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.     

甲烷单加氧酶的催化性能和活性中心结构

甲烷单加氧酶是甲烷利用细菌代谢甲烷过程中的重要酶系,它能够催化烷烃羟基化和烯烃环氧化反应;还能催化降解氯代烃类,可用于环境中氯代烃类化合物污染的治理,是具有广泛应用前景的生物催化剂．甲烷单加氧酶是含有μ－氧桥双核铁催化活性中心的蛋白,它的研究对分子氧的活化、化学催化剂的设计具有重要意义．文章介绍了甲烷单加氧酶催化性能和机理的最新研究进展．     

外源电子给体对甲烷单加氧酶催化丙烯环氧化反应活性的影响

以丙烯氧化反应为指标研究了不同外源电子给体对甲烷细菌(Methylomonas sp．GYJ30)休止细胞催化活性的影响。结果表明甲烷、甲醇、甲醛和甲酸盐作为电子给体加入反应中,将甲烷单加氧酶催化丙烯环氧化反应活性分别提高5．3,12．7,10和12．4倍。以甲烷和甲醛作为外源电子给体时提高初始浓度对甲烷单加氧酶具有抑制作用;而以甲醇和甲酸盐作为电子给体时提高初始浓度对甲烷单加氧酶催化活性无明显抑制作用。研究了甲醇作为电子给体时它的代谢、环氧丙烷的积累以及催化反应活性与反应时间的关系     

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.     

甲烷氧化菌Methylomonas sp.GYJ3中甲烷单加氧酶基因与16S rRNA序列分析

甲烷氧化细菌中的关键酶系甲烷单加氧酶是一个含双核铁的多组份氧化酶,常温、常压下能够催化甲烷转化为甲醇。对甲烷氧化细菌Methylomonas sp.GYJ3中溶解性甲烷单加氧酶基因和16SrDNA进行了测序与分析。利用已知相关基因数据库信息,设计了PCR引物和测序引物,获得了满意的测序结果。全长的溶解性甲烷单加氧酶基因为5690bp,部分16S rDNA的序列长度为1280bp。与已发表的甲烷氧化细菌中甲烷单加氧酶进行了比较,结果表明MMOX组份中氨基酸序列的同一性为78%到99%,基因序列的同一性为71%到97%,6个组份中orfY片段的同一性相对较低。MMOX氨基酸序列的多序列联配表明,MMOX序列具有高度保守性,特别是在双核铁中心区域。16S rDNA进化分析显示Methylomonas sp.GYJ3与γ蛋白细菌是相关联的,基于MMOX氨基酸序列的进化分析证明,与Methylomonas sp.GYJ3最近似的菌株是Ⅰ型甲烷氧化细菌Methylomonas sp.KSWⅢ。综合分析表明,菌株GYJ3属于Ⅰ型甲烷氧化细菌Methylomonas sp.属。这个结果为Ⅰ型甲烷氧化细菌也能表达溶解性甲烷单加氧酶提供了新的证据。羟基化酶的理论等电点是6.28,理论分子量为248874.41Da。     

专一营养与兼性甲烷氧化菌降解氯代烃的研究现状、动力学分析及展望

生物修复技术被认为是氯代烃类污染物处理处置的最有效途径之一,而甲烷氧化菌在该领域表现出较大的应用潜力。近期研究发现,突破了仅能利用单碳化合物的局限,兼性甲烷氧化菌能够利用多种底物降解氯代烃,这一独特的新陈代谢特性,使其在污染物生物处置领域逐渐受到关注。结合本课题组研究成果,对甲烷氧化菌降解氯代烃进行了全面总结,主要包括:分析了不同菌株(纯菌株和混合菌株)对不同氯代烃的降解效果;比较了不同类型甲烷单加氧酶在不同底物体系中的活性表达和催化特性;总结了模型菌株甲基弯菌Methylosinus trichosporium OB3b降解氯代烃的动力学特性;概述了兼性甲烷氧化菌株降解氯代烃的特性及其应用潜力;最后讨论了甲烷氧化菌降解氯代烃存在的问题及未来发展方向。     

甲烷氧化细菌Methylosinus trichosporium 3011甲醇累积条件的研究

本文考察了甲烷氧化细菌Methylosi nus trichosporium 3011的生理特性及反应条件对甲烷单加氧酶和甲醇累积的影响。M.3011菌株在4℃保存30～40天内,菌株的细胞生长量和甲烷单加氧酶活性均不受影响。在生长对数期收获细胞,其甲烷单加氧酶活力最高可达125nmol甲醇/mg(细胞干重)·min。在M.3011菌株的生长对数期后期收集细胞,将反应菌悬液浓度控制在0.15—0.3mg(细胞干重/ml,pH为6.7,反应温度为35℃,甲醇累积量可达3.1μmol甲醇/mg(细胞干重)·h。反应液的磷酸缓冲液的最适浓度为60mmol/L。     

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。     

甲烷单加氧酶的研究进展

甲烷氧化细菌在转化甲烷制造新型燃料、单细胞蛋白和新功能酶生产、污水处理等方面有着潜在的应用前景,因此,甲烷单加氧酶作为其代谢过程中重要的酶系也受到人们的广泛关注。我们简要综述了近年来对甲烷单加氧酶的性质、结构、催化机理等方面的研究,特别是对颗粒性甲皖单加氧酶的相关性质进行了详细的阐述。     

颗粒状甲烷单加氧酶异源表达方法

由于甲烷氧化菌只能利用甲烷作为唯一的碳源和能源,存在生长缓慢、细胞密度低、培养困难等问题,限制了其工业应用。解决该问题的有效途径之一是在容易实现高密度培养的异源宿主菌中表达甲烷单加氧酶(Methane monooxygenase,MMO)。本实验室前期首次在一种红球菌中成功地表达了来自于甲烷氧化菌(Methylosinus trichosporium)OB3b的pMMO(颗粒状甲烷单加氧酶),但比酶活较原始菌低很多。本实验在该结果的基础上,通过选用不同的启动子和宿主细胞探索表达pMMO的可能性,结果得到了具有氧化甲烷活性的重组菌,但是产物检测到乙醇的生成,且该重组菌的pMMO活性不稳定,暗示pMMO的催化特性可能发生了变化。另外,很多重组菌检测到pMMO蛋白的表达,但没有催化活性,说明pMMO在宿主细胞中的正确组装是其功能表达的关键。     

Methane emissions from rice microcosms: The balance of production, accumulation and oxidation

In rice microcosms (Oryza sativa, var. Roma, type japonica),CH₄ emission, CH₄ production, CH₄oxidation and CH₄ accumulation were measured over an entirevegetation period. Diffusive CH₄ emission was measured inclosed chambers, CH₄ production was measured in soil samples,CH₄ oxidation was determined from the difference between oxicand anoxic emissions, and CH₄ accumulation was measured byanalysis of porewater and gas bubbles. The sum of diffusiveCH₄ emission, CH₄ oxidation, andCH₄ accumulation was only 60% of the cumulativeCH₄ production. The two values diverged during the first 50days (vegetative phase) and then again during the last 50 days (latereproductive phase and senescence) of the 150 day vegetation period. Duringthe period of day 50–100 (early reproductive phase/flowering), theprocesses were balanced. Most likely, gas bubbles and diffusion limitationare responsible for the divergence in the early and late phases. The effectof rice on CH₄ production rates and CH₄concentrations was studied by measuring these processes also in unplantedmicrocosms. Presence of rice plants lowered the CH₄concentrations, but had no net effect on the CH₄ productionrates.     

Processes involved in formation and emission of methane in rice paddies

The seasonal change of the rates of production and emission of methane were determined under in-situ conditions in an Italian rice paddy in 1985 and 1986. The contribution to total emission of CH₄ of plant-mediated transport, ebullition, and diffusion through the flooding water was quantified by cutting the plants and by trapping emerging gas bubbles with funnels. Both production and emission of CH₄ increased during the season and reached a maximum in August. However, the numbers of methanogenic bacteria did not change. As the rice plants grew and the contribution of plant-mediated CH₄ emission increased, the percentage of the produced CH₄ which was reoxidized and thus, was not emitted, also increased. At its maximum, about 300 ml CH₄ were produced per m² per hour. However, only about 6% were emitted and this was by about 96% via plant-mediated transport. Radiotracer experiments showed that CH, was produced from H₂/CO₂. (30–50%) and from acetate. The pool concentration of acetate was in the range of 6–10 mM. The turnover time of acetate was 12–16 h. Part of the acetate pool appeared to be not available for production of CH₄ or CO₂     

盐分对湿地甲烷排放影响的研究进展

当前在全球气候变化和人类活动双重作用下,湿地正在或者将要面临着显著的盐分变化形势,尤其是内陆和滨海咸化湿地。湿地是大气甲烷的重要排放源。甲烷排放是甲烷产生、氧化和传输过程综合作用的结果。盐分变化将影响湿地水-土环境,降低植物群落初级生产力和有机物积累速率,改变微生物主导的有机物矿化速率和途径等,进而改变湿地生态系统的结构和功能,影响湿地甲烷产生、氧化、传输和排放系列过程。本文综述了盐分(浓度与组成)对湿地甲烷产生与排放的影响结果,从底物供给、微生物(产甲烷菌和甲烷氧化菌等)数量、活性与群落组成、酶活性、植物、电子受体、p H和氧化还原电位等几个关键方面分析了盐分影响湿地甲烷排放过程的内在机制。在此基础上提出了今后需重点关注的5个方面:1)加强盐分浓度与组成对湿地甲烷产生、氧化、传输与排放影响的系统性、框架性研究;2)深入探讨盐分背景、变化幅度与速率的耦合如何影响湿地甲烷系列过程;3)不同离子组成及其交互效应如何影响湿地甲烷动态过程;4)结合生物学、基因组学及同位素技术等,加强湿地产甲烷菌与甲烷氧化菌与盐分的关系及其响应研究;5)湿地甲烷对盐分变化响应的时空分异规律。     

Bacterial Processes of the Methane Cycle in Bottom Sediments of Lake Baikal

The activity of methanogenic and methanotrophic bacteria was evaluated in bottom sediments of Lake Baikal. Methane concentration in Baikal bottom sediments varied from 0.0053 to 81.7 ml/dm³. Bacterial methane was produced at rates of 0.0004–534.7 l CH₄/(dm³ day) and oxidized at rates of 0.005–1180 l CH₄/(dm³ day). Peak methane production and oxidation were observed in Frolikha Bay near a methane vent. Methane was emitted into water at rates of 49.2–4340 l CH₄/(m² day). Rates of bacterial methane oxidation in near-bottom water layers ranged from 0.002 to 1.78 l/(l day). Methanogens and methanotrophs were found to play an important role in the carbon cycle through all layers of sediments, particularly in the areas of methane vent and gas-hydrate occurrence.     

The geochemistry of methane in Lake Fryxell,an amictic,permanently ice-covered,antarctic lake

The abundance and distribution of dissolved CH₄ were determined from 1987–1990 in Lake Fryxell, Antarctica, an amictic, permanently ice-covered lake in which solute movement is controlled by diffusion. CH₄ concentrations were < 1 υM in the upper oxic waters, but increased below the oxycline to 936 μM at 18 m. Sediment CH₄ was 1100 μmol (1 sed)⁻¹ in the 0–5 cm zone. Upward flux from the sediment was the source of the CH₄, NH₄ ⁺, and DOC in the water column; CH₄ was 27% of the DOC+CH₄ carbon at 18 m. Incubations with surficial sediments indicated that H¹⁴CO₃ ⁻ reduction was 0.4 μmol (1 sed)⁻¹ day⁻¹ or 4× the rate of acetate fermentation to CH₄. There was no measurable CH₄ production in the water column. However, depth profiles of CH₄, NH₄, and DIC normalized to bottom water concentrations demonstrated that a significant CH₄ sink was evident in the anoxic, sulfate-containing zone of the water column (10–18 m). The δ¹³CH₄ in this zone decreased from −72 % at 18 m to −76% at 12 m, indicating that the consumption mechanism did not result in an isotopic enrichment of ¹³CH₄. In contrast, δ¹³CH₄ increased to −55 % at 9 m due to aerobic oxidation, though this was a minor aspect of the CH₄ cycle. The water column CH₄ profile was modeled by coupling diffusive flux with a first order consumption term; the best-fit rate constant for anaerobic CH₄ consumption was 0.012 yr⁻¹. On a total carbon basis, CH₄ consumption in the anoxic water column exerted a major effect on the flux of carbonaceous material from the underlying sediments and serves to exemplify the importance of CH₄ to carbon cycling in Lake Fryxell.     

Oxidation of methane in boreal forest soils: a comparison of seven measures

Methane oxidation rates were measured in boreal forest soils using seven techniques that provide a range of information on soil CH₄ oxidation. These include: (a) short-term static chamber experiments with a free-air (1.7 ppm CH₄) headspace, (b) estimating CH₄ oxidation rates from soil CH₄ distributions and (c)²²²Rn-calibrated flux measurements, (d) day-long static chamber experiments with free-air and amended (+20 to 2000 PPM CH₄) headspaces, (e) jar experiments on soil core sections using free-air and (f) amended (+500 ppm CH₄) headspaces, and (g) jar experiments on core sections involving tracer additions of¹⁴CH₄. Short-term unamended chamber measurements,²²²Rn-calibrated flux measurements, and soil CH₄ distributions show independently that the soils are capable of oxidizing atmospheric CH₄ at rates ranging to < 2 mg m^–2 d^–1. Jar experiments with free-air headspaces and soil CH₄ profiles show that CH₄ oxidation occurs to a soil depth of 60 cm and is maximum in the 10 to 20 cm zone. Jar experiments and chamber measurements with free-air headspaces show that CH₄ oxidation occurs at low (< 0.9 ppm) thresholds. The¹⁴CH₄-amended jar experiments show the distribution of end products of CH₄ oxidation; 60% is transformed to CO₂ and the remainder is incorporated in biomass. Chamber and jar experiments under amended atmospheres show that these soils have a high capacity for CH₄ oxidation and indicate potential CH₄ oxidation rates as high as 867 mg m^–2 d^–1. Methane oxidation in moist soils modulates CH₄ emission and can serve as a negative feedback on atmospheric CH₄ increases.     

Methane consumption in two temperate forest soils

Forest soils are thought to be an important sink for atmospheric methane. To evaluate methane consumption,¹⁴C-labeled methane was added to the headspace of intact soil cores collected from a mixed mesophytic forest and from a red spruce forest located in the central Appalachian Mountains. Both soils consumed the added methane at initially high rates that decreased as the methane mixing ratio of the air decreased. The mixed mesophytic forest soil consumed an average of 2 mg CH₄ m^–2 d^–1 versus 1 mg CH, m^–2 d^–1 for the spruce forest soil. The addition of acetylene to the headspace completely suppressed methane consumption by the soils, suggesting that an aerobic methane-consuming microorganism mediated the process. At both forest sites, methane mixing ratios in soil air spaces were greater than that in the air overlying the soil surface, indicating that these soils had the ability to produce methane. Models of methane emission from forest soils to the atmosphere must represent methane flux as the balance between production and consumption of methane, which are controlled by very different factors     

Soil environmental variables affecting the flux of methane from a range of forest, moorland and agricultural soils

Measurements of the net methane exchange over a range of forest, moorland, and agricultural soils in Scotland were made during the period April to June 1994 and 1995. Fluxes of CH₄ ranged from oxidation –12.3 to an emission of 6.8 ng m^–2 s^–1. The balance between CH₄ oxidation and emission depended on the physical conditions of the soil, primarily soil moisture. The largest oxidation rates were found in the mineral forest soils, and CH₄ emission was observed in several peat soils. The smallest oxidation rate was observed in an agricultural soil. The relationship between CH₄ flux and soil moisture observed in peats (Flux_{CH 4} = 0.023 × %H₂O (dry weight) – 7.44, p > 0.05) was such that CH₄ oxidation was observed at soil moistures less than 325%( ± 80%). CH₄ emission was found at soil moistures exceeding this value. A large range of CH₄ oxidation rates were observed over a small soil moisture range in the mineral soils. CH₄ oxidation in mineral soils was negatively correlated with soil bulk density (Flux_{CH 4} = –37.35 × bulk density (g cm^–3) + 48.83, p > 0.05). Increased nitrogen loading of the soil due to N fixation, atmospheric deposition of N, and fertilisation, were consistently associated with decreases in the soil sink for CH₄, typically in the range 50 to 80%, on a range of soil types and land uses.     

CH4 emission from a hollow-ridge complex in a raised bog: The role of CH4 production and oxidation

The aim of this study was to correlate magnitude andcontrols of CH₄ fluxes with the microtopographyand the vegetation in a hollow-ridge complex of araised bog. High CH₄ emission rates were measuredfrom hollows and mud-bottom hollows, while hummocksconsumed atmospheric CH₄ at a low rate. Thehighest emissions were measured from plots with Eriophorum vaginatum and Scheuchzeriapalustris. CH₄ emission ceased after Scheuchzeria had been clipped below the water table,indicating the importance of this aerenchymatic plantas a conduit for CH₄.Peat in the upper catotelm of hollows was younger andless decomposed than in hummocks. Potential CH₄production in vitro was higher and themethanogenic association was better adapted to highertemperatures in hollow than in hummock peat. Highertemperatures in hollows resulted in a strongerCH₄ source in hollows than in hummocks. Negativefluxes from hummocks indicated that even in wetlandsmethanotrophic bacteria exist that are able to oxidizeCH₄ at atmospheric mixing ratios, and thatoxidation controls CH₄ emission completely. TheCH₄ mixing ratio was low in the acrotelm, but itincreased within the catotelm. Comparing fluxesmeasured in static chambers with fluxes calculatedfrom the porewater CH₄ profiles it was deducedthat the zone of methane oxidation was located closeto the water table.In hollows, CH₄ production at in situtemperature was far higher than emission into theatmosphere, corresponding to an oxidation rate ofnearly 99%. The CH₄ flux between the catotelmand the acrotelm of hollows was also higher than theemission, indicating the importance of CH₄oxidation in the aerobic acrotelm, too. CH₄microprofiles showed that CH₄ oxidation inmud-bottom hollows was confined to the topmost 2 mm,and that in Sphagnum-covered hollows CH₄oxidation occurred at the lower edge of green Sphagnum-parts.