Influence of temperature and pH on CO2 fixation by a non-photosynthetic microbial community and its mechanism analysis

CO2是主要的温室气体,也是地球上最丰富的碳源和可再生资源,本实验通过研究温度和pH对非光合固碳微生物固碳效率的影响,以期获得非光合固碳微生物适宜的固碳条件,并通过16S rDNA序列分析,研究了不同条件对固碳微生物群落结构的影响及其与固碳效率的相关性.结果表明,好氧/厌氧固碳微生物菌群生长的最适pH均在7左右.好氧固碳微生物菌群可在较大的温度范围(10℃～40℃)内良好生长,但其固碳速率在10℃左右时最高,之后微生物固碳速率随温度上升而下降,在40℃时,微生物固碳速率开始回升.厌氧时微生物的固碳效率随温度的变化情况与好氧时相似,但总体效果要比其在好氧时低20％～25％.由DGGE图谱及戴斯系数分析可得培养温度和pH对群落结构有显著影响,不同pH和温度条件下优势菌差异显著,这可能是固碳效率变化的重要原因之一.     

非光合微生物菌群好氧固定CO2的研究

微生物固定CO2在环境、资源方面具有重要意义.然而,通常具有较高固碳能力的光合细菌和氢-氧化细菌由于需要光照/严格厌氧和供氢,限制了其在反应器或土壤中的应用.本研究通过生物技术手段从海水及其沉积物中选育到在普通好氧条件下具有固碳能力的非光合微生物菌群,并通过电子供体和无机碳源结构的优化,显著提高了其对无机碳的同化能力,好氧条件下固碳菌液的最高无机碳同化效率可达110 mgCO2 / L·d,为实现普通环境条件下的微生物固碳奠定了基础.同时,通过分子生物学手段研究了不同环境条件下固碳微生物菌群的群落结构,以期为进一步优化固碳微生物群落结构,提升固碳效率提供理论依据.结果发现在不同培养条件下,菌群的群落结构发生了很大改变,表明不同条件下的固碳优势菌属于不同的种属,但通过测序、序列比对及构建系统发育树后发现,在已测序的16个显著条带中,11个是不可培养微生物,即其只能以共生方式存在,混合培养时,固定CO2的效果可能是多种菌共同作用的结果.这意味着单一纯种微生物的固碳效率可能较低,研究与优化固碳微生物菌群的结构和配比将有利于固碳效率的提升.     

非光合固碳微生物的碳源组合优化

二氧化碳(CO_2)过量排放而引起的全球变暖是目前全球面临的重大环境问题。微生物固碳是实现二氧化碳资源利用的一种重要方式。探索无需光照的高效固碳微生物对于更广泛环境条件下的微生物固碳具有重要意义。在从全球各大海域筛选富集出非光合微生物茵群的基础上,本文构建了在不同的电子供体条件下(铵和氢气)促进非光合微生物茵群生长的最佳混合碳源组合,得到如下结果:在以NH_4~+为电子供体的条件下,优化后的碳源组合为374.24 mgC/L碳酸钠(Na_2CO_3),54.76 mgC/L碳酸氢钠(NaHCO_3)和0 mgC/LCO_2时,最佳响应值TOC为3.06 mg/L。最佳响应值TOC低于以Na_2CO_3为单一碳源时,但高于以CO_2或NaHCO_3为单一碳源时;在以H_2为电子供体条件下,使用优化后的混合碳源为0.26 mg/L Na_2CO_3、0.59mg/L NaHCO_3和71.48mL/L CO_2时,非光合微生物菌群的固碳效率可达27.62 mg/L,较以CO_2为单一碳源提高35%左右。这可能意味着有H_2条件下非光合微生物菌群中的微生物可能以羟基丙酸固碳途径为主,而且多条固碳途径均能被混合菌群利用。     

非光合固碳微生物菌群的耐盐特性及其影响因素

从海洋中分离驯化得到的非光合固碳微生物菌群(NPMC)是无需光照和供氢的化能自养微生物,若能用于贫瘠盐碱地改良,实现其在盐碱土壤中的二次固碳,对于盐碱土壤的低碳化改良具有重要的意义.本实验初步验证了NPMC的耐盐特性,以及微量元素和磷酸盐缓冲液两单因素在次高盐条件下对NPMC固碳效率的影响.并通过响应面法研究了微量元素与盐浓度对NPMC固碳效率的交互作用.结果表明,非光合固碳微生物拥有耐受高盐浓度的特性,可耐受高达100g/L以上的总盐度,因此可用于重盐碱土壤的改良.微量元素和磷酸盐缓冲液浓度的增加,都可增强NPMC的固碳效率,微量元素的促进效应高于磷酸盐缓冲液.微量元素和盐度对NPMC固碳效率的影响存在交互作用.     

三株铜绿微囊藻对外源无机碳利用的研究

本文研究了三株铜绿微囊藻在不同条件下光合放氧对外源溶解无机碳的响应。当温度从20℃上升到30℃时,三株铜绿微囊藻的光合放氧速率都显著增加。随着反应介质pH值从7．0上升到9．0,铜绿微囊藻的光合放氧逐渐增强,其最大光合放氧速率显著增大。胞外碳酸酐酶抑制剂乙酰唑胺（acetazolamide,AZ）对铜绿微囊藻的光合放氧无显著影响,而碳酸酐酶抑制剂乙氧苯丙噻唑磺胺（ethoxyzolamide,EZ）则抑制其光合放氧。通过酶动力学方程拟合,得出这三株铜绿微囊藻的表观无机碳亲和力常数K0.5（DIC）均低于50Lmol／L,显示出铜绿微囊藻对外源无机碳的亲和力较高。在20-30℃范围内,温度的变化对微囊藻无机碳亲和力无显著影响,表明环境温度不影响其对外源无机碳利用的能力。当反应介质pH值升高,铜绿微囊藻FACHB905和PCC7806的K0.5（DIC）增大,而铜绿微囊藻FACHB469的K0.5（DIC）值却减小。在加入AZ和EZ后铜绿微囊藻的K0.5（DIC）值均无显著变化。本文还比较了铜绿微囊藻与莱氏衣藻在介质pH值变化和加入碳酸酐酶抑制剂条件下光合放氧的不同响应。结果表明,莱氏衣藻的光合放氧随反应介质pH值的升高而减弱,而AZ和EZ均抑制莱氏衣藻的光合放氧。当反应介质pH值升高以及加入AZ或EZ后,莱氏衣藻的K0.5（DIC）值均减小。     

藏北高原草甸土壤固碳微生物群落特征随海拔和季节的变化

研究土壤固碳微生物丰度、群落结构、多样性差异及其影响因子对了解青藏高原土壤碳循环和固碳潜力具有重要意义。采用定量PCR(qPCR)、末端限制性片段分析(T-RFLP)、克隆文库和测序方法,研究了青藏高原草甸土壤固碳微生物丰度与群落结构随海拔和季节的变化,主要结果如下:1)随海拔升高高寒草甸土壤固碳微生物丰度显著升高,但季节变化不明显,不同类别微生物固碳基因cbbL丰度依次为:Form ICForm IABForm ID,其中Form IC类固碳微生物可达10~8拷贝数/g土壤,cbbL基因丰度与海拔、土壤含水量和铵态氮含量(NH_4~+-N)呈正相关关系,与土壤温度和pH值负相关;2)固碳微生物多样性和丰富度随海拔升高而升高,在4800m达到最大,且二者受季节影响较小,其群落结构随海拔升高而逐渐变化,主要受土壤pH值、海拔和土壤水分影响;3)Form IC类固碳微生物主要包括放线菌门和和变形菌门,其中α变形菌门是高寒草甸土壤优势固碳微生物类群。本研究有助于理解土壤微生物群落功能及其在土壤碳循环过程中的作用,为更准确评估高寒草甸土壤碳循环过程提供了科学依据。     

沉积物中菲高效降解菌群的筛选鉴定及降解特性

以河流表层沉积物为菌群来源,富集分离出一组新型好氧微生物降解菌群,并对其进行微生物群落结构鉴定和降解特性探究。采用16S rRNA基因高通量测序方法对该菌群进行微生物群落结构分析,并设置单因素实验确定最佳生长及降解条件。结果表明,菌群主要包括硝基黄杆菌(Diaphorobacter)83.35%,假单胞杆菌属(Pseudomonas)7.46%,反硝化卡斯特兰尼氏菌(Castellaniella)1.67%,水微菌属(Aquamicrobium)1.65%等;菌群可在60 h内对100 mg/L菲降解率到达95.78%,且在pH5至pH11之间生长并保持高效降解率,最适生长温度为35℃,当盐度为1%时对菌群影响较小,降解率依旧保持在97.26%;可在7 d内降解浓度为400 mg/L的菲,第7天降解率为96.36%。菌群不仅具有高效降解能力且生长条件适应广泛、抗压能力强,可为多环芳烃生物修复提供新的种质资源。     

混合电子供体促进非光合微生物菌群固碳效率的分子生态机制

前期研究已从全国各地的表层海水中分离驯化得到了能有效固碳的非光合固碳微生物菌群(Non-photosynthetic microbial community,NPMC),并发现在好氧条件下,混合电子供体(Mixture of Electron Donors,MEDs)(配比为0.46%NaNO_2、0.50%Na_2S_2O_3和1.25%Na_2S)能显著提高其固碳效率。但其与最佳H_2作为电子供体相比促进效果如何,其促进机制是什么,尚未见有报道。本文在比较了NPMC以MED和H_2为电子供体的固碳效率基础上,从NPMC的固碳基因丰度和表达活性角度分析了MED促进NPMC固碳效率的分子生态学机制。结果表明:(1) MED系统的固碳效率可以达到H_2系统的水平,并显著高于传统的NH_4~+系统的固碳效率;(2)卡尔文循环关键基因cbbL和cbbM在MED系统中的较高基因丰度和同时较高的表达可能是MED促进NPMC固碳效率的一个重要原因;(3)由于MED系统含有不同价态的N和S,有利于多种微生物生长,丰富了微生物多样性,为形成不同菌种之间的相互作用促进cbbL和cbbM的同时高表达提供了条件。     

荚膜红假单胞菌的基因转移子

紫色非硫细菌(Athiorhodaceae)可以在光照条件上厌氧生长,也可以在暗处好氧生长或厌氧生长。在光照条件下,它们可以行光合自养生长,也可以行光合异养生长。大多数光合细菌还能固氮,利用大气中的氮为生长的唯一氮源,同时能由固氮酶催化放氢。因此这类微生物在光合作用、固氮作用和生物力能学等     

细菌与古菌之间的直接电子传递

冰川占地球陆地表面的11%,储存了约10⁴ Pg有机碳。随着冰川消融有机碳被释放至下游生态系统中,刺激海洋、湖泊和径流的初级生产力进而影响其生态系统。微生物参与的固碳过程决定了冰川有机碳储量及向下游输出碳量。研究冰川固碳微生物群落构成及其生态功能,可为估算冰川碳积累量和保护下游生态系统提供数据基础。本文综述了冰川碳储量和释放量、冰川生态系统主要固碳途径、固碳微生物群落组成、固碳速率以及影响固碳速率的环境因素。最后基于研究现状展望了冰川生态系统固碳微生物的未来研究和发展方向。     

非光合固碳微生物菌群最佳组合氮源的正交实验分析

与光合微生物相比,非光合微生物固定CO2具有不用光照,可昼夜持续的优点.氮源是影响微生物固碳效率的重要因素.本研究围绕非光合固碳微生物培养中的氮源进行单因素实验,并在此基础上设计正交交互实验以确定最优的氮源组合.结果表明,实验中采用的三种无机氮对微生物固碳效率均有提升,其中( NH4)2SO4约在浓度为8g/L时达到最大值,NaNO3约在浓度为5 g/L时达到最大值而NaNO2约在浓度为5 g/L时达到最大值.根据单因素实验的结果设计L27(313)的正交交互实验表,得到不同氮源组合影响固碳效率的主次顺序:NaNO3和(NH4)2SO4是影响微生物固定CO2的主要因素,其余依次为NaNO3和(NH4)2SO4的交互作用,(NH4)2SO4和NaNO2的交互作用以及NaNO2等.同时三种氮源的最优组合为(NH4)2SO4浓度9 g/L,NaNO3浓度5 g/L和NaNO2浓度8 g/L,并得到三种因素影响的直观图.     

A microbial consortium isolated from a crude oil sample that uses asphaltenes as a carbon and energy source

A microbial consortium capable of mineralizing asphaltenes was obtained from the Maya crude oil. The enrichment system was built with a glass column reactor containing mineral medium supplied with asphaltenes as energy and carbon source. The consortium growth was evaluated in Casoy agar during 40 weeks. The steady-state phase of the enriched bacterial community was observed after 10 weeks when the culture reach 10(5) to 10(6) CFU ml(-1). The isolates belong to bacterial genus reported for degradation of other hydrocarbons and they were identified as Corynebacterium sp., Bacillus sp., Brevibacillus sp. and Staphylococcus sp. The bacterial consortium growth was evaluated by a viable counts during 14 days exposed to different aeration, temperature, salinity, and pH conditions. The ability of the consortium to mineralize asphaltenes was evaluated using the method of ISO 9439 in glass column reactors of 20 x 3.2 cm during 13 days. Temperatures of 55 degrees C and salinity of 1.8% were growth limiting. The respiration of the microbial consortium using asphaltenes as a sole carbon source (800 micromoles CO2 in 13 days) was significantly higher than those of the samples containing only the microbial consortium (200 micromoles CO2) or only asphaltenes (300 micromoles CO2). These results indicated the existence of asphaltenes-degradating microbes in the crude oil and confirmed that the consortium could mineralize asphaltenes in conditions of room temperature, salinity of 100 ppm, aeration of 1 l min(-1) and pH of 7.4.     

The response of soil microorganisms and roots to elevated CO2 and temperature in a terrestrial model ecosystem

We investigate the response of soil microorganisms to atmospheric CO2 and temperature change within model terrestrial ecosystems in the Ecotron. The model communities consisted of four plant species (Cardamine hirsuta, Poa annua, Senecio vulgaris, Spergula arvensis), four herbivorous insect species (two aphids, a leaf-miner, and a whitefly) and their parasitoids, snails, earthworms, woodlice, soil-dwelling Collembola (springtails), nematodes and soil microorganisms (bacteria, fungi, mycorrhizae and Protista). In two successive experiments, the effects of elevated temperature (ambient plus 2 °C) at both ambient and elevated CO2 conditions (ambient plus 200 ppm) were investigated. A 40:60 sand:Surrey loam mixture with relatively low nutrient levels was used. Each experiment ran for 9 months and soil microbial biomass (Cmic and Nmic), soil microbial community (fungal and bacterial phospholipid fatty acids), basal respiration, and enzymes involved in the carbon cycling (xylanase, trehalase) were measured at depths of 0–2, 0–10 and 10–20 cm. In addition, root biomass and tissue C:N ratio were determined to provide information on the amount and quality of substrates for microbial growth.Elevated temperature under both ambient and elevated CO2 did not show consistent treatment effects. Elevation of air temperature at ambient CO2 induced an increase in Cmic of the 0–10 cm layer, while at elevated CO2 total phospholipid fatty acids (PLFA) increased after the third generation. The metabolic quotient qCO2 decreased at elevated temperature in the ambient CO2 run. Xylanase and trehalase showed no changes in both runs. Root biomass and C:N ratio were not influenced by elevated temperature in ambient CO2. In elevated CO2, however, elevated temperature reduced root biomass in the 0–10 cm and 30–40 cm layers and increased N content of roots in the deeper layers. The different response of root biomass and C:N ratio to elevated temperature may be caused by differences in the dynamics of root decomposition and/or in allocation patterns to coarse or fine roots (i.e. storage vs. resource capture functions). Overall, our data suggests that in soils of low nutrient availability, the effects of climate change on the soil microbial community and processes are likely to be minimal and largely unpredicatable.     

Radioisotopic tracing of carbon monoxide conversion by anaerobic thermophilic prokaryotes

The rate of CO conversion by a pure culture of a thermophilic CO-oxidizing, H₂-producing bacterium Carboxydocella sp. strain 1503 was determined by the radioisotopic method. The overall daily uptake of ¹⁴CO by the bacterium was estimated at 38–56 μmol CO per 1 ml of the culture. A radioisotopic method was developed to separate and quantitatively determine the products of anaerobic CO conversion by microbial communities in hot springs. The new method was first tested on the microbial community from a sample obtained from a hot spring in Kamchatka. The potential rate of CO conversion by the anaerobic microbial community was found to be 40.75 nmol CO/cm³ sediment per day. 85% of the utilized ¹⁴CO was oxidized to carbon dioxide; 14.5% was incorporated into dissolved organic matter, including 0.2% that went into volatile fatty acids; 0.5% was used for cell biomass production; and only just over 0.001% was converted to methane.     

澳大利亚亚热带不同森林土壤微生物群落对碳源的利用

为阐明土壤微生物群落对碳源利用类型和强度,采用MicroRespTM方法研究3种森林类型不同土壤含水量微生物群落对不同类型碳源的利用情况,结果表明：湿地松（Pinus elliottii Engelm. var. elliotttii）林地土壤微生物碳源利用率依次为60%WHC>20%WHC>40%WHC,南洋杉（Araucaria cunninghamii）和贝壳杉（Agathis australis）林地土壤微生物对碳源利用的格局相似,为20%WHC>60%WHC>40%WHC。南洋杉和贝壳杉林地土壤对碳源利用率趋势相同,主要是对L-苹果酸、草酸和L-赖氨酸利用比较高。在40%WHC处理中,3种树种对碳源的利用均很低,差异不明显。除精氨酸和L-赖氨酸外,60%WHC处理土壤微生物利用单一碳源能力的大小顺序为：南洋杉>湿地松>贝壳杉。3种树种土壤Shannon多样性指数(H’)、Shannon均匀度(E)和Simpson指数(D)均无显著差异。土壤pH值影响微生物对L-丙氨酸、精氨酸、D-( )-葡萄糖、N-乙酰基-氨基葡萄糖的利用率较大,这些类群的微生物主要分布在贝壳杉林地;分布在南洋杉林地的微生物对柠檬酸、L-苹果酸和γ-酪氨酸利用率较大,且主要是受TP的影响;D-( )-果糖、柠檬酸和L-半胱氨酸-盐酸等受水分、TN和TC等影响较大,这类微生物类群主要分布在湿地松林地。     

16S rDNA-RFLP分析六氯苯好氧降解菌群的结构及其多样性

从某化工厂排水沟底泥中取样,经2个月的富集驯化得到六氯苯好氧降解菌群。通过测定该微生物菌群在降解六氯苯过程中累积耗氧量、微生物生长曲线及Cl-浓度的变化,证明在好氧条件下该微生物菌群能够以六氯苯为唯一碳源和能源生长。当培养温度为30℃,pH为7.0时,该菌群能在18d内将无机盐培养基中浓度为4.5mg/L的六氯苯降解55%以上,降解速率达到137.5μg/(L.d)。对降解菌群提取总DNA,选择性扩增细菌16S rDNA片段,建立克隆文库。通过限制性内切酶(限制性内切酶HaeⅢ和RsaⅠ)分析,得到9种不同的谱型,其中3种谱型是主要谱型。对主要谱型的克隆子测序,结果表明,它们分别与Alcaligenes和Azospirillum菌属相似性最高。该菌群在去除环境中难降解的有机氯污染物方面具有应用前景。     

土壤微生物群落构建理论与时空演变特征

土壤微生物作为陆地生态系统的重要组成部分,直接或间接地参与几乎所有的土壤生态过程,在物质循环、能量转换以及污染物降解等过程中都发挥着重要作用。对土壤微生物时空演变规律及其形成机制的研究,不仅是微生物演变和进化的基础科学问题,也是预测微生物及其所介导的生态功能对环境条件变化响应、适应和反馈的理论依据。讨论了土壤微生物群落的定义、测度方法和指标,认为群落是联系动植物宏观生态学与微生物生态学的基础,群落构建机制是宏观和微观生态学都需要研究的核心科学问题;从生态学的群落构建理论出发,阐述了包括生态位理论/中性理论、过程理论和多样性-稳定性理论在土壤微生物时空演变研究中的应用,以及微生物群落在时间和空间上的分布特征及其尺度效应;确立了以微生物群落构建理论为基础、不同时空尺度下土壤微生物群落演变特征为主要内容的微生物演变研究的基本框架。     

The microbial gene diversity along an elevation gradient of the Tibetan grassland

Tibet is one of the most threatened regions by climate warming, thus understanding how its microbial communities function may be of high importance for predicting microbial responses to climate changes. Here, we report a study to profile soil microbial structural genes, which infers functional roles of microbial communities, along four sites/elevations of a Tibetan mountainous grassland, aiming to explore the potential microbial responses to climate changes via a strategy of space-for-time substitution. Using a microarray-based metagenomics tool named GeoChip 4.0, we showed that microbial communities were distinct for most but not all of the sites. Substantial variations were apparent in stress, N and C-cycling genes, but they were in line with the functional roles of these genes. Cold shock genes were more abundant at higher elevations. Also, gdh converting ammonium into urea was more abundant at higher elevations, whereas ureC converting urea into ammonium was less abundant, which was consistent with soil ammonium contents. Significant correlations were observed between N-cycling genes (ureC, gdh and amoA) and nitrous oxide flux, suggesting that they contributed to community metabolism. Lastly, we found by Canonical correspondence analysis, Mantel tests and the similarity tests that soil pH, temperature, NH₄⁺–N and vegetation diversity accounted for the majority (81.4%) of microbial community variations, suggesting that these four attributes were major factors affecting soil microbial communities. On the basis of these observations, we predict that climate changes in the Tibetan grasslands are very likely to change soil microbial community functional structure, with particular impacts on microbial N-cycling genes and consequently microbe-mediated soil N dynamics.     

降解水稻秸秆的复合菌系及其微生物群落结构演替北大核心CSCD

【目的】比较和分析从堆肥中富集的水稻秸秆降解菌系F1和F2的纤维素分解能力、微生物群落结构及其在秸秆降解过程中的演替,从而探究微生物群落结构与秸秆降解效率的相关性。【方法】采用DNS(3,5-二硝基水杨酸,3,5-dinitrosalicylic acid)定糖法测定发酵液中的外切纤维素酶活;采用范氏(Van Soest)洗涤纤维分析法测定发酵前与发酵后的秸秆纤维素、半纤维素、木质素的含量,并计算降解率;采用16S r RNA基因序列分析和实时荧光定量PCR(Quantitative real-time PCR,Q-PCR)对秸秆降解过程中的微生物物种组成及特定的功能微生物进行定性和定量分析。【结果】复合菌系F1的水稻秸秆总降解率、纤维素降解率、半纤维素降解率显著高于复合菌系F2;2种复合菌系的外切纤维素酶活性与cel48基因的拷贝数变化趋势一致;复合菌系F1的物种较丰富,优势物种是好氧细菌,复合菌系F2的物种组成较单一,培养后期具有较高比例的厌氧纤维素分解菌;培养前4天,复合菌系F1和F2的优势物种均为Unclassified Bacillales和Bacillus;第4天之后,不同复合菌系的优势物种及丰度出现差异,F1的优势物种主要属于Bacteroidetes,F2的优势物种主要属于Firmicutes;虽然Petrimonas和Pusillimonas是培养后期的共有优势物种,但是Petrimonas在复合菌系F2中的相对丰度(38.30%)显著高于F1(9.47%),且培养第8天的F2中的Clostridiales OPB54增加至14.85%。【结论】cel48基因拷贝数变化与秸秆纤维素的降解效率、外切纤维素酶活性变化具有一定的相关性,cel48基因可作为潜在的生物分子标记监测秸秆纤维素的降解过程;微生物群落结构对秸秆纤维素的降解效率具有显著影响,Unclassified Bacillales,Bacillus,Petrimonas,Pusillimonas是复合菌系F1和F2降解秸秆纤维素过程中的重要物种。