Methane oxidation is a key process controlling methane emission from anoxic habitats into the atmosphere. Methanotrophs, responsible for aerobic methane oxidation, do not only oxidize but also assimilate methane. Once assimilated, methane carbon may be utilized by other organisms. Here we report on a microbial food web in a rice field soil driven by methane. A thin layer of water-saturated rice field soil was incubated under opposing gradients of oxygen and (13)C-labelled methane. Bacterial and eukaryotic communities incorporating methane carbon were analysed by RNA-stable isotope probing (SIP). Terminal restriction fragment length polymorphism (T-RFLP) and cloning showed that methanotrophs were the most prominent group of bacteria incorporating methane carbon. In addition, a few Myxobacteria-related sequences were obtained from the 'heavy' rRNA fraction. Denaturing gradient gel electrophoresis (DGGE) targeting 18S rRNA detected various groups of protists in the 'heavy' rRNA fraction including naked amoeba (Lobosea and Heterolobosea), ciliates (Colpodea) and flagellates (Cercozoa). Incubation of soil under different methane concentrations in air resulted in the development of distinct protozoan communities. These results suggest that methane carbon is incorporated into non-methanotrophic pro- and microeukaryotes probably via grazing, and that methane oxidation is a shaping force of the microeukaryotic community depending on methane availability.

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Reduced fertilizer usage is one of the objectives of field management in the pursuit of sustainable agriculture. Here, we report on shifts of bacterial communities in paddy rice ecosystems with low (LN), standard (SN), and high (HN) levels of N fertilizer application (0, 30, and 300 kg N ha−1, respectively). The LN field had received no N fertilizer for 5 years prior to the experiment. The LN and HN plants showed a 50% decrease and a 60% increase in biomass compared with the SN plant biomass, respectively. Analyses of 16S rRNA genes suggested shifts of bacterial communities between the LN and SN root microbiomes, which were statistically confirmed by metagenome analyses. The relative abundances of Burkholderia, Bradyrhizobium and Methylosinus were significantly increased in root microbiome of the LN field relative to the SN field. Conversely, the abundance of methanogenic archaea was reduced in the LN field relative to the SN field. The functional genes for methane oxidation (pmo and mmo) and plant association (acdS and iaaMH) were significantly abundant in the LN root microbiome. Quantitative PCR of pmoA/mcrA genes and a 13C methane experiment provided evidence of more active methane oxidation in the rice roots of the LN field. In addition, functional genes for the metabolism of N, S, Fe, and aromatic compounds were more abundant in the LN root microbiome. These results suggest that low-N-fertilizer management is an important factor in shaping the microbial community structure containing key microbes for plant associations and biogeochemical processes in paddy rice ecosystems.

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Methane oxidation was studied in mesotrophic lake water (Lake Biwa, Japan) under thermally stratified conditions. Methane oxidation rates at in situ concentrations were very low in lake water from the epilimnion and thermocline but were high in hypolimnetic water. Incubation under light conditions ranging from 4.1 to 57 µmol photons m−2 s−1 resulted in decreased methane oxidation in hypolimnetic water. This inhibition was more severe as the light intensity increased. Addition of inorganic nitrogen (ammonium and nitrate) did not promote methane oxidation in the thermocline but inhibited it in the hypolimnion. Methane oxidation activity in the thermocline was observed after 1 month of incubation under dark conditions. Our results suggest that the inhibitory effect of light was bacteriostatic for the methanotrophic population. The different rates of methane oxidation between the hypolimnion and epilimnion/thermocline may explain the surface maximum of dissolved methane during the period of thermal stratification.

Flooded rice fields have become a model system for the study of soil microbial ecology. In Italian rice fields, in particular, aspects from biogeochemistry to molecular ecology have been studied, but the impact of protistan grazing on the structure and function of the prokaryotic community has not been examined yet. We compared an untreated control soil with a ␥-radiation-sterilized soil that had been reinoculated with a natural bacterial assemblage. In order to verify that the observed effects were due to protistan grazing and did not result from sterilization, we set up a third set of microcosms containing sterilized soil that had been reinoculated with natural assemblage bacteria plus protists. The spatial and temporal changes in the protistan and prokaryotic communities were examined by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis, respectively, both based on the small-subunit gene. Sequences retrieved from DGGE bands were preferentially affiliated with Cercozoa and other bacteriovorous flagellates. Without protists, the level of total DNA increased with incubation time, indicating that the level of the microbial biomass was elevated. Betaproteobacteria were preferentially preyed upon, while low-G؉C-content gram-positive bacteria became more dominant under grazing pressure. The bacterial diversity detectable by T-RFLP analysis was greater in the presence of protists. The level of extractable NH 4 ؉ was lower and the level of extractable SO 4 2؊ was higher without protists, indicating that nitrogen mineralization and SO 4 2؊ reduction were stimulated by protists. Most of these effects were more obvious in the partially oxic surface layer (0 to 3 mm), but they could also be detected in the anoxic subsurface layer (10 to 13 mm). Our observations fit well into the overall framework developed for protistan grazing, but with some modifications pertinent to the wetland situation: O 2 was a major control, and O 2 availability may have limited directly and indirectly the development of protists. Although detectable in the lower anoxic layer, grazing effects were much more obvious in the partially oxic surface layer.

Biological methane oxidation is a key process in the methane cycle of wetland ecosystems. The methanotrophic biomass may be grazed by protozoa, thus linking the methane cycle to the soil microbial food web. In the present study, the edibility of different methanotrophs for soil protozoa was compared. The number of methanotroph-feeding protozoa in a rice field soil was estimated by determining the most-probable number (MPN) using methanotrophs as food bacteria; naked amoebae and flagellates were the dominant protozoa. Among ten methanotrophic strains examined as a food source, seven yielded a number of protozoa comparable with the yield with Escherichia coli [10(4) MPN (g soil dry weight)(-1)], and three out of four Methylocystis spp. yielded significantly fewer numbers [10(2)-10(3) MPN (g soil dry weight)(-1)]. The lower edibility of the Methylocystis spp. was not explained either by their growth phase or by harmful effects on protozoa. Incubation of the soil under methane resulted in a higher number of protozoa actively grazing on methanotrophs, especially on the less-edible group. Protozoa isolated from the soil demonstrated a grazing preference on the different methanotrophs consistent with the results of MPN counts. The results indicate that selective grazing by protozoa may be a biological factor affecting the methanotrophic community in a wetland soil.

The spatial distribution and seasonal variation in the concentration and carbon isotopic composition of dissolved methane in a river-lake ecosystem were studied in Lake Biwa, Japan, and its tributary rivers. Methane concentrations in all subsystems examined were supersaturated with respect to the atmosphere. The epilimnion showed higher concentrations of dissolved methane than the hypolimnion in the pelagic zone. Peak methane concentrations were observed at the thermocline. The largest amount of methane in the pelagic water column was recorded at the end of a stagnant period, at which the bottom water of the sublittoral zone (30 m in depth) exhibited increased methane concentration. Transect observation of dissolved methane revealed three methane peaks at different water depths in the lake, and river water and the sediments in littoral and sublittoral zones were suggested to be the corresponding sources. Water at the river mouth was replete with dissolved oxygen but also contained a high concentration of methane. The present results suggest that river water and littoral sediment are potential sources of dissolved methane in Lake Biwa, and other sources, such as internal waves, are responsible for increased methane in the pelagic zone at the end of stagnant periods. Carbon stable isotope analysis indicated that there were different sources of dissolved methane, although it was difficult to identify the origins due to high variation of the isotopic composition of methane from different sources.