Biogeochemical and omic evidence for paradoxical methane production via multiple co-occurring mechanisms in aquatic ecosystems

Aquatic ecosystems are globally significant sources of the greenhouse gas methane (CH4) to the atmosphere. However, CH4 is produced ‘paradoxically’ in oxygenated water via poorly understood mechanisms, fundamentally limiting our understanding of overall CH4 production. Here we resolve paradoxical CH4 production mechanisms through CH4 measurements, δ 13CH4 analyses, 16S rRNA sequencing, and metagenomics/metatranscriptomics applied to freshwater incubation experiments with multiple time points and treatments (addition of a methanogenesis inhibitor, dark, high-light). We captured significant paradoxical CH4 production, as well as consistent metabolism of methylphosphonate by abundant bacteria—resembling observations from marine ecosystems. Metatranscriptomics and δ 13CH4 analyses applied to experimental treatments identified an additional CH4 production mechanism associated with (bacterio)chlorophyll metabolism and photosynthesis by Cyanobacteria, and especially by Proteobacteria. Both mechanisms occured together within metagenome-assembled genomes, and appear widespread in freshwater. Our results indicate that multiple, co-occurring, and broadly-distributed bacterial groups and metabolic pathways produce CH4 in aquatic ecosystems. ### Competing Interest Statement The authors have declared no competing interest.