Identification of the bacterial community involved in methane-dependent denitrification in activated sludge using DNA stable-isotope probing

Methane is used as an alternative carbon source in the denitrification of wastewater lacking organic carbon sources because it is nontoxic and may be efficiently produced by anaerobic biological processes. Methane-dependent denitrification (MDD) in the presence of oxygen requires the co-occurrence of methanotrophy and denitrification. Activated sludge was incubated with 13C-labeled methane in either a nitrate-containing medium or a nitrate-free medium. Then, bacterial and methanotrophic populations were analyzed by cloning analysis and terminal restriction fragment length polymorphism analysis targeting 16S rRNA gene and cloning analysis targeting pmoA genes. DNA-based stable-isotope probing (DNA-SIP) analysis of the 16S rRNA gene revealed an association of the Methylococcaceae and the Hyphomicrobiaceae in a MDD ecosystem. Furthermore, supplementation of nitrate stimulated methane consumption and the activity of methanotrophic populations (i.e. the stimulation of uncultivated relatives of distinct groups of the Methylococcaceae). In particular, uncultured type-X methanotrophs of Gammaproteobacteria were dominant when nitrate was added, i.e. in the MDD incubations. On the other hand, most methanotrophs (types I, II, and X methanotrophs) were found to have been labeled with 13C under nitrate-free conditions. This DNA-SIP study identifies key bacterial populations involved in a MDD ecosystem.     

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.
     

Comparison of Methanotrophic Community and Methane Oxidation between Rhizospheric and Non-Rhizospheric Soils

Using particulate methane monooxygenase (pMMO) encoding gene, pmoA-based terminal-restrict fragment length polymorphism (T-RFLP), the methanotrophic communities between rhizospheric soils (RSs) and non-rhizospheric soil (NRSs) of landfill cover (LC), riparian wetland (RW) and rice paddy (RP) were compared before and after pre-incubation of 90 days. The ultimate potential of methane oxidation rate (UPMOR) and gene copy number of pmoA were evaluated in the soil samples after pre-incubation. Compared to the methanotrophic community in the soil samples before pre-incubation, type II methanotrophs, the Methylocystis-Methylosinus group, was mostly increased after pre-incubation, regardless of the soil type. The UPMOR (11.82 ± 0.27 μmol-CH₄· g^?1 _soil-DW·h^?1) in the LC-RS was significantly higher than that (9.57 ± 0.14 μmol-CH₄· g^?1 _soil-DW·h^?1) in the LC-NRS. However, no significant difference was found between RSs and NRSs in the RW (15.28 ± 0.91 and 13.23 ± 0.69 μmol-CH₄· g^?1 _soil-DW·h^?1, respectively) and RP (13.81 ± 1.04 and 12.81 ± 2.40 μmol-CH₄· g^?1 _soil-DW·h^?1, respectively) soils. There was no significantly difference in the gene copy numbers of pmoA in the RSs compared with those in the NRSs at all of the sampling sites. This study provides basic metagenomic information about both rhizospheric and non-rhizospheric methanotrophs, which will be helpful in developing a better strategy of biological methane removal from both natural and anthropogenic major methane sources.     

Potential activity of methane and ammonium oxidation by methanotrophic communities from the soda lakes of southern Transbaikal

Radioisotopic measurements of the methane consumption by mud samples taken from nine Southern Transbaikal soda lakes (pH 9.5–10.6) showed an intense oxidation of methane in the muds of Lakes Khuzhirta, Bulamai Nur, Gorbunka, and Suduntuiskii Torom, with the maximum oxidation rate in the mud of Lake Khuzhirta (33.2 nmol/(ml day)). The incorporation rate of the radioactive label from¹⁴CH₄ into¹⁴CO₂ was higher than into acid-stable metabolites. Optimum pH values for methane oxidation in water samples were 7–8, whereas mud samples exhibited two peaks of methane oxidation activity (at pH 8.15–9.4 and 5.8–6.0). The majority of samples could oxidize ammonium to nitrites; the oxidation was inhibited by methane. The PCR amplification analysis of samples revealed the presence of genes encoding soluble and paniculate methane monooxygenase and methanol dehydrogenase. Three alkaliphilic methanotrophic bacteria of morphotype I were isolated from mud samples in pure cultures, one of which, B5, was able to oxidize ammonium to nitrites at pH 7–11. The data obtained suggest that methanotrophs are widely spread in the soda lakes of Southern Transbaikal, where they can actively oxidize methane and ammonium.     

Trichloroethylene oxidation by the membrane-associated methane monooxygenase in type I,type II and type X methanotrophs

Trichloroethylene (TCE) oxidation was examined in 9 different methanotrophs grown under conditions favoring expression of the membrane associated methane monooxygenase. Depending on the strain, TCE oxidation rates varied from 1 to 677 pmol/min/mg cell protein. Levels of TCE in the reaction mixture were reduced to below 40 nmolar in some strains. Cells incubated in the presence of acetylene, a selective methane monooxygenase inhibitor, did not oxidize TCE.Cultures actively oxidizing TCE were monitored for the presence of the soluble methane monooxygenase (sMMO) and membrane associated enzyme (pMMO). Transmission electron micrographs revealed the cultures always contained the internal membrane systems characteristic of cells expressing the pMMO. Naphthalene oxidation by whole cells, or by the cell free, soluble or membrane fractions was never observed. SDS denaturing gels of the membrane fraction showed the polypeptides associated with the pMMO. Cells exposed to ¹⁴C-acetylene showed one labeled band at 26 kDa, and this protein was observed in the membrane fraction. In the one strain examined by EPR spectroscopy, the membrane fraction of TCE oxidizing cells showed the copper complexes characteristic of the pMMO. Lastly, most of the strains tested showed no hybridization to sMMO gene probes. These findings show that the pMMO is capable of TCE oxidation; although the rates are lower than those observed for the sMMO.     

Biphasic kinetics of a methanotrophic community is a combination of growth and increased activity per cell

Because methane-oxidizing bacteria (MOB) are the only biological sink for the greenhouse gas methane, knowledge of the functioning of these bacteria in various ecosystems is needed to understand the dynamics observed in global methane emission. The activity of MOB is commonly assessed by methane oxidation assays. The resulting methane depletion curves often follow a biphasic pattern of initial and induced methane oxidation activity, often interpreted as representing the in situ active and total MOB community, respectively. The application of quantitative-PCR on soil incubations, which were stopped before, at and after the transition point in the methane-depletion curve, demonstrated that both pmoA -mRNA was produced as well as substantial cell growth took place already in the initial phase. In addition, type Ia and II MOB displayed markedly different behaviour, which can be interpreted as ecologically different strategies. For the correct interpretation of methane oxidation assays, the use of small time windows is recommended to calculate methane oxidation activities to avoid substantial cell growth.     

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.     

好氧反硝化菌代谢机制及其与重金属相互作用的代谢特征

好氧反硝化是在好氧条件下将NO3--N最终转化为N2的过程.好氧反硝化菌不仅表现出优秀的脱氮性能,可在许多极端条件下生存,还拥有多种重金属耐受性.本文总结了不同重金属Cr(Ⅵ)、Cu(Ⅱ)和Cd(Ⅱ)对好氧反硝化菌脱氮效率的影响,同时整理了好氧反硝化菌对不同重金属的耐受或去除机制.分析了好氧反硝化菌在工业废水处理中的应...     

Coupling of Microbial Processes of Methane and Ammonium Oxidation in Soils

The effect of ammonium ions on the activity of methane oxidation in soils was studied. The degree of inhibition of the methanotrophic activity in the presence of ammonium in the soil solution was quantitatively assessed as dependent on ammonium concentration and the properties of different types of soils of the European part of Russia.     

Biochemical and molecular characterization of methanotrophs in soil from a pristine New Zealand beech forest

Methane (CH4) oxidation and the methanotrophic community structure of a pristine New Zealand beech forest were investigated using biochemical and molecular methods. Phospholipid-fatty acid-stable-isotope probing (PLFA-SIP) was used to identify the active population of methanotrophs in soil beneath the forest floor, while terminal-restriction fragment length polymorphism (T-RFLP) and cloning and sequencing of the pmoA gene were used to characterize the methanotrophic community. PLFA-SIP suggested that type II methanotrophs were the predominant active group. T-RFLP and cloning and sequencing of the pmoA genes revealed that the methanotrophic community was diverse, and a slightly higher number of type II methanotrophs were detected in the clone library. Most of the clones from type II methanotrophs were related to uncultured pmoA genes obtained directly from environmental samples, while clones from type I were distantly related to Methylococcus capsulatus. A combined data analysis suggested that the type II methanotrophs may be mainly responsible for atmospheric CH4 consumption. Further sequence analysis suggested that most of the methanotrophs detected shared their phylogeny with methanotrophs reported from soils in the Northern Hemisphere. However, some of the pmoA sequences obtained from this forest had comparatively low similarity (<97%) to known sequences available in public databases, suggesting that they may belong to novel groups of methanotrophic bacteria. Different methods of methanotrophic community analysis were also compared, and it is suggested that a combination of molecular methods with PLFA-SIP can address several shortcomings of stable isotope probing.     

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.     

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.     

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。     

准噶尔盆地东南部基于Thom-Thony-Vauclin模型的蒸发皿蒸发量估算

应用阜康荒漠生态站实际观测辐射和E601B型蒸发皿蒸发数据,分别校正了Angstrom-Prescott和Thom-Thony-Vauclin模型的参数,并探讨了在过去50年气候变化背景下,蒸发皿蒸发量下降的原因。当经验参数a_s=0.3134、b_s=0.5357,以及a=0.6844、c=1.4123时,Angstrom-Prescott模型和Thom-Thony-Vauclin模型分别达到最佳优合度。ThomThony-Vauclin模型计算表明:2005—2011年4—10月植物生长季蒸发皿蒸发量为954.2~1120 mm,平均值为1028.4 mm。风速下降对准噶尔盆地东南部蒸发皿蒸发量变化贡献最大(66.2%),其次为温度升高和辐射下降,分别贡献21.2%和8.0%,而水汽压饱和亏下降贡献最少(5.9%)。除了相对敏感度随净辐射升高而下降外,其他3项指标与之相反。可见,风速下降是准噶尔盆地东南部蒸发皿蒸发量下降的最主要原因。     

生物分子机器——甲烷单加氧酶的研究进展

甲烷氧化菌中的甲烷单加氧酶能够在生理条件下选择性地以甲烷和氧气为底物生成甲醇,麻省理工学院的Lippard教授称它为\"神奇的生物分子机器\"。本文重点对生物分子机器甲烷单加氧酶的结构、编码基因及调控机制、催化反应机理等进行了综述,此外也简要介绍了甲烷单加氧酶的产生菌甲烷氧化菌的研究历史及分类。生物分子机器甲烷单加氧酶可催化甲烷氧化成甲醇,不仅为甲醇的生产提供了一种新颖的生产方法,而且对生物分子机器的设计也有借鉴意义。