• Journal of Microbiology and Biotechnology
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• v.3 no.2
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• pp.123-128
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• 1993

The polyesters (polyhydroxyalkanoates; PHAs) production capability in a two-step cultivation of Alcaligenes eutrophus H16(ATCC 17699) was investigated by using various organic carbon sources. The carbon sources used included linear $C_2~C_10$ monocarboxylic acids, $C_3~C_10$ dicarboxylic acids, crotonic acid, and several linear vicinal and $\omega$-diols. The polyesters synthesized were characterized by 500 MHz $^1 H-NMR$ spectroscopy, intrinsic viscosity$[\eta]$ measurement in chloroform and differential scanning calorimetry (DSC). The PHAs synthesis data showed that the use of C-odd ($C_3, C_5, and C_7$) monocarboxylic acids resulted in poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(P(3HB-co-3HV) (3HV content ranging 40 to 70 mol%) while the use of $C_9$ substrate gave the copolyester containing only 4 mol% of 3HV. All culture products obtained on $C_3$~C$_{10}$ dicarboxylic acids gave exclusively P(3HB). 500 MHz $^1 H-NMR$ analysis showed that all polyesters synthesized generally contained 1~2 mol% 3HV even for the unrelated substrates such as the carboxylic acids with even number of carbon. When $\alpha, \omega$-diols with even number of carbon were used as substrates, 4-hydroxybutyrate(4HB) was inserted into the polyester chain composed of P(3HB-co-4HB). Vicinal diols were generally not utilized by the bacterium for polyester production.n.

• Journal of Microbiology and Biotechnology
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• v.3 no.2
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• pp.115-122
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• 1993

Poly(3-hydroxybutyrate)(P(3HB)) accumulation under nutrient-rich condition with various amounts of $(NH_4)_2 SO_4$ was systematically investigated. The results of the electron-microscopy and the solvent extraction showed that the P(3HB) accumulation is unavoidable even under nutrient-rich condition. This indicates that in a two-step culture of Alcaligenes eutrophus H16, the researches should be careful in interpreting the data of polyhydroxyalkanoates(PHAs) accumulation in terms of the carbon-source fed in the second step because the two-step culture product contains the P(3HB) produced under nutrient-rich condition. The polyester production capability in a two-step batch culture of A. eutrophus H16(ATCC 17699) was also investigated using various saccharides and their derivatives such as glucose, fructose, gluconic acid, glucaric acid, sorbitol, lactose, galactose, and mannose. The polyesters synthesized were characterized by 500 MHz$^{1}H-NMR$ spectroscopy, intrinsic viscosity$[\eta]$ measurement in chloroform and differential scanning calorimetry(DSC). 500 MHz $^{1}H-NMR$ analysis showed that all polyesters synthesized generally contained 1~2 mol% of 3HV. Another finding is that the glucose utilization can be increased by changing the autoclaving procedure of the substrate to enhance the P(3HB) production yield up to 46 wt% of P(3HB) in dry cells.

• Journal of Microbiology and Biotechnology
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• v.16 no.6
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• pp.901-910
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• 2006

Poly(3-hydroxybutyrate) [P(3HB)] is a microbial polyester intracellularly accumulated as distinct granules in numerous microorganisms as an energy and carbon storage material. Recombinant Escherichia coli harboring the heterologous P(3HB) biosynthesis genes accumulates large amounts of P(3HB) granules, yet the granule-associated proteins have not been identified. Therefore, this study reports on an analysis of the P(3HB) granule-associated proteome in recombinant E. coli. Fiye proteins out of 7 spots identified were found to be involved in functions of translation, heat-stress responses, and P(3HB) biosynthesis. Two of the major granule-associated proteins, IbpA/B, which are already known to bind to recombinant proteins forming inclusion bodies in E. coli, were further analyzed. Immunoblotting and immunoelectron microscopic studies with IbpA/B antibodies clearly demonstrated the binding and localization of IbpA/B to P(3HB) granules. IbpA/B seemed to play an important role in recombinant E. coli producing P(3HB) by stabilizing the interface between the hydrophobic P(3HB) granules and the hydrophilic cytoplasm. Thus, IbpA/B were found to act like phasins in recombinant E. coli, as they are the major proteins bound to the P(3HB) granules, affect the morphology of the granules, and reduce the amount of cytosolic proteins bound to the P(3HB) granules.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.847-853
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• 1999

A Pseudomonas sp. strain that is capable of utilizing dicarboxylic acids as a sole carbon source was isolated from activated sludge by using the enrichment culture technique. This organism accumulated polyhydroxyalkanoates (PHAs) with an unusual pattern of monomer units that depends on the carbon sources used. Polyhydroxybutyrate (PHB) homopolyester was synthesized from glucose or small $C_{-even}$ alkanoic acids, such as butyric acid and hexanoic acid. Accumulation of PHB homopolyester was also observed in the cells grown on $C_{-odd}$ dicarboxylic acids, such as heptanedioic acid and nonanedioic acid as the sole carbon sources. In contrast, a copolyester consisting of 6 mol% 3-hydroxybutyrate (3HB) and 94 mol% 3-hydroxyvalerate (3HV) was produced with a PHA content of as much as 36% of the cellular dry matter. This strain produced PHAs consisting both of the short-chain-length (SCL) and the medium-chain-length (MCL) 3-hydroxyacid units when heptanoic acid to undecanoic acid were fed as the sole carbon sources. Most interestingly, polyester consisting of significant amount of relevant fractions, 3HB, 3HV, and 3-hydroxyheptanoate (3HHp), was accumulated from heptanoic acid. According to solvent fractionation experiments, the polymer produced from heptanoic acid was a blend of poly(3HHp) and of a copolyester of 3HB, 3HV, and 3HHp units. The hexane soluble fractions contained only 3HHp units while the hexane-insoluble fractions contained 3HB and 3HV units with a small amount of 3HHp unit. The copolyester was an elastomer with unusual mechanical properties. The maximum elongation ratio of the copolyester was 460% with an ultimate strength of 10 MPa, which was very different from those of poly(3HB-co-3HV) copolyesters having similar compositions produced from other microorganisms.