Diversity of methanotrophs in Zoige wetland soils under both anaerobic and aerobic conditions

Zoige wetland is one of the most important methane emission centers in China. The oxidation of methane in the wetland a ects global warming, soil ecology and atmospheric chemistry. Despite their global significance, microorganisms that consume methane in Zoige wetland remain poorly characterized. In this study, we investigated methanotrophs diversity in soil samples from both anaerobic site and aerobic site in Zoige wetland using pmoA gene as a molecular marker. The cloning library was constructed according to the pmoA sequences detected. Four clusters of methanotrophs were detected. The phylogenetic tree showed that all four clusters detected were a liated to type I methanotrophs. Two novel clusters (cluster 1, cluster 2) were found to relate to none of the recognized genera of methanotrophs. These clusters have no cultured representatives and reveal an ecological adaptation of particular uncultured methanotrophs in Zoige wetland. Two clusters were belonging to Methylobacter and Methylococcus separately. Denaturing gradient gel electrophoresis gel bands pattern retrieved from these two samples revealed that the community compositions of anaerobic soil and aerobic soil were di erent from each other while anaerobic soil showed a higher metanotrophs diversity. Real-time PCR assays of the two samples demonstrated that aerobic soil sample in Zoige wetland was 1.5 times as much copy numbers as anaerobic soil. These data illustrated that methanotrophs are a group of microorganisms influence the methane consumption in Zoige wetland.     

Do land utilization patterns a ect methanotrophic communities in a Chinese upland red soil?

Soil samples were collected from three plots under di erent land utilization patterns including degradation, farming, and restoration. The abundances of methanotrophs were quantified using real-time polymerase chain reaction (PCR) based on the pmoA and 16S rRNA genes, and the community fingerprint was analyzed using denaturing gradient gel electrophoresis (DGGE) aiming at pmoA gene. Significantly lower 16S rRNA and pmoA genes copies were found in the degradation treatment than in farming and restoration. Higher abundances of Type I than those of Type II methanotrophs were detected in all treatments. The treatment of farming was clearly separated from degradation and restoration according to the DGGE profile by cluster analysis. The lowest diversity indices were observed in the F (farming plot), suggesting that the community structure was strongly a ected by farming activities. There were significantly positive correlations between the copy numbers of pmoA also Type II-related 16S rRNA genes and soil available K content. Strong negative and positive correlations were found between Type I and soil pH, and available P content, respectively. We concluded that the vegetation cover or not, soil characteristics including pH and nutrients of P and K as a result of anthropogenic disturbance may be key factors a ecting methanotrophic communities in upland soil.     

Do land utilization patterns affect methanotrophic communities in a Chinese upland red soil?

Soil samples were collected from three plots under different land utilization patterns including degradation,farming,and restoration.The abundances of methanotrophs were quantified using real-time polymerase chain reaction(PCR)based on the pmoA and 16S rRNA genes,and the community fingerprint was analyzed using denaturing gradient gel electrophoresis(DGGE)aiming at pmoA gene.Significantly lower 16S rRNA and pmoA genes copies were found in the degradation treatment than in farming and restoration.Higher abundances of Type Ⅰ than those of Type Ⅱ methanotrophs were detected in all treatments.The treatment of farming was clearly separated from degradation and restoration according to the DGGE profile by cluster analysis.The lowest diversity indices were observed in the F(farming plot),suggesting that the community structure was strongly affected by farming activities.There were significantly positive correlations between the copy numbers of pmoA also Type Ⅱ-related 16S rRNA genes and soil available K content.Strong negative and positive correlations were found between Type Ⅰ and soil pH,and available P content,respectively.We concluded that the vegetation cover or not,soil characteristics including pH and nutrients of P and K as a result of anthropogenic disturbance may be key factors affecting methanotrophic communities in upland soil.     

Interaction and independence on methane oxidation of landfill cover soil among three impact factors: water, oxygen and ammonium

To understand the influence patterns and interactions of three important environmental factors, i.e. soil water content, oxygen concentration, and ammonium addition, on methane oxidation, the soils from landfill cover layers were incubated under full factorial parameter settings. In addition to the methane oxidation rate, the quantities and community structures of methanotrophs were analyzed to determine the methane oxidation capacity of the soils. Canonical correspondence analysis was utilized to distinguish the important impact factors. Water content was found to be the most important factor influencing the methane oxidation rate and Type II methanotrophs, and the optimum value was 15% (w/w), which induced methane oxidation rates 10- and 6- times greater than those observed at 5% (w/w) and 20% (w/w), respectively. Ambient oxygen conditions were more suitable for methane oxidation than 3% oxygen. The addition of 100 mg-N·kg_drysoil⁻¹ of ammonium induced different effects on methane oxidation capacity when conducted at low or high water content. With regard to the methanotrophs, Type II was sensitive to the changes of water content, while Type I was influenced by oxygen content. Furthermore, the methanotrophic acidophile, Verrucomicrobia, was detected in soils with a pH of 4.9, which extended their known living environments.     

长期不同施肥对暗棕壤甲烷氧化菌群落特征与功能的影响

不同施肥方式对我国旱地农田土壤甲烷氧化影响的微生物机制尚不明确.本研究利用PCR-DGGE和实时荧光定量PCR技术,结合甲烷氧化速率和土壤性质测定,探索了长期不同施肥条件下暗棕壤的"土壤性质-甲烷氧化菌群落特征-土壤甲烷氧化速率"关系.结果表明,有机肥和无机肥配施处理显著降低了土壤甲烷氧化速率,降幅为61.2%,而单独施用有机肥或无机肥对暗棕壤甲烷氧化速率的影响不显著;与对照相比,有机肥处理土壤甲烷氧化菌多样性指数增加91.9%,有机肥和无机肥配施处理增加102.5%,而单施无机肥后土壤甲烷氧化菌多样性指数变化不明显;有机肥处理土壤的pmoA基因丰度显著增加,平均pmoA基因丰度为不施用有机肥的12.7倍;土壤甲烷氧化速率与甲烷氧化菌的群落结构和比活性呈显著正相关,相关系数分别为0.363和0.684,但与甲烷氧化菌群落丰度和多样性不相关;甲烷氧化菌的群落结构和比活性与土壤pH值、全氮和有机质含量呈显著正相关.上述结果说明,长期不同施肥可以通过改变暗棕壤的pH值、全氮和有机质含量等土壤性质,改变甲烷氧化菌群落结构和比活性,进而影响土壤甲烷氧化速率;有机肥和无机肥配施土壤甲烷氧化菌多样性和丰度大幅度增加,而甲烷氧化速率却显著降低,说明有机肥和无机肥配施土壤中只有部分微生物发挥了甲烷氧化活性,但有待进一步研究.     

Effect of bio-column composed of aged refuse on methane abatement – A novel configuration of biological oxidation in refuse landfill

An experimental bio-column composed of aged refuse was installed around the exhaust pipe as a new way to mitigate methane in refuse landfill.One of the objectives of this work was to assess the effect of aged refuse thickness in bio-column on reducing CH 4 emissions.Over the study period,methane oxidation was observed at various thicknesses,5 cm (small size),10 cm (middle size) and 15 cm (large size),representing one to three times of pipeline diameters.The middle and large size both showed over 90% methane conversion,and the highest methane conversion rate of above 95% occurred in the middle-size column cell.Michaelis-Menten equation addressed the methanotrophs diffusion in different layers of the bio-columns.Maximum methanotrophic activity (V max) measured at the three thicknesses ranged from 6.4 × 10 3 to 15.6 × 10 3 units,and the half-saturation value (K M) ranged from 0.85% to 1.67%.Both the highest V max and K M were observed at the middle-size of the bio-column,as well as the largest methanotrophs population,suggesting a significant efficiency of methane mitigation happened in the optimum zone with greatest affinity and methanotrophic bacteria activities.Therefore,bio-column is a potential style for methane abatement in landfill,and the aged refuse both naturally formed and artificially placed in the column plays a critical role in CH 4 emission.     

生活垃圾填埋场甲烷自然减排的新途径:厌氧与好氧的共氧化作用

采用暴雨过后垃圾填埋表层30～60 cm的覆土、表层1.5 m以下的垃圾,以及刚刚开挖出来的9年矿化垃圾进行硫酸盐还原菌阳性反应试验,结果表明生活垃圾填埋体不同填埋层都存在不同数量级的硫酸盐还原菌,且底层矿化垃圾中的硫酸盐还原菌的数量最多,表层覆土中最少.颗粒大小比例为50%:50%的垃圾样品表现出最佳的甲烷好氧与厌氧...     

甲烷氧化菌在填埋场覆盖层中的工程应用(Ⅱ):基质选择性及长效性

针对已筛选获得的甲烷氧化混合菌,选取填埋场覆盖土(LCS)、矿化垃圾(AR)和塘渣(TZ)3种填埋场周边易得的材料为供试生存基质,从基质选择性及长效性角度进行了甲烷减排应用条件的探究及使用效能评估.结果表明,在TZ、LCS、AR、TZ-AR和LCS-AR这5种生存基质中,TZ-AR最适合甲烷氧化混合菌的生长,且TZ与AR的复配比例以5∶5为最佳.甲烷氧化混合菌在TZ-AR的粒径≤4 mm和含水率为20%时具有最高甲烷氧化能力.一次性接种甲烷氧化混合菌在静态体系中的最佳使用有效期为31 d.其在接种量为0.08、0.16、0.20 m L·g-1和0.25m L·g-1时甲烷氧化速率无明显差异,从工程应用角度而言,8%的接种量为最佳.     

Characterization of Methylocystis strain JTA1 isolated from aged refuse and its tolerance to chloroform

To accelerate the efficiency of methane biodegradation in landfills,a Gram-negative,rod-shaped,non-motile,non-spore-forming bacterium,JTA1,which can utilize methane as well as acetate,was isolated from the Laogang MSW landfills,Shanghai,China.Strain JTA1 was a member of genus Methylocystis on the basis of 16S rRNA and pmoA gene sequence similarity.The maximum specific cell growth rates(μmax=0.042 hr-1,R2=0.995) was derived through Boltzmann simulation,and the apparent half-saturation constants(Km(app)=7.08mmol/L,R2=0.982) was calculated according to Michaelis-Menton hyperbolic model,indicating that Methylocystis strain JTA1 had higher-affinity potential for methane oxidation than other reported methanotrophs.By way of adding the strain JTA1 culture,the methane consumption of aged refuse reached 115mL,almost two times of control experiment.In addition,high tolerance of Methylocystis strain JTA1 to chloroform could facilitate the methane oxidation of aged refuse bio-covers.At the chloroform concentration of 50mg/L,the methane-oxidation rate of bio-cover reached 0.114mL/(day·g),much higher than the highest rate,0.0135mL/(day·g),of reported bio-covers.In conclusion,strain JTA1 opens up a new possibility for environmental biotechnology,such as soil or landfills bioremediation and wastewater decontamination.     

富铁炭对填埋覆土层甲烷氧化主导微生物活动的影响

为了进一步明晰甲烷氧化菌群与环境间的相关关系,通过比较其甲烷氧化量和胞外聚合物(EPS)2种重要的微生物生命活动来探讨不同基质浓度与不同炭组间交互产生的环境差异对微生物群落的影响.结果表明,不同基质浓度对微生物多样性和种群差异影响最大.在甲烷与氧气(15％,V/V)均充足的情况下甲烷氧化累积量主要由Ⅰ型甲烷氧化菌贡献;...     

浑河底泥反硝化厌氧甲烷氧化菌群落结构时空特征

为了解在反硝化厌氧甲烷氧化反应过程中起主要作用的微生物——Candidatus Methylomirabilis oxyfera(M. oxyfera)在河流底泥环境中的时空变化特征,利用克隆文库及实时荧光定量PCR技术考察了浑河流域部分底泥中M. oxyfera-like的16S rRNA和pmoA功能基因的多样性及丰度. 结果表明:从浑河上游至下游丰水期各采样点底泥中M. oxyfera-like的16S rRNA的多样性无明显地域性差异,其OTU(operational taxonomic unit,独立操作单元)数变幅不大;pmoA功能基因的多样性则存在较为明显的地域性差异,上游采样点A(章党大桥)底泥中OTU数为5,下游采样点E(三岔河大桥)底泥中仅为1. 采样点A底泥中M. oxyfera-like的16S rRNA和pmoA功能基因的多样性均存在一定的季节性差异,平水期16S rRNA和pmoA功能基因的Shannon-Wiener多样性指数分别为1.48和2.19,在各期中最高;而枯水期时二者均最低,分别为0.83和1.26. 丰度分析结果显示,所有采样点底泥中M. oxyfera-like的16S rRNA丰度均表现为平水期(2.42×106~1.74×107 copies/g)(以干质量计)最高、枯水期(2.87×104~4.19×106 copies/g)居中、丰水期(1.29×103~2.04×104 copies/g)最低,说明浑河底泥中M. oxyfera-like的丰度亦存在一定的季节性差异.      

Diversity of methanotrophs in a simulated modified biocover reactor

A simulated landfill biocover microcosm consisting of a modifying ceramsite material and compost were investigated. Results show that the mixture can improve the material porosity and achieve a stable and highly efficient (100%) methane oxidation over an extended operating period. The diversity of the methanotrophic community in the microcosm was assessed. Type I methanotrophs were enhanced in the microcosm due to the increased air diffusion and distribution, whereas the microbial diversity and population density of type II methanotrophs were not significantly affected. Moreover, the type I methanotrophic community structure significantly varied with the reactor height, whereas that of type II methanotrophic communities did not exhibit a spatial variation. Phylogenetic analysis showed that type I methanotroph-based nested polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) resulted in the detection of eight different populations, most of which are related to Methylobacter sp., whereas that of type II resulted in the detection of nine different populations, most of which are related to Methylocystaceae. Methanotrophic community analysis also indicated that a number of new methanotrophic genera not closely related to any known methanotrophic populations were present.     

湿地甲烷代谢微生物产甲烷菌和甲烷氧化菌的研究进展

湿地作为一种独特的生态系统,是大气中重要温室气体甲烷（CH₄）的"源",湿地CH₄的排放是CH₄的产生、传输和氧化三个过程的最终结果,其排放量与产甲烷菌（methanogens）和甲烷氧化菌（methanotrophs）的活动密切相关。本文对湿地甲烷代谢重要微生物产甲烷菌和甲烷氧化菌的分类、特点、代谢途径、分子生态学研究现状及影响湿地产甲烷菌和甲烷氧化菌的重要环境因子等方面进行了综述,旨在促进我国湿地产甲烷及甲烷氧化菌群的领域更加系统和深入的研究,为有效调控湿地CH₄代谢,减少CH₄排放通量提供参考。     

Excavation and characterization of refuse in closed landfill

Refuse in landfills becomes stabilized as organic matter in refuse degrades and soluble inorganic substances dissolve during their longterm stabilization process.In this paper,this process is also referred to as mineralization process and the resultant stabilized refuse referred to as aged refuse.Aged refuse contains a wide spectrum and huge quantity of microorganisms with strong decomposition capability for refractory organic matter present in some wastewater such as leachate.In this study,aged refuse excavated from 2 to 10 years old closed landfill compartments in Shanghai Refuse Landfill is characterized in terms of particulate distribution by screening,total nitrogen,total phosphorus,biodegradable matter.The approaches for redevelopment of both land and aged refuse in the stabilized landfills are proposed.     

上海老港生活垃圾填埋场陈垃圾的基本特性研究

对上海老港生活垃圾填埋场6年填埋龄和10年填埋龄陈垃圾的基本特征进行了测试研究，这些特性包括粒径分布，臭味、微生物数量、含水率、总磷、总氮、饱和水力渗透系数等。研究结果：上海老港生活垃圾填埋场中6年以上填埋龄陈垃圾已可以开采用使用；对其多种用途的开发主要限于陈垃圾中粒径小于15mm的细料部分；此细料是极为优良的污染物生物处理基质。     

硝酸盐对矿化垃圾中兼/厌氧甲烷氧化的影响

设计全因子实验研究了NO-3-N对填埋10~12 a的矿化垃圾中兼/厌氧甲烷氧化作用的影响.结果表明,兼/厌氧条件下,NO-3-N能促进矿化垃圾中CH4的去除.初始CH4和NO-3-N对CH4去除和N2产生有明显影响,且两者具有交互作用(P<0.05).CH4去除量随着初始CH4体积分数的增加而增加,添加一定含量的NO-3-N能促进CH4去除,同时通入一定体积分数CH4可以明显促进反硝化作用,说明矿化垃圾中NO-3-N还原能与兼/厌氧甲烷氧化耦合.本实验条件下,初始CH4体积分数为30%,NO-3-N含量为110 mg·kg-1时耦合效应较好.     

生活垃圾填埋场垃圾腐殖质组成和变化规律的表征

通过分析上海老港生活垃圾填埋场填埋垃圾的腐殖质组成和光密度,考察了填埋垃圾的腐殖质组成和光学特性随填埋龄的变化规律.研究结果表明,填埋龄在1～14 a间的填埋垃圾中腐殖质组分的有机碳绝对含量的波动幅度很大,而腐殖质组分相对含量具有比较明显的变化规律,其中腐殖质总可提取率和wHA/wFA比值分别随填埋龄增加呈线性下降和上升的趋势.另一方面,填埋垃圾腐殖质光学特性研究表明,填埋垃圾可提取腐殖质和富里酸组分的E4/E6比值随填埋龄变化无明显关系,而胡敏酸的E4/E6比值则随填埋龄的增加呈线性下降趋势.因此,填埋垃圾腐殖质的总可提取率、wHA/wFA比值以及胡敏酸组分的E4/E6比值可有效地表征填埋垃圾和填埋场的稳定化进程.     

矿化垃圾处理垃圾渗滤液的试验研究

试验研究了矿化垃圾细料与土壤作为生物反应床填料处理渗滤液效果的差异,以及加管通风条件对矿化垃圾柱和土壤柱处理渗滤液效果的影响. 结果表明:矿化垃圾细料对渗滤液中COD_Cr和NH₄＋-N的去除效果明显好于土壤. 在水力负荷为40 L/(m3·d)和布水时间为2.0 h的条件下,矿化垃圾细料对渗滤液中的COD_Cr和NH₄＋-N有良好的去除效果,去除率分别达73%和66%;而土壤对COD_Cr和NH₄＋-N的去除率分别只有52%和42%;加管通风条件使矿化垃圾对渗滤液的处理效果明显提高,对COD_Cr和NH₄＋-N的去除率分别提高了9%和26%.      

内蒙古高原干涸湖泊反硝化及甲烷氧化细菌的群落分析

以内蒙古高原半干旱草原区干涸湖泊为研究对象,应用末端限制性片段长度多态性(T-RFLP)技术对湿地土壤中的反硝化和甲烷氧化关联菌群的关键功能基因nosZ和pmoA进行了研究,根据末端限制性片段(T-RFs)分析了两种菌群的群落结构及多样性变化,以及环境因素所产生的影响,并探讨了土壤微生物群落对湖泊干涸的响应机制.结果表明:干涸湖泊的湖心、湖底土壤反硝化和甲烷氧化细菌多样性指数较高,群落结构较复杂;而较早干涸的湖坡以及相邻草原对照区,两个菌群多样性指数较低,群落结构相对单一.从湖心到相邻草原,反硝化和甲烷氧化细菌的群落相似度逐渐降低,显示两个菌群处于动态演替中.湖泊干涸过程中,水分含量和有机质含量对反硝化和甲烷氧化细菌的群落结构影响显著;高pH值和高铵态氮分别对反硝化和甲烷氧化细菌有抑制作用.随着湖泊干涸,土壤微生物群落因环境扰动而受到胁迫,这一过程会加剧CO2等温室气体的排放.     

垃圾填埋场覆盖黄土的甲烷氧化能力及其影响因素研究

利用西安江村沟填埋场不同覆盖时间的临时覆盖黄土和新鲜黄土掺堆肥配置的土样,开展了甲烷氧化培养瓶试验,培养历时最长达22d,研究了不同覆盖时间的覆盖黄土和不同堆肥掺量的新鲜黄土的甲烷氧化能力,以及含水量对覆盖黄土甲烷氧化能力的影响,分析了甲烷氧化过程中各组分气体体积变化关系.研究结果表明:不同覆盖时间的覆盖黄土的甲烷氧化能力相差很大,覆盖3~5年的黄土最大甲烷氧化能力达26.05~53.95μg CH4/(g×h), 而未覆盖的新鲜黄土几乎没有甲烷氧化能力.在新鲜黄土中掺入堆肥,能有效提高其甲烷氧化能力,且在堆肥掺量小于50%时,土样的最大甲烷氧化能力与堆肥掺量呈正比例关系.含水量对覆盖土的甲烷氧化能力有很大影响,黄土甲烷氧化的最适宜含水量约为20% ~ 30%.在甲烷氧化过程中,甲烷氧化菌将CH4中约44%的碳氧化为CO2,其余碳转化为甲烷氧化菌胞内物质.实际工程中进行覆盖层设计时,需要根据填埋场实际产气量和覆盖层导气性确定与甲烷通量相匹配的甲烷氧化速率,进而合理确定堆肥掺量.