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.     

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.     

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。     

土壤大气甲烷氧化菌研究进展

土壤微生物催化是大气中痕量甲烷(约1.8ppmv)氧化的唯一生物途径。目前的研究表明好氧土壤中存在专性和选择性大气甲烷氧化菌2种类型:前者(USCα和USCγ)广泛分布于各种好氧旱地土壤,其甲烷氧化酶对低浓度甲烷亲和力极高,属真正的寡营养型,但至今尚未获得该种类的纯培养菌株。后者属于传统甲烷氧化菌Methylocystis/Methylosinus属,广泛分布于各种周期性排放高浓度甲烷的土壤环境中。该属大部分菌株含有亲和力不同的2套甲烷单加氧酶系统,其中的高亲和力甲烷单加氧酶使这些菌株可以在相当长的时间内(3个月)保持大气浓度甲烷氧化活性,但其生长和繁殖还需依赖于土壤内部阶段性产生的高浓度甲烷。本文详细阐述了2类大气甲烷氧化菌的发现历程及其可能的生存策略,最后系统梳理了几种关键的环境因子(土壤温度及湿度、土壤pH、植被、土地利用及氮输入)对大气甲烷氧化菌群落结构和甲烷氧化活性的影响,提出并展望了土壤大气甲烷氧化菌研究的重要方向。     

甲烷氧化菌研究进展

甲烷氧化菌以甲烷为其唯一的碳源和能源 ,在全球大气甲烷平衡中起着重要的作用 ,它还可以降解卤代化合物 ,在污染治理方面具有潜在价值。本文从甲烷氧化菌的分类出发 ,对甲烷氧化菌氧化甲烷的机理及影响因素、甲烷氧化菌的生理、生态分布及检测方法、甲烷氧化菌降解有机污染物的潜在应用等作一综述 ,分析目前研究中存在的问题 ,并指出今后应加强研究的方面。     

基于甲烷氧化菌的脱氮技术研究进展

甲烷氧化菌利用甲烷作为唯一碳源和能源,在氧化甲烷的过程中能有效地实现脱氮,该过程分为好氧甲烷氧化耦合反硝化(aerobic methane oxidation coupled to denitrification,AME-D)和厌氧甲烷氧化耦合反硝化(anaerobic methane oxidation coupled to denitrification,ANME-D),在碳循环和氮循环的研究中具有重要意义。本文通过总结近年来有关甲烷氧化菌的分类与分布,阐述AME-D和ANME-D的基本原理、影响因素和应用情况,提出相应的研究方向,以期为甲烷氧化菌在污水脱氮中的应用提供参考。     

含有甲烷氧化菌的混合菌群特性研究

为获得高效甲烷氧化微生物体系,从农业土壤中采样,以甲烷作为唯一碳源进行好氧选择性传代培养,得到生长性能稳定、生长优于Methylosinus trichosporium OB3b纯培养的具有甲烷单加氧酶(Methane Monooxygenase,MMO)活性甲烷氧化混合菌。利用MMO的共代谢特性,分别以苯酚和环氧丙烷作为目标对象,考察该混合菌对有机污染物的降解及用于生产有用化学物质的催化特性。结果表明,所得混合菌具有高效降解苯酚能力,对初始浓度为600mg/L的苯酚,经过11h培养,苯酚降解率可达99%。另外,以该混合菌为催化剂可以实现丙烯氧化生产环氧丙烷。通过降低磷酸盐浓度可以有效提高环氧丙烷的积累浓度,最大可至5mmol/L。此外,采用纯种分离方法结合PCR扩增、16SrRNA和MMO功能基因分析技术对混合菌群结构进行解析。结果表明,该混合菌群由Ⅱ型甲烷氧化菌及其它至少4种非甲烷氧化菌组成,它们分别属于Methylosinus trichosporium和Acinetobacter junii、Cupriavidusme tallidurans、Comamonas testosteroni和Stenotrophomonas maltophilia。采用PCR方法从混合菌及纯化菌株M.trichosporiums Y9总DNA中都能扩增得到mmoB、mmoX和pmoA基因片段,表明该甲烷氧化菌同时具有sMMO和pMMO两种形式的MMO。通过对从甲烷氧化混合菌中分离纯化得到的甲烷氧化菌进行PCR产物测序,结果发现其与Methylosinus trichosporium的同源性为99.9%。     

贡嘎山海拔梯度上不同植被类型土壤甲烷氧化菌群落结构及多样性

甲烷是仅次于CO₂的第二大温室气体.森林表层土壤中甲烷好氧氧化作用是大气甲烷重要的汇,在碳循环和减缓全球变暖方面起着重要作用.研究不同植被类型土壤中甲烷氧化菌的群落结构及多样性,有助于更好地理解植被演替、人为干扰和不同土地利用背景下甲烷氧化菌群落组成和多样性变化与地上植被之间的相互关系.本研究在贡嘎山东坡海拔梯度上的4种不同植被类型中采集了92个土壤样品,利用Miseq测序技术和生物信息学方法评估了甲烷氧化菌群落组成及多样性在4种不同植被类型间的变化,并探讨了其变异的潜在原因.结果表明: 常绿阔叶林和针阔叶混交林土壤中甲烷氧化菌的群落结构较为相似,暗针叶林和灌丛草甸土壤甲烷氧化菌的群落结构较为相似.4种不同植被生态系统中,针阔叶混交林土壤中的甲烷氧化菌α多样性显著高于其他3种植被生态系统(P＜0.001),且暗针叶林和灌丛草甸土壤中甲烷氧化菌β多样性显著高于常绿阔叶林和针阔叶混交林(P＜0.001).Spearman相关分析表明,不同类型甲烷氧化菌的相对丰度对环境变化的响应模式不同.造成α多样性差异的主要因子可能是土壤总氮、电导率和土壤温度.偏Mantel检验分析和冗余分析(RDA)表明,常绿阔叶林和针阔叶混交林土壤甲烷氧化菌多样性受环境因子的影响较大,而暗针叶林和灌丛草甸土壤中甲烷氧化细菌多样性变化可能存在其他潜在的影响因素或者机制.降水可能是造成低海拔常绿阔叶林和针阔叶混交林与高海拔暗针叶林和灌丛草甸土壤甲烷氧化细菌群落结构差异的主要原因.贡嘎山海拔梯度上不同植被类型土壤中甲烷氧化菌的群落结构和多样性变化可能主要是由于土壤理化性质和气候变化综合作用的结果.     

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。     

Response of methanotrophic activity and community structure to temperature changes in a diffusive CH4/O2 counter gradient in an unsaturated porous medium

Microbial methane oxidation is a key process in the global methane cycle. In the context of global warming, it is important to understand the responses of the methane-oxidizing microbial community to temperature changes in terms of community structure and activity. We studied microbial methane oxidation in a laboratory-column system in which a diffusive CH₄/O₂ counter gradient was maintained in an unsaturated porous medium at temperatures between 4 and 20 °C. Methane oxidation was highly efficient at all temperatures, as on average 99 ± 0.5% of methane supplied to the system was oxidized. The methanotrophic community that established in the model system after initial inoculation appeared to be able to adapt quickly to different temperatures, as methane emissions remained low even after the system was subjected to abrupt temperature changes. FISH showed that Type I as well as Type II methanotrophs were probably responsible for the observed activity in the column system, with a dominance of Type I methanotrophs. Cloning and sequencing suggested that Type I methanotrophs were represented by the genus Methylobacter while Type II were represented by Methylocystis . The results suggest that in an unsaturated system with diffusive substrate supply, direct effects of temperature on apparent methanotrophic activity and community may be of minor importance. However, this remains to be verified in the field.     

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.     

互花米草入侵对滩涂湿地甲烷排放的影响

在浙江省台州市附近滩涂湿地设置3个不同互花米草入侵密度梯度,即仅有本土植物样地、互花米草与本土植物混生样地和互花米草单优群落样地,研究互花米草入侵对滩涂湿地CH₄排放的影响.结果表明: 3个样地CH₄排放通量为0.68～5.88 mg·m^-2·h^-1,CH₄排放通量随着互花米草入侵梯度的增加而显著升高,互花米草单优群落样地CH₄排放通量分别为本土植物样地和混生样地的8.7和2.3倍.互花米草入侵显著提高了产甲烷菌数量、产甲烷潜力、甲烷氧化菌数量、甲烷氧化潜力、植物生物量、土壤有机碳含量和土壤pH,降低了土壤全氮含量.CH₄排放通量与土壤全氮呈显著负相关,与产甲烷菌数量、产甲烷潜力、甲烷氧化菌数量、甲烷氧化潜力、植物生物量和土壤pH呈显著正相关.互花米草的入侵提高了滩涂湿地植物群落生物量和土壤pH,促进了产甲烷菌数量和产甲烷潜力,从而提高了滩涂湿地的CH₄排放.     

The regulation of photosynthetic rate and activation of extracellular carbonic anhydrase under CO2-limiting conditions in the marine diatom Skeletonema costatum

In the marine diatom Skeletonema costatum , carbonic anhydrase activity exterior to the plasma membrane (CA_ext) was detected only when the available CO₂ concentration was less than 5·0 mmol m^–3, this activity being unaffected by the total dissolved inorganic carbon concentration. The inhibition of CA_ext by dextran bound sulphonamide (DBS) demonstrated the key role of this enzyme in maintaining photosynthetic rate under CO₂-limited conditions. Treatment with trypsin followed by affinity chromatography on p-aminomethylbenzene-sulphamide agarose and subsequent SDS-PAGE analysis revealed a polypeptide from carbon-replete cells of identical molecular mass to the CA_ext released by trypsin from CO₂-limited cells. Redox activity in the plasma membrane of intact cells was measured by following the light-dependent reduction of ferricyanide or NADP, the greatest activity being shown by CO₂-limited cells. Overall the results suggest that high rates of redox activity under conditions of CO₂-limitation were required for the activation of CA_ext.     

Activity and composition of methanotrophic bacterial communities in planted rice soil studied by flux measurements, analyses of pmoA gene and stable isotope probing of phospholipid fatty acids

Methanotrophs in the rhizosphere of rice field ecosystems attenuate the emissions of CH₄ into the atmosphere and thus play an important role for the global cycle of this greenhouse gas. Therefore, we measured the activity and composition of the methanotrophic community in the rhizosphere of rice microcosms. Methane oxidation was determined by measuring the CH₄ flux in the presence and absence of difluoromethane as a specific inhibitor for methane oxidation. Methane oxidation started on day 24 and reached the maximum on day 32 after transplantation. The total methanotrophic community was analysed by terminal restriction fragment length polymorphism (T-RFLP) and cloning/sequencing of the pmoA gene, which encodes a subunit of particulate methane monooxygenase. The metabolically active methanotrophic community was analysed by stable isotope probing of microbial phospholipid fatty acids (PLFA-SIP) using ¹³C-labelled CH₄ directly added to the rhizospheric region. Rhizospheric soil and root samples were collected after exposure to ¹³CH₄ for 8 and 18 days. Both T-RFLP/cloning and PLFA-SIP approaches showed that type I and type II methanotrophic populations changed over time with respect to activity and population size in the rhizospheric soil and on the rice roots. However, type I methanotrophs were more active than type II methanotrophs at both time points indicating they were of particular importance in the rhizosphere. PLFA-SIP showed that the active methanotrophic populations exhibit a pronounced spatial and temporal variation in rice microcosms.     

甲烷及三氯乙烯驯化对垃圾填埋场覆盖土细菌群落结构的影响

对典型垃圾填埋覆盖土进行CH₄原位富集和三氯乙烯(TCE)驯化,研究了其生物氧化能力和微生物群落结构变化.覆盖土CH₄氧化速率为0.20～0.87 μmol·g^-1 soil·h^-1,TCE降解速率为0.009～0.013 mg·L^-1·h^-1,其中山东垃圾填埋场覆盖土土样甲烷氧化活性高于广东、上海和重庆地区土样.通过Illumina MiSeq测序技术分析了α多样性和驯化前后微生物菌群结构变化规律.结果表明: 在所有被注释的操作分类单元聚类结果中,细菌OTUs分配为39个门,85个纲,562个属,富集驯化后变形杆菌门、拟杆菌门、绿弯菌门和酸杆菌门仍为各土样的优势菌群,所占比例之和高于77.4%;γ-变形杆菌纲、β--变形杆菌纲、α-变形杆菌纲、放线菌纲和酸杆菌纲所占比例之和高于26.5%.嗜甲基菌属、厌氧绳菌属、节杆菌属和假单胞菌属经TCE驯化后,其相对丰度呈增加趋势.表明在覆盖土氯代烃生物降解过程中,除了被广泛认可的甲烷氧化菌异养共代谢机制以外,还存在非甲烷共代谢机制和氯代烃自养降解机制.