Seasonal variation in pathways of CH4 production and in CH4 oxidation in rice fields determined by stable carbon isotopes and specific inhibitors

Abstract Flooded rice fields, which are an important source of the atmospheric methane, have become a model system for the study of interactions between various microbial processes. We used a combination of stable carbon isotope measurements and application of specific inhibitors in order to investigate the importance of various methanogenic pathways and of CH 4 oxidation for controlling CH 4 emission. The fraction of CH 4 produced from acetate and H 2 /CO 2 was calculated from the isotopic signatures of acetate, carbon dioxide (CO 2 ) and methane (CH 4 ) measured in porewater, gas bubbles, in the aerenchyma of the plants and/or in incubation experiments. The calculated ratio between both pathways reflected well the ratio determined by application of methyl fluoride (CH 3 F) as specific inhibitor of acetate‐dependent methanogenesis. Only at the end of the season, the theoretical ratio of acetate: H 2 = 2 : 1 was reached, whereas at the beginning H 2 /CO 2 ‐dependent methanogenesis dominated. The isotope discrimination was different between rooted surface soil and unrooted deep soil. Root‐associated CH 4 production was mainly driven by H 2 /CO 2 . Porewater CH 4 was found to be a poor proxy for produced CH 4 . The fraction of CH 4 oxidised was calculated from the isotopic signature of CH 4 produced in vitro compared to CH 4 emitted in situ , corrected for the fractionation during the passage from the aerenchyma to the atmosphere. Isotope mass balances and in situ inhibition experiments with difluoromethane (CH 2 F 2 ) as specific inhibitor of methanotrophic bacteria agreed that CH 4 oxidation was quantitatively important at the beginning of the season, but decreased later. The seasonal pattern was consistent with the change of potential CH 4 oxidation rates measured in vitro . At the end of the season, isotope techniques detected an increase of oxidation activity that was too small to be measured with the flux‐based inhibitor technique. If porewater CH 4 was used as a proxy of produced CH 4 , neither magnitude nor seasonal pattern of in situ CH 4 oxidation could be reproduced. An oxidation signal was also found in the isotopic signature of CH 4 from gas bubbles that were released by natural ebullition. In contrast, bubbles stirred up from the bulk soil had preserved the isotopic signature of the originally produced CH 4 .