Environmental and microbial regulation of multi-temporal scale methane flux dynamics in a shallow karst lake

Lakes are increasingly recognized as hotspots for methane (CH₄) emissions, yet high-frequency (hourly-scale) measurements of CH₄ fluxes throughout all seasons and a clear understanding of their underlying environmental control mechanisms are still lacking. Here, we explore the dynamics of CH₄ flux and its hydrological and biogeochemical mechanisms in a karst shallow lake ecosystem, based on eddy covariance (EC), stable carbon isotope, and metagenomic sequencing techniques. Our 13-month EC monitoring shows that the lake was a CH₄ source to the atmosphere, with average emission rate being 2.07 ± 1.20 mmol CH₄ m⁻² d⁻¹, with the highest emissions in autumn (3.63 ± 0.5 mmol m^-2 d^-1), accounting for more than twice those of winter. Seasonal CH₄ flux variations were primarily driven by water temperature, water level, and electrical conductivity. Elevated temperature stimulated methanogenesis, water level changes altered redox gradients shaping methanogen activity, and electrical conductivity regulated substrate availability favoring acetoclastic methanogens. At the diurnal scale, CH₄ emissions were higher at night than during the day, with diel flux variations were mainly controlled by water temperature, which enhanced methanogenesis while suppressing oxidation. These environmental controls on CH₄ flux are consistent with the structure of the methanogenic community and support the dominance of acetoclastic methanogenesis. Methanothrix was found to be the dominant (∼65%) methanogenic microbe in this carbonate-rich alkaline karst lake. Additionally, the dominant pathway of CH₄ production in the lake was acetoclastic methanogenesis, with the apparent fractionation factor of δ¹³C-CH₄ being 1.041 ± 0.002. We emphasize the importance of integrating physicochemical variability with microbial functional potential to advance understanding of biogeochemical feedbacks in carbonate-rich karst systems and improve the accuracy of CH₄ emission estimates across scales.