Methylocella species are facultatively methanotrophic

All aerobic methanotrophic bacteria described to date are unable to grow on substrates containing carbon-carbon bonds. Here we demonstrate that members of the recently discovered genus Methylocella are an exception to this. These bacteria are able to use as their sole energy source the one-carbon compounds methane and methanol, as well as the multicarbon compounds acetate, pyruvate, succinate, malate, and ethanol. To conclusively verify facultative growth, acetate and methane were used as model substrates in growth experiments with the type strain Methylocella silvestris BL2. Quantitative real-time PCR targeting the mmoX gene, which encodes a subunit of soluble methane monooxygenase, showed that copies of this gene increased in parallel with cell counts during growth on either acetate or methane as the sole substrate. This verified that cells possessing the genetic basis of methane oxidation grew on acetate as well as methane. Cloning of 16S rRNA genes and fluorescence in situ hybridization with strain-specific and genus-specific oligonucleotide probes detected no contaminants in cultures. The growth rate and carbon conversion efficiency were higher on acetate than on methane, and when both substrates were provided in excess, acetate was preferably used and methane oxidation was shut down. Our data demonstrate that not all methanotrophic bacteria are limited to growing on one-carbon compounds. This could have major implications for understanding the factors controlling methane fluxes in the environment.     

Environmental distribution and abundance of the facultative methanotroph Methylocella

Methylocella spp. are facultative methanotrophs, which are able to grow not only on methane but also on multicarbon substrates such as acetate, pyruvate or malate. Methylocella spp. were previously thought to be restricted to acidic soils such as peatlands, in which they may have a key role in methane oxidation. There is little information on the abundance and distribution of Methylocella spp. in the environment. New primers were designed, and a real-time quantitative PCR method was developed and validated targeting Methylocella mmoX (encoding the α-subunit of the soluble methane monooxygenase) that allowed the quantification of Methylocella spp. in environmental samples. We also developed and validated specific PCR assays, which target 16S rRNA genes of known Methylocella spp. These were used to investigate the distribution of Methylocella spp. in a variety of environmental samples. It was revealed that Methylocella species are widely distributed in nature and not restricted to acidic environments.     

The processes of methane formation and oxidation in the soils of the Russian arctic tundra

Methane emission from the following types of tundra soils was studied: coarse humic gleyey loamy cryo soil, peaty gley soil, and peaty gleyey midloamy cryo soil of the arctic tundra. All the soils studied were found to be potential sources of atmospheric methane. The highest values of methane emission were recorded in August at a soil temperature of 8-10 degrees C. Flooded parcels were the sources of atmospheric methane throughout the observation period. The rates of methane production and oxidation in tundra soils of various types at 5 and 15 degrees C were studied by the radioisotope method. Methane oxidation was found to occur in bog water, in the green part of peat moss, and in all the soil horizons studied. Methane formation was recorded in the horizons of peat, in clay with plant roots, and in peaty moss dust of the bogey parcels. At both temperatures, the methane oxidation rate exceeded the rate of methane formation in all the horizons of the mossy-lichen tundra and of the bumpy sinkhole complex. Methanogenesis prevailed only in a sedge-peat moss bog at 15 degrees C. Enrichment bacterial cultures oxidizing methane at 5 and 15 degrees C were obtained. Different types of methanotrophic bacteria were shown to be responsible for methane oxidation under these conditions. A representative of type I methylotrophs oxidized methane at 5 degrees C, and Methylocella tundrae, a psychroactive representative of an acidophilic methanotrophic genus Methylocella, at 15 degrees C.     

Aerobic methanotrophic bacteria of cold ecosystems

This review summarizes the recent advances in understanding the ecophysiological role and structure-function features of methanotrophic bacteria living in various cold ecosystems. The occurrence of methanotrophs in a majority of psychrosphere sites was verified by direct measurement of their methane-utilizing activity, by electron microscopy and immunofluorescent observations, and analyses of specific signatures in cellular phospholipids and total DNAs extracted from environmental samples. Surprisingly, the phenotypic and genotypic markers of virtually all extant methanotrophs were detected in various cold habitats, such as underground waters, Northern taiga and tundra soils, polar lakes and permafrost sediments. Also, recent findings indicated that even after long-term storage in permafrost, some methanotrophs can oxidize and assimilate methane not only at positive but also at subzero temperatures. Pure cultures of psychrophilic and psychrotolerant methanotrophs were isolated and characterized as new genera and species: Methylobacter psychrophilus, Methylosphaera hansonii, Methylocella palustris, Methylocella silvestris, Methylocella tundrae, Methylocapsa acidiphila and Methylomonas scandinavica. However, our knowledge about their adaptive mechanisms and survival in cold ecosystems remains limited and needs to be established using both traditional and molecular microbiological methods.     

Methanotrophic bacteria of acid sphagnum bogs

Acid sphagnum bogs cover a considerable part of the territory of Russia and are an important natural source of biogenic methane, which is formed in their anaerobic layers. A considerable portion of this methane is consumed in the aerobic part of the bog profile by acidophilic methanotrophic bacteria, which comprise the methane filter of sphagnum bogs and decrease CH4 emission to the atmosphere. For a long time, these bacteria escaped isolation, which became possible only after the elucidation of the optimal conditions of their functioning in situ: pH 4.5 to 5.5; temperature, from 15 to 20 degrees C; and low salt concentration in the solution. Reproduction of these conditions and rejection of earlier used media with a high content of biogenic elements allowed methanotrophic bacteria of two new genera and species--Methylocella palustris and Methylocapsa acidophila--to be isolated from the peat of sphagnum bogs of the northern part of European Russia and West Siberia. These bacteria are well adapted to the conditions in cold, acid, oligotrophic sphagnum bogs. They grow in a pH range of 4.2-7.5 with an optimum at 5.0-5.5, prefer moderate temperatures (15-25 degrees C) and media with a low content of mineral salts (200-500 mg/l), and are capable of active nitrogen fixation. Design of fluorescently labeled 16S rRNA-targeted oligonucleotide probes for the detection of Methylocella palustris and Methylocapsa acidophila and their application to the analysis of sphagnum peat samples showed that these bacteria represent dominant populations of methanotrophs with a density of 10(5)-10(6) cells/g peat. In addition to Methylocella and Methylocapsa populations, one more abundant population of methanotrophs was revealed (10(6) cells/g peat), which were phylogenetically close to the genus Methylocystis.     

Identity of active methanotrophs in landfill cover soil as revealed by DNA-stable isotope probing

A considerable amount of methane produced during decomposition of landfill waste can be oxidized in landfill cover soil by methane-oxidizing bacteria (methanotrophs) thus reducing greenhouse gas emissions to the atmosphere. The identity of active methanotrophs in Roscommon landfill cover soil, a slightly acidic peat soil, was assessed by DNA-stable isotope probing (SIP). Landfill cover soil slurries were incubated with (13)C-labelled methane and under either nutrient-rich nitrate mineral salt medium or water. The identity of active methanotrophs was revealed by analysis of (13)C-labelled DNA fractions. The diversity of functional genes (pmoA and mmoX) and 16S rRNA genes was analyzed using clone libraries, microarrays and denaturing gradient gel electrophoresis. 16S rRNA gene analysis revealed that the cover soil was mainly dominated by Type II methanotrophs closely related to the genera Methylocella and Methylocapsa and to Methylocystis species. These results were supported by analysis of mmoX genes in (13)C-DNA. Analysis of pmoA gene diversity indicated that a significant proportion of active bacteria were also closely related to the Type I methanotrophs, Methylobacter and Methylomonas species. Environmental conditions in the slightly acidic peat soil from Roscommon landfill cover allow establishment of both Type I and Type II methanotrophs.     

Microbial acetate conversion to methane: kinetics,yields and pathways in a two-step digestion process

Summary Organic waste is converted in a two-stage process to methane and carbon dioxide by mixed cultures of microorganisms. Acetate, a product of acidogenic and acetogenic bacteria and the main substrate for methanogenic bacteria, is an important intermediate of the anaerobic degradation process, which results in the generation of methane. It was shown by labelling experiments using (U-¹⁴C) acetate that as much as 65%–96% of the total methane produced came from the acetate. The first order utilization rate for acetate in the methanogenic stages of a two-stage digestion process was between 0.17 h^-1 and 0.5 h^-1. The kinetics as well as the mass flow and yields of acetate and the methyl group of acetate were determined by pulse-labelling experiments with (U-¹⁴C) acetate and (2-¹⁴C) acetate without a significant rise of the total concentrations. Up to 58% of the acetate carbon was transformed to methane, and about 30% to carbon dioxide; only 4%–15% was incorporated into the biomass. There are at least two parallel degradation mechanisms in the metabolic transformation of acetate to methane: acetate is cleaved either to form methane and carbon dioxide or to form hydrogen and carbon dioxide, which can be transformed by an additional reaction to methane. Labelling experiments with (2-¹⁴C) acetate show that both mechanisms took place at similar order.     

Acetate threshold values and acetate activating enzymes in methanogenic bacteria

Abstract The minimum threshold concentrations of acetate utilization and the enzymes responsible for acetate activation of several methanogenic bacteria were investigated and compared with literature data. The minimum acetate concentrations reached by hydrogenotrophic methane bacteria, which require acetate as carbon source, were between 0.4 and 0.6 mM. The acetoclastic Methanosarcina achieves acetate concentrations between 0.2 and 1.2 mM and Methanothrix between 7 and 70 μM. For the activation of acetate most of the hydrogenotrophic methane bacteria investigated use an acetyl-CoA synthetase with a relatively low K _m (40–90 μM) for acetate. although the affinity for acetate was high, the hydrogenotrophic methane bacteria were not able to remove acetate to lower concentrations than the acetoclastic methane bacteria, neither in pure cultures nor in anaerobic granular sludge samples. Based on these observations, it is not likely that hydrogenotrophic methanogens compete strongly for acetate with the acetoclastic methane bacteria.     

Influence of temperature and high acetate concentrations on methanogenesis in lake sediment slurries

Methanogenesis from main methane precursors H(2)/CO(2) and acetate was investigated in a temperature range of 2-70 degrees C using sediments from Lake Baldegg, Switzerland. Psychrophilic, psychrotrophic, mesophilic, and thermophilic methanogenic microbial communities were enriched by incubations for 1-3 months of nonamended sediment slurries at 5, 15, 30, and 50 degrees C. Isotope experiments with slurries amended with (14)C-labeled bicarbonate and (14)C-2-acetate showed that in the psychrophilic community (enriched at 5 degrees C), about 95% of methane originated from acetate, in contrast to the thermophilic community (50 degrees C) where up to 98% of methane was formed from bicarbonate. In the mesophilic community (30 degrees C), acetate was the precursor of about 80% of the methane produced. When the hydrogen-carbon dioxide mixture (H(2)/CO(2)) was used as a substrate, it was directly converted to methane under thermophilic conditions (70 and 50 degrees C). Under mesophilic conditions (30 degrees C), both pathways, hydrogenotrophic and acetoclastic, were observed. At low temperatures (5 and 15 degrees C), H(2)/CO(2) was converted into methane by a two-step process; first acetate was formed, followed by methane production from acetate. When slurries were incubated at high partial pressures of H(2)/CO(2), the high concentrations of acetate produced of more than 20 mM inhibited acetoclastic methanogenesis at a temperature below 15 degrees C. However, slow adaptation of the psychrophilic microbial community to high acetate concentrations was observed.     

Changes in proportions of acetate and carbon dioxide used as methane precursors during the anaerobic digestion of bovine waste.

In an anaerobic digestor which was fed daily with bovine waste, during the early stages after feeding (4 to 7 h) acetate (via the methyl group) accounted for almost 90% of the methane produced. As time after feeding increased, acetate declined as a precursor so that in the 12- to 14-h and 21- to 23-h periods, after feeding the methyl group accounted for 80 and 73% of the methane produced, respectively. Measurements of methane production from CO2 reduction showed that in the 2- to 12-h period after feeding, CO2 accounted for 14% of the methane produced, whereas in the 12- to 24-h period it accounted for 27-5%. These results show that the percentages of methane accounted for by acetate and CO2 vary with time after feeding the digestor.     

Changes in proportions of acetate and carbon dioxide used as methane precursors during the anaerobic digestion of bovine waste

In an anaerobic digestor which was fed daily with bovine waste, during the early stages after feeding (4 to 7 h) acetate (via the methyl group) accounted for almost 90% of the methane produced. As time after feeding increased, acetate declined as a precursor so that in the 12- to 14-h and 21- to 23-h periods, after feeding the methyl group accounted for 80 and 73% of the methane produced, respectively. Measurements of methane production from CO2 reduction showed that in the 2- to 12-h period after feeding, CO2 accounted for 14% of the methane produced, whereas in the 12- to 24-h period it accounted for 27-5%. These results show that the percentages of methane accounted for by acetate and CO2 vary with time after feeding the digestor.     

A stable isotope titration method to determine the contribution of acetate disproportionation and carbon dioxide reduction to methanogenesis

A novel stable isotope titration approach was developed to determine the contributions to total methane production made by CO(2) reduction and the disproportionation of acetate in anoxic environments. (13)CH(4), (12)CH(4), (13)CO(2) and (12)CO(2) production rates were measured in the head space of replicate anaerobic microcosms titrated with increasing amounts of (13)C-labelled substrates. The contribution of CO(2) reduction was calculated from the linear relationship between ratios of labelled and total CH(4) production and ratios of labelled and total CO(2) after the addition of (13)C-bicarbonate. In the case of acetoclastic methanogenesis rates of (13)CH(4) and (12)CH(4) production were fitted to a model based on an assumption that the relationship between the concentration of (13)C-labelled acetate and the rates of labelled and unlabelled methane production followed Michaelis-Menten kinetics. A comparison of the raw data with the model supported the assumption and provided both an estimate of the contribution of acetate to methane production and an estimate of the size of the indigenous acetate pool without the need to measure acetate directly. The method was applied to a freshwater sediment in the English Lake District where it was found that 66.3% (se 4.9) of methane production was due to acetate disproportionation and 28.9% (se 1.9) of methane production resulted from CO(2) reduction. This is in agreement with theoretical predictions and other empirical measurements of methanogenesis.     

Competitive Mechanisms for Inhibition of Sulfate Reduction and Methane Production in the Zone of Ferric Iron Reduction in Sediments

Mechanisms for inhibition of sulfate reduction and methane production in the zone of Fe(III) reduction in sediments were investigated. Addition of amorphic iron(III) oxyhydroxide to sediments in which sulfate reduction was the predominant terminal electron-accepting process inhibited sulfate reduction 86 to 100%. The decrease in electron flow to sulfate reduction was accompanied by a corresponding increase in electron flow to Fe(III) reduction. In a similar manner, Fe(III) additions also inhibited methane production in sulfate-depleted sediments. The inhibition of sulfate reduction and methane production was the result of substrate limitation, because the sediments retained the potential for sulfate reduction and methane production in the presence of excess hydrogen and acetate. Sediments in which Fe(III) reduction was the predominant terminal electron-accepting process had much lower concentrations of hydrogen and acetate than sediments in which sulfate reduction or methane production was the predominant terminal process. The low concentrations of hydrogen and acetate in the Fe(III)-reducing sediments were the result of metabolism by Fe(III)-reducing organisms of hydrogen and acetate at concentrations lower than sulfate reducers or methanogens could metabolize them. The results indicate that when Fe(III) is in a form that Fe(III)-reducing organisms can readily reduce, Fe(III)-reducing organisms can inhibit sulfate reduction and methane production by outcompeting sulfate reducers and methanogens for electron donors.     

Methane formation and methane oxidation by methanogenic bacteria.

Methanogenic bacteria were found to form and oxidize methane at the same time. As compared to the quantity of methane formed, the amount of methane simultaneously oxidized varied between 0.3 and 0.001%, depending on the strain used. All the nine tested strains of methane producers (Methanobacterium ruminantium, Methanobacterium strain M.o.H., M. formicicum, M. thermoautotrophicum, M. arbophilicum, Methanobacterium strain AZ, Methanosarcina barkeri, Methanospirillum hungatii, and the "acetate organism") reoxidized methane to carbon dioxide. In addition, they assimilated a small part of the methane supplied into cell material. Methanol and acetate also occurred as oxidation products in M. barkeri cultures. Acetate was also formed by the "acetate organism," a methane bacterium unable to use methanogenic substrates other than acetate. Methane was the precursor of the methyl group of the acetate synthesized in the course of methane oxidation. Methane formation and its oxidation were inhibited equally by 2-bromoethanesulfonic acid. Short-term labeling experiments with M. thermoautotrophicum and M. hungatii clearly suggest that the pathway of methane oxidation is not identical with a simple back reaction of the methane formation process.     

Comparison of Acetate Turnover in Methanogenic and Sulfate-Reducing Sediments by Radiolabeling and Stable Isotope Labeling and by Use of Specific Inhibitors: Evidence for Isotopic Exchange

Acetate turnover in the methanogenic freshwater anoxic sediments of Lake Vechten, The Netherlands, and in anoxic sediments from the Tamar Estuary, United Kingdom, and the Grosser Jasmunder Bodden, Germany, the latter two dominated by sulfate reduction, was determined. Stable isotopes and radioisotopes, inhibitors (chloroform and fluoroacetate), and methane flux were used to provide independent estimates of acetate turnover. Pore water acetate pool sizes were determined by gas chromatography with a flame ionization detector, and stable isotope-labeled acetate was determined by gas chromatography-mass spectrometry. The appearance of acetates with a different isotope labeling pattern from that initially added demonstrated that isotopic exchange occurred during methanogenic acetate metabolism. The predominant exchange processes were (i) D-H exchange in the methyl group and (ii) (sup13)C-(sup12)C exchange at the carboxyl carbon. These exchanges are most probably caused by the activity of the enzyme complex carbon monoxide dehydrogenase and subsequent methyl group dehydrogenation by tetrahydromethanopterine or a related enzyme. The methyl carbon was not subject to exchange during transformation to methane, and hence acetate with the methyl carbon labeled will provide the most reliable estimate of acetate turnover to methane. Acetate turnover rate estimates with these labels were consistent with independent estimates of acetate turnover (acetate accumulation after inhibition and methane flux). Turnover rates from either radioisotope- or stable isotope-labeled methyl carbon isotopes are, however, dependent on accurate determination of the acetate pool size. The additions of large amounts of stable isotope-labeled acetate elevate the acetate pool size, stimulating acetate consumption and causing deviation from steady-state kinetics. This can, however, be overcome by the application of a non-steady-state model. Isotopic exchange in sediments dominated by sulfate reduction was minimal.     

Anaerobic Microflora of Everglades Sediments: Effects of Nutrients on Population Profiles and Activities

Everglades sediments (wetland soils) near sources of agricultural runoff had low redox potentials, were blackened with sulfide, and displayed high porewater phosphorus (total) concentrations and high water column conductivities. These sediments yielded 10(sup3)- to 10(sup4)-fold-higher numbers of culturable anaerobes, including methanogens, sulfate reducers, and acetate producers, than did sediments from Everglades and Lake Okeechobee comparative control sites not as directly associated with agricultural runoff. These observations demonstrated that there was a general, rather than specific, enhancement of the anaerobic microflora in the sediments most likely influenced by agricultural runoff. Despite these differences in microfloral patterns, methylmercury and total mercury levels were similar among these contrasting sediments. Although available sulfate and phosphorus appeared to stimulate the productivity of sulfate reducers in Everglades sediments, the number of culturable sulfate reducers did not directly correspond to the concentration of sulfate and phosphorus in porewaters. Microcosms supplemented with sulfate, nitrate, and phosphate altered the initial capacities of the sediment microflora to produce acetate and methane from endogenous matter. For sediments nearest sources of agricultural runoff, phosphorus temporarily enhanced acetate formation and initially suppressed methane production, sulfate enhanced acetate formation but did not significantly alter the production of methane, and nitrate totally suppressed the initial production of both methane and acetate. In regards to the latter, microbes capable of dissimilating nitrate to ammonium were present in greater culturable numbers than denitrifiers. In microcosms, acetate was a major source of methane, and supplemental hydrogen was directed towards the synthesis of acetate via CO(inf2)-dependent acetogenesis. These findings demonstrate that Everglades sediments nearest agricultural runoff have enhanced anaerobic microbial profiles and that the anaerobic microflora are poised to respond rapidly to phosphate, sulfate, and nitrate input.     

Methanotrophic Bacteria of Acidic Sphagnum Peat Bogs

Acidic Sphagnum peat bogs cover a considerable part of the territory of Russia and are an important natural source of biogenic methane, which is formed in their anaerobic layers. A considerable portion of this methane is consumed in the aerobic part of the bog profile by acidophilic methanotrophic bacteria, which comprise the methane filter of Sphagnum peat bogs and decrease CH₄ emission to the atmosphere. For a long time, these bacteria escaped isolation, which became possible only after the elucidation of the optimal conditions of their functioning in situ: pH 4.5–5.5; temperature, from 15 to 20°C; and low salt concentration in the solution. Imitation of these conditions and rejection of earlier used media with a high content of biogenic elements allowed methanotrophic bacteria of two new genera and species—Methylocella palustris and Methylocapsa acidiphila—to be isolated from the peat of Sphagnum peat bogs of European northern Russia and western Siberia. These bacteria are well adapted to the conditions in cold, acidic, oligotrophic Sphagnum peat bogs. They grow in a pH range of 4.2–7.5 with an optimum at 5.0–5.5, prefer moderate temperatures (15–25°C) and media with a low content of mineral salts (200–500 mg/l), and are capable of active dinitrogen fixation. Design of fluorescently labeled 16S rRNA–targeted oligonucleotide probes for the detection of Methylocella palustris and Methylocapsa acidiphila and their application to the analysis of sphagnum peat samples showed that these bacteria represent dominant populations of methanotrophs with a density of 10⁵–10⁶ cells/g peat. In addition to Methylocella and Methylocapsa populations, one more abundant population of methanotrophs was revealed (10⁶ cells/g peat), which were phylogenetically close to the genus Methylocystis.