中国希瓦氏菌D14T的厌氧腐殖质呼吸

实验证明,希瓦氏菌新种（Shewanella cinicaD14T）在厌氧条件下可以利用多种有机酸盐和甲苯等环境有毒污染物作为电子供体,以腐殖质作为唯一末端电子受体进行厌氧呼吸（即醌呼吸）。电子在细胞膜呼吸链的传递过程中,偶联能量的产生来支持菌体的生长,1mmol/L AQDS可支持细胞增殖约60倍。电子供体的氧化和唯一电子受体腐殖质还原之间存在着动态的偶联过程,随着电子供体量的增加腐殖质还原的量也随之增加。典型呼吸链抑制剂诸如：抑制FeS中心的Cu2+ ,甲基萘醌类似物标桩菌素,抑制甲基萘醌氧化型向还原型转化的双香豆素和细胞色素P450的专一抑制物甲吡酮等对腐殖质的还原有着极为显著的抑制作用,为进一步证明希瓦氏菌（Shewanella cinica）D14T可利用腐殖质进行厌氧呼吸提供了有力的佐证。而D14T在进行腐殖质呼吸的同时,对于甲苯,苯胺等环境有毒物质的有效降解则具有着重要的环境学意义。     

腐殖质在环境污染物生物降解中的作用研究进展*

腐殖质物质在地球的生态环境中大量存在,它不仅可以在有毒化合物的生物降解和生物转化过程中起到氧化还原中间体的作用,加速有毒物质的降解和转化。也可以作为唯一末端电子受体,接受来自一些有机酸或者甲苯等环境中有毒物质提供的电子,偶联能量的产生,支持菌体的生长,形成一种新的细菌厌氧呼吸形式——腐殖质呼吸。因此,对腐殖质在环境有毒物质的生物降解和生物转化过程中的作用进行研究,不仅对于深入理解细菌呼吸的本质具有重要的理论意义,而且对于环境有毒物质的降解和转化以及元素的生物地球化学循环具有重要的生态学意义,同时对地球表面     

腐殖质在环境污染物生物降解中的作用研究进展

腐殖质物质在地球的生态环境中大量存在,它不仅可以在有毒化合物的生物降解和生物转化过程中起到氧化还原中间体的作用,加速有毒物质的降解和转化。也可以作为唯一末端电子受体,接受来自一些有机酸或者甲苯等环境中有毒物质提供的电子,偶联能量的产生,支持菌体的生长,形成一种新的细菌厌氧呼吸形式——腐殖质呼吸。因此,对腐殖质在环境有毒物质的生物降解和生物转化过程中的作用进行研究,不仪对于深入理解细菌呼吸的本质具有重要的理论意义,而且对于环境有毒物质的降解和转化以及元素的生物地球化学循环具有重要的生态学意义,同时对地球表面的有毒物质进行更有效的生物降解具有重要的现实意义。     

腐殖质呼吸作用及其生态学意义

腐殖质呼吸是厌氧环境中普遍存在的一种微生物呼吸代谢模式.自1996年发现以来,日益成为生态学与环境科学领域的研究热点.在厌氧条件下,一些微生物能以腐殖质作为唯一电子受体,氧化环境中的有机质或者甲苯等环境有毒物质,产生CO2,参与碳循环;同时,腐殖质呼吸作用产生的还原态腐殖质可以还原环境中的一些氧化态物质,如Fe(III)、Mn(IV)、Cr(VI)、U(VI) 、硝基芳香化合物和多卤代污染物.因此,腐殖质呼吸能够影响环境中C、N、Fe、Mn以及一些痕量金属元素的生物地球化学循环,并且能够促进重金属以及有机污染物的脱毒,在水体自净、污染土壤原位修复、污水处理等方面具有积极作用.     

厌氧条件下希瓦氏菌腐殖质还原对偶氮还原的影响

以希瓦氏菌属的3个代表种为研究对象,研究了在厌氧条件下腐殖质的存在对偶氮还原的影响。实验结果表明:3个代表菌株在厌氧条件下都有高效的偶氮还原和腐殖质还原功能,1mmol/L偶氮染料在24h内完全脱色,并且偶氮还原与电子供体氧化存在着紧密的偶联关系。腐殖质物质模式物2-磺酸蒽醌AQS在小于1~2mmol/L条件下能显著加速偶氮还原,12h就完全脱色,3mmol/L时18h完全脱色。但当浓度大于3mmol/L时则对偶氮还原产生明显抑制作用。另一腐殖质模式物2,6-双磺酸蒽醌AQDS其浓度在1~3mmol/L以内亦使脱色在12h内完成,4~6mmol/L时15h左右完成脱色。7~12mmol/L仍有一定的脱色促进作用,但随着浓度的提高,其促进作用也逐渐减弱。这说明腐殖质的确可以作为氧化还原中间体穿梭于电子供体与染料的偶氮双键之间促进偶氮还原。但当其浓度达到某一阈值时它就显出与偶氮键竞争电子的本质,从而使偶氮还原速率下降。原因在于他们的氧化还原电势的差异,导致细菌呼吸链的电子递体对腐殖质物质和偶氮键的亲和力不同,从而使不同腐殖质浓度对偶氮键还原产生了不同的影响。     

一株嗜水气单胞菌HS01的偶氮还原脱色特性

[目的]研究嗜水气单胞菌HS01的偶氮染料还原脱色特性.[方法]建立HS01/偶氮染料/电子供体序批式厌氧反应体系,研究Fe(Ⅲ)/腐殖质还原菌HS01以偶氮染料为电子受体的厌氧呼吸特性及影响因素;并构建HS01/偶氮染料/电子供体/铁氧化物体系,探讨铁氧化物对HS01偶氮还原的影响.[结果]HS01可将金橙Ⅰ迅速还原,菌体增殖;柠檬酸、丙三醇、蔗糖和葡萄糖体系中,16h金橙Ⅰ的脱色率分别达87％、85％、88％、90％;不同pH和金橙Ⅰ初始浓度条件下的脱色率不同;在反应体系中加入α-FeOOH,脱色率从90％增加至95％,Fe(Ⅱ)生成量与无染料对照体系相当.[结论]HS01能以葡萄糖为电子供体,金橙Ⅰ为唯一电子受体,进行厌氧呼吸;蔗糖、柠檬酸、丙三醇也可作为有效的电子供体,脱色率依次递减;甲酸、乙酸、乳酸、乙醇及丙酸不能作为HS01厌氧呼吸的电子供体.金橙Ⅰ脱色的最佳pH范围为6.0-8.0;高浓度(2.0 mmol/L)金橙Ⅰ负荷下,HS01仍保持高脱色率(＞85％).在HS01/α-FeOOH/金橙Ⅰ体系中,异化铁还原作用与偶氮呼吸作用同时发生,异化铁还原能促进偶氮脱色,而脱色对Fe(Ⅲ)还原没有明显影响.这可为铁/腐殖质还原菌在环境修复和废水处理等领域的应用提供研究积累.     

脱色希瓦氏菌(Shewanella decolorationis) S12还原不同电子受体的厌氧发酵罐培养方法

脱色希瓦氏菌Shewanella decolorationisS12在厌氧环境下能够使用多种电子受体进行厌氧呼吸。为了取得足够的细胞量用于膜蛋白质组学等科学研究的需要,本研究选取无机小分子(硝酸钠)、金属离子(柠檬酸铁)和有机大分子(偶氮染料苋菜红)作为电子受体,在使用确定成分的无机盐培养基条件下,使用不同浓度的电子供体和碳源对S12进行厌氧条件下静置和发酵罐的优化培养,采用连续补充电子受体的培养方式,确认了电子供体和碳源的合适浓度,建立了S12厌氧发酵罐培养方法。相比传统的静置厌氧培养,厌氧发酵罐培养方法在保证了严格厌氧条件下高效率还原电子受体的同时,还极大的提高了细胞生长密度。连续补充电子受体的厌氧发酵罐培养的S12最大细胞密度最大分别可达到静置厌氧培养细胞密度的325,304,369倍,而生长时间也比静置厌氧培养分别缩短了26.5%,17.6%,7.5%。这为需要大量细胞和蛋白的细菌厌氧呼吸生长实验建立了可行方法,对于进行兼性厌氧呼吸的微生物的大规模厌氧培养具有借鉴意义。     

厌氧氨氧化菌的中心代谢研究进展

摘要: 厌氧氨氧化是以NH +4为电子供体,以NO－2为电子受体产生N2的生物反应。厌氧氨氧化菌是厌氧氨氧化过程的执行者,在废水生物脱氮和地球氮素循环中扮演着重要角色。研究厌氧氨氧化菌的代谢特性,将有助于理解厌氧氨氧化过程,开发厌氧氨氧化工艺。厌氧氨氧化菌是化能自养型细菌,以CO2或HCO－3为碳源,并通过偶联NH+4氧化和NO －2还原的生物反应获得能量。在NH+4/NO－2的生物氧化还原反应过程中,检出了中间产物N2H4,但未检出其他中间产物(如NH2OH、NO)。此外,由基因组信息推断,厌氧氨氧化菌     

奥奈达希瓦氏菌CctA介导周质甲基橙还原的电子传递机理

电活性微生物奥奈达希瓦氏菌的胞外电子传递（extracellular electron transfer,EET）在污染物降解、环境修复、生物电化学传感、能源利用等方面具有广泛的应用潜力;四血红素细胞色素CctA （small tetraheme cytochrome）是希瓦氏菌周质空间中最丰富的蛋白质之一,能够参与多种氧化还原过程,但目前对CctA在EET中的行为和机理认识仍然有限。【目的】研究阐明CctA蛋白在希瓦氏菌模式菌株MR-1周质空间以偶氮染料作为电子受体的EET中的作用,补充和拓展希瓦氏菌的厌氧呼吸产能机制。【方法】以周质还原型偶氮染料甲基橙（methyl orange,MO）作为电子受体,在mteal reduction （Mtr）蛋白缺失菌株Δmtr中研究MO的周质还原特点,并通过基因敲除和回补表达研究CctA蛋白在周质电子传递中的作用。【结果】在缺失Mtr通道的情况下,细胞色素CctA可以介导周质空间的电子传递而还原MO。重组表达CctA在低水平时,MO在周质空间中的还原速率与其表达水平呈正相关,更高水平的CctA表达无助于进一步提高MO的还原速率。蛋白膜伏安结果展示了CctA与周质空间内其他高电位氧化还原蛋白的显著区别,可能参与构成一条低电位的MO还原通道。【结论】从分子动力学层面揭示了CctA在周质MO还原中的独特电子传递行为,为进一步推进对细菌周质电子传递机制的理解,以及通过合成生物学设计或改造胞外氧化还原系统、强化生物电化学在污染物降解中的应用提供了重要信息。     

Mcc在脱色希瓦氏菌S12电极呼吸中的作用

【目的】研究脱色希瓦氏菌S12周质空间c型细胞色素Mcc的功能,进一步探索和补充微生物胞外电子传递过程的机制。【方法】借助自杀质粒敲除mcc基因,通过细胞浓度测定和激光共聚焦显微镜比较分析突变株和野生株之间的浮游细胞和生物膜的生长情况,并比较分析二者在微生物燃料电池电极还原、铁还原和胞外偶氮染料还原过程中的功能。【结果】Mcc缺失对铁还原和偶氮还原没有影响,但却造成电极呼吸活性下降34.1%;与野生株相比,mcc突变株的好氧生长和厌氧浮游细胞生长无明显影响,但却显著抑制了电极表面生物膜的形成。【结论】Mcc是希瓦氏菌S12电极呼吸过程中周质空间电子传递的重要组分之一,缺失会显著抑制其电极呼吸效率以及生物膜的形成。     

Competition between methanogenesis and quinone respiration for ecologically important substrates in anaerobic consortia

Anaerobic consortia obtained from a wide variety of environments were tested for oxidizing several ecologically significant substrates with the humic model compound, anthraquinone-2,6-disulfonate (AQDS), as terminal electron acceptor. All the substrates, including hydrogen, acetate, propionate, methanol and lactate, were completely or partially converted to methane when bicarbonate was the only electron acceptor available. Addition of AQDS (20 mM) to the cultures prevented methanogenesis in most cases and AQDS reduction became the preferred pathway. AQDS was shown to be toxic for methanogenesis and this effect played an important role in enabling quinone-respiring bacteria to outcompete methanogens. Furthermore, AQDS respiration is thermodynamically more favorable than methanogenesis. All the consortia evaluated were capable of oxidizing hydrogen linked to the reduction of AQDS. Most inocula tested were also able to oxidize acetate and lactate in the same way. When methanol was provided as an electron donor competition between methanogenesis and acetogenesis occurred. Acetate accumulated from the latter process was responsible for quinone respiration. These results suggest that quinone-respiring bacteria are ubiquitous and that quinones in humus may significantly contribute to carbon cycling process by serving as a terminal electron acceptor for the anaerobic microbial oxidation of a wide variety of ecologically important substrates.     

Contribution of quinone-reducing microorganisms to the anaerobic biodegradation of organic compounds under different redox conditions

The capacity of two anaerobic consortia to oxidize different organic compounds, including acetate, propionate, lactate, phenol and p-cresol, in the presence of nitrate, sulfate and the humic model compound, anthraquinone-2,6-disulfonate (AQDS) as terminal electron acceptors, was evaluated. Denitrification showed the highest respiratory rates in both consortia studied and occurred exclusively during the first hours of incubation for most organic substrates degraded. Reduction of AQDS and sulfate generally started after complete denitrification, or even occurred at the same time during the biodegradation of p-cresol, in anaerobic sludge incubations; whereas methanogenesis did not significantly occur during the reduction of nitrate, sulfate, and AQDS. AQDS reduction was the preferred respiratory pathway over sulfate reduction and methanogenesis during the anaerobic oxidation of most organic substrates by the anaerobic sludge studied. In contrast, sulfate reduction out-competed AQDS reduction during incubations performed with anaerobic wetland sediment, which did not achieve any methanogenic activity. Propionate was a poor electron donor to achieve AQDS reduction; however, denitrifying and sulfate-reducing activities carried out by both consortia promoted the reduction of AQDS via acetate accumulated from propionate oxidation. Our results suggest that microbial reduction of humic substances (HS) may play an important role during the anaerobic oxidation of organic pollutants in anaerobic environments despite the presence of alternative electron acceptors, such as sulfate and nitrate. Methane inhibition, imposed by the inclusion of AQDS as terminal electron acceptor, suggests that microbial reduction of HS may also have important implications on the global climate preservation, considering the green-house effects of methane.     

Alkaline extracellular reduction: isolation and characterization of an alkaliphilic and halotolerant bacterium, Bacillus pseudofirmus MC02

Aims: To isolate an alkaliphilic bacterium and to investigate its ability of extracellular reduction. Methods and Results: An alkaliphilic and halotolerant humus‐reducing anaerobe, Bacillus pseudofirmus MC02, was successfully isolated from a pH 10·0 microbial fuel cell. To examine its ability of extracellular reduction, AQDS (anthraquinone‐2, 6‐disulfonae), humic acids (HA) and Fe(III) oxides were chosen as representative electron acceptors. All the experiments were conducted in a pH 9·5 carbonate buffer. The results are as follows: (i) Sucrose, lactate, glucose and glycerol were the favourable electron donors for AQDS reduction by the strain MC02; (ii) The strain had the ability of reducing HA in the presence of sucrose; (iii) It could effectively reduce Fe(III) oxides coupled with sucrose fermentation when AQDS was added as electron shuttle and its Fe(III) reducing capacity ranked as: lepidocrocite (γ‐FeOOH) > goethite (α‐FeOOH) > haematite(α‐Fe₂O₃); (iv) The strain could decolourize azo dye Orange I. Conclusions: Bacillus pseudofirmus MC02 was capable of extracellular reduction in AQDS, HA and Fe(III) oxides, and it can be used for decolourizing azo dye (Orange I) in alkaline conditions. Significance and Impact of the Study: This is the first report of an alkaliphlic strain of B. pseudofirmus capable of extracellular reduction in AQDS, HA, Fe(III) oxides and decolourization of Orange I. This study could provide valuable information on alkaline biotransformation in the printing and dyeing wastewater and saline‐alkali soil.     

Humic analog AQDS and AQS as an electron mediator can enhance chromate reduction by Bacillus sp. strain 3C3

Humus as an electron mediator is recognized as an effective strategy to improve the biological transformation and degradation of toxic substances, yet the action of humus in microbial detoxification of chromate is still unknown. In this study, a humus-reducing strain 3C₃ was isolated from mangrove sediment. Based on the analyses of morphology, physiobiochemical characteristics, and 16S rRNA gene sequence, this strain was identified Bacillus sp. Strain 3C₃ can effectively reduce humic analog anthraquinone-2,6-disulfonate (AQDS) and anthraquinone-2-sulfonate (AQS) with lactate, formate, or glucose as electron donors. When the cells were killed by incubation at 95°C for 30 min or an electron donor was absent, the humic reduction did not occur, showing that the humic reduction was a biochemical process. However, strain 3C₃ had low capability of chromate reduction under anaerobic conditions, despite of having strong tolerance of the toxic metal. But in the presence of humic substances AQDS or AQS, we found that chromate reduction by strain 3C₃ was enhanced greatly. Because strain 3C₃ is an effective humus-reducing bacterium, it is proposed that humic substances could serve as electron mediator to interact with chromate and accelerate chromate reduction. Our results suggest that chromate contaminations can be detoxified by adding humic analog (low to 0.1 mM) as an electron mediator in the microbial incubation.     

Anaerobic mineralization of toluene by enriched sediments with quinones and humus as terminal electron acceptors

The anaerobic microbial oxidation of toluene to CO(2) coupled to humus respiration was demonstrated by use of enriched anaerobic sediments from the Amsterdam petroleum harbor (APH) and the Rhine River. Both highly purified soil humic acids (HPSHA) and the humic quinone moiety model compound anthraquinone-2,6-disulfonate (AQDS) were utilized as terminal electron acceptors. After 2 weeks of incubation, 50 and 85% of added uniformly labeled [(13)C]toluene were recovered as (13)CO(2) in HPSHA- and AQDS-supplemented APH sediment enrichment cultures, respectively; negligible recovery occurred in unsupplemented cultures. The conversion of [(13)C]toluene agreed with the high level of recovery of electrons as reduced humus or as anthrahydroquinone-2,6-disulfonate. APH sediment was also able to use nitrate and amorphous manganese dioxide as terminal electron acceptors to support the anaerobic biodegradation of toluene. The addition of substoichiometric amounts of humic acids to bioassay reaction mixtures containing amorphous ferric oxyhydroxide as a terminal electron acceptor led to more than 65% conversion of toluene (1 mM) after 11 weeks of incubation, a result which paralleled the partial recovery of electron equivalents as acid-extractable Fe(II). Negligible conversion of toluene and reduction of Fe(III) occurred in these bioassay reaction mixtures when humic acids were omitted. The present study provides clear quantitative evidence for the mineralization of an aromatic hydrocarbon by humus-respiring microorganisms. The results indicate that humic substances may significantly contribute to the intrinsic bioremediation of anaerobic sites contaminated with priority pollutants by serving as terminal electron acceptors.     

Microbially Mediated Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5- Triazine by Extracellular Electron Shuttling Compounds

The potential for humic substances to stimulate the reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was investigated. This study describes a novel approach for the remediation of RDX-contaminated environments using microbially mediated electron shuttling. Incubations without cells demonstrated that reduced AQDS transfers electrons directly to RDX, which was reduced without significant accumulation of the nitroso intermediates. Three times as much reduced AQDS (molar basis) was needed to completely reduce RDX. The rate and extent of RDX reduction differed greatly among electron shuttle/acceptor amendments for resting cell suspensions of Geobacter metallireducens and G. sulfurreducens with acetate as the sole electron donor. AQDS and purified humic substances stimulated the fastest rate of RDX reduction. The nitroso metabolites did not significantly accumulate in the presence of AQDS or humic substances. RDX reduction in the presence of poorly crystalline Fe(III) was relatively slow and metabolites transiently accumulated. However, adding humic substances or AQDS to Fe(III)-containing incubations increased the reduction rates. Cells of G. metallireducens alone reduced RDX; however, the rate of RDX reduction was slow relative to AQDS-amended incubations. These data suggest that extracellular electron shuttle-mediated RDX transformation is not organism specific but rather is catalyzed by multiple Fe(III)- and humic-reducing species. Electron shuttle-mediated RDX reduction may eventually become a rapid and effective cleanup strategy in both Fe(III)-rich and Fe(III)-poor environments.     

Microbially mediated biodegradation of hexahydro-1,3,5-trinitro-1,3,5- triazine by extracellular electron shuttling compounds

The potential for humic substances to stimulate the reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was investigated. This study describes a novel approach for the remediation of RDX-contaminated environments using microbially mediated electron shuttling. Incubations without cells demonstrated that reduced AQDS transfers electrons directly to RDX, which was reduced without significant accumulation of the nitroso intermediates. Three times as much reduced AQDS (molar basis) was needed to completely reduce RDX. The rate and extent of RDX reduction differed greatly among electron shuttle/acceptor amendments for resting cell suspensions of Geobacter metallireducens and G. sulfurreducens with acetate as the sole electron donor. AQDS and purified humic substances stimulated the fastest rate of RDX reduction. The nitroso metabolites did not significantly accumulate in the presence of AQDS or humic substances. RDX reduction in the presence of poorly crystalline Fe(III) was relatively slow and metabolites transiently accumulated. However, adding humic substances or AQDS to Fe(III)-containing incubations increased the reduction rates. Cells of G. metallireducens alone reduced RDX; however, the rate of RDX reduction was slow relative to AQDS-amended incubations. These data suggest that extracellular electron shuttle-mediated RDX transformation is not organism specific but rather is catalyzed by multiple Fe(III)- and humic-reducing species. Electron shuttle-mediated RDX reduction may eventually become a rapid and effective cleanup strategy in both Fe(III)-rich and Fe(III)-poor environments.     

Reduction of humic substances by halorespiring, sulphate-reducing and methanogenic microorganisms

Physiologically distinct anaerobic microorganisms were explored for their ability to oxidize different substrates with humic acids or the humic analogue, anthraquinone-2,6-disulphonate (AQDS), as a terminal electron acceptor. Most of the microorganisms evaluated including, for example, the halorespiring bacterium, Desulfitobacterium PCE1, the sulphate-reducing bacterium, Desulfovibrio G11 and the methanogenic archaeon, Methanospirillum hungatei JF1, could oxidize hydrogen linked to the reduction of humic acids or AQDS. Desulfitobacterium dehalogenans and Desulfitobacterium PCE1 could also convert lactate to acetate linked to the reduction of humic substances. Humus served as a terminal electron acceptor supporting growth of Desulfitobacterium species, which may explain the recovery of these microorganisms from organic rich environments in which the presence of chlorinated pollutants or sulphite is not expected. The results suggest that the ubiquity of humus reduction found in many different environments may be as a result of the increasing number of anaerobic microorganisms, which are known to be able to reduce humic substances.     

Reduction of Fe(III) oxide by methanogens in the presence and absence of extracellular quinones

Five methanogens (Methanosarcina barkeri MS, Methanosphaera cuniculi 1R7, Methanobacterium palustre F, Methanococcus voltaei A3 and Methanolobus vulcani PL-12/M) were investigated for their ability to reduce Fe(III) oxide and the soluble quinone anthraquinone-2,6-disulphonate (AQDS). Two species (M. barkeri and M. voltaei) reduced significant amounts of Fe(III) oxide using hydrogen as the electron donor, and 0.1 mM AQDS greatly accelerated Fe(III) reduction by these organisms. Although Fe(III) appeared to inhibit growth and methanogenesis of some strains, hydrogen partial pressures under donor-limited conditions were much lower (<0.5 Pa) in the presence of Fe(III) than in normal media (1-10 Pa) for all species except for M. vulcani. These results demonstrate that electrons were transferred to Fe(III) by hydrogen-utilizing methanogens even when growth and methanogenesis were inhibited. All species except the obligate methylotroph M. vulcani were able to reduce AQDS when their growth substrates were present as electron donors, and rates were highest when organisms used hydrogen as the electron donor. Purified soil humic acids could also be reduced by the AQDS-reducing methanogens. The ability of methanogens to interact with extracellular quinones, humic acids and Fe(III) oxides raises the possibility that this functional group of organ-isms contributes to Fe(III) and humic acid reduction under certain conditions in the environment and provides an alternative explanation for the inhibition of methanogenesis in some Fe(III)-containing ecosystems.