The Biology and Osmoadaptation of Haloalkaliphilic Methanotrophs

There is increasing evidence for the presence and activity of methanotrophic bacteria in saline and alkaline aquatic environments located in different ecogeographical regions. Alkalitolerant halophilic and alkaliphilic halotolerant methanotrophs of type I were found to be able to utilize methane and methanol, to oxidize ammonium ions, and to transform various organic compounds in a wide range of water salinities (up to 12% NaCl) and pH values (from 5 to 11). The ecophysiological importance of methanotrophs in microbial communities inhabiting saline and alkaline aquatic environments is due to their involvement in the global cycles of methane and major bioelements (C, N, and S). Specific cyto- and biochemical properties of haloalkaliphilic methanotrophs—the synthesis of osmoprotectants (ectoine, 5-oxoproline, and sucrose), the accumulation of potassium ions, the formation of glycoprotein S-layers on the outer surface of their cell walls, and the modification of the chemical composition of their membranes—allow them to adapt to highly saline and alkaline habitats. Due to their specific properties, haloalkaliphilic methanotrophs may be of use in modern biotechnology.     

Diversity and activity of methanotrophs in alkaline soil from a Chinese coal mine

Culture-independent molecular biological techniques, including 16S rRNA gene and functional gene clone libraries and microarray analyses using pmoA (encoding a key subunit of particulate methane monooxygenase), were applied to investigate the methanotroph community structure in alkaline soil from a Chinese coal mine. This environment contained a high diversity of methanotrophs, including the type II methanotrophs Methylosinus / Methylocystis , type I methanotrophs related to Methylobacter / Methylosoma and Methylococcus , and a number of as yet uncultivated methanotrophs. In order to identify the metabolically active methane-oxidizing bacteria from this alkaline environment, DNA stable isotope probing (DNA-SIP) experiments using ¹³CH₄ were carried out. This showed that both type I and type II methanotrophs were active, together with methanotrophs related to Methylocella , which had previously been found only in acidic environments. Methylotrophs, including Methylopila and Hyphomicrobium , were also detected in soil DNA and after DNA-SIP experiments. DNA sequence information on the most abundant, active methanotrophs in this alkaline soil will facilitate the design of oligonucleotide probes to monitor enrichment cultures when isolating key alkaliphilic methanotrophs from such environments.     

低氧生境中好氧甲烷氧化菌的缺氧耐受机理及种群结构研究进展

甲烷生物氧化在全球大气甲烷平衡和温室气体的控制中起着重要作用.氧气是甲烷生物氧化过程中的重要影响因素之一.生境中氧浓度不仅影响好氧甲烷氧化菌的种群结构、活性及甲烷碳的分配,而且好氧甲烷氧化菌在不同氧浓度下具有不同的代谢途径.理解低氧生境中好氧甲烷氧化菌的缺氧耐受机理和甲烷生物氧化过程,对甲烷驱动型生态系统的碳循环和生物多样性有着重要意义.本文以好氧甲烷氧化菌为对象,综述了低氧生境中好氧甲烷氧化菌的活性及其种群结构、好氧甲烷氧化菌的缺氧耐受机理以及低氧生境中甲烷氧化菌与非甲烷氧化菌的关系,并对今后的研究方向进行了展望.     

Production and optimization of a commercially viable alkaline protease from a haloalkaliphilic bacterium

Twenty five haloalkaliphilic bacterial strains were isolated from sea water along the Coastal Gujarat (India) and screened for their ability to secret alkaline proteases. Among them, a potent strain S-20-9 (GenBank accession number EU118360), resembling to Halophilic Bacterium MBIC3303 on the basis of 16S rRNA gene sequencing, was selected for the optimization of enzyme production. S-20-9 produced protease optimally, under aerobic conditions during mid-stationary phase over a broad range of salt (5∼25%, w/v) and pH (7∼10). The optimum production was at pH 9 and 15% (w/v) NaCl. The production was suppressed by lactose, maltose, sucrose, and inorganic nitrogen sources, especially ammonium ions. Further, the production was significantly stimulated by KH₂PO₄ and suppressed by glucose. Similarly, the production was also suppressed at higher concentrations of gelatin, yeast extract, peptone, and casamino acids, indicating towards a threshold value for nitrogen requirement. The growth and protease production were enhanced by mono-valent cation (KCl), while the divalent cations acted as inhibitors. The study holds significance as only few reports are available on the alkaline proteases from haloalkaliphilic bacteria, particularly those from moderate saline habitats.     

Detection of methanotrophic bacteria in environmental samples with the PCR.

We designed PCR primers by using the DNA sequences of the soluble methane monooxygenase gene clusters of Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and these primers were found to be specific for four of the five structural genes in the soluble methane monooxygenase gene clusters of several methanotrophs. We also designed primers for the gram-negative methylotroph-specific methanol dehydrogenase gene moxF. The specificity of these primers was confirmed by hybridizing and sequencing the PCR products obtained. The primers were then used to amplify methanotroph DNAs in samples obtained from various aquatic and terrestrial environments. Our sequencing data suggest that a large number of different methanotrophs are present in peat samples and also that there is a high level of variability in the mmoC gene, which codes for the reductase component of the soluble methane monooxygenase, while the mmoX gene, which codes for the alpha subunit of the hydroxylase component of this enzyme complex, appears to be highly conserved in methanotrophs.     

极端环境下嗜热酸甲烷营养细菌研究进展

甲烷营养细菌能够将温室气体甲烷(CH4)转化为CO2或生物质,在碳生物地球化学循环及缓解由温室气体导致的全球气候变化方面发挥着重要的作用.甲烷营养细菌生存的条件范围较为广泛,但在中性pH (5～8)和中温(20～35℃)范围内生长最佳.系统进化分析认为,它们均属于γ-或α-变形菌门(Proteobacteria).最近3项独立完成的研究从极端热酸(pH接近1,温度高于50℃)环境中分离获得了具有甲烷氧化(营养)功能的微生物,经鉴定均属于疣微菌门(Verrucomicrobia).这些全新的、不同于以往的研究结果不仅是对现有关于甲烷营养细菌生态学认知的进一步拓展,同时也暗示着可能存在着新型的、由微生物介导的CH4氧化途径与机制. 因此,特就极端环境中嗜热嗜酸甲烷营养细菌的最新研究进展作一概述.     

好氧甲烷氧化菌生态学研究进展

好氧甲烷氧化菌是一群以甲烷为碳源和能源的细菌。好氧甲烷氧化菌在自然环境中分布广泛,人类已从土壤、淡水和海洋沉积、泥炭沼泽、热泉、海水和南极环境分离到甲烷氧化菌的纯培养。好氧甲烷氧化菌可分为14个属,包括研究较为深入的隶属于变形菌门Alpha和Gamma纲的细菌,以及属于疣微菌门的极端嗜热嗜酸甲烷氧化菌。最近,好氧甲烷氧化菌还被发现存在于苔藓类植物（尤其是泥炭苔藓）共生体中,兼性营养好氧甲烷氧化菌也被发现。本文通过对好氧甲烷氧化菌的分类、生理生化特征、分子生物学检测方法以及微生物生态学中的研究成果的总结与分析,以及对甲烷氧化菌研究所面临的问题进行讨论,以期为今后进一步开展好氧甲烷氧化菌及其在碳循环中的作用研究提供参考。     

自然湿地土壤产甲烷菌和甲烷氧化菌多样性的分子检测

自然湿地是CH4排放的重要来源之一。产甲烷菌和甲烷氧化菌是介导自然湿地甲烷循环的重要功能菌群。开展产甲烷菌和甲烷氧化菌多样性的检测研究有助于揭示微生物介导的甲烷循环以及自然湿地甲烷排放的时空异质性。传统基于培养的检测方法已被证实无法充分描述产甲烷菌和甲烷氧化菌的多样性,而分子检测方法为自然湿地土壤产甲烷菌和甲烷氧化菌的多样性检测提供了一种更准确和科学的工具。本文综述了自然湿地土壤产甲烷菌和甲烷氧化菌的定性和定量分子检测方法,包括末端限制性片段长度多态性（T-RFLP）、变性梯度凝胶电泳（DGGE）、荧光原位杂交（FISH）和实时定量PCR（real-time qPCR）,重点分析了分子检测中两类重要的标记基因,总结了不同类型自然湿地产甲烷菌和甲烷氧化菌群落多样性的最新成果,提出了我国在该领域今后应深入研究探讨的一些问题及建议。     

Large‐scale biogeography and environmental regulation of methanotrophic bacteria across boreal inland waters

Aerobic methanotrophic bacteria (methanotrophs) use methane as a source of carbon and energy, thereby mitigating net methane emissions from natural sources. Methanotrophs represent a widespread and phylogenetically complex guild, yet the biogeography of this functional group and the factors that explain the taxonomic structure of the methanotrophic assemblage are still poorly understood. Here, we used high‐throughput sequencing of the 16S rRNA gene of the bacterial community to study the methanotrophic community composition and the environmental factors that influence their distribution and relative abundance in a wide range of freshwater habitats, including lakes, streams and rivers across the boreal landscape. Within one region, soil and soil water samples were additionally taken from the surrounding watersheds in order to cover the full terrestrial–aquatic continuum. The composition of methanotrophic communities across the boreal landscape showed only a modest degree of regional differentiation but a strong structuring along the hydrologic continuum from soil to lake communities, regardless of regions. This pattern along the hydrologic continuum was mostly explained by a clear niche differentiation between type I and type II methanotrophs along environmental gradients in pH, and methane concentrations. Our results suggest very different roles of type I and type II methanotrophs within inland waters, the latter likely having a terrestrial source and reflecting passive transport and dilution along the aquatic networks, but this is an unresolved issue that requires further investigation.     

Aerobic methane oxidation and methanotroph community composition during seasonal stratification in Mono Lake, California (USA)

Patterns of aerobic methane (CH4) oxidation and associated methanotroph community composition were investigated during the development of seasonal stratification in Mono Lake, California (USA). CH4 oxidation rates were measured using a tritiated CH4 radiotracer technique. Fluorescence in situ hybridization (FISH), denaturing gradient gel electrophoresis (DGGE) and sequence analysis were used to characterize methanotroph community composition. A temporally shifting zone of elevated CH4 oxidation (59-123 nM day(-1)) was consistently associated with a suboxycline, microaerophilic zone that migrated upwards in the water column as stratification progressed. FISH analysis revealed stable numbers of type I (4.1-9.3 x 10(5) cells ml(-1)) and type II (1.4-3.4 x 10(5) cells ml(-1)) methanotrophs over depth and over time. Denaturing gradient gel electrophoresis and sequence analysis indicated slight shifts in methanotroph community composition despite stable absolute cell numbers. Variable CH4 oxidation rates in the presence of a relatively stable methanotroph population suggested that zones of high CH4 oxidation resulted from an increase in activity of a subset of the existing methanotroph population. These results challenge existing paradigms suggesting that zones of elevated CH4 oxidation activity result from the accumulation of methanotrophic biomass and illustrate that type II methanotrophs may be an important component of the methanotroph population in saline and/or alkaline pelagic environments.     

Anaerobic methanotrophy is stimulated by graphene oxide in a brackish urban canal sediment

Anthropogenic activities are influencing aquatic environments through increased chemical pollution and thus are greatly affecting the biogeochemical cycling of elements. This has increased greenhouse gas emissions, particularly methane, from lakes, wetlands, and canals. Most of the methane produced in anoxic sediments is converted into carbon dioxide by methanotrophs before it reaches the atmosphere. Anaerobic oxidation of methane requires an electron acceptor such as sulphate, nitrate, or metal oxides. Here, we explore the anaerobic methanotrophy in sediments of three urban canals in Amsterdam, covering a gradient from freshwater to brackish conditions. Biogeochemical analysis showed the presence of a shallow sulphate–methane transition zone in sediments of the most brackish canal, suggesting that sulphate could be a relevant electron acceptor for anaerobic methanotrophy in this setting. However, sediment incubations amended with sulphate or iron oxides (ferrihydrite) did not lead to detectable rates of methanotrophy. Despite the presence of known nitrate-dependent anaerobic methanotrophs (Methanoperedenaceae), no nitrate-driven methanotrophy was observed in any of the investigated sediments either. Interestingly, graphene oxide stimulated anaerobic methanotrophy in incubations of brackish canal sediment, possibly catalysed by anaerobic methanotrophs of the ANME-2a/b clade. We propose that natural organic matter serving as electron acceptor drives anaerobic methanotrophy in brackish sediments.     

Termites Facilitate Methane Oxidation and Shape the Methanotrophic Community

Termite-derived methane contributes 3 to 4% to the total methane budget globally. Termites are not known to harbor methane-oxidizing microorganisms (methanotrophs). However, a considerable fraction of the methane produced can be consumed by methanotrophs that inhabit the mound material, yet the methanotroph ecology in these environments is virtually unknown. The potential for methane oxidation was determined using slurry incubations under conditions with high (12%) and in situ (∼0.004%) methane concentrations through a vertical profile of a termite (Macrotermes falciger) mound and a reference soil. Interestingly, the mound material showed higher methanotrophic activity. The methanotroph community structure was determined by means of a pmoA-based diagnostic microarray. Although the methanotrophs in the mound were derived from populations in the reference soil, it appears that termite activity selected for a distinct community. Applying an indicator species analysis revealed that putative atmospheric methane oxidizers (high-indicator-value probes specific for the JR3 cluster) were indicative of the active nest area, whereas methanotrophs belonging to both type I and type II were indicative of the reference soil. We conclude that termites modify their environment, resulting in higher methane oxidation and selecting and/or enriching for a distinct methanotroph population.     

Electroporation-Based Genetic Manipulation in Type I Methanotrophs

Methane is becoming a major candidate for a prominent carbon feedstock in the future, and the bioconversion of methane into valuable products has drawn increasing attention. To facilitate the use of methanotrophic organisms as industrial strains and accelerate our ability to metabolically engineer methanotrophs, simple and rapid genetic tools are needed. Electroporation is one such enabling tool, but to date it has not been successful in a group of methanotrophs of interest for the production of chemicals and fuels, the gammaproteobacterial (type I) methanotrophs. In this study, we developed electroporation techniques with a high transformation efficiency for three different type I methanotrophs: Methylomicrobium buryatense 5GB1C, Methylomonas sp. strain LW13, and Methylobacter tundripaludum 21/22. We further developed this technique in M. buryatense, a haloalkaliphilic aerobic methanotroph that demonstrates robust growth with a high carbon conversion efficiency and is well suited for industrial use for the bioconversion of methane. On the basis of the high transformation efficiency of M. buryatense, gene knockouts or integration of a foreign fragment into the chromosome can be easily achieved by direct electroporation of PCR-generated deletion or integration constructs. Moreover, site-specific recombination (FLP-FRT [FLP recombination target] recombination) and sacB counterselection systems were employed to perform marker-free manipulation, and two new antibiotics, zeocin and hygromycin, were validated to be antibiotic markers in this strain. Together, these tools facilitate the rapid genetic manipulation of M. buryatense and other type I methanotrophs, promoting the ability to perform fundamental research and industrial process development with these strains.     

Molecular diversity of methanotrophs in Transbaikal soda lake sediments and identification of potentially active populations by stable isotope probing

Soda lakes are an environment with an unusually high pH and often high salinity. To identify the active methanotrophs in the Soda lake sediments, sediment slurries were incubated with a 10% (v/v) (13)CH(4) headspace and the (13)C-labelled DNA was subsequently extracted from these sediments following CsCl density gradient centrifugation. This DNA was then used as a template for PCR amplification of 16S rRNA genes and genes encoding PmoA and MmoX of methane monooxygenase, key enzymes in the methane oxidation pathway. Phylogenetic analysis of 16S rRNA genes, PmoA and MmoX identified that strains of Methylomicrobium, Methylobacter, Methylomonas and 'Methylothermus' had assimilated the (13)CH(4). Phylogenetic analysis of PmoA sequences amplified from DNA extracted from Soda lake sediments before Stable Isotope Probing (SIP) treatment showed that a much wider diversity of both type I and type II methanotroph sequences are present in this alkaline environment. The majority of methanotroph sequences detected in the (13)C-DNA studies were from type I methanotrophs, with 50% of 16S rRNA clones and 100% of pmoA clones from both Lake Suduntuiskii Torom and Lake Gorbunka suggesting that the type I methanotrophs are probably responsible for the majority of methane oxidation in this environment.     

Planktonic and sediment-associated aerobic methanotrophs in two seep systems along the North American margin

Methane vents are of significant geochemical and ecological importance. Notable progress has been made toward understanding anaerobic methane oxidation in marine sediments; however, the diversity and distribution of aerobic methanotrophs in the water column are poorly characterized. Both environments play an essential role in regulating methane release from the oceans to the atmosphere. In this study, the diversity of particulate methane monooxygenase (pmoA) and 16S rRNA genes from two methane vent environments along the California continental margin was characterized. The pmoA phylotypes recovered from methane-rich sediments and the overlying water column differed. Sediments harbored the greatest number of unique pmoA phylotypes broadly affiliated with the Methylococcaceae family, whereas planktonic pmoA phylotypes formed three clades that were distinct from the sediment-hosted methanotrophs and distantly related to established methanotrophic clades. Water column-associated phylotypes were highly similar between field sites, suggesting that planktonic methanotroph diversity is controlled primarily by environmental factors rather than geographical proximity. Analysis of 16S rRNA genes from methane-rich waters did not readily recover known methanotrophic lineages, with only a few phylotypes demonstrating distant relatedness to Methylococcus. The development of new pmo primers increased the recovery of monooxygenase genes from the water column and led to the discovery of a highly diverged monooxygenase sequence which is phylogenetically intermediate to Amo and pMMO. This sequence potentiates insight into the amo/pmo superfamily. Together, these findings lend perspective into the diversity and segregation of aerobic methanotrophs within different methane-rich habitats in the marine environment.     

Life in the extreme: thermoacidophilic methanotrophy

Aerobic methane-oxidizing bacteria (methanotrophs) have a key role in the global carbon cycle, converting methane to biomass and carbon dioxide. Although these bacteria have been isolated from many environments, until recently, it was not known if they survived, much less thrived in thermoacidic environments, that is, locations with pH values of approximately 1 and temperatures greater than 50 degrees C. Recently, three independent studies have isolated unusual methanotrophs from such extreme environments, expanding the known functional and phylogenetic diversity of methanotrophs.     

Detection of osmoprotector ectoine content in methylotrophic bacteria using of normal-phase high performance liquid chromatography

Detection and quantitative analysis of ectoine in bacterial biomass were performed by normal-phase high performance liquid chromatography with ultraviolet detection at 230 nm. Quantitative analysis was not hindered by glutamate and sucrose accumulation in bacteria. Measurement of ectoine concentration in haloalkaliphilic methanotrophs Methylobacter marinus 7C and Methylomicrobium alcaliphilum 5S showed that ectoine accumulation reached maximum (5 and 12% of dry cell weight) in the presence of NaCl at concentrations of 4 and 6%, respectively.     

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。