Abstract: Methane (CH₄) is an important greenhouse gas, the formation and emission mechanism of which in water have a profound impact on global carbon cycle and climate change. Isotope technology is an effective means to examine the formation pathway, emission mechanism, and environmental effects of CH₄. We reviewed the main pathways of CH₄ production and their isotopic characteristics in water, with emphasis on the diversity of biogenic CH₄ metabolic pathways, including acetate fermentation  (δ¹³C of about -50‰ to -60‰) and CO₂ reduction (δ¹³C of about -110‰ to -60‰). Microorganisms participate in CH₄ cycle in a complex redox environment in different ways. Isotope fractionation is regulated by methanogenic bacterial community and substrate availability, and later processes such as oxidation, migration and transformation lead to obvious isotope fractionation. CH₄ emission processes (diffusion, ebullition, plant transport) in aquatic ecosystems are accompanied by significant isotopic effects. The diffusion pathway leads to residual CH₄ enrichment of ¹³C (up to +15‰) due to oxidation while ebullition preferentially retains the deficient isotope signal. Physical (such as water temperature), chemical (such as oxygen solubility), and biological factors (such as microbial community composition and expression) of the water environment have important effects on CH₄ generation and emission, driving the temporal and spatial heterogeneity of CH₄ isotope composition. Therefore, the source and transformation process of CH₄ can be well retrieved from its isotope characteristics in aquatic systems. In addition, the collaborative application of cluster isotopes and microbial analysis techniques enables the multi-scale analysis of isotope co-use, which can reveal the key processes of CH₄ cycle in water at the level of the natural abundance of multiple substituted isotopologues, providing key parameters for quantifying carbon cycling mechanism. However, the mechanism of sediment-water-atmosphere interface fractionation and the flux contribution and control mechanism of the atypical methanogenic pathway are still unknown. Future studies should explore the mechanism of CH₄ generation and emission and the dynamic response of ecosystems in the context of climate change by combining field observation, experimental simulation, and model analysis.

Key words: methane, aquatic ecosystem, greenhouse gas, isotope, carbon cycle