Biogeochemical and omic evidence for multiple paradoxical methane production mechanisms in freshwater lakes

Aquatic ecosystems are globally significant sources of the greenhouse gas methane (CH) to the atmosphere. However, CH is produced ‘paradoxically’ in oxygenated water via at least three mechanisms, fundamentally limiting our understanding of overall CH production. Here we resolve these CH production mechanisms through CH measurements, CH analyses, 16S rRNA sequencing, and metagenomics/transcriptomics applied to freshwater incubation experiments with multiple time points and treatments (addition of a methanogenesis inhibitor, dark, high-light). We captured significant paradoxical CH production, but show that methanogenesis was an unlikely CH source. In contrast, abundant freshwater bacteria metabolized methylphosphonate—similar to observations in marine ecosystems. Metatranscriptomics and stable isotopic analyses applied to experimental treatments also identified a potential CH production mechanism linked to (bacterio)chlorophyll metabolism by Cyanobacteria and especially Proteobacteria. Variability in these mechanisms across experiments indicates that multiple, widely-distributed bacterial groups and pathways can produce substantial quantities of CH in aquatic ecosystems.