INTRODUCTION

An increasing need for new energy sources due to the concerns of the limited availability of fossil fuels has motivated the development of alternate sources of energy. Microorganisms are potential sources for development of such alternative fuels from biomass which are also called as biofuels. Biofuels such as biodiesel (Ibeto et al., 2011; Igwenyi et al., 2011; Motojesi et al., 2011; Alkabbashi et al., 2009; Wan Omar et al., 2009), bioalcohols (Asad-ur-Rehman et al., 2008), biohydrogen (Nazlina et al., 2009; Yusoff et al., 2009) have been well described in literature.

Apart from biofuels, microorganisms have also been effectively used as a source of electricity by the use of Microbial fuel cell technology (Ho et al., 2009; Lim et al., 2010). Microbial fuel cells are the devices which convert energy associated with organic substrates into electricity using microorganisms as catalysts. In normal fuel cells, chemical oxidants oxidize the fuel and generated electrons travel across anode to cathode where an electron acceptor gets reduced thereby generating an electric current. Similarly in a microbial fuel cell, the oxidation of the fuel (or substrate) is brought about as a result of microbial metabolism. The primary mechanisms for electrochemically active microorganisms which are the key biocatalysts involved in electricity generation in MFCs, to transfer electrons to the electrodes, metabolic or physiological characteristics and the fundamentals of the anodic or cathodic reactions are well understood (Schroder, 2007). MFC-microbial communities have been known to degrade a wide range of substrates and pollutants. MFCs have operational and functional advantage over other technologies used for generating current from organic material. Firstly, direct conversion of substrate to electricity enables high conversion efficiency. Secondly, MFCs operate efficiently at ambient and even at low temperature, thus distinguishing them from current bio-energy processes. Thirdly, they don’t require gas treatment, as off gasses from MFCs are enriched with CO₂ with no useful energy content. Fourthly, they don’t require energy input for aeration and lastly MFC technology has widespread application in locations lacking electrical infrastructure.

Many anaerobic bacteria (DiChristina et al., 2002; Lovley, 2006) are known to produce electricity when supplied with an efficient electron acceptor or some mediators which assist microbes for electron transfer. These bacteria are known to be electrochemically active and are usually known as exoelectrogens.

Exoelectrogens are mostly metal reducing anaerobic bacteria which utilize metal ions as substrate for transfer of electrons from their surface to outside the bacterial cell. Many anaerobes can only transfer electrons to soluble compounds such as nitrate or sulphate that can diffuse across the cell membrane and into the cell. Exoelectrogenic bacteria are distinguished from these anaerobes by their ability to directly transport electrons outside of the cell which permits them to function in an MFC.

Microbial Fuel Cells (MFC) are electrochemical devices that convert the chemical energy contained in organic matter into electricity by means of the catalytic (metabolic) activity of living microorganisms (Mathuriya and Sharma, 2008; Bennetto, 1990; Kim et al., 2002; Rosenbaum et al., 2007). Microorganisms oxidize the substrate and produce electrons and protons in the anode chamber. Electrons, collected on the anode, are transported to cathode by external circuit and protons are transferred through the membrane internally. Thus, potential difference is produced between anode chamber and cathode chamber due to dissimilar liquid solutions. Electrons and protons are consumed in the cathode chamber by reducing oxygen, usually from water. MFCs can be classified into two types. One type generates electricity from the addition of artificial electron shuttles (mediators) to accomplish electron transfer to the electrode. The other type does not require these additions of exogenous chemicals and can be loosely defined as a mediator-less MFC. Mediators can divert electrons from the respiratory chain by entering the outer cell membrane, becoming reduced and then leaving in a reduced state to shuttle the electron to the electrode. 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 electrode.

In the present study two-compartment MFCs have been constructed with different enriched environmental samples and their efficiency have been analysed for power generation.

MATERIALS AND METHODS

Medium composition and inoculum source: The four collected samples were inoculated in enrichment medium for culturing anaerobic bacteria with composition peptone (15 g L^-1), yeast extract (5 g L^-1), D-glucose(5.5 g L^-1), sodium chloride (2.5 g L^-1), cysteine hydrochloride (0.5 g L^-1) and agar (0.75 g L^-1). Cysteine hydrochloride is a common reducing agent used to depress the redox potential of the medium which is usually required for the growth of anaerobes. Addition of agar increased the viscosity of medium which reduced the diffusion of atmospheric oxygen into medium. The above cited four samples contained two samples each of soil and water. Soil samples were collected from Mahamaya Ispat industry and Abhishek industry. Both of these are iron-ore industries located in Raipur. These sites were selected because of the probability of presence of iron reducing bacteria or exoelectrogens in these sites. Water samples were collected from two sources, first, from industrial waste water line of Urla industrial region and second from sewage tank of Amleshwar locality, both from Raipur.

Cultivation: One percent of each of the four samples was inoculated in 20 mL enrichment medium described above and incubated at room temperature without agitation under anaerobic conditions. The anaerobic conditions were maintained by sealing the culture tube after flushing with nitrogen gas and applying a layer (approx. 2 cm) of autoclaved oil over the surface of medium. This ensured anaerobic conditions and therefore, allowed the growth of only anaerobes. At the end of 24 h of incubation the enriched cultures were analysed for bioelectricity generation.

Microbial fuel cell construction and operation: Various forms of two cell compartment electrochemical cell may be devised to demonstrate microbial electricity generation and the one described here is one of them. A Microbial Fuel Cell (MFC) constructed in present study is a two-compartment structure divided by a salt bridge. The electrons available through the metabolism of the electron donors by microorganisms are transferred to the anode of the fuel cell and then to the cathode through the circuit, where they reduce the oxidant (Allen and Bennetto, 1993), consuming protons available through the membrane from the anode.

MFCs were constructed using two glass test tubes (35 mL capacity) connected with a salt bridge (5 mm diameter). Salt bridge was constructed using plastic U-tube filled with KNO₃ (saturated) and agar (20 g L^-1). Three different electrodes made up of graphite rod (14.828 sq. cm), carbon paper (34.2 sq. cm) and copper plate (15.68 sq. cm) were used. Copper wires of resistance 3.4x10^-3 Ω m^-1 were used for connecting circuits. The electrodes were soaked in phosphate buffer (50 mM) before placing in MFC. Different cathodic electrolytes were used to compare the performance of MFC. The cathodic electrolytes used were tap water, NaCl (10 g L^-1) with tap water, NaCl (10 g L^-1) with aerated tap water and KMnO₄ (0.2 g L^-1) with tap water. The enriched cultures were directly taken as anode. Methylene blue (0.1 mM) was used as mediator to study the performance of MFC. Potential (V volt) and current (I amp) were measured using a digital multimeter (Mastech, M-830BZ).

Calculation: Current density of MFC was calculated using i (mA m^-2) I/A where I is the current measured (mA) and A is the geometric surface area of anode (m²). The power density of the MFC was calculated using formula: P (mW m^-2) = IV where I is the current density and V is the voltage measured (mV).

RESULTS AND DISCUSSION

Enrichment of anaerobic exoelectron generating bacteria has been reported by Bond and Lovley (2003), Kim et al. (2002). In the present study glucose was used in the medium as carbon source in construction of fuel cell. Natural micro-flora of Palm Oil Mill Effluent (POME) sludge was also grown in dual-chamber Microbial Fuel Cells (MFC) to produce electricity by providing glucose at different concentration (Lim et al., 2010). Scott and Murano (2007) have also constructed the carbohydrate utilizing MFC. The comparative production of electricity from all four different samples with all variables has been shown in Table 1 and 2. Similar electricity generation from waste water sample have been done by Patil et al. (2011).

Table 1:	Measure of voltage, current, current density and power density in all samples with graphite as electrode

Table 2:	Measure of voltage, current, current density and power density in all four samples with copper as electrode

Fig. 1:	Microbial fuel cell

Presence of mediator (methylene blue) does not execute the enhanced current production. Table 1 and 2 show that cathodic electrolyte has significant effects on power density. In all the samples tap water with KMnO₄ resulted in maximum current density (Table 1, 2). Jadhav and Ghangrekar (2008) found the effect of catholyte for improving the performance in fuel cell.

The maximum current density was found with graphite electrode water sample from Amleshwar locality (108.57 mA m^-2) and with carbon electrode from water sample from industrial waste water line of urla (84.82 mA m^-2). The enriched exoelectron generating bacteria demonstrates the electricity generation which can be further improved and may have other application too as reported by Kim et al. (2008).

Figure 1 shows the construction of microbial fuel cell using the enriched anaerobic bacteria from soil and water samples for exoelectron generation and electricity production.

CONCLUSION

The maximum power density obtained was 48.85 mW m^-2 with graphite rod and 42.59 mW m^-2 with copper electrode and current density obtained was 108.57 mA m^-2 with graphite rod and 88.01 mA m^-2 with copper electrode all obtained with KMnO₄ solution.