Biosynthesis of Polyhydroxyalkanoate by a Marine Bacterium Vibrio sp

Downloaded by MICHIGAN STATE UNIV on February 18, 2015 | http://pubs.acs.org Publication Date (Web): November 22, 2013 | doi: 10.1021/bk-2013-1144.ch015

Chapter 15

Biosynthesis of Polyhydroxyalkanoate by a Marine Bacterium Vibrio sp. Strain Using Sugars, Plant Oil, and Unsaturated Fatty Acids as Sole Carbon Sources Satoshi Tomizawa,1 Jo-Ann Chuah,1 Misato Ohtani,2 Taku Demura,2 and Keiji Numata*,1 1Enzyme

Research Team, Biomass Engineering Program, RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan 2Cellulose Production Research Team, Biomass Engineering Program, RIKEN, Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan *E-mail: [email protected]

This is a mini review of our recent work and update on polyhydroxyalkanoate (PHA) production by marine bacteria using sugars, plant oils and three types of unsaturated fatty acids as sole carbon sources. Marine bacteria have recently attracted attention as potentially useful candidates for the production of practical materials from marine ecosystems, including the oceanic carbon dioxide cycle. A newly-identified marine bacterium, Vibrio sp. strain KN01, was characterized with respect to PHA productivity using various carbon sources under aerobic and aerobic-anaerobic marine conditions. The produced PHA was further analyzed with respect to its monomer composition. The influence of unsaturated fatty acids as sole carbon sources as well as aerobic-anaerobic conditions on PHA compositions is summarized and discussed in this chapter.

© 2013 American Chemical Society In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


Introduction Polyhydroxyalkanoate (PHA), one of the eco-friendly, bio-based and biodegradable plastics, is produced by various bacteria as an intracellular storage material of carbon and energy. PHA also shows excellent biodegradability and biocompatibility, and diverse mechanical properties related to the chemistry of secondary monomer units (1–3). In the PHA biosynthetic pathway of microbial cells, the provision and polymerization of the hydroxyalkanoate (HA) monomers are performed by various enzymes. Typical PHA synthesis genes such as the genes from Ralstonia eutropha (also known as Cupriavidus necator) within an operon including a beta-ketothiolase (phaA), an acetoacetyl-coenzyme A (CoA) reductase (phaB) and a synthase (phaC) (4). The thiolase and reductase synthesize a monomer substrate, 3-hydroxybutyryl CoA (3HBCoA), and the synthase polymerizes the monomers to PHA (4). Also, the other proteins related to PHA synthesis are an intracellular PHA depolymerase, a phasin protein, and a regulatory protein, which are often encoded near phaA, phaB, and phaC. With respect to PHA synthesis via the beta-oxidation pathway, the phaJ and fabG genes encoding (R)-specific 2-enoyl-CoA hydratase and 3-ketoacyl-acyl carrier protein (ACP) reductase are also known to be responsible for converting 3-enoyl-CoA and 3-ketoacyl-CoA into (R)-3-hydroxyacyl-CoA, respectively (5, 6). The substrate specificity differs depending on the species of PHA synthase, PhaC, thus, the monomeric compositions of the resultant PHA copolymers are influenced by the PhaC activities and/or combination of these related proteins (7). Poly[(R)-3-hydroxybutyrate] [P(3HB)], the most ubiquitous of PHAs, is one of the most studied bio-based and biodegradable plastics. P(3HB) was first isolated as a microbial reserve polyester from Bacillus megaterium (8). Following the discovery of P(3HB), many types of bacteria, such as Bacillus spp., Pseudomonas spp., Cupriavidus spp. and Aeromonas spp., have been investigated for their potential use in producing PHA more efficiently for industrial use (Table 1) (9–12). A few kinds of marine bacteria have also been investigated for the production of PHA under marine conditions, but the resultant PHAs have not been characterized in detail (13–15). The advantages of biosynthesizing PHA under marine conditions include avoiding contamination with bacteria that lack salt-water resistance, and the ability to use filtered seawater as a culture medium, which would enable large-scale industrial production of PHA. Further, marine bacteria have recently attracted attention as candidates for the production of practical materials from marine ecosystems, including the oceanic carbon dioxide cycle (16–18). These make it attractive to find or construct other marine bacteria that are able to produce PHA efficiently enough to be used for the industrial production of PHA. Research on the medical, environmental and taxonomic aspects of Vibrio species, which are typical marine bacteria, has expanded in the last several decades, because several species of Vibrio, i.e., V. cholerae, V. parahaemolyticus and V. vulnificus, are clinically important human pathogens. On the other hand, some species of Vibrio, such as V. fischeri, have not been shown to be pathogens. With respect to PHA productivity, V. harveyi was shown to form PHA granules at a high cell density during its development of luminescence (19). Recently, a novel 212 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

type of marine bacterium, Vibrio sp. strain KN01 (Figure 1), was isolated and characterized with respect to its PHA productivity using various carbon sources under aerobic and aerobic-anaerobic marine conditions (20). In this minireview, we provide our recent works of PHA production by marine bacteria, based on the results of feeding experiments on the strain KN01, using sugars, plant oils and unsaturated fatty acids as sole carbon sources.


Table 1. Types of PHA production

Figure 1. Atomic force microscopy amplitude image of Vibrio sp. strain KN01 cast on mica in air at 25°C (A). Vibrio sp. strain KN01 shows a flagellum. The strain was isolated from seawater of Hizushi beach (B).

PHA Production Using Sugars and Organic Acid The isolated strain, Vibrio sp. KN01, was investigated with respect to PHA productivity from well-known carbon sources, namely, glucose, fructose or gluconate at 30°C for 48 h under an aerobic condition (Table 2) (20). The strain exhibited cell growth ranging from approximately 1.4 to 1.7 g/L. PHA accumulation was observed with all three carbon sources, and PHA contents (wt%) were determined by the weight of the produced PHA. The 1H-NMR 213 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


spectrum revealed that all of these products were composed of 3HB without any major second monomer units, such as medium chain-length hydroxyalkanoates (HA). The PHA productivity of Vibrio sp. KN01 under an aerobic-anaerobic condition was also examined, because Vibrio spp. are known as facultative anaerobes that can grow under both aerobic and anaerobic conditions. Lee and coworkers reported that the reduction in dissolved oxygen concentration from 40% to 5% inhibited cell growth of recombinant Escherichia coli harboring PHA-synthesis genes and enhanced PHA contents from 10 to 27 wt% (21), demonstrating that PHA production could be enhanced under conditions similar to the aerobic-anaerobic ones used here. In our study, the strain cultured under the aerobic-anaerobic condition demonstrated constant cell growth irrespective of whether glucose, fructose or gluconate was used as the sole carbon source (Table 2) (20). Here, an aerobic–anaerobic condition denotes that the strain was prepared and sealed under nitrogen atmosphere and cultured. PHA accumulation was observed under the aerobic-anaerobic condition with each of the three kinds of carbon sources, and the average PHA content of the isolate cultured with fructose and gluconate was over 10%. Also, the PHA contents of the isolate cultured with glucose and fructose were significantly higher compared with the PHA contents from the isolate cultured under an aerobic condition. The number-average molecular weights of the PHAs synthesized by Vibrio sp. KN01 under the aerobic condition ranged from 110 × 103 to 140 × 103 g/mol. On the other hand, the number-average molecular weights of the PHAs synthesized under the aerobic-anaerobic condition ranged from 86 × 103 to 100 × 103 g/mol, which were lower in comparison to the PHA produced under the aerobic condition (20).

Table 2. PHA accumulations using different carbon sources under aerobic and aerobic-anaerobic conditions. Data from Numata and Doi, 2012 (20)

Culture Condition

Carbon Source

Dry Cell Weight, g/L

PHA Content, wt%

PHA Composition, mol% a 3HB

Aerobic

Aerobic-Anaerobic

Glucose

1.7 ± 0.2

0.06 ± 0.01

100

Fructose

1.6 ± 0.1

0.3 ± 0.1

100

Gluconate

1.4 ± 0.1

14 ± 2

100

Glucose

1.6 ± 0.2

5±2

100

Fructose

1.3 ± 0.2

17 ± 3

100

Gluconate

1.1 ± 0.2

15 ± 2

100

a

PHA composition determined by 1H NMR. The data of dry cell weight and PHA content denote averages of three replicates and their standard deviations.

214 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


PHA Production Using Plant Oil Plant oil is also one of the most important carbon sources in nature. Here, we focused on soybean oil, a typical industrial plant oil, and Jatropha oil, which is getting much attentions as next-generation material for bio-diesel. The fatty acid compositions of these oils have been described by several groups as shown in Table 3 (22, 23) .Many types of bacteria have been reported to use soybean oil as a sole carbon source via the beta oxidation pathway to produce PHA containing medium-chain length units such as 3-hydroxyhexanoate (24–27). P(3HB) homopolymer production with plant oils such as soybean oil is a rare characteristic among PHA-producing bacteria, including Pseudomonas spp. and Cupriavidus spp. (Cupriavidus necator was formally known as Ralstonia eutropha). The composition of plant oil has been investigated and described by several groups (Table 3) (22, 23). The PHA productivity of the isolated strain, Vibrio sp. KN01, was characterized using soybean oil and jatropha oil, respectively, as sole carbon sources at 30°C for 48 h under aerobic and aerobic-anaerobic conditions (Table 4) (20). The cell growth when soybean oil was used as the sole carbon source was relatively low and its dry cell weight was approximately 0.4 g/L. PHA accumulation was observed with both plant oils, however the PHA content (wt%) of the culture using jatropha oil was significantly lower. Under the aerobic-anaerobic condition, the average PHA content of the isolate cultured with soybean oil was over 40%, which is a high level of PHA productivity in comparison to the other native bacteria (1, 3). The PHA contents of all the samples under the aerobic-anaerobic condition were enhanced by limiting the amount of dissolved oxygen, especially when soybean oil was used as the sole carbon source, under which condition the limitation of dissolved oxygen increased the PHA contents from 8 ± 2% to 40 ± 6%. The 1H-NMR spectrum revealed that only the PHA obtained from the isolate cultured with soybean oil under the aerobic-anaerobic condition contained 3-hydroxypropionate (3HP) and 5-hydroxyvalerate (5HV) as monomer units (Figure 2A). Furthermore, gas chromatography measurement using methylated 5-hydroxyvalerate as a standard confirmed the presence of the 5HV unit. PHA containing 5HV units was recently reported to show lipase-mediated degradation and supported cell viability better than the other PHAs (28). The PHA synthesized from soybean oil under the aerobic-anaerobic condition was composed of 3HP and 5HV, in addition to 3HB, according to the assignment based on the 1H-NMR spectrum and gas chromatography data (Figure 2A). The additional monomer units (3HP and 5HV) have odd-numbered carbon atoms, even though the major fatty acids in soybean oil have even-numbered carbon atoms. These fatty acids might be metabolized inefficiently under the aerobic-anaerobic condition and converted into 3HPCoA and 5HVCoA from acetyl-CoA and acylCoA via the beta-oxidation pathway or another pathway by carboxylase, based on findings in the literature (29). The chromosomes of Vibrio sp. Ex25, which shows over 99% identity to Vibrio sp. KN01 in the 16S rDNA sequence, have been revealed to contain genes encoding acetyl-CoA carboxylase, 3-polyprenyl-4hydroxybenzoate carboxylase, succinylbenzoate-CoA synthase, and 2,4-dienoylCoA reductase, in addition to FabG, PhaJ, PhaA, PhaB and PhaC. Furthermore, 215 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

the substrate specificities of the carboxylases, CoA synthase and CoA reductase are not known and might be broad enough to process C5 substrates. Although the pathways to produce 3HPCoA and 5HVCoA were not confirmed in the present study, a possible pathway for production of these monomers is the conversion from acetyl-CoA and acyl-CoA by carboxylase.


Table 3. Fatty acids compositions of plant oil Fatty acid (wt%)

a

Saturated acid

Unsaturated acid

C14:0 (Myristic acid)

C16:0 (Palmitic acid)

C18:0 (Stearic acid)

C16:1 (Palmitoleic acid)

C18:1 (Oleic acid)

C18:2 (Linoleic acid)

C18:3 (Linolenic acid)

Soybean oil a

-

10

5

-

23

51

11

Jatropha oil b

0.1

17.1

4.3

1.2

42

34.8

0.1

Liu et al. Catalysis Commu 2007 (22).

b

Ng et al. Polym. Degrad. Stab. 2010 (23).

Table 4. PHA accumulations in Vibrio sp. KN01 using plant oils as sole carbon sources. The data are partially from Numata and Doi, 2012 (20) PHA Composition, mol% a

Culture Condition

Carbon Source


PHA Content, wt%

3HB

HA

Aerobic

Soybean oil

2.4

8

100

-

b

Jatropha oil

1.2

0.3

100

-

b

Soybean oil

0.4

40

83

Jatropha oil

0.8

0.1

100

AerobicAnaerobic

3HP: 14 5HV: 3

a

-

b

PHA composition determined by 1H NMR. b Not detected. The data of dry cell weight and PHA content denote averages of three replicates and their standard deviations.



Figure 2. 1H-NMR spectrum of PHA produced by the isolate under the aerobic–anaerobic condition using soybean oil (A), oleic acid (B), linoleic acid (C) and jatropha oil (D) as sole carbon sources. The data are partially from Numata and Doi, 2012 (20).

PHA Production Using Unsaturated Fatty Acids Plant oils, such as soybean oil and jatropha oil, contain several saturated and unsaturated fatty acids (Table 3) (22, 23). To clarify the influence of unsaturated acids as carbon sources in PHA production, PHA synthesis from three types of unsaturated fatty acids by Vibrio sp. KN01 was performed at 30°C for 48 h under aerobic and aerobic-anaerobic conditions (Table 5). The cell growth under the aerobic condition when unsaturated fatty acids were used as sole carbon sources was significantly lower than when soybean oil was used. PHA accumulation was also detected with three of the unsaturated fatty acids, and the PHA contents (wt%) were relatively low (0.1-3.2 wt%). Under the aerobic-anaerobic condition, the average PHA content of the isolate cultured with oleic acid was slightly higher than that under the aerobic condition. The 1H-NMR spectra revealed that the polymers from the isolate cultured with the unsaturated fatty acids were P(3HB) homopolymers (Figure 2B, C and D). Based on the present results, the pure unsaturated fatty acids are not good candidates to produce a novel type of PHA, which suggests that another biomass such as lignin and seaweed, containing chemicals other than fatty acids, would be a more promising carbon source than fatty acids from animals and plants.



Table 5. PHA accumulations using three types of unsaturated fatty acids as a sole carbon source under aerobic and aerobic-anaerobic conditions PHA Composition, mol% a

Culture Condition

Carbon Source


PHA Content, wt%

3HB

HA

Aerobic

Oleic acid

0.7

0.3

100

-

c

Linoleic acid

0.6

0.4

100

-

c

Linolenic acid

1.5

0.2

100

-

c

Oleic acid

0.9

3.2

100

-

c

Linoleic acid

0.3

0.2

100

-

c

Linolenic acid

0.2

0.1

100

-

c

AerobicAnaerobic

a

PHA composition determined by 1H NMR. Not detected.

b

b

HA denotes hydroxyalkanoate units.

c

PHA Synthase from Marine Bacteria Our results presented here indicate that PHA production is dependent on the culture conditions, i.e. carbon sources and aerobic/anaenobic condition, in Vibrio sp. KN01. The data concerning the PHA produced with soybean oil suggest critical roles of metabolic regulation in the endogenous production of monomer units, to response with the level of environmental oxygen. In other words, one of key targets for engineering should be the supply of unique monomer units, which are provided endogenously and/or exogenously, to produce novel types of PHA (20). As the other important target, PHA synthase, the key enzyme for biosynthesis of PHA, has been classified into 4 classes with respect to their primary structures (7). The relationship between the structure and function of PHA synthases has been discussed based on their primary structures, because the crystal structures of PHA synthases (i.e., PhaCs), are currently unavailable. V. cholera, V. parahaemolyticus and V. harveyi have been reported to possess the class I PHA synthases (7, 19). Similarly, Vibrio sp. Ex25, which shows over 99% similarity to Vibrio sp. KN01 in 16S rDNA sequence, was found to possess a PHA synthase belonging to the class I, according to the revealed chromosome sequences (20). Class I PhaC is composed of a single subunit with molecular weights between 61 and 68 kDa. The PhaCs from R. eutropha and A. caviae are known to be typical class I PhaCs that exhibit strict and broad substrate specificities, respectively (7, 30, 31). The PhaC from A. caviae is one of the exceptional PhaCs in Class I, because of its low similarity in amino acid sequence (approximately 45%) to the other Class I PHA synthases (32, 33). Multiple alignments of the amino acid sequences of PhaC from Vibrio sp. Ex25, R. eutropha and A. caviae are shown in Figure 3. The amino acid sequence of the PhaC from Vibrio sp. Ex25 showed 31% and 54% identities to those from R. eutropha and A. caviae, respectively. Inspection of the protein model of PhaC from R. eutropha revealed that the active 218 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


site Cys-319, the conserved Asp-485 and His-513 are adjacent and conceivably form a catalytic triad (7, 34) (see the arrows in Figure 3). The amino acid sequence of PhaC from Vibrio sp. Ex25 showed high identity to that from A. caviae, especially around those three amino acid residues, which could form the catalytic triad, in comparison to that from R. eutropha. This would suggest the potential of wide substrate specificity of PhaC from Vibrio sp. Ex25, much as in the case of the PhaC from A. caviae.

Figure 3. Multiple alignment of amino acid sequences of PHA synthases originating from the isolate Vibrio sp. Ex25, Ralstonia eutropha, and Aeromonas caviae. The arrows denote Cys-319, Asp-485 and His-513 for PhaC from R. eutropha. Modified data from Numata and Doi, 2012 (20).

Conclusion The newly-identified marine facultative anaerobe Vibrio sp. KN01 produced PHA under aerobic and aerobic-anaerobic conditions when artificial seawater is used as the culture medium. The PHA production from plant oil and unsaturated fatty acids demonstrated no significant difference from that shown by some previously reported bacteria. However, the amino acid sequence of PHA synthase of the isolated strain seemed to be different from that of the typical soil bacteria. This new insight could lead to marine biotechnology for the production of various polymeric materials, including PHA, by marine bacteria to alleviate the problem of carbon dioxide emissions.

References 1. 2. 3. 4.

Doi, Y. Microbial Polyesters; VCH Publishers: New York, 1990. Doi, Y.; Steinbüchel, A. Biopolymers; Wiley-VCH: Weinheim, Germany, 2001; Vol. 3. Lenz, R. W.; Marchessault, R. H. Biomacromolecules 2005, 6, 1–8. Pohlmann, A.; Fricke, W. F.; Reinecke, F.; Kusian, B.; Liesegang, H.; Cramm, R.; Eitinger, T.; Ewering, C.; Potter, M.; Schwartz, E.; 219 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.

5. 6. 7. 8. 9.


10. 11. 12. 13.

14. 15. 16. 17. 18.

19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.

Strittmatter, A.; Voss, I.; Gottschalk, G.; Steinbüchel, A.; Friedrich, B.; Bowien, B. Nat. Biotechnol. 2006, 24, 1257–1262. Fiedler, S.; Steinbüchel, A.; Rehm, B. H. Arch. Microbiol. 2002, 178, 149–160. Tsuge, T.; Fukui, T.; Matsusaki, H.; Taguchi, S.; Kobayashi, G.; Ishizaki, A.; Doi, Y. FEMS Microbiol. Lett. 2000, 184, 193–198. Rehm, B. H. Biochem. J. 2003, 376, 15–33. Lemoignei, M. Bull. Soc. Chim. Biol. 1926, 8, 770–782. Shimamura, E.; Kasuya, K.; Kobayashi, G.; Shiotani, T.; Shima, Y.; Doi, Y. Macromolecules 1994, 27, 878–880. Abe, H.; Doi, Y.; Fukushima, T.; Eya, H. Int. J. Biol. Macromol. 1994, 16, 115–119. Saito, Y.; Doi, Y. Int. J. Biol. Macromol. 1994, 16, 99–104. Fuchtenbusch, B.; Wullbrandt, D.; Steinbüchel, A. Appl. Microbiol. Biotechnol. 2000, 53, 167–172. Gonzalez-Garcia, Y.; Nungaray, J.; Cordova, J.; Gonzalez-Reynoso, O.; Koller, M.; Atlic, A.; Braunegg, G. J. Ind. Microbiol. Biotechnol. 2008, 35, 629–633. Wang, Q.; Zhang, H. X.; Chen, Q.; Chen, X. L.; Zhang, Y. Z.; Qi, Q. S. World J. Microbiol. Biotechnol. 2010, 26, 1149–1153. Lopez, N. I.; Pettinari, M. J.; Stackebrandt, E.; Tribelli, P. M.; Potter, M.; Steinbüchel, A.; Mendez, B. S. Curr. Microbiol. 2009, 59, 514–519. Karl, D. M. Nat. Rev. Microbiol. 2007, 5, 759–769. Moran, M. A.; Miller, W. L. Nat. Rev. Microbiol. 2007, 5, 792–800. Umani, S. F.; Del Negro, P.; Larato, C.; De Vittor, C.; Cabrini, M.; Celio, M.; Falconi, C.; Tamberlich, F.; Azam, F. Aquat. Microb. Ecol. 2007, 46, 163–175. Sun, W. Q.; Teng, K.; Meighen, E. Can. J. Microbiol. 1995, 41, 131–137. Numata, K.; Doi, Y. Mar. Biotechnol. 2012, 14, 323–331. Park, S. J.; Ahn, W. S.; Green, P. R.; Lee, S. Y. Biomacromolecules 2001, 2, 248–254. Liu, X. J.; He, H. Y.; Wang, Y. J.; Zhu, S. L. Catal. Commun. 2007, 8, 1107–1111. Ng, K. S.; Ooi, W. Y.; Goh, L. K.; Shenbagarathai, R.; Sudesh, K. Polym. Degrad. Stab. 2010, 95, 1365–1369. de Smet, M. J.; Eggink, G.; Witholt, B.; Kingma, J.; Wynberg, H. J. Bacteriol. 1983, 154, 870–878. Fukui, T.; Ohsawa, K.; Mifune, J.; Orita, I.; Nakamura, S. Appl. Microbiol. Biotechnol. 2011, 89, 1527–1536. Timm, A.; Steinbüchel, A. Appl. Environ. Microbiol. 1990, 56, 3360–3367. Tsuge, T.; Yamamoto, T.; Yano, K.; Abe, H.; Doi, Y.; Taguchi, S. Macromol. Biosci. 2009, 9, 71–78. Chuah, J. A.; Yamada, M.; Taguchi, S.; Sudesh, K.; Doi, Y.; Numata, K. Polym. Degrad. Stab. 2013, 98, 331–338. Fukui, T.; Suzuki, M.; Tsuge, T.; Nakamura, S. Biomacromolecules 2009, 10, 700–706. Fukui, T.; Doi, Y. J. Bacteriol. 1997, 179, 4821–4830. 220 In Green Polymer Chemistry: Biocatalysis and Materials II; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.


31. Fukui, T.; Yokomizo, S.; Kobayashi, G.; Doi, Y. FEMS Microbiol. Lett. 1999, 170, 69–75. 32. Steinbüchel, A.; Hustede, E.; Liebergesell, M.; Pieper, U.; Timm, A.; Valentin, H. FEMS Microbiol. Rev. 1993, 10, 347–350. 33. Liebergesell, M.; Steinbüchel, A. Appl. Microbiol. Biotechnol. 1993, 38, 493–501. 34. Qi, Q.; Rehm, B. H. Microbiology 2001, 147, 3353–3358.


Biosynthesis of Polyhydroxyalkanoate by a Marine Bacterium Vibrio sp

Recommend Documents