基于组装基因组解码和高分辨率显微镜技术研究互营乙酸氧化菌群及种间电子传递模式

During anaerobic digestion of biowaste, syntrophic acetate-oxidizing bacteria (SAOB) and the cooperating methanogens are demonstrated to be the core microbial populations for maintaining the process stability which can tolerate the stress from accumulated organic acids and ammonia. Interspecies electron transfer (IET) between these bacteria and archaea is the key step for the conversion of acetate to methane. However, the diversity of this functional group and the syntrophy mechanism are still unclear. This project aims to identify the predominating bacterial and archaeal species capable of syntrophic acetate oxidation (SAO) function and investigate the IET models between them. SAO functional group will be enriched from industrial anaerobic digesters in China, the diversity will be investigated and the dominant species with high abundance will be identified. To study the physiology of such key populations, high quality metagenome-assembled genomes (MAG) will be constructed and annotated for them. Specific probes will be designed to target them which will be used together with fluorescence in situ hybridization and high resolution microscopy, the detailed structure of the microbial cells and the contacting status between the bacterial and archaeal cells will be observed in situ. The IET models will then be illustrated from the aspects including genetic potential, metabolic pathways and morphology. A complete syntrophy mechanism between bacteria and methanogens involved in SAO will be established, which will help to develop technologies focusing on enhancing and maintaining high SAO activities in anaerobic digestion of biowaste.

生物质废物厌氧消化过程中，互营乙酸氧化细菌和伴生产甲烷菌可耐受高浓度酸和氨胁迫，是维持工艺稳定运行的核心微生物类群。两种群间的电子传递过程是乙酸向甲烷转化的关键步骤。但该功能类群的多样性及种间互营机制尚不清晰。本项目以互营乙酸氧化菌群的多样性分析、优势种类识别及其与伴生古菌之间的电子传递模式为研究出发点，拟从工业规模反应器中富集该功能类群，识别关键细菌和古菌种类，并对其生理生态进行深入分析。将采用宏基因组-组装基因组技术构建其高质量基因组并解码；设计特异性探针对其进行靶向定位，结合荧光原位杂交和高分辨率显微镜对其细胞的细微结构、细菌和古菌间的接触形态进行原位观测；对互营细菌和古菌间的电子传递机制从基因、代谢途径和形态学角度进行全方位解读。本项目拟为互营乙酸氧化菌群的互营生存机制建立完整的理论体系，可为发展以提高和维持高活性互营乙酸氧化产甲烷能力为核心目标的技术方法提供理论依据。

厌氧消化是易腐有机废弃物减量化与资源化处理的主流技术工艺。互营乙酸氧化细菌和伴生产甲烷古菌可耐受该过程所累积的高浓度氨和有机酸，是维持工艺稳定运行的核心微生物类群。但尚缺乏对实际工程系统中该功能类群物种多样性和碳转化机制的深入研究。本项目以阐明其物种多样性，精准识别未培养关键物种，分析菌种间的互营机制为目标，(1)系统比较了14个工业反应器中驱动餐厨垃圾和剩余活性污泥厌氧消化产甲烷的微生物群落，识别了七十余个属级别的核心种群，发现了多样化﹑具有互营乙酸氧化功能潜力的菌种；结合气体稳定碳同位素分析，揭示了优势产甲烷途径和产甲烷菌种类；聚焦于氨﹑pH﹑盐度﹑有机酸等环境因子的影响，阐明了导致优势种群和代谢途径差异化的驱动因子。(2)从餐厨垃圾厌氧消化微生物群落中，以乙酸为有机碳源，富集培养出乙酸氧化耦合氢或甲酸营养型产甲烷功能类群，并组装出关键物种的基因组；基于基因解码和代谢途径重构，发现系统内共存着两种乙酸氧化代谢方式(独立的或者结合甘氨酸裂解的逆向Wood-Ljungdahl途径)，功能菌种多为未培养新型种类(如DTU022)，且新型种类的丰度高于已知种类。结合组装基因组引导的宏转录组分析，展示了乙酸氧化、产甲烷相关代谢途径和种内、种间电子传递功能基因的赋存与表达。(3)利用多种同位素标记示踪方法，对中温和高温﹑驯化与未驯化﹑不同氨浓度下，乙酸向甲烷的转化途径与过程进行量化分析；阐明了乙酸氧化互营菌群逐渐替代乙酸发酵型产甲烷古菌成为优势种群的动态过程和主控因子，揭示了引发优势种群演替的氨浓度阈值为200mg/L,并精准识别了新型物种；应用FISH与NanoSIMS技术，展示了多种功能菌种的细胞形态﹑空间分布和对乙酸﹑无机碳﹑氮的同化过程。成果为基于生物强化，优化易腐有机废弃物厌氧消化工艺提供了方法基础和理论依据，对新型互营菌种的发现和特征研究也具有重要的生物学意义。