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.

The activity and community structure of methanotrophs in compartmented microcosms were investigated over the growth period of rice plants. In situ methane oxidation was important only during the vegetative growth phase of the plants and later became negligible. The in situ activity was not directly correlated with methanotrophic cell counts, which increased even after the decrease in in situ activity, possibly due to the presence of both vegetative cells and resting stages. By dividing the microcosms into two soil and two root compartments it was possible to locate methanotrophic growth and activity, which was greatest in the rhizoplane of the rice plants. Molecular analysis by denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH) with family-specific probes revealed the presence of both families of methanotrophs in soil and root compartments over the whole season. Changes in community structure were detected only for members of the Methylococcaceae and could be associated only with changes in the genus Methylobacter and not with changes in the dominance of different genera in the family Methylococcaceae. For the family Methylocystaceae stable communities in all compartments for the whole season were observed. FISH analysis revealed evidence of in situ dominance of the Methylocystaceae in all compartments. The numbers of Methylococcaceae cells were relatively high only in the rhizoplane, demonstrating the importance of rice roots for growth and maintenance of methanotrophic diversity in the soil.Wetland rice fields are an important source of the greenhouse gas CH 4 . They contribute up to 20% (20 to 50 Tg year Ϫ1

Dissolved methane was investigated in the water column of eutrophic Lake Plußsee and compared to temperature, oxygen, and sulfide profiles. Methane concentrations and ␦-13 C signatures indicated a zone of aerobic methane oxidation and additionally a zone of anaerobic methane oxidation in the anoxic water body. The latter coincided with a peak in hydrogen sulfide concentration. High cell numbers of aerobic and anaerobic methane-oxidizing microorganisms were detected by fluorescence in situ hybridization (FISH) or the more sensitive catalyst-amplified reporter deposition-FISH, respectively, in these layers.Methane emissions from lakes contribute 6 to 16% of global methane emissions (3). Consequently, methane oxidation in lakes is an important process for the mitigation of methane emissions. Hitherto, only aerobic methane oxidation has been described for freshwater systems, which preferentially occurs at oxic-anoxic interfaces (17), where methane and oxygen are available. The anaerobic oxidation of methane (AOM) has so far only been described in marine environments (7), even though indications for its occurrence in other habitats exist (6).Lake Plußsee is well studied and has been described in detail elsewhere (14). It has a stable thermal stratification during the summer and regularly occurring anoxia in the hypolimnion, leading to high methane concentrations in the water column. Profiles of methane, oxygen, and hydrogen sulfide concentrations and ␦-13 C signatures of dissolved methane were measured to localize methane oxidation activity in the water column. Water samples for measurements of methane were taken as described by Bastviken et al. (3). Methane concentrations were determined by gas chromatography, and stable carbon isotopes using gas chromatograph-combustion-isotope ratio mass spectrometry (10). Temperature and oxygen were measured in situ with an EOT 190 oxygen probe (WTW Germany). These profiles revealed an anoxic hypolimnion for both sampling time points in June and September 2004 ( Fig. 1A and B). Oxygen was not detectable below 8 m in June and 6 m in September. Methane concentrations (Fig. 1C and D) first increased below the oxocline but then showed a layer of decreasing concentrations in the anoxic hypolimnion, located between 12 and 16 m in June and between 8 and 12.5 m in September. Below, a strong increase in methane concentration towards the sediment was detected. Both methane and oxygen concentration profiles indicate a layer of aerobic methane oxidation in the 9-m depth in June and 6 to 7 m in September. The second decrease in methane concentration detected at both sampling time points was located in the anoxic water body and can therefore not be explained by aerobic methane oxidation. The maximum in methane concentration between the two layers of methane oxidation could be explained by high methane production rates in this layer. These might be caused by a high availability of substrates for methanogens. The sulfate originating from the sediment, reaching 300 M in the bottom water in Septem...

Recent investigations have shown that biogenic methane can be a carbon source for macro invertebrates in freshwater food webs. Stable carbon isotopic signatures, used to infer an organism's food source, indicated that methane can play a major role in the nutrition of chironomid larvae. However, the pathway of methane-derived carbon into invertebrate biomass is still not confirmed. It has been proposed that chironomid larvae ingest methane-oxidizing bacteria (MOB), but this has not been experimentally demonstrated to date. Using (13)C-labelled methane we could show for the first time that chironomid larvae assimilate methane-derived carbon through MOB. Chironomid larval biomass was significantly (13)C-enriched after dwelling for 10 days in lake sediment enriched with labelled methane. Moreover, phospholipid fatty acids diagnostic for MOB were detected in larval tissue and were significantly (13)C-enriched, which encompasses the (13)C-uptake predicted for a methane-based nutrition. Additionally, chironomid larvae fed on sediment and water-column derived MOB biomass.

Stable carbon isotope analysis of chironomid larvae gave rise to the hypothesis that methane-oxidizing bacteria can provide an important food source for higher trophic levels in lakes. To investigate the importance of the methane cycle for the larval stable carbon signatures, isotope analysis and microbiological and biogeochemical investigations were combined. The study was based on comparison of a dimictic lake (Holzsee) and a polymictic, shallow lake (Grosser Binnensee), both located in northern Germany. Both lakes are inhabited by Chironomus plumosus larvae, which exhibited a stronger (13)C-depletion in Holzsee than in Grosser Binnensee, indicating a greater contribution of methane-carbon in the former. Indeed, the processes involved in the microbial methane cycle were found to be more active in Holzsee, showing higher potential methane production and methane oxidation rates. Consistently, cell numbers of methane-oxidizing bacteria were with 0.5 - 1.7 x 10(6) cells g(dw)(-1) about one order of magnitude higher in Holzsee than in Grosser Binnensee. Molecular analysis of the microbial community structure revealed no differences in the methanotrophic community between the two lakes, with a clear dominance of type I methanotrophs. The methanogenic population seemed to be adapted to the prevailing substrate in the respective lake (H(2)/CO(2) in Holzsee and acetate in Grosser Binnensee), even though differences were minor. In conclusion, the stronger larval (13)C-depletion in Holzsee was not reflected in differences in the microbial community structure, but in the activity and size of the methanogenic and methanotrophic populations in the lake sediment.