Anaerobic oxidation of methane (AOM) and sulphate reduction were examined in sediment samples from a marine gas hydrate area (Hydrate Ridge, NE Pacific). The sediment contained high numbers of microbial consortia consisting of organisms that affiliate with methanogenic archaea and with sulphate-reducing bacteria. Sediment samples incubated under strictly anoxic conditions in defined mineral medium (salinity as in seawater) produced sulphide from sulphate if methane was added as the sole organic substrate. No sulphide production occurred in control experiments without methane. Methane-dependent sulphide production was fastest between 4 degree C and 16 degree C, the average rate with 0.1 MPa (approximately 1 atm) methane being 2.5 micro mol sulphide day(-1) and (g dry mass sediment)(-1). An increase of the methane pressure to 1.1 MPa (approximately 11 atm) resulted in a four to fivefold increase of the sulphide production rate. Quantitative measurements using a special anoxic incubation device without gas phase revealed continuous consumption of dissolved methane (from initially 3.2 to 0.7 mM) with simultaneous production of sulphide at a molar ratio of nearly 1:1. To test the response of the indigenous community to possible intermediates of AOM, molecular hydrogen, formate, acetate or methanol were added in the absence of methane; however, sulphide production from sulphate with any of these compounds was much slower than with methane. In the presence of methane, such additions neither stimulated nor inhibited sulphate reduction. Hence, the experiments did not provide evidence for one of these compounds acting as a free extracellular intermediate (intercellular shuttle) during AOM by the presently investigated consortia.

Anaerobic oxidation of methane (AOM) in marine sediments is an important microbial process in the global carbon cycle and in control of greenhouse gas emission. The responsible organisms supposedly reverse the reactions of methanogenesis, but cultures providing biochemical proof of this have not been isolated. Here we searched for AOM-associated cell components in microbial mats from anoxic methane seeps in the Black Sea. These mats catalyse AOM rather than carry out methanogenesis. We extracted a prominent nickel compound displaying the same absorption spectrum as the nickel cofactor F430 of methyl-coenzyme M reductase, the terminal enzyme of methanogenesis; however, the nickel compound exhibited a higher molecular mass than F430. The apparent variant of F(430) was part of an abundant protein that was purified from the mat and that consists of three different subunits. Determined amino-terminal amino acid sequences matched a gene locus cloned from the mat. Sequence analyses revealed similarities to methyl-coenzyme M reductase from methanogenic archaea. The abundance of the nickel protein (7% of extracted proteins) in the mat suggests an important role in AOM.

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 anaerobic oxidation of methane (AOM) is one of the major sinks for methane on earth and is known to be mediated by at least two phylogenetically different groups of anaerobic methanotrophic Archaea (ANME-I and ANME-II). We present the first comparative in vitro study of the environmental regulation and physiology of these two methane-oxidizing communities, which occur naturally enriched in the anoxic Black Sea (ANME-I) and at Hydrate Ridge (ANME-II). Both types of methanotrophic communities are associated with sulfate-reducing-bacteria (SRB) and oxidize methane anaerobically in a 1:1 ratio to sulfate reduction (SR). They responded sensitively to elevated methane partial pressures with increased substrate turnover. The ANME-II-dominated community showed significantly higher cell-specific AOM rates. Besides sulfate, no other electron acceptor was used for AOM. The processes of AOM and SR could not be uncoupled by feeding the SRB with electron donors such as acetate, formate or molecular hydrogen. AOM was completely inhibited by the addition of bromoethanesulfonate in both communities, indicating the participation of methanogenic enzymes in the process. Temperature influenced the intensity of AOM, with ANME-II being more adapted to cold temperatures than ANME-I. The variation of other environmental parameters, such as sulfate concentration, pH and salinity, did not influence the activity of both communities. In conclusion, the ecological niches of methanotrophic Archaea seem to be mainly defined by the availability of methane and sulfate, but it remains open which additional factors lead to the dominance of ANME-I or -II in the environment.

We investigated the effect of seasonal environmental changes on the rate and distribution of anaerobic oxidation of methane (AOM) in Eckernförde Bay sediments (German Baltic Sea) and identified organisms that are likely to be involved in the process. Surface sediments were sampled during September and March. Field rates of AOM and sulfate reduction (SR) were measured with radiotracer methods. Additional parameters were determined that potentially influence AOM, i.e., temperature, salinity, methane, sulfate, and chlorophyll a. Methanogenesis as well as potential rates of AOM and aerobic oxidation of methane were measured in vitro. AOM changed seasonally within the upper 20 cm of the sediment, with rates being between 1 and 14 nmol cm Ϫ3 d Ϫ1 . Its distribution is suggested to be controlled by oxygen and sulfate penetration, temperature, as well as methane supply, leading to a shallow AOM zone during the warm productive season and to a slightly deeper AOM zone during the cold winter season. Rising methane bubbles apparently fed AOM above the sulfate-methane transition. Methanosarcinales-related anaerobic methanotrophs (ANME-2), identified with fluorescence in situ hybridization, is suggested to mediate AOM in Eckernförde Bay. These archaea are known also from other marine methane-rich locations. However, they were not directly associated with sulfate-reducing bacteria. AOM is possibly mediated solely by these archaea that show a mesophilic physiology according to the seasonal temperature changes in Eckernförde Bay.

Summary Irrigated rice fields are an important source of atmospheric methane. In order to improve our understanding of the controlling processes, we measured in situ CH4 emission and CH4 oxidation in an Italian rice field in 1998 and 1999, and studied CH4 production in soil and root samples. The CH4 emission rates were correlated with diurnal temperature variations and showed pronounced seasonal and interannual variations. The contribution of CH4 oxidation to total CH4 flux, determined by specific inhibition with difluoromethane, decreased from 40% at the beginning to zero at the end of the season. The stable carbon isotopic composition of the emitted CH4 also decreased. The CH4‐oxidizing bacteria probably became limited by nitrogen as indicated by the seasonal decrease of NH4+. Thus, CH4 oxidation had little effect on CH4 emission. Methane production on rice roots was relatively constant over the season. Methane production in soil slowly increased after flooding and was highest in the middle of the season. Pore water concentrations of CH4 showed a similar seasonal pattern. In 1999, CH4 production increased later in the season and reached lower rates than in 1998. An additional drainage in 1999 resulted in higher ferric iron concentrations, higher soil redox potentials and lower acetate concentrations. As a result, acetate‐utilizing methanogens were probably out‐competed by iron‐reducers so that a larger percentage of [2–14C]acetate was converted to 14CO2 instead of 14CH4. The residual CH4 production was relatively low and was mainly due to H2/CO2‐dependent methanogenesis. Experiments with radioactive bicarbonate and with methyl fluoride as specific inhibitor showed that the theoretical ratio of 7:3 of methanogenesis from acetate vs. H2/CO2 was only reached later in the season when total CH4 production was at the maximum. In conclusion, our results give a mechanistic explanation for the intraseasonal and interannual differences in CH4 emission.

Present address: Bundesanstalt für Geowissenschaften und Rohstoffe, Stilleweg 2, 30655 Hannover, GermanyABSTRACT: Deep-sea whale falls create sulfidic habitats supporting chemoautotrophic communities, but microbial processes underlying the formation of such habitats remain poorly evaluated. Microbial degradation processes (sulfate reduction, methanogenesis) and biogeochemical gradients were studied in a whale-fall habitat created by a 30 t whale carcass deployed at 1675 m depth for 6 to 7 yr on the California margin. A variety of measurements were conducted including photomosaicking, microsensor measurements, radiotracer incubations and geochemical analyses. Sediments were studied at different distances (0 to 9 m) from the whale fall. Highest microbial activities and steepest vertical geochemical gradients were found within 0.5 m of the whale fall, revealing ex situ sulfate reduction and in vitro methanogenesis rates of up to 717 and 99 mmol m -2 d -1, respectively. In sediments containing whale biomass, methanogenesis was equivalent to 20 to 30% of sulfate reduction. During in vitro sediment studies, sulfide and methane were produced within days to weeks after addition of whale biomass, indicating that chemosynthesis is promoted at early stages of the whale fall. Total sulfide production from sediments within 0.5 m of the whale fall was 2.1 ± 3 and 1.5 ± 2.1 mol d -1 in Years 6 and 7, respectively, of which ~200 mmol d -1 were available as free sulfide. Sulfate reduction in bones was much lower, accounting for a total availability of ~10 mmol sulfide d Skeleton of a whale on the deep-sea floor, covered by chemoautotrophic bacterial mats, anemones, and bone-eating worms Osedax spp., 6 yr after arrival at the seafloor.

The efficient representation of all species in conservation planning is problematic. Often, species distribution is assessed by dividing the land into a grid; complementary sets of grids, in which each taxon is represented at least once, are then sought. To determine if this approach provides useful surrogate information, species and higher taxon data for South African plants and animals were analyzed. Complementary species sets did not coincide and overlapped little with higher taxon sets. Survey extent and taxonomic knowledge did not affect this overlap. Thus, the assumptions of surrogacy, on which so much conservation planning is based, are not supported.