Research Journal of Biotechnology

Review Paper:

Vol. 8 (3) March (2013) Res. J. Biotech

Bioelectricity Production using Microbial Fuel Cell

Shah Chirag K. and Yagnik B.N.*
Department of Biotechnology, Government Science College, K.K Shastri Educational Campus, Maninagar (E), Ahmedabad, INDIA *yagnikbn@gmail.com

Abstract Energy with its ever increasing demand is becoming more and more scarce resource. Microbial Fuel Cells (MFC) hold promise as an alternative not only to meet the widening gap in the energy sector but can be an effective mean for the sustainable utilization of reduced compounds in an eco-friendly way. The bacteria within the MFC oxidize complex substrates within the waste by removing electrons. The electrons generated by the bacteria growing in the oxygendeficient anodic chamber of the MFC, are captured by the cathode exposed to the air. The potential difference generated can be optimized and developed into an independent electricity production unit. For this reason, better understanding of the electron coupling reactions and carriers within the bacteria is attaining wider attention. The MFC can be customized to suite diverse substrate range by choosing specific bacterial species having matching metabolic repertoire and accordingly chosen operational parameters of the MFC.
Keywords: Bacteria, bioelectricity, Microbial Fuel Cell, Energy, Rhodoferax.

solution to the cathode where electrons combine with protons and oxygen to form water6. Microorganisms can transfer electrons to the anode electrode in three ways: Exogenous mediators (external to the cell) such as thionine or neutral red; using mediators produced by the bacteria or by direct transfer of electrons from the respiratory enzymes (i.e. Cytochromes) to the electrode2. These mediators trap the electrons from the respiratory chain and get reduced to transfer the electron to the ultimate acceptor the cathode via outer cell membrane1. Bacterial species such as Clostridium butyricum15, Saccharomyces cerevisae20 and Proteus vulgaris1 are reported to transfer electrons in mediated MFC while Shewanella putrefaciens, Geobacter sulfurreducens, Geobacter metallireducens and Rhodoferax ferrireducens have been shown to generate electricity in a mediator-less MFC2. Bacteria present in mediator-less MFCs have electrochemically active redox enzymes on their outer membranes that transfer the electrons to external materials and therefore do not require exogenous chemicals to accomplish electron transfer to the electrode16. An overview of different bacterial species and their electron transport pathway is given in table 1. MFCs have operational and functional advantages over the technologies currently used for generating energy from organic matter. The apparent advantages of the MFC over the conventional sources are (1) Direct conversion of substrate energy to electricity enables high conversion efficiency, (2) Operates efficiently at ambient temperature, (3) Does not require gas treatment because the off-gases of MFCs are enriched in carbon dioxide and normally have no useful energy content (4) No need of energy input for aeration provided the cathode is passively aerated12, (5) Potential for wide-spread application at locations lacking electrical infrastructures and (6) Diverse substrate range including environmental wastes. Recent developments could achieve high conversion rates and efficiencies using simple carbohydrates like glucose19, complex carbohydrate like starch15 and cellulose. Although MFCs generate a lower amount of energy than hydrogen fuel cells, a combination of both electricity production and waste-water treatment would reduce the cost of treating primary effluent waste-water. Presently, research on MFCs using waste-water as substrate is in the initial stages of laboratory evaluation around the world. The reported work so far is mainly based on using the monoculture at laboratory level11. Substantial technical
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Introduction
With the ever-increasing global population, advancement in science and technology, humankind is achieving better living standard and comfort but at the cost of growing energy requirements. The present energy scenario in India and around the globe is precarious enough to thrust the search for an alternate to fossil fuels depleting at an alarming rate. Increased energy consumption requires power sources that are able to sustain for longer periods. Trapping renewable energy from waste organic sources is the present trend in the quest for energy research. Microbial fuel cells (MFC) are not new the concept of using microorganisms as catalysts in fuel cells was explored from the 1970s21 and microbial fuel cells treating domestic waste-water were presented in 19917. An MFC is a device that uses bacteria to catalyze the conversion of chemical energy in the bio-convertible organic matter into electricity (Figure 1). This can be achieved when bacteria switch over from using the natural electron acceptor such as oxygen or nitrate, to an insoluble acceptor, such as the MFC anode. Bacteria generate electrons and protons at the anode by oxidizing the substrate. The electrons then flow through a resistor to a cathode, at which the electronacceptor is reduced while the protons diffuse through the

Research Journal of Biotechnology

and engineering challenges still remain to resolve for the viable electricity production at full scale. The function and efficiency of MFCs with respect to power generation are generally dependent on factors such as nature of carbon source used, fuel-cell configuration (single/multiple chamber), dimensions and volume, nature and type of electrode, electron acceptors (mediators) present in the cathode chamber, electrolytes used, operating temperature, nature of inoculum (biocatalyst) used in the anode chamber and nature of the proton exchange membrane.

Vol. 8 (3) March (2013) Res. J. Biotech

total electrons are possibly available for electricity generation because the hydrogenases, which generally use the electrons to produce hydrogen gas, are often situated at places on the membrane surface accessible from outside, or by mobile electron shuttles that connect directly to the electrode. As repeatedly observed, this metabolic type can imply a high acetate or butyrate production. Several organisms that are known to produce fermentation products belonging to the genera Clostridium, Alcaligenes, Enterococcus have been isolated17 from MFCs. Fermentation products such as acetate can be oxidized at low anode potential by anaerobic bacteria such as Geobacter23 species, capable of withdrawing electrons from acetate under the MFC conditions.

Metabolism in MFC
Assessing the generation of bacterial electricity requires determining the metabolic pathways governing microbial electron and proton flows. In addition to the influence of the substrate9, the potential of the anode will also determine the bacterial metabolism. Increasing MFC current will concomitantly decrease the potential of the anode, forcing the bacteria to deliver the electrons through more-reduced complexes. The potential of the anode will therefore determine the redox potential of the final bacterial electron shuttle and therefore, the type of metabolism. Several metabolism routes have been distinguished on the basis of the anode potential viz. high redox oxidative metabolism; medium to low redox oxidative metabolism and fermentative metabolism. Therefore, the organisms found to be compatible for MFC till to-date encompasses aerobes, facultative anaerobes to strict anaerobes. At high anodic potentials, bacteria can use the respiratory chain in an oxidative metabolism. Electrons and concomitantly, protons can be transported through the NADH dehydrogenase, ubiquinone, coenzyme Q or cytochrome9. Processes using oxidative phosphorylation have regularly been observed in MFCs, yielding high energy efficiencies of up to 65%19. Examples are consortia containing aerobes Pseudomonas aeruginosa, Enterococcus faecium and Rhodoferax ferrireducens. If the anode potential decreases in the presence of alternative electron acceptors such as sulphate, the electrons are likely to be deposited onto these components. Methane production has repeatedly been observed when the inoculum is an anaerobic sludge9, indicating that the bacteria does not use the anode. If no sulphate, nitrate or other electron acceptors are present, fermentation will be the main process when the anode potential remains low. For example, during fermentation of glucose, possible reactions can be: C6H12O6 + 2H2O C6H12O6 4H2 + 2CO2 + 2C2H4O2 2H2 + 2CO2 + C4H8O2

Anodic Electron Transfer Mechanism in MFC

The electrons to reach the electrode require a physical transport system to facilitate electron transfer across the membrane. This can either occur through the use of soluble electron shuttles6 or through membrane-bound electron shuttling compounds10. The oxidative, membrane-associated electron transfer is thought to occur through compounds that belong to the respiratory chain. Bacteria known to use this pathway are for example Geobacter metallireducens, Aeromonas hydrophila and Rhodoferax ferrireducens18. The main requirement for a component to act as an electron gateway seems to be the steric accessibility17. The potential of the gateway in relation to the anode will determine whether the gateway is actually used (an electron will not be transferred to a more reduced electrode). Many fermentative organisms identified in MFCs possess a hydrogenase, for example Clostridium butyricum and Enterococcus faecium. Hydrogenases could be directly involved in electron transfer towards electrodes. Recent internet communiqu suggested possibility of electron transfer, although in combination with a mobile redox shuttle. Hydrogenases have a role in reducing neutral red at the bacterial surface. Bacteria can use soluble components that physically transport the electron from an intracellular compound which becomes oxidized, to the electrode surface. In several studies, redox mediators such as neutral red17, thionin and methyl viologen4 were added to the reactor. The addition of these mediators often seemed to be essential. However, bacteria can also produce redox mediators themselves, which can occur through the production of organic, reversibly reducible compounds produced as secondary metabolites. This method was suggested for many bacteria, such as Shewanella putrefaciens and Pseudomonas aeruginosa. It was recently shown that these microbial mediators influence the performance of an MFC or more generally
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This shows that a maximum of one-third of a hexose substrate electrons can theoretically be used to generate current, whereas two-third remain in the fermentation products such as acetate and butyrate. The one-third of the

Research Journal of Biotechnology

interfere in extracellular electron transfer8. Inactivation of the genes responsible for mediator production in an MFCisolate Pseudomonas aeruginosa reduced the current generation by a factor of 20. The redox mediators produced by one bacterium can be used by other bacterial species to reach the electrode.

Vol. 8 (3) March (2013) Res. J. Biotech

conditions. The potential for the oxidation of acetate (1M) at the anode and the reduction of protons to H2 at the cathode are -0.28 and -0.42V, respectively. Therefore, H2 can theoretically be produced at the cathode by applying a voltage difference in the magnitude of -0.42 - (-0.28) V = > 0.14 V. Interestingly, the voltage requirement of 0.14 V is substantially lower than that needed to produce H2 by the direct electrolysis of water carried out at 1.23 V (pH 7). This is possible with the energy aided from the substrate oxidation process in the anode chamber. The richness and feasibility for storage of hydrogen makes this alternative more viable especially for transportation fuel. The modified MFCs have other advantages over the conventional technologies for generation of H2 from the organic matter. This includes: (1) High conversion efficiency (2) High yield (8-9 against 4 mol H2 mol-1 glucose conventionally) as suggested in a web communiqu (3) Elimination of electron loss due to oxygen diffusion in the cathode and (4) High purity of H2 against mixture of diverse biogases along with amended impurities (CH4, CO2, H2S, NH3) by the traditional process requiring expensive purification. Waste-water Treatment and Cathodic Denitrification: An MFC can act as a sustainable technology for the simultaneous waste-water treatment for Biological Oxygen Demand (BOD), carbon and sulfide in addition to production of electricity. Electricity generation using a variety of biodegradable materials include not only pure chemicals such as acetate, glucose, cysteine and ethanol, but also waste-water from swine, food processing, domestic sources and corn-stover. Furthermore, MFCs yield 50-90% less excess sludge, which eventually reduces the cost for sludge disposal. Despite the rapid advances in MFCs, this technology is still facing considerable challenges before its full-scale application, which are mainly related to materials such as electrodes and ion exchange membrane. The singlechambered MFC with a continuous flow is considered an alternative system because ion-exchange membrane associated proton transfer problems no longer apply. In particular, continuous up-flow membrane-less MFC is attractive for waste-water treatment due to its easy scale-up and free of ion exchange membrane associated limitations. For nutrient removal, MFC cathodes can be driven effectively by biological nitrate reduction to nitrite or to nitrogen gas. This type of cathode is referred to as a biocathode when bacteria are used as catalysts to assist in cathodic reduction. A bacterial culture enriched with Geobacter species has ability to reduce nitrate to nitrite using the cathode as the sole electron donor. Thus a full biological denitrification is possible at a biocathode in the acetate-fed MFC. It was shown to have an effective application such as biocathode-based MFC for simultaneous organic removal, power production and
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MFC Construction at Laboratory Level

The MFC is expected to act as a bioreactor that converts the energy stored in chemical bonds of organic compounds directly into current through catalytic reactions of microorganisms under anaerobic conditions. Hence, it should consist of two chambers, an anaerobic anode chamber and an aerobic cathodic chamber separated by an ion-conducting membrane usually a proton exchange membrane (PEM). At the anode organic substrates will be oxidized by bacteria. The produced electrons will then be transferred from the anodic electrode (negative terminal) through an external circuit to the cathodic electrode (positive terminal) where the electron acceptor will be reduced. Protons produced at the anode will migrate through the solution of the anode across the proton exchange membrane (PEM) to the cathode where they will combine with oxygen and electrons to form water. Therefore, MFC set up would require two hard borosilicate 500 ml glass bottles as shown in figure 2. An agar-salt bridge instead of proton exchange membrane can be an economical alternative. The salt-bridge will act as an electrolytic contact of the solutions in the two bottles, in which the two electrodes (ca. 12cm x 1.5cm each) made up of carbon are immersed. The cell is connected to the external circuit by single-stranded copper wires (0.8mm id). A hot agar solution (2-5% w/v in 1M potassium chloride) is introduced into the glass U tube having 12mm id. The glass tube is stored in 1M KCl solution when not in use. The anodic chamber is filled with sample like wastewater inoculated with micro-organisms to study, whereas the cathodic chamber is filled with distilled water. Strict anaerobic condition at the anode and physical aeration over the cathode chamber are maintained. The electricity generated can be read over an ammeter.

Applications Generation

of

MFC

beyond

Electricity

Biohydrogen Production via Bioelectrolysis: Possibility for generating hydrogen by MFC is attracting the researchers attention. Modification of the MFC (Figure 3) for biocatalyzed electrolysis or a bio-electrochemically assisted microbial reactor (BEAMR) process or electrohydrogenesis, can be a promising technology for hydrogen production in the near future, although presently anaerobic degradation of a substrate by electrochemically active bacteria seems thermodynamically unfavorable. This thermodynamic barrier can be overturned by applying an external mild electrical potential. In this system, the protons and electrons produced by the anodic reaction migrate and combine at the cathode to form H2 under anaerobic

Research Journal of Biotechnology

complete denitrificaion without relying on H2-formation or external added power. Bioremediation: A MFC can be modified in an attractive way for bioremediation because bacteria such as Geobacter species can not only donate electrons to the anode but can also accept electrons from the cathode if the cathode is poised at a negative potential. For successful bioremediation of contaminated environments, the addition of an electron acceptor or donor to promote the desired biodegradation is the most common methodology. Electrodes can potentially serve as electron donors or acceptors for the bioremediation of contaminants such as organics or metals in contaminated groundwater. For example, a typical strategy of removing chlorinated solvents from contaminated groundwater is to add organic electron donors to stimulate biological reductive dechlorination. Supplying the electron donors to the target site in the correct amount is difficult task; however, considerable progress has been made in this amendment by right level of electron donor. Microbial reduction of the oxidized form of soluble uranium U6+ to insoluble U4+ and the precipitation of U4+ onto a cathode by poising the electrodes with a negative potential (-500 mV) will take place. This poised-electrode system can act as a potential and preferable strategy for the bioremediation of uranium contamination by simply placing electrodes at the site. Nitrate can also be converted to nitrite when electrodes are used as electron donors. On the other hand, electrodes can also act as a more convenient electron acceptor for the bioremediation of hydrocarbons compared with diverse electron acceptors such as oxygen, Fe3+ and sulfate. Geobacter metallireducens oxidizes aromatic hydrocarbon contaminants with an electrode serving as the sole electron acceptor. Considering that the optimum delivery of electron acceptors or donors to promote the desired biodegradation is the main difficulty in the bioremediation of contaminated subsurface areas with organic pollutants or metals, the electrodes in the modified MFC can be used as an effective alternative for an electron source or sink. Biosensors: One of the most common is the use of MFCs as biosensors for pollutant analysis and in situ process monitoring due to the instant and reliable current response during the degradation of electron donors. An MFC using Shewanella as a biocatalyst was demonstrated to be an effective sensor for quantifying BOD. Microbial Fuel Cellbased BOD sensors have advantages of being operationally stable, reproducible and have long maintenance free working life of 5 years. Furthermore, an easy and effective expansion of MFC based sensors to detect other candidates that can act as electron donors for electricity generation is expected.
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In-situ Power Source for Remote Areas: Another application of MFCs that has attracted increasing interests is the recovery of electricity from sediments. The MFC based technology can be an in situ power source for electronic devices (e.g. environmental sensors) in remote areas such as the ocean and the bottom of deep-water where it is difficult to routinely access to replace traditional batteries. A good example is sediment MFC with the most well known being the benthic unattached generator (BUG) in which the anode is buried in anoxic or anaerobic marine sediments connected to a cathode suspended in the overlying aerobic water. Organic matter in the sediments was used to produce electricity in BUG. A mud battery can serve not only as a power source for the sensors in remote locations but also as a bioremediation method. However, even with this conceptual advantage, sediment MFC has a technical challenge, such as low power density due to both the low organic matter concentration and its high intrinsic internal resistance.

Technological Challenges
In MFC-like systems, installation of an ion-exchange membrane is critical step to separate the anode from the cathode and allows the transfer of electrons and protons produced in the anode chamber. There are still a number of problems associated with them like biofouling, changes in pH, such as decrease at the anode but increase at the cathode chambers, can inhibit the growth of electrochemically active bacteria and many more. Furthermore, the following problems need to be solved in order to achieve viable MFCs for full scale applications: the increasing biomass density at both the anode and cathode; the suppression of competitive metabolisms such as methanogenesis and sulfate reduction and the reduction of capital costs. For successful commercial uses, much concern should be paid for finding not only alternative membranes to the expensive membrane, but sufficiently sturdy materials instead of conventional anodes, such as carbon felt and carbon paper.

Future Trends and Conclusion

The Plant Microbial Fuel Cell (plant-MFC) concept that can overcome all the drawbacks related to MFC, is what we can look forward to in the future as the most promising and the best natural device for production of renewable energy by exploiting biological entities and their cellular processes. The plant-MFC aims to transform solar radiation into green electricity in a clean and efficient manner by integrating the roots of a living plant in the anode compartment of a microbial fuel cell. The plant-MFC is based on two proven processes, rhizodeposition of organic compounds by living plants and electricity generation from organic compounds in the MFC. The living plant is photosynthesizing in its leaves whereby solar energy is used to fix carbon dioxide in the form of carbohydrates. Depending on plant species, age and environmental conditions up to 60% of the net fixed

Research Journal of Biotechnology

carbon can be transferred from its leaves to the roots. The plant root system produces and releases different types of organic compounds into the soil which include (1) Exudates: sugars, organic acids etc.; (2) Secretions: polymeric carbohydrates and enzymes; (3) Lysates: dead cell materials; (4) Gases: ethylene and CO2. The total of these release processes is called plant rhizodeposition and its products, rhizodeposits, are used in the plant-MFC as a renewable bioenergy substrate. The produced rhizodeposits can total up to 40% (or more) of the plants photosynthetic productivity. Rhizodeposits play many roles in the rhizosphere. Carbonic acids have been implied in the mobilization of soil-bound cationic nutrients for uptake by the plants. Rhizodeposits contain carbon and a part of this carbon can be utilized by micro-organisms in the rhizosphere which can lead to mutually beneficial interactions between plants and micro-organisms. Bacteria, for example, can positively interact with plant roots by

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forming protective biofilms or by producing antibiotics as biocontrols against potential pathogens. The largest fraction of rhizodeposits consists of small molecules which are efficiently synthesized by the plant and efficiently metabolized by bacteria. In the plant-MFC, the principal idea is that plant rhizodeposits will be utilized as substrates by the bacteria to generate electricity in the MFC (Figure 4). Microbial Fuel Cells are evolving to become a simple and robust technology. Certainly in the field of wastewater treatment, middle term application can be foreseen at market value prices. However, to increase the power output, many technological improvements are needed. With the better understanding of the biological electron passage and electrochemical technology advances and more new concepts like that of plant-MFC evolves, this technology might qualify as a new core technology for conversion of bio-convertible substrates to electricity in years to come.

Figure 1: Working of an MFC

Figure 2: Diagram of simple two-chambered MFC

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Figure 3: Hypothetical production of hydrogen through the biocatalyzed electrolysis of acetate using power assisted-MFC

Figure 4: Flow chart showing production of electricity through plant supported MFC Table 1 Bacterial species feasible for MFC Transfer type Membranedriven Bacterial species Mediator Oxidative metabolism Rhodoferax ferrireducens3 Unknown Geobacter sulfurreducens2 89 kDa c-type Cytochrome Aeromonas hydrophila10 c-type Cytochrome Escherichia coli13 Hydrogenase 14 Shewanella putrefaciens Quinones Pseudomonas aeruginosa19 Pyocyanin, phenazine Erwinia dissolvens22 Unknown Desulfovibrio desulfuricans5 S2Fermentative metabolism Clostridium butyricum6 Cytochromes Enterococcus faecium19 Unknown
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External Mediator Neutral red Fe(III)CyDTA (an iron chelator) Pyocyanin

Mediator-driven

Membranedriven Mediator-driven

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39, 658662 (2005) 13. McKinlay J.B., Extracellular iron reduction is mediated in part by neutral red and hydrogenase in Escherichia coli, Appl. Environ. Microbiol., 70, 34673474 (2004) 14. Newman D.K., Extracellular electron transfer, Cell. Mol. Life Sci., 58, 15621571 (2001) 15. Niessen J., Exploiting complex carbohydrates for microbial electricity generation - a bacterial fuel cell operating on starch, Electrochem. Commun., 6, 955958 (2004) 16. Oh S., Cathode performance as a factor in electricity generation in microbial fuel cells, Environ. Sci. Technol., 38, 49004904 (2004) 17. Park D.H., Electricity production in biofuel cell using modified graphite electrode with neutral red, Biotechnol. Lett., 22, 13011304 (1999, 2001) 18. Pham T.H., Improvement of cathode reaction of a mediatorless microbial fuel cell, J. Microbiol. Biotechnol., 14, 324329 (2004) 19. Rabaey K., A microbial fuel cell capable of converting glucose to electricity at high rate and efficiency, Biotechnol. Lett., 25, 15311535 (2003) 20. Reed G. and Nagodawithana T.W., Yeast Technology, 89-95 (1991) 21. Suzuki S., Fuel cells with hydrogen-forming bacteria, Hospital hygiene, Gesundheitswesen and desinfektion, 159 (1976) 22. Vega C.A., Mediating effect of ferric chelate compounds in microbial fuel-cells with Lactobacillus plantarum, Streptococcus lactis and Erwinia dissolvens, Bioelectrochem. Bioenerg., 17, 217222 (1987) 23. Verstraete W., Other treatment techniques in sludge into biosolids, Spinosa L. and Vesilind P.A., eds., IWA Publishing, 364383 (2001). (Received 29th July 2012, accepted 15th November 2012)

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