Novel Electrochemically Active Bacterium Phylogenetically Related to Arcobacter butzleri,Isolated from a Microbial Fuel Cell

Exoelectrogenic bacteria are organisms that can transfer electrons to extracellular insoluble electron acceptors and have the potential to be used in devices such as microbial fuel cells (MFCs). Currently, exoelectrogens have been identified in the Alpha-, Beta-, Gamma- and Deltaproteobacteria, as well as in the Firmicutes and Acidobacteria. Here, we describe use of culture-independent methods to identify two members of the genus Arcobacter in the Epsilonproteobacteria that are selectively enriched in an acetate-fed MFC. One of these organisms, Arcobacter butzleri strain ED-1, associates with the electrode and rapidly generates a strong electronegative potential as a pure culture when it is supplied with acetate. A mixed-community MFC in which ∼90% of the population is comprised of the two Arcobacter species generates a maximal power density of 296 mW/liter. This demonstration of exoelectrogenesis by strain ED-1 is the first time that this property has been shown for members of this genus.A microbial fuel cell (MFC) is a mimic of a biological system in which microorganisms transfer electrons from organic compounds to a conductive external electron acceptor under anaerobic conditions (6). In an MFC, the electron acceptor is provided by an artificial anode, which is connected to an electric circuit. Although the basic processes involved in the generation of electricity by bacteria have been known for many years, recent interest in MFC development has been stimulated by the need to find alternative, carbon-neutral sources of energy generation. MFCs are particularly useful for breakdown of organic matter in wastewater treatment plants, in which production of electricity as a by-product can be used to power the process or can be sold to offset the cost of operation (6). At present, although the key principles of MFC design and operation are well understood (19), the technical aspects and particularly the microbiological aspects (18) are still in development. Further optimization of the design and microbial composition of these devices is desirable as current MFCs achieve power densities of no more than 1,550 mW/liter (7), which limits their real-world applications (6).The basic microbiological characteristics which influence the efficiency of an MFC are bacterial metabolism and bacterial electron transfer. Although most current MFCs perform optimally when they contain a mixed microbial community, some pure cultures that exhibit strong electrogenic activity in the MFC environment have been characterized (19). The electrogenic properties and some aspects of extracellular electron transfer have been defined for pure cultures of organisms such as Geobacter sulfurreducens (2, 3), Escherichia coli (27), Shewanella putrefaciens (15, 16), Rhodoferax ferrireducens (5), Rhodopseudomonas palustris DX-1 (40), and Ochrobactrum anthropi YZ-1 (41). The current list of confirmed exoelectrogens includes representatives of four of the five classes of Proteobacteria (only the Epsilonproteobacteria are not represented), as well as representatives of the Firmicutes and Acidobacteria (18). However, it is likely that novel electrogenic bacteria remain to be discovered.The metabolic characteristics required for an electrogenic bacterium depend upon the specific application for which an MFC is used, because not all electrogenic bacteria are able to fully oxidize several substrates. For example, Shewanella oneidensis oxidizes lactate to acetate under anaerobic conditions, while G. metallireducens oxidizes acetate but not glucose (20). R. ferrireducens can oxidize acetate, lactate, and glucose but does not degrade ethanol, another common fermentation end product (11). For this reason, MFCs which are employed in wastewater treatment when complex compounds have to be degraded are often inoculated with a diverse microbial community (for example, methanogenic sludge [30]). Degradation of acetate is a key bacterial characteristic because acetate is a primary organic intermediate in the degradation of organic matter in anoxic aquatic sediments. Moreover, the ability to use artificial electron acceptors (anodic electrodes) provides bacteria such as Geobacteraceae with a competitive advantage over other microorganisms under such conditions. Analysis of the microbial community firmly attached to anodes harvesting electricity from a variety of sediments demonstrated that microorganisms in the family Geobacteraceae were highly enriched on these anodes (2, 35). Moreover, it was shown that an MFC initially inoculated with methanogenic sludge failed to consume acetate in the absence of anodic electrodes over a 1-year period (8).Arcobacter spp. are inhabitants of human and animal hosts (14, 37) and also occur in various environments, including wastewater (24), surface water (21), seawater (9), and groundwater (32). Arcobacter spp. belong to the Epsilonproteobacteria, which includes pathogens (e.g., Campylobacter jejuni and Helicobacter pylori), opportunistic pathogens, and nonpathogenic environmental isolates (4). Typically, these bacteria have genomes with low G+C contents (27 to 30%), although some Epsilonproteobacteria, such as Wolinella spp. and Campylobacter curvus, have higher G+C contents. The environmental bacteria group into four clusters: Nautiliales, Arcobacter, Sulfurospirillum, and Thiovulgaceae. The genus Arcobacter comprises Arcobacter butzleri, Arcobacter cryaerophilus, Arcobacter skirrowii, and Arcobacter cibarius, all of which have been isolated from animals or food (particularly poultry), as well as Arcobacter halophilus, Arcobacter nitrofigilis, “Candidatus Arcobacter sulfidicus,” and a number of species characterized so far only at the 16S rRNA gene level (4). A feature of both “Ca. Arcobacter sulfidicus” and Arcobacter sp. strain FWKBO is autotrophic metabolism under microaerobic conditions, in contrast to the heterotrophic growth of A. butzleri. Both of these organisms use oxidation of sulfide to sulfur and are obligate autotrophs. Some Arcobacter spp. may be capable of Mn and Fe reduction; isolates from Black Sea sediments (36) oxidized acetate in the presence of Mn oxide. This was the first evidence of Mn or Fe reduction in nitrate-reducing Arcobacter microaerophiles and nitrate reducers; previously, the only other epsilonproteobacterium identified with this ability was Sulfurospirillum barnesii. Thus, organisms related to Arcobacter comprise an ecologically significant new group of dissimilatory Fe- and Mn-reducing bacteria.In the present study we isolated and characterized two strains phylogenetically related to Arcobacter spp. which are selectively enriched in an acetate-fed MFC. One of these strains, A. butzleri strain ED-1, which specifically associates with the MFC electrode, shows electrochemical activity when it is grown on acetate, and hence it is the first example of an exoelectrogenic epsilonproteobacterium.     

A Novel Electrochemically Active and Fe(III)-reducing Bacterium Phylogenetically Related to Clostridium butyricum Isolated from a Microbial Fuel Cell

An obligatory anaerobic bacterium was isolated from a mediator-less microbial fuel cell using starch processing wastewater as the fuel and designated as EG3. The isolate was Gram-positive, motile and rod (2.8–3.0 μm long, 0.5–0.6 μm wide). The partial 16S rRNA gene sequence and analysis of the cellular fatty acids profile suggested that EG3 clusters with Clostridium sub-phylum and exhibited the highest similarity (98%) with Clostridium butyricum. The temperature and pH optimum for growth were 37°C and 7.0, respectively. The major products of glucose and glucose/Fe(O)OH metabolism were lactate, formate, butyrate, acetate, CO₂and H₂. Growth was faster at the initial phase and the cell yield was higher when the medium was supplemented with Fe(O)OH than without Fe(O)OH. These results suggest that Fe(III) ion is utilised as an electron sink. Cyclic voltammetry showed that Clostridium butyricum EG3 cells were electrochemically active. It is a novel characteristic of strict anaerobic Gram-positive bacteria.     

微生物燃料电池发展态势分析

随着世界经济的高速发展和人口的不断增长,能源短缺和环境污染问题日益成为制约发展的瓶颈。微生物燃料电池(microbial fuel cell,MFC)能将污染物中蕴含的化学能直接转化为电能,实现同步污水处理和电能回收,是一种极具前景的可持续污水处理技术。同时,MFC在污泥处理、生物修复、环境监测、海水淡化等方面也展示了诱人的前景。基于科睿唯安Web of Science数据库和德温特专利检索分析平台(Derwent Innovation,DI),对MFC领域1990～2018年的论文和专利数据进行统计分析,得出全球MFC领域的发展趋势、国际分布、研发热点和技术格局。在此基础上,对未来MFC领域的发展做出了展望,对中国MFC产业化发展提出了思考和建议。     

微生物燃料电池发展态势分析

随着世界经济的高速发展和人口的不断增长,能源短缺和环境污染问题日益成为制约发展的瓶颈。微生物燃料电池(microbial fuel cell,MFC)能将污染物中蕴含的化学能直接转化为电能,实现同步污水处理和电能回收,是一种极具前景的可持续污水处理技术。同时,MFC在污泥处理、生物修复、环境监测、海水淡化等方面也展示了诱人的前景。基于科睿唯安Web of Science数据库和德温特专利检索分析平台(Derwent Innovation, DI),对MFC领域1990~2018年的论文和专利数据进行统计分析,得出全球MFC领域的发展趋势、国际分布、研发热点和技术格局。在此基础上,对未来MFC领域的发展做出了展望,对中国MFC产业化发展提出了思考和建议。     

人工湿地-微生物燃料电池耦合系统的研究进展

人工湿地-微生物燃料电池耦合系统(CW-MFC)是一种将人工湿地技术(CW)和微生物燃料电池技术(MFC)结合在一起的新型污水处理系统,其产电机理是产电微生物在底层湿地(阳极)的厌氧条件下生成电子,通过外电路传递到表面湿地(阴极)完成氧化还原反应。但是,近几年来,关于CW-MFC研究的文章较少且研究深度较浅。综述了电极材料、水力条件、湿地植物及微生物等条件对CW-MFC污水处理能力和产电能力的影响。在电极材料方面,选用导电性、吸附性及有效面积大的材料作为电极可有效提高CW-MFC产电与去污能力;在水利条件方面,在HRT为2-3 d的条件下,应选用升流式或升流-降流式的入水方式;湿地植物方面,种植湿地植物的CW-MFC在去污和产电能力上都要优于未种植植物的CW-MFC;微生物方面,阴极与阳极的微生物群落结构存在明显的差异,但存在的产电菌的种类却十分相似。CW-MFC中存在的常见产电微生物主要包括地杆菌属(Geobacter)、脱硫叶菌属(Desulfobulbus)、假单胞菌属(Pseudomona)和脱硫弧菌属(Desulfovibrio)等。最后对CW-MFC的研究方向进行了分析,以期为CW-MFC的实际应用提供理论依据。     

微生物燃料电池中产电微生物的研究进展

产电微生物是微生物燃料电池系统的核心组成, 本文从生物学角度介绍了几种产电微生物的分类学地位、形态特征、生理生化特征及在微生物燃料电池中的产电机理和产电能力, 分析了利用产电微生物进行废水处理同时生物发电的应用前景, 提出产电微生物在MFC系统中的进一步研究方向为微生物的富集、驯化、改造和多种菌种优化组合等。     

微生物燃料电池中产电微生物的系统发育分析及筛选

对污水处理厂曝气池的产电微生物进行富集并利用纯培养法筛选,采用基于16S rRNA基因序列的系统发育分析方法研究了产电微生物的生物多样性,并基于三电极体系绘制出的循环伏安曲线鉴别出产电性能较强的纯菌株。结果表明,菌株F003、F042和F050与其系统发育关系最密切的有效发表种的典型菌株的16S rRNA基因序列存在较大差异,分别代表新的分类单元。之后又对所获得的38株菌株进行电化学测试活性,得出4株活性较强的菌株,其中菌株F010和F017的电化学活性比菌株F007和F051更为显著。     

微生物燃料电池中脱色希瓦氏菌S12的产电特性研究

为了确定脱色希瓦氏菌S12的电化学活性,采用循环伏安法(cyclic voltammograms, CV)对厌氧培养的菌株S12进行曲线扫描,所得曲线表明S12具有一定的电化学活性,可以用来进行产电实验.研究了不同电子供体和供体浓度对菌株S12产电的影响,结果表明,以浓度为10mmol/L的不同有机酸(甲酸钠、乳酸钠和丙酮酸钠)分别作为电子供体时,乳酸钠产电量最大,其最大功率密度Pmax为21.93mW/m2增加乳酸钠的浓度,菌株S12的产电量也相应增加,当乳酸钠的浓度为20mmol/L时,所产生的最大功率密度达55.72 mW/m2.     

Study of Cell Migration in Microfabricated Channels

The method described here allows the study of cell migration under confinement in one dimension. It is based on the use of microfabricated channels, which impose a polarized phenotype to cells by physical constraints. Once inside channels, cells have only two possibilities: move forward or backward. This simplified migration in which directionality is restricted facilitates the automatic tracking of cells and the extraction of quantitative parameters to describe cell movement. These parameters include cell velocity, changes in direction, and pauses during motion. Microchannels are also compatible with the use of fluorescent markers and are therefore suitable to study localization of intracellular organelles and structures during cell migration at high resolution. Finally, the surface of the channels can be functionalized with different substrates, allowing the control of the adhesive properties of the channels or the study of haptotaxis. In summary, the system here described is intended to analyze the migration of large cell numbers in conditions in which both the geometry and the biochemical nature of the environment are controlled, facilitating the normalization and reproducibility of independent experiments.     

微生物燃料电池中脱色希瓦氏菌S12的产电特性研究

为了确定脱色希瓦氏菌S12的电化学活性, 采用循环伏安法(cyclic voltammograms, CV)对厌氧培养的菌株S12进行曲线扫描, 所得曲线表明S12具有一定的电化学活性, 可以用来进行产电实验。研究了不同电子供体和供体浓度对菌株S12产电的影响, 结果表明, 以浓度为10 mmol/L 的不同有机酸(甲酸钠、乳酸钠和丙酮酸钠)分别作为电子供体时, 乳酸钠产电量最大, 其最大功率密度Pmax为21.93 mW/m2, 增加乳酸钠的浓度, 菌株S12的产电量也相应增加, 当乳酸钠的浓度为20 mmol/L时, 所产生的最大功率密度达55.72 mW/m2。     

Application of Microbes and Microbial Esterases to the Preparation of Optically Active N-Acetylindoline-2-Carboxylic Acid

Methylotrophic bacteria, isolated from soil samples or from sewage sludge, proved to be useful sources of esterases for catalyzing the enantioselective hydrolysis of racemic N-acetyl-indoline-2-carboxylic acid methyl ester (7) to the corresponding (25) or (2R)-N- acetyl amino acid (6) with high optical yields. From the DMF-utilizer Pseudomonas DMF 5/8 and the methanol-utilizer Isolate EE 210, the corresponding esterases were isolated. Reactions with whole cells as well as with the purified enzymes are described.     

Metagenomic Analyses Reveal the Involvement of Syntrophic Consortia in Methanol/Electricity Conversion in Microbial Fuel Cells

Methanol is widely used in industrial processes, and as such, is discharged in large quantities in wastewater. Microbial fuel cells (MFCs) have the potential to recover electric energy from organic pollutants in wastewater; however, the use of MFCs to generate electricity from methanol has not been reported. In the present study, we developed single-chamber MFCs that generated electricity from methanol at the maximum power density of 220 mW m⁻² (based on the projected area of the anode). In order to reveal how microbes generate electricity from methanol, pyrosequencing of 16S rRNA-gene amplicons and Illumina shotgun sequencing of metagenome were conducted. The pyrosequencing detected in abundance Dysgonomonas, Sporomusa, and Desulfovibrio in the electrolyte and anode and cathode biofilms, while Geobacter was detected only in the anode biofilm. Based on known physiological properties of these bacteria, it is considered that Sporomusa converts methanol into acetate, which is then utilized by Geobacter to generate electricity. This speculation is supported by results of shotgun metagenomics of the anode-biofilm microbes, which reconstructed relevant catabolic pathways in these bacteria. These results suggest that methanol is anaerobically catabolized by syntrophic bacterial consortia with electrodes as electron acceptors.     

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.Download video file.^{(96M, mov)}     

基于微电极阵列的微生物电融合

利用自主研制的梳状交叉微电极阵列细胞电融合芯片系统,研究微生物电融合。在酶解浓度1．5％、酶解温度33℃、酶解时间3h、酶解pH值6．0、0．8mol／L山梨醇的渗透压条件下,获得生成率和再生率分别为95．2％和8．9％的微生物细胞原生质体。在脉冲峰值电压50V、持续时间80μs、8个脉冲、间隔1s的电融合条件下,该原生质体通过电融合获得一株菌株其乳化性能从62％提高到85％。     

Modeling the Impact of Interspecies Competition on Performance of a Microbial Fuel Cell

Previous models of biofilms growing in a microbial fuel cell (MFC) have primarily focused on modeling a single growth mechanism: growth via a conductive biofilm matrix, or growth utilizing diffusible electron shuttles or mediators. In this work, we implement both flavors of models in order to explore the competition for space and nutrients in a MFC biofilm populated by both species types. We find that the optimal growth conditions are for bacteria that utilize conductive EPS provided a minimal energy used to create the EPS matrix. Mediator-utilizing bacteria do have favorable niche regions, most notably close to the anode and where exposed to the bulk inflow, where oxidized mediator is readily available.     

微生物燃料电池中假单胞菌F026的产电性能研究

基于微生物燃料电池的反应装置,从污水处理厂曝气池的污泥中通过富集,筛选和基于16S rRNA基因序列的系统发育分析等手段驯化出1株高效产电假单胞菌F026。以F026为阳极产电菌制作微生物燃料电池,考察了底物种类、温度和p H值等因素对微生物燃料电池产电性能的影响。结果表明,F026最适合在以可溶性淀粉为底物,p H为中性偏碱性,温度在30~35℃的环境下生长。在此条件下,微生物燃料电池的最高电压达到500 m V,体积功率密度达到2 W/m3。     

Controlled One-on-One Encounters between Immune Cells and Microbes Reveal Mechanisms of Phagocytosis

Among many challenges facing the battle against infectious disease, one quandary stands out. On the one hand, it is often unclear how well animal models and cell lines mimic human immune behavior. On the other hand, many core methods of cell and molecular biology cannot be applied to human subjects. For example, the profound susceptibility of neutropenic patients to infection marks neutrophils (the most abundant white blood cells in humans) as vital immune defenders. Yet because these cells cannot be cultured or genetically manipulated, there are gaps in our understanding of the behavior of human neutrophils. Here, we discuss an alternative, interdisciplinary strategy to dissect fundamental mechanisms of immune-cell interactions with bacteria and fungi. We show how biophysical analyses of single-live-cell/single-target encounters are revealing universal principles of immune-cell phagocytosis, while also dispelling misconceptions about the minimum required mechanistic determinants of this process.     

Simultaneous Cellulose Degradation and Electricity Production by Enterobacter cloacae in a Microbial Fuel Cell

Electricity can be directly generated by bacteria in microbial fuel cells (MFCs) from many different biodegradable substrates. When cellulose is used as the substrate, electricity generation requires a microbial community with both cellulolytic and exoelectrogenic activities. Cellulose degradation with electricity production by a pure culture has not been previously demonstrated without addition of an exogenous mediator. Using a specially designed U-tube MFC, we enriched a consortium of exoelectrogenic bacteria capable of using cellulose as the sole electron donor. After 19 dilution-to-extinction serial transfers of the consortium, 16S rRNA gene-based community analysis using denaturing gradient gel electrophoresis and band sequencing revealed that the dominant bacterium was Enterobacter cloacae. An isolate designated E. cloacae FR from the enrichment was found to be 100% identical to E. cloacae ATCC 13047^T based on a partial 16S rRNA sequence. In polarization tests using the U-tube MFC and cellulose as a substrate, strain FR produced 4.9 ± 0.01 mW/m², compared to 5.4 ± 0.3 mW/m² for strain ATCC 13047^T. These results demonstrate for the first time that it is possible to generate electricity from cellulose using a single bacterial strain without exogenous mediators.Exoelectrogenic microorganisms can release electrons to electron acceptors outside the cell, such as iron oxides or carbon anodes in microbial fuel cells (MFCs). Members of many genera, including Rhodoferax (6), Shewanella (13, 14), Pseudomonas (29), Aeromonas (28), Geobacter (2), Geopsychrobacter (10), Desulfuromonas (1), Desulfobulbus (9), Clostridium (27), Geothrix (3), Ochrobactrum (40), and Rhodopseudomonas (38), have been shown to produce electricity in an MFC. These bacteria have been grown on simple soluble substrates, such as glucose or acetate, that can be directly taken into the cell and used for energy production.Cellulose is the most abundant biopolymer in the world, and there is great interest in using this material as a substrate in an MFC. However, use of a particulate substrate in an MFC has not been well investigated. Cellulose must first be hydrolyzed to a soluble substrate that can be taken up by the cell. In previous MFC tests this has required the use of enzymes to hydrolyze the cellulose into sugars or the use of cocultures or mixed cultures (32, 33, 35). For example, Ren et al. (32) used a coculture of the cellulose fermentor Clostridium cellulolyticum and the exoelectrogen Geobacter sulfurreducens to generate electricity in an MFC fed with cellulose. Analysis of the anode microbial communities in other studies of cellulose-fed MFCs showed that Clostridium spp. (in a biofilm) and Comamonadaceae (in suspension) were predominant when rumen contents were used as an inoculum (35), while a rice paddy soil inoculum (12) converged to a Rhizobiales-dominated anode community (more than 30% of the population). To date, it has not been demonstrated that a single microbe can both degrade cellulose and generate current.Conventional methods of isolating exoelectrogenic microorganisms are based primarily on identifying microorganisms that can respire using soluble or insoluble metal oxides in agar plates (20-22). However, not all dissimilatory metal oxide-reducing bacteria are capable of producing electricity in an MFC, and not all bacteria that produce current in an MFC can grow using metal oxides (5, 34). Therefore, these methods may miss important electrochemically active strains of microorganisms. A new method to isolate exoelectrogenic microorganisms was recently developed (40); this method is based on dilution to extinction and a specially designed U-tube MFC that enriches exoelectrogenic bacteria on the anode. Using this method, a bacterium that could produce electricity in an MFC but not respire using iron was isolated (40).The main objective of this study was to isolate a bacterium capable of producing current from particulate cellulose. A cellulose-degrading consortium was diluted and serially transferred into U-tube MFCs using cellulose as the sole electron donor. Community analysis demonstrated the predominance of a single bacterium, which was isolated and compared to a culture collection strain for generation of current in an MFC.     

Bioinformatic Approaches Reveal Metagenomic Characterization of Soil Microbial Community

As is well known, soil is a complex ecosystem harboring the most prokaryotic biodiversity on the Earth. In recent years, the advent of high-throughput sequencing techniques has greatly facilitated the progress of soil ecological studies. However, how to effectively understand the underlying biological features of large-scale sequencing data is a new challenge. In the present study, we used 33 publicly available metagenomes from diverse soil sites (i.e. grassland, forest soil, desert, Arctic soil, and mangrove sediment) and integrated some state-of-the-art computational tools to explore the phylogenetic and functional characterizations of the microbial communities in soil. Microbial composition and metabolic potential in soils were comprehensively illustrated at the metagenomic level. A spectrum of metagenomic biomarkers containing 46 taxa and 33 metabolic modules were detected to be significantly differential that could be used as indicators to distinguish at least one of five soil communities. The co-occurrence associations between complex microbial compositions and functions were inferred by network-based approaches. Our results together with the established bioinformatic pipelines should provide a foundation for future research into the relation between soil biodiversity and ecosystem function.