合成生物学方法构建电活性大肠杆菌

电活性微生物具有独特的在细胞内外环境之间传递电子的能力。在对天然电活性微生物电子传递机制充分研究的基础上,通过合成生物学方法异源构建天然电活性微生物电子传递结构基础也可以将遗传背景清晰的非电活性大肠杆菌改造为电活性微生物。构建获得的工程化电活性大肠杆菌可以直接应用于微生物燃料电池和生物传感器等领域,同时也可以作为底盘细胞整合相应的目标产物合成通路实现电能驱动的生物合成。本文以合成生物学方法构建电活性大肠杆菌为主题,详细阐述天然电活性微生物电子传递的机理及结构基础,总结了工程化电活性大肠杆菌的构建策略、成功案例以及应用领域,并对合成生物学方法构建电活性大肠杆菌未来的研究方向进行了展望。     

生物工程强化微生物电合成转化CO_2的研究进展

微生物电合成(Microbial electrosynthesis,MES)可直接利用电能驱动微生物还原固定CO_2合成多碳化合物,为可再生新能源转化、精细化学品制备和生态环境保护提供新机遇。但是,微生物吸收胞外电极电子速率慢、产物合成效率低和产品品位不高,限制了MES实现工业化应用。在概述阴极电活性微生物吸收胞外电子的分子机制的基础上,重点综述近5年应用生物工程的理论和技术强化MES用于CO_2转化的策略与研究进展,包括改造和调控胞外电子传递通路和胞内代谢途径以及定向构建有限微生物混合培养菌群三方面,阐明了生物工程可有效突破MES中电子传递慢和可用代谢途径相对单一等瓶颈。针对目前生物工程在改进MES所面临的主要问题,从胞外电子传递机理研究、基因工具箱开发、组学技术与现代分析技术联用等角度展望了今后的研究方向。     

奥奈达希瓦氏菌电活性生物被膜的研究进展

面对日益严峻的能源紧缺与环境污染形势,电活性微生物(electroactive microorganisms)的电催化过程为实现绿色生产提供了新的思路。奥奈达希瓦氏菌具有独特的呼吸方式和电子传递能力,在微生物燃料电池、增值化学品的生物电合成、金属废物处理和环境修复系统等领域有着广泛的应用。奥奈达希瓦氏菌(Shewanella oneidensis MR-1)电活性生物被膜是实现电活性微生物电子传递过程的优良载体,其形成过程十分复杂且受到多种因素的影响和调控,在增强细菌环境抗逆性、提高电子传递效率等多方面发挥着十分重要的作用。本文较为系统地综述了奥奈达希瓦氏菌生物被膜的形成过程、影响因素及其在生物能源、生物修复和生物传感中的相关应用,为进一步实现其在更多领域的应用提供了理论基础。     

电活性微生物胞外聚合物的特征与应用

电活性微生物是一类能够通过直接接触、导电菌毛或氧化还原介质与电极或者其他细胞进行胞外电子传递的微生物。而在这个过程中,胞外聚合物(extracellular polymeric substances, EPS)扮演着重要的角色。EPS是微生物生长过程中通过细胞裂解、水解分泌的高分子聚合物的混合物,主要由蛋白质、多糖和腐殖质等物质组成。来自电活性微生物的EPS的不同组成成分和特性会对EPS的电活性以及电活性微生物胞外电子传递产生一定的影响,同时在环境应用方面发挥重要作用。因此,为了更全面了解电活性微生物EPS的电活性及其对电活性微生物胞外电子传递的作用,本文总体介绍了电活性微生物EPS的电活性的直接表征方法,再从组成成分、化学性质、物理性质和空间分布4个方面综述了其对EPS电活性的影响及其在电子传递中的作用,介绍了当前电活性微生物EPS在染料废水脱色、重金属吸附、有机污染物的生物转化和渗滤液管理等方面的环境应用,并从表征方法、试验规模和互作机理研究等角度展望了未来的研究方向。     

分子生物学方法提高电活性微生物胞外电子传递效率的研究进展*

微生物细胞与电极之间的胞外电子传递效率是限制微生物电化学技术发展的关键因素,而分子生物学的发展为提高胞外电子传递效率带来了光明前景。从四种具有代表性的纯培养电活性微生物(奥奈达希瓦氏菌、铜绿假单胞菌、硫还原地杆菌和工程大肠杆菌)和混合培养电活性微生物出发,综述了利用分子生物学手段改造几种电活性微生物的研究成果,阐明了针对特异的电活性微生物,如何采取相应的分子生物学手段提高其胞外电子传递的效率,并展望了未来的研究方向。     

基于细胞色素c的胞外电子传递过程

电活性微生物具有独特的胞外电子传递功能,在地球化学循环和环境污染修复中起着重要作用。细胞色素c在电活性微生物胞外电子传递过程中扮演了重要角色,不仅参与直接电子传递途径,还参与电子媒介介导的间接电子传递。其电子传递功能不仅对地球环境中铁、锰、碳等元素的循环具有重要作用,还应用于能源生产、废水处理、生物修复等众多领域,具有良好的应用潜力。本文以电活性微生物的2个模式菌属(希瓦氏菌属和地杆菌属)为例,综述了电活性微生物将电子由胞内转移至胞外的方式和途径,详细阐述了细胞色素c在该胞外电子传递过程中的重要作用,总结了细胞色素c介导的胞外电子传递过程所涉及的分析方法,并对微生物胞外电子传递未来的研究方向提出了展望。     

基于电活性微生物的芳香烃类污染物转化机制研究进展

芳香烃类化合物(aromatic hydrocarbon compounds)是一类基于苯环结构的有机物,广泛分布在自然环境中,难以自然降解、易被生物积累,且有很大的环境危害性。生物法是有机化合物转化降解的主流工艺,而电活性微生物(electroactive microorganisms, EAM)因其独特的胞外电子传递(extracellular electron transfer, EET)能力和生理代谢模式在芳香烃类化合物污染修复领域具有巨大的应用潜力。电活性微生物可以通过还原脱卤、脱硝与氧化开环过程相结合的方式,最终实现芳香烃类污染物的降解矿化。本文重点综述了电活性微生物降解芳香烃类污染物过程中主要还原/氧化反应机理,归纳了电活性微生物高效还原脱卤、脱硝的关键酶活、代谢途径及转化机理,分析了不同含氧条件下电活性微生物开环方式及降解代谢途径,并通过调控微生物胞外聚合物与添加导电材料等途径来提升电活性微生物的胞外电子传递过程,总结了电极电位、电极材料、电解液性质及温度等环境因子对芳香烃类化合物降解的影响,探讨了芳香烃类污染物的强化生物降解策略的可行性。最后,展望了电活性微生物降解技...     

强化产电微生物与电极间电子传递速率的研究进展

产电微生物是微生物燃料电池、电解池和电合成等微生物电化学技术(Microbial electrochemical technologies,METs)的研究基础。产电微生物与电极界面间的胞外电子传递(Extracellular electron transfer,EET)效率低以及生物被膜形成能力弱限制了METs在有机物降解、电能生产、海水淡化、生物修复和生物传感等方面的应用。因此,强化产电微生物与电极界面间的相互作用是过去几年的主要研究热点。针对近年的研究,本文系统概述了通过改造产电微生物来增强微生物-电极间相互作用的各种策略,重点分析了这些策略的适用性和局限性,并展望了强化产电微生物-电极界面作用在微生物电化学技术利用方面的研究前景。     

黄素介导的胞外电子转移研究与工程改造*

产电微生物的胞外电子转移在能源、环境等诸多领域有着非常重要的应用价值。希瓦氏菌(Shewanella oneidensis)作为模式产电微生物,其电催化系统引起了广泛的研究。黄素作为S. oneidensis重要的电子载体,其介导的胞外电子转移是电子传递过程中的一个限速步骤。然而自然环境中野生型S. oneidensis的黄素分泌量极低,对其工程改造也存在一定的局限性,因而严重阻碍了胞外电子的传递过程,这已成为限制其电子转移的主要瓶颈。基于S. oneidensis黄素介导的电子转移机制,系统地从黄素的合成路径及转录调控的角度阐明了黄素合成的调控因素,并综述近年来利用代谢工程、合成生物学以及电极材料修饰等方法来提高黄素介导电子转移的工程化策略,未来可利用一些系统的研究方法和表达工具来加速产电微生物黄素介导的胞外电子转移。     

一株促甲烷氧化假单胞菌Pseudomonas putida P7的分离及电活性特征

微生物胞外呼吸是厌氧环境中控制性能量代谢方式,直接驱动着C、N、S、Fe等关键元素的生物地球化学循环。微生物纳米导线（Microbial nanowires）的发现,被认为是微生物胞外呼吸的里程碑事件,推动了电微生物学（Electromicrobiology）的形成与发展。微生物纳米导线是一类由微生物合成的,具有导电性的纤维状表面附属结构。通过细菌纳米导线,微生物胞内代谢产生的电子可以长距离输送到胞外受体或其他微生物,改变了电子传递链仅仅局限于细胞胞内的认识,从而大大拓展了微生物-胞外环境互作的范围。微生物纳米导线的良好导电性,赋予了其作为天然纳米材料的广阔应用前景。目前,微生物纳米导线的导电机制、生态功能及其在生物材料、生物能源、生物修复及人体健康多领域的应用,已经成为新兴电微生物学的前沿与热点。然而,微生物纳米导线的生物学、生态学功能尚不清楚,它的电子传递机制仍存在分歧。本文在系统性总结微生物纳米导线性质、功能的基础上,以Geobacter sulfurreducens和Shewanella oneidensis纳米导线为模型,详细阐述了纳米导线的组成与结构、表征与测量方法、导电理论（类金属导电学说与电子跃迁学说）及其潜在的应用,最后提出了未来微生物纳米导线研究的重点方向、挑战与机遇。     

Microbial extracellular electron transfer and its relevance to iron corrosion

Extracellular electron transfer (EET) is a microbial metabolism that enables efficient electron transfer between microbial cells and extracellular solid materials. Microorganisms harbouring EET abilities have received considerable attention for their various biotechnological applications, including bioleaching and bioelectrochemical systems. On the other hand, recent research revealed that microbial EET potentially induces corrosion of iron structures. It has been well known that corrosion of iron occurring under anoxic conditions is mostly caused by microbial activities, which is termed as microbiologically influenced corrosion (MIC). Among diverse MIC mechanisms, microbial EET activity that enhances corrosion via direct uptake of electrons from metallic iron, specifically termed as electrical MIC (EMIC), has been regarded as one of the major causative factors. The EMIC‐inducing microorganisms initially identified were certain sulfate‐reducing bacteria and methanogenic archaea isolated from marine environments. Subsequently, abilities to induce EMIC were also demonstrated in diverse anaerobic microorganisms in freshwater environments and oil fields, including acetogenic bacteria and nitrate‐reducing bacteria. Abilities of EET and EMIC are now regarded as microbial traits more widespread among diverse microbial clades than was thought previously. In this review, basic understandings of microbial EET and recent progresses in the EMIC research are introduced.     

产电微生物的筛选方法研究进展

产电微生物是一类具有胞外电子转移能力的微生物,能够将有机物中储存的化学能转化为电能,其作为微生物电催化系统的催化剂,已经成为环境和能源领域的研究热点。但目前所发现的产电菌,产电机制有所差异,产电能力参差不齐,菌株的性能从根本上影响了其产电能力,其产电能力不足成为限制微生物燃料电池在工业上广泛应用的主要瓶颈。目前,通过理性设计或定向进化等改造方法,难以实现产电微生物在复杂多样环境中的广泛应用。通过定向筛选策略,建立一套快速、高效的筛选鉴定技术,挖掘环境中性能优异的产电微生物,是促进其广泛应用的有效途径。文中基于产电微生物的种类,总结回顾了现有的产电微生物的筛选鉴定方法,并对其研究前景进行了展望。     

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。     

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.     

Waste Water Derived Electroactive Microbial Biofilms: Growth,Maintenance, and Basic Characterization

The growth of anodic electroactive microbial biofilms from waste water inocula in a fed-batch reactor is demonstrated using a three-electrode setup controlled by a potentiostat. Thereby the use of potentiostats allows an exact adjustment of the electrode potential and ensures reproducible microbial culturing conditions. During growth the current production is monitored using chronoamperometry (CA). Based on these data the maximum current density (j_max) and the coulombic efficiency (CE) are discussed as measures for characterization of the bioelectrocatalytic performance. Cyclic voltammetry (CV), a nondestructive, i.e. noninvasive, method, is used to study the extracellular electron transfer (EET) of electroactive bacteria. CV measurements are performed on anodic biofilm electrodes in the presence of the microbial substrate, i.e. turnover conditions, and in the absence of the substrate, i.e. nonturnover conditions, using different scan rates. Subsequently, data analysis is exemplified and fundamental thermodynamic parameters of the microbial EET are derived and explained: peak potential (E_p), peak current density (j_p), formal potential (E^f) and peak separation (ΔE_p). Additionally the limits of the method and the state-of the art data analysis are addressed. Thereby this video-article shall provide a guide for the basic experimental steps and the fundamental data analysis.     

Rapid and highly efficient genomic engineering with a novel iEditing device for programming versatile extracellular electron transfer of electroactive bacteria

The advances in synthetic biology bring exciting new opportunities to reprogram microorganisms with novel functionalities for environmental applications. For real-world applications, a genetic tool that enables genetic engineering in a stably genomic inherited manner is greatly desired. In this work, we design a novel genetic device for rapid and efficient genome engineering based on the i ntron-encoded homing-endonuclease empowered genome editing (iEditing). The iEditing device enables rapid and efficient genome engineering in Shewanella oneidensis MR-1, the representative strain of the electroactive bacteria group. Moreover, combining with the Red or RecET recombination system, the genome-editing efficiency was greatly improved, up to approximately 100%. Significantly, the iEditing device itself is eliminated simultaneously when genome editing occurs, thereby requiring no follow-up to remove the encoding system. Then, we develop a new extracellular electron transfer (EET) engineering strategy by programming the parallel EET systems to enhance versatile EET. The engineered strains exhibit sufficiently enhanced electron output and pollutant reduction ability. Furthermore, this device has demonstrated its great potential to be extended for genome editing in other important microbes. This work provides a useful and efficient tool for the rapid generation of synthetic microorganisms for various environmental applications.     

Isolation of the exoelectrogenic bacterium Ochrobactrum anthropi YZ-1 by using a U-tube microbial fuel cell

Exoelectrogenic bacteria have potential for many different biotechnology applications due to their ability to transfer electrons outside the cell to insoluble electron acceptors, such as metal oxides or the anodes of microbial fuel cells (MFCs). Very few exoelectrogens have been directly isolated from MFCs, and all of these organisms have been obtained by techniques that potentially restrict the diversity of exoelectrogenic bacteria. A special U-tube-shaped MFC was therefore developed to enrich exoelectrogenic bacteria with isolation based on dilution-to-extinction methods. Using this device, we obtained a pure culture identified as Ochrobactrum anthropi YZ-1 based on 16S rRNA gene sequencing and physiological and biochemical characterization. Strain YZ-1 was unable to respire using hydrous Fe(III) oxide but produced 89 mW/m(2) using acetate as the electron donor in the U-tube MFC. Strain YZ-1 produced current using a wide range of substrates, including acetate, lactate, propionate, butyrate, glucose, sucrose, cellobiose, glycerol, and ethanol. Like another exoelectrogenic bacterium (Pseudomonas aeruginosa), O. anthropi is an opportunistic pathogen, suggesting that electrogenesis should be explored as a characteristic that confers advantages to these types of pathogenic bacteria. Further applications of this new U-tube MFC system should provide a method for obtaining additional exoelectrogenic microorganisms that do not necessarily require metal oxides for cell respiration.     

A kinetic perspective on extracellular electron transfer by anode-respiring bacteria

In microbial fuel cells and electrolysis cells (MXCs), anode-respiring bacteria (ARB) oxidize organic substrates to produce electrical current. In order to develop an electrical current, ARB must transfer electrons to a solid anode through extracellular electron transfer (EET). ARB use various EET mechanisms to transfer electrons to the anode, including direct contact through outer-membrane proteins, diffusion of soluble electron shuttles, and electron transport through solid components of the extracellular biofilm matrix. In this review, we perform a novel kinetic analysis of each EET mechanism by analyzing the results available in the literature. Our goal is to evaluate how well each EET mechanism can produce a high current density (>10 A m⁻²) without a large anode potential loss (less than a few hundred millivolts), which are feasibility goals of MXCs. Direct contact of ARB to the anode cannot achieve high current densities due to the limited number of cells that can come in direct contact with the anode. Slow diffusive flux of electron shuttles at commonly observed concentrations limits current generation and results in high potential losses, as has been observed experimentally. Only electron transport through a solid conductive matrix can explain observations of high current densities and low anode potential losses. Thus, a study of the biological components that create a solid conductive matrix is of critical importance for understanding the function of ARB.