Organic Aggregate Microniches Govern Oxic Methane Emissions via Distinct Methanogenic Archaea and Bacteria Pathways

Lakes are hotspots for natural methane (CH₄) emissions. However, the microbial processes driving CH₄ production in oxygenated surface waters remain unclear. We investigated CH₄-producing microorganisms associated with organic aggregates (OAs) fractionated into four sizes (>112, 64-112, 5-64, and 0.2-5 μm) across five ecologically diverse lakes. Using qPCR and amplicon sequencing, we quantified methanogenic archaea (mcrA gene) and bacteria (phnJ gene) and identified their environmental drivers. Gene absolute abundances were significantly affected by the lake type, OAs size, and their interaction. Both mcrA and phnJ increased with lake trophic status, following a size-dependent pattern: decreasing from >112 to 64-112 μm, then increasing to peak in the 0.2-5 μm. Overall, mcrA absolute abundance (2.17-4.23 × 10⁵ copies/mL) exceeded phnJ (0.98-1.16 × 10⁵ copies/mL), particularly in the >112 and 0.2-5 μm fractions. Organic matter characteristics, nutrient levels, and physicochemical conditions collectively shaped microbial distributions. These findings support a dual-pathway for oxic methane production: (1) classical archaeal methanogenesis within anoxic microniches embedded in OAs, and (2) bacterial CH₄ generation via methylphosphonate (MPn) degradation triggered by phosphorus limitation or algal-derived methylated substrates. This mechanistic framework explains persistent methane supersaturation in oxic waters and improves predictions of freshwater-methane emissions and carbon cycling under environmental change.