甲烷代谢古菌分离培养研究进展

全球变暖是全人类面临的一个巨大挑战,而温室气体排放持续上升是全球变暖的关键因素,并引发一系列生态环境问题。甲烷是第二温室气体,对全球变暖的贡献达20%。然而,在甲烷代谢中发挥重要作用的产甲烷古菌和厌氧甲烷氧化古菌(anaerobic methanotroph,ANME)较难培养,极大地限制了人们对甲烷代谢及其影响碳源-汇关系与机制的研究。本文综述了最新产甲烷古菌和ANME富集、分离和培养方法,包括富集培养、原位培养、共培养、微流控技术、稀释分离和固体分离技术、ANME反应器和培养瓶富集培养,以及宏基因组预测和反向基因组学,并对这些方法的优缺点进行了评估,对未来甲烷代谢古菌的富集、分离和培养提出新的建议。     

有氧产甲烷研究进展

甲烷是温室气体的一种,对全球气候变化和人们的生活都有着重要的影响。全球绝大部分甲烷来自于产甲烷古菌的代谢,因此主要发生在厌氧环境之中。然而一些最新的研究发现,自然界的有氧环境中同样存在着甲烷产生现象,而产甲烷微生物也不仅限于产甲烷古菌。本文从微生物的产甲烷作用出发,对有氧环境中的甲烷产生进行整体的归纳,总结已有的研究结果,为以后产甲烷方面的研究提供新的思路和方向。     

陆地生态系统甲烷产生和氧化过程的微生物机理

陆地生态系统存在许多常年性或季节性缺氧环境,如:湿地、水稻土、湖泊沉积物、动物瘤胃、垃圾填埋场和厌氧生物反应器等。每年有大量有机物质进入这些环境,在缺氧条件下发生厌氧分解。甲烷是有机质厌氧分解的最终产物。产生的甲烷气体可通过缺氧-有氧界面释放到大气,产生温室效应,是重要的温室气体。产甲烷过程是缺氧环境中有机质分解的核心环节,而甲烷氧化是缺氧-有氧界面的重要微生物过程。甲烷的产生和氧化过程共同调控大气甲烷浓度,是全球碳循环不可分割的组成部分。对陆地生态系统甲烷产生和氧化过程的微生物机理研究进展进行了概要回顾和综述。主要内容包括:新型产甲烷古菌即第六和第七目产甲烷古菌和嗜冷嗜酸产甲烷古菌的发现;短链脂肪酸中间产物互营氧化过程与直接种间电子传递机制;新型甲烷氧化菌包括厌氧甲烷氧化菌和疣微菌属好氧甲烷氧化菌的发现;甲烷氧化菌生理生态与环境适应的新机制。这些研究进展显著拓展了人们对陆地生态系统甲烷产生和氧化机理的认识和理解。随着新一代土壤微生物研究技术的发展与应用,甲烷产生和氧化微生物研究领域将面临更多机遇和挑战,对未来发展趋势做了展望。     

产甲烷菌研究进展

产甲烷菌是重要的环境微生物,在自然界的碳素循环中起重要作用。迄今已有5种产甲烷菌基因组测序完成。基因组信息使人们对产甲烷茵的细胞结构、进化、代谢及环境适应性有了更深的理解。目前已知的甲烷生物合成途径有3种,它们以乙酸、甲基化合物、氢/二氧化碳为起始,通过不同的反应途径都形成了甲基辅酶M,在甲基辅酶M还原酶的催化下最终形成甲烷。     

反硝化型甲烷厌氧氧化的研究进展

反硝化型甲烷厌氧氧化(denitrifying anaerobic methane oxidation,DAMO)即甲烷厌氧氧化耦合反硝化,是指在厌氧条件下以甲烷作为电子供体,NO2-/NO3-作为电子受体的反硝化过程。甲烷是一种温室气体,其引起的温室效应是等物质量CO2的20~30倍。DAMO过程利用甲烷代替常规碳源进行脱氮,有利于减少温室效应,并改善氮循环。研究发现,Candidatus Methylomirabilis oxyfera细菌和Candidatus Methanoperedens nitroreducens古菌是参与DAMO过程的2类主要功能微生物,前者通过内部好氧机制耦合亚硝酸盐还原与甲烷的厌氧氧化,后者则通过逆向产甲烷途径耦合硝酸盐还原与甲烷的厌氧氧化。本文详细阐述了M.oxyfera细菌和M.nitroreducens古菌细胞内代谢途径,着重总结了甲烷、NO2-/NO3-、反应器构型、温度等因素对DAMO性能的影响。并对DAMO实际应用方面的研究现状做了调研。在DAMO功能微生物作用机制、快速富集及影响因素的进一步深入研究的基础上,推进DAMO污水脱氮工艺的应用是未来的主要研究热点和发展方向。     

新型产甲烷古菌研究进展

产甲烷古菌是一类能利用简单化合物产生甲烷气体的厌氧菌。近年来,随着测序技术的不断发展,科学家结合宏基因组学和其他技术先后发现了众多之前未被报道的新型产甲烷古菌。基因组分析等研究发现这几类新型产甲烷古菌具有独特的甲烷代谢通路以及广泛的生态分布,科学家推测它们在全球生态调节以及碳循环中可能起到了不可忽视的作用。然而,这些新型产甲烷古菌大部分尚未通过传统培养方法获得纯培养菌株,其确切的生理代谢机制和生态功能还有待深入研究。为了更加系统地了解这些新型产甲烷古菌,本文从它们的分类、系统发育地位、代谢机制、生态分布以及分离培养等方面进行了综述,并对新型产甲烷古菌未来的研究方向进行了展望。     

乙酸型甲烷八叠球菌细胞工厂的设计与构建研究进展

在整个地球演化历史过程中,产甲烷古菌在生物地球碳循环中一直扮演着重要角色。据报道,约三分之二的地球生物甲烷通量来自乙酸型产甲烷途径,乙酸型甲烷八叠球菌(Methanosarcina acetivorans)是目前发现为数不多的可以进行乙酸型产甲烷途径的模式产甲烷古菌,对M.acetivorans代谢途径的解析、改造和应用可为温室气体甲烷的减排与其作为能源的合理利用提供新思路。本文综述了M.acetivorans的产甲烷代谢途径、遗传改造策略、细胞工厂构建3个方面的研究进展,分析了M.acetivorans与其他进行乙酸型产甲烷代谢的产甲烷古菌在以上三方面的异同,并对进一步设计和构建其作为微生物细胞工厂所面临的问题与挑战进行了展望。     

不同抑制剂对乙酸降解产甲烷及产甲烷菌群结构的影响

摘要:【目的】研究不同温度条件下的石油烃降解产甲烷菌系中是否存在乙酸互营氧化产甲烷代谢途径。【方法】以3个不同温度条件的正十六烷烃降解产甲烷菌系Y15(15℃)、M82(35℃)和SK(55℃)作为接种物,通过乙酸喂养实验、并添加乙酸营养型产甲烷古菌的选择性抑制剂NH4Cl和CH3F,结合末端限制性片段长度多态性(terminal restriction fragment length polymorphism,T-RFLP)和克隆文库技术,分析乙酸产甲烷潜力及产甲烷古菌群落的演替趋势,推测产甲烷代谢途径的变化趋势。【结果】无论是否添加NH4Cl和CH3 F,这3个菌系都可以利用乙酸生长并产生甲烷,但是添加NH4Cl和CH3 F后产甲烷延滞期增加,最大比甲烷增长速率降低;只添加乙酸后,3个不同温度的菌系的古菌群落主要由乙酸营养型产甲烷古菌甲烷鬃毛菌属(Methanosaeta)组成,其丰度分别为92.8±1.4%、97.3±2.4%和82.8±9.0%;当添加选择性抑制剂NH4Cl,3 个菌系中的Methanosaeta的丰度分别变为98.5±0.7%、87.4±4.8%和6.1±8.6%,中温菌系M82中氢营养型产甲烷古菌甲烷袋装菌属(Methanoculleus)的相对丰度增加到12. 6±4.0%,高温菌系SK中另一类氢营养型产甲烷古菌甲烷热杆菌属(Methanothermobacter)增至84.3±1.5%;当添加选择性抑制剂CH3 F,Methanosaeta丰度分别降至77.1 ± 14.5%,86.4±6.1%和35.8±7.8%,低温菌系Y15中的甲烷八叠球菌属(Methanosarcina)增高(15.7±21%),这类产甲烷古菌具有多种产甲烷代谢途径,M82中Methanoculleus丰度上升到13.6±13.1%,SK中Methanothermobacter丰度增大到48.5±11.2%。【结论】在低温条件下,菌系Y15可能主要通过乙酸裂解完成产甲烷代谢,在中高温条件下,菌系M82和SK中可能存在乙酸互营氧化产甲烷代谢途径,并且甲烷的产生分别通过不同种群的氢营养型产甲烷古菌来完成。     

氢营养型产甲烷代谢途径研究进展

产甲烷古菌是一类极端厌氧的古菌域微生物,可以利用CO_2、甲醇、乙酸等简单化合物产甲烷并获得能量。目前能够培养的氢营养型(CO_2/H_2)产甲烷古菌的种类较多,而且在三类产甲烷代谢类型中,氢营养型产甲烷途径的产能效率最高,并具有多种模式的特殊能量利用系统。近年来,随着质谱、光谱和晶体技术的发展与运用,人们对产甲烷代谢途径的研究进一步深入,尤其是对氢营养型产甲烷途径的生化机制有了新的认识,揭示了产甲烷古菌在能量极限条件下独特、高效的能量利用模式。本文从能量储存、代谢途径、蛋白功能与催化机制等方面概述产甲烷古菌利用CO_2/H_2产甲烷的详细过程,并对产甲烷古菌代谢途径的研究方向与技术发展进行展望。     

人肠道梭菌和甲烷马赛球菌协同代谢甜菜碱和胆碱产甲烷研究

【目的】根据人肠道富含胆碱和甜菜碱,同时肠道微生物组中具有裂解胆碱和还原甜菜碱产三甲胺的细菌,以及利用三甲胺产甲烷的古菌,本研究探讨肠道细菌与古菌协同代谢甜菜碱和胆碱产甲烷的可能性。【方法】调查不同年龄段人群粪便中的16S rRNA基因多样性,分析肠道中古菌的菌群组成;利用定量PCR(quantitativePCR,qPCR)定量甲烷马赛球菌(Methanomassiliicoccus)特异的甲醇甲基转移酶基因mtaB和甲烷八叠球菌(Methanosarcina)及细菌的16SrRNA基因拷贝数,分析肠道中甲基营养型产甲烷古菌及总细菌的含量;宏基因组组装基因组(metagenome-assembled genomes, MAGs)分析携带甜菜碱还原酶基因grdH和胆碱裂解酶基因cutC的细菌组成。从粪便中分离代谢甜菜碱及胆碱产生三甲胺的细菌,并与分离自人肠道的甲烷马赛球菌构建共培养物,测定其协同转化甜菜碱和胆碱产甲烷的能力。【结果】年轻人粪便中含有甲烷杆菌科(Methanobacteriaceae,82.16%)的甲烷短杆菌属(Methanobrevibacter,49.18%)和甲烷...     

Methyl/alkyl-coenzyme M reductase-based anaerobic alkane oxidation in archaea

Methyl-coenzyme M reductase (MCR) has been originally identified to catalyse the final step of the methanogenesis pathway. About 20 years ago anaerobic methane-oxidizing archaea (ANME) were discovered that use MCR enzymes to activate methane. ANME thrive at the thermodynamic limit of life, are slow-growing, and in most cases form syntrophic consortia with sulfate-reducing bacteria. Recently, archaea that have the ability to anaerobically oxidize non-methane multi-carbon alkanes such as ethane and n-butane were described in both enrichment cultures and environmental samples. These anaerobic multi-carbon alkane-oxidizing archaea (ANKA) use enzymes homologous to MCR named alkyl-coenzyme M reductase (ACR). Here we review the recent progresses on the diversity, distribution and functioning of both ANME and ANKA by presenting a detailed MCR/ACR-based phylogeny, compare their metabolic pathways and discuss the gaps in our knowledge of physiology of these organisms. To improve our understanding of alkane oxidation in archaea, we identified three directions for future research: (i) expanding cultivation attempts to validate omics-based metabolic models of yet-uncultured organisms, (ii) performing biochemical and structural analyses of key enzymes to understand thermodynamic and steric constraints and (iii) investigating the evolution of anaerobic alkane metabolisms and their impact on biogeochemical cycles.     

Diversity and three-dimensional structures of the alpha Mcr of the methanogenic Archaea from the anoxic region of Tucuruí Lake, in Eastern Brazilian Amazonia

Methanogenic archaeans are organisms of considerable ecological and biotechnological interest that produce methane through a restricted metabolic pathway, which culminates in the reaction catalyzed by the Methyl-coenzyme M reductase (Mcr) enzyme, and results in the release of methane. Using a metagenomic approach, the gene of the α subunit of mcr (mcrα) was isolated from sediment sample from an anoxic zone, rich in decomposing organic material, obtained from the Tucuruí hydroelectric dam reservoir in eastern Brazilian Amazonia. The partial nucleotide sequences obtained were 83 to 95% similar to those available in databases, indicating a low diversity of archaeans in the reservoir. Two orders were identified - the Methanomicrobiales, and a unique Operational Taxonomic Unit (OTU) forming a clade with the Methanosarcinales according to low bootstrap values. Homology modeling was used to determine the three-dimensional (3D) structures, for this the partial nucleotide sequence of the mcrα were isolated and translated on their partial amino acid sequences. The 3D structures of the archaean Mcrα observed in the present study varied little, and presented approximately 70% identity in comparison with the Mcrα of Methanopyrus klanderi. The results demonstrated that the community of methanogenic archaeans of the anoxic C1 region of the Tucurui reservoir is relatively homogeneous.     

Hydroxylation of methane through component interactions in soluble methane monooxygenases

Methane hydroxylation through methane monooxygenases (MMOs) is a key aspect due to their control of the carbon cycle in the ecology system and recent applications of methane gas in the field of bioenergy and bioremediation. Methanotropic bacteria perform a specific microbial conversion from methane, one of the most stable carbon compounds, to methanol through elaborate mechanisms. MMOs express particulate methane monooxygenase (pMMO) in most strains and soluble methane monooxygenase (sMMO) under copper-limited conditions. The mechanisms of MMO have been widely studied from sMMO belonging to the bacterial multicomponent monooxygenase (BMM) superfamily. This enzyme has diiron active sites where different types of hydrocarbons are oxidized through orchestrated hydroxylase, regulatory and reductase components for precise control of hydrocarbons, oxygen, protons, and electrons. Recent advances in biophysical studies, including structural and enzymatic achievements for sMMO, have explained component interactions, substrate pathways, and intermediates of sMMO. In this account, oxidation of methane in sMMO is discussed with recent progress that is critical for understanding the microbial applications of C-H activation in one-carbon substrates.     

Oxidation of deuterated compounds by high specific activity methane monooxygenase from Methylosinus trichosporium. Mechanistic implications

Hydrocarbon oxidations catalyzed by methane monooxygenase purified to high specific activity from the type II methanotroph Methylosinus trichosporium OB3b were compared to the same reactions catalyzed by methane monooxygenase from the type I methanotroph Methylococcus capsulatus Bath and liver microsomal cytochrome P-450. The two methane monooxygenases produced nearly identical product distributions, in accord with physical studies of the enzymes which have shown them to be very similar. The products obtained from the oxidation of a series of deuterated substrates by the M. trichosporium methane monooxygenase were very similar to those reported for the same reaction catalyzed by liver microsomal cytochrome P-450, suggesting that the enzymes use similar mechanisms. However, differences in the product distributions and other aspects of the reactions indicated the mechanisms are not identical. Methane monooxygenase epoxidized propene in D2O and d6-propene in H2O without exchange of substrate protons or deuterons with solvent, in contrast to cytochrome P-450 (Groves, J. T., Avaria-Neisser, G. E., Fish, K. M., Imachi, M., and Kuczkowski, R. L. (1986) J. Am. Chem. Soc. 108, 3837-3838), suggesting that the mechanism of epoxidation of olefins by methane monooxygenase differs at least in part from that of cytochrome P-450. Hydroxylation of alkanes by methane monooxygenase revealed close similarities to hydroxylations by cytochrome P-450. Allylic hydroxylation of 3,3,6,6-d4-cyclohexene occurred with approximately 20% allylic rearrangement in the case of methane monooxygenase, whereas 33% was reported for this reaction catalyzed by cytochrome P-450 (Groves, J. T., and Subramanian, D. V. (1984) J. Am. Chem. Soc. 106, 2177-2181). Similarly, hydroxylation of exo,exo,exo,exo-2,3,5,6-d4-norbornane by methane monooxygenase occurred with epimerization, but to a lesser extent than reported for cytochrome P-450 (Groves, J. T., McClusky, G. A., White, R. E., and Coon, M. J. (1978) Biochem. Biophys. Res. Commun. 81, 154-160). A large intramolecular isotope effect, kH,exo/kD,exo greater than or equal to 5.5, was calculated for this reaction. However, the intermolecular kinetic isotope effect on Vm for methane oxidation was small, suggesting that steps other than C-H bond breakage were rate limiting in the overall enzymatic reaction. Similar isotope effects have been observed for cytochrome P-450. These observations indicate a stepwise mechanism of hydroxylation for methane monooxygenase analogous to that proposed for cytochrome P-450.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enzymatic C-H activation by metal-superoxo intermediates

The mechanisms of four enzymes that initiate oxidation of their substrates by using mid-valent metal-superoxo intermediates, rather than the more frequently described high-valent iron-oxo complexes, to cleave relatively strong C-H bonds have come into focus in the past several years. In two of these reactions, the alternative manifold for O2 and C-H activation enables unique four-electron oxidation reactions, thus significantly augmenting Nature's arsenal for transformation of aliphatic carbon compounds. General principles of this alternative manifold, including common kinetic characteristics and thermodynamic limitations, are emerging. Recent, combined experimental and computational studies on other systems have shown how a more thorough understanding of the structures of the metal-superoxo intermediates and the mechanisms by which they cleave C-H bonds might be achieved.     

甲烷氧化菌及其在环境治理中的应用

甲烷的生物氧化包括好氧氧化和厌氧氧化两种,分别由好氧甲烷氧化菌和厌氧甲烷氧化菌完成.由于该过程是减少自然环境中温室气体甲烷排放的重要途径,越来越受到各国学者的重视.本文主要对当前甲烷氧化菌的研究现状进行了综述,对好氧甲烷氧化菌的种类、参与氧化甲烷的关键酶,厌氧甲烷氧化菌的种类、参与的微生物菌种以及氧化机理进行了论述,并对这两类微生物在温室气体减排、污染物治理、废水生物脱氮、硫及金属元素回收等方面的应用现状及前景进行了分析.     

甲烷氧化菌及甲烷单加氧酶的研究进展

甲烷氧化菌是以甲烷作为唯一碳源和能源进行同化和异化代谢的微生物,其关键酶之一是甲烷单加氧酶(MMOs),可以在氧气的作用下催化甲烷等低碳烷烃或烯烃羟基化或环氧化,甲烷氧化菌在自然界碳循环和工业生物技术中具有重要的应用价值.因此,近20年来对于甲烷氧化菌和MMOs的研究一直倍受生物学家的关注.以下从现代生物技术的角度,对近年来国内外在甲烷氧化菌的分类与分布,MMOs的结构与功能、甲烷氧化菌与MMOs的基因工程等方面取得的研究成果进行了总结,全面综述了甲烷氧化菌及MMOs的应用基础研究现状,并对其今后的研究和应用方向提出了展望.     

Regulation of methane monooxygenase catalysis based on size exclusion and quantum tunneling

The hydroxylase component (MMOH) of the soluble form of methane monooxygenase (sMMO) isolated from Methylosinus trichosporium OB3b catalyzes both the O2 activation and the CH4 oxidation reactions at the oxygen-bridged dinuclear iron cluster present in its buried active site. During the reaction cycle, the diiron cluster forms a bis-mu-oxo-(Fe(IV))2 intermediate termed compound Q (Q) that reacts directly with methane. Many adventitious substrates also react with Q, most at a relatively slow rate. We have proposed that Q reacts preferentially with CH4 because the sMMO regulatory component MMOB induces a size selective pore into the MMOH active site as the two components form a complex. Support for this proposal has come through the observation of a nonlinear Arrhenius plot for the CH4 oxidation, presumably due to a shift in rate-limiting step from substrate binding at low temperature to C-H bond cleavage at high temperature. Reactions of all substrates other than CH4 fail to exhibit a break in the Arrhenius plot because binding is always rate limiting in the temperature range explored. Here we show that it is possible to induce a break in the Arrhenius plot for the ethane reaction with Q by using an MMOB mutant termed DBL2 (S109A/T111A) in which residues at the MMOH-MMOB interface are reduced in size. We hypothesize that this increases the ethane binding rate and shifts the Arrhenius breakpoint into the observable temperature range. As a result of this shift, the kinetic and activation parameters of the C-H bond breaking reaction for both methane and ethane can be observed using the DBL2 mutant. A 2H-KIE is observed for both substrate oxidation reactions when using DBL2, whereas only CH4 oxidation exhibits an effect when using wild type MMOB, consistent with the C-H bond cleaving reaction becoming at least partially rate limiting for ethane. Analysis of the temperature dependence of the 2H-KIE for ethane and methane for reactions using both mutant and wild type forms of MMOB suggests that quantum tunneling plays a significant role in methane oxidation but not ethane oxidation.     

Immunological Localization of Coenzyme M Reductase in Anaerobic Methane-Oxidizing Archaea of ANME 1 and ANME 2 Type

The Black Sea is a large, euxinic marine basin, in which the anaerobic oxidation of methane (AOM) plays an important role in the carbon cycle. Methane-related carbonate build-ups, found on the NW' Black Sea shelf are part of an unique microbial ecosystem. Two archaeal guilds are mainly responsible for the AOM: ANME-1 (anaerobic-methane-oxidizing communities)/DSS consortia and ANME-2/greigite-bearing DSS-consortia. These microorganisms constitute a significant sink of methane on earth, but despite their relevance for the global carbon cycle little is known about the biology of AOM. Phylogenetic and biochemical analyses suggested that ANME-archaea have supposedly reversed the methanogenic pathway. Here, we were able to localize methyl-coenzyme M reductase (MCR), which catalyzes the final step of the methane formation, in ultrathin sections. The result was obtained by the immunogold labeling technique using a specific antiserum against the MCR. This technique revealed that the MCR is located in both ANME-1- and ANME-2-archaea. The data also show that MCR-like enzymes are not only encoded in the genomes of ANME-1 and ANME-2, but are, in fact, expressed as cellular proteins at high levels.     

Low-temperature biological activation of methane: structure,function and molecular interactions of soluble and particulate methane monooxygenases

Mechanistic aspects of oxidation of methane to methanol by methanotrophic bacteria via methane monooxygenase (MMO) is still not well understood. Elucidating how various molecules pertinent to methane oxidation interact with each other at the MMO active site offers critical insights on low-temperature activation of methane, which is one of the greatest technical challenges in hydrocarbon chemistry. In this review, most recent contributions to the area are analyzed comparing soluble (sMMO) and particulate (pMMO) forms. Initially, the taxonomical, morphological and physiological differences of different methanotrophs are discussed. Then, the structural and functional differences of sMMO and pMMO are analyzed while considering substrate/product-cofactor-active site interactions. A docking analysis was performed using Autodock Vina to uncover interactions between cofactors and corresponding enzymes. With natural gas becoming a significant contributor to the energy continuum, this literature analysis and molecular simulations conducted brings new insights to low-temperature activation of methane.