【作者】 董静；

【导师】 辛嘉英；

【作者基本信息】 哈尔滨商业大学 ， 食品科学， 2013， 博士

【摘要】 聚p-羟基丁酸酯(PHB)是大多数细菌细胞内碳源和能源的储备物,具有生物可降解性,热塑性,且透氧率低,用作食品包装用膜、袋,饮料用包装内衬层和生鲜食品盘等,更利于食品的储藏运输,在食品包装领域具有广阔的应用前景,日益受到人们的关注。目前,PHB的商业化生产主要基于野生菌和基因工程菌的纯菌种发酵工艺,严格控制的无菌底物和生产环境导致了较高的生产成本,从而限制了生物塑料PHB的大规模应用,特别是在食品包装中的应用。富集高产PHB、组成稳定的混合菌在开放条件下,以甲烷甲醇等一碳化合物生产PHB成为目前实现PHB在食品包装领域中规模化应用最具前景的研究课题。本学位论文利用从黑龙江省大庆油田富含沼气地区的土壤中培养筛选获得的甲烷氧化混合菌,以甲烷为碳源通过好氧瞬时供料方式(充盈-饥饿交替),筛选掉其中PHB含量少的菌株,对菌群中开放条件下稳定高产PHB的甲烷氧化菌进行了富集。通过甲烷氧化混合菌的结构解析,对菌群中分离纯化的非甲烷氧化菌和甲烷氧化菌的生长和催化性能的初步探索,以及优化细胞培养环境,高密度甲烷氧化混合菌发酵培养方式的探索,建立了开放条件下以廉价碳源稳定合成PHB的方法。(1)根据甲烷氧化混合菌细胞生长规律,建立基于Logistic模型的细胞生长动力学模型：经对比验证结果表明,对于不同的培养条件,该动力学模型对细胞生长密度(用吸光度OD600表示)的计算值与实验值之间都具有较好的相关性,相关系数均达到92%以上。这表明应用" Logistic模型”建立的动力学模型对甲烷氧化混合菌具有较好的适用性,可以进行实际工作中的模拟研究,该模型中各参数和特征值随培养条件而变化,适用于发酵条件优化控制,为后续细胞高密度培养提供指导。(2)采用充盈-饥饿(feast-famine cycle)模式间歇供料,以甲烷为底物好氧开放式培养甲烷氧化混合菌,利用苏丹黑染色法动态检测充盈和饥饿阶段胞内PHB含量变化,实现了菌群中高PHB存储能力的甲烷氧化菌的富集和稳定传代。结果显示,在充分供氧条件下,充盈培养5d,饥饿培养15d,循环培养5次,PHB含量从10.8%增加到36.6%,且富集后甲烷氧化混合菌经过开放条件下连续传代培养,细胞生长和PHB合成稳定,表明甲烷氧化混合菌具有菌群组成稳定性,适于PHB工业化生产。(3)通过对富集后的甲烷氧化混合菌进行菌群结构及生长生长特性分析,推测了混合菌体系中不同微生物间的协同作用机理：甲基弯菌JW和JC能以甲烷、甲醇作为碳源生长,并为伴生菌提供从甲烷、甲醇得到的代谢碳供伴生菌生长,伴生菌的存在可以及时利用代谢中间产物有效避免产物抑制,同时为甲基弯菌JW和JC提供生长因子,促进细胞生长。甲烷氧化混合菌分离出来的重要成员甲基弯菌JC能高效利用甲醇生长,能增强甲烷氧化混合菌对甲醇的耐受性,促使混合菌体系无需驯化直接利用甲醇为碳源生长,避免由于甲烷在培养基中溶解度低而导致细胞生长慢的问题,为高密度培养打下基础。基于物种间的自然选择和竞争,以碳饥饿作为选择压力,通过甲烷为碳源短期充盈(5d)长期饥饿(15d)循环交替的富集方式,形成了能长期耐受碳饥饿的高PHB存储能力的甲烷氧化菌-伴生菌共生的微生态系统,其在开放条件下也不易受到杂菌的污染,更具环境适应性而长期稳定生长并积累PHB。(4)在掌握了细胞的基本生长规律和胞内PHB积累能力的基础上,利用富集后的甲烷氧化混合菌以廉价碳源甲烷、甲醇生长的优势,通过调整碳源供给、培养基组成等手段优化胞外生长环境促进胞内PHB积累,以少量甲醇的加入在原有基础上大幅提高了细胞产量及胞内PHB的积累能力。对营养平衡-营养受限两阶段培养的最佳培养基组分和培养时间的实验结果表明,在相同的碳源补加方式下,优化后的营养平衡(培养6d)-营养受限(培养6d)的两段式培养方式不仅可以明显提高PHB的含量,最高可达61.5%,而且并没有完全抑制细胞生长,细胞干重达到1.35g/L,实现了细胞生长密度和单位细胞PHB积累量的提高,在一定程度上解决了细胞生长与PHB积累的矛盾。(5)甲烷氧化混合菌可在开放条件下利用甲醇代替甲烷做为唯一碳源生长并在细胞内合成PHB,且甲醇能够较大程度的促进菌体生长和PHB的胞内积累。通过对富集后甲烷氧化混合菌在发酵罐培养过程中细胞生长动力学的研究,以延滞期和最大比增长速度为依据,对发酵罐的碳源补加量和补加方式进行优化,建立提高PHB产量为目的甲醇流加策略,通过分批补加不同浓度碳源(甲醇),解除高浓度甲醇的抑制作用,为菌体生长和PHB的胞内合成提供适宜的碳源利用环境,从而明显缩短发酵时间,提高甲烷氧化混合菌胞内PHB的积累量。根据发酵罐培养细胞的生长规律,建立了开放条件下一段式的发酵罐发酵生产PHB的方法,简化了操作步骤,提高了生产效率。经过10d的分批发酵培养,细胞干重可达3.1g/L, PHB含量提升至78.4%。更多还原

【Abstract】 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.更多还原