Groundwater Nitrogen Fixation Is Associated with Methane and Sulfur Cycling

Nitrogen fixation is an energy-intensive process critical for microbial survival in nitrogen-depleted environments. Groundwater, the Earth's largest accessible freshwater reservoir, is typically oligotrophic. However, recent studies have identified productive groundwater habitats where nitrogen fixation may become important to support the productivity. This study investigated the diversity, activity, and metabolisms of diazotrophs in groundwater through 25 metagenomes and five proteomes. We identified 203 nonredundant nitrogenase iron proteins (NifH) and 140 diazotroph genomes. They were affiliated with diverse bacterial and archaeal taxa and could comprise half of the community. Methanotrophic <i>Methylomonadaceae</i> emerged as the most abundant and active diazotrophs in methane-rich groundwater systems. These microorganisms, along with diazotrophic methanogens, anaerobic methanotrophs, and methylotrophs, highlighted a strong linkage between nitrogen fixation and the methane cycle. In addition, sulfur-cycling diazotrophs, including chemoautotrophic and heterotrophic sulfur oxidizers and sulfate reducers, were ubiquitous and showed metabolic versatility in varying geochemical conditions. Proteomes further suggested active nitrogen fixation by both methane- and sulfur-associated diazotrophs. This study highlights nitrogen fixation as an important process in groundwater, especially in aquifers where nitrogen-depleted methane, produced by methanogens, serves as a major carbon source.