Distribution of dissimilatory enzymes in methane and methanol oxidizing bacteria

Methanol oxidation was studied in several RuMP and serine type methylotrophic bacteria. On the basis of the distribution of the dissimilatory enzymes and the electrophoretic mobility of the methanol dehydrogenases, the methanol and methane oxidizers of the RuMP type belong to two different taxonomic groups. The pink pigmented facultative serine type methylotrophs represent another taxon.     

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.     

甲醇生物转化合成化学品的研究进展

甲醇来源丰富、价格低廉,已成为生物制造行业极具吸引力的底物之一。构建微生物细胞工厂实现甲醇到增值化学品的生物转化,具有过程绿色、条件温和、产品体系多样等优势,不仅能拓展基于甲醇的产品链,还能缓解当前生物制造“与民争粮、与粮争地”的问题,是实现绿色生物制造的重要手段。因此,阐明不同天然甲基营养菌中涉及甲醇氧化、甲醛同化和异化途径对于后续基因工程改造工作至关重要,也更有利于构建新型非天然甲基营养菌。本文讨论了甲基营养菌中甲醇代谢途径的研究现状,并结合近年来天然和人工合成甲基营养菌在甲醇生物转化中的应用进展及面临的挑战。     

Genetics of carbon metabolism in methylotrophic bacteria

Abstract The application of genetic techniques to the methylotrophic bacteria has greatly enhanced studies of these important organisms. Two methylotrophic systems have been studied in some detail, the serine cycle for formaldehyde assimilation and the methanol oxidation system. In both cases, genes have been cloned and mapped in Methylobacterium species (facultative serine cycle methanol-utilizers). In addition, methanol oxidation genes have been studied in an autotrophic methanol-utilizer ( Paracoccus denitrificans ) and three methanotrophs ( Methylosporovibrio methanica, Methylomonas albus and Methylomonas sp. A4). Although much remains to be learned in these systems, it is becoming clear that the order of C₁ genes has been conserved to some extent in methylotrophic bacteria, and that many C₁ genes are loosely clustered on the chromosome. Operons appear to be rare, but some examples have been observed. The extension of genetic approaches to both the obligate and facultative methylotrophs holds much promise for the future in understanding and manipulating the activities of these bacteria.     

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。     

The mechanism of inhibition by EDTA and EGTA of methanol oxidation by methylotrophic bacteria

Ethyleneglycol (aminoethylether) tetra-acetic acid (EGTA) was shown to be a potent competitive inhibitor of electron transfer between methanol dehydrogenase (MDH) and its electron acceptor cytochrome cL. Addition of Ca2+ ions relieved the inhibition by removal of the inhibitory EGTA. Removal of EGTA by gel filtration completely relieved the inhibition. EGTA did not remove the tightly bound Ca2+ present in the MDH. Indo-1, a fluorescent analogue of EGTA, bound tightly to MDH in a 1:1 ratio but not to cytochrome cL; binding was prevented by EGTA. It was concluded that EGTA inhibits methanol oxidation by binding to lysyl or arginyl residues on MDH thus preventing docking with cytochrome cL.     

氮依赖型甲烷厌氧氧化菌的整合

氮依赖型甲烷厌氧氧化菌(nitrite-dependent anaerobic methane oxidation bacteria,n-damo细菌,属于NC10门)是最近10年来微生物生态学领域的研究热点。然而,对该类群基于现有数据的生态分布、群落结构和系统进化的整合分析还未见报道。【目的】为了更好地将近年来针对该类群的研究做一次全面梳理,本文通过整合前人已有发表数据和结合自身实验数据两方面进行。【方法】一方面,利用NCBI数据库(数据搜集到2016年11月)中所有n-damo细菌序列对其进行生物信息学分析;另一方面,对大九湖泥炭地表层泥炭利用16S rRNA二代测序技术对该类群进行检测,并同前人数据进行对比。【结果】n-damo细菌主要在沉积物、湿地和水稻土检出;基于pmo A基因的n-damo细菌的平均检出率是基于16S rRNA基因检出率的7倍,但是这两类基因分子标记物所得到的多样性指数保持相对稳定(1.4-3.4);贫氮的大九湖泥炭其NC10的丰度仅为0.067%。【结论】n-damo类群种群相对稳定,暗示其行使的生态功能相对单一;贫氮的大九湖泥炭其极低的NC10丰度暗示氮对NC10是限制因子;具有真正氮依赖型甲烷厌氧氧化细菌的Group A可能只占很少的一部分(小于20%),暗示出该类群真正的生态潜能需要进一步评估。本次整合分析为更好的理解n-damo细菌的生活环境、评估不同基因分子标记物下n-damo细菌的检出率、不同亚类群比如Group A和Group B等的丰度和真正的潜在生态功能提供参考。     

New unified nomenclature for genes involved in the oxidation of methanol in Gram-negative bacteria

Abstract The system involving the oxidation of methanol to formaldehyde in Gram-negative methylotrophic bacteria is complex. A total of 32 genes have been reported, termed mox , for methanol oxidation, and it is possible that more will be identified. Some mox genes carrying out completely different functions have been given the same designations by different laboratories and others have been given separate designations that were later discovered to be the same. It is now important to change the mox nomenclature to remedy this confusing situation. This communication proposes a new nomenclature for genes involved in methanol oxidation based on currently known linkage groups.     

自然生境中亚硝酸盐型厌氧甲烷氧化细菌研究进展

随着功能微生物介导的亚硝酸盐型厌氧甲烷氧化(nitrite-dependent anaerobic methane oxidation,N-DAMO)过程被发现,人们对自然界的碳氮循环有了全新的认识,该过程成为自然生态系统中温室气体甲烷的汇,同时还是氮污染的消减途径。本文系统介绍了N-DAMO过程反应机理以及参与该过程的亚硝酸盐型厌氧甲烷氧化细菌(Candidatus Methylomirabilis oxyfera)的生理生化特征,并对研究该功能菌的分子微生物方法进行了汇总。通过对不同自然生境中该细菌的研究报道进行总结分析,揭示各生境中年均降水量、年均温度、所处不同自然区等大尺度宏观环境因子及碳源、氮源、pH和氧气含量等生存因子对其群落结构的潜在影响,最后在展望中提出此功能菌在未来可深入研究的方向,期望能厘清厌氧甲烷氧化过程及其功能菌在碳、氮循环中的生态学功能。     

Investigation of the methyl fluoride technique for determining rhizospheric methane oxidation

Methane oxidation rates in the rhizosphere of Pontederia cordata,Sagittaria lancifolia, and Typha latifolia were quantified in fieldstudies using the methyl fluoride inhibition technique. An averageoxidation of 22.9 ± 17.7% (sd,n = 44) was found for all field experiments (oxidation is expressedas a % of total potential emission in the presumedabsence of oxidation). Greenhouse experiments using the same techniquegave an average rhizospheric oxidation of 64.9 ±17.0% (sd, n = 44). Comparison of a subset ofgreenhouse plants with the methyl fluoride (MF) and a light oxic/darkanoxic (LO/DA) technique for suppressing CH₄ oxidationyielded similar percentages (57.7 ±15.0% for MF and 58.5 ±13.9% for LO/DA, n = 11). Rhizospheric oxidationdisplayed a seasonal trend in Typha latifolia with decreasingoxidation percentages during warmer months as the importance ofrhizospheric CH₄ oxidation declined relative toCH₄ emission (46.5 ±13.8% in December and 13.5 ±1.7% in July). However, the absolute rateof methane oxidation was highest during the warmer months (44.2± 3.4 mg m^-2 d^-1 inDecember and 318.7 ± 151.4 mg m^-2d^-1 in July). As methane emission rates increased,the sensitivity of the methyl fluoride technique decreased dueto the larger error between replicate flux measurements.     

Action of polycarbon compounds on the oxidation of methanol and other Cl compounds by methylotrophic bacteria

Cells of the facultative methylotrophic bacteria Achromobacter parvulus 1T, Pseudomonas sp AM1, Ps. methylica 2K, Ps. methylica 20T, Ps. oleovorans 52Z, Ps. fluorescens 45K exhibit the ability to oxidize methanol only on the medium with methanol. At the growth of various methylotrophic bacteria on the media containing both methanol and succinate their ability to methanol oxidation is lost, and oxidation of formaldehyde and formate occur at a low rate. Glucose, citrate, acetate and glycerol produce no repressive effect on the ability of the cells to oxidize methanol and other C1-compounds.     

Quantification of methane oxidation in the rhizosphere of emergent aquatic macrophytes: defining upper limits

Rates of rhizospheric methane oxidation were evaluated by aerobic incubations of subcores collected in flooded anoxic soils populated by emergent macrophytes, by greenhouse whole plant incubations, and by CH₄ stable isotopic analysis. Subcore incubations defined upper limits for rhizospheric methane oxidation on an areal basis which were equal to or greater than emission rates. These rates are considered upper limits because O₂ did not limit CH₄ uptake as is likely to occur in situ. The ratio of maximum potential methane oxidation (MO) to methane emission (ME) ranged from 0.7 to 1.9 in Louisiana rice (Oryza sativa), from 1.0 to 4.0 in a N. Florida Sagittaria lancifolia marsh, and from 5.6 to 51 in Everglades Typha domingensis and Cladium jamaicense areas. Methane oxidation/methane emission ratios determined in whole plant incubations of Sagittaria lancifolia under oxic and anoxic conditions ranged from 0.5 to 1.6. Methane oxidation activity associated with emergent aquatic macrophytes was found primarily in fine root material. A weak correlation was observed between live root biomass and CH₄ uptake in Typha. Rhizomes showed small or zero rates of methane uptake and no uptake was associated with plant stems. Methane stable isotope data from a S. lancifolia marsh were as follows: CH₄ emitted from plants: −61.6 ± 0.3%; CH₄ within stems: −42.0 ± 0.2%; CH₄ within sedimentary bubbles: −51.7 ± 0.3%). The ¹³C enrichment observed relative to emitted CH₄ could be due to preferential mobilization of CH₄ containing the lighter isotope and/or the action of methanotrophic bacteria.     

微生物甲烷氧化反硝化耦合反应研究进展

甲烷氧化反硝化耦合过程是连接碳循环和氮循环的重要桥梁.该过程的深入研究有助于完善人们对全球碳氮生物化学循环的认识.甲烷作为反硝化外加气体碳源,既能调控大气甲烷平衡,有效减缓由甲烷引起的温室效应,又能降低反硝化工艺中因投入外加碳源带来的成本.因此近年来甲烷氧化反硝化耦合反应及其机理研究倍受关注.本文主要讨论了好氧和厌氧两种类型的甲烷氧化反硝化过程,重点对其微生物耦合反应机理及其影响因素进行了综述,同时指出了其工程化应用存在的问题,并对其应用前景提出展望.
     

Cofactor-dependent pathways of formaldehyde oxidation in methylotrophic bacteria

Methylotrophic bacteria can grow on a number of substrates as energy source with only one carbon atom, such as methanol, methane, methylamine, and dichloromethane. These compounds are metabolized via the cytotoxin formaldehyde. The formaldehyde consumption pathways, especially the pathways for the oxidation of formaldehyde to CO(2) for energy metabolism, are a central and critical part of the metabolism of these aerobic bacteria. Principally, two main types of pathways for the conversion of formaldehyde to CO(2) have been described: (1) a cyclic pathway initiated by the condensation of formaldehyde with ribulose monophosphate, and (2) distinct linear pathways that involve a dye-linked formaldehyde dehydrogenase or C(1) unit conversion bound to the cofactors tetrahydrofolate (H(4)F), tetrahydromethanopterin (H(4)MPT), glutathione (GSH), or mycothiol (MySH). The pathways involving the four cofactors have in common the following sequence of events: the spontaneous or enzyme-catalyzed condensation of formaldehyde and the respective C(1) carrier, the oxidation of the cofactor-bound C(1) unit and its conversion to formate, and the oxidation of formate to CO(2). However, the H(4)MPT pathway is more complex and involves intermediates that were previously known solely from the energy metabolism of methanogenic archaea. The occurrence of the different formaldehyde oxidation pathways is not uniform among different methylotrophic bacteria. The pathways are in part also used by other organisms to provide C(1) units for biosynthetic reactions (e.g., H(4)F-dependent enzymes) or detoxification of formaldehyde (e.g., GSH-dependent enzymes).     

好氧甲烷氧化菌及其工程应用进展

好氧甲烷氧化菌能以甲烷作为碳源和能源,对全球甲烷消除的贡献率高达10%–20%,还能有效地合成有价值的甲烷来源生物产品。文中介绍了好氧甲烷氧化菌的甲烷氧化代谢机理,总结了好氧甲烷氧化菌在填埋场甲烷减排、煤矿通风气治理、合成生物产品、油气藏勘探等领域的实际应用功效和研究热点,即污染物去除和产品合成效率的影响因素。基于对甲烷氧化菌规模化培养方法的研究,本文认为加强培养过程中代谢产物对甲烷氧化菌活性和种群结构影响的研究,以及开发经济高效的替代培养基和培养技术的研究将有利于好氧甲烷氧化菌生物技术的应用推广。     

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.     

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.