In-Situ Ammonia Recovery in Anaerobic digestion via Gas Permeable Membranes: Reshaping Dominant Methanogens under Ammonia Stress through Divergent Insights from Microbiomics, Metagenomics, and Metaproteomics

Mechanistically, this study reconstructs the dominant methanogens under ammonia stress through insights from microbiomics, metagenomics, and metaproteomics. The research identifies Methanobacterium as a "pseudo dominant genus" under ammonia stress—large in population but low in contribution. The significant downregulation of electron transfer efficiency and methyl-reducing protein expression in Methanobacterium may be key factors limiting its activity. Conversely, the study confirms Methanothrix as the "true dominant genus" under ammonia inhibition—reduced in population but undiminished in contribution. Methanothrix expresses all proteins required for acetoclastic and hydrogenotrophic methanogenesis and upregulates functional proteins related to post-translational modifications, tRNA activation, and initiation complex formation during enzyme synthesis. Finally, metaproteomics unveiled the presence of "exceptionally active genera" with remarkably low community abundance—specifically, Methanolinea, Methanospirillum, and unclassified_f__Methanoregulaceae. Their protein expression levels in the methanogenesis pathway surpassed those of Methanobacterium, establishing them as the primary drivers of hydrogenotrophic methanogenesis.从机制层面，本研究通过微生物组学（microbiomics）、宏基因组学（metagenomics）及宏蛋白质组学（metaproteomics）的研究视角，重构了氨胁迫下的优势产甲烷菌（methanogens）群落。研究发现，氨胁迫下Methanobacterium属为“假优势属”——种群数量庞大但贡献度较低。该属中电子传递效率显著降低，且甲基还原蛋白表达量大幅下调，这可能是限制其活性的关键因素。相反，研究证实氨抑制条件下Methanothrix属为“真优势属”——种群数量减少但贡献度未受影响。Methanothrix属可表达产乙酸型和氢营养型产甲烷作用所需的全部蛋白质，并上调与翻译后修饰、tRNA激活及酶合成过程中起始复合物形成相关的功能蛋白。最后，宏蛋白质组学揭示了群落丰度极低但“异常活跃”的菌属存在——具体包括Methanolinea属、Methanospirillum属及未分类的Methanoregulaceae科（unclassified_f__Methanoregulaceae）。这些菌属在产甲烷途径中的蛋白质表达水平超过Methanobacterium属，成为氢营养型产甲烷作用的主要驱动者。