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 CH4 oxidation for controlling CH4 emission. The fraction of CH4 produced from acetate and H2/CO2 was calculated from the isotopic signatures of acetate, carbon dioxide (CO2) and methane (CH4) 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 (CH3F) as specific inhibitor of acetate-dependent methanogenesis. Only at the end of the season, the theoretical ratio of acetate: H2 = 2 : 1 was reached, whereas at the beginning H2/CO2-dependent methanogenesis dominated. The isotope discrimination was different between rooted surface soil and unrooted deep soil. Root-associated CH4 production was mainly driven by H2/CO2. Porewater CH4 was found to be a poor proxy for produced CH4.
The fraction of CH4 oxidised was calculated from the isotopic signature of CH4 produced in vitro compared to CH4 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 (CH2F2) as specific inhibitor of methanotrophic bacteria agreed that CH4 oxidation was quantitatively important at the beginning of the season, but decreased later. The seasonal pattern was consistent with the change of potential CH4 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 CH4 was used as a proxy of produced CH4, neither magnitude nor seasonal pattern of in situ CH4 oxidation could be reproduced. An oxidation signal was also found in the isotopic signature of CH4 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 CH4.
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