Sulfide stress tolerance as a controller of methane production in temperate wetlands.

Wetlands are a major source of methane emissions and contribute to the observed increase in atmospheric methane over the last 20 years. Methane production in wetlands is the final step of carbon decomposition performed by anaerobic archaea. Although hydrogen/carbon dioxide and acetate are the substrates most often attributed to methanogenesis, other substrates-such as methylated compounds-may additionally play important roles in driving methane production in wetland systems. Here we conducted mesocosm experiments combined with genome-resolved metatranscriptomics to investigate the impact of diverse methanogenic substrate amendment on methanogenesis in two high methane-emitting wetlands with distinct geochemistry, termed P7 and P8. Methanol amendment resulted in high methane production at both sites, whereas acetate and formate amendment only stimulated methanogenesis in P7 mesocosms, where aqueous sulfide concentrations were lower. In P7 sediments, formate amendment fueled acetogenic microbes that produced acetate, which was subsequently utilized by acetoclastic methanogens. In contrast to expression profiles in P7 mesocosms, active methylotrophic methanogen genomes from P8 showed increased expression of genes related to membrane remodeling and DNA damage repair, indicative of stress tolerance mechanisms to counter sulfide toxicity. Methylotrophic methanogenesis generates higher free energy yields than acetoclastic methanogenesis, which likely enables allocation of more energy toward stress responses. These findings contribute to the growing body of literature highlighting methylotrophic methanogenesis as an important methane production pathway in wetlands. By using less competitive substrates like methanol that provide greater energy yields, methylotrophic methanogens may invest in physiological strategies that provide competitive advantages across a range of environmental stresses.