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 (<i>mcrA</i> gene) and bacteria (<i>phnJ</i> gene) and identified their environmental drivers. Gene absolute abundances were significantly affected by the lake type, OAs size, and their interaction. Both <i>mcrA</i> and <i>phnJ</i> 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, <i>mcrA</i> absolute abundance (2.17-4.23 × 10⁵ copies/mL) exceeded <i>phnJ</i> (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.