Geobacter应用于重金属污染生物修复的研究进展

随着现代工农业的发展,环境中的重金属污染问题日益突出。传统的重金属去除方法价格昂贵,还会产生有毒副产物,对环境产生负面影响。生物修复技术因其去除效率高、成本低以及可用性高逐渐成为了治理重金属污染的常用手段。对于重金属等不易降解的有毒污染物,细菌生物修复是最安全的处理方法,其中地杆菌(Geobacter)作为地下电极菌群中的关键物种,在生物修复中表现出极高的应用价值。从Geobacter的分类、重金属移除机制及细菌内部解毒机制3个方面,深入阐述了其在重金属修复中的应用价值与发展前景。

阳极电势对Geobacter sulfurreducens产电性能的影响

以产电模式菌Geobacter sulfurreducens为研究对象接种两瓶型微生物燃料电池(MFC)阳极室,利用恒电位仪控制阳极电势,考察了7种电势条件下MFC的启动期、最大功率密度和阳极生物量的变化情况.研究结果表明,当阳极电势为-250,-100和50 mV(vs.SCE)时,MFC启动较快,CV曲线和极化曲线表明,在这3种电势条件下,MFC产电性能增强,其中阳极电势为-100 mV时,MFC最大功率密度为1.67 W/m3,比固定外阻条件下启动的MFC最大功率密度提高了5倍.控制合适的阳极电势可以使阳极生物量提高2.5～3倍.

动态磁场对Geobacter sulfurreducens产电特性的影响

以Geobacter sulfurreducens为产电菌构建双室微生物燃料电池(MFC)。产电菌液分别在0、100、200mT的垂直磁场下动态处理1h,然后接种到MFC1、MFC2和MFC3中,研究动态磁场处理对MFC产电性能的影响。实验结果表明:动态磁场处理使产电菌反应器的启动时间延长、稳定电压降低、表观内阻增大,MFC1、MFC2和MFC3的表观内阻依次为329、507、353Ω;通过电化学阻抗谱测试可知,相比对照组MFC活化内阻,经磁场处理的产电菌MFC全电池的活化内阻变大,其中MFC1、MFC2和MFC3的活化内阻依次为12.34、28.29、16.87Ω;循环伏安测试发现经过动态磁场处理的产电菌其电化学活性降低。

Inocula selection in microbial fuel cells based on anodic biofilm abundance of Geobacter sulfurreducens

Microbial fuel cells （MFCs） rely on microbial conversion of organic substrates to electricity. The optimal perfor- mance depends on the establishment of a microbial community rich in electrogenic bacteria. Usually this micro- bial community is established from inoculation of the MFC anode chamber with naturally occurring mixed inocula. In this study, the electrochemical performance of MFCs and microbial community evolution were eval- uated for three inocula including domestic wastewater （DW）, lake sediment （LS） and biogas sludge （BS） with varying substrate loading （Lsub） and external resistance （Rext） on the MFC. The electrogenic bacterium Geobacter sulfurreducens was identified in all inocula and its abundance during MFC operation was positively linked to the MFC performance. The IS inoculated MFCs showed highest abundance （18% ± 1%） of G. sulfurreducens, maximum current density [Imax = （690 ± 30） mA.m 2] and coulombic efficiency （CE = 29% ±1%） with acetate as the substrate./max and CE increased to （1780 ± 30） mA.m-2 and 58%± 1%, respectively, after decreasing the Rext from 1000 Ωto 200 Ω, which also correlated to a higher abundance ofG. sulfurreducens （21% ±0.7%） on the MFC anodic biofilm. The data obtained contribute to understanding the microbial community response to Lsub and Roy, for of timizing electricity eneration in MFCs.

汞胁迫对Geobacter sulfurreducens PCA汞甲基化作用的代谢组影响

采用新兴的代谢组学技术,筛选差异性细胞代谢物,分析甲基汞生成和调控的关键代谢通路。在典型环境污染浓度(0~100μg/L) Hg(Ⅱ)的胁迫下,汞甲基化微生物Geobacter sulfurreducens PCA吸附/吸收的Hg(Ⅱ)成为参与汞还原和甲基化的主要反应物质。初始Hg(Ⅱ)浓度为10μg/L时, G. sulfurreducens PCA达到最高汞甲基化效率3.09%±0.16%。代谢组学数据显示,Hg(Ⅱ)胁迫对胞内的碳水化合物代谢、核苷酸代谢和氨基酸代谢造成干扰。为了应对Hg(Ⅱ)胁迫, G. sulfurreducens PCA增大了对能量的需求,用来调控汞生物甲基化反应,并对受损DNA进行修复。

Geobacter sulfurreducens promoted the biosynthesis of reduced graphene oxide and coupled it for nitrobenzene reduction

In order to explore an efficient and green method to deal with nitrobenzene(NB)pollutant,reduced graphene oxide(r GO)as an electron shuttle was applied to enhance the extracellular electron transfer(EET)process of Geobacter sulfurreducens,which was a typical electrochemically active bacteria(EAB).In this study,r GO biosynthesis was achieved via the reduction of graphene oxide(GO)by G.sulfurreducens PCA within 3 days.Also,the r GOPCA combining system completely reduced 50-200μmol/L of NB to aniline as end product within one day.SEM characterization revealed that PCA cells were partly wrapped by rGO,and therefore the distance of electron transfer between strain PCA and r GO material was reduced.Beside,the ID/IGof GO,r GO,and r GO-PCA combining system were 0.990,1.293 and 1.31,respectively.Moreover,highest currents were observed in r GO-PCA-NB as 12.950μA/-12.560μA at -408 m V/156 m V,attributing to the faster electron transfer efficiency in EET process.Therefore,the NB reduction was mainly due to:(I)direct EET process from G.sulfurreducens PCA to NB;(II)r GO served as electron shuttle and accelerated electron transfer to NB,which was the main degradation pathway.Overall,the biosynthesis of r GO via GO reduction by Geobacter promoted the NB removal process,which provided a facile strategy to alleviate the problematic nitroaromatic pollution in the environment.

Effect of exogenous flavins on the microbial corrosion by Geobacter sulfurreducens via iron-to-microbe electron transfer

Microbes can cause or accelerate metal corrosion,leading to huge losses in corrosion damages each year.Geobacter sulfurreducens is a representative electroactive bacterium in many soils,sediments,and wastew-ater systems.It has been confirmed to directly extract electrons from elemental metals.However,little is known about the effect of electron shuttles in G.sulfurreducens corrosion on stainless steel.In this study,we report that exogenous flavins promote iron-to-microbe electron transfer,accelerating micro-bial corrosion.G.sulfurreducens caused 1.3 times deeper pits and increased electron uptake(with 2 times increase of i_(corr))from stainless steel when riboflavin was added to the culture medium.OmcS-deficient mutant data suggest that G.sulfurreducens utilizes riboflavin as a bound-cofactor in outer membrane c-type cytochromes.The finding that,in the presence of microbes,riboflavin can substantially accelerate corrosion highlights the role of flavin redox cycling for enhanced iron-to-microbe electron transfer by G.sulfurreducens and provides new insights in microbial corrosion.

Geobacter sulfurreducens对汞的甲基化及其影响因素研究

在实验室模拟条件下,研究了铁还原菌Geobacter sulfurreducens对汞的甲基化作用及其影响因素.结果表明,G.sulfurreducens在低浓度汞溶液中能够生长,但生长受到一定程度的抑制,主要表现在菌株生长曲线迟缓期的延长.G.sulfurreducens在生长过程中能同时将溶液中无机汞转化为甲基汞,甲基化过程受多种环境因素的制约.在初始HgCl2浓度为1 mg.L-1、温度35℃、pH 6.0、0.9%盐度的厌氧环境条件下,G.sulfurreducens对汞的甲基化率可达38%.适当增加HgCl2初始浓度与盐度能提高甲基汞的转化率,但过高汞浓度和盐度会造成微生物的死亡;温度在4～35℃范围内,温度越高甲基汞转化率越高;弱酸性环境比酸性或中碱性环境更利于汞的甲基化.此外,腐殖酸和半胱氨酸等均影响甲基汞的转化效率,其中腐殖酸对汞甲基化有一定的抑制作用,而半胱氨酸有较强的促进作用.该研究为自然水体生态系统中铁还原菌参与汞甲基化的过程提供了直接证据.

微生物纳米导线的结构与功能:争议及进展

土壤胞外呼吸是驱动元素生物地球化学循环的引擎,而微生物纳米导线是实现土壤胞外呼吸的重要途径。微生物纳米导线是一类生长于微生物表面,可长达数十微米的具有导电性的纤维状结构。它直接作用于微生物与土壤矿物、产甲烷与甲烷氧化微生物间的电子传递,从而影响了土壤矿物的迁移转化及温室气体减排。Geobacter sulfurreducens是研究微生物纳米导线的模式微生物。长久以来,基于分子生物学实验证据表明,G.sulfurreducens纳米导线是PilA-N菌毛。而最近基于冷冻电镜技术的纳米导线结构分析发现,G.sulfurreducens实际上表达着各种形式的细胞色素c纳米导线。自此,关于“纳米导线本质”的问题成为学术界争论的焦点。以G.sulfurreducens纳米导线理论发展为主线,综述了不同时期对纳米导线结构与功能的认识,并系统分析了作为“纳米导线本质”争议的证据基础,将推动该争议的早日解决,并助力土壤胞外呼吸理论的成熟及微生物纳米导线的应用研究。

Maghemite(γ-Fe_2O_3) nanoparticles enhance dissimilatory ferrihydrite reduction by Geobacter sulfurreducens: Impacts on iron mineralogical change and bacterial interactions

Microbially mediated bioreduction of iron oxyhydroxide plays an important role in the biogeochemical cycle of iron.Geobacter sulfurreducens is a representative dissimilatory ironreducing bacterium that assembles electrically conductive pili and cytochromes.The impact of supplementation withγ-Fe_2O_3 nanoparticles(NPs)(0.2 and 0.6 g)on the G.sulfurreducens-mediated reduction of ferrihydrite was investigated.In the overall performance of microbial ferrihydrite reduction mediated byγ-Fe_2O_3 NPs,stronger reduction was observed in the presence of direct contact withγ-Fe_2O_3 NPs than with indirect contact.Compared to the production of Fe(Ⅱ)derived from biotic modification with ferrihydrite alone,increases greater than 1.6-and 1.4-fold in the production of Fe(Ⅱ)were detected in the biotic modifications in which direct contact with 0.2 g and 0.6 gγ-Fe_2O_3 NPs,respectively,occurred.X-ray diffraction analysis indicated that magnetite was a unique representative iron mineral in ferrihydrite when active G.sulfurreducens cells were in direct contact withγ-Fe_2O_3 NPs.Because of the sorption of biogenic Fe(Ⅱ)ontoγ-Fe_2O_3 NPs instead of ferrihydrite,the addition ofγ-Fe_2O_3 NPs could also contribute to increased duration of ferrihydrite reduction by preventing ferrihydrite surface passivation.Additionally,electron microscopy analysis confirmed that the direct addition ofγ-Fe_2O_3 NPs stimulated the electrically conductive pili and cytochromes to stretch,facilitating long-range electron transfer between the cells and ferrihydrite.The obtained findings provide a more comprehensive understanding of the effects of iron oxide NPs on soil biogeochemistry.

Electromicrobiology and biotechnological applications of the exoelectrogens Geobacter and Shewanella spp.

Electromicrobiology is a sub-discipline of microbiology that investigates electrical interplay between microorganisms and redox active materials, such as electrodes and solid-phase minerals, and the mechanisms underlying microbial ability to exchange electrons with the redox active materials that are external to the microbial cells. The microorganisms with extracellular electron transfer capability are often referred to as exoelectrogens. Although exoelectrogens were documented in early 1900’s, discovery of the dissimilatory metal-reducing microorganisms Geobacter and Shewanella spp. in late 1980’s marked the beginning of modern electromicrobiology. Since then, thorough and rigorous studies have made Geobacter and Shewanella spp. the two best characterized groups of exoelectrogens. These include identification and characterization of the molecular mechanisms for exchanging electrons with electrodes by Geobacter sulfurreducens and Shewanella oneidensis. In addition, a variety of applications of Geobacter and Shewanella spp. in microbial fuel cells and electrobiosynthesis, such as maintenance of redox balance during fermentations and bioremediations, have also been developed. This review briefly discusses the molecular mechanisms by which G. sulfurreducens and S. oneidensis exchange electrons with electrodes and then focuses on biotechnological applications of Geobacter and Shewanella spp. in microbial fuel cells and electrobiosynthesis as well as the future directions of this research area.

Reductive degradation of chloramphenicol by Geobacter metallireducens

Geobacter metallireducens is known to be capable of removing nitroaromatic compounds via an oxidation mode. However, little attention has been paid to investigate the reductive removal of chlorinated nitroaromatic compounds by G. metallireducens. In this study, G. metallireducens was used to reduce chloramphenicol(CAP), a typical chlorinated nitroaromatic antibiotic. Cyclic voltammograms and chronoamperometry highlighted a higher peak current for CAP reduction by G. metallireducens compared to the control without bacteria. G. metallireducens efficiently reduced CAP(20 mg/L) with acetate as the sole electron donor, and the removal efficiency reached(97.6±4.9)% within 6 d. Aromatic amine(AMCl2), AMCl(dechlorinated AMCl2) and AM(dechlorinated AMCl) were identified as reduction products by liquid chromatography-mass spectrometry. However, the removal efficiency declined to(25.0±3.6)% when the CAP dosage increased to 80 mg/L. Transcriptomic analysis indicated the significant upregulation of genes related to electron transfer, such as pilus assembly protein gene(2.8 folds), NADH-quinone oxidoreductase subunit K2 gene(4.5 folds) and many c-type cytochrome genes such as cytochrome c biogenesis protein Res B(Gmet 2901, 4.6 folds), cytochrome c(Gmet 0335, 4.4 folds) and cytochrome c7(Gmet 2902, 3.4 folds). Furthermore, a gene related to chlorinated contaminant removal(Gmet 1046, 5.4 folds) was also upregulated, possibly resulting in enhanced CAP reduction. This work deepened our knowledge of the bioremediation ability of G. metallireducens with respect to environmental contaminants and provided a potential strategy to treat antibiotics with electrochemically active bacteria.

Comparative transcriptomic insights into the mechanisms of electron transfer in Geobacter co-cultures with activated carbon and magnetite

Both activated carbon and magnetite have been reported to promote the syntrophic growth of Geobacter metallireducens and Geobacter sulfurreducens co-cultures, the first model to show direct interspecies electron transfer (DIET); however, differential transcriptomics of the promotion on co-cultures with these two conductive materials are unknown. Here, the comparative transcriptomic analysis of G. metallireducens and G. sulfurreducens co-cultures with granular activated carbon (GAC) and magnetite was reported. More than 2.6-fold reduced transcript abundances were determined for the uptake hydrogenase genes of G. sulfurreducens as well as other hydrogenases in those co-cultures to which conductive materials had been added. This is consistent with electron transfer in G. metallireducens-G. sulfurreducens co-cultures as evinced by direct interspecies electron transfer (DIET). Transcript abundance for the structural component of electrically conductive pili (e-pili), PilA, was 2.2-fold higher in G. metallireducens, and, in contrast, was 14.9-fold lower in G. sulfurreducens in co-cultures with GAC than in Geobacters co-cultures without GAC. However, it was 9.3-fold higher in G. sulfurreducens in co-cultures with magnetite than in Geobacters co-cultures. Mutation results showed that GAC can be substituted for the e-pili of both strains but magnetite can only compensate for that of G. sulfurreducens, indicating that the e-pili is a more important electron acceptor for the electron donor strain of G. metallireducens than for G. sulfurreducens. Transcript abundance for G. metallireducens c-type cytochrome gene GMET_RS14535, a homologue to c-type cytochrome gene omcE of G. sulfurreducens was 9.8-fold lower in co-cultures with GAC addition, while that for OmcS of G. sulfurreducens was 25.1-fold higher in co-cultures with magnetite, than in that without magnetite. Gene deletion studies showed that neither GAC nor magnetite can completely substitute the cytochrome (OmcE homologous) of G. metallireducens but compensate for the cytochrome (OmcS) of G. sulfurreducens. Moreover, some genes associated with central metabolism were up-regulated in the presence of both GAC and magnetite; however, tricarboxylic acid cycle gene transcripts in G. sulfurreducens were not highly-expressed in each of these amended co-cultures, suggesting that there was considerable redundancy in the pathways utilised by G. sulfurreducens for electron transfer to reduce fumarate with the amendment of GAC or magnetite. These results support the DIET model of G. metallireducens and G. sulfurreducens and suggest that e-pili and cytochromes of the electron donor strain are more important than that of the electron acceptor strain, indicating that comparative transcriptomics may be a promising route by which to reveal different responses of electron donor and acceptor during DIET in co-cultures.

Enhanced methane production in an anaerobic digestion and microbial electrolysis cell coupled system with co-cultivation of Geobacter and Methanosarcina

The anaerobic digestion（AD）and microbial electrolysis cell（MEC）coupled system has been proved to be a promising process for biomethane production.In this paper,it was found that by co-cultivating Geobacter with Methanosarcina in an AD–MEC coupled system,methane yield was further increased by 24.1%,achieving to 360.2 m L/g-COD,which was comparable to the theoretical methane yield of an anaerobic digester.With the presence of Geobacter,the maximum chemical oxygen demand（COD）removal rate（216.8 mg COD/（L·hr））and current density（304.3 A/m3）were both increased by 1.3 and 1.8 fold compared to the previous study without Geobacter,resulting in overall energy efficiency reaching up to 74.6%.Community analysis demonstrated that Geobacter and Methanosarcina could coexist together in the biofilm,and the electrochemical activities of both were confirmed by cyclic voltammetry.Our study observed that the carbon dioxide content in total gas generated from the AD reactor with Geobacter was only half of that generated from the same reactor without Geobacter,suggesting that Methanosarcina may obtain the electron transferred from Geobacter for the reduction of carbon dioxide to methane.Taken together,Geobacter not only can improve the performance of the MEC system,but also can enhance methane production.

Mechanisms behind the accelerated extracellular electron transfer in Geobacter sulfurreducens DL-1 by modifying gold electrode with self-assembled monolayers

Modification of electrode surface with carboxylic acid terminated alkanethiol self-assembled monolayers （SAMs） has been found to be an effective approach to improve the extracellular electron transfer （EET） of electrochemically active bacteria （EAB） on electrode surface, but the underlying mechanism behind such enhanced EET remains unclear. In this work, the gold electrodes modified by mercapto-acetic acid and mercapto- ethylamine （Au-COOH, Au-NH2） were used as anodes in microbial electrolysis cells （MECs） inoculated with Geobacter sulfurreducens DL- 1, and their electrochemical performance and the bacteria-electrode interactions were investigated. Results showed that the Fe（CN）6^3-/4^- redox reaction occurred on the Au-NH2 with a higher rate and a lower resistance than that on the Au or the Au-COOH. Both the MECs with the Au-COOH and Au-NH2 anodes exhibited a higher current density than that with a bare Au anode. The biofilm formed on the Au-COOH was denser than that on bare Au, while the biofilm on the Au-NH2 had a greater thickness, suggesting a critical role of direct EET in this system. This work suggests that functional groups such as --COOH and-NH2 could promote electrode performance by accelerating the direct EET of EAB on electrode surface.

Development of a whole-cell biosensor based on an ArsR-Pars regulatory circuit from Geobacter sulfurreducens

In this study,an Escherichia coli(E.coli)whole-cell biosensor for the specific detection of bioavailable arsenic was developed by placing a green fluorescent protein(GFP)reporter gene under the control of the ArsR1(GSU2952)regulatory circuit from Geobacter sulfurreducens.E.coli cells only emitted green fluorescence in the presence of arsenite and were more sensitive to arsenite when they were grown in M9 supplemented medium compared to LB medium.Under optimal test conditions,the Geobacter arsR1 promoter had a detection limit of 0.01 mM arsenite and the GFP expression was linear within a range of 0.03-0.1 mM(2.25-7.5 mg/l).These values were well below World Health Organization’s drinking water quality standard,which is 10 mg/l.The feasibility of using this whole-cell biosensor to detect arsenic in water samples,such as arsenic polluted tap water and landfill leachate was verified.The biosensor was determined to be just as sensitive as atomic fluorescence spectrometry.This study examines the potential applications of biosensors constructed with Geobacter ArsR-Pars regulatory circuits and provides a rapid and cost-effective tool that can be used for arsenic detection in water samples.

Different outer membrane c‐type cytochromes are involved in direct interspecies electron transfer to Geobacter or Methanosarcina species

Direct interspecies electron transfer(DIET)may be most important in methanogenic environments,but mechanistic studies of DIET to date have primarily focused on cocultures in which fumarate was the terminal electron acceptor.To better understand DIET with methanogens,the transcriptome of Geobacter metallireducens during DIET‐based growth with G.sulfurreducens reducing fumarate was compared with G.metallireducens grown in coculture with diverse Methanosarcina.The transcriptome of G.metallireducens cocultured with G.sulfurreducens was significantly different from those with Methanosarcina.Furthermore,the transcriptome of G.metallireducens grown with Methanosarcina barkeri,which lacks outer‐surface c‐type cytochromes,differed from those of G.metallireducens cocultured with M.acetivorans or M.subterranea,which have an outer‐surface c‐type cytochrome that serves as an electrical connect for DIET.Differences in G.metallireducens expression patterns for genes involved in extracellular electron transfer were particularly notable.Cocultures with c‐type cytochrome deletion mutant strains,ΔGmet_0930,ΔGmet_0557 andΔGmet_2896,never became established with G.sulfurreducens but adapted to grow with all three Methanosarcina.Two porin–cytochrome complexes,PccF and PccG,were important for DIET;however,PccG was more important for growth with Methanosarcina.Unlike cocultures with G.sulfurreducens and M.acetivorans,electrically conductive pili were not needed for growth with M.barkeri.Shewanella oneidensis,another electroactive microbe with abundant outer‐surface c‐type cytochromes,did not grow via DIET.The results demonstrate that the presence of outer‐surface c‐type cytochromes does not necessarily confer the capacity for DIET and emphasize the impact of the electron‐accepting partner on the physiology of the electron‐donating DIET partner.

直接微生物燃料电池的构建及初步研究

利用Geobactermetallireducens能够以Fe(OH)3固体作电子受体进行呼吸的特性,用其构建直接微生物燃料电池,初步考察了产电情况和产电原理.实验证明,Geobactermetallireducens直接微生物燃料电池的电能产出主要依赖于吸附在电极上的细菌.燃料醋酸钠可以完全氧化至CO2,反应结束后其浓度低于检测下限(<10μmol/L).电子回收率达80%,电流密度达704.4mA/m2.

异化Fe(Ⅲ)还原微生物研究进展

铁是地壳中含量第四丰富的元素,微生物介导的异化铁还原是自然界中Fe(Ⅲ)还原的主要途径。介绍了Fe(Ⅲ)还原菌的分类及多样性,总结了Fe(Ⅲ)还原菌还原铁氧化物机制及其产能代谢机制,概述了Fe(Ⅲ)还原菌的生态环境意义,并对未来Fe(Ⅲ)还原菌的分子生态学研究方向提出了探索性的建议。

典型胞外呼吸细菌的胞内电子转移机制研究进展

胞外呼吸在污染物的降解转化和微生物产电过程中具有重要作用。微生物进行胞外呼吸时,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经细胞周质转移到外膜。S.oneidensis MR-1与G.Sulfurreducens作为微生物燃料电池中最常用的模式菌株,是现阶段研究最深入和系统的胞外呼吸细菌,其胞内电子传递过程目前研究最为清楚。这两种胞外呼吸细菌的电子传递需多种细胞色素c的参与,S.oneidensis MR-1位于内膜及周质上的细胞色素c-Cym A和MtrA可将电子由内膜上的醌池通过周质到外膜蛋白MtrC和OmcA,MtrC和OmcA接收电子后可直接还原胞外受体,Type Ⅱ secretion system对外膜蛋白中的MtrC和OmcA起到了转运及定位的作用。而在G.sulfurreducens中,电子由MacA传递到PpcA,最终由外膜蛋白OmcB、OmcE、OmcS及OmcZ接受电子,并在Type Ⅳ pili的共同作用下将电子传递到胞外电子受体。本文最后指出目前对Shewanella与Geobacter胞内电子转移研究尚不清楚的地方提出展望。