高-中温两相厌氧发酵体系中的微生物群落解析及互营关系研究

Anaerobic fermentation of organic matters to produce biogas is a complex biological conversion which is driven by the synergism of various microorganisms. The lack microscopic understanding of this process has become the bottleneck that limits the development of anaerobic fermentation technology. In this research, corn stalks and chicken manure are used as the mixed substrates and the process of thermophilic-mesophilic temperature phased anaerobic fermentation is applied as the experimental system. During the startup stage and long-term stable operation, the dynamic changes of the fermentative anaerobes in acidification phase, the acetogens and methanogens in methanation phase are quantitatively analyzed by real-time quantitative PCR, T-RFLP and 454 sequencing technology, combined with bioinformatics methods. The responses of the microorganisms to environmental factors are studied. The research emphasis is focused on exploring the key microbial interaction: the syntrophism between acetogens and methanogens. Syntrophism is strictly regulated by metabolic substrates. The effects of the important metabolic factors, hydrogen partial pressure, VFA as well as other factors are analyzed, and the dynamic changes of acetogens and methanogens are monitored. Based on the above results, the critical factors for the syntrophism can be determined and the syntrophic metabolism could be revealed. This research project will help to understand the anaerobic fermentation system from macro to micro, from the process conditions to chemical properties and further to microbiology. The conclusions can provide the scientific basis for the development of new technologies, and then promote the industry of anaerobic fermentation as well as natural gas production from biogas.

有机质厌氧发酵产沼气是由多种微生物协同作用的复杂的生物转化过程，对这一过程微观认识的不足已成为制约厌氧发酵技术发展的瓶颈问题。本项目以玉米秸秆和鸡粪的高-中温两相厌氧发酵作为实验体系，采用实时定量PCR、T-RFLP等分子生态学手段和454测序技术，结合生物信息学方法，定量的解析启动和长期稳定运行阶段产酸相的发酵细菌、产甲烷相的产乙酸菌和产甲烷菌的动态变化，研究它们对环境因子的响应规律，重点探究关键微生物的关键相互作用，即产乙酸菌和产甲烷菌的互营关系。互营受代谢底物的严格调控，本项目从代谢入手，研究氢分压、VFA等因素对互营关系的影响，并通过监测产乙酸菌和产甲烷菌的动态变化，分析互营的关键因素，揭示其作用机制。本项目的研究有助于建立对厌氧发酵体系从宏观到微观，从工艺条件到化学特性再到微生物学的全面认识，为发展新技术提供科学基础，推动厌氧发酵消纳有机废弃物并生产天然气产业的发展。

有机质厌氧发酵产沼气是由多种微生物协同作用的复杂的生物转化过程，对这一过程微观认识的不足已成为制约厌氧发酵技术发展的瓶颈问题。本项目从高-中温两相厌氧发酵工艺切入，并相继开展了两相独立运行实验、干发酵产酸-生物膜产甲烷两相厌氧发酵工艺研究，然后将重点放在产甲烷相的研究上，以氢气和二氧化碳为底物，强化氢营养型产甲烷途径，实现了高浓度甲烷的稳定连续生产。在这些实验体系的运行中，监测产酸相和产甲烷相的特性，解析微生物群落的变化演替，分析互营机制，调控代谢途径，形成对厌氧发酵从宏观到微观的全面认识。取得的主要研究成果有三点。一是在产甲烷相实现高浓度甲烷的稳定连续生产。从高-中温两相厌氧发酵、干发酵产酸-生物膜产甲烷两相厌氧发酵、氢气/二氧化碳生物甲烷化等实验体系，将产品气中的甲烷浓度由60%提高至80%，再继续升高至90%，最终实现浓度为99%甲烷气的稳定连续生产。这项技术既可以原位提纯厌氧发酵工程的沼气，也可以在二氧化碳固定、废氢利用等领域进行应用。二是完成多个产酸相和产甲烷相的微生物解析。解析了细菌和古菌的群落结构，研究启动阶段、稳定阶段及其他操作条件下的微生物和代谢途径的动态变化，了解它们对温度、VFA、氢分压等环境因子的响应规律。三是研究关键微生物的相互作用，调控代谢途径。在不同的厌氧发酵体系中，结合工艺特点，运用多种方法研究关键微生物的相互作用，调控并强化目标代谢途径。在高-中温两相厌氧发酵中，初步研究了多种导电纳米颗粒对微生物互营和直接电子传递的影响；在产酸相中，通过pH变化实现对不同产酸类型的调控；在产甲烷相中，联合稳定同位素探测技术，分析反应各阶段的甲烷化途径；对氢营养型产甲烷菌进行驯化富集，强化氢气和二氧化碳转化为甲烷的代谢途径，实现高浓度甲烷气的生产。