微生物种间直接电子传递机理及应用研究进展

微生物胞内产生的电子转移到其他电子受体而获得能量的过程称为微生物胞外电子传递,其中,另一微生物作为电子受体时发生的电子传递称为微生物种间电子传递。根据微生物种间电子传递机制,可分间接种间电子传递和种间直接电子传递。由于种间直接电子传递不需要其他物质介导,因此较间接种间电子传递效率更高、能量利用更高。本文系统阐述了微生物进行胞外电子传递的机理及应用,重点分析了种间直接电子传递机理,并概述种间直接电子传递应用领域,为寻找更多电连接的微生物群落以及应用微生物提供参考。     

微生物种间直接电子传递研究进展

一直以来氢气和甲酸被认为是微生物间电子传递的中间电子传递体.近年来的研究发现,微生物之间可以通过种间直接电子传递(DIET)来替代氢气/甲酸传递.DIET作为一种新发现的微生物间电子传递途径,其电子传递效率要高于传统的种间氢气/甲酸传递.DIET这一新发现改变了微生物互营生长代谢必须依赖氢气或甲酸等电子载体的传统认识,...     

Evidence for the coexistence of direct and riboflavin-mediated interspecies electron transfer in Geobacter co-culture

Geobacter species can secrete free redox-active flavins, but the role of these flavins in the interspecies electron transfer (IET) of Geobacter direct interspecies electron transfer (DIET) co-culture is unknown. Here, we report the presence of a new riboflavin-mediated interspecies electron transfer (RMIET) process in a traditional Geobacter DIET co-culture; in this process, riboflavin contributes to IET by acting as a free-form electron shuttle between free Geobacter species and serving as a bound cofactor of some cytochromes in Geobacter co-culture aggregates. Multiple lines of evidence indicate that RMIET facilitates the primary initiation of syntrophic growth between Geobacter species before establishing the DIET co-culture and provides additional ways alongside the DIET to transfer electrons to achieve electric syntrophy between Geobacter species. Redox kinetic analysis of riboflavin on either Geobacter species demonstrated that the Gmet_2896 cytochrome acts as the key riboflavin reduction site, while riboflavin oxidation by Geobacter sulfurreducens is the rate-limiting step in RMIET, and the RMIET makes only a minor contribution to IET in Geobacter DIET co-culture. The discovery of a new RMIET process in Geobacter DIET co-culture suggests the complexity of IET in syntrophic bacterial communities and provides suggestions for the careful examination of the IET of other syntrophic co-cultures.     

Improving anaerobic digestion via direct interspecies electron transfer requires development of suitable characterization methods

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互营氧化产甲烷微生物种间电子传递研究进展

甲烷是重要的温室气体,也是典型的可再生性生物质能源。目前约70%的大气甲烷排放来源于产甲烷微生物过程。在产甲烷环境中,产甲烷菌与互营细菌形成互营关系,从而克服有机质厌氧分解反应的热力学能垒,实现短链脂肪酸和醇类物质的互营氧化产甲烷过程。该过程中,种间电子传递是关键步骤。本文首先概述了甲烷的研究意义及微生物互营降解有机质产甲烷的过程,然后分别综述了种间H2转移、种间甲酸转移和种间直接电子传递这3种种间电子传递机制的起源、发展、研究现状和未来所需要解决的研究问题。     

Modeling of interspecies electron transfer in anaerobic microbial communities

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Fold versus sequence effects on the driving force for protein‐mediated electron transfer

Electron transport chains composed of electron transfer reactions mainly between proteins provide fast efficient flow of energy in a variety of metabolic pathways. Reduction potentials are essential characteristics of the proteins because they determine the driving forces for the electron transfers. As both polar and charged groups from the backbone and side chains define the electrostatic environment, both the fold and the sequence will contribute. However, although the role of a specific sequence may be determined by experimental mutagenesis studies of reduction potentials, understanding the role of the fold by experiment is much more difficult. Here, continuum electrostatics and density functional theory calculations are used to analyze reduction potentials in [4Fe‐4S] proteins. A key feature is that multiple homologous proteins in three different folds are compared: six high potential iron‐sulfur proteins, four bacterial ferredoxins, and four nitrogenase iron proteins. Calculated absolute reduction potentials are shown to be in quantitative agreement with electrochemical reduction potentials. Calculations further demonstrate that the contribution of the backbone is larger than that of the side chains and is consistent for homologous proteins but differs for nonhomologous proteins, indicating that the fold is the major protein factor determining the reduction potential, whereas the specific amino acid sequence tunes the reduction potential for a given fold. Moreover, the fold contribution is determined mainly by the proximity of the redox site to the protein surface and the orientation of the dipoles of backbone near the redox site. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

产甲烷分离物中Clostridium spp.与Methanosarcina barkeri潜在的种间直接电子传递

【目的】革兰氏阴性菌Geobacter metallireducens可以与乙酸型产甲烷菌Methanosaeta harundinacea或Methanosarcina barkeri通过种间直接电子传递(DIET)还原CO2产甲烷。本实验室前期的研究发现Methanosarcina mazei和Geobacteraceae在铁还原富集培养中形成团聚体,可能存在直接电子传递。然而,革兰氏阳性菌(如Clostridium spp.)与产甲烷菌是否存在种间直接电子传递尚不明确。【方法】采用Hungate厌氧滚管法,以乙醇为唯一电子供体从铁还原富集培养体系中获得产甲烷分离物(S6)。通过T-RFLP及克隆文库分析群落多样性,结合循环伏安法等电化学方法研究产甲烷分离物的电活性。【结果】Clostridium spp.(与C.tunisiense相似性最高)和M.barkeri分别在S6细菌和古菌群落中占优势。S6与G.metallireducens共培养后铁还原和产甲烷能力未明显增加,Clostridium spp.可能与G.metallireducens类似,将电子直接传递给产甲烷菌M.barkeri产甲烷。此外,电化学检测发现,在用透析袋包裹电极阻碍微生物与电极表面通过直接接触形成生物膜的条件下,电流密度显著降低,并且循环伏安扫描无明显氧化还原峰。【结论】产甲烷分离物S6中存在直接电子传递途径。本工作提出在产甲烷分离物中占优势的革兰氏阳性菌Clostridium spp.和M.barkeri之间可能存在种间直接电子传递。     

Advances in direct interspecies electron transfer and conductive materials: Electron flux,organic degradation and microbial interaction

Direct interspecies electron transfer (DIET) via electrically conductive pili (e-pili) and c-type cytochrome between acetogens and methanogens has been proposed as an essential pathway for methane production. Supplements of conductive materials have been extensively found to promote methane production in microbial anaerobic treatment systems. This review comprehensively presents recent findings of DIET and the addition of conductive materials for methanogenesis and summarizes important results through aspects of electron flux, organic degradation, and microbial interaction. Conductive materials improve DIET and methanogenesis by acting as either substitute of e-pili or electron conduit between e-pili and electron acceptors. Other effects of conductive materials such as the change of redox potential may also be important factors for the stimulation. The type and organic loading rate of substrates affect the occurrence of DIET and stimulating effects of conductive materials. Geobacter, which can participate in DIET, were less enriched in anaerobic systems cultivated with non-ethanol substrates, suggesting the existence of other syntrophs with the capability of DIET. The coupling of communication systems such as quorum sensing may be a good strategy to achieve the formation of biofilm or granule enriched with syntrophic partners capable of DIET.     

基于直接接触的微生物胞外电子传递

微生物电子传递在微生物的代谢繁殖和物质的生物地球化学循环中发挥着关键作用。其中基于直接接触的微生物胞外电子传递(Direct extracellular electron transfer,DEET)已成为微生物学、地球化学和生物物理学等学科共同关注的焦点,并在近几年取得了一系列重要发现和理论突破,包括微生物纳米导线、电缆细菌、微生物种间DEET等。伴随着这些新进展,更多的问题也需要研究者们在进一步的研究中解决,包括DEET的分子机制及其相关功能微生物种群等。不同学科理论和技术的交叉是进一步揭示DEET过程的关键。     

Biosensors based on novel peroxidases with improved properties in direct and mediated electron transfer.

Native horseradish peroxidase (HRP) on graphite has revealed approximately 50% of the active enzyme molecules to be in direct electron transfer (ET) contact with the electrode surface. Some novel plant peroxidases from tobacco, peanut and sweet potato were kinetically characterised on graphite in order to find promising candidates for biosensor applications and to understand the nature of the direct ET in the case of plant peroxidases. From measurements of the mediated and mediatorless currents of hydrogen peroxide reduction at the peroxidase-modified rotating disk electrodes (RDE), it was concluded that the fraction of enzyme molecules in direct ET varies substantially for the different plant peroxidases. It was observed that the anionic peroxidases (from sweet potato and tobacco) demonstrated a higher percentage of molecules in direct ET than the cationic ones (HRP and peanut peroxidase). The peroxidases with a high degree of glycosylation demonstrated a lower percentage of molecules in direct ET. It could, thus, be concluded that glycosylation of the peroxidases hinders direct ET and that a net negative charge on the peroxidase (low pI value) is beneficial for direct ET. Especially noticeable are the values obtained for sweet potato peroxidase (SPP), revealing both a high percentage in direct ET and a high rate constant of direct ET. The peroxidase electrodes were used for determination of hydrogen peroxide in RDE mode (mediatorless). SPP gave the lowest detection limit (40 nM) followed by HRP and peanut peroxidase.     

Potential-dependent extracellular electron transfer pathways of exoelectrogens

Exoelectrogens are distinct from other bacteria owing to their unique extracellular electron transfer (EET) abilities that allow for anaerobic respiration with various external redox-active surfaces, including electrode and metal oxides. Although the EET process is known to trigger diverse extracellular redox reactions, the reverse impact has been long overlooked. Recent evidences show that exoelectrogens can sense the potential changes of external surfaces and alter their EET strategies accordingly, which imparts them remarkable abilities in adapting to diverse and redox-variable environment. This mini-review provides a condensed overview and critical analysis about the recent discoveries on redox-dependent EET pathways of exoelectrogens, with focus on Geobacter sulfurreducens and Shewanella oneidensis. We summarize the detailed responses of various EET components, analyze the drives and mechanisms of such responses, highlight the diversity of EET dynamics among different bacterial species and under integrated effects of redox potential and surface chemistry, and discusses the future research needs.     

Bacterial extracellular electron transfer in bioelectrochemical systems

Bioelectrochemical systems (BES), typically microbial fuel cells (MFCs), have attracted increasing attention in the past decade due to their promising applications in many fields, such as bioremediation, energy generation and biosynthesis. Current-generating microorganisms play a key role in BES. The process of transferring electrons to electrode has been considered as a novel anaerobic bacteria respiration, and more and more bacteria capable of exchanging electrons with electrodes have been isolated. Among those bacteria, Shewanella and Geobacter genera are the most frequently used model organisms in the studies of BES, as well as the bacteria-electrode electron transfer mechanisms. Many significant new findings in the field of the bacterial extracellular electron transfer in BES have been reported recently. A better understanding of the mechanisms of bacterial extracellular electron transfer would provide more efficient strategies to enhance the applicability of BES. This review summarizes the recent advances of extracellular electron transfer mechanisms with foci on Shewanella and Geobacter species in BES.     

Microbial electrocatalysis: Redox mediators responsible for extracellular electron transfer

Redox mediator plays an important role in extracellular electron transfer (EET) in many environments wherein microbial electrocatalysis occurs actively. Because of the block of cell envelope and the low difference of redox potential between the intracellular and extracellular surroundings, the proceeding of EET depends mainly on the help of a variety of mediators that function as an electron carrier or bridge. In this Review, we will summarize a wide range of redox mediators and further discuss their functional mechanisms in EET that drives a series of microbial electrocatalytic reactions. Studying these mediators adds to our knowledge of how charge transport and electrochemical reactions occur at the microorganism-electrode interface. This understanding would promote the widespread applications of microbial electrocatalysis in microbial fuel cells, bioremediation, bioelectrosynthesis, biomining, nanomaterial productions, etc. These improved applications will greatly benefit the sustainable development of the environmental-friendly biochemical industries.     

Mechanically mediated electron transfer in model metallo-enzyme interfaces

In this paper, we develop a physical analysis of charge transfer in the model 'metallo-enzyme' complex which consists of a synthetic redox-addressed assembly (a 'reaction center') hybridized with a quantum dot (a gold nanoparticle) and attached via molecular bridge (a spacer) to the electrode. This artificial system allows us to model electronic transduction in experimental redox ezyme-gold nanoparticle hybrid structure recently reported by Xiao et al. [Xiao, Y., Patolsky, F., Katz, E., Hainfeid, J.F., Willner, I., 2003. Science 299, 1877-1881]. We consider a photosensitive spacer that allows us to control the conductivity of the bridge by light. In this paper we will focus on a special type of nanoelectro-mechanical processes in this system and investigate single electronic effects in there.     

Direct interspecies electron transfer accelerates syntrophic oxidation of butyrate in paddy soil enrichments

Syntrophic interaction occurs during anaerobic fermentation of organic substances forming methane as the final product. H₂ and formate are known to serve as the electron carriers in this process. Recently, it has been shown that direct interspecies electron transfer (DIET) occurs for syntrophic CH₄ production from ethanol and acetate. Here, we constructed paddy soil enrichments to determine the involvement of DIET in syntrophic butyrate oxidation and CH₄ production. The results showed that CH₄ production was significantly accelerated in the presence of nanoFe₃O₄ in all continuous transfers. This acceleration increased with the increase of nanoFe₃O₄ concentration but was dismissed when Fe₃O₄ was coated with silica that insulated the mineral from electrical conduction. NanoFe₃O₄ particles were found closely attached to the cell surfaces of different morphology, thus bridging cell connections. Molecular approaches, including DNA‐based stable isotope probing, revealed that the bacterial Syntrophomonadaceae and Geobacteraceae, and the archaeal Methanosarcinaceae, Methanocellales and Methanobacteriales, were involved in the syntrophic butyrate oxidation and CH₄ production. Among them, the growth of Geobacteraceae strictly relied on the presence of nanoFe₃O₄ and its electrical conductivity in particular. Other organisms, except Methanobacteriales, were present in enrichments regardless of nanoFe₃O₄ amendment. Collectively, our study demonstrated that the nanoFe₃O₄‐facilitated DIET occurred in syntrophic CH₄ production from butyrate, and Geobacter species played the key role in this process in the paddy soil enrichments.     

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。     

Control of interspecies electron transfer flow during anaerobic digestion: dynamic diffusion reaction models for hydrogen gas transfer in microbial flocs

Dynamic reaction diffusion models were used to analyze the consequences of aggregation for syntrophic reactions in methanogenic ecosystems. Flocs from a whey digestor were used to measure all model parameters under the in situ conditions of a particular defined biological system. Fermentation simulations without adjustable parameters could precisely predict the kinetics of H(2) gas production of digestor flocs during syntrophic methanogenesis from ethanol. The results demonstrated a kinetic compartmentalization of H(2) metabolism inside the flocs. The interspecies electron transfer reaction was mildly diffusion controlled. The H(2) gas profiles across the flocs showed high H (2) concentrations inside the flocs at any time. Simulations of the syntrophic metabolism at low substrate concentrations such as in digestors or sediments showed that it is impossible to achieve high H(2) gas turnovers at simultaneously low steady-state H(2) concentrations. This showed a mechanistic contradiction in the concept of postulated low H(2) microenvironments for the anaerobic digestion process. The results of the computer experiments support the conclusion that syntrophic H(2) production may only be a side reaction of H(2) independent interspecies electron transfer in methanogenic ecosystems.     

Radiation damage to dinucleoside monophosphates: mediated versus direct damage

The mediation of radiation-induced damage to dinucleoside monophosphate by oxygen and by glutathione was studied. The sequence isomers d(TpA) and d(ApT) were X-irradiated in aqueous solutions and the products isolated by reverse-phase high-performance liquid chromatography. The main products were characterized by proton NMR spectroscopy. In the presence of oxygen the principal products are the formamido derivative formed by breakdown of thymine and the aldehyde derivative formed at the 5' end of the dinucleoside monophosphate, both nucleoside monophosphates and free bases. In the presence of glutathione, the two stereoisomers of the 5,6-dihydrothymine derivatives are prominent. Radiation-induced damage to d(TpA) and d(ApT) in the solid state was also studied.     

微生物胞外电子传递效率的合成生物学强化

电活性微生物(产电微生物和亲电微生物)通过与外界环境进行双向电子和能量传递来实现多种微生物电催化过程(包括微生物燃料电池、微生物电解电池、微生物电催化等),从而实现在环境、能源领域的广泛应用,并为开发有效且可持续性生产新能源或大宗精细化学品的工艺提供了新机会。但是,电活性微生物的胞外电子传递效率比较低,这已经成为限制微生物电催化系统在工业应用中的主要瓶颈。以下综述了近年来利用合成生物学改造电活性微生物的相关研究成果,阐明了合成生物学如何用于打破电活性微生物胞外电子传递途径低效率的瓶颈,从而实现电活性微生物与环境的高效电子传递和能量交换,推动电活性微生物电催化系统的实用化进程。