Characterization of electrochemical activity of a strain ISO2‐3 phylogenetically related to Aeromonas sp. isolated from a glucose‐fed microbial fuel cell

The microbial communities associated with electrodes in closed and open circuit microbial fuel cells (MFCs) fed with glucose were analyzed by 16S rRNA approach and compared. The comparison revealed that bacteria affiliated with the Aeromonas sp. within the Gammaproteobacteria constituted the major population in the closed circuit MFC (harvesting electricity) and considered to play important roles in current generation. We, therefore, attempted to isolate the dominant bacteria from the anode biofilm, successfully isolated a Fe (III)‐reducing bacterium phylogenetically related to Aeromonas sp. and designated as strain ISO2‐3. The isolated strain ISO2‐3 could grow and concomitantly produce current (max. 0.24 A/m²) via oxidation of glucose or hydrogen with an electrode serving as the sole electron acceptor. The strain could ferment glucose, but generate less electrical current. Cyclic voltammetry supported the strain ISO2‐3 was electrically active and likely to transfer electrons to the electrode though membrane‐associated compounds (most likely c‐type cytochrome). This mechanism requires intimate contact with the anode surface. Scanning electron microscopy revealed that the strain ISO2‐3 developed multiplayer biofilms on the anode surface and also produced anchor‐like filamentous appendages (most likely pili) that may promote long‐range electron transport across the thick biofilm. Biotechnol. Bioeng. 2009; 104: 901–910. © 2009 Wiley Periodicals, Inc.     

Electricity generation and microbial community analysis of alcohol powered microbial fuel cells

Two different microbial fuel cell (MFC) configurations were investigated for electricity production from ethanol and methanol: a two-chambered, aqueous-cathode MFC; and a single-chamber direct-air cathode MFC. Electricity was generated in the two-chamber system at a maximum power density typical of this system (40+/-2 mW/m2) and a Coulombic efficiency (CE) ranging from 42% to 61% using ethanol. When bacteria were transferred into a single-chamber MFC known to produce higher power densities with different substrates, the maximum power density increased to 488+/-12 mW/m2 (CE = 10%) with ethanol. The voltage generated exhibited saturation kinetics as a function of ethanol concentration in the two-chambered MFC, with a half-saturation constant (Ks) of 4.86 mM. Methanol was also examined as a possible substrate, but it did not result in appreciable electricity generation. Analysis of the anode biofilm and suspension from a two-chamber MFC with ethanol using 16S rDNA-based techniques indicated that bacteria with sequences similar to Proteobacterium Core-1 (33.3% of clone library sequences), Azoarcus sp. (17.4%), and Desulfuromonas sp. M76 (15.9%) were significant members of the anode chamber community. These results indicate that ethanol can be used for sustained electricity generation at room temperature using bacteria on the anode in a MFC.     

Use of acetate for enrichment of electrochemically active microorganisms and their 16S rDNA analyses

A fuel cell-type electrochemical device has been used to enrich microbes oxidizing acetate with concomitant electricity generation without using an electron mediator from activated sludge. The device generated a stable current of around 5 mA with complete oxidation of 5 mM acetate at the hydraulic retention time of 2.5 h after 4 weeks of enrichment. Over 70% of electrons available from acetate oxidation was recovered as current. Carbon monoxide or hydrogen did not influence acetate oxidation or current generation from the microbial fuel cell (MFC). Denaturing gradient gel electrophoresis showed that DNA extracted from the acetate-enriched MFC had different 16S rDNA patterns from those of sludge or glucose+glutamate-enriched MFCs. Nearly complete 16S rDNA sequence analyses showed that diverse bacteria were enriched in the MFC fed with acetate. Electron microscopic observations showed biofilm developed on the electrode, but not microbial clumps observed in MFCs fed with complex fuel such as glucose and wastewater from a corn-processing factory.     

Evaluation of procedures to acclimate a microbial fuel cell for electricity production

A microbial fuel cell (MFC) is a relatively new type of fixed film bioreactor for wastewater treatment, and the most effective methods for inoculation are not well understood. Various techniques to enrich electrochemically active bacteria on an electrode were therefore studied using anaerobic sewage sludge in a two-chambered MFC. With a porous carbon paper anode electrode, 8 mW/m² of power was generated within 50 h with a Coulombic efficiency (CE) of 40%. When an iron oxide-coated electrode was used, the power and the CE reached 30 mW/m² and 80%, respectively. A methanogen inhibitor (2-bromoethanesulfonate) increased the CE to 70%. Bacteria in sludge were enriched by serial transfer using a ferric iron medium, but when this enrichment was used in a MFC the power was lower (2 mW/m²) than that obtained with the original inoculum. By applying biofilm scraped from the anode of a working MFC to a new anode electrode, the maximum power was increased to 40 mW/m². When a second anode was introduced into an operating MFC the acclimation time was not reduced and the total power did not increase. These results suggest that these active inoculating techniques could increase the effectiveness of enrichment, and that start up is most successful when the biofilm is harvested from the anode of an existing MFC and applied to the new anode.     

Continuous power generation and microbial community structure of the anode biofilms in a three-stage microbial fuel cell system

A mediator-less three-stage two-chamber microbial fuel cell (MFC) system was developed and operated continuously for more than 1.5 years to evaluate continuous power generation while treating artificial wastewater containing glucose (10 mM) concurrently. A stable power density of 28 W/m³ was attained with an anode hydraulic retention time of 4.5 h and phosphate buffer as the cathode electrolyte. An overall dissolved organic carbon removal ratio was about 85%, and coulombic efficiency was about 46% in this MFC system. We also analyzed the microbial community structure of anode biofilms in each MFC. Since the environment in each MFC was different due to passing on the products to the next MFC in series, the microbial community structure was different accordingly. The anode biofilm in the first MFC consisted mainly of bacteria belonging to the Gammaproteobacteria, identified as Aeromonas sp., while the Firmicutes dominated the anode biofilms in the second and third MFCs that were mainly fed with acetate. Cyclic voltammetric results supported the presence of a redox compound(s) associated with the anode biofilm matrix, rather than mobile (dissolved) forms, which could be responsible for the electron transfer to the anode. Scanning electron microscopy revealed that the anode biofilms were comprised of morphologically different cells that were firmly attached on the anode surface and interconnected each other with anchor-like filamentous appendages, which might support the results of cyclic voltammetry. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electricity generation by anaerobic bacteria and anoxic sediments from hypersaline soda lakes

Anaerobic bacteria and anoxic sediments from soda lakes produced electricity in microbial fuel cells (MFCs). No electricity was generated in the absence of bacterial metabolism. Arsenate respiring bacteria isolated from moderately hypersaline Mono Lake (Bacillus selenitireducens), and salt-saturated Searles Lake, CA (strain SLAS-1) oxidized lactate using arsenate as the electron acceptor. However, these cultures grew equally well without added arsenate using the MFC anode as their electron acceptor, and in the process oxidized lactate more efficiently. The decrease in electricity generation by consumption of added alternative electron acceptors (i.e. arsenate) which competed with the anode for available electrons proved to be a useful indicator of microbial activity and hence life in the fuel cells. Shaken sediment slurries from these two lakes also generated electricity, with or without added lactate. Hydrogen added to sediment slurries was consumed but did not stimulate electricity production. Finally, electricity was generated in statically incubated “intact” sediment cores from these lakes. More power was produced in sediment from Mono Lake than from Searles Lake, however microbial fuel cells could detect low levels of metabolism operating under moderate and extreme conditions of salt stress.     

The Performance of a Microbial Fuel Cell Depends Strongly on Anode Geometry: A Multidimensional Modeling Study

A multidimensional biofilm model is developed to simulate biofilm growth on the anode of a Microbial Fuel Cell (MFC). The biofilm is treated as a conductive material, and electrons produced during microbial growth are assumed to be transferred to the anode through a conductive biofilm matrix. Growth of Geobacter sulfurreducens is simulated using the Nernst–Monod kinetic model that was previously developed and later validated in experiments. By implementing a conduction-based biofilm model in two dimensions, we are able to explore the impact of anode density and arrangement on current production in a MFC.     

New exoelectrogen Citrobacter sp. SX-1 isolated from a microbial fuel cell

Aims: Isolation, identification and characterization of a new exoelectrogenic bacterium from a microbial fuel cell (MFC). Methods and Results: Exoelectrogenic bacterial strain SX‐1 was isolated from a mediator‐less MFC by conventional plating techniques with ferric citrate as electron acceptor under anaerobic condition. Phylogenetic analysis of the 16S rDNA sequence revealed that it was related to the members of Citrobacter genus with Citrobacter sp. sdy‐48 being the most closely related species. The bacterial strain SX‐1 produced electricity from citrate, acetate, glucose, sucrose, glycerol and lactose in MFCs with the highest current density of 205 mA m^?2 generated from citrate. Cyclic voltammetry analysis indicated that membrane‐associated proteins may play an important role in facilitating the electrons transferring from bacteria to electrode. Conclusions: This is the first study that demonstrates that Citrobacter species can transfer electrons to extracellular electron acceptors. Citrobacter strain SX‐1 is capable of generating electricity from a wide range of substrates in MFCs. Significance and Impact of the Study: This finding increases the known diversity of power generating exoelectrogens and provided a new strain to explore the mechanisms of extracellular electron transfer from bacteria to electrode. The wide range of substrate utilization by SX‐1 increases the application potential of MFCs in renewable energy generation and waste treatment.     

产电微生物及微生物燃料电池最新研究进展

新型产电微生物(Electricigens)的发现,使得微生物燃料电池概念的内涵发生了根本性的变化,展现了广阔的应用前景。这种微生物能够以电极作为唯一电子受体,把氧化有机物获得的电子通过电子传递链传递到电极产生电流,同时微生物从中获得能量而生长。这种代谢被认为是一种新型微生物呼吸方式。以这种新型微生物呼吸方式为基础的微生物燃料电池可以同时进行废水处理和生物发电,有望可以把废水处理发展成一个有利可图的产业,是MFC最有发展前景的方向。     

Thionine increases electricity generation from microbial fuel cell using Saccharomyces cerevisiae and exoelectrogenic mixed culture

Microbial fuel cells (MFCs) have been shown to be capable of clean energy production through the oxidation of biodegradable organic waste using various bacterial species as biocatalysts. In this study we found Saccharomyces cerevisiae, previously known electrochemcially inactive or less active species, can be acclimated with an electron mediator thionine for electrogenic biofilm formation in MFC, and electricity production is improved with facilitation of electron transfer. Power generation of MFC was also significantly increased by thionine with both aerated and non-aerated cathode. With electrochemically active biofilm enriched with swine wastewater, MFC power increased more significantly by addition of thionine. The optimum mediator concentration was 500 mM of thionine with S. cerevisae in MFC with the maximum voltage and current generation in the microbial fuel cell were 420 mV and 700 mA/m(2), respectively. Cyclic voltametry shows that thionine improves oxidizing and reducing capability in both pure culture and acclimated biofilm as compared to non-mediated cell. The results obtained indicated that thionine has great potential to enhance power generation from unmediated yeast or electrochemically active biofilm in MFC.     

Understanding the degradation of Congo red and bacterial diversity in an air–cathode microbial fuel cell being evaluated for simultaneous azo dye removal from wastewater and bioelectricity generation

We investigated the mechanism of Congo red degradation and bacterial diversity in a single-chambered microbial fuel cell (MFC) incorporating a microfiltration membrane and air–cathode. The MFC was operated continuously for more than 4 months using a mixture of Congo red and glucose as fuel. We demonstrated that the Congo red azo bonds were reduced at the anode to form aromatic amines. This is consistent with the known mechanism of anaerobic biodegradation of azo dyes. The MFC developed a less dense biofilm at the anode in the presence of Congo red compared to its absence indicating that Congo red degradation negatively affected biofilm formation. Denaturing gradient gel electrophoresis and direct 16S ribosomal DNA gene nucleotide sequencing revealed that the microbial communities differed depending on whether Congo red was present in the MFC. Geobacter-like species known to generate electricity were detected in the presence or absence of Congo red. In contrast, Azospirillum, Methylobacterium, Rhodobacter, Desulfovibrio, Trichococcus, and Bacteroides species were only detected in its presence. These species were most likely responsible for degrading Congo red.     

Effect of enrichment procedures on performance and microbial diversity of microbial fuel cell for Congo red decolorization and electricity generation

The purpose of this study was to determine the effect of enrichment procedure on the performance and microbial diversity of an air-cathode microbial fuel cell (MFC) which was explored for simultaneous azo dye decolorization and electricity generation. Two different enrichment procedures in which glucose and Congo red were added into the MFCs sequentially (EP1) or simultaneously (EP2) were tested by operating parallel MFCs independently for more than 6 months. The power density, electrode potential, Congo red decolorization, biofilm morphology, and bacterial diversity of the MFCs under the two enrichment procedures were compared and investigated. The results showed that the enrichment procedures have a negligible effect on the dye decolorization, but significantly affected the electricity generation. More than 90% decolorization at dye concentration of 300 mg/L was achieved within 170 h for the two tested enrichment procedures. However, the MFC with EP2 achieved a maximum power density of 192 mW/m², which was 75% higher than that of the MFC with EP1 (110 mW/m²). The depressed surfaces of the bacteria in the MFC with EP1 indicated the allergic response caused by the subsequent addition of Congo red. 16S rRNA sequencing analysis demonstrated a phylogenetic diversity in the communities of the anode biofilm and showed clear differences between the anode-attached populations in the MFCs with a different enrichment procedure. This study suggests that the enrichment procedure is important for the MFC explored for simultaneous dye decolorization and electricity generation.     

Enhancement of electricity production in a mediatorless air–cathode microbial fuel cell using <Emphasis Type="Italic">Klebsiella</Emphasis> sp. IR21

A novel dissimilatory iron-reducing bacteria, Klebsiella sp. IR21, was isolated from the anode biofilm of an MFC reactor. Klebsiella sp. IR21 reduced 27.8 % of ferric iron to ferrous iron demonstrating that Klebsiella sp. IR21 has electron transfer ability. Additionally, Klebsiella sp. IR21 generated electricity forming a biofilm on the anode surface. When a pure culture of Klebsiella sp. IR21 was supplied into a single chamber, air–cathode MFC fed with a mixture of glucose and acetate (500 mg L^?1 COD), 40–60 mV of voltage (17–26 mA m^?2 of current density) was produced. Klebsiella sp. IR21 was also utilized as a biocatalyst to improve the electrical performance of a conventional MFC reactor. A single chamber, air–cathode MFC was fed with reject wastewater (10,000 mg L^?1 COD) from a H₂ fermentation reactor. The average voltage, current density, and power density were 142.9 ± 25.74 mV, 60.5 ± 11.61 mA m^?2, and 8.9 ± 3.65 mW m^?2, respectively, in the MFC without inoculation of Klebsiella sp. IR21. However, these electrical performances of the MFC were significantly increased to 204.7 ± 40.24 mV, 87.5 ± 17.20 mA m^?2, and 18.6 ± 7.23 mW m^?2, respectively, with inoculation of Klebsiella sp. IR21. The results indicate that Klebsiella sp. IR21 can be utilized as a biocatalyst for enhancement of electrical performance in MFC systems.     

Power generation from cellulose using mixed and pure cultures of cellulose-degrading bacteria in a microbial fuel cell

Microbial fuel cells (MFCs) have been used to generate electricity from various organic compounds such as acetate, glucose, and lactate. We demonstrate here that electricity can be produced in an MFC using cellulose as the electron donor source. Tests were conducted using two-chambered MFCs, the anode medium was inoculated with mixed or pure culture of cellulose-degrading bacteria Nocardiopsis sp. KNU (S strain) or Streptomyces enissocaesilis KNU (K strain), and the catholyte in the cathode compartment was 50mM ferricyanide as catholyte. The power density for the mixed culture was 0.188mW (188mW/m(2)) at a current of 0.5mA when 1g/L cellulose was used. However, the power density decreased as the cellulose concentration in the anode compartment decreased. The columbic efficiencies (CEs) ranged from 41.5 to 33.4%, corresponding to an initial cellulose concentration of 0.1-1.0g/L. For the pure culture, cellobioase enzyme was added to increase the conversion of cellulose to simple sugars, since electricity production is very low. The power densities for S and K strain pure cultures with cellobioase were 162mW/m(2) and 145mW/m(2), respectively. Cyclic voltammetry (CV) experiments showed the presence of peaks at 380, 500, and 720mV vs. Ag/AgCl for the mixed bacterial culture, indicating its electrochemical activity without an external mediator. Furthermore, this MFC system employs a unique microbial ecology in which both the electron donor (cellulose) and the electron acceptor (carbon paper) are insoluble.     

Light/electricity conversion by a self-organized photosynthetic biofilm in a single-chamber reactor

Biological energy-conversion systems are attractive in terms of their self-sustaining and self-organizing nature and are expected to be applied to low-cost and environment-friendly processes. Here we show a biofilm-based light/electricity-conversion system that was self-organized from a natural microbial community. A bioreactor equipped with an air cathode and graphite-felt anode was inoculated with a green hot-spring microbial mat. When the reactor was irradiated with light, electric current was generated between the anode and cathode in accordance with the formation of green biofilm on the anode. Fluorescence microscopy of the green biofilm revealed the presence of chlorophyll-containing microbes of ∼10 μm in size, and these cells were abundant close to the surface of the biofilm. The biofilm community was also analyzed by sequencing of polymerase chain reaction-amplified small-subunit rRNA gene fragments, showing that sequence types affiliated with Chlorophyta, Betaproteobacteria, and Bacteroidetes were abundantly detected. These results suggest that green algae and heterotrophic bacteria cooperatively converted light energy into electricity.     

Comparative Metagenomics of Anode-Associated Microbiomes Developed in Rice Paddy-Field Microbial Fuel Cells

In sediment-type microbial fuel cells (sMFCs) operating in rice paddy fields, rice-root exudates are converted to electricity by anode-associated rhizosphere microbes. Previous studies have shown that members of the family Geobacteraceae are enriched on the anodes of rhizosphere sMFCs. To deepen our understanding of rhizosphere microbes involved in electricity generation in sMFCs, here, we conducted comparative analyses of anode-associated microbiomes in three MFC systems: a rice paddy-field sMFC, and acetate- and glucose-fed MFCs in which pieces of graphite felt that had functioned as anodes in rice paddy-field sMFC were used as rhizosphere microbe-bearing anodes. After electric outputs became stable, microbiomes associated with the anodes of these MFC systems were analyzed by pyrotag sequencing of 16S rRNA gene amplicons and Illumina shotgun metagenomics. Pyrotag sequencing showed that Geobacteraceae bacteria were associated with the anodes of all three systems, but the dominant Geobacter species in each MFC were different. Specifically, species closely related to G. metallireducens comprised 90% of the anode Geobacteraceae in the acetate-fed MFC, but were only relatively minor components of the rhizosphere sMFC and glucose-fed MFC, whereas species closely related to G. psychrophilus were abundantly detected. This trend was confirmed by the phylogenetic assignments of predicted genes in shotgun metagenome sequences of the anode microbiomes. Our findings suggest that G. psychrophilus and its related species preferentially grow on the anodes of rhizosphere sMFCs and generate electricity through syntrophic interactions with organisms that excrete electron donors.     

Sustained generation of electricity by the spore-forming,Gram-positive, <Emphasis Type="Italic">Desulfitobacterium hafniense</Emphasis> strain DCB2

Desulfitobacterium hafniense strain DCB2 generates electricity in microbial fuel cells (MFCs) when humic acids or the humate analog anthraquinone-2,6-disulfonate (AQDS) is added as an electron-carrying mediator. When utilizing formate as fuel, the Gram-positive, spore-forming bacterium generated up to 400 mW/m² of cathode surface area in a single-chamber MFC with a platinum-containing air-fed cathode. Hydrogen, lactate, pyruvate, and ethanol supported electricity generation, but acetate, propionate, and butyrate did not. Scanning electron microscopy indicated that strain DCB2 colonized the surface of a current-generating anode but not of an unconnected electrode. The electricity was recovered fully within minutes after the exchange of the medium in the anode chamber and within a week after an exposure of a colonized anode to 90°C for 20 min. Of the six strains of Desulfitobacteria tested, all of which would reduce AQDS, only D. hafniense strain DCB2 continued to reduce AQDS and generate electricity for more than 24 h, indicating that reduction of the humate analog alone is insufficient to sustain electrode reduction.     

Analysis of ammonia loss mechanisms in microbial fuel cells treating animal wastewater

Ammonia losses during swine wastewater treatment were examined using single- and two-chambered microbial fuel cells (MFCs). Ammonia removal was 60% over 5 days for a single-chamber MFC with the cathode exposed to air (air-cathode), versus 69% over 13 days from the anode chamber in a two-chamber MFC with a ferricyanide catholyte. In both types of systems, ammonia losses were accelerated with electricity generation. For the air-cathode system, our results suggest that nitrogen losses during electricity generation were increased due to ammonia volatilization with conversion of ammonium ion to the more volatile ammonia species as a result of an elevated pH near the cathode (where protons are consumed). This loss mechanism was supported by abiotic tests (applied voltage of 1.1 V). In a two-chamber MFC, nitrogen losses were primarily due to ammonium ion diffusion through the membrane connecting the anode and cathode chambers. This loss was higher with electricity generation as the rate of ammonium transport was increased by charge transfer across the membrane. Ammonia was not found to be used as a substrate for electricity generation, as intermittent ammonia injections did not produce power. The ammonia-oxidizing bacterium Nitrosomonas europaea was found on the cathode electrode of the single-chamber system, supporting evidence of biological nitrification, but anaerobic ammonia-oxidizing bacteria were not detected by molecular analyses. It is concluded that ammonia losses from the anode chamber were driven primarily by physical-chemical factors that are increased with electricity generation, although some losses may occur through biological nitrification and denitrification.     

Electricity generation from glucose by a Klebsiella sp. in microbial fuel cells

As electrochemically active bacteria play an important role in microbial fuel cells (MFCs), it is necessary to get a comprehensive understanding of their electrogenesis mechanisms. In this study, a new electrochemically active bacterium, Klebsiella sp. ME17, was employed into an “H” typed MFC for electrogenesis, with glucose as the electron donor. The maximum power density was 1,209 mW/m² at a resistance of 340 Ω and the maximum current was 1.47 mA. Given the original anode medium, fresh medium, and the supernatant of the anode medium in the same MFC, respectively, the polarization curves illustrated that the strain produced mediators to promote extracellular electron transfer. The anode medium supernatant was electrochemically active, based on cyclic voltammogram, and the supernatant was very likely to contain quinone-like substances, as indicated by spectrophotometric and excitation–emission matrix fluorescence spectroscopy analysis. Further investigation on the color and ultraviolet absorbance at 254 nm of the filtered anode medium showed that the redox states of mediators strongly associated with the electricity generation states in MFCs.     

Characterization of Exoelectrogenic Bacteria Enterobacter Strains Isolated from a Microbial Fuel Cell Exposed to Copper Shock Load

Microorganisms capable of generating electricity in microbial fuel cells (MFCs) have gained increasing interest. Here fourteen exoelectrogenic bacterial strains were isolated from the anodic biofilm in an MFC before and after copper (Cu) shock load by Hungate roll-tube technique with solid ferric (III) oxide as an electron acceptor and acetate as an electron donor. Phylogenetic analysis of the 16S rRNA gene sequences revealed that they were all closely related to Enterobacter ludwigii DSM 16688^T within the Enterobacteriaceae family, although these isolated bacteria showed slightly different morphology before and after Cu shock load. Two representative strains R2B1 (before Cu shock load) and B4B2 (after Cu shock load) were chosen for further analysis. B4B2 is resistant to 200 mg L⁻¹ of Cu(II) while R2B1 is not, which indicated the potential selection of the Cu shock load. Raman analysis revealed that both R2B1 and B4B2 contained c-type cytochromes. Cyclic voltammetry measurements revealed that strain R2B1 had the capacity to transfer electrons to electrodes. The experimental results demonstrated that strain R2B1 was capable of utilizing a wide range of substrates, including Luria-Bertani (LB) broth, cellulose, acetate, citrate, glucose, sucrose, glycerol and lactose to generate electricity, with the highest current density of 440 mA·m⁻² generated from LB-fed MFC. Further experiments indicated that the bacterial cell density had potential correlation with the current density.