Enrichment of electrochemically active bacteria using a three-electrode electrochemical cell

Electrochemically active bacteria were successfully enriched in an electrochemical cell using a positively poised working electrode. The positively poised working electrode (+0.7 V vs. Ag/AgCl) was used as an electron acceptor for enrichment and growth of electrochemically active bacteria. When activated sludge and synthetic wastewater were fed to the electrochemical cell, a gradual increase in amperometric current was observed. After a period of time in which the amperometric current was stabilized (generally 8 days), linear correlations between the amperometric signals from the electrochemical cell and added BOD (biochemical oxygen demand) concentrations were established. Cyclic voltammetry of the enriched electrode also showed prominent electrochemical activity. When the enriched electrodes were examined with electron microscopy and confocal scanning laser microscopy, a biofilm on the enriched electrode surface and bacterium-like particles were observed. These experimental results indicate that the electrochemical system in this study is a useful tool for the enrichment of an electrochemically active bacterial consortium and could be used as a novel microbial biosensor.     

Continuous electricity production from artificial wastewater using a mediator-less microbial fuel cell

A microbial fuel cell (MFC) was optimized in terms of MFC design factors and operational parameters for continuous electricity production using artificial wastewater (AW). The performance of MFC was analyzed through the polarization curve method under different conditions using a mediator-less MFC. The highest power density of 0.56 W/m2 was achieved with AW of 300 mg/l fed at the rate of 0.53 ml/min at 35 degrees C. The power per unit cell working volume was 102 mW/l, which was over 60 times higher than those reported in the previous mediator-less MFCs which did not use a cathode or an anode mediator. The power could be stably generated over 2 years.     

Enrichment of microbial electrolysis cell biocathodes from sediment microbial fuel cell bioanodes

Electron-accepting (electrotrophic) biocathodes were produced by first enriching graphite fiber brush electrodes as the anodes in sediment-type microbial fuel cells (sMFCs) using two different marine sediments and then electrically inverting the anodes to function as cathodes in two-chamber bioelectrochemical systems (BESs). Electron consumption occurred at set potentials of -439 mV and -539 mV (versus the potential of a standard hydrogen electrode) but not at -339 mV in minimal media lacking organic sources of energy. Results at these different potentials were consistent with separate linear sweep voltammetry (LSV) scans that indicated enhanced activity (current consumption) below only ca. -400 mV. MFC bioanodes not originally acclimated at a set potential produced electron-accepting (electrotrophic) biocathodes, but bioanodes operated at a set potential (+11 mV) did not. CO(2) was removed from cathode headspace, indicating that the electrotrophic biocathodes were autotrophic. Hydrogen gas generation, followed by loss of hydrogen gas and methane production in one sample, suggested hydrogenotrophic methanogenesis. There was abundant microbial growth in the biocathode chamber, as evidenced by an increase in turbidity and the presence of microorganisms on the cathode surface. Clone library analysis of 16S rRNA genes indicated prominent sequences most similar to those of Eubacterium limosum (Butyribacterium methylotrophicum), Desulfovibrio sp. A2, Rhodococcus opacus, and Gemmata obscuriglobus. Transfer of the suspension to sterile cathodes made of graphite plates, carbon rods, or carbon brushes in new BESs resulted in enhanced current after 4 days, demonstrating growth by these microbial communities on a variety of cathode substrates. This report provides a simple and effective method for enriching autotrophic electrotrophs by the use of sMFCs without the need for set potentials, followed by the use of potentials more negative than -400 mV.     

Treatment of biodiesel production wastes with simultaneous electricity generation using a single-chamber microbial fuel cell

Biodiesel production through transesterification of lipids generates large quantity of biodiesel waste (BW) containing mainly glycerin. BW can be treated in various ways including distillation to produce glycerin, use as substrate for fermentative propanediol production and discharge as wastes. This study examined microbial fuel cells (MFCs) to treat BW with simultaneous electricity generation. The maximum power density using BW was 487 ± 28 mW/m² cathode (1.5 A/m² cathode) with 50 mM phosphate buffer solution (PBS) as the electrolyte, which was comparable with 533 ± 14 mW/m² cathode obtained from MFCs fed with glycerin medium (COD 1400 mg/L). The power density increased from 778 ± 67 mW/m² cathode using carbon cloth to 1310 ± 15 mW/m² cathode using carbon brush as anode in 200 mM PBS electrolyte. The power density was further increased to 2110 ± 68 mW/m² cathode using the heat-treated carbon brush anode. Coulombic efficiencies (CEs) increased from 8.8 ± 0.6% with carbon cloth anode to 10.4 ± 0.9% and 18.7 ± 0.9% with carbon brush anode and heat-treated carbon brush anode, respectively.     

Online monitoring of yeast cultivation using a fuel-cell-type activity sensor

A microbial fuel-cell type activity sensor integrated into 500 mL and 3.2 L bioreactors was employed for ampero- (μA) and potentiometric (mV) measurements. The aim was to follow the microbial activity during ethanol production by Saccharomyces cerevisiae and to detect the end of carbohydrate consumption. Three different sensor setups were tested to record electrochemical signals produced by the metabolism of glucose and fructose (1:1) online. In a first setup, a reference electrode was used to record the potentiometric values, which rose from 0.26 to 0.5 V in about 10 h during the growth phase. In a second setup, a combination of ampero- and pseudo-potentiometric measurements delivered a maximum voltage of 35 mV. In this arrangement, the pseudo-potentiometric signal changed in a manner that was directly proportional to the amperometric signals, which reached a maximum value of 32 μA. In a third type of arrangement, a reference electrode was added to the anodic bioreactor compartment to carry out ampero- and potentiometric measurements; this is made possible by the high internal resistance of the cultivation. In this case, the reference potential rose to 0.44 V while the current maximum recorded by the working electrodes reached 27 μA. Reference and pseudo-reference electrodes were in all cases K₃Fe(CN)₆/carbon. Electrodes were made of 9 cm² woven graphite. To compare the electrochemical signals with established values, the metabolism was also monitored for optical density (at 600 nm) indicating biomass production. For fructose and glucose conversion, HPLC with an Aminex column and RI detector was used, and ethanol production was analyzed by GC with methanol as internal standard. The combination of amperometric and potentiometric recordings was found to be an ideal setup and was successfully used in reproducible cultivations.     

Enrichment and characterization of a microbial community from tannery effluent for degradation of pentachlorophenol

A bacterial community obtained by continuous enrichment from the microbial population of tannery effluent using pentachlorophenol (PCP) as sole source of carbon and energy, contained four different bacterial species including Serratia marcescens (three isolates, TE₁, TE₂ and TE₄) and Pseudomonas fluorescens (one isolate, TE₃). The members of the community grew separately on various chlorinated compounds, carbon and nitrogen sources and exhibited a remarkable ability to utilize PCP. Biodegradation studies revealed a time-dependent disappearance of PCP and its intermediary metabolites, tetrachloro-p-hydroquinone and chlorohydroquinone, and indicated the individual role of members of the community in the degradation of PCP.     

Experimental evaluation of influential factors for electricity harvesting from sediment using microbial fuel cell

The aim of this study was to evaluate limiting factors affecting electricity output from sediment microbial fuel cells (sediment MFCs). In laboratory tests, various factors likely to be encountered in field application were divided into controllable and uncontrollable ones. Based on the findings, it could be suggested that the sediment MFCs can be operated with an anode to cathode area ratio of at least 5:1 and at high external loads (1000 Ω) when the cathode is closely placed to the anode, though DO concentration at the cathode must be kept above 3 mg/l. Furthermore, no significant effect on current production over a prolonged period was observed within the sediment temperature range of 20–35 °C, but was negatively affected by lower temperatures (10 °C). These observations provide important factors with respect to the construction and operation of sediment MFCs at field sites, which will aid in maximizing electricity output.     

Effect of bacterial cell size on electricity generation in a single-compartmented microbial fuel cell

A single-compartmented microbial fuel cell composed of a graphite felt anode modified with Neutral Red (NR-anode) and a porous Fe(II)-carbon cathode (FeC-cathode) were compared for electricity generation from Microbacterium sp. and Pseudomonas sp. under identical conditions. Pseudomonas sp. was more than four times the size of Microbacterium sp. based on SEM images. In cyclic voltammetry, the redox reaction between Microbacterium sp and electrode was three times the rate observed between Pseudomonas sp. and the electrode based on the Y-axis (current) variation of cyclic voltammogram. The electric power generated by Microbacterium sp. was approx 3–4 times higher than that with Pseudomonas sp. during incubation for more than 150 days in the fuel cell.     

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.     

Analysis of a microbial electrochemical cell using the proton condition in biofilm (PCBIOFILM) model

Common to all microbial electrochemical cells (MXCs) are the anode-respiring bacteria (ARB), which transfer electrons to an anode and release protons that must transport out of the biofilm. Here, we develop a novel modeling platform, Proton Condition in BIOFILM (PCBIOFILM), with a structure geared towards mechanistically explaining: (1) how the ARB half reaction produces enough acid to inhibit the ARB by low pH; (2) how the diffusion of alkalinity carriers (phosphates and carbonates) control the pH gradients in the biofilm anode; (3) how increasing alkalinity attenuates pH gradients and increases current; and (4) why carbonates enable higher current density than phosphates. Analysis of literature data using PCBIOFILM supports the hypothesis that alkalinity limits the maximum current density for MXCs. An alkalinity criterion for eliminating low-pH limitation - 12 mg CaCO₃/mg BOD - implies that a practical MXC can achieve a maximum current density with an effluent quality comparable to anaerobic digestion.     

微生物燃料电池利用乳酸产电性能与微生物群落分布特征

【目的】为探讨以乳酸为基质的微生物燃料电池(Microbial fuel cell,MFC)产电性能以及微生物群落在阳极膜、悬浮液、阳极沉淀污泥中的分布特征,【方法】试验建立了双室MFC,以乳酸为阳极主要碳源,研究了反应器的启动过程及产电效能,同时以电镜和PCR-变性梯度凝胶电泳(Denaturing gradient gelelectrophoresis,DGGE)技术解析了微生物群落的空间分布特征。【结果】结果表明,反应器启动第7天时外电压达到0.56 V,当外阻为80Ω时,电流密度为415 mA/m2,MFC的功率密度达到最大值82 mW/m2。电镜观察发现大量杆菌附着在阳极表面,结合较为紧密;DGGE图谱显示阳极膜表面微生物与种泥最为相似,与阳极悬浮液、底部沉淀污泥中的主要菌群一致,条带序列与睾丸酮丛毛单胞菌(Comamonas testosteroni)和布氏弓形菌(Arcobacter butzleri)等最为相似。【结论】本研究表明以乳酸为基质MFC可产生较高的功率密度,阳极附着的优势菌与接种污泥来源密切相关。     

Analysis of the microbial community of the biocathode of a hydrogen-producing microbial electrolysis cell

The microbial electrolysis cell (MEC) is a promising system for hydrogen production. Still, expensive catalysts such as platinum are needed for efficient hydrogen evolution at the cathode. Recently, the possibility to use a biocathode as an alternative for platinum was shown. The microorganisms involved in hydrogen evolution in such systems are not yet identified. We analyzed the microbial community of a mixed culture biocathode that was enriched in an MEC bioanode. This biocathode produced 1.1 A m⁻² and 0.63 m³ H₂ m⁻³ cathode liquid volume per day. The bacterial population consisted of 46% Proteobacteria, 25% Firmicutes, 17% Bacteroidetes, and 12% related to other phyla. The dominant ribotype belonged to the species Desulfovibrio vulgaris. The second major ribotype cluster constituted a novel taxonomic group at the genus level, clustering within uncultured Firmicutes. The third cluster belonged to uncultured Bacteroidetes and grouped in a taxonomic group from which only clones were described before; most of these clones originated from soil samples. The identified novel taxonomic groups developed under environmentally unusual conditions, and this may point to properties that have not been considered before. A pure culture of Desulfovibrio strain G11 inoculated in a cathode of an MEC led to a current development from 0.17 to 0.76 A m⁻² in 9 days, and hydrogen gas formation was observed. On the basis of the known characteristics of Desulfovibrio spp., including its ability to produce hydrogen, we propose a mechanism for hydrogen evolution through Desulfovibrio spp. in a biocathode system.     

Enzymatic hydrolysis of cellulose coupled with electricity generation in a microbial fuel cell

Electricity can be directly generated by bacteria in microbial fuel cells (MFCs) from a variety of biodegradable substrates, including cellulose. Particulate materials have not been extensively examined for power generation in MFCs, but in general power densities are lower than those produced with soluble substrates under similar conditions likely as a result of slow hydrolysis rates of the particles. Cellulases are used to achieve rapid conversion of cellulose to sugar for ethanol production, but these enzymes have not been previously tested for their effectiveness in MFCs. It was not known if cellulases would remain active in an MFC in the presence of exoelectrogenic bacteria or if enzymes might hinder power production by adversely affecting the bacteria. Electricity generation from cellulose was therefore examined in two-chamber MFCs in the presence and absence of cellulases. The maximum power density with enzymes and cellulose was 100 +/- 7 mW/m(2) (0.6 +/- 0.04 W/m(3)), compared to only 12 +/- 0.6 mW/m(2) (0.06 +/- 0.003 W/m(3)) in the absence of the enzymes. This power density was comparable to that achieved in the same system using glucose (102 +/- 7 mW/m(2), 0.56 +/- 0.038 W/m(3)) suggesting that the enzyme successfully hydrolyzed cellulose and did not otherwise inhibit electricity production by the bacteria. The addition of the enzyme doubled the Coulombic efficiency (CE) to CE = 51% and increased COD removal to 73%, likely as a result of rapid hydrolysis of cellulose in the reactor and biodegradation of the enzyme. These results demonstrate that cellulases do not adversely affect exoelectrogenic bacteria that produce power in an MFC, and that the use of these enzymes can increase power densities and reactor performance.     

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.     

Effect of separator and inoculum type on electricity generation and microbial community in single-chamber microbial fuel cells

Single-chamber microbial fuel cell (SMFC)-I consisted of 4 separator-electrode assemblies (SEAs) with two types of cation exchange membrane (CEM: Nafion and CMI 7000) and an anion exchange membrane (AEM: AMI 7001). SMFC-II consisted of 4 SEAs with Nafion and three types of nonwoven fabric. SMFC-I and -II were inoculated with anaerobic digested and activated sludge, respectively, and operated under fed-batch mode. In SMFC I, AEM-SEA showed a maximum power density (PD_max). Nafion-SEA showed a PD_max in SMFC II, which was similar to that of Nafion–SEA of SMFC I. Although different bacteria were developed in SMFC-I (Deltaproteobacteria and Firmicutes) and SMFC-II (Gammaproteobacteria, Betaproteobacteria and Bacteroidetes), the inoculum type little affects electricity generation. Variations of pH and oxygen in biofilm have influenced microbial community structure and electricity generation according to the electrode and separator material. Although the electricity generation of non-woven fabric-SEA was less than that of Nafion-SEA, the use of non-woven fabrics is expected to reduce the construction and operating costs of MFCs.     

Enhancement of hexavalent chromium reduction and electricity production from a biocathode microbial fuel cell

Enhancement of Cr (VI) reduction rate and power production from biocathode microbial fuel cells (MFCs) was achieved using indigenous bacteria from Cr (VI)-contaminated site as inoculum and MFC architecture with a relatively large cathode-specific surface area of 340–900 m² m⁻³. A specific Cr (VI) reduction rate of 2.4 ± 0.2 mg g⁻¹VSS h⁻¹ and a power production of 2.4 ± 0.1 W m⁻³ at a current density of 6.9 A m⁻³ were simultaneously achieved at an initial Cr (VI) concentration of 39.2 mg L⁻¹. Initial Cr (VI) concentration and solution conductivity affected Cr (VI) reduction rate, power production and coulombic efficiency. These findings demonstrate the importance of inoculation and MFC architecture in the enhancement of Cr (VI) reduction rate and power production. This study is a beneficial attempt to improve the efficiency of biocathode MFCs and provide a good candidate of bioremediation process for Cr (VI)-contaminated sites.     

Analysis of methanotroph community composition using a pmoA-based microbial diagnostic microarray

Microbial diagnostic microarrays (MDMs) are highly parallel hybridization platforms containing multiple sets of immobilized oligonucleotide probes used for parallel detection and identification of many different microorganisms in environmental and clinical samples. Each probe is approximately specific to a given group of organisms. Here we describe the protocol used to develop and validate an MDM method for the semiquantification of a range of functional genes--in this case, particulate methane monooxygenase (pmoA)--and we give an example of its application to the study of the community structure of methanotrophs and functionally related bacteria in the environment. The development and validation of an MDM, following this protocol, takes ～6 months. The pmoA MDM described in detail comprises 199 probes and addresses ～50 different species-level clades. An experiment comprising 24 samples can be completed, from DNA extraction to data acquisition, within 3 d (12-13 h bench work).     

Spatial and temporal analysis of the microbial community in the tailings of a Pb-Zn mine generating acidic drainage

Analysis of spatial and temporal variations in the microbial community in the abandoned tailings impoundment of a Pb-Zn mine revealed distinct microbial populations associated with the different oxidation stages of the tailings. Although Acidithiobacillus ferrooxidans and Leptospirillum spp. were consistently present in the acidic tailings, acidophilic archaea, mostly Ferroplasma acidiphilum, were predominant in the oxidized zones and the oxidation front, indicating their importance to generation of acid mine drainage.     

Characterization of a filamentous biofilm community established in a cellulose-fed microbial fuel cell

Background  

Microbial fuel cells (MFCs) are devices that exploit microorganisms to generate electric power from organic matter. Despite the development of efficient MFC reactors, the microbiology of electricity generation remains to be sufficiently understood.