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 (CH 4 ) to the atmosphere. However, CH 4 is produced ‘paradoxically’ in oxygenated water via poorly understood mechanisms, fundamentally limiting our understanding of overall CH 4 production. Here we resolve paradoxical CH 4 production mechanisms through CH 4 measurements, δ 13 CH 4 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 CH 4 production, as well as consistent metabolism of methylphosphonate by abundant bacteria—resembling observations from marine ecosystems. Metatranscriptomics and δ 13 CH 4 analyses applied to experimental treatments identified an additional CH 4 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 CH 4 in aquatic ecosystems.