Biosynthesis Of Poly (β-Hydroxybutaryte)by Methane-Utilizing Mixed Culture

Poly-β-hydroxybutyrate (PHB) can be produced by various species of bacteria as carbon and energy storage materials. This biopolyester has attracted increasing attentions as biodegradable plastics not only for its similar material properties to conventional plastics but also for its biodegradable properties. PHB has thermoplastic, can be used for food packaging film bag, packaging lining layer for drinks and fresh food tray, etc. Furthermore, PHB has lower oxygen and moisture permeability, which is benefit for food storage and transportation, so that it has a broad application prospect in the field of food packaging. Commercial production of PHB is currently based on pure culture processes employing either natural PHB producers or genetically modified bacteria. Pure culture processes use generally pure sterile substrates and axenic reactors, leading to high production costs and thus relatively expensive products, which have prevented the use of this material on a real industrial scale, especially in the field of food packaging. Much effort needs to be devoted to develop a process for the enrichment of a mixed bacterial culture with a high PHB storage capacity to produce PHB in an open system efficiently and stably, which is great important for the large-scale application of PHB in the field of food packaging.In this dissertation, Methane-utilizing mixed culture with a high PHB storage capacity was gained from the biogas soil at DA Qing oil field as enriching source after domestication with the approach of the aerobic dynamic feeding(ADF, feast-famine condition). The community structure of Methane-utilizing mixed culture and the catalytic performance of the heterotrophs and methanotrophic bacteria isolated from the mixed culture were investigated. In order to increase the yield of biomass and yield of PHB, optimized culture condition for the cell, the fermentation way to product PHB were developed. New and efficient methods for biosynthesis of PHB with Methane-utilizing mixed culture, which can utilize low-cost Cl carbon sources to accumulate PHB intracellular, were established in an open system.(1)The growth kinetic model was constructed according to the growth regularity of Methane-utilizing mixed culture and Logistic model and showed as follows:The result of contrasting experimental values to the simulation data with the above formula of cell density (indicated with Absorbance OD600) under different culture condition indicated that the simulation data in the kinetic model had good relevance with the experimental values. All the relevant coefficients were higher than0.92. Such results demonstrated that the kinetic model constructed based on the "Logistic model" was valuably applied to the Methane-utilizing mixed culture. Because the characteristic values of the parameters in the model changed with the culture conditions, which is suitable for optimization control of fermentation condition, the simulation formula could be realized in the practical and could offer some guidance for high cell density cultivation.(2)The approach of the aerobic dynamic feeding(ADF, feast-famine condition) was used to enrich Methane-utilizing mixed culture with a high PHB storage capacity and Sudan black was used to detect the content of PHB. The results show the content of PHB in biomass increased from10.8%to36.6%after five feast-famine cycles of5d feast and15d famine cultivation under aerobic condition. The enriched Methane-utilizing mixed culture can grow and produce PHB stably for a long time in an open system, which is suitable for PHB industrialized production.(3)Through analyzing the community structure of Methane-utilizing mixed culture and the catalytic performance of methanotrophic bacteria and heterotrophs isolated from the mixed culture, it is speculated that Methylosinus trichosporium JW, JC can utilize methane or methanol as carbon source for growth and provide carbon metabolized from methane or methanol for accompanying bacteria in mixed culture. The presence of accompanying bacteria in mixed culture improve the physiological growth conditions for Methylosinus trichosporium JW, JC and maintain the stability of community structure and function through both the removal of toxic metabolites excreted by Methylosinus trichosporium JW, JC and the provision of essential growth supplements. Methylosinus trichosporium JC were identified as potentially important members of the community, which utilize methanol as carbon source with high efficiency. It was found the presence of Methylosinus trichosporium JC make Methane-utilizing mixed culture utilize methanol as carbon source to grow without any domestication, which lay the foundation for high density culture, avoiding the low cell growth due to low methane solubility in the medium. The repeatedly alternating presence (feast phase) and absence of the methane (famine phase), as is typically established in a repeated batch cultivation mode engineer a methanotrophic-heterotrophic community based on natural selection and competition, which do not seem to be susceptible to invasions by contaminating strains although the cultivation is carried out in an open system.(4) Taking advantage of the ability of enriched Methane-utilizing mixed culture utilizing low-cost Cl carbon source for growth and accumulation of PHB, an optimized condition for the strains to increase biomass and PHB accumulation in cell was developed. By optimizing the supply of carbon sources and the composition of the culture medium, high cell density and yield of PHB can be obtained with small amount of methanol addition. The medium and culturing time under nutrients sufficient-nutrients deficiency condition were optimized. Under optimized two-step culturing condition, biomass and PHB content increased to1.35g dry wt/L,61.5%(w/w) respectively, which solved the contradictions of cell growth and PHB accumulation to a certain extent.(5)Methane-utilizing mixed culture could use methanol as sole carbon source for cell gowth and PHB accumulation in an open system. And the concentrations of cells and PHB were both increased by using methanol as carbon source. Based on the important characteristic parameter (lag phase and maximum specific growth rate) in cell growth kinetic model, the optimal methanol feeding strategy was established, which would be beneficial to shorten fermentation time and increase the yield of PHB. With the results of the study in the fermentation of Methane-utilizing mixed culture, a new fermentation strategy, one-step fed-batch fermentation was developed for the production of PHB with high content and good productivity in a simplified way.The biomass of3.1g dry wt/L and PHB content of78.4(w/w) were obtained in the10d’s fed-batch fermentation.