Thermophilic and thermotolerant aerobic methanotrophs

The review generalizes the modern data on the taxonomic, structural, and functional diversity of aerobic methanotrophs growing at 25–50°C (Methylococcus capsulatus), 30–62°C (Methylocaldum szegediense, Methylocaldum gracile, and Methylocaldum tepidum), and 50–65°C (Methylothermus thermalis), which belong mainly to the Gammaproteobacteria. The specific features of adaptation of these methanotrophs to the temperature influences are considered on the metabolic and genetic levels. The recent sensational reports on the discovery and primary characterization of thermoacidophilic methanotrophs of the phylum Verrucomicrobia surviving at extreme pH (1–2) and temperature (65°C) values, corresponding to extremely low levels of CH₄ and O₂ solubility, are analyzed. The possibilities of implementation of the biotechnological potential of thermophilic and thermotolerant methanotrophs are discussed.     

植物建植对垃圾填埋场生物覆盖层甲烷氧化及其微生物群落的影响

利用柱试验模拟填埋场生物覆盖层,研究了白三叶和苜蓿建植对增强覆盖层甲烷(CH4)氧化能力及保持甲烷氧化优势菌群的影响。结果表明:植物建植能明显降低基质含水率,提高氮含量,改善O2和CH4扩散,提高基质CH4氧化能力;在CH4氧化的高速期和下降期,植物建植的CH4氧化速率显著高于对照,白三叶和苜蓿处理之间无显著差异;在CH4氧化的低速期,对照与植物建植之间的CH4氧化速率无显著差异,而苜蓿处理显著高于白三叶处理。基于磷脂脂肪酸(PLFA)的微生物群落结构分析表明,植物建植有利于Ⅰ型菌在深层的分布,随着CH4氧化速率逐渐下降,柱体底部甲烷氧化细菌群落由Ⅰ型为主向Ⅱ型为主转变。     

Do nitrogen fertilizers stimulate or inhibit methane emissions from rice fields?

In rice cultivation, there are controversial reports on net impacts of nitrogen (N) fertilizers on methane (CH ₄) emissions. Nitrogen fertilizers increase crop growth as well as alter CH ₄ producing (Methanogens) and consuming (Methanotrophs) microbes, and thereby produce complex effects on CH ₄ emissions. Objectives of this study were to determine net impact of N fertilizers on CH ₄ emissions and to identify their underlying mechanisms in the rice soils. Database was obtained from 33 published papers that contained CH ₄ emissions observations from N fertilizer (28–406 kg N ha^?1) treatment and its control. Results have indicated that N fertilizers increased CH ₄ emissions in 98 of 155 data pairs in rice soils. Response of CH ₄ emissions per kg N fertilizer was significantly (P < 0.05) greater at < 140 kg N ha^?1 than > 140 kg N ha^?1 indicating that substrate switch from CH ₄ to ammonia by Methanotrophs may not be a dominant mechanism for increased CH ₄ emissions. On the contrary, decreased CH ₄ emission in intermittent drainage by N fertilizers has suggested the stimulation of Methanotrophs in rice soils. Effects of N fertilizer stimulated Methanotrophs in reducing CH ₄ emissions were modified by the continuous flood irrigation due to limitation of oxygen to Methanotrophs. Greater response of CH ₄ emissions per kg N fertilizer in urea than ammonia sulfate probably indicated the interference of sulfate in the CH ₄ production process. Overall, response of CH ₄ emissions to N fertilizers was correlated with N‐induced crop yield (r = +0.39; P < 0.01), probably due to increased carbon substrates for Methanogens. Using CH ₄ emission observations, this meta‐analysis has identified dominant microbial processes that control net effects of N fertilizers on CH ₄ emissions in rice soils. Finally, we have provided a conceptual model that included microbial processes and controlling factors to predict effects of N fertilizers on CH ₄ emissions in rice soils.     

On the Problem of Anaerobic Methane Oxidation

To clarify the biological mechanism of anaerobic methane oxidation, experiments were performed with samples of the Black Sea anaerobic sediments and with the aerobic methane-oxidizing bacterium Methylomonas methanica strain 12. The inhibition–stimulation analysis did not allow an unambiguous conclusion to be made about a direct and independent role of either methanogenic or sulfate-reducing microorganisms in the biogeochemical process of anaerobic methane oxidation. Enrichment cultures obtained from samples of water and reduced sediments oxidized methane under anaerobic conditions, primarily in the presence of acetate or formate or of a mixture of acetate, formate, and lactate. However, this ability was retained by the cultures for no more than two transfers on corresponding media. Experiments showed that the aerobic methanotroph Mm. methanica strain 12 is incapable of anaerobic methane oxidation at the expense of the reduction of amorphous FeOOH.     

Taxonomic Characterization of New Alkaliphilic and Alkalitolerant Methanotrophs from Soda Lakes of the Southeastern Transbaikal Region and description of Methylomicrobium buryatense sp.nov.

Five strains of obligate methanotrophic bacteria (4G, 5G, 6G, 7G and 5B) isolated from bottom sediments of Southeastern Transbaikal soda lakes (pH 9.5–10.5) are taxonomically described. These bacteria are aerobic, Gram-negative monotrichous rods having tightly packed cup-shaped structures on the outer cell wall surface (S-layers) and Type I intracytoplasmic membranes. All the isolates possess particulate methane monooxygenase (pMMO) and one strain (5G) also contains soluble methane monooxygenase (sMMO). They assimilate methane and methanol via the ribulose monophosphate pathway (RuMP). The isolates are alkalitolerant or facultatively alkaliphilic, able to grow at pH 10.5–11.0 and optimally at pH 8.5–9.5. These organisms are obligately dependent on the presence of sodium ions in the growth medium and tolerate up to 0.9–1.4 M NaCl or 1 M NaHCO₃. Although being mesophilic, all the isolates are resistant to heating (80 °C, 20 min), freezing and drying. Their cellular fatty acids profiles primarily consist of C_16:1. The major phospholipids are phosphatidylethanolamine and phosphatidylglycerol. The main quinone is Q-8. The DNA G+C content ranges from 49.2–51.5 mol%. Comparative 16S rDNA sequencing showed that the newly isolated methanotrophs are related to membres of the Methylomicrobium genus. However, they differ from the known members of this genus by DNA-DNA relatedness. Based on pheno- and genotypic characteristics, we propose a new species of the genus Methylomicrobium - Methylomicrobium buryatense sp. nov.     

Pristine New Zealand forest soil is a strong methane sink

Methanotrophic bacteria oxidize methane (CH₄) in forest soils that cover ～30% of Earth's land surface. The first measurements for a pristine Southern Hemisphere forest are reported here. Soil CH₄ oxidation rate averaged 10.5±0.6 kg CH₄ ha^?1 yr^?1, with the greatest rates in dry warm soil (up to 17 kg CH₄ ha^?1 yr^?1). Methanotrophic activity was concentrated beneath the organic horizon at 50–100 mm depth. Water content was the principal regulator of (r²=0.88) from the most common value of field capacity to less than half of this when the soil was driest. Multiple linear regression analysis showed that soil temperature was not very influential. However, inverse co‐variability confounded the separation of soil water and temperature effects in situ. Fick's law explained the role of water content in regulating gas diffusion and substrate supply to the methanotrophs and the importance of pore size distribution and tortuosity. This analysis also showed that the chambers used in the study did not affect the oxidation rate measurements. The soil was always a net sink for atmospheric CH₄ and no net CH₄ (or nitrous oxide, N₂O) emissions were measured over the 17‐month long study. For New Zealand, national‐scale extrapolation of our data suggested the potential to offset 13% of CH₄ emissions from ca. 90 M ruminant animals. Our average was about 6.5 times higher than rates reported for most Northern Hemisphere forest soils. This very high was attributed to the lack of anthropogenic disturbance for at least 3000–5000 years and the low rate of atmospheric nitrogen deposition. Our truly baseline data could represent a valid preagricultural, preindustrial estimate of the soil sink for temperate latitudes.     

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。     

土壤中甲烷厌氧氧化菌多样性的分子检测

甲烷厌氧氧化作用是减少海洋底泥甲烷释放的重要生物地球化学过程,然而在陆地生态系统中甲烷厌氧氧化作用及其功能菌群的生态功能仍然不确定。对甲烷厌氧氧化菌多样性的研究可为减少甲烷排放提供重要科学依据。与传统的分离培养方法比较,分子检测方法是一种更为快速和高效的研究手段,可直接和全面的反映参与甲烷厌氧氧化作用的功能微生物。以DNA分子标记物为研究对象,重点探讨三类主要的分子标记基因,即16S rRNA,mcr A和pmo A,所采用的相关探针和引物信息,同时从定性和定量两个角度综述土壤甲烷厌氧氧化菌的多样性研究的主要进展,最后提出厌氧甲烷氧化菌多样性研究中存在的一些问题和相应的解决思路。     

Methane Oxidation in Landfill Cover Soil

Methane oxidation in the cover soil of the Khmet'evo municipal landfill in Moscow oblast was investigated. Methane emission from the experimental site of the landfill was highly heterogeneous. At a depth of 45–60 cm, the pore gas mainly consisted of CH₄ (60–70%) and CO₂ (30–40%). In the upper layers of the cover soil, the concentration of these gases sharply decreased. Methods for estimation of the methane-oxidizing activity in the cover soil of the landfill were tested. The rate of methane oxidation in the soil correlated with the cell number of culturable methanotrophic bacteria and was the factor limiting methane emission from the surface of the landfill. The method of indirect immunofluorescence revealed ten known species of methanotrophic bacteria in enrichment cultures obtained from samples of the cover soil. Our results also indicate the presence of unknown psychrotolerant methanotrophs that are active at the low temperatures characteristic of Moscow oblast.     

New Thermophilic Methanotrophs of the Genus Methylocaldum

Two pure cultures of obligate methanotrophs, strains H-11 and O-12, growing in the temperature range from 30 to 61°C with a optimum at 55°C were isolated from samples of silage and manure. Based on the results of analysis of the 16S rRNA genes and genes of membrane-bound methane monooxygenase, as well as on phenotypic properties, the isolates were assigned to the genus Methylocaldum. Significant temperature-dependent variations in morphology and phospholipid and fatty acid composition were revealed. Both strains assimilated methane carbon via the ribulose monophosphate, serine, and ribulose bisphosphate pathways. The activity of hexulosephosphate synthase was independent of the cultivation temperature; however, the activities of hydroxypyruvate reductase and ribulose bisphosphate carboxylase were higher in cells grown at 55°C than in cells grown at 37°C, indicating the important roles of the serine and ribulose bisphosphate pathways in the thermoadaptation of the strains under study. NH₄ ⁺ assimilation occurred through reductive amination of -ketoglutarate and via the glutamate cycle. The relationship between the physiological and biochemical peculiarities of the isolates and their thermophilic nature is discussed.