Production of PHA Copolymers

The homopolymer of 3-hydroxybutyric acid or poly(3HB) is the prototype biodegradable PHA that is naturally produced by many bacteria. Because poly(3HB) is a crystalline and relatively brittle substance, it is not a suitable substitute for the commonly used thermoplastics manufactured from petrochemicals. Copolymers of hydroxyalkanoic acids, on the other hand, are less brittle and more elastic. Therefore, a major area of research on PHA is to develop organisms that can produce PHA copolymers with better mechanical properties and biodegradabflity. 

The three key enzymes for PHA synthesis, 3-ketothiolase, 3-ketoacyl-CoA reductase, and PHA polymerase, can usually accommodate substrates with a shghtly different chain length. Therefore, two or three kinds of hydroxyalkanoic adds can be incorporated during PHA synthesis to form a copolymer or terpolymer, if the additional kinds of CoA thioesters of hydroxyalkanoic acids can be made available to PHA polymerase through a special feeding regimen or metabolic engineering or a combination of both. 

The first PHA copolymer to be produced commercially is poly(3HB-co-3HV), which was produced by R. eutropha when fed with glucose and propionic acid (Byrom 1992). The production strain is a glucose-utilizing mutant of R. eutropha A. eutrophus) strain H16 that also can assimilate propionate more efficiently than the wild type. Propionic acid is 

Organic Acid and Solvent Production Acetic, Lactic, Gluconic, Succinic, and Polyhydroxyalkanoic Acids 

5- 30 mol%. The PHA polymerase of R. eutropha accepts CoA thioesters of 3-, 4-, or 5-hydroxyalkanoic acid with 3-5 carbon atoms (Steinbiichel 1996), and PHA copolymers consisting of these monomers may be produced. 

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The key metabolic pathways utilized in the production of PHA copolymer are shown in Figure 1. Two units of acetyl CoA form acetoacetyl CoA with phaA thiolase, which is then converted to 3-hydroxybutyryl CoA with phaB reductase. Parallel to these steps are the other metabolic pathways involving fatty acid biosynthesis (phaG) and fatty acid oxidation (phaJ, OAR, MFP), leading to the other larger 3-hydroxyacyl CoA units. Finally, the copolymerization of 3HB CoA and 3HA CoA with phaC PHA synthase results in the production of NodcuP PHA copolymers. 

Various types of uncharacterized spent cooking oils had been previously investigated for the production of PHA (Taguchi and Doi 2004 Fernandez et al. 2005 Haba et al. 2007 Song et al. 2008). They were found to be a feasible yet inexpensive source of carbon feedstock for PHA synthesis. Recently, Rao and co-workers used mixtures of PO-based spent cooking oil and 1,4-butanediol for the biosynthesis of P(3HB-co-4HB) copolymer by C. necator (Rao et al. 2010). They reported that P(3HB-co-4HB) copolymer with 15 mol% of 4HB was produced and the copolymer showed good biocompatibUity to be developed as an absorbable biomaterial. 

The thermal properties of moderately high Mw P(3HB-co-3HV) produced from jatropha oil and precursor were comparable to those produced from other plant oils (Lee et al. 2008). Jatropha oil was found compatible to be used in combination with precursor carbon sources for the biosynthesis of PHA copolymers by C. necator H16. The compositions of the P(3HB-co-3HV) copolymers were controllable by varying the concentration of the 3HV precursors. Jatropha oil supported both good cell growth and the biosynthesis of PHA copolymers by C. necator H16. In conclusion, Jatropha oil is a potential feedstock for the large-scale production of various types of PHAs. 

The uses of low specificity PHA synthases for production of PHA with functional groups for chemical modifications, and PHA with controllable compositions, including block PHA copolymers The use of special strain and mutated PHA synthases The making of PHA diols and the block copolymerization with other polymers... 

Because of the improved properties of P(HB-HV) copolymers over PHB, bacterial production of P(HB-HV), also known under the trade name Biopol , was central to the marketing and commercial production of PHA by Monsanto in the mid-1990s. It was therefore natural that after the demonstration of high-level PHB synthesis in the plastids, efforts would be focused on the synthesis of PHA copolymers, such as P(HB-HV). 

Fig. 16 Product design space of PHA copolymers (Noda et al. 2005a)...

The properties of PHA copolymers depend strongly on the type, level, and distribution of comonomer units comprising the polymer chains. While PHAs have been investigated by various researchers from academic and industrial laboratories as an interesting class of materials, the successful commercial utilization of PHAs has been slow. The production cost of PHAs by conventional fermentation processes was initially high, making the entry of this material into the commodity plastics market difficult. Limited availability also did not contribute favorably toward the establishment of a robust cost structure and product development. Most importantly, the physical properties of earlier commercial PHAs, like PHBV, were inadequate for many of the applications envisioned for the replacement of commodity plastics. 

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