Search

Aquatic ecosystems are globally significant sources of the greenhouse gas methane (CH4) to the atmosphere. However, CH4is produced ‘paradoxically’ in oxygenated water via poorly understood mechanisms, fundamentally limiting our understanding of overall CH4production. Here we resolve paradoxical CH4production mechanisms through CH4measurements,δ13CH4analyses, 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 CH4production, as well as consistent metabolism of methylphosphonate by abundant bacteria—resembling observations from marine ecosystems. Metatranscriptomics andδ13CH4analyses applied to experimental treatments identified an additional CH4production 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 CH4in aquatic ecosystems.