污泥固体厌氧发酵反应器内甲烷累积的微生态机理研究

Biomethane, as a clean renewable energy that promises to be a good substitution for traditional fossil fuels in the future, has become an increasingly attraction due to global energy crisis. In various technologies for producing biomethane, solid-state fermentation technology has increasingly attracted some researchers in some developed countries interests. Solid-state fermentation is characterized by the high solid content of the feed stocks to be digested, which is typically greater than 20%. However, anaerobic efficiency of biomethane production is still not high due to the lack of understanding the accumulative methane mechanism during the whole solid-state fermentation. Hence, to improve biomethane yield through solid-state fermentation in a biomethane reactor, a better understanding of microbial community composition and interactions between these and other microorganisms as well as methanogenic mechanism is required. Based on the above analyse, this project will firstly build a total DNA extraction method for metagenomic analyses in a solid-state anaerobic reactor, and this method will able to differentiate between live and dead anaerobic microbiology. And then Metagenomics which is the latest high-throughput sequencing technology (454 sequencing) will be used to clarify the molecular microbial ecology mechanism of biomethane accumulation in the whole solid-state fermentation process. By analyzing the relationship between the number, structure and activity of anaerobic microorganisms and the accumulation of biomethane during the whole solid-state fermentation, the contribution of each functional anaerobic microorganisms on the accumulation of biomethane will be clarified, and the original state of anaerobic microorganisms in the process of solid-state fermentation will also be accurately restored. Meanwhile, the main metabolic pathway and metabolic flux for producing biomethane will be investigated by combining the means of stoichiometry, and finally biomethane production and accumulative mechanism will be explored.

厌氧发酵技术生产清洁的生物质能源即甲烷是实现污泥中有机质资源化再利用的一条新途径，业已引起一些发达国家研究者的高度重视。为提高污泥中有机质转化为甲烷的效率并增加甲烷的累积量，需要对污泥固体厌氧消化反应器中甲烷的累积机理进行详细的研究。本项目在构建能够区分活细胞和死亡细胞DNA片段以及适合于宏基因组学研究的总DNA提取方法基础上，采用基于最新的高通量测序技术（454测序）的宏基因组学研究方法阐明污泥固体厌氧发酵生产甲烷过程的微生物分子生态学机理，通过分析污泥固体厌氧发酵反应器内微生物的数量、结构以及活性和甲烷累积之间的关系，阐明它们对甲烷累积的贡献，并准确还原发酵过程厌氧微生物的原始状态。同时，结合化学计量学手段研究污泥固体厌氧反应器中的主要代谢途径和代谢通量，阐明固体发酵过程污泥厌氧反应器内甲烷的产生和累积机理。

城市污泥产生总量随着国民经济持续快速发展迅速地增加，因此发展经济有效的污泥处理处置技术迫在眉睫。生物甲烷作为一种清洁能源，被视为未来传统的石油燃料的替代品，其生产已备受关注。固体厌氧发酵技术被认识是一种有效的从废弃有机物中生产甲烷的工艺。然而，由于甲烷累积机理尚不明了，固体厌氧发酵产甲烷效率有待提高。本项目在确定适合于宏基因组学研究的总DNA提取方法基础上，采用高通量测序技术阐明污泥固体厌氧发酵生产甲烷过程的微生物分子生态学机理，通过分析污泥固体厌氧发酵反应器内微生物的数量、结构以及活性和甲烷累积之间的关系，阐明它们对甲烷累积的贡献。同时，结合化学计量学手段研究了污泥固体厌氧反应器中的主要代谢途径和代谢通量，阐明固体发酵过程污泥厌氧反应器内甲烷的产生和累积机理。通过比较手提法、试剂盒直接提取法和手提结合试剂盒提取方法对污泥固体厌氧反应器内发酵产甲烷微生物样品总DNA的提取效率与提取质量，确定手提结合试剂盒提取法为后续固体厌氧发酵产甲烷样品微生物总DNA提取方法。虽然乙酸、总酸、Na+、NH4-N、Cl-及SO42-浓度在固体厌氧发酵条件下达到最大，分别为9.7g/L、17.3g/L、249 mg/L、6111 mg/L、1656 mg/L及1802 mg/L，但厌氧发酵产甲烷过程仍维持稳定。高通量测序结果表明，污泥固体质量百分比可显著影响产甲烷菌群的结构，但对厌氧细菌菌群结构的影响并不显著。在TS为20%时，Methanosarcina属成为厌氧体系中占绝对优势的产甲烷菌属。而，在其它固体质量百分比条件下，除Methanosarcina属，Methanosaeta属和Methanobacterium属亦成为产甲烷的优势菌属。组间差异显著性检验结果进一步表明，Methanosarcina属的产甲烷古菌在不同固体质量百分比条件下呈现显著的丰富度差异。结合葡萄糖酵解产乙酸途径中的关键酶即乙酸激酶及磷酸转乙酰酶比酶活与蛋白质转换为乙酸的化学计量学分析结果，污泥固体厌氧发酵过程中产生的乙酸可能主要是来源于蛋白质降解形成的氨基酸之间的Stickland反应。而，高通量测序结果分析表明固体厌氧发酵过程产生的甲烷途径包括还原CO2途径和乙酸途径。