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.     

Electrochemical activity of an Fe(III)-reducing bacterium, Shewanella putrefaciens IR-1, in the presence of alternative electron acceptors

Cyclic voltammetry demonstrated that cells of Shewanella putrefaciens grown under anaerobic conditions without nitrate were electrochemically active. The electrochemical activity was inactivated reversibly by exposure to air, but not by nitrate. Lactate and an applied potential at +200 mV against an Ag/AgCl reference electrode restored the electrochemical activity. These findings can be used to improve the performance of a mediator-less microbial fuel cell using electrochemically active bacteria in the presence of nitrate.     

Mediatorless electrocatalytic reduction of hydrogen peroxide at graphite electrodes chemically modified with peroxidases

The electrocatalytic reduction of H₂O₂ was studied for carbonaceous electrodes modified with horse-radish peroxidase (HRP), microperoxidase (MP), and lactoperoxidase (LP). The carbonaceous electrodes were of three different graphites, carbon and glassy carbon. The peroxidase modified electrode was inserted as the working electrode in a flow through amperometric cell of the wall jet type and connected to a flow injection system. The effect of different pretreatments of the electrode surface prior to adsorption of the enzyme was investigated. Heating the electrodes in a muffle furnace at 700°C for 1.5 min was found to yield the highest currents. The electrocatalytic current for HRP-modified electrodes starts at about +600 mV vs. Ag/AgCl (pH 7.0) and reaches a maximum value at about −200 mV. For MP- and LP-modified electrodes the currents start at a lower potential (≈ 300 mV). For the best electrode material for HRP, straight calibration curves were obtained between 1 and 500 μM H₂O₂ at 0 mV. The mechanism for the electron transfer from the electrode to the adsorbed peroxidase is discussed. Deliberate modification of the electrode surface with quinoid type electroactive species was found to mediate the reaction. It is proposed that spontaneously occurring electrochemically active surface groups mediate the electron transfer to the adsorbed enzyme. However, a contribution to the observed current from a direct electron transfer cannot be ruled out.     

Mediation of glycosylated and partially-deglycosylated glucose oxidase of Aspergillus niger by a ferrocene-derivatised detergent.

A ferrocene-derivatised detergent, (11-ferrocenylundecyl) trimethylammonium bromide (FTMAB), when oxidised to the corresponding ferricinium ion, was found by electrochemical studies to be an effective electron acceptor for reduced glucose oxidase of Aspergillus niger (EC 1.13.4) and thus acts as a electron-transfer mediator between glucose oxidase and a working electrode held at a potential sufficiently positive to reoxidise reduced FTMAB. An increase in mediating activity was produced when FTMAB was present in concentrations above its critical micelle concentration. An 'enzyme electrode' was formed by adsorption of glucose oxidase and FTMAB surfactant on a graphite rod. The electrode functioned as an amperometric biosensor for glucose in phosphate-buffered saline solution. A mixed micelle of glucose oxidase and FTMAB, probably adsorbed on the electrode surface, appears to be advantageous for the amperometric determination of glucose. Additionally, glucose oxidase was treated with alpha-mannosidase. When this partially-deglycosylated glucose oxidase was incorporated in an enzyme electrode, a 100-fold increase in the second-order rate constant (k) for electron transfer between the enzyme and FTMAB was observed, together with increased current densities, with respect to the equivalent values for FTMAB and commercial glucose oxidase. The use of deglycosylated enzymes in biosensors is suggested.     

Light‐responsive current generation by phototrophically enriched anode biofilms dominated by green sulfur bacteria

The objective of this study was to employ microbial electrochemical cells (MXCs) to selectively enrich and examine anoxygenic photosynthetic bacteria for potential anaerobic respiration capabilities using electrodes. In the process, we designed a novel enrichment strategy that manipulated the poised anode potential, light, nitrogen availability, and media supply to promote growth of phototrophic bacteria while minimizing co‐enrichment of non‐phototrophic anode‐respiring bacteria (ARB). This approach resulted in light‐responsive electricity generation from fresh‐ and saltwater inocula. Under anoxic conditions, current showed a negative light response, suggesting that the enriched phototrophic consortia shifted between phototrophic and anaerobic respiratory metabolism. Molecular, physical, and electrochemical analyses elucidated that anode biofilms were dominated by green sulfur bacteria, and biofilms exhibited anode respiration kinetics indicative of non‐mediated electron transfer, but kinetic parameters differed from values previously reported for non‐phototrophic ARB. These results invite the utilization of MXCs as microbiological tools for exploring anaerobic respiratory capabilities among anoxygenic photosynthetic bacteria. Biotechnol. Bioeng. 2013; 110: 1020–1027. © 2012 Wiley Periodicals, Inc.     

An electrochemical thin-layer cell for spectroscopic studies of photosynthetic electron-transport components

An electrochemical thin-layer cell, 0.5 ml in volume and 0.25 mm in pathlength, employing an optically transparent gold minigrid electrode has been constructed and used in redox studies of photosynthetic electron-transport components. Light-induced absorption changes accompanying P700 photo-oxidation in photosystem-I subchloroplasts were measured at a series of electrochemically poised potentials at 88°K. The fluorescence-yield changes in photosystem-II subchloroplasts were measured as the poised potential was varied electrochemically. The utility of the cell was further demonstrated by a redox titration of soluble (spinach) ferredoxin using circular dichroism to monitor changes in the redox state. One important advantage of electrochemical vs chemical titration of photosynthetic electron-transport components is that it is possible to attain much more negative potentials than is thermodynamically possible in chemical titrations. This permits the exhaustive titration of certain components at physiologically reasonable pH values not possible chemically.     

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.     

Electrochemical assay of endo-depolymerase activity of cellulases

A method for determination of endo-1,4-beta-D-glucanase activity of cellulase samples based on the indirect measurement of decrease in viscosity of a carboxymethylcellulose solution in an electrochemical cell in the presence of an electron carrier was developed. A rotating disk electrode is used as the working electrode. When two reactions (enzymatic and electrochemical) proceeded in the cell simultaneously, the limiting diffusion current at a constant applied potential increases as the viscosity of the solution decreases. Conditions where the initial rate of change of diffusion current (dI/dt) is proportional to the enzyme concentration were found. A good correlation between the new method and a previously known viscometric method for determination of endoglucanase activity was observed.     

Bacterial community structure, compartmentalization and activity in a microbial fuel cell

AIMS: To characterize bacterial populations and their activities within a microbial fuel cell (MFC), using cultivation-independent and cultivation approaches. METHODS AND RESULTS: Electron microscopic observations showed that the fuel cell electrode had a microbial biofilm attached to its surface with loosely associated microbial clumps. Bacterial 16S rRNA gene libraries were constructed and analysed from each of four compartments within the fuel cell: the planktonic community; the membrane biofilm; bacterial clumps (BC) and the anode biofilm. Results showed that the bacterial community structure varied significantly between these compartments. It was observed that Gammaproteobacteria phylotypes were present at higher numbers within libraries from the BC and electrode biofilm compared with other parts of the fuel cell. Community structure of the MFC determined by analyses of bacterial 16S rRNA gene libraries and anaerobic cultivation showed excellent agreement with community profiles from denaturing gradient gel electrophoresis (DGGE) analysis. CONCLUSIONS: Members of the family Enterobacteriaceae, such as Klebsiella sp. and Enterobacter sp. and other Gammaproteobacteria with Fe(III)-reducing and electrochemical activity had a significant potential for energy generation in this system. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that electrochemically active bacteria can be enriched using an electrochemical fuel cell.     

Characterization of microbial fuel cells enriched using Cr(VI)-containing sludge

Microbial fuel cells (MFCs) were successfully enriched using sludge contaminated with Cr(VI) and their characteristics were investigated. After enrichment, the charge of the final 10 peaks was 0.51 C +/- 1.16%, and the anodic electrode was found to be covered with a biofilm. The enriched MFCs removed 93% of 5 mg/l Cr(VI) and 61% of 25 mg/l Cr(VI). 16S rDNA DGGE profiles from the anodic electrode indicated that beta-Proteobacteria, Actinobacteria, and Acinetobacter sp. dominated. This study is the first to report that electrochemically active and Cr(VI)-reducing bacteria could be enriched in the anode compartment of MFCs using Cr(VI)-containing sludge and demonstrates the Cr(VI) removal capability of such MFCs.     

Enrichment of microbial community generating electricity using a fuel-cell-type electrochemical cell

A fuel cell was used to enrich a microbial consortium generating electricity, using organic wastewater as the fuel. Within 30 days of enrichment the maximum current of 0.2 mA was generated with a resistance of 1 k. Current generation was coupled to a fall in chemical oxygen demand from over 1,700 mg l^–1 down to 50 mg l^–1. Denaturing gradient gel electrophoresis showed a different microbial population in the enriched electrode from that in the sludge used as the inoculum. Electron microscopic observation showed a biofilm on the electrode surface and microbial clumps. Nanobacteria-like particles were present on the biofilm surface. Metabolic inhibitors and electron acceptors inhibited the current generation. 16S ribosomal RNA gene analysis showed a diverse bacterial population in the enrichment culture. These findings demonstrate that an electricity-generating microbial consortium can be enriched using a fuel cell and that the electrochemical activity is a form of anaerobic electron transfer.     

利用异化金属还原菌构建含糖微生物燃料电池

环境中的一些微生物通过还原金属氧化物进行无氧呼吸,而石墨电极与金属氧化物相似,也可以作为这类微生物呼吸作用的最终电子受体,利用这类微生物构建微生物燃料电池,以糖类物质为燃料,对电池产电情况、产电原理进行研究。实验结果表明,以Rhodoferaxferrireducens为产电微生物,在外接电阻510Ω条件下,以葡萄糖为燃料,常温下产生的电流密度达158mAm2(平台电压为0.46V,电极有效接触表面积为57cm2),且循环性能良好。更换燃料为其它糖,发现微生物可以利用多种糖进行产电;通过SEM观察发现大量微生物吸附在石墨电极上,用Bradford法对运行20d后电池的细胞量进行定量,测得悬浮细胞蛋白浓度为140mgL,吸附在电极上的生物量为1180mgm2。通过数据采集分析和细菌还原实验,发现吸附在电极上的微生物对电压的产生贡献最大,具有电化学和生物学活性;悬浮细胞对产电贡献很小,不具有电化学和生物学活性。     

An amperometric xanthine oxidase enzyme electrode based on hydrogen peroxide electroreduction

A xanthine oxidase enzyme electrode (xanthine oxidase immobilized on electrochemically modified graphite and conveniently coated with gelatine electrode working surface) for quantitative analysis of xanthine is proposed. The detection of thus developed electrochemical system is based on the electroreduction of hydrogen peroxide generated in enzyme layer and offered L-ascorbic and uric acid reducing interference effect on the substrate determination. At a working potential -50 mV (vs. Ag/AgCl) the detection limit of 4.5 microM and the linearity of the amperometric signal up to substrate concentration of about 40 microM were found. At that working potential, the electrode is practically inert towards L-ascorbic- and uric acid present. The response time did not exceed 2 min.     

Development of a molecular diagnosis assay based on electrohybridization at plastic electrodes and subsequent PCR

Technologies enabling specific recognition of medically relevant nucleic acid sequences will play a pivotal role in future medical diagnosis. Whereas many approaches to molecular diagnosis systems include DNA microarrays on chips and fluorometric detection, the basis of our approach is the use of inexpensive components like plastic or metal thin film electrodes with low multiplexing and an electrochemical detection unit. To increase the sensitivity, PCR can be used as an intermediate step. For selective enrichment, specific nucleic acid probes were covalently attached at their 5′-ends to conducting polycarbonate/carbon fiber electrodes. Complementary oligonucleotides were enriched at the electrodes by cyclic inversion of an electrochemical potential, transferred into a PCR vial and thermally or electrochemically desorbed. The analysis of the PCR product shows the efficiency and selectivity of the electrochemical enrichment. Hybridization of DNA was shown by electrochemical methods, in this work especially by differential pulse voltammetry (DPV) using the single strand specific hybridization redox indicator osmium(VIII)-tetroxide, and potentiometric stripping analysis (PSA). This combination of experimental methods is the basis for a molecular diagnosis system including a disposable nucleic acid modified working electrode for specific enrichment, detection and quantification, and an optional capillary PCR module for fast amplification.     

Enhanced Propionate Formation by Propionibacterium freudenreichii subsp. freudenreichii in a Three-Electrode Amperometric Culture System

In order to influence the fermentation pattern of Propionibacterium freudenreichii towards enhanced propionate formation, growth and product formation with glucose and lactate as energy sources were studied in a three-electrode poised-potential amperometric culture system. With anthraquinone 2,6-disulfonic acid (E(0)' = -184 mV; poised electron potential = -224 mV) or cobalt sepulchrate (E(0)' = -350 mV; -390 mV) as mediator and an activated platinum working electrode, reduction of bacterially oxidized mediator occurred fast enough to keep more than 50% of the respective mediator (in minimum 0.4 mM) in the reduced state, up to a current of 2 mA. With glucose as substrate, 90.0 or 97.3% propionate was formed during exponential growth in the presence of 0.5 mM anthraquinone 2,6-disulfonic acid or 0.4 mM cobalt sepulchrate, respectively. Growth yields of 56.3 or 53.8 g of cell material per mol of substrate degraded were calculated, respectively, and the electrons were transferred quantitatively from the working electrode to the bacterial cells. With l-lactate, only 68.6 or 72.9% propionate was formed with the same mediators. The results are discussed with respect to energetics, electron transfer potentials, and potential application of the new technique in technical propionate production.     

Simultaneous topographic and amperometric membrane mapping using an AFM probe integrated biosensor

The investigation of the plasma membrane with intercorrelated multiparameter techniques is a prerequisite for understanding its function. Presented here, is a simultaneous electrochemical and topographic study of the cell membrane using a miniaturized amperometric enzymatic biosensor. The fabrication of this biosensor is also reported. The biosensor combines a scanning force microscopy (AFM) gold-coated cantilever and an enzymatic transducer layer of peroxidases (PODs). When these enzymes are brought in contact with the substrate, the specific redox reaction produces an electric current. The intensity of this current is detected simultaneously with the surface imaging. For sensor characterization, hydroquinone-2-carboxylic acid (HQ) is selected as an intrinsic source of H(2)O(2). HQ has been electrochemically regenerated by the reduction of antraquinone-2-carboxylic acid (AQ). The biosensor reaches the steady state value of the current intensity in 1 ± 0.2s.     

Electrochemical disinfection of bacteria in drinking water using activated carbon fibers

A novel electrochemical reactor employing activated carbon fiber (ACF) electrodes was constructed for disinfecting bacteria in drinking water. Escherichia coli adsorbed preferentially onto ACF rather than to carbon-cloth or granular-activated carbon. E. coli cells, which adsorbed onto the ACF, were killed electrochemically when a potential of 0.8 V vs. a saturated calomel electrode (SCE) was applied. Drinking water was passed through the reactor in stop-flow mode: 2mL/min for 12 h, o L/min for 24 h, and 1 mL/min for 6 h. At an applied potential of 0.8 V vs, SCE, viable cell concentration reamined below 30 cells/mL. In the absence of an applied potential, bacteria grew to a maximum concentration of 9.5 x 10(3) cells/mL. After continuous operation at 0.8 V vs. SCE, cells adsorbed onto the ACF could not be observed by scanning electron microscopy. In addition, chlorine in drinking water was completely removed by the reactor. Therefore, clean and efficient inactivation of bacteria in drinking water was successfully performed. (c) 1994 John Wiley & Sons, Inc.     

Electricity production by Geobacter sulfurreducens attached to electrodes

Previous studies have suggested that members of the Geobacteraceae can use electrodes as electron acceptors for anaerobic respiration. In order to better understand this electron transfer process for energy production, Geobacter sulfurreducens was inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor. The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at +0.2 V versus an Ag/AgCl reference electrode (poised potential). When a small inoculum of G. sulfurreducens was introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism. When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks. This represents the first report of microbial electricity production solely by cells attached to an electrode. Electrode-attached cells completely oxidized acetate to levels below detection (<10 micro M), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 micro mol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (E(o)' =+0.37 V). The production of current in microbial fuel cell (65 mA/m(2) of electrode surface) or poised-potential (163 to 1,143 mA/m(2)) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds. Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells. These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such as G. sulfurreducens that can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode.     

Real-Time Dynamic Adsorption Processes of Cytochrome c on an Electrode Observed through Electrochemical High-Speed Atomic Force Microscopy

An understanding of dynamic processes of proteins on the electrode surface could enhance the efficiency of bioelectronics development and therefore it is crucial to gain information regarding both physical adsorption of proteins onto the electrode and its electrochemical property in real-time. We combined high-speed atomic force microscopy (HS-AFM) with electrochemical device for simultaneous observation of the surface topography and electron transfer of redox proteins on an electrode. Direct electron transfer of cytochrome c (cyt c) adsorbed on a self-assembled monolayers (SAMs) formed electrode is very attractive subject in bioelectrochemistry. This paper reports a real-time visualization of cyt c adsorption processes on an 11-mercaptoundecanoic acid-modified Au electrode together with simultaneous electrochemical measurements. Adsorbing cyt c molecules were observed on a subsecond time resolution simultaneously with increasing redox currents from cyt c using EC-HS-AFM. The root mean square roughness (R_RMS) from the AFM images and the number of the electrochemically active cyt c molecules adsorbed onto the electrode (Γ) simultaneously increased in positive cooperativity. Cyt c molecules were fully adsorbed on the electrode in the AFM images when the peak currents were steady. This use of electrochemical HS-AFM significantly facilitates understanding of dynamic behavior of biomolecules on the electrode interface and contributes to the further development of bioelectronics.