【作者】 刘英杰；

【导师】 班睿； 贾晓强；

【作者基本信息】 天津大学 ， 生物化工， 2014， 硕士

【摘要】 基于环境资源方面的考虑，利用微生物混合菌群以木糖为底物发酵生产PHA越来越受到人们的关注。混合菌群由于成员间的分工合作，能够承受较高的代谢负荷和具有较高的鲁棒性，在低值和复杂原料利用方面与纯培养微生物相比具有优势。木糖是木质纤维素中第二丰富的糖类，开展利用木糖发酵生产PHA的研究，可以为木质纤维素原料资源化奠定基础。传统的混合菌群合成PHA的三段式工艺过程较为繁琐，加之无法建立有效的筛选压力，造成运行周期长，底物转化率低，无法达到工业化的要求。针对上述问题，本课题以木糖为例，探究混合菌群一步生产PHA的方法。首先以木糖为底物，使用饱食饥饿模式结合尼罗蓝染色的筛选方法进行木糖产PHA混合菌群的驯化，最终获得具有较强PHA合成能力的混菌体系。通过参数优化，发现该体系在温度为33℃、pH为8、木糖浓度为2.4g/L、碳氮比为160以及碳磷比为125时具有最大的PHA生产能力，最终积累的PHA占到了细胞干重的31%。继而通过DGGE分析了驯化过程中菌落结构的变化，阐明了混合菌群演变的过程，DGGE条带测序结果表明最终的稳定体系中主要存在γ-Proteobacteria、Cellvibrio sp.、Uncultured bacterium和Pseudomonasputida等4种优势微生物。为所筛选得到混合菌群的进一步优化奠定了基础。为深入研究体系中混合菌群间的相互作用关系，以驯化结束的混菌体系为样本，筛选得到γ-Proteobacteria、Cellvibrio sp.、Pseudomonas sp.、Enterobacter cloacae sp.和Bacillus sp.5种类型的微生物，但是由于不可纯培养等原因，体系中一些重要的具有PHA合成能力微生物无法分离得到，导致这些菌株混合后所形成混合菌群的PHA合成能力十分有限。继而研究尝试将一些优良的木糖利用基因和PHA合成基因导入到大肠杆菌等宿主细胞中构建基因工程菌株，从而构建利用木糖合成PHA的人工混合菌群，初步发酵结果显示其具备一定的PHA合成能力，该方法为利用木糖为底物生产PHA以及研究混菌体系中细胞相互作用关系提供了新的思路。更多还原

【Abstract】 The production of PHA from cellulose by using mixed cultures gains more andmore attention, based on the consideration of environment and resources. Xylose isthe second most abundant carbohydrate in lignocellulose materials, ranking onlysecond to glucose, the research of PHA production from which could lay a solidfoundation for reclamation of the cellulose-based substrates. However, thetraditional three-stage process for PHA synthesis by mixed cultures is cumbersome,coupled with the lackage of effective screening pressure, which combined resultingin a long running period as well as a low substrate conversion rate. Thus it isunable to meet the requirements of industrialization. Focusing on the problem, thisstudy takes xylose as an example for exploring direct PHA production by mixedcultures.Taking xylose as substrate, Feast-famine processes combining Nile bluestaining method were used for screening mixed cultures for PHA production. Themixed cultures possesses strong PHA synthesis ability was obtained ultimately. Byoptimizing the parameters, it was found that the optimal conditions for maximumPHA production capacity of the mixed cultures were as follows: temperature33°C,pH8, xylose concentration2.4g/L, C/N ratio160and C/P ratio125. The finalPHA accumulation was up to31%of dry cell weight. Then the structure changesof mixed microbial cultures were analyzed by DGGE, and the evolution process ofmixed cultures was clarified. Sequencing results of DGGE bands showed that thefinal stable system mainly existed four kinds of advantage microorganisms:γ-Proteobacteria, Cellvibrio sp., Uncultured bacterium and Pseudomonas putida.To further research the interaction among mixed cultures, the system afterdomestication was used as sample to screen microorganisms and γ-Proteobacteria,Cellvibrio sp., Pseudomonas sp., Enterobacter cloacae sp. and Bacillus sp. wereobtained finally. However, some important microorganisms with high ability forPHA synthesis were failure to be separated due to the reason that thesemicroorganisms could not be successfully cultured, which led to the limitless PHAsynthesize ability of these microorganisms after being mixed. Further research hadbeen performed for transforming excellent xylose utilization genes and PHA synthesis genes into hosts (E. coli et al) to construct genetically engineered strains.We tried to mix them together to build artificial microbial consortia for PHAproduction using xylose as sunstrate. Preliminary fermentation results showed thatit possessed the certain PHA production ability. This method provided a new wayfor PHA production from xylose and the research of interaction relationship amongmixed cultures in systems.更多还原