Potential activity of methane and ammonia oxidation by methanotropic communities from soda lakes of the southern Transbaikal

Radioisotopic measurements of the methane consumption by mud samples taken from nine Southern Transbaikal soda lakes (pH 9.5-10.6) showed an intense oxidation of methane in the muds of lakes Khuzhirta, Bumalai Nur, Gorbunka, and Suduntuiskii Torom, with the maximum oxidation rate in the mud of lakes Khuzhirta (33.2 nmol/(ml day)). The incorporation rate of the radioactive label from 14CH4 into 14CO2 was higher than into acid-stable metabolites. Optimum pH values for methane oxidation in water samples were 7-8, whereas mud samples exhibited two peaks of methane oxidation activity (at pH 8.15-9.4 and 5.8-7.0). The majority of samples could oxidize ammonium to nitrites; the oxidation was inhibited by methane. The PCR amplification analysis of samples revealed the presence of genes encoding soluble and particulate methane monooxygenase and methanol dehydrogenase. Three alkaliphilic methanotrophic bacteria of morphotype I were isolated from mud samples in pure cultures, one of which, B5, was able to oxidize ammonium to nitrites at pH 7-11. The data obtained suggest that methanotrophs are widely spread in the soda lakes of Southern Transbaikal, where they actively oxidize methane and ammonium.     

Response of methanotrophic communities to afforestation and reforestation in New Zealand

Methanotrophs use methane (CH₄) as a carbon source. They are particularly active in temperate forest soils. However, the rate of change of CH₄ oxidation in soil with afforestation or reforestation is poorly understood. Here, soil CH₄ oxidation was examined in New Zealand volcanic soils under regenerating native forests following burning, and in a mature native forest. Results were compared with data for pasture to pine land-use change at nearby sites. We show that following soil disturbance, as little as 47 years may be needed for development of a stable methanotrophic community similar to that in the undisturbed native forest soil. Corresponding soil CH₄-oxidation rates in the regenerating forest soil have the potential to reach those of the mature forest, but climo-edaphic fators appear limiting. The observed changes in CH₄-oxidation rate were directly linked to a prior shift in methanotrophic communities, which suggests microbial control of the terrestrial CH₄ flux and identifies the need to account for this response to afforestation and reforestation in global prediction of CH₄ emission.     

Trichloroethylene oxidation by the membrane-associated methane monooxygenase in type I,type II and type X methanotrophs

Trichloroethylene (TCE) oxidation was examined in 9 different methanotrophs grown under conditions favoring expression of the membrane associated methane monooxygenase. Depending on the strain, TCE oxidation rates varied from 1 to 677 pmol/min/mg cell protein. Levels of TCE in the reaction mixture were reduced to below 40 nmolar in some strains. Cells incubated in the presence of acetylene, a selective methane monooxygenase inhibitor, did not oxidize TCE.Cultures actively oxidizing TCE were monitored for the presence of the soluble methane monooxygenase (sMMO) and membrane associated enzyme (pMMO). Transmission electron micrographs revealed the cultures always contained the internal membrane systems characteristic of cells expressing the pMMO. Naphthalene oxidation by whole cells, or by the cell free, soluble or membrane fractions was never observed. SDS denaturing gels of the membrane fraction showed the polypeptides associated with the pMMO. Cells exposed to ¹⁴C-acetylene showed one labeled band at 26 kDa, and this protein was observed in the membrane fraction. In the one strain examined by EPR spectroscopy, the membrane fraction of TCE oxidizing cells showed the copper complexes characteristic of the pMMO. Lastly, most of the strains tested showed no hybridization to sMMO gene probes. These findings show that the pMMO is capable of TCE oxidation; although the rates are lower than those observed for the sMMO.     

甲烷氧化过程中铜的作用研究进展

甲烷生物氧化在全球甲烷平衡和温室效应控制中扮演着重要的角色,而铜是甲烷生物氧化过程中的重要影响因子.一方面,铜是调控不同类型甲烷单加氧酶表达的主要影响因子,是组成颗粒性甲烷单加氧酶的必需金属元素;另一方面,在自然环境体系中,铜含量及其形态的变化对甲烷氧化菌的分布、代谢甲烷和非甲烷类有机化合物的能力以及甲烷氧化菌的特异性铜捕获系统也会产生较大影响.准确把握铜在甲烷生物氧化过程中发挥的作用将有助于全面了解甲烷生物氧化过程,进而更好地指导甲烷氧化微生物在温室气体减排及非甲烷有机物污染修复中的应用.本文主要从铜的角度,概述了铜对甲烷氧化菌的分布和活性的影响,介绍了铜在调控甲烷单加氧酶的表达和活性以及调节甲烷氧化菌铜捕获系统方面的作用,并展望了其研究方向.     

Simultaneous nitrification and denitrification in a mixed methanotrophic culture

Simultaneous nitrification and denitrification using a mixed methanotrophic culture was investigated. When both NO₃ ^–-N (108 mg l^–1) and NH₃-N (59 mg l^–1) were added into batch reactors, nitrate removal was complete within 10 h at the rate of 47 mg NO₃ ^–-N g VSS^–1 day^–1 when dissolved oxygen (DO) concentration was maintained at 2 mg DO l^–1. Ammonia removal started simultaneously with nitrate removal at a slower rate of 14 NH₃-N g VSS^–1 day^–1. No significant accumulation of nitrite or nitrate during ammonia utilization suggested the occurrence of simultaneous nitrification and denitrification.     

Carbonic anhydrase activity in halophilic anaerobes from soda lakes

The activity and cellular localization of carbonic anhydrase (CA) in two alkaliphilic anaerobes growing in soda lakes at pH 9–10 were studied. CA activity in the cell extracts of the acetogenic bacterium Natroniella acetigena was comparable to that of neutrophilic acetogens. Hydrogenotrophically grown cells of Desulfonatronum lacustre exhibited higher CA activity compared to the cells grown on medium with formate. High CA activity in the cytoplasmic fraction and the absence of high activity in the extracellular fraction were demonstrated. We propose that the cytoplasmic CA in alkaliphilic sulfate-reducers participates in conversion of bicarbonate to CO₂, which is reduced in the cell to acetate via the acetyl-CoA pathway.     

Biphasic kinetics of a methanotrophic community is a combination of growth and increased activity per cell

Because methane-oxidizing bacteria (MOB) are the only biological sink for the greenhouse gas methane, knowledge of the functioning of these bacteria in various ecosystems is needed to understand the dynamics observed in global methane emission. The activity of MOB is commonly assessed by methane oxidation assays. The resulting methane depletion curves often follow a biphasic pattern of initial and induced methane oxidation activity, often interpreted as representing the in situ active and total MOB community, respectively. The application of quantitative-PCR on soil incubations, which were stopped before, at and after the transition point in the methane-depletion curve, demonstrated that both pmoA -mRNA was produced as well as substantial cell growth took place already in the initial phase. In addition, type Ia and II MOB displayed markedly different behaviour, which can be interpreted as ecologically different strategies. For the correct interpretation of methane oxidation assays, the use of small time windows is recommended to calculate methane oxidation activities to avoid substantial cell growth.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^-1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^-1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

Production of methanol from methane by methanotrophic bacteria

Methanotrophs can oxidize methane to carbon dioxide through sequential reactions catalyzed by a series of enzymes including methane monooxygenase, methanol dehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase. When suspensions of methanotrophic bacteria of Methylosinus trichosporium IMV 3011 were incubated at 32°C with methane and oxygen, there was an extracellular accumulation of methanol from methane oxidation in response to carbon dioxide addition. Maximal accumulation of methanol was achieved with 40% carbon dioxide in the mixed reaction gases. A continuous experiment was performed in a continuous ultrafiltration reactor. The optimum gas mixture containing 20% (v v^?1) methane, 20% oxygen, 20% nitrogen and 40% carbon dioxide was used to provide substrates and to maintain the transmembrane pressure. The product (methanol) was removed in the eluate buffer. The initial methanol concentration in the eluate buffer was 8.22 μmol L^?1. The bioreactor was operated continuously for 198 h without obvious loss of productivity.     

The structure of phototrophic communities of soda lakes of the southeastern Transbaikal region

The structure of benthic phototrophic communities of 24 soda lakes of the southeastern Transbaikal Region was studied. The physicochemical properties of the lakes were determined. The results of the cell count of anoxygenic phototrophic bacteria (APB) belonging to various groups are presented. The influence of salinity on the structure of APB communities was investigated. The APB reaction to environmental conditions was determined. Massive development of phototrophic microorganisms in the form of mats and films was observed in the majority of the investigated lakes. The APB communities were characterized by a wide diversity of species and evenness of species composition. Purple sulfur bacteria of the families Ectothiorhodospiraceae and Chromatiaceae were predominant. Purple nonsulfur bacteria of the family Rhodobacteraceae, green filamentous bacteria Oscillochloris sp., and heliobacteria were also detected. According to preliminary data, no less than 15 species of APB occur in the studied lakes. Among them, three novel genera and four species have already been described. Identification of other isolates is still in progress. The lakes make an almost continuous series of fresh, brackish, and saline water bodies, varying in their degree of mineralization. It was demonstrated that the structure of APB communities was unaffected by salinity ranging from 5 to 40 g/l. At salt concentrations of lower than 5 g/l, the level of water mineralization became a limiting factor. Experiments with the isolated cultures showed that the APB were obligately dependent on the presence of carbonate ions in the medium. They were haloalkalitolerant or haloalkaliphilic. Thus, they are well adapted to the conditions of soda lakes with a high mineralization. It was demonstrated that soda lakes of the southeastern Transbaikal Region represent a special type of habitat which harbors a peculiar autochthonous microflora and differs from both highly mineralized soda lakes and shallow saline water bodies of the sea origin.     

The structure of phototrophic communities of soda lakes of the southeastern Transbaikal Region

The structure of benthic phototrophic communities of 24 soda lakes of the southeastern Transbaikal Region was studied. The physicochemical properties of the lakes were determined. The results of enumeration of anoxygenic phototrophic bacteria (APB) belonging to various groups are presented. The influence of salinity on the structure of APB communities was investigated. The APB reaction to environmental conditions was determined. Massive development of phototrophic microorganisms in the form of mats and films was observed in the majority of the investigated lakes. The APB communities were characterized by a wide diversity and evenness of species composition. Purple sulfur bacteria of the families Ectothiorhodospiraceae and Chromatiaceae were predominant. Purple nonsulfur bacteria of the family Rhodobacteraceae, green filamentous bacteria Oscillochloris sp., and heliobacteria were also detected. According to preliminary data, no less than 15 species of APB occur in the studied lakes. Among them, three novel genera and four species have already been described. Identification of other isolates is still in progress. The lakes make an almost continuous series of fresh, brackish, and saline water bodies, varying in their degree of mineralization. It was demonstrated that the structure of APB communities was unaffected by changes in salinity from 5 to 40 g/l. At salt concentrations of lower than 5 g/l, the level of water mineralization became a limiting factor. Experiments with the isolated cultures showed that the APB were obligately dependent on the presence of carbonate ions in the medium. They were haloalkalitolerant or haloalkaliphilic. Thus, they are well adapted to the conditions of soda lakes with a low of moderate mineralization. It was demonstrated that soda lakes of the southeastern Transbaikal Region represent a special type of habitat which harbors a peculiar autochthonous microflora and differs from both highly mineralized soda lakes and shallow saline water bodies of the sea origin.     

Mass spectrometric measurements of methane and oxygen utilization by methanotrophic bacteria

Abstract A mass spectrometer with membrane inlet was used to measure methane and oxygen utilization rates at various methane concentrations in Methylosinus trichosporium and a locally isolated strain of a methane-oxidizing coccus (OU-4-1). The apparent K _m for methane was found to be 2 μM for M. trichosporium and 0.8 μM for strain OU-4-1. These K _m-values are 10–30 times lower than most previously reported values. The ratio of oxygen to methane utilization rates was 1.7 for M. trichosporium and 1.5 for strain OU-4-1 corresponding to a growth yield of 0.38 and 0.63 g dry weight/g methane, respectively.     

Coupling of Microbial Processes of Methane and Ammonium Oxidation in Soils

The effect of ammonium ions on the activity of methane oxidation in soils was studied. The degree of inhibition of the methanotrophic activity in the presence of ammonium in the soil solution was quantitatively assessed as dependent on ammonium concentration and the properties of different types of soils of the European part of Russia.     

Molecular characterization of methanotrophic communities in forest soils that consume atmospheric methane

Methanotroph abundance was analyzed in control and long-term nitrogen-amended pine and hardwood soils using rRNA-targeted quantitative hybridization. Family-specific 16S rRNA and pmoA/amoA genes were analyzed via PCR-directed assays to elucidate methanotrophic bacteria inhabiting soils undergoing atmospheric methane consumption. Quantitative hybridizations suggested methanotrophs related to the family Methylocystaceae were one order of magnitude more abundant than Methyloccocaceae and more sensitive to nitrogen-addition in pine soils. 16S rRNA gene phylotypes related to known Methylocystaceae and acidophilic methanotrophs and pmoA/amoA gene sequences, including three related to the upland soil cluster Alphaproteobacteria (USCalpha) group, were detected across different treatments and soil depths. Our results suggest that methanotrophic members of the Methylocystaceae and Beijerinckiaceae may be the candidates for soil atmospheric methane consumption.     

The absolute requirement for carbon dioxide for aerobic methane oxidation by a methanotrophic-heterotrophic soil community of bacteria

An aerobic methanotrophic-heterotrophic soil community has been characterised when growing with different partial pressures of CO₂. The methanotrophic population using methane as carbon source reached 3 × 10⁷ cfu ml^–1 with one of the major methanotrophs being of type II which uses the serine pathway for C assimilation. Optimal methanotrophic activity required the addition of CO₂, and in the absence of CO₂ no methane oxidisers grew. Partial pressures of CO₂ from 1.6 to 11.6 kPa gave optimal cell growth and production of soluble organic compounds. Biomass yield, soluble organics and CO₂ production were 0.36, 0.15, and 0.48 mg mg^–1 methane uptake, respectively, with CO₂ at 11.6 kPa. The results presented here may have important implications for the use of methane-oxidising bacteria in bioremedial applications.     

长期不同施肥下水稻土甲烷氧化能力及甲烷氧化菌多样性的变化

稻田内源甲烷的氧化是稻田甲烷减排的重要途径。而甲烷氧化菌是土壤中甲烷氧化的主要施动者,在长期不同施肥条件下,土壤微生物群落的演变是否影响到土壤甲烷氧化菌群落结构及其活性,进而影响到田土壤CH4向大气的实际排放强度还不清楚。为此,选择太湖地区一个长期肥料试验的稻田土壤为研究对象,分析长期不同肥料施用对土壤甲烷氧化能力的影响及其与土壤中甲烷氧化菌群落结构变化的可能关系。结果表明,长期不同的施肥措施下稻田土壤对甲烷的氧化能力产生了明显差异,伴随着土壤中甲烷氧化菌（MOBI和MOBII）的基因群落多样性的显著变化。长期单一施用氮肥为主的化肥显著降低了土壤对甲烷的氧化能力,同时显著降低了稻田土壤甲烷氧化菌的多样性和丰富度;不同施肥下甲烷氧化菌多样性的变化与土壤的甲烷氧化能力的变化趋势相一致。因此,研究显示长期不同施肥处理下甲烷氧化菌群落结构的改变可能是引起水稻土甲烷氧化能力变化的一个主要因素,有机无机配合施用可以明显降低稻田土壤甲烷的大气释放潜能。但长期不同施肥处理下甲烷氧化菌活性的变化还有待于进一步研究。     

Microbiology of Lonar Lake and other soda lakes

Soda lakes are saline and alkaline ecosystems that are believed to have existed throughout the geological record of Earth. They are widely distributed across the globe, but are highly abundant in terrestrial biomes such as deserts and steppes and in geologically interesting regions such as the East African Rift valley. The unusual geochemistry of these lakes supports the growth of an impressive array of microorganisms that are of ecological and economic importance. Haloalkaliphilic Bacteria and Archaea belonging to all major trophic groups have been described from many soda lakes, including lakes with exceptionally high levels of heavy metals. Lonar Lake is a soda lake that is centered at an unusual meteorite impact structure in the Deccan basalts in India and its key physicochemical and microbiological characteristics are highlighted in this article. The occurrence of diverse functional groups of microbes, such as methanogens, methanotrophs, phototrophs, denitrifiers, sulfur oxidizers, sulfate reducers and syntrophs in soda lakes, suggests that these habitats harbor complex microbial food webs that (a) interconnect various biological cycles via redox coupling and (b) impact on the production and consumption of greenhouse gases. Soda lake microorganisms harbor several biotechnologically relevant enzymes and biomolecules (for example, cellulases, amylases, ectoine) and there is the need to augment bioprospecting efforts in soda lake environments with new integrated approaches. Importantly, some saline and alkaline lake ecosystems around the world need to be protected from anthropogenic pressures that threaten their long-term existence.     

好氧甲烷氧化菌生理生态特征及其在自然湿地中的群落多样性研究进展

甲烷氧化菌是一类可以利用甲烷作为唯一碳源和能源的细菌,在全球变化和整个生态系统碳循环过程中起着重要的作用。近年来,对甲烷氧化菌的生理生态特征及其在自然湿地中的群落多样性研究取得了较大进展。在分类方面,疣微菌门、NC10门及两个丝状菌属甲烷氧化菌的发现使其分类体系得到了进一步的完善;在单加氧酶方面,发现甲烷氧化菌可以利用pM MO和sM MO两种酶进行氧化甲烷的第一步反应,Ⅱ型甲烷氧化菌中pM MO2的发现证实甲烷氧化菌可以利用这种酶氧化低浓度的甲烷;在底物利用方面,已经发现了越来越多的兼性营养型甲烷氧化菌,证实它们可以利用的底物比之前认为的更广泛,其中包括乙酸等含有碳碳键的化合物;在生存环境方面,能在不同温度、酸度和盐度的环境中生存的甲烷氧化菌不断被分离出来。全球自然湿地甲烷氧化菌群落多样性的研究目前主要集中在北半球高纬度的酸性泥炭湿地,Ⅱ型甲烷氧化菌Methylocystis、Methylocella和Methylocapsa是这类湿地主要的甲烷氧化菌类群,尤其以Methylocystis类群最为广泛,而Ⅰ型甲烷氧化菌尤其是Methylobacter在北极寒冷湿地中占优势。随着高通量测序时代的到来和新的分离技术的发展,对甲烷氧化菌的现有认识将面临更多的挑战和发展。     

Anaerobic methane oxidation potential and bacteria in freshwater lakes: Seasonal changes and the influence of trophic status

Nitrite-dependent anaerobic methane oxidation (n-damo), mainly carried out by n-damo bacteria, is an important pathway for mitigating methane emission from freshwater lakes. Although n-damo bacteria have been detected in a variety of freshwater lakes, their potential and distribution, and associated environmental factors, remain unclear. Therefore, the current study investigated the potential and distribution of anaerobic methanotrophs in sediments from Erhai Lake and Dianchi Lake, two adjacent freshwater lakes in the Yunnan Plateau with different trophic status. Both lakes showed active anaerobic methane oxidation potential and harbored a high density of n-damo bacteria. Based on the n-damo pmoA gene, sediment n-damo bacterial communities mainly consisted of Candidatus Methylomirabils oxyfera and Candidatus Methylomirabils sinica, as well as novel n-damo organisms. Sediment anaerobic methane oxidation potential and the n-damo bacterial community showed notable differences among seasons and between lakes. The environmental variables associated with lake trophic status (e.g. total nitrogen, ammonia nitrogen, nitrate nitrogen, and total organic carbon) might have significant impacts on the anaerobic methane oxidation potential, as well as the abundance and community structure of n-damo bacteria. Therefore, trophic status could determine the n-damo process in freshwater lake sediment.