The Generation of Electricity in Microbial Fuel Cell (MFC) Using Eschericia Coli as Micro Organism

Farida Nur CahyaniGerard Markx

ABSTRACT

Microbial fuel cell (MFC) is a promising device for generating bioelectricity from waste water. This project explores further application of MFC in waste water treatment. As parameter, it is evaluated the voltage and current produced during MFC operation to determine the power density and volumetric power optimum achieved.

A batch system of MFC was used. As microbes, the pure strain of Eschericia coli, was fed to the anode using artificial waste water as substrate/fuel which consisted of glucose-glutamic acid in range of BOD content 100-400 mg/l. In the cathode, Potassium hexacyanoferrate was immersed as oxidizing agent.. It was also investigated the application of microbial aggregation in MFC. For this purpose, aggregation of pure strain Eschericia coli was constructed using dielectrophoresis method which immobilized using polyethyleneamine (PEI) in the microelectrodes. The aggregation was enriched with FAB solution and glucose before inserted to MFC. The electricity produced from aggregation was compared to the non-aggregation.

As the result the optimum condition for Eschericia coli is at concentration7 mg/10 ml. The value of power density and volumetric power in this condition are 1384.1 mW/m2;152.3 mW/l. For microbial aggregation, the voltage and current obtained are 0.21 V and 0.3mA, respectively

.Keywords: artificial waste water, Microbial fuel cell, aggregation, Eschericia coli

INTRODUCTION

Microbial fuel cell (MFC) is a device that converts chemical energy to electrical energy by the catalytic reaction of microorganisms. The generation of electricity is driven by the bacteria when switch from the natural electron acceptor, such as oxygen or nitrate, to an insoluble acceptor, such as the MFC anode.

MFC has advantages over the technologies currently used for generating energy from organic matter. First, the direct conversion of substrate energy to electricity enables high conversion efficiency. Second, MFC operates efficiently at ambient temperature and even at low temperature distinguishing it from all current bio-energy processes. Third, an MFC does not require gas treatment because the off-gases of MFC are enriched in carbon dioxide and normally have no useful energy content. Fourth, MFC does not need energy input for aeration provided the cathode is passively aerated. Fifth, MFC has potential for widespread application in location lacking electrical infrastructure and also to expand the diversity of fuels to satisfy the energy requirements (Rabaey, et al., 2005).

The technology of MFC also enables to produce bioelectricity from waste water that is rich of organic matter. The using of microbial fuel cell to treat domestic waste water were presented in 1991(Haberman,et al., 1991). However, it

is only recent that microbial fuel cells with an enhanced power output have been developed providing possible opportunities for practical applications.

Waste water treatment using a MFC is promising since this process converts the major part of the chemical energy of the contaminants to electricity thereby reducing the generation of excess sludge (Jang, et al., 2004; Kim, et al.., 2004).

This paper presents bioelectricity generation using microorganism Eschericia coli and its microbial aggregation with artificial waste water as substrate (fuel) to explore the further application of mediatorless MFC for waste water treatment.

METABOLISM IN MICROBIAL FUEL CELL

The metabolism of microorganism in microbial fuel cell can be classified into several ways: high redox oxidative metabolism; medium to low redox oxidative metabolism; and fermentation. At high anodix potentials, bacteria can use the respiratory chain in an oxidative metabolism. Electron and concomitantly protons can be transported through NADH dehydrogenase, ubiquinone, coenzyme Q or cytochrome. Processes using oxidative phosphorylation have been observed in MFCs, yielding high energy efficiencies of up to 65%. Examples are consortia containing Enterococcus faecium and Rhodoferax ferrireducens.

If the anode potential decreases in the presence of alternative electron acceptors such as sulphate, the electrons are to be deposited onto these components.

If there is no sulphate, nitrate or other electron present, fermentation will be the main process when the anode potential remains low. Several organisms that are known to produce fermentation products and belong to the genus Clostridium, Alcaligens, Enterococcus, have been isolated from MFCs. Fermentation products such as acetate can be oxidized at low anode potential by anaerobic bacteria such as Geobacter species, which is capable of withdrawing electrones from acetate in MFC conditions.

MICROORGANISM

So far, most of the studies used well-defined pure strains, such as Shewanella putrefaciens or Escherichia coli. Recently, two bacteria that exhibited high coulomb efficiency have been described; these bacteria, Geobacter sulfurreducens and Rhodoferax ferrireducens (Rabaey, et al., 2005) are capable of transferring the majority of the electrons gained from the carbon sources acetate and glucose to the electrode. These studies implied that there was high coulomb efficiency, meaning that there was high electron transfer efficiency. However, this does not imply that there was high energy transfer efficiency, which is dependent on the product of current and potential, both of which determine the energy, expressed in joules. In a limited number of cases, mixed consortia obtained from wastewater treatment plants have been used in flow through systems.

Bond and Lovley (2003) showed the potential of Geobacter sulfureducens fermentans as biocatalyst for an MFC. When a small inoculum of G. sulfurreducens was introduced into electrode-containing chambers, the suspension of this organism could oxidize acetate in a two-electrode fuel cells that stimulated

the marine sediment fuel cells, with no mediator compounds addition. Further research involving different microbia-Geothrix fermentans was conducted by Bond and Lovley (2005). In their latest experiment, the Fe (III) reducing organism Geotrix fermentans conserved energy to support growth by coupling the complete oxidation of acetate to reduction of graphite electrode. This is the first report of complete oxidation of organic compounds linked to electrode reduction.

MICROBIAL AGGREGATION

Aggregation or cellular adhesion was defined as the gathering together of cells to form fairly stable, contiguous, multicellular associations under physiological condition. Aggregation is a microbial concortia which in nature typically form biofilm, normally heterogeneous and often complex in structures. A kinds of methods have been developed which potentially could be used to create biofilms. Prominents of these methods is the use of electrokinetics, and in particular dielectrophoresis (Markx et al., 2004).

Dielectrophoresis is electric phenomenon and occurs when a particle is placed in a non-uniform electric field, which induces the particle to form an electric dipole. Because the electric field is non-uniform, the one side of dipole is stronger than the other. The result is a net force on particle, causing it to move. The direction movement of the particle/cell depends on the properties of the particle relative to the medium (Marxk et al., 2004). The phenomenon of dielectropohresis has recently been exploited to develop novel techniques for rapidly constructing structured biomaterials (Alp et al., 2002, 2003; Verduzco-Luque et al., 2003; Markx et al., 2005). In this approach, cells are dispersed in low conductivity medium, and guided to spesific areas of a microeletrode array, by dielectrophoresis, by energising selected electrodes using low (typically 5-20 V peak to peak) high frequency (typically 1 MHz) AC signals. Once the cells have reached their respective positions, the cells are immobilised on the electrodes to prevent redistribution of the cells by Brownian motion or other forces once the electric field is removed. Verduzco-Luque et al. (2003) demostrated that the use of polyethylenimine (PEI) as flocculating agent allowed better maintenance of cell viability, whilst also providing sufficient immobilisation to prevent redistribution of the cells.

PERFORMANCE DATA

The amount of energy (Joules) gained out of an electrochemical process can be calculated based on power output and process duration:E = P×t ………….........................................................................(1)with P the power (Watts) and t time (s). The power depends both on the voltage V and the current I: P=V×I …....................................................................………….(2)

The latter factors are linked by the fuel cell resistance, by Ohm’s law V=IxR in which R represents the resistance (Ohm).EXPERIMENTAL

This research was carried out in Laboratory of Microbiology, University of Manchester.

Strains and mediaFor non aggregation system, the pure strain of Eschericia coli is inoculated

in nutrient broth which consisted of lab-lemco powder (1.0), yeast extract (2,0), peptone (5.0) and sodium chloride (5.0) over period of 24 hours at 37C.

Microbial fuel cellThe MFC consists of Perspex fuel cell, carbon fiber tissue electrodes,

double neoprene gaskets, and cation exchange membrane.In the cathode, it is immersed oxidizing solution that is Potassium

hexacyanoferrate 0.01 M in phosphate buffer solution (in range area of pH 7). The separation of the anode and cathode solutions is achieved with the aid of a cation exchange membrane. The second electrode (anode) contains micro organism and substrate (artificial waste water). The contact between two electrodes should be avoided to prevent from possibility a short circuit. For electrical measurement, the multi meter was connected via crocodile clips to the electrode terminals and it is made sure that the electrodes do not touch the cation membrane exchange. The reading of electrical current generated is based on the multi meter measurement.

Formation of biofilmsCells of Eschericia coli was inoculated in nutrient broth and growth

overnight in shaker 230-260 rpm. The cells centrifuged down at 10 000 rpm and washed five times using distilled water, in order to reduce the medium conductivity. A low medium conductivity increases the attraction of the cells to high electric field regions by positive dielectrophoresis. The cells were introduced into the chamber and drawn over the electrodes using tissue paper as a wick. The cells were attracted to the electrodes using a non-uniform electric field (1 MHz, 20 V peak-peak), supplied by a Thurlby-Thandar TG 120 signal generator. In these conditions, the cells formed aggregates between castellation, in areas of high electrical field strength, in a matter of a few seconds. Cells not attracted to the areas of high field strength between electrodes were washed away using gentle flow of sterile distilled H2O through chamber. This gentle flow was created using tissue paper wicks to draw 1 ml of distilled H2O through the chamber over 10 min.

It was necessary to immobilise the cells to avoid redistribution over the electrode surface. The cells that remained attached to the electrodes after gentle washing with distilled H2O were immobilised using the flocculating agent polyethylenimine (PEI)(Sigma) at a concentration of 0.025% w/v in sterile distilled H2O. It was about two hundred micro litre of PEI solution was applied to the chamber and drawn over the cells using a tissue paper wick. To remove excess PEI after immobilisation the cells were washed with deionised water (Markx et al., 2006). The aggregation formed is shown on Figure 1.

Figure 1. Microbial Aggregation before Washing Process and Immobilization.

The immobilized cells were fed with FAB solution enriched with glucose 25% mmol and left overnight in incubator at 37C. FAB solution consists of FB solution and A-10 solution with ration 9 and 1, respectively. To prepare FB solution (pH 6.4), it was used (NH4)2SO4 (0.15 mM); Na2HPO4.2H2O (0.33 mM); KH2PO4 (0.2 mM); NaCl (0.5 mM) and for A-10, it was made from MgCl2 (1 mM); CaCl2 (0.1 mM); and Fe-EDTA (0.01 mM). The enriched cells then embedded in anode chamber to substitute the presence of micro organism in the anode.

Substrate/Artificial waste waterGlucose and glutamic acid test solution for biochemical oxygen demand

(BOD) test standard was modified and used as artificial waste (AW) water through out the study. The AW was autoclaved at 121C for 15 min before being added with the filter sterilized glucose and glutamic acid solutions to the final BOD values of 100-400 mg/l. This solution played as substrate for micro organism fed in the anode chamber.

RESULT AND DISCUSSION

The data of voltage and current generated for Eschericia coli and its energy calculation are shown on Table 1. And for microbial aggregation are presented on Table 2.

Table 1. Data of Power Density and Volumetric Power for Eschericia coli at Various Concentrations

Concentration, mg/10 ml

V, Volt I, mAPower

density, mW/m2

Volumetric power, mW/l

3 0.68 1.6 985.8 108.44 0.61 1.2 665.4 73.25 0.63 2.2 1264.0 139.06 0.61 2.1 1140.5 125.57 0.67 2.5 1384.1 152.38 0.59 1.2 763.6 84.0

Table 2. Data of Power Density and Volumetric Power for Bacterial Aggregation of Eschericia coli

Microbial aggregation

V, Volt I, mAPower

density, mW/m2

Volumetric power, mW/l

Eschericia coli 0.21 0.3 57.27 6.3

Microbial fuel cell operated in this research works in a batch system and uses Eschericia coli as biocatalyst and artificial waste water as a fuel. Eschericia coli is a bacterium which can grow in aerobic condition. Previous experiment implied that it was suitable for generating electricity using glucose as substrate. In this research, it was used artificial waste water containing glucose-glutamic acid as a source of organic matter. In the anode chamber of a MFC, the bacteria will oxidize organic content of artificial waste water and transfer electrons to an electrode. Electrons flow from the anode to the cathode electrode through the clip and conductive wire generating current. A volt meter equipped with plotter and multi meter were used for electricity measurement. During the measurement of current, a low current was produced, which was not sustained. It rapidly fell as the system become polarized, an indication that there was no effective coupling of the source of the electrons with the electrode.

Regarding with variables used in this research, various concentration of micro organism for constant ratio of micro organism/substrate (fuel) applied. For this purpose, different concentration was obtained by diluting micro organisms with different volume of buffer solution pH 7. The difference in concentration will give different reducing action of the micro organism.

The second variable is variation in microbial system used. Eschericia coli embedded in anode chamber were varied in two forms, in its liquid media and in its microbial aggregation. In the aggregation, cells were concentrated, constructed as consortia and immobilized to avoid redistribution using PEI. The reducing action of micro organism in this system surely will be different from that in non-aggregation.

And based on the experiment results, it can be seen that for variation of micro organism concentration, at concentration 7 mg/10 ml, the voltage and current is at maximum value. In this condition, the power density and volumetric power produced are 1384.1 mW/m2; 152.3 mW/l (see Table 1).

For microbial aggregation, to construct the microbial aggregation, 1.2 ml of bacteria in nutrient broth were centrifuged and washed 5 times. The volume of substrate used was 8 ml. The voltage and current data achieved is very low (see Table 2). The voltage, which is 0.21 V, is almost similar with the one with only substrate fed in anode chamber (0.191 V). In this case, the little volume of bacteria used could not give a significant rise to voltage. However, from the current measurement it can be proved that electricity is generated (0.27-0.3mA; 0.14mA for substrate only). The construction of cells aggregation was carried out through injecting 100 l of concentrated bacteria to the microelectrodes. The concentrated bacteria were centrifuged from 1.2 ml bacteria in nutrient broth. Therefore, the volume of bacteria used in aggregation is less than those on non-aggregation which in range of 3-10 ml.

An ideal comparison between aggregation and non aggregation system could be developed through extrapolation data of voltage for non microbial aggregation of Eschericia coli. However, the result seems practically

inappropriate because the voltage obtained from this method is only 0.13 V that is lower than the system with substrate. Therefore, the value of 0.2 V for aggregation system is more reasonable result than the extrapolated value.

For the future experiments, it can be suggested that more extended microelectrodes surface and larger volume of MFC compartments should be used to explore more advanced application of microbial aggregation for electricity generation in MFC, in order to produce more electricity.

CONCLUSION

Based on the research conducted, there are several points that can be concluded:1. Eschericia coli can be used as biocatalyst in MFC operation using BOD

standard test (glucose-glutamic acid) solution as substrate/fuel.2. The maximum energy achieved of Eschericia coli in this operation for

concentration variation is at 7 mg/10ml which gives the value of power density and volumetric power 1384.1 mW/m2; 152.3 mW/l.

3. The further experiment emphasized on application of microbial aggregation in MFC will be better conducted in large volume of anode-cathode chambers to enable more surface of microelectrode that can be embedded in electrodes chambers. The more surface of microelectrode used, the larger number of cells aggregation will be involved to construct the consortia, which will produce more electricity.

REFERENCES

Bond, D.R. and Lovley, D.R.. 2003. Electricity Production by Geobacter sulfurreducens Attached to Electrodes. Appl. Environ. Microbiol. 69: 1548 - 1555

Chaudhuri, S.K., and Lovley, D.R. 2003. Electricity Generation by Direct Oxidation of Lucose in Mediatorless Microbial Fuel Cells. Nat. Biotechnol. 21: 1229-1232

Haberman, W., and Pommer, E.H., 1991. Biological Fuel Cells with Sulphide Storage Capacity. Appl.Microbiol.Biotechnol. 35: 128-133

Jang, J.K., Pham, T.H., Chang, I.S., Kang, K.H., Moon, H., Cho, K.S., Kim, B.H. 2004. Construction and operation of a novel mediator and membrane-less microbial fuel cell. Process Biochem. 39: 1007-1012

Kim, B.H., Park, H.S., Kim, H.J., Kim G.T., Chang I.S., Lee, J., Phung, N.T. 2004. Enrichment of Microbial Community Generating Electricity Using a Fuel Cell Type Electrochemical Cell. Appl.Microbiol. Biotechnol. 63(6): 672-681

Liu, H. and Logan, B.E. 2004. Electricity Generation Using an Air-Cathode Single Chamber Microbial Fuel Cell in The Presence and Absence of a Proton Exchange Membrane. Environ,Sci.Technol. 38: 4040-4046

Markx, G.H., Andrewss, J.S., Mason, V.P. 2004. Towards Microbial Tissue Engineering. Trends Biotechnol. 22: 417-422

McKinley, J.B and Zeikus, J.G. 2004. Extracellular Iron reduction is Mediated in Part by Neutral Red and Hydrogenase in Escherichia coli. Appl.Environ.Microbiol. 70: 3467-3474G

Rabaey, K., and Verstraete, W. 2005. Microbial Fuel Cells: Novel Biotechnology For Energy Generation. Trends in Biotechnology. 23 (6): 291- 298

Verduzco-Luque, C.E., Alp, B., Stephens, G.M., Markx, G.H. 2003. Construction of Biofilmss with Defined Internal Architecture Using Dielectrophoresis and Flocculation. Biotechnol. Bioeng. 83: 39-44