second monomer unit when the values were compared among the copolymers with an identical composition of the 6 mol% (R)-3HA unit [82]. In the melting endotherm of melt-crystallised films for P(3HB)-based copolymers, a broad endothermal peak starting around room temperature was detected in addition to a sharper endothermal peak starting above the isothermal crystallisation temperature [81, 831. By microscopic analyses of lamellar crystals formed in a relatively thin layer (-100 nm thickness), the formation of thin crystals of thickness 1-7 nm during storage at room temperature after the primary crystallisation was confirmed (Figure 6.10).Therefore, it has been proposed that the long sequences of (R)-3HBunits in a random copolyester form relatively thick P(3HB) crystalline lamellae during primary crystallisation at a given crystallisation temperature, whereas the shorter sequences of (R)-3HB units (which cannot crystallise at a given crystallisation temperature) form relatively thin crystalline lamellae during the subsequent crystallisation process at room temperature [83,84]. The formation of such thin lamellar crystals is a characteristic of P(3HB)-based random copolymers, and the subsequent crystallisation at room temperature occurs on the surface and in the interlamellar amorphous regions of copolymer films.

Figure 6.10 Atomic force microscopy deflection images of P(3HB-co-10 mol% 6HHx) thin films crystallised at 110 "C for 3 days: (A) without storage at room temperature; (B) with storage at room temperature for 24 days, and (C) with reheating at 110 "C for 1 h after storage at room temperature for 24 days 43

Practical Guide to Microbial Polyhydroxyalkanoates

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49

7

Physical Properties of Poly hyd roxyalkanoates (PHA)

7.1 Crystallisation Kinetics The crystallisation rate of samples of PHA can be determined by monitoring the timedependent changes in the proportion of crystalline region using X-ray diffraction and Fourier-transform infrared (FTIR)spectroscopy [ 13 or in the crystallisation exotherm using differential scanning calorimetry (DSC) under isothermal and non-isothermal conditions [2]. These analyses relate to the overall crystallisation kinetic, which is governed by the primary nucleation rate and crystal growth rate. The primary nucleation and crystal growth behaviours from the melt are usually observed by polarised optical microscopy [2-51. If the PHA polymer crystallises isothermally from the melt, the radius of the spherulite formed increases linearly with time. The radial growth rate of spherulites (G) is derived from the slope of the line obtained by plotting the spherulite radius against time. The kinetic of growth rate of poly(3hydroxybutyrate) (P(3HB))spherulite has been evaluated at various crystallisation temperatures. The spherulite growth rate of P(3HB) shows a convex curve against the crystallisation temperature and a maximum value of about 3 - 4 pm/s at -90 "C (Figure 7.l(a)) [3]. The radial growth rate of spherulites is similar to polypropylene, nylon 6 , and polyethylene terephthalate (PET). Three types of crystallisation behaviours (regimes I, 11, and 111) have been observed in crystallisable polymers according to Hoffman's crystallisation theory. The radial crystal growth rate (G) is is governed by two important processes and is a function of the rate (i) at which the secondary nucleus is formed and of the velocity ( g )with which the surface nucleus spreads on the crystal surface. If the secondary nucleation rate, i, is very small compared with g, a single nucleus completes one layer before another nucleus is formed. This is regime I of crystallisation. If the nucleation rate, i, is high and it spreads slowly at a velocity, g, multiple nucleation occurs before the completion of one layer, and this type of crystallisation is called regime 11. Regime I11 is a case where the nucleation rate, i, is much higher than the velocity, g. In the case of P(3HB) homopolymer, crystallisation at high crystallisation temperature shows regime-I1 behaviour, whereas regime I11 appears at a low crystallisation temperature. The transition from regime I1 to 111occurs at -130 "C for P(3HB) [3]. Regime-I growth has yet to be observed in P(3HB).

51

0

OO

3 0 O O

0

0

0

0

0 3

0

O

0

0

O

8

3

O

Practical Guide to Microbial Polyhydroxyalkanoates

52

i

Physical Properties of Polyhydroxyalkanoates (PHA) The primary nucleation rate is obtained by counting the number of spherulites formed during the isothermal crystallisation process. The complete melts of P(3HB) may undergo homogeneous nucleation under the condition without addition of nucleating agents. The primary nucleation rate of P(3HB) also shows a convex curve against the crystallisation temperature. The maximum of primary nucleation for P(3HB) homopolymer is shown at a lower temperature region compared with the temperature observed in the maximum spherulite growth rate (Figure 7.l(b))[4]. As a result, the overall crystallisation rate of P(3HB)typically shows a maximum in the temperature range 60-70 "C [l]. The maximum rate of primary nucleation for P(3HB) homopolymer is of the order of several events/s/mm3 161. The nucleation density for P(3HB) is about one magnitude less than for polyethylene and polypropylene. Such a low level of primary nucleation is responsible for the slow rate of overall crystallisation in P(3HB).

P(3HB)molecules within the native granules are present in a rubbery amorphous form. de Koning and Lemstra [7] claimed that the amorphous state of native PHA granules in vivo can be explained by a straightforward application of crystallisation kinetic concepts. Subsequently, Horowitz and Sanders [ 81 explained the observed failure of nascent granules to crystallise was due to the slow nucleation kinetics operative for small isolated particles. Lauzier and co-workers [9] attribute the failure to crystallise to a network of hydrogen bonds between PHA and bound water which effectively inhibits the nucleation. This concept has been supported by computer simulation analyses for in vitro formation of PHA granules [lo]. The overall rate of crystallisation can be accelerated by the use of nucleating agents that promote crystallisation of the molten or glassy resin by increasing the number of nuclei within P(3HB). The increased number of nuclei leads to small-diameter spherulites and reduced cycle times in thermal processes. Nucleating agents such as saccharin, talc, micronised mica, boron nitride (BN), chalk, calcium hydroxyapatite, calcium carbonate, ammonium chloride, cyclohexyl phosphonic acid and zinc stearate mixtures, I-hydroxyethyiidene diphosphonic acid, and P(3HB)seed crystals [4,11,12] have been effective for the crystallisation of P(3HB)and other PHA. BN is commonly used as the nucleant for P(3HB) and other PHA. The kinetics of the growth rate of spherulites have also been evaluated for P(3HB)based copolymers [2,5,13-151. Even though the second monomer units are introduced into (R)-3HB sequences, the radius of copolymer spherulites increases linearly with time. The rates of spherulite growth are dependent on copolymer composition and crystallisation temperature. As the second monomer component increases, spherulitic growth rates are markedly reduced (Figure 7.2(a)).In addition, the crystallisation curves are shifted toward lower temperatures. The randomly distributed co-monomer units in the P(3HB)-basedcopolymers are responsible for the remarkable decrease in the rate of

53

Practical Guide to Microbial Polyhydroxyalkanoates (R)-3HBsegments at the growth front of crystalline lamellae. Such a tendency holds for any P(3HB)-based copolymers independently of the chemical structure of the second monomer units, but the values of growth rate at the same temperature vary with the second monomer structure among the copolymers with an identical composition. The considerable reduction in the crystal growth rate is detected even for P(3HB-co-3HV) copolymers, suggesting that the (R)-3HVunits (which are co-crystallisable with the P(3HB)crystalline phase) have to be regarded as energetically unfavourable defects in the P(3HB)crystal. If the spherulitic growth rates are compared at the same temperature among the P(3HB)-basedcopolymers containing 6 mol% of (R)-3HAunits with different lengths of crystallised side chains, values are reduced as the side-chain length of the second monomer increases (Figure 7.2(b))[ 151. In addition, the crystallisation curves of copolymers are shifted towards lower temperatures with an increase in the side-chain length of the second monomer. The observed shift of the spherulite growth curves depending on the side-chain length of the second monomer is attributed to depression of the equilibrium melting temperature for P(3HB)-based copolymers.

10

.

,

,

lo 5

,

I' 2! e!

I

I

t

loSl

o

O** 1

I

I

I

I

I

I

ao 80 l o o 120 140 Crystalllsatlon temperature f'C) 20

40

"..

t

~~

~

0

20

40

60

80

100 120 140

Crystallisation temperature ('C)

Figure 7.2 Radial growth rates of spherulites for P(3HB)-based copolymers as a function of crystallisation temperature. (a) P(3HB-co-3HHx) with various 3HHx composition, and (b) P(3HB)-based copolymers containing 6 mol% of second monomer units with different side-chain length

54

Physical Properties of Polyhydroxyalkanoates (PHA) Reduction of crystallisation by copolymerisation is reflected not only in the spherulitic growth rates, but also in the primary nucleation rates. As a result, the overall crystallisation rates of P(3HB)-based copolymers are extremely slow. Addition of the nucleating agents is usually required for the thermal processing of P(3HB)-based copolymers. In the case of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) i.e., P(~ H B - c o - ~ H B ) , the rates of crystal growth decrease with the 4HB fraction, suggest that the 4HB units are excluded from the P(3HB) crystalline phase [16, 171. However, the overall crystallisation rate of P(3HB-co-94 mol% 4HB) at room temperature from the melt is almost identical to that of P(3HB) homopolymer [18].

7.2 Thermal Properties 7.2.1 Melting Temperature PHA materials behave like thermoplastics with melting temperatures of 50-1 80 ".The melting endotherms of crystallised PHA samples are usually characterised by DSC. The typical melting temperature (T,,,)of P(3HB) homopolymer samples prepared by solution-cast is observed at -180 "C [19]. For solution-cast films of poly(3hydroxybutyrate-co-3-hydroxyvalerate)i.e., P(3HB-co-3HV) copolymers, the Tn, decreases from 177 "C t o 64 "C with increasing R)-3HV fraction, and then increases to 102 "C as the (R)-3HVfraction is increased from 42 mol% to 88 mol% (Figure 7.3). The T,,,of solution-cast films of P(3HB-co-3HHx)copolymers decreases from 177 "C to 52 "C as the (R)-3HHx fraction is increased from 0 mol% to 25 mol% [13, 201. The T,,, value of solution-cast films of P(3HB-co-mcl-3HA) copolymers also decreases from 177 to 112 "C as the mcl-(R)-3HAcontent is increased from 0 mol% to 18 mol%. For solution-cast films of P(3HB-co-3HP) copolymer, the T,,, decreases to 44 "C with the 3HP fraction and then increases to 77 "C as the 3HP fraction is increased from 6 7 mol% to 100 mol% [21, 221. The T,,, of P(3HB-co4HB) solution-cast films drops from 178 "C to 45 "C as the 4HB content increases from 0 mol% to 45 mol%, and the P(3HB-co-4HB) copolymers with 4HB content in the range of 45 mol% to 100 mol% have melting temperatures ranging from 45 "C to 53 "C [16, 171. Poly(3-hydroxyoctanoate)i.e., P(3HO) has a melting temperature of 61 "C [23].

55

Practical Guide to Microbial Polyhydroxyalkanoates

*0°

1 0

0

i 0

a

0

0

A O8

a 0

Lp

20

0

40

0

A

0

60

0

A AAA A

80

100

Second monomer fraction (molY') Figure 7.3 Relationship between melting temperatures ( Tm)and the fraction of second monomer unit for random copolymers of (R)-3HBwith different HA units. ( 0 ): P(3HB-co-3HV),(A):P(3HB-co-3HHx),( rn ): P(3HB-co-mcl-3HA), ( 0 ) : P(3HB-co-3HP),and (A): P(3HB-co-4HB)

As mentioned above, PHA polymers organise into lamellar crystals, and the thickness of lamellae varies with the crystallisation conditions. It is well-known that the T,,, values of crystals depend on the thickness of crystalline lamellae. Therefore, the melting temperature detected by DSC is varied with crystallisation conditions such as crystallisation temperature, crystallisation time, and annealing treatment. The observed melting temperatures of P(3HB)homopolymer isothermally crystallised from the melt increase from 166 "C to 180 "C with a rise in crystallisation temperature from 60 "C to 140 "C [24]. The melting temperature of a stretched P(3HB) film

56

Physical Properties of Polyhydroxyalkanoates (PHA) with annealing treatment was reached at around 190 "C [25].To determine the equilibrium melting temperature ( Tmo),the Hoffman-Weeks method is widely used. The Hoffman-Weeks method involves linear extrapolation of experimental melting temperatures ( Tm)observed for various crystallisation temperatures ( Tc)toward the equilibrium line T , = T,. If the Hoffman-Weeks method is applied for the evaluation of P(3HB) homopolymer, the Tmovalue is of equilibrium melting temperature (Tm0) calculated to be around 190 "C (Figure 7.4) [6, 15, 261.

200

180

-

o3 2

160

3 +-

E

140 Q) * cn c

- 120 3

= *

100

80 20

40

60

80 100 120 140 160 180 200

Crystallization temperature ("C) Figure 7.4 Melting temperatures ( T,) of melt-crystallised P(3HB)-based copolymer films crystallised at various temperatures. ( 0 ) :P(3HB), ( 0 ): P(3HB-60-6 mol% 3HV), (A):P(3HB-co-6 mol% 3HHx), and (A): P(3HB-co-6 mol% mcl3HA)

57

Practical Guide to Microbial Polyhydroxyalkanoates value is the Gibbs-Thompson method. In this The other method to determine the Tmo method, the reduction in melting temperature from an equilibrium value is accounted for in terms of the limited lamellar thickness, and the melting temperature of infinite crystal thickness is extrapolated using the proportional relationship between the inverse lamellar thickness and the observed melting temperature. For P(3HB) homopolymer, the plot of melting temperature ( Tm)against inverse lamellar thickness (l/lc)shows a linear relationship, and the equilibrium melting temperature (Tm0) can be evaluated from the intercept of the line to be 194-198 "C (Figure 7.5) [3, 15,24, 261.

200

180

160

140

120

100

0

0.1

0.2

0.3

0.4

0.5

0.6

Inverse lamellar thickness (nm-1) Figure 7.5 Variation in melting temperature (T,) as a function of the inverse lamellar thickness ( l/lc) for melt-crystallised polyester films. ( 0 ) : P(3HB), ( ): P(3HB-co-6 mol% 3HV), (0): P(3HB-co-16 mol% 3HV), (A):P(3HB-co-6 mol% 3HHx), (0): P(3HB-co-8 mol% 3HHx), (V):P(3HB-co-8 mol% 4HB), (V): P(3HB-co-10 mol% 4HB), and (A): P(3HB-co-6 mol% mcl-3HA)

It is known that two apparent endothermic peaks are detected in the thermograms for P(3HB)-based copolyester samples. Based on the DSC curves of melt-crystallised

58

0.7

Physical Properties of Polyhydroxyalkanoates (PHA) copolyester films recorded at heating rates of 5 - 40 "C/min, the higher temperature peak became smaller as the heating rate increased, whereas the lower temperature peak area increased. This result indicates that the higher melting endothermic peak is caused by rearrangement of the initial crystal morphology of the copolyester. The lower melting temperature represents the melting of original crystals formed at the crystallisation temperature [26, 271. Also, for P(3HB)-based copolymer samples, the melting temperature corresponding to the original crystals increases with crystallisation temperature, suggesting that the thickness of crystalline lamellae in melt-crystallised polyester films increase with an increase in the crystallisation temperature. If the Hoffman-Weeks method is applied for P(3HB)-based copolymers [15], the Tmovalues of P(3HB)-based copolymers are reduced by the introduction of second monomer units, including the (R)-3HVunit. The melting temperatures of melt-crystallised films for random copolymers vary with the chemical structure of second monomer units when the values are compared among copolymers with an identical copolymer composition crystallised at the same temperature. The melting temperatures of P(3HB-co-6 mol% 3HHx) films are higher than those of P(3HB-co-6 mol% mcl-3HA) films, whereas these are lower than the values of P(3HB-co-6 mol% 3HV) films, indicating that the melting temperature decreases with the side-chain length of the second monomer unit (as shown in Figure 7.4) [15]. If the Gibbs-Thompson method is applied for P(3HB)-based copolymers, there is an interesting tendency for the relationship between the lamellar thickness (lc)and the melting temperature ( Tm)of melt-crystallised P(3HB)-based copolymer samples [ 15, 261. Figure 7.5 shows the relationship between the inverse lamellar thickness ( l/lc)and the melting temperature ( T,) of melt-crystallised P(3HB)-based copolymer samples. For P(3HB-co-3HV)samples, the lines shift to lower melting temperatures at the same lamellar thickness with an increase in the (R)-3HVfraction. The plots of T, against 1/ lcfor the other copolyester samples fit with the line obtained from plotting the T, and l/lcof P(3HB)homopolymer samples extrapolating toward thinner lamellar thickness. This result indicates that the crystalline structure of lamellae for copolyesters except for P(3HB-co-3HV) is essentially the same as that for P(3HB) homopolymer [15,26]. It has been concluded that the randomly distributed second monomer units except for (R)-3HVin P(3HB) copolyesters act as defects in the P(3HB)crystal and are excluded from the P(3HB)crystalline lamellae, whereas the (R)-3HVunits are partially included into the P(3HB) crystalline lattice for the P(3HB-co-3HV) samples.

7.2.2 GIass Transition Temperature (TJ The Tgof PHA is also determined by DSC using melt-quenched amorphous samples. The Tgof P(3HB) homopolymer is about 4 "C. P(3HB-co-3HV) copolymers display an almost linear depression in Tg value with an increase in the (R)-3HV fraction

59

Practical Guide to Microbial Polyhydroxyalkanoates (Figure 7.6). The Tgof P(3HB-co-3HHx) also decreased from 4 "C to - 4 "C as the (R)-3HHx fraction was increased from 0 mol% to 25 mol% [13, 281. From the exploration of lines, the Tgvalues for P(3HV) and P(3HHx) homopolymers can be calculated to be -17 "C and -27 "C, respectively. P(3HO) samples containing 10 mol% of (R)-3HHx and 4 mol% of (R)-3HD unit have a Tgof -35 "C [29]. Thus, an increase in the average side-chain length of P(3HA)tends to decrease the Tgvalue. In addition, at an identical second monomer composition, the Tgof P(3HB) is more efficiently depressed by the introduction of a (R)-3HA unit with a longer side-chain as a second monomer.

v) v)

m

G

-60-

I

I

I

Figure 7.6 Relationship between Tgand the fraction of second monomer unit for random copolymers of (A)-3HBwith different HA units. ( ): P(3HB-co-3HV), (A):P(3HB-co-3HHx), ( rn ): P(3HB-co-mcl-3HA), ( 0 ) : P ~ ~ H B - c o - ~ Hand P ) , (A): P(3HB-co-4HB)

60

Physical Properties of Polyhydroxyalkanoates (PHA)

P(3HP) without side chains has a Tgvalue of -19 "C, and the value is lower than that of P(3HB) with a methyl side chain. P(4HB)homopolymer shows a Tgat around - 48 "C. Thus, the Tgvalue of PHAs without a side chain decreases with an increase in the main-chain length. The Tgvalues of P(3HB-co-4HB) or P(3HB-co-3HP)copolymers vary continuously between the Tgvalues of both homopolymers [16,17,21,30].

7.2.3 Thermal Degradation Temperature Many studies on the thermal stability of PHA, especially P(3HB), have been conducted for understanding their processes and applications [31- 401. From the standpoint of chemical recycling of polymer materials, understanding of the thermal stability of PHA is regarded as being important. The pioneering works on the thermal degradation of P(3HB) were done by Grassie and co-workers [41-431. Many researchers investigated the thermal degradation behaviour of P(3HB) using various analytical techniques. From the extensive data reported in the literature, it is widely understood that during the early stage of thermal degradation at temperatures >160 "C, the degradation of P(3HB) occurs exclusively via random chain scission reaction (cis-elimination), which has a six-membered ring ester intermediate (Figure 7.7). There is no conspicuous difference in the manner of degradation according to the nature of the atmosphere (under nitrogen, air, vacuum condition), indicating a non-radical or non-oxidative thermal degradation. As a result, a drastic reduction in the molecular weight of P(3HB) occurs during processing at temperatures above the melting temperature. Thermal decomposition of polymers has been investigated by non-isothermal techniques such as differential thermogravimetry (DTG) and differential scanning calorimetry (DSC). Figure 7.8a shows the typical thermogravimetric (TG) curve of a P(3HB) sample at a heating rate of 10 "C/min under a nitrogen atmosphere. The weight loss starts at around 275 "C under a nitrogen atmosphere, and no carbonaceous residue is left at around 305 "C. The DTG curve of purified P(3HB) reveals a single peak at 293 "C. Volatile products have been analysed by pyrolysis-gas chromatography/massspectrometry (Py-MS) techniques, nuclear magnetic resonance (NMR)analysis, and/ or infrared (IR) spectroscopy. The major volatile products from P(3HB) are crotonic acid and the oligomers of (R)-3HBcontaining crotonyl chain-ends produced by the cis-elimination reaction [3 1,321. Small quantities of isocrotonic acid are also evolved if the polymer is heated to around 300 "C. Secondary products such as propene, ketene, ethanol, aceteldehyde, and CO, were also formed by further decomposition of the primary products at the higher temperature region.

61

Practical Guide to Microbial Polyhydroxyalkanoates

CH3

I

-0-CH-CH2-

F 0

CH3 0 I -O-CH-CH2-C4

I

\I

OH

CH3 CH3 CH I I CH-C-0-CH-CH2-CII II 0 0

' +

CH3 FH3 I CH=CH-C-0-C H-CH2-C-

II 0

II

0

Figure 7.7 Scheme of the cis-elimination reaction for P(3HB) thermal degradation

To obtain information regarding the elementary reactions of thermal degradation processes, isothermal degradation experiments and analyses of residual molecules have also been done. During isothermal degradation at a given temperature from 170 "C to 190 "C for 30 min, the sample weight of P(3HB) was almost unchanged, whereas the number-average molecular weight (M,) of P(3HB) decreased nonlinearly with time. When the inverse of number-average degree of polymerisation (P,) was plotted against the reaction time, the plot revealed a linear relationship, indicating that the thermal degradation of P(3HB) proceeds via a random scission of the polymer chain. When the constant rate of thermal degradation (k,) was determined from the slope of a line plotting U P , against time (Figure 7.8(b)),the k, value of pure P(3HB) under the isothermal condition at 180 "C was calculated to be 3.0 * 0.3 x 10" min-' 1331. However, the degradation of P(3HB) followed a kinetic model of random scission during the first few hours of isothermal degradation at 190 "C, whereas eventually auto-acceleration of the pyrolysis due to the function of low-molecular-weight compounds was detected, as reported by Nguyen and co-workers [40].

62

Physical Properties of Polyhydroxyalkanoates (PHA)

Temperature ("C) 4

(B) 3 n

'?

0 F

x

v

2 -

1

0

0

5

10

15

20

25

30

Time (min) Figure 7.8 Thermogravimetric curve of P(3HB) recorded at a heating rate of 10 "C/min under a nitrogen atmosphere (a). Changes in the inverse of the numberaverage degree of polymerisation (l/Pn)during isothermal degradation of P(3HB) at various degradation temperatures (b). Isothermal degradation temperature: 170 "C ( ), 180 "C ( ), and 190 "C ( A )(331 63

Practical Guide to Microbial Polyhydroxyalkanoates The apparent activation energy (E,) of thermal degradation for P(3HB) samples can be determined from the data obtained by TG, DSC, Py-MS, and gel permeation chromatography (GPC). However, the E , values obtained for P(3HB) varied widely, from 109+13 to 380220 kJ/mol [32, 33, 35, 36, 38, 41-43], depending on the analytical technique used and the kinetic model applied, and there was no consistency in the values. When the apparent E , values for P(3HB)samples were determined from TG data according to the Ozawa model, the values were calculated to be 109213 kJ/mol, 111i7 kJ/mol, and 140+4 kJ/mol by Mitomo and Ota [38], Aoyagi and coworkers [32], and Kim and co-workers [33], respectively. Grassie and co-workers [41- 431, Kunioka and co-workers [35], and Kim and co-workers [33] reported that the activation energies ( E , ) of the isothermal degradation of P(3HB)were 247210 kJ/ mol, 212+10 kJ/mol, and 182k23 kJ/mol, respectively, using molecular-weight data from GPC and the Arrhenius equation. The estimated Ea values from isothermal degradation processes (Ea = 182+23-247+10 kJ/mol) are apparently larger than those from heating processes (Ea = 109213-140i4 kJ/mol). Under the isothermal degradation condition, Ea values are determined from the rate constant (k,) of the chain scission for P(3HB)molecules. In contrast, the values for heating processes are estimated from the production rate of volatile components such as monomers and oligomers. Therefore, the activation energies obtained from the heating process and isothermal process should denote the energies at different processes in the thermal degradation of P(3HB).

P(3HB) is synthesised by fermentation of microorganisms and does not depend on the participation of metal compounds in the polymerisation reaction between the monomer and polymerising enzyme. Therefore, there are only few reports on the effects of residual metal contents on the thermal degradation of bacterial P(3HB) compared with the synthetic polymers with metal catalyst residues. However, various metal compounds are added as essential elements to the cultivation medium of microorganisms, and the isolated P(3HB) from bacterial cells sometimes contains certain amounts of metal compounds. The amounts of metallic residual contaminants are dependent upon the purification procedures. Kim and co-workers [33] reported that commercial-grade P(3HB) has a relatively large amount of calcium (Ca)compounds (-400 ppm) after crude purification. They demonstrated that the presence of Ca compounds enhances the depolymerisation reaction of P(3HB) molecules. When the P(3HB) sample with -400 ppm of Ca compound was subjected to TG measurement, the TG curve was shifted to a temperature region lower by about 20 "C as compared with pure P(3HB).Moreover, the rate constants (k,) of chain scission for the P(3HB) with Ca compounds was several-times higher than those of pure P(3HB) when the values were compared at an identical degradation temperature. A similar acceleration effect on a random chain scission of P(3HB) molecules was detected with the addition of compounds of

64

Physical Properties of Pofyhydroxyaikanoates (PHA) magnesium (Mg) or sodium (Na), but not with compounds of aluminium (Al), tin (Sn) and zinc (Zn) [44]. Therefore, they proposed that the metal compounds which belong to metal ions of hard acid (Lewis acid) act as an acid catalyst. It interacts with carboxyl group to facilitate the elimination of the P-hydrogen. Recently, Kawalec and co-workers [45]reported that a random chain scission of P(3HB) molecules was enhanced by the conversion of the carboxylic acid chain-end into a carboxylate group with cationic counterions of Na+,K', and (C,H,),N+. They proposed that the a-carboxylate chain-end induced the intermolecular a-deprotonation reaction of the 3HB unit during thermal degradation with the formation of a crotonyl chain-end. Melchiors and co-workers [46] reported the depolymerisation reaction of P(3HB) in the melt yielded crotonic acid and linear oligomers. This depolymerisation reaction is independent from the presence of metal and acid catalysts. O n the other hand, the back-biting reactions of P(3HB) molecules progressed in organic solvent in the presence of those catalysts to give rise to cyclic trimers of (R)-3HB units. Although it is difficult to define the role of metal compounds in the thermal degradation of P(3HB), the metal compounds participate in the catalytic reaction in the thermal degradation of P(3HB) molecules, and the catalytic effect is strongly dependent on the species of metal compound. The thermal stability of P(3HB)-based copolymers has also been examined by several researchers. When P(3HB-co-3HV)copolymers with (R)-3HVcontents ranging from 0 mol% to 20 mol% underwent TG measurement, the maximum decomposition temperature shifted slightly towards the higher temperature region with an increase in (R)-3HVcontent [47]. In addition, the apparent activation energy ( E , ) of thermal degradation for P(3HB-co-3HV) copolymers obtained from TG data was higher than that of P(3HB) homopolymer [38]. Therefore, it seems that the thermal stability of P(3HB-co-3HV)copolymers is enhanced by the introduction of (R)-3HV units. In contrast to such non-isothermal degradation conditions, it has been reported that the thermal degradation of P(3HB-co-3HV) copolymers under isothermal conditions progresses in an analogous mechanism with that of P(3HB) homopolymer, with a similar activation energy and degradation kinetics that are independent from the (R)-3HVcomposition [35]. Nguyen and co-workers [40] reported that the P(3HV) homopolymer was observed to thermally degrade via random chain scission at a faster rate than P(3HB)homopolymer under isothermal degradation at 190 "C.As mentioned above, the TG data represent the production rate of the volatile components such as monomers and oligomers in the heating process. It is expected that the volatility of a compound with a similar chemical structure decreases with an increase in molecular mass. As a result, the TG curve of P(3HB-co-3HV) copolymers shifts towards the

65

Practical Guide to Microbial Polyhydroxyalkanoates higher temperature region with increasing (R)-3HV content. However, the kinetic constants of thermal chain scission (k,) may be very similar among the P(3HB-co3HV) copolymers. In contrast to P(3HB-co-3HV) copolymers, the rate of random chain scission is strongly influenced by the compositions of P(3HB-co-3HP) copolymer under isothermal degradation at temperatures <180 "C [21]. The rate constant of chain scission at 180 "C for P(3HB-co-71mol% 3HP) was about ten-times larger than that of P(3HB)homopolymer, whereas the value of P(3HB-co-11 mol% 3HP) was almost identical to P(3HB).It is known that the cis-elimination reaction occurs for the thermal degradation reaction of P(3HP) homopolymer [48, 491. The DTA curve of P(3HP) recorded at a heating rate of 10 "C/min under a nitrogen atmosphere showed one peak at 230 "C, and the peak temperature of P(3HP)was apparently lower than that of P(3HB) (293 "C). Unfortunately, the rate constants of chain scission for P(3HP) homopolymer have not been reported, but the thermal decomposition of P(3HB-co3HP) copolymer may be accelerated by the introduction of 3HP units. Doi and co-workers reported that the thermal degradation of P(3HB-co-4HB) containing 4HB units ranging from 11 mol% to 94 mol% occurs by a random chain scission process involving cis-elimination at temperatures <200 "C and is independent of copolymer composition [35]. Abate and co-workers reported that the main degradation mechanism of P(3HB-co-4HB) containing 97 mol% of 4HB unit is intramolecular transesterification 150,5 11. In addition, they suggested that the existence of a (R)3HBunit of 3-34 mol% would cause competition between ciselimination of the (R)-3HBunit and the intramolecular transesterification reaction, resulting in the rate of chain scission being influenced by the copolymer composition, and cyclic and linear chain oligomers being generated as degradation products. Kim and co-workers investigated the thermal degradation mechanism of P(4HB) homopolymer using samples with different molecular weight and chain-end structure under isothermal and non-isothermal conditions [33]. Based on TG analyses, it was found that two distinct processes occurred at temperatures below and above 350 "C during the non-isothermal degradation for P(4HB) samples depending on the molecular weight and heating rate. From the analysis of residual P(4HB) molecules after isothermal degradations at different temperatures, it was confirmed that the o-hydroxyl chain-end remained unchanged in the residual P(4HB) molecules at temperatures <300 "C, whereas the w-chain-ends of P(4HB) molecules were converted to 3-butenoyl units at temperatures >300 "C. In contrast, most of the volatile products evolved during the thermal degradation of P(4HB) were y-BL regardless of the degradation temperature. From these results, it is concluded that during the thermal degradation of P(4HB), the selective formation of y-BL via an unzipping reaction from the a-hydroxyl chain-end predominantly occurs at temperatures <300 "C.At

66

Physical Properties of Polyhydroxyalkanoates (PHA) temperatures >300 "C, the cis-elimination reaction of the 4HB unit and the formation of cyclic macromolecules of P(4HB)via intramolecular transesterification take place in addition to the unzipping reaction from the a-hydroxyl chain-end. Finally, the primary reaction of thermal degradation for P(4HB) at temperatures >350 "C progresses by cyclic rupture via the intramolecular transesterification of P(4HB) molecules with the release of yBL as a volatile product. From TGA data, the Ea value of the unzipping reaction as the first degradation stage at the lower temperature region were calculated to be 78210 kJ/mol. The intramolecular transesterification as the second stage at the higher temperature region proceeded with an Ea value of 1 5 3 d kJ/mol. Lee and co-workers investigated the effect of side-chain structure on the thermal stability of mcl-P(3HA) using TG and DSC [36]. The TG curves of P(3HO) and P(3HO)-based copolymer containing 22 mol% of mcl-(R)-3HA units with an unsaturated group in the side chain revealed only one-step degradation process with almost the same maximum decomposition temperatures as P(3HB) homopolymer, while these mcl-P(3HA) samples had higher Ea values on a thermal decomposition as compared with P(3HB). The P(3HO)-based copolymer containing 22 mol% of mcl(R)-3HA units with an epoxy group in the side chain had higher thermal stability than the P(3HO)-basedcopolymer with an unsaturated side chain owing to the crosslinking reactions by the epoxy group during the heating process. The decomposition pattern of mcl-P(3HA) containing 38 mol% of mcl-(R)-3HA units with a hydroxyl group in the side chain was more complicated, and showed reduced thermal stability.

7.3 Mechanical Properties 7.3.1 Films and Fibres of P(3HB) Homopolymers The Young's modulus (3.5 GPa) and tensile strength (43 MPa) of P(3HB) material are close to those of isotactic polypropylene. The extension to break (5%) for P(3HB) is, however, markedly lower than that of polypropylene (400%). Therefore, P(3HB) material is considered to be a plastic with poor properties of stiffness and brittleness. When the spherulites of P(3HB) homopolymer are grown from the melt, large cracks are often observed in spherulites. The formation of such cracks in P(3HB) spherulites is one of the causes of embrittlement, as suggested by Barham and co-workers [2,52, 531. To produce non-brittle P(3HB) materials, it is necessary to seal the cracks in the spherulitic films or to crystallise the material. The sealing of cracks can be achieved by rolling the spherulitic films. Spherulites without cracks can be made by increasing the nucleation density until the spherulites become sufficiently small to prevent cracks. 67

Practical Guide to Microbial Polyhydroxyalkanoates P(3HB) films without cracks can be obtained when the spherulite size is <20 pm. It is achieved by using the self-seeding procedure, and the P(3HB) films are slowly cooled after melting at a relatively low temperature for a short time, and then isothermally crystallised >130 "C. The P(3HB) film obtained by this method shows ductility. de Koning and Lemstra [54] correlated the crystallinity and mechanical properties of as-molded P(3HB) as a function of storage time at room temperature. They reported that the embrittlement of P(3HB) materials occurs due to the progress of secondary crystallisation during storage at room temperature. This secondary crystallisation has been argued to be involved with the reorganisation of lamellar crystals formed at initial crystallisation, which tightly constrains the amorphous chains between crystals. They demonstrated that as-molded P(3HB) materials can be toughened by annealing after initial crystallisation [ 5 5 ] . Some research groups attempted to improve the mechanical properties of P(3HB)films by the drawing technique. Holmes tried to obtain uniaxially and biaxially orientated films of P(3HB)to improve the mechanical properties [56]. However, the P(3HB) films were difficult to draw uniaxially and biaxially. An alternative method to improve the embrittlement of P(3HB) films is a cold drawing procedure, as reported by Barham and Keller [52]. However, the authors did not report on the reproducibility of the cold rolling procedure. Iwata and co-workers applied a hot drawing technique to obtain stretched films of P(3HB) [25, 57, 581. They used P(3HB) samples with different weight-average molecular weight (Mw)in the range from 0 . 6 0 ~ 1 0to~ llx106, and demonstrated that the solvent-cast films of ultra-high-molecular-weight-P(3HB) (Mw> 3 . 3 ~ 1 0samples ~) were orientated easily and reproducibly up to almost ten-times their initial length, in a silicone oil bath at 165 "C, by the loosely-hanging-weight method [25,57,58]. The mechanical properties of the stretched P(3HB) film were markedly improved relative to those of non-stretched film, and the improvement in the tensile strength was due to the orientation of P(3HB) molecular chains, facilitated through the increasing draw ratio. The tensile strength of the ten-times drawn ultra-high-molecularweightT(3HB)film was as high as 277 MPa (see Table 7.1). Further improvement of the mechanical properties for P(3HB) films was achieved by subsequent drawing against the nine- or ten-times hot-drawn ultra-high-molecular-weight?( 3HB) film (i.e., two-step drawing) [25]. The tensile strength increased drastically with an increase in the second-step draw ratio, and the highest value of tensile strength reached was 388 MPa, with elongation to break of 25% and Young's modulus of 2.94 GPa (Table 7.1). In addition, it was found that the mechanical properties of the stretched P(3HB) film did not deteriorate during storage at room temperature for 6 months.

68

Physical Properties of Polyhydroxyalkanoates (PHA)

Table 7.1 Mechanical properties of P(3HB) and copolymer films and fibres Sample

Drawing method

1

Draw Tensile Elongation Young's :atio (h) strength to break modulus (MPa) (%) (GPa)

(Films) Ultra-highmolecularweight P(3HB)3

1

36

Hot-drawing

10

277

Two step hotdrawing ( l o x 1.5)

15

388

Cold-drawing

10

237

25 112

Two step colddrawing (10x15)

15

287

53

1

1

14

10

195

1

19

Cold-drawing

10

117

109

1

Two step colddrawing (10~1.4)

14

185

P(3HB)" Cold-drawing P(3HB-CO-8 mol% 3HVy

P(3HB-CO-5 mol% ~ H H x ) ~ Two step colddrawing (4x25)

63 1

32

267 10

140 116

II

2.9 1.5 1.8

0.5

I

I

I

OS

lS

(Fibres 1

38

Cold-drawing

6

121

Two step colddrawing (6x3)

18

497

Ultra-highmolecularweight P(3HB)e

6

1.9

69

Practical Guide to Microbial Polyhydroxyalkanoates

P(3HB)

Two step colddrawing (6x5)

30

625

69

4.5

Two step colddrawing (6x10)

60

1320

35

18.1

Melt-spinning'

190

54

5.6

Melt-spinningg

330

37

7.7

Hot-drawingh

6

310

60

3.8

Cold-drawing'

14.5

416

24

5.2

4

740

26

10.7

1

27

15

1.2

Cold-drawing

10

90

76

2.0

Isothermal nucleation and one-step drawing

10

1065

40

8.0

Isothermal nucleation and one-step drawing'

P(3HB-CO-8 mol% 3HV)k

I

I

I

I

I

I

"321, h[591,"601, d[61],"621, f[63],g[64], h[65],"661, i[67], "681

The stability and reproducibility of the hot drawing process is controlled by the 0 a~ molecular weight of P(3HB).The weight-average molecular weight ( M w )of 3 . 3 ~ 1 is critical value as to whether hot drawing to >400%of the initial length is reproducible. Iwata and co-workers also developed a new stretching method of cold drawing of typical molecular weight P(3HB)produced by wild-type bacteria [SS]. In this method, the drawing of melt-quenched films (amorphous pre-form) of P(3HB) was carried out in iced water and succeeded easily and reproducibly at a temperature below, but near to, the glass transition temperature of 4 "C,independent of the molecular weight of the polymer. The elastic recovery of stretched film occurred on release from the stretching machine, so annealing is required for fixing the extended polymer chains. The tensile strength of P(3HB) film with a M w of 0 . 6 ~ 1 0was ~ drastically increased up to 195 MPa when the film was stretched to a draw ratio of 10 and annealed at 75 "C. The cold drawing procedure is also available for ultra-high-molecular-weightP(3HB),and the tensile strength of cold-drawn ultra-high-molecular-weight-P(3HB) increased up to 237 MPa. Furthermore, the cold drawing technique is available for

70

Physical Properties of Polyhydroxyalkanoates (PHA) processing uniaxially and biaxially orientated films. The same authors also applied two-step drawing against cold-drawn films. Cold drawing to 5 - or 10-times was run at the temperature of iced water for the amorphous pre-form, and then cold drawn film was kept at room temperature for several minutes to increase the crystallinity. This slightly crystalline film was then further stretched to between 1.4- and 2.5-times at room temperature, and then annealed. The tensile strength of ultra-high-molecularweight-P(3HB)increased in this two-step drawing procedure to reach 287 MPa, with an elongation to break of 53%. Thus, P(3HB) homopolymer which was initially understood to be a brittle material with poor physical properties, was now a potential candidate for further commercial exploitation. The X-ray fibre pattern obtained from the two-step-drawn P(3HB) films applied to the hot-drawn and cold-drawn procedures showed a new equatorial reflection derived from the planar zigzag conformation (p-form),together with a 2, helix conformation (a-form) (Figure 7.9).It has been considered that the p-form be introduced by the orientation of free molecular chains in amorphous regions between a-form lamellar crystals [25, 69, 701. In particular, tie molecules between the lamellar crystals are important in the generation of high mechanical properties. By two-step drawing at room temperature, the tie molecules are strongly extended and, as a result, the planar zigzag conformation is generated. The planar zigzag conformation is responsible for the good mechanical properties of P(3HB) films.

u-form (2, helix)

f3-foI-m (planer zigzag)

Figure 7.9 Wide-angle X-ray diffractogram of ultra-high-molecular-weight P(3HB) two-step-drawn film (draw ratio = 15) [25]

71

Practical Guide to Microbial Polyhydroxyalkanoates Several research groups succeeded in obtaining melt-spun fibres from P(3HB) (Mw of 0 . 3 ~ 1 to 0 ~approximately 0 . 8 ~ 1 0produced ~) by wild-type bacteria [71]. Initially, Gordeyev and Nekrasov obtained P(3HB) fibres with a tensile strength of 190 MPa by annealing after melt spinning [63]. Then, Schmack and co-workers reported that the tensile strength of high-speed spun-P(3HB) fibres increased to 330 MPa by annealing [64]. Yamane and co-workers obtained P(3HB)fibres with a tensile strength of 310 MPa by a two-step annealing process of melt-spun fibres 1651. Subsequently, Furuhashi and co-workers obtained P(3HB) fibres with a tensile strength of 420 MPa by a combination of cold drawing and two-step drawing methods [ 6 6 ] .In this process, fibres were drawn in an amorphous state at the temperature above the Tg and then further drawn at a higher temperature [66]. Strong P(3HB)fibres with tensile strength were first produced by Iwata and co-workers ~ ) Amorphous fibres using an ultra-high-molecular-weight P(3HB) ( M w= 5 . 3 ~ 1 0[62]. were obtained by quenching the melt-spun fibres of ultra-high-molecular-weight P(3HB) into ice water. The cold drawing of amorphous fibres of ultra-high-molecularweight P(3HB)was achieved easily and reproducibly in ice water with two sets of rolls. The cold-drawn amorphous fibres were kept at room temperature for several minutes to generate the crystal nucleus, and then two-step drawing applied using a stretching machine at room temperature. The tensile strength and elongation to break of as-spun fibres was only 38 MPa and 6%, respectively. After cold drawing six times in ice water, the tensile strength increased to 121 MPa. The tensile strength of two-step-drawn and annealed fibres increased linearly in the same ratio of two-step drawing. When the total drawn ratio reached 60-times (cold-drawn for six times and two-step-drawn for ten times), the tensile strength reached 1.3 GPa (Table 7.1). This value is higher than those of polyethylene, polypropylene, poly(ethy1ene terephthalate), and poly(viny1 alcohol) of industrial level, and poly(glyco1icacid) used as sutures [72]. If the bundle of P(3HB) fibres in which the a-and p-forms of P(3HB) crystals coexist is applied for X-ray analysis in the laboratory (beam size: 300 pm), the obtained X-ray fibre patterns include reflections from both types of molecular conformations simultaneously [62]. Iwata and co-workers carried out the micro-beam X-ray diffraction experiments (beam size: 0.5 pm)for the mono-filament of two-step-drawn ultra-high-molecular-weight-P(3HB) fibre to reveal the detailed fibre structure and the distribution of the two types of molecular conformations using synchrotron radiation [62,73]. In the micro-beam X-ray fibre diffraction pattern from the edge of the monofilament, all the reflections were indexed with only a-form crystals with a 2, helix conformation. However, in the patterns from the centre part, the other reflection, indexed by the p-form (planar zigzag conformation) was observed, together with a-form crystals with a 2, helix conformation (Figure 7.10). These results indicate that strong, two-step-drawn ultra-high-molecular-weight-I?(3HB) fibre has a core-sheath structure, with only a 2, helix conformation (a-form) in the sheath region, and with

72

Physical Properties of Polyhydroxyalkanoates (PHA) the planar zigzag conformation (p-form) and 2, helix conformation (a-form) in the core region.

X-ray diffi-action (beam size: 300 p i )

Micro-bran X-ray diffraction beaiti size: 0.5 pm) 1 I

?

. )

2

* t

*

JP

3 " . v

Core-sheath structure

Figure 7.10 Micro-beam X-ray fibre pattern of cold-drawn and two-step-drawn ultra-high-molecular-weight P(3HB)mono-filaments recorded from the three marked points in the microscope image, and a schematic display of the core-sheath structure revealed by microbeam X-ray diffraction [62] The fibre preparation method combining cold drawing and two-step drawing which had a M w of procedures was useful only for ultra-high-molecular-weight-P(3HB) over 3.3x106,as reported by Iwata and co-workers [62,72]. The same research group ~ ) by a succeeded in producing high-strength commercial P(3HB)(M, = 0 . 7 ~ 1 0fibres new drawing method. In this process, amorphous fibres of P(3HB)were obtained by quenching the melt-spun fibre in iced water, and keeping it in iced water for several hours to prevent rapid crystallisation and allow the growth of small crystal nuclei. One-step drawing at room temperature was applied to the amorphous fibres after isothermal nucleation, and then an annealing procedure was done under constant tension to increase the crystallinity [67]. The tensile strength of as-spun fibres was approximately 20 MPa, independent of isothermal nucleation. The tensile strength of the 10-times drawn fibre without isothermal nucleation was 75 MPa. However, only 4-times drawn, one-step-drawn fibres with isothermal crystallisation for 72 hours had a tensile strength of 740 MPa, elongation to break of 26%, and Young's modulus of

73

Practical Guide to Microbial Polyhydroxyalkanoates 10.7 GPa [67]. In this drawing method, small crystal nuclei grow initially during the isothermal nucleation process. Then, the molecular chains between the small crystal nuclei that acted as the entanglement points are oriented by stretching.

7.3.2 Films and Fibres of P(3HB)-based Copolymers

Introduction of co-monomers into a P(3HB) chain is a useful technique to improve its mechanical properties. The PHA family of polyesters offers a wide variety of polymeric materials properties, from hard crystalline plastics to elastic rubbers. Mitomo and co-workers reported the mechanical properties of cast films for P(3HBco-3HV) copolymers containing from 0 mol% to 28 mol% (R)-3HV[74]. The tensile strength and Young’s modulus of the films decreased from 44 MPa to 26 MPa and from 3.8 GPa to 1.9 GPa, respectively, as the (R)-3HV fraction was increased from 0 mol% to 20 mol%. The elongation to break gradually increased from 3 % to 27% as the (R)-3HV fraction was increased from 0 mol% to 20 mol%. As mentioned above, P(3HB-co-3HV) copolymers show a high degree of crystallinity due to cocrystallisation. Therefore, the mechanical characteristics of P(3HB-co-3HV) with (R)-3HV content up to 20 mol% are not significantly improved in comparison with the P(3HB) homopolymer. However, if sufficient (R)-3HV units are incorporated into P(3HB) molecules, the films show high elongation to break, for example, that of P(3HB-co-28 mol% 3HV) reached up to 700%. In contrast to P(3HB-co-3HV) copolymers, P(3HB-co-3HHx) copolymers do not display co-crystallisation, and the crystallinity of P(3HB-co-3HHx) is steeply reduced by the introduction of (R)-3HHx units. The tensile strength of the films decreased from 43 MPa to 20 MPa as the (R)-3HHx fraction was increased from 0 mol% to 17 mol%. The elongation to break dramatically increased from 6% to 850%. Thus, the P(3HB-co-3HHx) becomes a soft and flexible material by copolymerising with small amount of the (R)-3HHx unit [13]. Furthermore, a random copolymer of (R)-3HBand mcl-(R)-3HA of carbon number ranging from 6 to 12, P(3HB-co-mcl-3HA), exhibits an effective improvement of the brittleness of P(3HB) film [75]. In the case of P(3HB-co-6 mol% mcl3HA)-cast film, the elongation to break reached 680% by introduction of only 6 mol% of mcl-(R)3HA, and the tensile strength was 17 MPa. The mechanical properties of P(3HB-co-6 mol% mcl3HA) were very similar to those of low-density polyethylene. The mechanical properties of P(3HB-co-4HB) copolymer have been determined using solution-cast films with a wide range of compositions from 0 mol% to 100 mol% 4HB. The tensile strength of P(3HB-co-4HB) films with compositions of 0-16 mol% 4HB decreased from 43 MPa to 26 MPa with an increase in the 4HB fraction, whereas

74

Physical Properties of Polyhydroxyalkanoates (PHA) the elongation to break increased from 5% to 444%. The tensile strength of the films with compositions of 64-100 mol% 4HB increased from 17 MPa to 104 MPa with increasing 4HB fraction. The true tensile strength of P(4HB) homopolymer was calculated to be as high as 1 GPa if the cross-section was corrected. Thus, P(3HB-co4HB) copolymers exhibit a wide range of material properties [16, 171. Even though P(3HB)-based copolymers have slow crystallisation rates and exhibit soft and flexible material properties, secondary crystallisation occurs during storage at room temperature to bring about the embrittlement of materials similar to P(3HB) homopolymer. In contrast to the secondary crystallisation of P(3HB) homopolymer involving the reorganisation of lamellar crystals, the small and thin crystallites produced at the interlamellar amorphous regions (see Section 6.7) reduce the mobility of molecular segments with the progress of secondary crystallisation. Iwata and co-workers applied a cold drawing technique for copolymer samples of P(3HB-co-8 mol% 3HV) ( M w = 1 . 0 ~ 1 0and ~ ) P(3HB-co-5 mol% 3HHx) ( M w = 0 . 8 ~ 1 0 [60, ~ ) 611. Independent of the co-monomer structure, the cold drawing of melt-quenched amorphous films of P(3HB-co-8 mol% 3HV) ( M w = 1 . 0 ~ 1 0and ~) P(3HB-co-5 mol% 3HHx) ( M w= 0 . 8 ~ 1 0succeeded ~) easily and reproducibly at the temperature of iced water. The tensile strength of P(3HB-co-8 mol% 3HV) films was drastically increased from 19 MPa to 117 MPa. In the case of P(3HB-co-5 mol% 3HHx) films, melt-quenched films in the rubber state could be stretched reproducibly. The 5-times cold-drawn films of P(3HB-co-5 mol% 3HHx) had a tensile strength of 80 MPa with a high elongation to break of 258%. By using two-step drawing for the cold drawn films, the tensile strength of P(3HB-co-8 mol% 3HV) and P(3HBco-5 mol% 3HHx) increased from 117 MPa to 185 MPa and from 80 MPa to 140 MPa, respectively. The X-ray fibre diagram of P(3HB-co-8 mol% 3HV) two-step-drawn film demonstrated the formation of p-form crystals, similar to those of the two-step-drawn films for P(3HB) homopolymer [60]. In contrast to P(3HB-co-8 mol% 3HV), the reflection from p-form crystals was absent in the X-ray fibre diagram of P(3HB-co-5 mol% 3HHx) two-step-drawn film [61]. This result indicates that tie molecules in the amorphous regions could not be strongly extended because of low crystallinity and because of the exclusion of (R)-3HHx units from the P(3HB) lamellar crystals. The nucleation rate of PHA copolymer is extremely low so, for the processing of P(3HB-co-3HV) fibres to be successful, a certain amount of nucleating agent is added to enhance the crystallisation rate. Yamamoto and co-workers [76] processed P(3HBco-8 mol% 3HV) fibres with a tensile strength of 210 MPa by the simultaneous methods of drawing and annealing after melt spinning. Tanaka and co-workers succeeded in producing P(3HB-co-8 mol% 3HV) fibres without adding a nucleating

75

Practical Guide to Microbial Polyhydroxyalkanoates

agent [68]. Similar to one-step drawn P(3HB) fibres, melt-spun fibres of P(3HB-co-8 mol% 3HV) ( M w = 1 . 0 ~ 1 0 were ~ ) drawn at room temperature after isothermal treatment in iced water to generate small crystal nuclei. An annealing procedure was then done under constant tension to increase crystallinity. The tensile strength of the 10-times drawn fibre without isothermal nucleation treatment was 90 MPa. However, 10-times, one-step-drawn P(3HB-co-8 mol% 3HV) fibres with isothermal nucleation treatment have a tensile strength of 1.06 GPa, elongation to break of do%, and a Young’s modulus of 8.0 GPa [68].

As mentioned above, the strong ultra-high-molecular-weight$( 3HB) fibre has a coresheath structure with only a 2, helix conformation (a-form) in the sheath region, and with the planar zigzag conformation (p-form) and a 2, helix conformation (a-form) in the core region. In the case of one-step-drawn P(3HB-co-8 mol% 3HV) mono-filament, the micro-beam X-ray fibre patterns obtained from each point of the mono-filament have reflections indexed by the a-form and the p-form simultaneously [ 6 8 ] .This result indicates that the strong one-step-drawn fibre does not have a coresheath structure, but a unique structure monotonously distributing the two types of molecular conformations. Furthermore, the three-dimensional (3D) analysis of onestep-drawn P(3HB-co-8 mol% 3HV) fibre was done using X-ray microtomography with synchrotron radiation [77]. There are many fine voids for one-step-drawn P(3HB-co-8 mol% 3HV) fibres after isothermal nucleation from the results of X-ray microtomography, and the polymer molecules occupied only a cross-section area of 52.7% in the mono-filament (Figure 7.11).Therefore, it is assumed that the true tensile strength of one-step-drawn P(3HB-co-8 mol% 3HV) fibre is calculated t o be as high as 2.02 GPa [77].

Figure 7.11 X-ray microtomography images and the stereoscopic model for the one-step-drawn P(3HB-co-8 mol% 3HV) fibre (draw ratio = 10): (a) cross-section perpendicular to the drawing direction, (b) cross-section parallel to the drawing direction, and (c) the stereoscopic model [77]

76

Physical Properties of Polyhydroxyalkanoates (PHA)

7.3.3 Mechanical Properties of mcl-P(3HA) and their Modification Techniques The crystallisation rate of P(3HO) is extremely slow, so the mechanical properties of P(3HO) are affected by the crystallisation condition. The tensile modulus ranged from 2.5 MPa to 9 MPa, the tensile strength at break from 6 MPa to 10 MPa, and the ultimate elongation from 300% to 450% depending on the crystallisation temperature and crystallisation time [78]. In addition, P(3HO) possesses very high permanent tensile sets under deformation. For example, the tensile sets are 40%, 80% and 230% for P(3HO) under strains of loo%, 150%, and 300%, respectively [78]. These behaviours limit its applicability as a practical thermoplastic elastomer. In addition to producing mcl-P(3HA), Pseudomonas strains can also utilise many functionalised organic substrates to produce mcl-P(3HA) with functional groups in the side chain. Unsaturated [79,80], halogenated [8 1-83], branched [84], and aromatic [85]side chains in mcl-(R)-JHA monomeric units have been found in the sequence of mcLP(3HA). The presence of functional groups in the mcl-P(3HA) provides sites for chemical modifications and can affect the physical properties of polymers. One of the most useful types of functional monomeric unit is mcl-(R)-3HA containing unsaturated [79, 801 or epoxy [86] groups in the side chain. It has been mentioned above that mcLP(3HA) has very low rates of crystallisation, and this limits the practicability of many processing techniques. The irradiation [87] or chemical [86] cross-linking reaction of mcl-P(3HA) employed by the unsaturated bonds or epoxy functions in side chains may solve this problem. The preparation and characterisation of nanocomposite materials using P( 3HO) latex as the matrix and polysaccharide fillers as the reinforcing phase has been reported [88, 891. Addition of a small amount of polysaccharide (-6 wt%) substantially improved the mechanical properties of P(3HO). The reinforcing effect of polysaccharide fillers was strongly related to the aspect ratio of the reinforcing phase, and the mechanical properties of composite materials improved with an increase in the amount of polysaccharide fillers. The presence of functional groups in mcl-P(3HA) is expected to allow favourable interactions with the hydroxyl groups of polysaccharides, and to improve the mechanical properties more efficiently by loading the polysaccharide fillers.

7.3.4 P(3HB)-based Polymer Blends To regulate the physical properties of P(3HB), the polymer blends of P(3HB) with biodegradable polymers have been investigated intensively. Polymer blends are physical

77

Practical Guide to Microbial Polyhydroxyalkanoates mixtures of structurally different polymers, and the mixture of two polymers forms the homogeneous or heterogeneous phase in the amorphous region on a microscopic scale at equilibrium. When a mixture of two polymers in the amorphous phase exists as a single phase, the blend is considered to be miscible in the thermodynamic sense. In contrast, a mixture of two polymers separates into two distinct phases consisting primarily of the individual components, and the blends are considered to be immiscible in the thermodynamic sense, The physical properties of a mixture are strongly dependent on the phase structures. Therefore, the miscibilities of P(3HB)based polymer blends have been evaluated extensively. Miscible blends containing P(3HB) have been formed with poly(ethy1ene oxide) [90-931, poly(viny1 alcohol) [94, 951, atactic P(3HB) [96-1001, poly(1actide) [101, 1021, poly(E-caprolactone-co-lactide)[103], poly(buty1ene succinate-co-butylene adipate) [ 1041, and poly(butylene succinate-co-e-caprolactone)[ 1041. The mixtures of P(3HB) with P(3HP) [ 1051, poly(ethy1ene adipate) [93], poly( butylene adipate) [93], and poly(e-caprolactone) [lo61 are immiscible. In the case of the miscible blends of P(3HB) with atactic P(3HB), the elongation to break was increased from 5% to 500% with an increase in the content of atactic P(3HB) content from 0 wt% to 76 wt%, whereas the Young’s modulus and tensile strength were reduced [98]. In contrast, addition of poly(E-caprolactone) to P(3HB) resulted in a decrease in the Young’s modulus and tensile strength without an increase in the elongation to break, owing to macroscopic phase separation [ 1061.

References 1.

S. Bloembergen, D.A. Holden, G.K. Hamer, T.L. Bluhm and R.H. Marchessault, Macromolecules, 1986, 19, 11,2865.

2.

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15. H. Abe and Y. Doi, Biomacromolecules, 2002,3, 1, 133. 16. Y. Saito and Y. Doi, International ]ournal of Biological Macromolecules, 1994,16, 2, 99. 17. Y. Saito, S. Nakamura, M. Hiramitsu, and Y. Doi, Polymer International, 1996,39,3, 169. 18. S . Nakamura, Y. Doi and M. Scandola, Macromolecules, 1992,25, 17,4237. 19. T.L. Bluhm, G.K. Hamer, R.H. Marchessault, C.A. Fyfe and R.P. Veregin, Macromolecules, 1986, 19, 11,2871. 20. L.D. Feng, T. Watanabe, Y. Wang, T. Kichise, T. Fukui, G.Q. Chen, Y. Doi and Y. Inoue, Biomacromolecules, 2002, 3,5, 1071. 21. E. Shimamura, M. Scandola and Y. Doi, Macromolecules, 1994,27, 16, 4429. 22. K. Kasuya, K. Takagi, S . Ishiwatari, Y. Yoshida and Y. Doi, Polymer Degradation and Stability, 1998, 59, 1-3, 327.

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8

lntracellular degradation (mobilisation) of Polyhyd roxyaIkanoates (PHA)

8.1 Endogeneous Degradation of PHA PHA can be degraded intracellularly in PHA-accumulating bacteria (by intracellular depolymerases) or extracellularly in PHA-degrading bacteria and fungi (by extracellular depolymerases). Unlike extracellular PHA depolymerase, intracellular PHA depolymerase, which functions directly in the degradation of any intracellular PHA that may be present as inclusion bodies (native PHA granules), has not been well studied. Bacteria usually accumulate PHA in response to nutritional limitations and in the presence of an excess of carbon source. Indeed, the biological role of the stored PHA seems to be to provide carbon for the organism when it cannot be obtained externally. Lemoigne [ 11observed that (R)-3-hydroxybutyrate i.e., (R)-3HBappeared upon anaerobic incubation of Bacillus. Macrae and Wilkinson [2] reported a decrease of the P(3HB) content in Bacillus megaterium during the incubation of P(3HB)rich cells in phosphate buffer, and proposed that the P(3HB) functions as a storage material. Hayward and co-workers [3] observed that the intracellular content of PHA in Rhizobium, Spirillum, and Pseudomonas species reached a maximum followed by a decrease in the stationary growth phase. Similar reports have been published for C. necator (formerly Ralstoniu eutropha) H16 [4, 51. In addition, intracellular mobilisation of P(3HB)was shown for Legionella pneurnophila and Hydrogenophagu pseudoflava in the absence of an exogeneous carbon source [6,7]. Survival of bacteria in the absence of exogeneous carbon sources was dependent on intracellular PHA content. Many reports have shown that the viability of P(3HB)-rich cells descends much slower than that of P(3HB)-poor cells. James and co-workers found that, for L. pneurnophila, the P(3HB)-accumulatedbacterium showed long-term survival for at least 600 days in the absence of an exogeneous carbon source [7]. It was shown that C. necator H16 could grow even in the absence of an exogeneous carbon source by utilising previously accumulated P(3HB) [8]. An outline of P(3HB) metabolism was first established in 1973 for Cuprimidus necator [9] and Azotobacter beijerinkii [lo]. A cyclic metabolic route (‘the P(3HB) circle’) from acetyl-CoA via acetoacetyl-CoA, (R)-3HB-CoA to P(3HB) (P(3HB) biosynthesis-sequence);and from P(3HB) via (R)-3HBand acetoacetate to acetoacetyl-

85

Practical Guide to Microbial Polyhydroxyalkanoates CoA and acetyl-CoA by intracellular P(3HB) depolymerase, 3HB dehydrogenase, acetoacetate:succinyI-CoAtransferase, and ketothiolase (P(3HB)degradation-sequence) was formulated (Figure 8.1) [9, lo]. ( R ) 3 H B released from P(3HB) is oxidised to acetoacetate, which is in turn converted to acetoacetyl-CoA by acetoacetatemccinylCoA transferase. The resultant acetoacetyl-CoA is cleaved by 3-ketothiolase to acetylCoA. Bacteria can utilise the acetyl-CoA in the tricarboxylic acid cycle. Recently, from the results of functional analyses of intracellular depolymerase, the presence of a direct pathway from P(3HB) to (R)-3HB-CoA was proposed [ll].

Tricarbaboxylicacid cycle

C

Pyruvate CaASH

NAUH AMP + PPI

\

Acetyl-CoA ’a...................1 ..

\

Succinate

\ \ NADH

Figure 8.1 P(3HB) cycle in bacteria. Numbers show enzymes as follows: (1) 3-ketothiolase, (2) acetoacetyl-CoA reductase, (3)P(3HB) synthase, (4) P(3HB) depolymerase, ( 5 ) 3HB oligomers hydrolase, (6) 3HB dehydrogenase, (7)succinylCoA transferase, and (8)acetoacetyl-CoA synthase Schlegel and co-workers [ 121 first demonstrated P(3HB) degradation in cells with addition of a nitrogen source, and suggested that P(3HB) can be used for protein synthesis in the presence of a nitrogen source. The mechanism of mobilisation of P(3HB) in the presence of nitrogen is still unknown, but the cooperation of P(3HB)

86

Intracellular degradation (mobilisation) of Polyhydroxyalkanoates (PHA) synthesis and mobilisation in C. necator has been suggested. Doi and coworkers [ 131 reported that the amount of P(3HB) accumulated in the cells decreases with time, whereas a concomitant increase in P(3HB-co-3HV)was noted when the cells were transferred into a nitrogen-free medium containing pentanoate as the sole carbon source. Conversely, when C. necator cells containing P(3HB-co-3HV)are incubated with butyrate, the content of P(3HB-co-3HV)decreases with a concomitant increase of P(3HB). These results indicate that P(3HB)and P(3HB-co-3HV)are degraded in C. necator cells. The kinetics of the synthesis and degradation of P(3HB) in such a two-step incubation system in C. necator have been studied further [14].The average molecular weight and content of P(3HB)in the cells decreased with time at almost the same rate during the degradation of P(3HB).This means that the number of P(3HB) molecules is almost constant during the degradation stage, and suggests that P(3HB) molecules are degraded from the terminus of the polymer chain via an exo-type hydrolysis reaction. Taidi and co-workers prepared P(3HB)that was newly synthesised from 14C-labeledglucose, and examined the specific radioactivity and molecular weight of the polymer in C. necator [15]. The specific activity of the polymer continued to increase by approximately 30% after cessation of P(3HB)accumulation, indicating that there was a turnover of P(3HB).Subsequent fractionation of the P(3HB)showed that the high-molecular-weight polymer was gradually being replaced by P(3HB) of lower molecular weight. It was concluded that the decrease in molecular weight of P(3HB)produced from glucose in batch culture represented the combined effects of a loss of high-molecular-weight polymer and the production of low-molecular-weight P(3HB). The specificity of the intracellular degradation of various PHA inclusions in the bacterial cells has been investigated for H. pseudoflava [6, 161. H. pseudoflava can accumulate different types of homopolymers (P(3HB) and P(4HB))and copolymers (P(3HB-co-3HV)and P(3HB-co-4HB))depending on the cultivation conditions. The H. pseudoflava cells with different types of PHA are transferred to and cultivated in a carbon-free mineral medium containing ammonium sulfate, and then the PHA in cells is degraded. By nuclear magnetic resonance (NMR)analysis of the intracellular polymers obtained before and after degradation, the sequence-specific intracellular degradation for the PHA can be estimated. Based on the magnitude of the degradation rate constants, the relative substrate specificity of intracellular degradation towards the constituting monomer units was shown to decrease in the order (R)-3HB>( R ) 3HV> 4HB.

8.2 lntracellular P(3HB) Depolymerases and Degradation Systems The intracellular mobilisation of PHA molecules is initiated by the function of intracellular PHA depolymerases to cleave ester bonds of PHA molecules. These 87

Practical Guide to Microbial Polyhydroxyalkanoates enzymes differ from extracellular PHA depolymerases in their inability to hydrolyse crystalline PHA. Intracellular PHA depolymerases are specific for the native, amorphous form of the polymer, and semi-crystalline PHA is not a substrate for these enzymes. The biochemical study on intracellular P(3HB) degradation was first done by Merrick and Doudoroff in 1964 [17]. Native P(3HB)granules of B. megaterium are hydrolysed by a crude extracts of polymer-depleted cells of Rhodospirillum rubru [17, 181. In the crude extracts of R . rubrum, two soluble components necessary for hydrolysis of the polymer were contained. One compound is heat-sensitive and is the intracellular P(3HB) depolymerase, and the second component is heat-stable and is named the ‘activator’ [17]. Under certain conditions, the activator can be replaced with a low concentration of trypsin [19], whereas treatment with heat, acid or organic solvent, repeated centrifugation, and freeze-thawing inactivate the native P(3HB) granules. The inactivated granules do not function as a substrate for P(3HB) depolymerase from R. rubrum. The principal product of the depolymerase action is (R)-3HB,together with small amounts of (R)-3HB-oligomers. Thus, hydrolysis of native P(3HB) granules requires pretreatment with activator or trypsin prior to hydrolysis by the depolymerase. In vivo, polymer molecules are in the amorphous state, and PHA granules are covered by a 4-nm-thick surface layer. The surface layer of isolated PHA granules consists of proteins and phospholipids [3,20,21]. Therefore, it has been expected that the activator is a protease that removes some protein of the surface layer of the native PHA, thus making the polyester molecules accessible for the depolymerase. Subsequently, mild alkaline-extracted, native P(3HB)granules have been reported to be susceptible to hydrolysis by depolymerase without pretreatment with activator or trypsin [19]. Furthermore, it was found that hydrolysis of artificial surfactant-coated amorphous P( 3HB)granules did not require treatment with activator [22]. However, the activator could not be inhibited by any protease inhibitor. The activator appeared in the high molecular fraction during gel filtration experiments and was sensitive to proteases. The activator was resistant to many solvents, including phenol and chloroform. More recently, the activator ApdA was purified [23] and its function studied [23]. The ApdA in R. rubrum in vivo was a P(3HB)-bound molecule with all the features of a phasin, and was similar to the PhaP of C. necator [23-251, not a protease. The ability of ApdA to bind to PhaP-containing C. necator native P(3HB) granules in vitro presumably disturbs the membranous or proteinaceous surface layer of native P(3HB)such that P(3HB) molecules become partially exposed and P(3HB) depolymerase can bind to the polymer surface. Treatment of native P(3HB) granules with small amounts of trypsin partially removes phasins such as PhaP, leading to an effect similar to that of ApdA (i.e., rapid hydrolysis of native P(3HB) by P(3HB) depolymerase), although the mechanisms by which trypsin and ApdA activate P(3HB) granules are clearly different from each other [23-251.

88

Intracellular degradation (mobilisation) o f Polyhydroxyalkanoates (PHA) The intracellular P(3HB) depolymerase PhaZl of R. rubrum has also been purified by Jendrossek and Handrick 1261. It consists of one polypeptide of 35 kDa and has a pH and temperature optimum of 9 and 50 "C, respectively. In contrast to extracellular PHA depolymerases, the purified intracellular P(3HB) depolymerase is inactive with semi-crystalline P( 3HB) and had no lipase, protease, or esterase activity with p-nitrophenylacyl esters of 2 to 8 carbon atoms. Trypsin-treated P(3HV) native granules were hydrolysed but native P(3HO) granules were not, indicating high substrate specificity. Isolated native P(3HB)granules of C. necator have a low rate of self-hydrolysis, which is about two orders of magnitude lower compared with the hydrolysis rates obtained by R. rubrum extracts. The endogeneous activity of C. necator granules can be enhanced by about threefold if P(3HB)-rich bacteria are exposed to carbon starvation before cell harvest (i.e., mobilisation conditions) [8]. The pH optimum of this endogeneous intracellular P(3HB) depolymerase activity was at pH 7. It was concluded that this activity represents the intracellular P( 3HB) depolymerase, and was responsible for mobilisation of the storage material during starvation. No significant intracellular P(3HB) depolymerase activity could be detected in soluble cell extracts of C. necator if native P(3HB) granules were used as a substrate [8]. However, soluble intracellular P(3HB) depolymerase activity and a second pH optimum at pH 9 have been reported for C. necator [27] when high-level trypsin-treated and artificial surfactant-coated P(3HB) granules were used. Intracellular P(3HB) depolymerase gene (phaZal ) was first cloned from C. necator using the 'shotgun method', and then sequenced and characterised [28]. The phaZal gene was shown to consist of 1257 nucleotides, specifying a protein of 419 amino acids with a predicted molecular weight of 47.3 kDa. The heterologous E. coli expressed depolymerase hydrolysed artificial amorphous P(3HB) granules but not freeze-dried crystallised P(3HB). The depolymerase was located in the P(3HB) granules fraction. Most interestingly, in phaZal ,there is no sequence corresponding to a classical lipase box [Gly-X,-Ser-X,-Gly]. Recently, by comparative study on the amino-acid sequence of an intracellular P(3HB) depolymerase (PhaZal) from C. necator H16 with the sequences of various proteins, it was found that the enzyme had relative similarities to the P(3HB) synthases [29]. In addition, the results on the intracellular P(3HB) depolymerase activity assay for the mutated enzymes indicated that the catalytic residues of PhaZal are C183, D355, and H388, and are located similarly to the amino acids of the catalytic triad of P(3HB) synthase. When the artificial protein-free P(3HB) is incubated with CoA and PhaZal, (R)-3HB-CoA is generated from P(3HB) as a degradation product [ l l ] . This result indicates that PhaZal depolymerises the P(3HB) molecules by thiolysis.

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Practical Guide to Microbial Polyhydroxyalkanoates

A putative intracellular P(3HB) depolymerase gene of Paracoccus denitrificans was characterised [30]. The heterologically expressed Escherichia coli gene product hydrolyses artificial P(3HB) granules. The DNA-deduced amino-acid sequence had close similarities to the intracellular P(3HB) depolymerase PhaZal from C. necator. Chromosomal knock-out mutants in the phaZal gene of C. necator showed a reduced (but still significant) self-hydrolyis activity. It was considered that the gene could be partially responsible for P(3HB)mobilisation, and that other depolymerase isoenzymes must be present in C. necator [8,31]. Indeed, recently, the presence of four isoenzymes of intracellular P(3HB)depolymerases (PhaZa2to PhaZaS) with similarity to PhaZal and two new types of putative intracellular P(3HB) depolymerase (PhaZdl and PhaZd2) were identified on the pHGl megaplasmid of R. eutropha H16 [32, 331. The latter two have amino-acid sequences that are significantly similar to those of the catalytic domain of extracellular P(3HB) depolymerases [34-361. The PhaZl isolated from R . rubrum as an intracellular P(3HB) depolymerase is certainly an enzyme with similarity to the catalytic domain of extracellular P(3HB) depolymerases. Sequence analysis of the cloned intracellular P(3HB) depolymerase gene (phaZ1) of R. rubrum revealed that the depolymerase (37.7 kDa) had high homologies to extracellular P(3HB) depolymerases with a catalytic triad (Ser42, Asp138, and His178) and a type-I1 catalytic domain, and it had a putative twodimensional structure related to a@-proteins [24,25]. Interestingly, a typical signal peptide sequence was found at the N-terminus of the predicted polypeptide, and the experimentally-determined N-terminus of the purified depolymerase coincided with the predicted signal peptidase cleavage site. However, PhaZl of R. rubrum cannot hydrolyse semi-crystalline P(3HB) and, because PhaZl of R . rubrum is not secreted into the environment under any of the culture conditions tested, an extracellular function of PhaZl is unlikely. Analyses of cell fractions unequivocally showed that PhaZl of R. rubrum is a periplasm-located enzyme not localised in the cytoplasm or at the surfaces of P(3HB) granules [8]. Therefore, the physiological function of PhaZl in vivo remains to be elucidated. Inspection of the R. rubrum genome revealed the presence of an open reading frame whose product showed significant homologies to intracellular P(3HB) depolymerases (PhaZal to PhaZaS) of C. necator and other P(3HB)-accumulating bacteria. No signal peptide was found in the deduced aminoacid sequence. Therefore, it has been assumed that this gene (phaZ2) is the true intracellular P(3HB) depolymerase of R . rubrum.

A similar intracellular P(3HB) depolymerase with a lipase box-like sequence was also identified from Bacillus thuringiensis [37]. Purified enzymes could efficiently degrade trypsin-activated native P(3HB) granules as well as artificial amorphous P(3HB) granules and release (R)-3HB monomer as a hydrolytic product, but they could not hydrolyse denatured semi-crystalline P(3HB).

90

Intracellular degradation (mobilisation) of Polyhydroxyalkanoates (PHA) It is therefore expected that two types of intracellular P(3HB) depolymerases are present within a single strain, but the advantage for having such isozymes is not known.

8.3 lntracellular 3HB Oligomer Hydrolases It has been confirmed that 3HB oligomer hydrolases are present in bacterial cells together with intracellular P(3HB) depolymerases. Intracellular 3HB oligomer hydrolase is likely to play an important part in the further degradation of (R)-3HB oligomers to monomers. Several intracellular 3HB oligomer hydrolases have been identified, the molecular mass of most being 30 kDa [38 - 431, except for PhaZb of C. necator (78 kDa) [44, 451. The intracellular 3HB oligomers hydrolases purified from Pseudomonas lemoignei [38] and Acidouorax sp. strain SA1 [41] hydrolyse the (R)-3HB dimer at a high rate, but show weak activity for (R)-3HBtrimer, indicating that these enzymes should be 3HB dimer hydrolase. The 3HB oligomer hydrolases purified from R. rubrum [40], C. necator [39], and Zoogloea ramigera [42] hydrolyse the 3HB oligomers more efficiently than the dimer. It is therefore feasible to refer to them as 3HB oligomer hydrolases, but they do not hydrolyse denatured crystalline

P(3HB). 8.4 lntracellular mcl-PHA Depolymerases Biochemical studies on the intracellular mcl-PHA depolymerase from Pseudomonas oteouorans have been reported [46,47]. When P(3HO) inclusion bodies produced by P. oleovorans grown on n-octanoate are freeze-thawed, the supernatant contains depolymerase activity against P(3HO) granules [48]. On gel filtration, a protein of about 32 kDa was found to be responsible for the activity. Experiments with inhibitors suggest that the enzyme probably has disulfide linkages and serine residues at its active site that are analogous to the extracellular P(3HB)depolymerase. The optimum pH for the granule-bound depolymerase is about 9.0. The solubilised depolymerase fraction obtained by freeze-thawing of P(3HO) granules is active in the presence of colloidal suspensions of amorphous P(3HO) granules, but shows no activity with crystalline, solvent-cast P(3HO) film. A putative intracellular mcl-PHA depolymerase gene (phaZ)was identified initially for P. oleovorans [49]. The intracellular mcl-PHA depolymerase gene is sandwiched between two PHA synthase genes (phaCI and phaC2). The phaZ gene (849 nucleotides) encodes a 31.5-kDa product (283 amino acids). The deduced aminoacid sequence contained a potential lipase box (Gly-X,-Ser-XI-Gly) but no significant homologies to any intracellular P(3HB) depolymerases.

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A similar PHA depolymerase-like sequence has been cloned and characterised from Pseudomonas aeruginosa P A 0 1 [SO], Pseudomonas sp. 61-3 [51], Pseudomonas putida U [52], and Pseudomonas resinovorans [53]. These PHA depolymerase genes are located in the PHA locus, and have a very similar gene arrangement to that in P. oleovorans. PHA depolymerase shows no significant homology to any extracellular P(3HB) depolymerases cloned, although extracellular P(3HB) depolymerases are also serine-dependent esterases with a lipase consensus sequence. The polymerising-depolymerising enzyme system has been extensively studied in P. putida U,which accumulates PHA comprising units of 3-hydroxy-n-phenylalkanoates [52]. Disruption of the gene encoding phaZ prevents mobilisation of the polymer accumulated intracellularly, and decreases the total PHA contents. This suggests that phaC2 located downstream of phaZ is not expressed because it must be expressed from a promoter located upstream of the gene encoding the depolymerase.

References 1.

M. Lemoigne, Annales d’lmmunologie, 1925,39, 144.

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R.M. Macrae and J.F. Wilkinson, Journal of General Microbiology, 1958, 19, 210.

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M.H. Choi, S.C. Yoon and R.W. Lenz, Applied and Environmental Microbiology, 1999,65,4,1570.

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V. Oeding and H.G. Schlegel, Biochemical Journal, 1973, 134,239.

10. P.J. Senior and E.A. Dawes, Biochemical]ournal, 1973,134,225.

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lntracellular degradation (mobilisation) of Polyhydroxyalkanoates (PHA) 11. K. Uchino, T. Saito, B. Gebauer and D. Jendrossek, Journal of Bacteriology, 2007,189,22,8250.

12. H.G. Schlegel, G. Gottschalk and R. Von Bartha, Nature, 1961, 191,4787, 463. 13. Y. Doi, A. Segawa and M. Kunioka, International Journal of Biological Macromolecules, 1990,12,2, 106. 14. Y. Kawaguchi and Y. Doi, Macromolecules, 1992,25,9,2324. 15. B. Taidi, D.A. Mansfield and A.J. Anderson, FEMS Microbiology Letters, 1995, 129,2,201. 16. S.C. Yoon and M.H. Choi,Journal of Biological Chemistry, 1999,274, 53, 37800. 17. J.M. Merrick and M. Doudoroff, Journal of Bacteriology, 1964,88, 1,60. 18. J.M. Merrick, D.G. Lundgren and R.M. Pfister, Journal of Bacteriology, 1965, 89, 1,234. 19. R.J. Griebel and J.M. Merrick, Journal of Bacteriology, 1971, 108,2, 782. 20. A.J. Anderson and E.A. Dawes, Microbiological Reviews, 1990,54, 1,450. 21. R.J. Griebel, Z. Smith and J.M. Merrick, Biochemistry, 1968, 7, 10, 3676. 22. J.M. Merrick, R. Steger and D. Dombroski, lnternational Journal of Biological Macromolecules, 1999,25, 1-3, 129. 23. R. Handrick, U. Technow, T. Reichart, S. Reinhardt, T. Sander and D. Jendrossek, FEMS Microbiology Letters, 2004,230,2,265. 24. R. Handrick, S. Reinhardt, P. Kimmig and D. Jendrossek, Journal of Bacteriology, 2004,186,21, 7243. 25. R. Handrick, S. Reinhardt, D. Schultheiss, T. Reichart, D. Schuler, V. Jendrossek and D. Jendrossek, Journal of Bacteriology, 2004,186, 8,2466. 26. D. Jendrossek and R. Handrick, Annual Review of Microbiology, 2002, 56, 403. 27. T. Saito, K. Takizawa and H. Saegusa, Canadian Journal of Microbiology, 1995, 41,2, 187.

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Practical Guide to Microbial Polyhydroxyalkanoates 28. H. Saegusa, M. Shiraki, C. Kanai and T. Saito,]ournal of Bacteriology, 2001, 183, 1, 94. 29. T. Kobayashi and T. Saito, Journal of Bioscience and Bioengineering, 2003, 96,5,487. 30. D. Gao, A. Maehara, T. Yamane and S. Ueda, FEMS Microbiology Letters, 2001,196,2,159. 31. G.M. York, J. Lupberger, J. Tian, A.G. Lawrence, J. Stubbe and A.J. Sinskey, Journal of Bacteriology, 2003,185,13,3788. 32. A. Pohlmann, W.F. Fricke, F. Reinecke, B. Kusian, H. Liesegang, R. Cramm, T. Eitinger, C. Ewering, M. Potter, E. Schwartz, A. Strittmatter, I. Voss, G. Gottschalk, A. Steinbuchel, B. Friedrich and B. Bowien, Nature Biotechnology, 2006,24, 10, 1257. 33. E. Schwartz, A. Henne, R. Cramm, T. Eitinger, B. Friedrich and G. Gottschalk, Journal of Molecular Biology, 2003,332,2,369. 34. T. Abe, T. Kobayashi and T. Saito,Journal of Bacteriology, 2005, 187,20, 6982. 35. D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H.D. Kratzin, T. Stanislawski and H.G. Schlegel, Journal of Bacteriology, 1995, 177,3,596. 36. T. Shinohe, M. Nojiri, T. Saito, T. Stanislawski and D. Jendrossek, FEMS Microbiology Letters, 1996, 141, 1, 103. 37. C.L. Tseng, H.J. Chen and G.C. Shaw, Journal of Bacteriology, 2006,188, 21,7592. 38. F.P. Delafield, K.E. Cooksey and M. Doudoroff, Journal of Biological Chemistry, 1965,240, 10,4023. 39. T. Kobayashi, K. Uchino, T. Abe, Y. Yamazaki and T. Saito,]ournal of Bacteriology, 2005, 187, 15, 5129. 40. J.M. Merrick and C.I. Yu, Biochemistry, 1966, 5, 11, 3563. 41. A. Sugiyama, M. Shiraki, T. Kobayashi, G. Morikawa, M. Yamamoto, M. Yamaoka and T. Saito, Current Microbiology, 2002,45,2, 123. 42. Y. Tanaka, T. Saito, T. Fukui, T. Tanio and K. Tomita, European Journal of Biochemistry, 1981, 118, 1, 177. 94

Intracellular degradation (mobilisation) of Polyhydroxyalkanoates (PHA) 43. K. Zhang, M. Shiraki and T. Saito,Journal of Bacteriology, 1997, 179, 1, 72. 44. T. Kobayashi, M. Shiraki, T. Abe, A. Sugiyama and T. Saito, Journal of Bacteriology, 2003, 185, 12, 3485. 45. H. Saegusa, M. Shiraki and T. Saito,Journal of Bioscience and Bioengineering, 2002,94,2, 106. 46. L.J.R. Foster, E.S. Stuart, A. Tehrani, R.W. Lenz, R.C. Fuller, International Journal of Biological Macromolecules, 1996, 19, 3, 177. 47. E.S. Stuart, L.J.R. Foster, W. Lenz and R.C. Fuller, ZnternationalJournal of Biological Macromolecules, 1996,19, 3, 171. 48. L.J.R. Foster, R.W. Lenz and R.C. Fuller, FEMS Microbiology Letters, 1994, 118, 3,279. 49. G.W. Huisman, E. Wonink, R. Meima, B. Kazemier, P. Terpstra and B. Witholt,Journal of Biological Chemistry, 1991,266,2191.

50. A. Timm and A. Steinbiichel, European Journal of Biochemistry, 1992,209, 1, 15. 51. H. Matsusaki, S. Manji, K. Taguchi, M. Kato, T. Fukui and Y. Doi,Jou.mal of Bacteriology, 1998, 180,24,6459. 52. B. Garcia, E.R. Olivera, B. Minambres, M. Fernandez-Valverde, L.M. Canedo, M.A. Prieto, J.L. Garcia, M. Martinez and J.M. Luengo, Journal of Biological Chemistry, 1999, 274,29228. 53. D.K.Y. Solaiman, Biotechnology Letters, 2000, 22, 9, 789.

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9

Extracellular Degradation of Polyhydroxyalkanoates (PHA)

PHA are solid polymers with a high molecular weight and cannot be transported into microorganisms through their cell wall. Therefore, several microorganisms such as bacteria and fungi excrete extracellular PHA-degrading enzymes to hydrolyse solid PHA into water-soluble oligomers and monomers, and then utilise the resulting products as nutrients within cells. Aerobic and anaerobic PHA-degrading microorganisms were isolated from various ecosystems such as soil, compost, aerobic and anaerobic sewage sludge, fresh and marine water, estuarine sediment and air [ l , 2-1 81. Thus, PHA-degrading microorganisms are present in nearly all terrestrial and aquatic ecosystems.

9.1 Effect of Environmental Conditions on the Degradation of PHA The biodegradability of PHA materials in natural environments has been evaluated by monitoring the properties of samples (sample dimension, molecular weight, mechanical strength) [ 13,19-221. To evaluate the biodegradability of PHA materials and the utilisability of degradable products, the modified Medical Interpreter Test (MITI), the Organisation for Economic Cooperation and Development (OECD) guideline for testing of chemicals number 301C [23], has been carried out in aquatic environments [ 19,24-261. The time-dependent changes in the biochemical oxygen demand (BOD), weight loss (erosion) of polyester sample, and dissolved organic carbon (DOC) concentration of test solution were measured to confirm that PHA materials are degraded completely in aquatic environments under aerobic conditions, and that oligomers of hydroxyalkanoate (HA) units from PHA are metabolised by microorganisms in the environment. The biodegradation of PHA materials is dependent upon many factors, notably those related to the environmental conditions (temperature, moisture level, pH, nutrient supply) and those related to the PHA materials (composition, crystallinity, additives, surface area). The environmental conditions strongly influence the growth of each of the PHA-degrading microorganisms, the production and secretion of PHA depolymerases, and the degradation activity of the enzymes.

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PHA-degrading microorganisms differ with respect to the type of polyester they can degrade. Most of the characterised microorganisms are specific for poly(3hydroxybutyrate) (P(3HB))and similar short-chain-length-PHA (scl-PHA)or for mclPHA such as poly(3-hydroxyoctanoate) (P(3HO)). However, some bacteria revealed a broad polyester specificity and could utilise a large variety of polymers, including scl-PHA and mcl-PHA [27,28]. A Xanthomonas-like bacterium which can degrade PHA with aromatic side chains [19,30] and a Comamonas sp. strain with scl-PHAand mcl-PHA-degrading capability [30] have been discovered. In general, production of PHA depolymerases in bacteria is repressed if suitable soluble carbon sources such as glucose or organic acids are present. However, after exhaustion of the soluble nutrients, synthesis of PHA depolymerases is not repressed in many strains [5]. At least in some bacteria, PHA depolymerase is expressed even in the absence of the polymer after cessation of growth, indicating that an induction mechanism by the polymer is not necessary. In most known PHA-degrading bacteria, high levels of PHA depolymerase are produced only during growth on PHA, but are repressed on succinate. In contrast to other PHA-degrading bacteria, production of scl-PHA depolymerases by Pseudomonas lemoignei is maximal during growth in batch culture on succinate. Therefore, isolation of scl-PHA depolymerases from l? lemoignei is usually from succinate-grown cells [l,31, 321. Synthesis of P(3HB) depolymerase on succinate is pH-dependent and occurs only above pH 7 [33]. Recently, the relationship between depolymerase synthesis and growth onhptake of succinate has been elucidated [34]. It was shown that transport of succinate into the bacteria is pH-dependent and does not work well at pH >7 in l? lemoignei. As a consequence, the bacteria starve even in the presence of residual succinate at pH > 7, and depolymerase synthesis is not repressed. Regulation of mcl-PHA depolymerase synthesis is apparently similar to that of scl-PHA depolymerases. High levels of P(3HO) depolymering activity were found during the growth of Pseudomonas fluorescens GK13 on mcl-PHA and on low concentrations of mcl-(R)-3HA monomers. The presence of sugars or fatty acids repressed synthesis of mcl-PHA depolymerase [35]. Similar results have been reported for a mcl-PHA depolymerase of Pseudomonas maculicola [36J.

9.2 Structure and Properties of PHA-degrading Enzymes Many extracellular PHA depolymerases have been purified from different microorganisms and/or characterised [5, 14, 15, 18, 37, 381. The purified PHA depolymerases consisted of a single polypeptide chain, and their molecular weights were in the range of 26,000-63,000 [6, 8 , 3 9 - 461. PHA depolymerases have high stability at a wide range of pH, temperature, and ionic strength, and the most of the 98

Extracellular Degradation of Polyhydroxyalkanoates (PHA) enzymes have optimum pH at alkaline (7.5-9.8) conditions. Most PHA depolymerases are inhibited by reducing agents (e.g., dithioerythritol (DTT)),which indicates the presence of essential disulfide bonds, and by serine hydrolase inhibitors such as diisopropylfluorylphosphate (DFP) or acylsulfonyl derivatives. The latter compounds bind covalently to the active site of serine hydrolases and irreversibly inhibit enzyme activity. PHA depolymerases have a pronounced affinity to hydrophobic materials. Most PHA-degrading bacteria apparently contain only one PHA depolymerase. P. lemoignei is unique among PHA-degrading bacteria because it can synthesise at least six extracellular depolymerases [47]. All purified PHA depolymerases are specific for scl-PHA or mcl-PHA. Even a P(3HB) depolymerase of Streptomyces exfoliates K10, a strain that degrades P(3HB) and P(3HO),is specific for scl-PHA, indicating at least one additional depolymerase with specificity for mcl-PHA in S. exfoliates [43]. Evidence for the presence of two distinct PHA depolymerases specific for hydrolysis of scl-PHA or mcl-PHA has been reported recently for a Comamonas sp. [30]. Several extracellular bacterial PHA depolymerase genes (phaZ)have been cloned and analysed. Except for the PHA depolymerase gene of S. exfoliates [43] and Streptomyces hygroscopius var. ascornyceticus [48], all other genes were cloned from Gramnegative bacteria: Acidovorax sp. [49], Alcaligenes faecalis AE122 [50],Comamonas acidovorans [42], Comamonas testosteroni [46], Comamonas sp. [41], Leptothrix sp. strain HS [Sl],Pseudomonas fluorescens [28, 521, P. lemoignei [6, 39, 40, 531, Pseudomonas stutzeri [ 16, 381, Ralstonia pickettii [45,54, 551, and Marinobacter sp. [56]. More than 20 genes coded for depolymerases with specificity for only sclPHA, especially P(3HB). Some of these depolymerase proteins have significant activity with P(3HV) homopolymer (especially PhaZ6 from P. lemoignei [53]), but none of the scl-PHA depolymerases have significant activity on mcl-P(3HA). The mcl-PHA depolymerases from several pseudomonads [35,36,52,57-621 are specific for P(3HO) and related mcl-P(3HA). The mcl-PHA depolymerase is completely inactive with P(3HB), but hydrolyses p-nitrophenylesters of fatty acids with six or more carbon atoms. PHA depolymerases are therefore considered to be highly specific with respect to the length of the carbon side chain of the PHA substrate.

9.2.1 Short-chain-length PHA Depolymerase Analyses of the structural genes of extracellular scl-PHA depolymerases (P(3HB) depolymerases) have shown that most enzymes comprise a N-terminal catalytic domain, a C-terminal putative substrate-binding domain, and a linker region connecting the two domains (Figure 9.1). The presence of catalytic and binding domains has been found in many depolymerising enzymes such as cellulase [63], xylanase [63,64], and chitinase [651, which hydrolyse water-insoluble polysaccharides.

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Practical Guide to Microbial Polyhydroxyalkanoates

P. lemvignei I'haZ I (PHVdapolymcrasc)

I

P. kmoignei Pha7.2

I

TY Pe I

117

I I I7

I 117

P. lemoignei PhaZ3

t', leinvignei Pha24 (PHV dKpOlymKraSK) P. lenioignei PhaZ5 P. lemvignei PhaZ6

I

I I37

1

I I38

1 I

I I36

I 137

R. pickafii TI

R.pickeitii

I I

I 140

I 131

S. h.ygroscopicu5 A. faecc~lisAE 122

1 I

-V

I 119

V

I

Z

Z

12s

Marinobactcrr sp. N K- 1 I25

P. sluizeri 20

Acidovorax sp.

Type T I

II ZQ

Cnmnmonns sp. 20

C.acidvvvrans

I I 20

I 20

Lepioihrix sp.

II 24

Cudherin-like ccgioii Fi bronrctin type 111 mgiun

Ttuconinc-rich

Linker region Strbshttte-binding domain

region

Figure 9.1 Domain structure and subclasses of extracellular P(3HB) depolymerases from various bacteria

100

Extracellular Degradation of Polybydroxyalkanoates (PHA) The catalytic domain contains a lipase box pentapeptide [Gly-X,-Ser-X,-Gly] as an active site which is present in almost all known serine hydrolases such as lipases, esterases, and serine-proteases [66-681. In contrast to most bacterial lipases, in which X, of the lipase-box is a histidine, a leucine is found in all scl-PHA depolymerases. The active site of serine forms a catalytic triad with an aspartate and a histidine, and these three amino acids are strictly conserved in all P(3HB) depolymerases (Table 9.1).

As shown in Figure 9.1, extracellular P(3HB) depolymerases are classified into two types according to the difference in the position of the lipase box in the catalytic domain. Type-I enzymes, in which a lipase box is located in the centre of catalytic domain, are represented by A. faeculis AE122, R . Picketti T1 (formerly A . fueculis Tl), P. lemoignei PhaZl, P. lemoignei PhaZ2, l? lemoignei PhaZ3, P. lernoignei PhaZ4, P. lemoignei PhaZ5, P. lemoignei PhaZ6, P. stutzeri, and Marinobacter sp. Type-I1 enzymes, in which a lipase box is adjacent to N-termini, are produced from Acidovorax sp., Comamonas sp., C. acidovorans, C. testosteroni, Leptotbrix sp., and S. exfoliatus. The deletion of about 60 amino acids at the C-terminal domain caused the P(3HB) depolymerase to lose its hydrolysing activity towards the water-insoluble P(3HB). Nevertheless, the truncated enzyme retained the activity towards water-soluble ( R ) 3HB oligomers and artificial water-soluble substrates such as p-nitrophenylesters [54, 69-72]. Based on these observations, it was suggested that the C-terminal domain acts as a substrate-binding domain for water-insoluble P(3HB) substrate. In addition, a fusion protein of the putative substrate-binding domain of P(3HB) depolymerase with glutathione S-transferase or maltose-binding protein demonstrated that the substratebinding domain moiety is essential for the adsorption of P( 3HB) depolymerase toward the surface of P(3HB) granules [42, 70-733. Recently, three P( 3HB) depolymerase sequences, namely those of A. fuecalis AE122 [50], P. stutzeri [38], and Marinobacter sp. [56], were shown to contain two instead of one P(3HB)-binding domain. It has recently been proposed that the substrate-binding domain has an additional and more active function of disrupting the structure of the polymer.

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Practical Guide to Microbial Polyhydroxyalkanoates

P. bmoignei

102

PhaZ7

136 TCKSQVDIVAH SMGVSMS 242 LSAGFK DQVG

306 GVGHFRTK TNT

Extracellular Degradation of Polyhydroxyalkanoates (PHA) The linker regions connecting catalytic and substrate-binding domains have been found in P(3HB) depolymerases, and show a fibronectin type-I11 (Fn3) module, a threonine-rich region, or a cadherin-like domain [39,40,54,72,74]. Cell-membrane proteins such as fibronectins and cadherins have been found in a linker region of waterinsoluble polymer hydrolases such as cellulases, chitinases and several glucoamylases [63,65,75,76]. The Fn3 domain of the P(3HB) depolymerase from R. pickettii T1 was essential for depolymerase activity because deletion of the Fn3 domain resulted in a protein which had absent P(3HB) depolymerase activity, but not 3HB dimer hydrolase activity [54]. Interestingly, the Fn3 domain of the depolymerase from R. pickettii T1 could be functionally replaced by a threonine-rich region of the P(3HB) depolymerase (PhaZ5)of P. lemoignei. A recent study of a homologous protein to the linker domain, the Fn3 homology domain of cellobiohydrolase ChbA of Clostridium thermocellum, indicates that the linker domain may also exhibit a disruptive function against crystalline substrates, analogous to the substrate-binding domain [77]. The role of the linker domain is incompletely understood, but it has been suggested that the linker regions may have a structural role in maintaining an optimal distance between the catalytic domain and substrate-binding domain. Each of the catalytic domains and substrate-binding domains function independently, but these two functional domains and linker region are essential for the enzymatic degradation of water-insoluble polymers. Recently, an extracellular P(3HB) depolymerase from the fungus Penicillium funiculosum was purified and characterised [3,78]. It has a molecular mass of 33 kDa, and is smaller than the other extracellular depolymerases (-50 kDa). It efficiently degrades P(3HB) and a trimer of (R)-3HB, although the degradation of P(3HB) is less efficient by two orders of magnitude than a multidomain enzyme from R . pickettii T1. Furthermore, when the enzyme is inactivated by the inhibitor of diisopropylfluorylphosphate (DFP), it retains the ability to bind to P(3HB). A sequence analysis indicates that the enzyme can be classified as having a type-I1 catalytic domain [79]. Three amino acids constituting a catalytic triad are conserved, and the lipase-box pentapeptide Gly-Leu-Ser-Ser-Glyis present. However, the amino acid sequences corresponding to the substrate-binding domain and linker region homologues are absent. Furthermore, the three-dimensional structural analysis indicates that the enzyme comprised a single domain, and suggests that 13 hydrophobic residues around the active site induce the enzyme to bind onto polymer chains without a substrate-binding domain [79]. A similar extracellular P(3HB) depolymerase composed of a single peptide with relatively low molecular mass of 35 kDa has been isolated from the fungus Penicillium pinophilum [80]. Although all known data on extracellular PHA depolymerases show that these enzymes are inactive towards rubbery amorphous polyesters such as native amorphous PHA granules and atactic P(3HB), unexpectedly high levels of the novel extracellular enzymatic activity of P. lemoignei that hydrolysed native scl-PHA granules and atactic

103

Practical Guide to Microbial Polyhydroxyalkanoates P(3HB)were detected [Sl]. The purified depolymerase (PhaZ7, M r = 36 kDa) is active on native P(3HB) granules and stable at high temperatures (60 "C) and at alkaline pH (9-12). The depolymerase has extremely low activities with p-nitrophenylesters, with a relative maximum for p-nitrophenyloctanoate, and is completely inactive with any crystalline PHA. Analyses of the DNA-deduced amino-acid sequence of the extracellular P(3HB) depolymerase PhaZ7 of P. lemoignei showed no sequence similarity to any P(3HB) depolymerase. Only weak similarities to lipase LipB of Bacillus subtilis and to other lipases of Bacillus sp. were found. PhaZ7 is different from known PHA depolymerases, but apparently is a member of the serine hydrolase family, having a catalytic triad of Ser, Asp, His [82]. While there are many, the three-dimensional structure has been obtained only for the enzyme from P . funiculosum [79]. The shape of the molecule is globular with approximate dimensions of 5.2 nm x 4.8 nm x 4.1 nm, showing that the enzyme comprises a single domain (Figure 9.2). The enzyme represents alp-type structure. It consists of an eight-stranded P-sheet composed of seven parallel and one anti-parallel P-strands, nine a-helices surrounding the P-sheet, two concomitant two-stranded antiparallel P-sheets, and six 3,,-helices (Figure 9.3). The topology of the secondary structure resembles that of the a l p hydrolase fold (Figure 9.3) [83]. A remarkable feature is that its polypeptide linkage is circularly permuted [84] in comparison with that of the a l p hydrolase fold. That is, the positions of the N and C termini of the depolymerase are different from those of the canonical a l p hydrolase fold. From the sequence analysis, it is found that the enzyme can be classified as a type-I1 enzyme. In the type-I1 depolymerase structure, the location of the nucleophilic serine residue in the lipase box pentapeptide at the N-terminal region of the protein results in discontinuity of the polypeptide linkage in the middle of the central P-sheet, whereas the polypeptide linkage is continued from one edge to the other edge of the P-sheet in the canonical a l p hydrolase fold. This finding reveals an interesting relationship between the two types of catalytic domains, which are not apparently similar, with respect to the primary structure. Comparison of the amino-acid sequences of the type-I enzymes with a modified sequence of P. funiculosum enzyme in which the circular permutation is cancelled show about a 28% homology, Thus, type-I and -11 enzymes are related by an unexpected evolutionary relationship. That is, the type-I1 depolymerase could have been derived from the type-I enzyme, probably by gene duplication. Therefore, it is very likely that the structure of the type-I catalytic domain resembles that of the P. funiculosum enzyme, and exhibits a l p hydrolase folding.

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Extracellular Degradation of Polyhydroxyalkanoates (PHA)

N

Figure 9.2 Ribbon plot of chain folding for P(3HB) depolymerase (type 11) from P. funiculosum 2791 The enzymatic hydrolysis experiments using water-soluble (R)-3HB oligomers provide significant information regarding substrate recognition of depolymerase. Hydrolysis of end-labelled (R)-3HB oligomers by the P(3HB) depolymerase from R. pickettii T1 showed that the enzyme mainly cleaved the second and third ester linkages from hydroxyl terminus [ 8 5 , 371. As a result, the enzymatic hydrolysis of P(3HB) by the P(3HB) depolymerase from R. pickettii T1 produces (R)-3HB dimer as a major product, and only a small amount of (R)-3HB monomer is produced. However, the enzyme also hydrolyses cyclic oligomers, so the P(3HB) depolymerase from R. pickettii T1 has endo-hydrolase activity in addition to exo-hydrolase activity [37, 861. The endo-hydrolase activity of P(3HB) depolymerase was confirmed by Brand1 and co-workers [87] from the results of a hydrolysis of cyclic oligomers with culture supernatant of Acidovorax delafieldii. Therefore, it is assumed that most extracellular P(3HB) depolymerases have endo-hydrolase activity in addition to exohydrolase activity. The hydrolysis products of depolymerases can consist of either only monomers, monomers and dimers, or a mixture of oligomers, depending on the original polymer. A study on the enzymatic hydrolysis of 3HB oligomers by the P(3HB) depolymerase from R. pickettii T1 demonstrated that the active site of depolymerase

105

Practical Guide to Microbial Polyhydroxyalkanoates recognises the sequential four monomeric units as substrate for the hydrolysis of ester bonds in a polymer chain [86]. Similarly, the P(3HB) depolymerase from P. stutzeri recognises the sequential plural monomeric units as substrate, whereas the hydrolysis reaction yields the (R)-3HBmonomer as a major product [26]. Such difference in the hydrolysis products among the depolymerases may be dependent on the oligomer- or dimer-hydrolase activities of the enzyme rather than substrate recognition.

Figure 9.3 Topology diagrams of (A) P(3HB) depolymerase (type 11) from I? funicdosum, and (B) the canonical cr/p hydrolase fold. Boxes and arrows represent a-helices and P-strands, respectively [79]

106

Extracellular Degradation of Polyhydroxyalkanoates (PHA) The enzymatic degradation rate of a solid P(3HB) can be determined by monitoring the changes in turbidity of a solution-suspended polymer granule, in the weight of films, and in the amounts of liberated water-soluble monomers and oligomers, as a function of reaction time. By using each detection system, it has been found that the enzymatic degradation rate of P(3HB) is strongly dependent upon the concentration of enzyme. The degradation rate increases to a maximum value with the concentration of P(3HB) depolymerase, followed by a gradual decrease, as shown in Figure 9.4 [15,88-901. A solid P(3HB) polymer is a water-insoluble substrate, whereas P(3HB) depolymerases are soluble in water, so the enzymatic degradation of P(3HB) material by P(3HB) depolymerase is a heterogeneous reaction. This reaction involves two steps: adsorption and hydrolysis. The first step is adsorption of the enzyme on the surface of P(3HB) material by the binding domain of enzyme. The second step is a hydrolysis of polymer chains by the active site of the enzyme. Accordingly, the kinetic behaviour of P(3HB) hydrolysis has been accounted for in terms of a surface-enzymatic reaction: the hydrolysis of P(3HB) chains takes place by the surface reaction between adsorbed enzymes and free adsorption points on the surface [15, 16, 89, 901. From kinetic analyses of the adsorption of the enzyme on the surface of P(3HB), it has been confirmed that the adsorption isotherms of P(3HB)depolymerase are expressed by the Langmuir adsorption equation [91]. It has been concluded that, at low concentrations of P(3HB)depolymerase, most catalytic domains of the adsorbed enzyme can hydrolyse P(3HB) chains on the surface. However at high concentrations of enzyme, most catalytic domains are not accessible to P(3HB)chains on the surface due to condensed coverage of the substrate-binding domains on the surface of P(3HB). Kasuya and co-workers investigated the kinetics and mechanism of surface hydrolysis of P(3HB) with P(3HB) depolymerase from R. pickettii T1 at different reaction temperatures and pH [89]. The rate constant of enzymatic hydrolysis increased with a rise in temperature, whereas the adsorption equilibrium constant decreased. From the obtained results, the activation energy of the hydrolysis by catalytic domain was found to be 82 kJ/mol. The heat of enzyme adsorption on the P(3HB) surface was 43 kJ/ mol, indicating a strong interaction between the enzyme and the polymer surface.

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Practical Guide to Microbial Polyhydroxyalkanoates

n

8

3.0

f a

7

2.5

6

n

2.0

cn 5 Eu

1.5

4

fn g

v

.-5

U

!

.-

I

E

m

5

I

3 c n

. I

1.0

e z. 0.5

2

s

1

0

a

d

c)

0

0

0

2

4

6

8

10

12

14

16

18

20

Concentration of P(3HB) depolymerase (pg1mI)

Figure 9.4 Effects of enzyme concentration on the rate of (R)-3HB unit liberation ( 0 ) and on weight loss ( ) of a film for 19 h in the enzymatic degradation of P(3HB) film by P(3HB) depolymerase from R. pickettii T1 [88]

Recently, Yamashita and co-workers determined the amount of enzyme molecules adsorbed on P(3HB) films using catalytically inactive mutant enzyme of R. pickettii T1 by the quartz crystal microbalance (QCM) technique [92]. They found that the adsorption isotherm of P(3HB)depolymerase with P(3HB)films could not be satisfied by any set of parameters for the Langmuir equation. Furthermore, they determined the changes in rate of enzymatic degradation of P(3HB) film by the replacement of the wild-type enzyme solution with a pure buffer solution or mutant enzyme solution. When the wild-type enzyme solution was replaced with the mutant enzyme solution in the middle of the reaction, the degradation rate was markedly reduced. Replacement with a pure buffer solution hardly affected the degradation rate. From these results, they suggested that each molecule of P(3HB) depolymerase is irreversibly adsorbed

108

Extracellular Degradation of Polyhydroxyalkanoates (PHA)

on the P(3HB)film surface, and that it is readily substituted by attack of the enzyme molecules in the solution. As a result, it seems that the apparent adsorption isotherms of P(3HB) depolymerase obey the Langmuir isotherm. As mentioned above, the P(3HB) depolymerase from P. funiculosum has a single domain structure lacking linker and substrate-binding domains, whereas the other enzymes contain three domains. It has been proposed that the P(3HB) depolymerase of P. funiculosum is adsorbed on the P(3HB) material by hydrophobic interaction through the hydrophobic amino-acid residues around the active site of the enzyme [79]. Numata and co-workers investigated the difference in the adsorption characteristics between two types of P(3HB) depolymerases with different domain structure by atomic force microscopy (AFM) and QCM measurements [93]. They demonstrated the irreversible adsorption characteristics of P(3HB)depolymerase from R. pickettii T1 on the P(3HB) crystals using AFM to visualise enzyme molecules. From the continuous AFM images of the reaction process, the enzyme molecule of P(3HB) depolymerase from R. pickettii T1 adsorbed on the P(3HB) single crystals remained at the same position during the observation. In contrast to the P(3HB) depolymerase from R. pickettii T1, visualising enzyme molecules from P. funiculosum in real-time AFM images was rarely successful. In addition, the enzyme molecule observed immediately disappeared from the surface of the single crystal during continuous imaging. When the enzyme molecules attached on the surface of P(3HB) single crystals were detected in the AFM images obtained in air after enzymatic treatment for P(3HB)depolymerases from I? funiculosum, the number of attached enzyme molecules of P. funiculosum was approximately the same with as that for R. pickettii T1. This result indicates that the P(3HB) depolymerase from P. funiculosum can adsorb onto the surface of P(3HB) single crystals. From the QCM measurement, it was found that replacement of the enzyme solution of P. funiculosum with a buffer solution induced a reduction of the degradation rate of P(3HB)film, whereas enzymatic erosion could be detected continuously. These results indicate that P(3HB) depolymerase from P. funiculosum adsorbs onto the P(3HB)surface to degrade P(3HB)molecules, whereas the adsorbed enzyme is readily released from P(3HB)under an aqueous condition. Such disparities in binding affinity of the two enzymes may be caused by the difference in domain structure, and is reflected in the adsorption characteristics of enzymes. The P(3HB) depolymerase from R. pickettii T1 is anchored to the substrate surface with the substrate-binding domain due to a strong affinity with P(3HB)molecules. The absence of a substrate-binding domain provides the reduction in binding affinity of the enzyme from l? funiculosum compared with the enzyme from R. pickettii T1, resulting in reversible adsorption and desorption of enzyme molecule. Thus, the presence of a substrate-binding domain of P(3HB)depolymerase is liable to interact with polyester chains for the catalytic domain.

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Practical Guide to Microbial Polyhydroxyalkanoates

9.2.2 Medium-chain-length PHA Depolymerase

The mcl-PHA depolymerases (P(3HO) depolymerases) from P. fluorescens GK13 [35, 521, Pseudomonas alcaligenes LB 19 [60] and M4-7 [59], and Pseudomonas luteola M13-4 [61,62] are known bacterial mcl-PHA depolymerases that have been purified and characterised. P(3HO) depolymerases differ from P(3HB) depolymerases in several ways, including structure and biochemical properties. This is specific for mcl-PHA and for artificial esters such as p-nitrophenylacylesters with six or more carbon atoms in the fatty-acid moiety. P(3HB) and other scl-PHA are not hydrolysed. The enzyme is not inhibited by DTT or by ethylenediamine tetra-acetic acid (EDTA) and therefore apparently does not contain essential disulfide bonds. The deduced amino-acid sequence of the cloned P(3HO) depolymerase genes does not exhibit significant homology to P(3HB) depolymerases, except for small regions in the neighborhood of a lipase-box, and aspartate and a histidine residues [28,59, 621. Therefore, the P(3HO) depolymerase apparently also belongs to the group of serine-hydrolases with a catalytic triad in the active centre. In contrast to all known P(3HB) depolymerases, the three amino acids of the putative active centre are located in the C-terminal region of the enzyme. In addition, an isoleucine residue is found as the XI of the lipase-box in P(3HO) depolymerase, instead of a leucine residue in all P(3HB) depolymerases [28,59,62]. From the gene analysis for the mutant enzymes of P. fluorescens GK13 which were impaired in P(3HO) depolymerase activity but still have almost normal levels of esterase activity with p-nitrophenyloctanoate, it has been found that most of the mutations were located in the N-terminal region of the mature depolymerase. Therefore, it is assumed that the N-terminal part of the protein constitutes a polymer-binding site, in particular, Leul5, Phe50, and Phe63 and neighbouring amino acids are involved in the interaction of the enzyme with its polymeric substrate [28]. Thus, the P(3HO) depolymerase differs considerably from P(3HB) depolymerases in terms of primary sequence and polymer-binding, though there is evidence that all PHA depolymerases cleave the polyesters by the same mechanism (catalytic triad). This may be due to different approaches of these enzymes to obtain access to the polymers, thereby reflecting the distinctive physicochemical properties of scl-PHA and mcl-PHA rather than co-evolution.

9.3 Effect of Chemical Structures on Enzymatic Degradability

9.3.1 Enantiomer Selectivity of PHA Depolymerase

Biological P(3HB) is composed of only the (R)-3HB unit. In contrast, chemosynthetic P(3HB) is prepared by the ring-opening polymerisation of P-butyrolactone (P-BL) 110

Extracellular Degradation of Poiyhydroxyalkanoates (PHA) and contains ( R ) - and (S)-3HB units. The stereocomposition and tacticity of chemosynthetic P(3HB) can be regulated by varying the feed ratio of (R)- and (S)P-BL and catalyst species. Several researchers have investigated the stereoselectivity of P(3HB) depolymerase for the hydrolysis of P(3HB) [94-1021. Chemosynthetic P(3HB) samples containing monomeric units of ( R ) -and (S)-3HB have been shown to be hydrolysed by P(3HB) depolymerases, whereas P(3HB) samples with a high (S)-3HB fraction (>92 mol%) were hardly hydrolysed by the P(3HB) depolymerases [96, 1011. The enzymatic hydrolysis of biological P(3HB) by P(3HB) depolymerases produces water-soluble products composed from monomer and/or dimer of (R)-3HB.In contrast, after enzymatic degradation, chemosynthetic P(3HB) samples gave 3HB oligomers as degradation products in addition to 3HB monomer and dimers, suggesting that the P(3HB) depolymerase barely hydrolyses the ester bonds connecting the (S)-3HBunits [95, 961. The rate of enzymatic erosion of chemosynthetic P(3HB) samples containing monomeric units of (R)-and (S)-3HBis strongly dependent on stereocomposition and tacticity. Bachmann and Seebach [86] investigated the stereoselectivity of P(3HB) depolymerase from R. pickettii T1 using the stereoisomers of 3HB oligomer. They demonstrated that the P(3HB) depolymerase hydrolyses only the ester bond between the sequential two (R)-3HB units.

9.3.2 Substrate Specificity of the Catalytic Domain The substrate specificity of P(3HB) depolymerases have been investigated using various PHA homopolymers [71,88]. Figure 9.5 shows the enzymatic degradabilities of 12 types of aliphatic polyester by three types of P(3HB) depolymerases. Though the rates of hydrolysis were different among the three P(3HB) depolymerases, all P(3HB) depolymerases hydrolysed the same kinds of films: P(3HB), P(3HP), P(4HB),poly(ethy1ene succinate), and poly(ethy1ene adipate) [71]. The enzymatically degradable five polyesters share some common chemical structures: the number of carbon and oxygen atoms between two carbonyl groups of backbone is 3 or 4, whereas the side chain is methyl carbon or hydrogen. Thus, P(3HB) depolymerases show relatively narrow substrate specificities for PHA hydrolysis.

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Practical Guide to Microbial Polyhydroxyalkanoates

P. slulzeri P(3HB) depolymerase (Type I) R. pickenii T1 P(3HB) depolymerase (Type I) C acidnvorans P(3HR) aepolymerase(Type It)

10-

P(6HHx)

{o-fi" 4-'& PESU

PEA

{o-+y

PBSU

( d " ' M o PBA> o ~ 0

0.05

0.10

0.15

0.20

0.25

0.30

Rate of erosion ( W c m l/h)

Figure 9.5 Rates of enzymatic erosion of 12 polyester films at 37 "C in aqueous solution (pH 7.4) containing P(3HB) depolymerases from I? stutzeri, R. pickettii T1, or C. acidovorans [71]

The effects of the chemical structure of second monomer units and copolymer compositions on the rate of enzymatic erosion have been examined through the enzymatic degradation of solution-cast films of random copolymers of (R)-3HBwith various HA units in the presence of P(3HB) depolymerase [103-1091. The enzymatic degradation of the solution-cast films of these PHA copolymers was done in aqueous solution of purified P(3HB) depolymerase from R. pickettii T1 at 37 "C. The rate of enzymatic erosion on the solution-cast PHA films increased markedly with an increase in the fraction of second monomer units up to 10-20 mol% to reach a maximum value followed by a decrease in the erosion rate (Figure 9.6).The highest rates of enzymatic erosion were 5- to 10-times higher than the rate of P(3HB) homopolymer film.

112

Extracellular Degradation of Polyhydroxyalkanoates (PHA)

0

20

40

60

80

100

Second monomer fraction (mot%) Figure 9.6 Relationship between the rate of enzymatic hydrolysis by P(3HB) depolymerase from R. pickettii T1 and the fraction of second monomer unit for random copolymers of (R)-3HBwith different HA units. (A):P(3HB-co-3HHx), (0): P(3HB-co-3HP), and (A): P(3HB-co-4HB)

The structures and compositions of water-soluble products of PHA copolymers during the enzymatic degradation by P(3HB) depolymerase from R. pickettii T1 have been characterised using high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) analyses [ 103, 106-1091. The water-soluble products of random copolymers by P(3HB) depolymerase showed a mixture of monomers and several oligomers of (R)-3HBand other HA units. The water-soluble degradation products of random copolymers have some common chemical structures: the carboxylterminus of water-soluble products was always monomeric units with a main-chain length of 3 or 4 carbon atoms, whereas the hydroxyl-terminus was always monomeric units with the side-chain length ranging from 0 to 2 carbon atoms. A schematic model for enzymatic hydrolysis of ester linkages in a polymer (PHA)chain by P(3HB) depolymerase from R. pickettii T1 has been proposed by Abe and co-workers [ 1071, as shown in Figure 9.7. The active site of the catalytic domain in P(3HB) depolymerase

113

Practical Guide to Microbial Polyhydroxyalkanoates recognises at least three monomeric units as a substrate. The rate of enzymatic hydrolysis of an ester bond may be strongly dependent on the chemical structure of the second and third monomeric units in a polyester chain. The catalytic site may bind the substrate by interacting with the carbonyl group in the hydroxyl-terminated monomer unit (unit 2) to assist the hydrolysis reaction of the second ester bond with the active site of the catalytic domain. The length of the main carbon chain (R,) of the second monomer unit (unit 1) may be limited to 3 or 4 carbon atoms because of the facile binding of substrate by the active site. The chemical structure (R,) of the third monomer unit (unit -1) may affect the accessibility of the ester bond to the active site of P(3HB)depolymerase. When the third monomeric unit has a sterically bulky side chain such as (R)-3HVand (R)-3HHxunits, P(3HB)depolymerase may barely attack the ester bond. Consequently, the hydrolysis rate of ester bonds by P(3HB) depolymerase decreases as the side-chain length of a HA unit increases.

9.3.3 Binding Specificity of the Substrate-binding Domain From adsorption studies using P(3HB) depolymerases and the fusion proteins of substrate-binding domains of P(3HB)depolymerases with glutathione S-transferase [42,46, 71-73], P(3HB) depolymerases can bind on the surfaces of enzymatically degradable and non-degradable polyesters, whereas they hardly bind to the semicrystalline polysaccharides of avicel or chitin [71]. These results suggest that there are some specific interactions based on molecular recognition between the substratebinding domain and the surface of polyesters, whereas the binding specificity of the substrate-binding domain is broad compared with the substrate specificity of an active site in the catalytic domain. Yamashita and co-workers studied the adsorption of P(3HB)depolymerase from R. pickettii T1 onto thin films of poly(L-lactic acid) (PLLA),poly(3-hydroxyoctanoate) (P(3HO)),polystyrene (PS), and polyethylene (PE), using QCM techniques [ l l l ] . They reported that the rate of adsorption of P(3HB) depolymerase decreases in the following order: PLLA >> P(3HO) > PS >> PE. It has been suggested that the binding domain has affinity not only for polyesters but also for PS, indicating that hydrophobic interaction partially contributes to the affinity of the binding domain. Indeed, the P(3HB)depolymerase adsorbs even on a surface of silicon wafers via a hydrophobic interaction, whereas the presence of surfactant inhibits the adsorption of enzyme on the surface of silicon wafers [112]. Many enzyme particles are detected on P(3HB) single crystals in the presence of surfactant, so it is suggested that the affinity of the binding domain of P(3HB) depolymerase onto P(3HB) single crystals is not only a hydrophobic interaction, but also another specific interaction.

114

Extracellular Degradation of Polyhydroxyalkanoates (PHA)

Active sequence structure Unit 2

G

v

0

0

RI rJ!

0

- O-RI-C-O-R~-C-O-R~-C-O-R~-C-O-

Unit 1

: (R)-3HB

-CHCHz-

, (R)-3HV

0

-CHCHZ- , (R)-3HHX -CHzCHz- : 3HP -CHzCHzCH2- :4HB

1

Catalytic (active) site Unit 2

= -CHCHz-

recognition

Unit -1

RJ 5

-CHCHZ- : (R)dHB

hydrolysis

0

-O-Ri-C-O-R2-C-O-R3-C 0

0

-RI-C-O-R~-C-OH - 0Carboxyi-terminus

0

0

-CHCHz- : (R)-BHV -CHzCHz- ; 3HP -CHzCHzCHa- ; 4HB

-0-R4-C-O-

0

+ HO-RI-C-O-RI-C-O-

9

Hydroxyl-terminus

Figure 9.7 A schematic model for the enzymatic hydrolysis of an ester bond in various sequences by P(3HB) depolymerase [107, 1101

Recently, the binding interaction of P(3HB) depolymerase from R. pickettii T1 on P(3HB) and PLLA thin films was investigated by AFM [113]. The adhesive forces between polyester substrates and the substrate-binding domain of P(3HB) depolymerase using AFM cantilever tips immobilised with the su bstrate-binding measured; the adhesive force of a single molecule of the substrate-binding domain was estimated to be about 100 pN for P(3HB) and PLLA [113]. Furthermore, it has been suggested that the P(3HB) depolymerase interacts specifically with polyester surfaces by electrostatic interaction and hydrogen bonding; in addition to hydrophobic interaction, some amino-acid residues of the substrate-binding domain take part in the adsorption reaction. In summary to this section, the substrate-binding domain of P(3HB) depolymerase interacts with polyester molecules via various affinities by several amino-acid residues, and hence the chain-folding surface of lamellar crystals is disordered and deformed by adsorption of enzyme molecules.

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Practical Guide to Microbial Polyhydroxyalkanoates

9.4 Effect of Solid-state Structures on Enzymatic Degradability P(3HB) is a semi-crystalline thermoplastic, and can be processed by conventional extrusion and moulding equipment. The melt-crystallised films of P(3HB) exhibit large-banded spherulites, and the spherulitic morphology and degree of crystallinity are dependent on crystallisation conditions (see Section 6.1). The rate of enzymatic hydrolysis for melt-crystallised P(3HB) film by P( 3HB) depolymerase from R. pickettii T1 decreased with an increase in the crystallinity of P(3HB) film, whereas the size of spherulites hardly affected the rate of hydrolysis [114]. In addition, it was demonstrated that the rate of enzymatic degradation for P(3HB) chains in an amorphous state was approximately 20-30-times higher than the rate for P(3HB) chains in a crystalline state [ 1141. As a result, the rate of biodegradability of P(3HB) materials can be regulated by varying the degree of crystallinity. The size of crystal for melt-crystallised P(3HB)films also varies with the crystallisation conditions. Tomasi and co-workers [ 1151 prepared melt-crystallised P(3HB) films with different crystal size, and examined the rate of enzymatic hydrolysis by P(3HB) depolymerase from P. lemoignei. They reported that the rate of enzymatic erosion of melt-crystallised P(3HB)films by the P(3HB) depolymerase decreased with an increase in the average size of P(3HB) crystals. Koyama and Doi [ 1161 prepared melt-crystallised films of various random copolymers of (R)-3HBwith different HA units, and studied the rate of enzymatic erosion on the surface of films by P(3HB)depolymerase from R. pickettii T1 (Figure 9.8).The erosion rates for P(3HB-co-3HV) films were several times higher than the rates of P(3HB) homopolymer films with the same degree of crystallinity. As a result, the significant difference in the erosion rates for melt-crystallised films of P(3HB) homopolymer and P(3HB-co-3HV) copolymers could not be explained only in terms of degree of crystallinity and the average size of crystal.

116

Practical Guide to Microbial Polyhydroxyalkanoates The thickness of crystalline lamellae for melt-crystallised films also varied with crystallisation conditions. The rates of enzymatic degradation for melt-crystallised films of PHA copolymers with different crystallinity and different lamellar thickness have been investigated by Abe and co-workers [ 1171. The rate of enzymatic erosion of melt-crystallised copolyester films was significantly decreased as the degree of crystallinity increased (Figure 9.8). It is suggested that the P(3HB) depolymerase predominantly hydrolyses polymer chains in an amorphous phase and subsequently eroded crystalline phase. In addition, the enzymatic erosion rate of crystalline phase in polyester films was determined from overall erosion rate and crystallinity. The enzymatic erosion rate of crystalline phase in polyester films decreased with an increase in the lamellar thickness (Figure 9.9).

3 nm

1

10

Lamellar thickness (nm) Figure 9.9 Relationship between the erosion rate of the crystalline phase and lamellar thickness (1J. (0):P(3HB), ( 0 ) :P(3HB-co-6mol% 3HV), (0): P(3HB-co16 mol% 3HV), ( A ) : P(3HB-co-6mol% 3HHx), ( 0 ) :P(3HB-co-8 mol% 3HHx), (V):P(3HB-co-8 mol% 4HB), (V): P(3HB-co-10mol% 4HB), and (A): P(3HBco-6 mol% mcl-3HA)

118

Extracellular Degradation of Polyhydroxyalkanoates (PHA) From scanning electron microscopic observations, the surface of melt-crystallised copolyester film after enzymatic degradation was apparently blemished by the action of P(3HB) depolymerase, and the ringed texture of spherulites detected. Two types of planes can be observed on the surface of PHA spherulites after enzymatic degradation; the smooth and rough planes which coexist alternately along the radial direction of the spherulite [ 1171. To elucidate the mechanism of enzymatic degradation in the crystalline region for P(3HB) by P(3HB) depolymerase, the enzymatic degradation of single crystals of P(3HB) with P(3HB) depolymerases from bacteria and fungi have been studied [118-1221. Marchessault and co-workers first carried out the enzymatic degradation of P(3HB) single crystals by PHA depolymerase from P. lernoignei. Hocking and co-workers carried out enzymatic degradation of P(3HB) single crystals by PHA depolymerase using turbidimetric and titrimetric assays and changes in molecular weight of polymers. Nobes and co-workers found that the PHA single crystals were converted into needlelike splinters after enzymatic degradation according to transmission electron microscopy (TEM). From these results, the Marchessault research group proposed that the single crystals were enzymatically hydrolysed preferentially at the crystal edges (ac plane) and ends (bc plane) rather than the chain-folding surfaces (ab plane) of single crystals because the lamellar thicknesses of single crystals and the molecular weight of P(3HB) chains remain unchanged during enzymatic degradation. Many narrow cracks and the small crystal fragments along the crystal long axis corresponding to the crystallographic a-axis from P(3HB) single crystals were produced by the enzymatic reaction. Subsequent to the publications by the Marchessault research group, Iwata and co-workers published a series of articles on the enzymatic degradation of P(3HB) and its copolymer single crystals [119, 123-1261. The P(3HB) and its copolymer single crystals partially degraded by PHA depolymerases from P. stutzeri and R . pickettii T1 were characterised using TEM, AFM, GPC, and immunogold labelling techniques. They confirmed that enzymatic degradation of single crystals occurred from the crystal edges and ends, generating a needlelike morphology, independent of the types of P(3HB) depolymerases. In addition, it was demonstrated that the adsorption of P(3HB) depolymerase showed a homogeneous distribution on the surfaces of single crystals. The binding of P(3HB) depolymerase on the edge of P(3HB) lamellar crystals may cause an increase in the mobility of polyester chains along the crystal edge, resulting in the formation of disordered P(3HB) chains like polymer chains in the amorphous phase which are facilely atracked by the active site of enzyme, as illustrated in Figure 9.10. In the cases of single crystals for the P(3HB)-basedcopolymers P(~ H B - c o - ~ H VP() ~HB-co-~HHx), , P(3HB-co-4HB), and P(~HB-co-~HH), the enzymatic degradation mechanism is the same as in the case of P(3HB) single crystals [119, 1231.

119

Practical Guide to Microbial Polyhydroxyalkanoates

Adsorption on ciystal snrfacc by binding Joniain

Hydrolysk 0 1 single cryat4

by catalytic dntnilitr

Figure 9.10 A schematic model of enzymatic hydrolysis of P(3HB)single crystals by P(3HB)depolymerase consisting of binding and catalytic domains Similar morphological changes in lamellar crystals by an enzymatic reaction have been confirmed for the flat-on lamellar crystals formed in melt-crystallised thin films of P(3HB)and its copolymers [127, 128-1311. After enzymatic degradation on thin film by a PHA depolymerase, the jagged texture along the crystal long axis could be observed at the ends of crystalline lamellae on the surface of the thin film. It is predicted that the polyester chains exposed at the edges and ends of relatively thin crystalline lamellae may form disordered polyester chains more easily than those at the edges of relatively thicker crystalline lamellae. Consequently, the erosion rate of the crystalline phase in melt-crystallised films may increase with a decrease in lamellar thickness, as described above. In addition, lamellar stacks on the surface of melt-crystallised films have also been hydrolysed preferentially at the crystal edge by P(3HB) depolymerase rather than the chain-folding surface. Such selective hydrolysis at the edge of lamellar crystals by P(3HB) depolymerase leads to formation of an alternating smooth (chain-folding surface) and rough (crystal edge) ringed texture on the surface of melt-crystallised copolyester film after enzymatic degradation.

120

Extracellular Degradation of Polyhydroxyalkanoates (PHA) Although the enzymatic degradation characteristics of P(3HB)-based copolymer single crystals is the same as that of P(3HB) single crystals, the needlelike morphology generated by enzymatic degradation among the P(3HB) and copolymers is varied [132, 1331. Numata and co-workers carried out direct imaging of the morphological changes in lamellar crystals during enzymatic degradation by real-time AFM to quantitatively estimate the erosion rates along the a- and 6-axis of crystals for P(3HB) and P(3HBco-3HV) (Figure 9.11) [131,133].

Figure 9.11 Real-time AFM height images of P(3HB) single crystals before (a) and during enzymatic degradation by the P(3HB) depolymerase from R. pickettii T1 at 37 "C fore 20 min (b), and 43 min (c) [112] Grooves were formed along the crystallographic a-axis of single crystals to generate the needlelike crystals at the ends of crystals, and the erosion of generated needlelike crystals progressed from the tips of crystals along the a-axis and the edges along the 6-axis. All the erosion rates at the point of the grooves and at the tip of needlelike crystals along the a-axis and at the edges along the b-axis increased with an increase in the (R)-3HVcomposition. However, the rates along the a-axis were significantly altered by the introduction of (R)-3HVunits. Therefore, the morphologies and sizes of needlelike crystals were predominantly governed by the erosion rates along the a-axis at the grooves and tip of the needlelike crystals. Furthermore, the same authors estimated the enzymatic erosion rate of PHA single crystals using the volumetric analysis from real-time AFM height images [133]. By considering the density of the P(3HB) crystalline region, the changes in volume of single crystals during enzymatic degradation can be converted into weight changes. For P(3HB) single crystals, the overall erosion rates increased with a decrease in the molecular weight of P(3HB), similar to the erosion rate at the grooves. The overall degradation rates of P(3HB-co3HV) single crystals were much faster than those of P(3HB) single crystals. As described above, the binding specificity of the substrate-binding domain is broad compared with the substrate specificity of the catalytic domain. However, the binding

121

Practical Guide to Microbial Polyhydroxyalkanoates of P(3HB) depolymerase to substrate is necessary for the degradation of polymer substrates. The solid-state structures of substrate also affect the adsorption reaction of P(3HB) depolymerase. Zinc-based catalysts produce an amorphous atactic P(3HB)

from racemic P-BL. Abe and co-workers reported that slight erosion occurred on the surface of amorphous atactic P(3HB) samples by P(3HB) depolymerase. However, when amorphous P(3HB) was blended with crystalline P(3HB), the enzymatic erosion of films was accelerated [ 1101. Water-soluble products liberated from P(3HB)/atactic P(3HB) blend film were a mixture of monomers and oligomers of (R)-and (S)3HB units, indicating that the atactic P(3HB) component is hydrolysed by P(3HB) depolymerase in the presence of a bacterial P(3HB) component. Therefore, they proposed that the binding domain of P(3HB) depolymerase adsorbs selectively to the crystalline phase on the surface of films, and then the catalytic domain hydrolyses predominantly P(3HB) chains in the amorphous phase on the surface. It has been confirmed that the presence of crystalline components such as P(~ H B - c o - ~ H V ) , poly(E-caprolactone), poly(lactide) and poly(pivalo1actone) by blending [134, 1351 or block-copolymerising [ 1361 with atactic P(3HB) induce the enzymatic hydrolysis of atactic P(3HB) molecule by P(3HB) depolymerase. These results suggest that the P(3HB) depolymerase is liable to adsorb to stable crystalline lamellae, whereas it barely binds to mobile polymer chains in an amorphous state. However, it was found that the P(3HB) depolymerase can bind on the surface of glassy amorphous polymers such as melt-quenched PLLA [137], poly(D-lactic acid) (PDLA) and poly(methy1 methacrylate) [138, 1391, which have a higher Tgthan the reaction temperature of degradation. Therefore, it is concluded that the P(3HB) depolymerase adsorbs tightly to the molecules with less mobility in the stable region such as the crystalline lamellar or glassy amorphous region. The adsorption of P(3HB) depolymerase to single crystals has been investigated using immunogold labelling techniques. In both cases of P(3HB) homopolymer [ 124, 1251 and copolymer [119, 1231 single crystals, the P(3HB) depolymerase adsorbed homogeneously onto the surfaces of single crystals, suggesting that the substratebinding domain contributes to the adsorption of enzyme on P(3HB) crystals without site specificity. However, the concentrations of adsorbed enzyme on the surface of copolymer single crystals are slightly lower than that of P(3HB) homopolymer single crystal. P(3HB) depolymerase molecules cannot bind tightly to the irregular surface of copolymer crystals by hydrophobic adsorption because the copolymer chains with second monomer units have loose loop foldings. A similar difference in the concentrations of adsorbed enzyme among the surfaces of homopolymer and copolymer single crystals has been confirmed by direct observations of adsorbed enzyme molecules using AFM [ 1121. It has been proposed that the substrate-binding domain has another function together with its adsorption role. Murase and co-workers studied the morphological change

122

Extracellular Degradation of Polyhydroxyalkanoates (PHA) in P(3HB) single crystals during the adsorption of a mutant P(3HB) depolymerase in which the hydrolysis site of the catalytic domain was inactivated [140, 1411. They showed that the adsorption of the mutant P(3HB) depolymerase caused some cracks along the long axis of the P(3HB) single crystal, and suggested that the adsorption of P(3HB)depolymerase disturbed the molecular chain packing in P(3HB) single crystals. Also, Kikkawa and co-workers observed two types of P(3HB) depolymerases adsorbed on amorphous PLLA films, and demonstrated that the small hollows were formed on the PLLA film after removal of the adsorbed enzyme [137]. Furthermore, Numata and co-workers investigated the changes in surface morphology and properties of P(3HB) single crystals by the adsorption of P(3HB) depolymerase using real-time AFM observations [ 1121 and frictional force microscopic (FFM) analyses [ 1421. Initially, they observed the P(3HB) depolymerase molecule on P(3HB) crystals. Upon treatments to remove the enzyme molecule, they observed a concave identation (25 nm in width) left by the enzyme molecule on the crystal surface. Based on the changes in the contrast of the AFM phase image to dark at the concave area, it seemed that the surface of the concave area became softer than the surface of the chain-folded crystal. In addition, the values of frictional force on the surface of P(3HB) single crystals after the enzymatic treatment apparently decreased in comparison with those before the treatment, so it could be deduced that the adhesion of PHA depolymerase onto the chain-folding surface caused a partial disordering of molecular folding. These results suggest that the adsorption of P(3HB) depolymerase provides non-hydrolytic disruption of the structure of the polymer on the surface of P(3HB) crystals.

9.5. Molecular Mechanisms of the Enzymatic Degradation of PHA It is known that type-I and type-I1 P(3HB) depolymerases contain lipase-like catalytic triads (serine, histidine, and aspartic acid residues). As mentioned above, Hisano and co-workers [79] obtained the three-dimensional structure of P(3HB) depolymerase from P. funiculosum. In the case of the P(3HB) depolymerase from P. funiculosum, the catalytic residues Ser39, Aspl21, and His155 are located after the first ( p l ) , third (p3), and fourth (p4) j3-strands (see Figure 9.3). Despite the circularly permuted fold, it has been reported that the spatial arrangement of the catalytic triad is very similar to that of lipases and other dp hydrolase fold enzymes. A crevice is formed on the surface of the enzyme, and the catalytic residues Ser39, Aspl21, and His155 are located in the crevice. The inside of the crevice is of sufficient length (-1.5 nm) and width (-0.6 nm) to permit the incorporation of a single chain of the polymer. Several hydrophobic residues are located inside the crevice to provide a hydrophobic environment favourable for the binding of polymer chains, and these residues are highly conserved in type-I and type-I1 P(3HB)depolymerases. Furthermore, the authors prepared a hydrolytic-activity-disrupted mutant protein in which the original Ser39

123

Practical Guide to Microbial Polyhydroxyalkanoates was replaced by Ala, and determined the structure of the mutant protein complexed with (R)-3HBtrimer to understand the mechanism of substrate recognition by the enzyme as well as the catalytic mechanism on the basis of protein structure. From these spatial arrangements of the catalytic residues and the complexed (R)-3HBtrimer, they indicate that the mechanism of the depolymerising reaction may be similar to that of a lipase or serine esterase, and that the catalytic reaction may proceed as shown in Figure 9.12. Ser39 has a central role in the catalytic reaction that participates in a nucleophilic attack on the carbonyl carbon atom of the P(3HB) chain. The Hisl55-Asp121 hydrogen bonding system enhances the nucleophilicity of the hydroxyl group of Ser39. Ser39 does not form a hydrogen bond to His155 in the ligand-free state. Ser39 probably changes its conformation upon binding of a substrate, forming a hydrogen bond with Hisl55, and increasing its nucleophilicity. P(3HB) chain is bound to the active site through its four sequential monomeric units. The carbonyl oxygen of unit 2 is hydrogen-bonded to the indolyl group of Trp307, which enables the carbonyl carbon of the scissile linkage to be located at a position that would permit it to be attacked by the hydroxyl group of Ser39. In addition, the carbonyl oxygen of unit 3 is hydrogen-bonded to a water molecule, which is further hydrogen-bonded to the carbamoyl oxygen of Asn300 and the main chain amide group of Asn302. A tetrahedral coordination at the carbonyl carbon atom of the scissional ester linkage is transiently formed which is stabilised by the oxyanion hole formed by the amide groups of Ser40 and Cys250. After rearrangement of the bond formation, a covalent enzyme-substrate intermediate is subsequently formed, releasing the first product of a molecule with a hydroxyl group at the chain-end. The intermediate is then hydrolysed by the nucleophilic attack of a water molecule activated by the His-Asp system. Thus, the hydroxyl group of Ser39 is regenerated and a second product of a molecule with a carboxyl group at the chain-end released. His248, which is generally believed to form part of the oxyanion hole based on sequence conservation and the mechanistic analogy of the depolymerase to lipase [143], is located at the C-terminal end of strand p9, and neighbours the active site; it is hydrogen-bonded to the main-chain carbonyl oxygen of Gly249 and the amide group of Ala283. Therefore, its side chain does not point to the active site, and it is unlikely that it would participate in catalysis. From the conformational data of (R)-3HBtrimer complexed with mutant enzyme, it is predicted that the trimer substrate of (R)-3HBwith a planar zigzag conformation is preferred to bind in the crevice for the active site over the helix conformer. Therefore, the enzyme readily degrades the molecules in the amorphous region with a higher degree of freedom for their conformational arrangement. Molecules in the crystalline regions can barely bind to the active site of the enzyme because they have a helix conformation. Non-hydrolytic disruption of the structure of the P(3HB) molecule in the crystalline regions by the adsorption of P(3HB) depolymerase enables the active site of enzyme to have access to the P(3HB) molecule. 124

Extracellular Degradation of Polyhydroxyalkanoates (PHA)

~ '0

o

~

o

~

o

~

o

,

f

f

t

Figure 9.12 A proposed mechanism for the hydrolysis reaction of P(3HB) molecule by the P(3HB) depolymerase from P. funiculosum [79]

125

Practical Guide to Microbial Polyhydroxyalkanoates

9.6 Conclusion and Future Perspectives Although the biological synthesis of PHA by microbial cells is incompletely understood, the material properties of the various types of PHA and especially the influence of second monomers are well established. Recent studies have shed some light on the molecular mechanism of PHA degradation by enzymatic reactions. It is also becoming increasingly clear that the key enzyme of PHA biosynthesis, PHA synthase, is a versatile enzyme capable of polymerising various natural and unnatural monomers. It can be anticipated that the mutants of PHA synthase will have unique capabilities to polymerise monomers that are completely novel. In comparison with petrochemical plastics that have superior properties that match their application need, the currently available PHA materials have properties that fall short in their applicability as commodity plastics. The main reason for this shortfall in PHA is because of their unsatisfactory physical and mechanical properties. Careful design and synthesis of PHA with the right monomer composition have been shown to partially alleviate these problems. In addition, new processing conditions are continuously being developed to improve and enhance the material properties of PHA. This leaves the production cost of PHA as the final hurdle to overcome. For PHA to find widespread applications, they have to be commercially viable. Much progress has been achieved in the efficient production of PHA by the microbial fermentation process. However, the currently available technologies for the extraction and purification of PHA from bacterial cell biomass are not only expensive, but also environmentally hazardous. Despite these shortcomings, much effort is in progress in several countries to produce PHA in large scales. The main reason for this optimistic outlook for PHA is because they can be produced from renewable resources. The fact that PHA are bio-based and biodegradable makes them very attractive as future materials for sustainable living.

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86. B.M. Bachmann and D. Seebach, Macromolecules, 1999,32,6, 1777. 87. H. Brandl, B. Aeberli, R. Bachofen, I. Schwegler, H.M. Muller, M.H. Burger, T. Hoffmann, U.D. Lengweiler and D. Seebach, Canadian Journal of Microbiology, 1995,41, 1, 180. 88. Y. Doi, K. Mukai, K. Kasuya and K. Yamada in Biodegradable Plastics and Polymers, Eds., Y. Doi and K. Fukuda, Elsevier, Amsterdam, 1994,39.

89. K. Kasuya, Y. Inoue, K. Yamada and Y. Doi, Polymer Degradation and Stability, 1995, 48, 1, 167. 90. M.R. Timmins, R.W. Lenz and R.C. Fuller, Polymer, 1997,38,3, 551. 91. K. Kasuya, Y. Inoue and Y. Doi, International Journal of Biological Macromolecules, 1996,19, 1,35. 92. K. Yamashita, T. Funato, Y. Suzuki, S. Teramachi and Y. Doi, Macromolecular Bioscience, 2003, 3, 11, 694. 93. K. Numata, K. Yamashita, M. Fujita, T. Tsuge, K. Kasuya, T.Iwata, Y. Doi and H. Abe, Biornacromolecules, 2007,8,7,2276. 94. H. Abe and Y. Doi, Macromolecules, 1996,29,27, 8683. 95. H. Abe, I. Matsubara, Y. Doi, Y. Hori and A. Yamaguchi, Macromolecules, 1994,27,21,6018.

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100. J.J. Jesudason, R.H. Marchessault and T. Saito, Journal of Environmental Polymer Degradation, 1993, 1,2, 89. 101. J.E. Kemnitzer, S.P. McCarthy and R.A. Gross, Macromolecules, 1992,25, 22,5927.

102. M.R. Timmins, R.W. Lenz, P.J. Hocking, R.H. Marchessault and R.C. Fuller, Macromolecular Chemistry and Physics, 1996,197,4, 1193. 103. Y. Doi, S. Kitamura and H. Abe, Macromolecules, 1995,28, 14,4822. 104. Y. Saito, S. Nakamura, M. Hiramitsu and Y. Doi, Polymer International, 1996, 39, 3, 169. 105. E. Shimamura, M. Scandola and Y. Doi, Macromolecules, 1994,27, 16, 4429. 106. H. Abe and Y. Doi, International Journal of Biological Macromolecules, 1999, 25, 1-3, 185. 107. H. Abe, Y. Doi, H. Aoki, T. Akehata, Y. Hori and A. Yamaguchi, Macromolecules, 1995,28,23,7630. 108. H. Abe, Y.T. Doi, Y. Hori and T. Hagiwara, Polymer, 1997, 39, 1, 59.

109. Y. Kanesawa, N. Tanahashi, Y. Doi and T. Saito, Polymer Degradation and Stability, 1994,45, 2, 179. 110. H. Abe, I. Matsubara and Y. Doi, Macromolecules, 1995,28,4, 844. 111. K. Yamashita, Y. Aoyagi, H. Abe and Y. Doi, Biornacromolecules, 2001,2, 1, 25.

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136

bbreviations

A

Tetrabutylammonium Pm

Micrometre

111,

Inverse lamellar thickness

I3C

Carbon-13

I4C

Carbon-14

3D

Three-dimensional

3HB

3-hydroxybutyrate

3HD

3-h ydroxydecanoate

3HHx

3-hydroxyhexanoate

3H0

3-hydroxyoctanoate

3HP

3-hydroxypropionate

3HV

3-hydroxyvalerate

4HB

4-hydroxybutyrate

AFM

Atomic force microscopy

Ala

Alanine

Ala283

Alanine at position 283

AOT

Sodium bis(2-ethy1hexyl)sulfosuccinate

ApdA

Activator protein

137

Practical Guide to Microbial Polyhydroxyalkanoates Asn300

Asparagine at position 300

Asn302

Asparagine at position 302

ASP

Aspartic acid

Asp121

Aspartic acid at position 121

p-BL

Beta-butyrolactone

p-PL

Beta-propiolactone

BN

Boron nitride

BOD

Biochemical oxygen demand

C183

Cysteine at position 183

Ca

Calcium

CHZ

Methylene group

COZ

Carbon dioxide

CoA

Coenzyme A

CTAB

Cetyl trimethylammonium bromide

cys250

Cysteine at position 250

D355

Aspartate at position 355

DFP

Diisopropylfluorylphosphate

DNA

Deoxyribonucleic acid

DOC

Dissolved organic carbon concentration

DSC

Differential scanning calorimetry

DTG

Differential thermogravimetry

DTT

Dithioerythritol

Ea

Activation energy

138

Abbreviations EDTA

Ethylenediamine tetra-acetic acid

FFM

Frictional force microscopic

Fn3

Fibronectin type I11

FTIR

Fourier-transform infrared

g

Gram

G

Growth rate of spherulites

g

Velocity

GlY

Glycine

Gly249

Glycine at position 249

GPa

Gigapascal

GPC

Gel permeation chromatography

HP4

Sulfuric acid

HC1

Hydrochloric acid

His

Histidine

His155

Histidine at position 155

His248

Histidine at position 248

HPLC

High-performance liquid chromatography

2

Function of the rate

Inc.

Incorporation

K

Potassium ion

kd

Thermal degradation

kd

Kinetic constants of thermal chain scission

kDa

Kilodalton 139

Practical Guide to Microbial Polyhydroxyalkanoates

kJ

Kilojoules

KOH

Potassium hydroxide

1‘

Lamellar core thickness

Leu

Leucine

LP

Long period distance

mcl

Medium chain-length

MDa

Megadalton

Mg

Magnesium

MITI

Medical Interpreter Test

M”

Number-average molecular weight

MPa

Mega pascal

Mr

Relative molecular mass

Mw

Weight-average molecular weight

Na

Sodium

Na‘

Sodium ion

NaOH

Sodium hydroxide

NH,OH

Ammonium hydroxide

nm

Nanometre

NMR

Nuclear magnetic resonance

OECD

Organisation for Economic Cooperation and Development

P(3HA)

Poly(3-hydroxyalkanoate)(s)

P(3HB)

Poly(3-hydroxybutyrate)

P(3HB-co-3HHx) Poly(3-hydroxy butyrate-co-3 -hydroxyhexanoate) 140

Abbreviations 3-hydroxybutyrate-co-3-hydroxyvalerate) P(~ H B - c o - ~ H VPoly( )

P(~ H B - c o - ~ H B )Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) 3-hydroxybutyrate-co-6-hydroxyheptanoate) P(~ H B - c o - ~ H HPoly( ) P(3HD)

Poly(3-hydroxydecanoate)

P(3HN)

Pol y (3-hydroxynonanoate)

P(3HO)

Poly (3-hydroxyoctanoate)

P(3HP)

Poly (3-hydroxypropionate)

P(3HV)

Poly ( 3-h ydroxyvalerate)

P(4HB)

Poly(4-hydroxybutyrate)

PDLA

Poly(D-lactic acid)

PE

Polyethylene

PET

Polyethylene terephthalate

PHA

Polyhydroxyalkanoate(s)

PhaP

Phasin

phaZ

P(3HB) depolymerase gene

PhaZ

P(3HB) depolymerase

PHB

Polyhy droxybutyrate

Phe

Pheny lalanine

PLA

Poly(1actic acid)(s)

PLLA

Poly(L-lactic acid)

P"

Number-average degree of polymerisation

pn

Number-average degree of polymerisation

PPm

Parts per million 141

Practical Guide to Microbial Polyhydroxyalkanoates

PS

Polystyrene

Py-MS

Pyrolysis-gas chromatography/mass-spectrometry

QCM

Quartz crystal microbalance

R

Enantiomer form

S

Second

scl

Short chain-length

SDS

Sodium dodecyl sulfate

Ser

Serine

Ser39

Serine at position 39

Ser4O

Serine at position 40

SP.

Species

Tc

Crystallisation temperature

TEM

Transmission electron microscopy

Tg

Glass transition temperature

TG

Thermogravimetry

Tm

Melting temperature

Tmo

Equilibrium melting temperature

Trp307

Tryptophan at position 307

uv

Ultraviolet

X-ray

X-radiation

y-BL

Gamma-butyrolactone

142

INDEX

Index Terms

Links

A Acidovorax sp Acidovorax delafieldii

91 105

Alcaligenes eutrophus H16

9

Alcaligenes faecalis AE122

99

Alcaligenes faecalis T1

99

101

Alcaligenes latus

33

36

Annealing

36

56

72

Arrhenius equation

64 115

119

Atomic force microscopy Azobacter beijerinkii

109 85

B Bacillus sp

85

104

Bacillus megaterium

85

87

Bacillus subtilis

104

Bacillus thuringiensis

90

Bacterial cells, cultivation of

17

Bacterial polyhydroxyalkanoates, depolymerase genes, extracellular Biomass, non-polyhydroxyalkanoate β-Butyrolactone Budding model, bacterial

99 21 110 7

122

121

Index Terms

Links

C Cell solubiliser

21

Cellulose

22

Chemo-stat method

17

Chemosynthetic

33

Clostridium thermocellum

103

Co-crystallisation

39

Cold drawing process

36

Comamonas sp

98

Comamonas acidovorans

99

Comamonas testosteroni

99

Comb-like model

32

Copolymerisation

55

Crystal morphology

59

Crystalline lamellae

54

71

75

112

Crystallisation kinetic

51

rate

51

Cultivation of bacterial cells

53

17

cell growth phase

17

two-stage

17

polyhydroxyalkanoate accumulation phase

17

Cupriavidus necator

7

33

Cupriavidus necator H16

6

9

103

107

85

87

111

116

D Degradation, enzymatic

123 Degradation, extracellular

97

Degradation (mobilisation), intracellular

85

Degradation, non-isothermal

66

120

Index Terms

Links

Delftia acidovorans

33

Differential scanning calorimetry

51

55

58

61

64

67 Differential thermogravimetry

61

E Electron microscopy, freeze-fracturing process

15

Electron microscopy, transmission

15

119

2

6

22

90

97

99

104

107

114

122

33

90

Enzyme

Enzyme recovery, heat shock treatment

21

Escherichia coli

21

Ethylenediamine tetra-acetic acid

22

F Fed-batch

17

cultivation mode

17

Fourier-transform infrared spectroscopy Frictional force microscopic analyses

38

51

123

G Gel permeation chromatography

64

Gibbs-Thompson method

58

Glass transition temperature

59

H Herringbone model

32

High-performance liquid chromatography

113

13

High-resolution solid-state C-NMR spectroscopy Hoffman’s crystallisation theory

51

Hoffman-Weeks method

57

119

Index Terms Hot drawing process two-step Hydrogenophaga pseudoflava

Links 68 71 85

Hydrolase, α/β folding

104

Hydrolysis, enzymatic

105

87

111

I Infrared spectroscopy

61

Isothermal crystallisation process

53

L Lamellar crystals lozenge shaped Langmuir adsorption equation

56 36 107

Legionella pneumophilia

85

Leptothrix sp

99

Light microscope, ultraviolet

13

Light microscopy, phase-contrast Lysozymes

68

2

14

22

M Marinobacter sp Marinobacter NK1

99 100

Melt spinning

72

Micelle model

7

Microbial fermentation polyhydroxyalkanoate

126 2

Microorganisms, polyhydroxyalkanoate producing

13

Molecular conformation

29

planar zigzag chain

36

101

113

117

120

Index Terms Monolamellar system

Links 27

Monomers, medium chain-length

9

monomers, short chain-length

8

Multi-lamella system

27

N Nocardia corollina

9

Nuclear magnetic resonance analysis

61

Nucleation

74

rate of

87

113

29

34

71

124

1

5

7

15

17

21

51

61

85

97

121

126

110

120

51

O Optical microscopy, phase-contrast

13

Oxazine dye, Nile Blue

13

Ozawa model

64

P Paracoccus denitrificans

90

Penicillium funiculosum

103

Penicillium pinophilium

103

Planar zigzag conformation (β-form) Polyhydroxyalkanoates

accumulating bacteria

85

biosynthesis of

17

degrading bacteria

85

degrading enzymes

98

depolymerase

98

103

depolymerase, extracellular

88

98

depolymerase, intracellular

88

Index Terms

Links

Polyhydroxyalkanoates (Cont.) depolymerase, medium chain-length

98

110

depolymerase, short chain-length

99

101

hydrolysis

111

intracellular degradation of

85

medium chain-length

30

67

77

99

110

99

110

5

7

9

18

5

15

21

25

28

40

43

51

53

56

60

65

68

72

77

78

85

91

98

101

103

107

111

115

119

25

26

29

30

32

33

36

71

bacterial

26

64

based copolymer

38

54

74

121

based polymer blends

77

monomers

5

naturally occurring

5

production of

17

purification of

21

short chain-length solubilisation of spherulites synthase

9 21 119 2 126

synthesis

13

unnatural

6

Poly(3-hydroxybutyrate)

123 21 helix conformation (α-form)

chemosynthetic

110

crystalline lamellae

43

crystalline lattice

43

crystalline phase

55

59

Index Terms

Links

Poly(3-hydroxybutyrate) (Cont.) degradation of

86

depolymerase

88

90

122

125

depolymerase, extracellular

100

103

depolymerase, intracellular

87

91

homopolymers

67

lamellar crystals

28

lath-shaped morphology

42

lattice

39

mobilisation

90

oligomer hydrolases, intracellular

91

production

18

single crystal

26

spherulite

28

tacticity of

111

Poly(3-hydroxyoctanoate) depolymerases

77

36

helical conformation

38

single crystals

37

single crystals

29

33

helical conformation

35

single crystals

35

Polymerisation

41

51

89

91

61

31

32

Poly(4-hydroxybutyrate)

2

bulk

36

ring-opening

36

Polymerising-depolymerising enzyme system

92

Polymers, synthetic

64

Proteases

22

101

119

110

Poly(3-hydroxypropionate)

Poly(3-hydroxyvalerate)

38

91

36

64

110

66

98

103

Index Terms

Links

Pseudomonas alcaligenes

110

Pseudomonas fluorescens GK13

98

110

Pseudomonas funiculosum

102

104

109

123

Pseudomonas funiculosum enzyme

104 98

116

119

Pseudotnonas lemoignei Pseudomonas luteola

91

125

110

Pseudotnonas maculicola

98

Pseudomonas oleovorans

30

Pseudomonas putida U

92

Pseudomonas resinovorans

92

Pseudomonas spp

77

Pseudomonas strains

77

Pseudomonas stutzeri Pyrolysis - gas chromatography - mass-spectrometry

91

85

92

99

106

112

61

64

108

114

Ralstonia eutropha

36

85

90

Ralstonia picketti

99

105

107

119

121

119

Q Quartz crystal microbalance technique

R

Rhizobium Rhodococcus sp NCIMB 40136

85 9

Rhodospirilium rubrum

88

Rietveld fitting method

25

S Serine hydrolases

101

Solubilisation, non-polyhydroxyalkanoate

22

Spherulites

42

51

67

111

114

Index Terms

Links

Spirillum

85

Streptomyces exfoliates

99

Streptomyces hygroscopicus

99

Streptomyces hygroscopicus var ascomyceticus

99

Surface hydrolysis

102

107

T Thermal degradation temperature

61

Thermogravimetric curve

61

63

Thermogravimetric analysis

64

67

Thiolysis

89

64

W Wide-angle X-ray diffraction

40

X Xanthomonas spp

98

X-ray diffraction analysis

25

X-ray fibre diagram

34

X-ray microtomography

76

36

Y Young’s modulus

67

Z Zoogloea ramigera

91

78

75

67