In many anoxic environments, syntrophic acetate oxidation (SAO) is a key pathway mediating the conversion of acetate into methane through obligate cross-feeding interactions between SAO bacteria (SAOB) and methanogenic archaea. The SAO pathway is particularly important in engineered environments such as anaerobic digestion (AD) systems operating at thermophilic temperatures and/or with high ammonia. Despite the widespread importance of SAOB to the stability of the AD process, little is known about their in situ physiologies due to typically low biomass yields and resistance to isolation. Here, we performed a long-term (300-day) continuous enrichment of a thermophilic (55 °C) SAO community from a municipal AD system using acetate as the sole carbon source. Over 80% of the enriched bioreactor metagenome belonged to a three-member consortium, including an acetate-oxidizing bacterium affiliated with DTU068 encoding for carbon dioxide, hydrogen, and formate production, along with two methanogenic archaea affiliated with Methanothermobacter_A. Stable isotope probing was coupled with metaproteogenomics to quantify carbon flux into each community member during acetate conversion and inform metabolic reconstruction and genome-scale modeling. This effort revealed that the two Methanothermobacter_A species differed in their preferred electron donors, with one possessing the ability to grow on formate and the other only consuming hydrogen. A thermodynamic analysis suggested that the presence of the formate-consuming methanogen broadened the environmental conditions where ATP production from SAO was favorable. Collectively, these results highlight how flexibility in electron partitioning during SAO likely governs community structure and fitness through thermodynamic-driven mutualism, shedding valuable insights into the metabolic underpinnings of this key functional group within methanogenic ecosystems.

在许多缺氧环境中，互养乙酸盐氧化 (SAO) 是通过 SAO 细菌 (SAOB) 和产甲烷古菌之间的专性交叉摄食相互作用介导乙酸盐转化为甲烷的关键途径。 SAO 途径在工程环境中尤其重要，例如在高温和/或高氨条件下运行的厌氧消化 (AD) 系统。尽管 SAOB 对 AD 过程的稳定性具有广泛的重要性，但由于通常较低的生物量产量和对分离的抵抗力，对其原位生理学知之甚少。在这里，我们使用醋酸盐作为唯一碳源，对市政 AD 系统中的嗜热 (55 °C) SAO 群落进行了长期（300 天）连续富集。超过 80% 的富集生物反应器宏基因组属于一个三成员联合体，其中包括一种与 DTU068 相关、编码二氧化碳、氢气和甲酸生产的乙酸盐氧化细菌，以及两种与Methanothermobacter_A相关的产甲烷古菌。稳定同位素探测与宏蛋白质组学相结合，可量化醋酸盐转化过程中进入每个群落成员的碳通量，并为代谢重建和基因组规模建模提供信息。这项工作表明，两种Methanothermobacter_A物种在其首选电子供体方面有所不同，其中一种具有在甲酸上生长的能力，而另一种仅消耗氢气。热力学分析表明，消耗甲酸的产甲烷菌的存在扩大了有利于 SAO 生产 ATP 的环境条件。 总的来说，这些结果凸显了 SAO 期间电子分配的灵活性如何通过热力学驱动的互利共生来控制群落结构和适应性，为产甲烷生态系统中这一关键功能组的代谢基础提供了宝贵的见解。