Methanol-utilizing bacteria, Methylobacterium organophilum and Methylobacterium sp. KCTC 0048 isolated from soil, were tested for the production of PHA and its copolyesters. These methylotrophs could synthesize a variety of copolyesters when the secondary carbon substrates were added to the nutrient-limited cultures containing methanol as major carbon and energy source.. Methylobacterium organophilum could produce PHB from methanol and P3HB-3HV from valeric acid. A strain, SH 1332, which was identified as Methylobacterium sp. KCTC 0048, was selected for further studies on PHA production considering its high PHA productivity, among several strains. This methylotroph could accumulate PHB, P3HB-3HV, P3HB-4HB and P3HB-3HP from methanol and the secondary carbon substrates. Methylobacterium sp. KCTC 0048 which was confirmed as a facultative methylotroph, could accumulate PHB by using methanol, ethanol, glycerol, and fructose as precursors, respectively. The copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate, P3HB-3HV was accumulated when valeric acid, pentanol or heptanoic acid was added to the nitrogen-limited medium containing methanol. The copolyester of 3-hydroxybutyrate and 4-hydroxybutyrate, P3HB-4HB was synthesized from 4-hydroxybutyrate, 1,4-butanediol, or $\gamma$-butyrolactone, and the copolyester of 3-hydroxybutyrate and 3-hydroxypropionate(P3HB-3HP), from 3-hydroxypropionate as the secondary carbon substrates, respectively. In case of P3HB-3HV production by Methylobacterium sp. KCTC 0048, the composition of copolyester varied from 10\% to 99.9\% of 3HV fraction. For the production of PHA and its copolyester in industrial scale, it is necessary to control preciesely PHAs composition which is important for the physical properties of PHAs polyesters. With respect to the change of P3HB-3HV composition, it was found that mole fraction of 3HV was dependent on initial nitrogen concentration. It seemed that 3HV synthesis from valeric acid by Methylobacterium sp. KCTC 0048, might be performed via NADH linked ketoacyl-CoA reductase in the cyclic $\beta$-oxidation pathway, which is independent of the presence of nitrogen source. Also, 3HV mole fraction was increased with culture temperature. By determination of the molecular weights and thermal properties of some PHAs produced by Methylobacterium sp. KCTC 0048 with GPC and DSC, those polyesters could be confirmed to be able to replace chemical plastics. And to improve productivity of PHAs by Methylobacterium sp. KCTC 0048, high cell density cultures with D.O STAT were carried out. The final cell concentration reached 78.3g/l and polymer contents in cells was 46.2\% at 93 hour, from valeric acid as precursor.
메탄올을 탄소원과 에너지원으로 이용하는 박테리아에 의해 생분해성 고분자 물질인 Polyhydroxyalkanoate(PHA)의 세포내 축적 가능성과 배양조건에 의한 PHAs의 조성의 영향, 그리고 생산성을 증대시키는 발효공정의 개발 및 물성실험등을 응용하고자 하였다. 사용된 균주는 Methylobacterium organophilum과 토양에서 분리, 선택하여 동정한 Methylobacterium sp. KCTC 0048이었다. Methylobacterium organophilum는 PHB를 생산할 수가 있었고 valeric acid를 전구체로 이용하여 P3HB-3HV 공중합체를 세포내에 축적하였다. 특히 Methylobacterium sp. KCTC 0048은 PHB는 물론, valeric acid와 pentanol 그리고 heptanoic acid를 전구체로 이용하여 P3HB-3HV를 생산할 수 있었고, 4-hydroxybutyrate, $\gamma$-butyrolactone, 1,4-butanediol을 이용하여 P3HB-4HB를 축적하였다. 또한 3-hydroxypropionic acid를 이용하여 P3HB-3HP를 생산하였다. P3HB-3HV 생산의 경우 조성의 변화가 크므로 P3HB-3HV 공중합체의 조성에 따른 물성에 대한 영향을 감안하여, 배양조건이 미치는 조성의 변화를 실험한 결과, 초기 질소원 농도와 배양온도에 따라 P3HB-3HV 공중합체의 조성이 영향 받음을 확인했다. 즉 초기질소원 농도가 증가함에 따라 3HV함량이 비례하여 증가하였고 이는 대사경로의 특이성에 기인하는 것으로 추론되었다. 또한 배양온도에 의해 3HV세포내의 함량이 증가하는 것으로 확인되었다. 이와 같이 배양조건에 의하여 공중합체 조성을 결정함으로써 고분자의 물성을 조절할 수 있음을 확인하였다. 그리고 생산성을 증대시키기 위하여 D.O. STAT를 이용한 고농도 세포배양 기술을 응용하여 건조세포 중량이 78.3g/1, 그리고 세포내 46.2\%의 P3HB-3HV 함량을 얻었다. 세포내에서 분리된 PHA의 분자량의 범위와 melting temperature를 측정함으로써 기존의 합성 플라스틱과 유사한 성질을 갖음을 확인하였다.
