厌氧条件下Shewanella oneidensis MR-1对2,4-二硝基甲苯的还原转化

【目的】研究Shewanella oneidensis MR-1厌氧生物转化2,4-二硝基甲苯(2,4-DNT)的能力、转化过程和影响因素。【方法】以乳酸钠为电子供体, 2,4-DNT为电子受体, S. oneidensis MR-1为降解菌, 黄素为胞外电子载体, 设立四个不同的对照体系并监测各体系在转化过程中2,4-DNT及其产物的动态变化。同时研究不同2,4-DNT浓度下细胞的生长情况, 以及不同黄素浓度下2,4-DNT的降解情况。【结果】S. oneidensis MR-1菌能够高效还原转化2,4-DNT为4-氨基-2-硝基甲苯(4A2NT)和2-氨基-4-硝基甲苯(2A4NT), 并将其进一步还原为2,4-二氨基甲苯(2,4-DAT), 黄素能加速转化过程。【结论】S. oneidensis MR-1菌具备高效还原转化2,4-DNT的能力, 为实际环境中硝基苯污染的原位修复提供科学依据。     

微生物胞外呼吸电子传递机制研究进展

胞外呼吸是近年来发现的新型微生物厌氧能量代谢方式,主要包括铁呼吸、腐殖质呼吸与产电呼吸3种形式。微生物胞外呼吸与传统的有氧呼吸、胞内厌氧呼吸存在显著差异。其电子受体多以固态形式存在于胞外;氧化产生的电子必须通过电子传递链从胞内转移到细胞周质和外膜,并通过外膜上的细胞色素c、纳米导线或自身产生的电子穿梭体等方式,最终将电子传递至胞外的末端受体。胞外呼吸的本质问题是微生物与胞外电子受体(铁/锰氧化物、固态电极或腐殖质等)的相互作用,即微生物如何将胞内电子传递至胞外受体。胞外呼吸的研究丰富了人们对微生物呼吸多样性的认识,同时在污染物原位修复及清洁生物能源提取方面具有重要应用前景,是当前研究的热点问题。总结了胞外呼吸类型和胞外呼吸菌的多样性,重点阐述了胞外呼吸的电子传递过程,并提出了其应用前景及今后的研究方向。     

金属还原菌希瓦氏菌厌氧呼吸能力及其在环境修复中的研究进展

Shewanella oneidensis MR-1是一种模式金属还原菌,它能够在厌氧条件下,将多种金属化合物和人工合成染料等作为电子受体还原代谢。因此,该菌常常被用于生态修复等研究。厌氧条件下,S.oneidensis MR-1能够将细胞质内或细胞内膜产生的电子通过定位于细胞内膜、细胞膜周质和细胞外膜上的c-血红色素蛋白或还原酶所组成的具有多样性的电子传递系统,最终传递到存在于细菌细胞外环境中的电子受体。通过对多种电子传递过程的介绍,进一步阐明其对污染物修复和纳米材料合成的机理,从而为未来对该类微生物的利用和开发提供更为充分的理论依据。     

微生物厌氧呼吸与有机污染水体沉积物修复

水体沉积物有机污染是当前全球关注的重要环境问题。微生物具有呼吸和代谢多样性,能以多种污染物作为厌氧呼吸的电子供体或受体,与周围环境中的生物和非生物因素组成代谢网络耦合有机污染物降解转化,是有机污染水体沉积物修复的重要驱动者。本文重点综述了微生物厌氧呼吸、电子传递网络及其对有机污染水体沉积物的修复机制研究进展,并对有机污染水体沉积物微生物修复理论和技术研究的问题和挑战进行了探讨。     

促进电活性微生物产电呼吸能力的方法研究进展

产电呼吸是指电活性微生物(electroactive microorganisms,EAMs)以胞外固体电极作为电子受体的一种呼吸形式,在可再生能源利用和环境修复方面具有广阔的应用前景。能否进一步提高EAMs的产电呼吸能力是相关技术能否从实验室走向实际应用的核心,而提高产电呼吸能力的关键是加强EAMs与胞外固体电极间的电子传递能力。目前总结如何促进EAMs产电呼吸能力的综述文献极少。因此,本文从投加化学试剂、施加物理作用及改造生物基因3个方面总结了现有的促进EAMs产电呼吸能力的方法,介绍了每种方法的优势与缺陷,重点阐述了每种手段的作用机理及促进效果,并从实际应用和机理研究的角度展望了今后的研究方向。     

一株海洋产电菌Shewanella sp. S2的筛选和产电分析

以厦门白城海域的潮间带表面沉积物为菌种来源筛选得到一株具有电催化活性的菌株S2,该菌株的16S rRNA和gyrB基因发育树与Shewanella oneidensis MR-1同支,相似性分别为98.5%和87%,葡萄糖、木糖、半乳糖等碳源利用及最佳生长的NaCl浓度与S.oneidensis MR-1有显著差别,因此初步鉴定为Shewanella属菌株,命名为Shewanella sp.S2。初步研究了菌株S2产电活性,在以乳酸作为碳源产电时,电压最高为150mV,相应的电流密度为66.1mA/m2。     

双氯芬酸对烟草BY-2细胞呼吸代谢的影响

双氯芬酸是一种新的全球性环境污染物,严重影响植物的生长发育,但其作用机制至今尚不清楚。本研究从双氯芬酸对烟草BY-2细胞呼吸代谢和活性氧代谢的影响入手,探讨双氯芬酸抑制植物细胞生长的作用机理。结果表明,双氯芬酸处理1 d后,显著抑制了烟草BY-2细胞的生长,引起细胞内活性氧(ROS)的爆发和积累,并导致烟草BY-2细胞死亡。双氯芬酸对烟草BY-2细胞的糖酵解途径(EMP)、三羧酸循环(TCA)和戊糖磷酸途径(PPP)三个碳代谢途径以及细胞色素氧化酶(COX)和交替氧化酶(AOX)参与的两条呼吸电子传递途径均有即时抑制作用。双氯芬酸可能通过抑制细胞线粒体呼吸电子传递链的活性,导致电子从呼吸链泄漏加速ROS的形成,并反馈抑制呼吸碳代谢途径,进而导致细胞内物质代谢和能量代谢紊乱,这些是双氯芬酸抑制烟草BY-2细胞生长、导致细胞死亡的重要原因。     

酮古龙酸菌与呼吸链偶联的2-KGA代谢途径研究进展

酮古龙酸菌可将底物L-山梨糖转化为维生素C的前体2-酮基-L-古龙酸(2-KGA)。该菌共存在5种反应参与2-KGA代谢,包括:1D-山梨醇氧化为L-山梨糖;2L-山梨糖氧化为L-山梨酮;3L-山梨酮(吡喃型)氧化为2-KGA;4L-山梨酮(呋喃型)氧化为维生素C。52-KGA还原为L-艾杜糖酸。其中L-山梨糖/L-山梨酮脱氢酶(SSDH)参与反应123,L-山梨糖脱氢酶(SDH)参与反应23,L-山梨酮脱氢酶(SNDH)参与反应34,醛脱氢酶(ALDH)参与反应3,2-KGA还原酶(2-KGR)参与反应5。SDH/SSDH/ALDH属于Ⅰ型醌酶,其辅酶为1分子PQQ;SNDH属Ⅱ型醌酶,与PQQ、heme C共同构成quinohemoproteins,2种醌酶均分布于周质空间中与呼吸链相偶联,意味着这种膜上直接氧化过程伴随ATP产生,使得菌体可以利用环境中的底物实现快速供能。     

厌氧产氢微生物研究进展

微生物是生物制氢的核心。本文论述了通过厌氧代谢途径产氢的微生物种类及高效产氢微生物选育和应用的研究趋势, 其中重点论述了中温和嗜热厌氧产氢微生物的产氢能力、底物利用范围及代谢特性, 简述了嗜热一氧化碳营养型产氢菌的种类及代谢特点。     

微生物燃料电池中阳极产电微生物的研究进展

微生物燃料电池（MFC）是利用阳极产电微生物为催化剂降解有机废物直接将化学能转化为电能的装置。在MFC系统中,产电微生物是影响产电性能的核心要素之一。介绍了MFC中产电微生物的最新研究现状,详细讨论了产电微生物的种类、产电机理和产电能力．为产电微生物的富集、驯化、改造和多种菌种优化组合提供思路。     

Anaerobic respiration of Shewanella putrefaciens requires both chromosomal and plasmid-borne genes

Abstract Pleiotropic respiratory mutants, incapable of growth on any electron acceptor other than oxygen, were isolated from two strains of Shewanella putrefaciens (MR-1 and sp200). All anaerobic respiratory functions were restored by complementation of the mutants with specific cloned DNA fragments. Southern hybridization experiments revealed that the fragment that complements the MR-1 mutant was localized on the megaplasmids of both strains, while the fragment that complements the sp200 mutant was chromosomal. Neither of these fragments hybridized with the anaerobic regulatory genes of S. putrefaciens ( etrA ) or E. coli ( fnr ).     

一株奥奈达希瓦氏菌烈性噬菌体的分离、纯化及鉴定

【目的】从环境中分离获得希瓦氏菌烈性噬菌体,并对其性质进行研究。【方法】以4株希瓦氏菌为宿主菌,采用双层平板法从污水样品中分离得到奥奈达希瓦氏菌MR-1烈性噬菌体M1;观察噬菌斑特征;利用超速离心法浓缩M1颗粒,进一步用氯化铯密度梯度离心纯化;采用透射电子显微镜观察纯化的M1颗粒;提取M1核酸,通过核酸酶处理分析其核酸类型及结构;绘制一步生长曲线。【结果】噬菌体M1在双层平板上形成圆形的噬菌斑,清晰透明,边缘光滑,直径为2.3 mm-2.5 mm;经电镜观察,噬菌体M1头部呈二十面体,直径约为55 nm,尾长约为170 nm,尾部可收缩,属于肌尾噬菌体科(Myoviridae);通过酶切分析表明噬菌体M1核酸为线形双链DNA;一步生长曲线显示该噬菌体感染后完成一个复制循环所需要的时间约为15-20 min。【结论】噬菌体M1属肌尾噬菌体科,研究结果为后续研究病毒在地球微生物成岩过程中所起的作用提供了实验材料。     

Isolation of U(VI) reduction-deficient mutants of Shewanella putrefaciens

A U(VI) reduction-deficient mutant (Urr) screening technique was developed and combined with chemical mutagenesis procedures to identify a Urr mutant of Shewanella putrefaciens strain 200. The Urr mutant lacked the ability to grow anaerobically on U(VI) and NO(2)(-), yet retained the ability to grow anaerobically on eight other compounds as terminal electron acceptor. All 11 members of previously isolated sets of Fe(III) and Mn(IV) reduction-deficient mutants of S. putrefaciens 200 displayed Urr-positive phenotypes with the Urr screen and were capable of anaerobic growth on U(VI). This is the first reported isolation of a respiratory mutant that is unable to grow anaerobically on U(VI) as terminal electron acceptor.     

中国希瓦氏菌D14^T的厌氧腐殖质呼吸

实验证明,希瓦氏菌新种(ShewanellacinicaD14T)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/LAQDS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(Shewanellacinica)D14T可利用腐殖质进行厌氧呼吸提供了有力的佐证。而D14T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。     

Linkage of formate hydrogenlyase with anaerobic respiration in Proteus mirabilis

The linkage between the enzyme system catalysing formate hydrogenlyase and reductases involved in anaerobic respiration in intact cells of anaerobically grown Proteus mirabilis was studied. Reduction of nitrate and fumarate by molecular hydrogen or formate was possible under all growth conditions; reduction of tetrathionate and thiosulphate occurred only in cells harvested at late growth phase from a pH-regulated batch culture and not in cells harvested at early growth phase or in cells grown in pH-auxostat culture. Under all conditions, cells possessed the enzyme tetrathionate reductase. We conclude that linkage between tetrathionate reductase (catalysing also reduction of thiosulphate) and the formate hydrogenlyase chain is dependent on growth conditions. During reduction of high-potential oxidants such as fumarate, tetrathionate (when possible) or the artificial electron acceptor methylene blue by formate, there was no simultaneous H₂ evolution due to the formate hydrogenlyase reaction. H₂ production started only after complete reduction of methylene blue or fumarate, in the case of methylene blue after a lag phase without gas production. In preparations with a low fumarate reduction activity this was accompanied by an acceleration in CO₂ production. During reduction of thiosulphate (a low-potential oxidant) or of tetrathionate in the presence of benzyl viologen (a low-potential mediator) by formate, H₂ was evolved simultaneously. From this we conclude that formate hydrogenlyase is regulated by a factor that responds to the redox state of any electron acceptor couple present such that lyase activity is blocked when the acceptor couple is oxidised to too great an extent.     

Influence of respiratory substrate on the cytochrome content of Shewanella putrefaciens

Shewanella putrefaciens can use trimethylamine oxide (TMAO) as electron acceptor under anoxic conditions. The associated cytochromes induced during growth under various respiratory conditions have been separated by liquid chromatography (DEAE Sepharose CL6b) and SDS-PAGE and characterized spectrophotometrically and by redox potentiometry. Two major low potential cytochromes and at least three minor low potential cytochromes, likely to be involved in TMAO reduction, were found. No cytochrome specific for TMAO reductase was found.     

Cytochromes of the trimethylamine N-oxide anaerobic respiratory pathway of Escherichia coli

Escherichia coli grown anaerobically with trimethylamine N-oxide (TMAO) as a terminal electron acceptor develops a new cytochrome pathway in addition to the aerobic respiratory pathways which are still formed. Formate, NADH, and possibly other substrates derived from glucose, supply electrons to this pathway. Cytochromes with α-absorption peaks at about 548, 552, 554 and 557 nm are rapidly reoxidized by TMAO in a reaction which is not inhibited by 2-n-heptyl-4-hydroxyquinoneN-oxide. CuSO₄ inhibits the reoxidation by TMAO of the first two of these cytochromes. This suggests that the pathway of electron transfer leading to the reduction of TMAO is: substrates → cytochromes 548,552 → cytochromes 554,557 → trimethylamine-N-oxide reductase → TMAO. These cytochromes, but not those of the aerobic respiratory pathways, are reoxidized by the membrane-impermeant oxidant ammonium persulfate in intact cells. This suggests that the cytochromes of the TMAO reduction pathway and / or trimethylamine-N-oxide reductase are situated at the periplasmic surface of the cytoplasmic membrane of E. coli.     

Microbial Reduction of Cr (VI)-loaded Schwertmannite by Shewanella oneidensis MR-1

Microbial transformation of sulfate minerals plays an important role in controlling the behavior of heavy metals in mining areas. Here, the anaerobic reduction of Cr (VI)-loaded schwertmannite by Shewanella oneidensis MR-1 (S. oneidensis MR-1) was investigated. The release of ferrous iron (Fe(II)) to the solution demonstrated the microbial reduction of structural Fe(III) from the schwertmannite to Fe(II). The concentration of Cr in solution decreased in all treatments, indicating that no Cr was released to the solution during this bio-reduction process of schwertmannite. The incorporation of chromate into the mineral structure of schwertmannite increased the microbial stability of the mineral, retarding the formation of secondary phases during bio-reduction process. Analysis of the XRD, SEM and fourier transform infrared spectroscopy (FT-IR) results further showed that goethite formed after 3 or 7 days with a lower content (0.22% or 0.37%) of Cr in schwertmannite, while no secondary mineral was observed with a higher concentration of Cr (0.6 wt%) incorporated in schwertmannite until 22 days. These results imply that microbial reduction of Cr(VI)-loaded schwertmannite does not lead to the release of Cr to the solution, and the microbial stability of schwertmannite will be increased by the incorporation of chromate.