1. INTRODUCTION:

In recent years, surge for fossil fuels and other nonrenewable resources has been seen due to explosion in worldwide population, modernization and boom in industrialization (Brown, 2001). The scenario has made the path for searching for alternative to non-renewable resources which should meet the criteria of being renewable and eco-friendly. Utilization of waste materials represents such alternative. In recent decades, impressive progress has been made in the field of development of clean and green energy technologies. Energy is the backbone for the development of any nation. Electricity is one of the major forms of energy resulting from many technologies like burning of coal and other fossil fuels; larger hydrothermal plants, nuclear power plants etc. which are associated with the adverse environmental consequences (Wei et al., 2010). With increasing industrialization, an ever seen amount of waste has been generated.

This led to economical challenges like production of bulk of the waste which led to high handling and management coast, sophisticated treatment and disposal operations as well as severe environmental consequences. The World Water Development Report (2003) estimated that near about 2 million tons of wastes per day are disposed to within receiving water bodies and rivers which contains domestic wastes, human excreta, agricultural wastes, industrial wastes in the various forms. Approximately, 22% of water is used for different industrial purposes worldwide and about 70% of industrial discharges is dumped untreated in water resources by developing countries. In India only, 68.5 million m³ per day wastewater from medium and large industries is dumped directly into local rivers and streams without any treatments whereas total amount of wastewater generated is about 55,000 million m³ per day (Pangare et al., 2006). Concerning the scenario, MFCs are of much important because it offers direct degradation of wide range of organic compounds for producing bioelectricity and its treatment (Logan, 2009; Kaushik and Jadhav, 2017).

MFCs are the bio-electrochemical devices that enable the diverse metabolic activity of the native microorganism to convert the biodegradable waste organic matters directly into electrical energy without any combustion (Ahn and Logan, 2010). In other words, MFCs enable the oxidation and reduction of waste matter at electrode surface of fuel cells where microbial entities perform catalytic activities to degrade the almost any organic compound into electricity (Bond and Lovley, 2003). In these electrochemical systems, the native microorganisms play crucial role in harnessing useful energy meanwhile treatment of the substrate in terms of normalization of pH (Kaushik and Jadhav, 2017), removal of odors (Kim et al., 2008, Kaushik and Jadhav, 2017), many dyes (Fernando et al., 2013) and significantly lowering the chemical oxygen demand (Jiang et al., 2012). Two types of basic deigns are there; one is single chambered microbial fuel cell (SCMFC) and other is dual chambered microbial fuel cell (DCMFC). Two of these basic configurations can further be modified say for plate (Min and Logan, 2004), tubular (Rabaey et al., 2005), stacked (Choi & Ahn 2013), up flow, with membrane (Bond and Lovley, 2003), membrane-less (Gajda et al., 2015) or consisting a salt bridge (Min et al., 2005), open air cathode or with additional catalyst, having single (Sund et al., 2007) or multiple electrodes (Liu et al., 2004) and so on.

Microbial fuel cells may employ simple fermentable substrates like pure chemicals, for example- glucose (Potter, 1911, Park and Zeikus, 2000), xylose, sucrose and maltose (Chaudhuri and Lovley, 2003), acetate (Pham et al., 2003), lactate (Ringeisen et al., 2007) etc as fuels while fermentative microorganisms as catalyst (Shukla et al., 2004; Lovley, 2006). After industrial revolution rapid growth in industrialization and diversity in industries came into existence, the by-products and waste generation also diversified. Nowadays MFCs have turned to be more realistic and variety of wastewater has been used as fuel in MFC such as domestic sewage (Min and Logan, 2004; Liu at et., 2004, Ahn and Logan, 2010), brewery wastewater (Feng et al., 2008), paper and pulp wastewater (Huang and Logan, 2008), swine wastewater (Kim et al., 2008), chocolate industry wastewater ( Patil et al., 2009), rice mill wastewater (Daniel et al., 2009; Behera et al., 2010), phenolic wastewater (Luo et al., 2009), real dye wastewater (Kalathil et al., 2012), and Oil refinery wastewater (Majumder et al., 2014) etc. These wastewaters are available in bulk, has high organic load and are rich in nutrients which may support the bacterial growth of microorganisms. Biodegradability of wastewater makes it possible to use it in MFCs as fuel. It took huge amount of energy to purify wastewater in treatment units. The MFCs can also be associated with these units to purify it to certain extend with electricity production.

The present work deals with performance of microbial fuel cells with low-cost material (zinc and carbon electrodes) as sole electron acceptors, optimization of operational conditions (pH and temperature), utilization of aerated cathode, and native bacteria to generate electricity from rice bran oil refinery wastewater.

2. MATERIALS AND METHODS:

2.1. MFC fabrication and operation:

Dual-chambered microbial fuel cells were fabricated using non-reactive, autoclavable, polyvinyl chloride (PVC) containers with working volume of 750 mL and operated in fed-batch mode (Kaushik and Jadhav, 2017). The fuel cells comprised of an anaerobic anode and an open air-Cathode chambers in order to minimize the possibility of diffusion of oxygen to nearby anode which may increase the efficiency of the system (Das and Mangwani, 2010) (Figure–01). The non-noble, inexpensive materials i.e. carbon rod and Zinc plate metals were chosen and Zn-C combination as anode and cathode were selected based on our previous studies (data not shown). Use of non-noble materials may replace the expensive electrode materials like platinum based electrodes which may lower the cost of MFCs (Liu et al., 2013). A UPVC pipe containing agar salt bridge was used to separate the chambers physically at distance of 3.2 cm. The aid of adhesive material (M-Seal) was used to fix both the chamber intact and other epoxy materials were used to seal the ports (Kumar et al., 2012; Kaushik and Jadhav, 2017). External copper wires were used to connect the electrodes to the digital multimeter by alligator clips. The open air-cathode chamber was filled with phosphate buffer and pH adjusted to 7.0 (Logan and Regan, 2006) 33. In this set up oxygen was employ as the final electron acceptor (Liu et al., 2013). MFCs were surface sterilized as described by Kaushik and Jadhav, 2017. All tests were performed under room temperature, 30±2 ⁰C, without pH control or any catalyst except where mentioned.

Figure (01) - Schematic diagram of dual chambered MFC

2.2 Isolation, Screening, biochemical characterization and Growth Condition of selected Electrogen:

2.2.1. Isolation and screening of electrogenic bacterial strain:

The bacterial isolates were isolated from anode of rice bran oil refinery wastewater fed MFC by serial dilution method. The dilutions were inoculated on plates containing nutrient agar medium (NAM). Mixed bacterial colonies were observed upon 48 h of incubation, which was further pure cultured through the streak plate method and aseptically transferred to NAM slants for further use (Prescott and Harley, 2002). Screening process for confirmation of the electrogenic properties were carried out in sterile wastewater fed MFCs. The most potent bacterial isolate was subjected to cross check in synthetically designed wastewater (glucose 3.0 g/l, FeCl₃ 25.0 mg/l, NH₄Cl 0.5 g/l, CaCl₂ 5.0 mg/l, KH₂PO₄ 0.25 g/l, K₂HPO₄ 0.25 g/l, CuCl₂ 10.5 mg/l, MgCl₂ 0.3 g/l, NiSO₄ 16.0 mg/ l, CoCl₂ 25.0 mg/l, ZnCl₂ 11.5 mg/l and MnCl₂ 15.0 mg/l) under anaerobic microenvironment (Aldrovandi et al., 2009).

2.2.2. Microscopic and Biochemical characterization of the most potent electrogenic bacterial isolate:

Microscopic study was performed by Gram’s staining, endospore staining and acid-fast staining. For biochemical characterization of selected bacteria, indole test, methyl red test, voges–proskauer test, citrate utilization test (IMViC test), fermentation of carbohydrate test and amylase production test were performed (Prescott and Harley, 2002).

2.2.3. Media, growth conditions and inoculum preparation:

Nutrient agar and broth (Hi Media Laboratory Pvt. Ltd., Mumbai, India), were used for inoculum preparation as well as maintenance of bacterial isolates. Ultraviolet-visible spectrophotometric analysis and serial dilution colony-counting method was used to estimate the cell concentration. The bacterial isolate was grown at 36±2⁰C temperature for 48 h. The cells were harvested by refrigerated centrifugation at 6000 rpm for 10 min (Liu et al., 2010). The cell concentrations were set at 0.2 (OD_600nm), inoculum accounting for 16±2 x 10⁴CFU/mL throughout the experiments.

2.3 Wastewater characterization:

The wastewater was collected from local rice bran oil refinery named Shree Sita Refiners Pvt. Ltd, Arasnara, Durg, Chhattisgarh, India designated as SSR. Sample was collected in sterile flask (volume 5 L) and stored at 4±1⁰C in a refrigerator for short term. Wastewater without modification in organic load or pH adjustments was used except where mentioned. Typical physicochemical characteristics of wastewater pH, color, odor, biological oxygen demand (BOD), chemical oxygen demand (COD), TDS and electrical conductivity (EC) were analyzed before and after the experiment for the monitoring of wastewater treatment progress (APHA, 1998).

2.4 Optimization of operating conditions:

Operating conditions are important factors for maximum bioelectricity production. Thus, incubation of bacterial culture in nutrient media for different time periods was tested as inoculum. 48 h incubated bacterial culture of isolate WRS-6 (most potent electrogenic bacterial strain) was studied at various incubation periods of 24, 48, 72, 96, and 120 h. It is evident from previous reports that growth of microorganisms is greatly affected by pH which directly influence the rate of bioelectricity production. Concerning this MFCs were operated at different pH range of 4.0 to 9.0. The operating temperature also influences the growth, rate of metabolic activity and survival of bacteria. Thus, a series of experiment at different temperature range of 20, 25, 30, 35 and 40⁰C was conducted.

2.5 Statistical Analysis:

The performance of MFCs were continuously operated and monitored. Electrode output was evaluated in terms of voltage (V) and current (mA) in the open circuit using auto-range digital multimeter (KUSAM-MECO 603) after one hour time intervals. The COD and BOD were determined by standard methods as per APHA. EC and TDS were analyzed using EC-TDS analyzer (ELICO CM-183 VER. 2.3), DO was measured using a DO analyzer (ELICO PE-135) and the pH was measured using a pH meter (ELICO LI-120). All experiments were performed in triplicate and repeated at least thrice to measure the reproducibility. Descriptive analysis of data was performed by one-way analysis of variance (ANOVA), least significant difference (LSD) for test of significant difference among means using Duncan’s multiple range test (DMRT). ˃ 0.05 (P values) were considered as significant. All the analysis was performed at the 0.05 probability level by SPSS version 16.0 and typical values are presented.

3. RESULTS AND DISCUSSION:

3.1. MFC Operation and Performance:

High cost of the electrode materials hinders the real field application of MFCs. In present study, MFCs were operated with the non-noble materials zinc and carbon because of their low cost and ubiquity and are implicated with 2- electron pathway for catalytic reduction of oxygen (Liu et al., 2013). Sterile wastewater was added to the anodic chamber and cathode chamber was filled with 50 mM phosphate buffer. MFCs were operated in batch mode at ambient room temperature (30 ± 2⁰C) and pressure. Anode and cathode was connected to external wiring to complete the circuit and voltage and current were measured via auto-range digital multimeter. The anode compartment was sealed completely to maintain anaerobic micro-environment. It is noted from previous studies that absence of oxygen or any terminal electron acceptor in anode chamber forces the exoelectrogens to transfer their electrons outside of the cell to the electrode, hence maintaining anaerobic condition is important to gain electricity (Lovely et al., 1993). The catholyte solution was exposed to ambient air for oxygen reduction reaction to occur where oxygen work as final electron acceptor. Air-cathode was chosen for the study as ambient oxygen has ubiquity and high standard equilibrium potential and is available for zero prices (Liu et al., 2013; Feng et al., 2008). In anode compartment, microbial entities oxidize fuel which result in production of electrons and protons (Jung and Regan, 2007). Protons are transferred to the cathode compartment through the salt bridge and the electrons travel through the external circuit resulting in electrolytic reduction of oxygen on cathode side. Thus, the overall performance is strongly affected by the oxygen reduction reaction efficiency especially where MFCs are being operated at neutral pH and room temperature (Gil et al., 2003, Gregory et al., 2004, Liu et al., 2013; Merino-Jimenez et al., 2017).

3.2. Isolation and screening of electrogenic bacterial strain:

Seven bacterial strains were isolated from anode of rice bran oil refinery wastewater fed MFC by serial dilution method during the month of June, 2015 to September, 2015. The dilutions were inoculated on plates containing nutrient agar medium (NAM). After 48 h of incubation, mixed bacterial colonies were observed which was further pure cultured through the streak plate method (Prescott and Harley, 2002). The isolates were named as WRS-1 to WRS-7. Screening process for confirmation of the electrogenic properties were carried out in sterile wastewater fed MFCs (figure -02). The electrogenic potentials of bacterial isolates were subjected to test in synthetically designed wastewater (glucose 3.0 g/l, FeCl₃ 25.0 mg/l, NH₄Cl 0.5 g/l, CaCl₂ 5.0 mg/l, KH₂PO₄ 0.25 g/l, K₂HPO₄ 0.25 g/l, CuCl₂ 10.5 mg/l, MgCl₂ 0.3 g/l, NiSO₄ 16.0 mg/ l, CoCl₂ 25.0 mg/l, ZnCl₂ 11.5 mg/l and MnCl₂ 15.0 mg/l) under anaerobic microenvironment for final selection of most potent isolate (Aldrovandi et al., 2009). The isolate WRS-6 was selected for further study based on power behavior (Figuure-03).

Figure (02) – Voltage-Time and Current-time profile of bacterial isolates on sterile wastewater

3.2. Characteristics of most potent bacterial isolate:

The colony characterization of selected bacterial species (WRS-6) was done and it was found to be irregular in form with lobate margin, convex elevation and opaque in optical character on Nutrient Agar plates at 37°C. The bacterial colony was cream-white and rough. After this, microscopic study was performed with different staining techniques such as Gram’s staining, Endospore staining and Acid-fast staining. It was found to be large rod-shaped, gram positive, acid-fast negative, and endospore forming. Different biochemical tests were performed. The isolate was positive for glucose and sucrose fermentation, amylase test, urease test, citrate test while found to be negative for lactose fermentation, methyl red, VP and Indol test.

3.5. Optimization of operational conditions:

3.5.1. Incubation period of inoculum:

Effect of incubation period of inoculum on bioelectricity production was studied with a range of 24 h to 120 h. Isolate WRS-6 was inoculated on nutrient broth (NB) for 48h and was used for making different inoculum. Inoculum incubated for different periods was added to the MFCs containing sterile wastewater and OCV and current were recorded using auto-range multimeter. All the experiments were repeated at least three times and typical values are represented. It was found that increasing the incubation period of bacterial culture used as inoculum led to increase in production to maximum 1.239±0.012V with corresponding current 9.535±0.148mA at 48 h (Figure -04). Further increase in incubation of inoculum of bacterial culture resulted in decreased OCV, however current at 72 h incubated cultures were comparable to current obtained from 48 h. It implies that 48 h to 72 h incubated culture of newly isolated electrogen WRS-6 would be optimum to produce maximum OCV and current in rice bran oil refinery wastewater fed MFC.

Figure (03) – Voltage-Time and Current-time profile of bacteria isolates on synthetic wastewater

Figure (04) –Effect of incubation period of inoculum (WRS – 6) on bioelectricity production for five consequent days

Significant impact on characteristics of wastewater was observed in terms of removal of BOD and COD. Up to 37.34 % removal of COD and 84.60 % removal of BOD were noted. It was noted for all the set up that there was slight lowering in pH which might be due to production of fermentative acids. Alteration in catholyte characteristics also noted in terms of EC, TDS and pH. Two to four fold increase in EC from 4.473 to 7.760±0.210; 8.950±0.244, 12.158±0.08, 16.55±0.658, and 15.158±0.08 mS/cm were noted for incubation period of 24, 48, 72, 96, 120 h respectively. Similar trend has been seen for TDS of catholyte.

4.1.1. Effect of operating temperature:

In MFC, the temperature may play crucial role as it directly influences the metabolic activity of the bacterial counter part of MFC, thus it is essentially required to maintain the optimal temperature condition inside the vessel in order to generate maximum amount of electricity. Therefore production of bioelectricity at different temperature ranges from 20 to 40⁰C was studied. In MFC assisted with WRS-6, bioelectricity production increased considerably with increase in temperature and reached a maximum OCV of 1.157±0.009 V and Current of 4.417±0.269 mA at 35⁰C followed by the lower temperatures (Table-01). After the completion of experimental run, it was noted that variation in temperature resulted in approximately two fold increase in electrical conductivity of anolyte as well as catholyte solution. COD removal efficiency reached to a maximum of 60.27% at 35⁰C and decreased with lower and higher operating temperature (20 ⁰C and 40 ⁰C).

Earlier report also suggests optimal temperature has significant role in higher electricity production (Pham et al., 2008; Kumar et al., 2012). Some researcher found that MFCs operated under 35⁰C temperatures can covert substrate efficiently in mediator-less MFC, although there was good power output at 40⁰C also (Kumar et al. 2012; Wei et al.,2013, Kaushik and Jadhav, 2017). Our findings suggest that MFCs are able to produce stable power (maximum current of 1.4 mA) at lower operating temperature (8–22 ⁰C) also (Pham et al., 2008; Jadhav and Ghangrekar, 2009). This shows the flexibility of these systems for operating it under diverse environmental temperature due to diversity of various electrochemically active bacteria making them eligible for degradation of substrate and production of electricity (Min et al., 2008).

Table (01) - Effect of operating temperature on electricity production for five consequent days by isolate WRS – 6

Time (days)

Operating temperature

20⁰C

25⁰C

30⁰C

35⁰C

40⁰C

Voltage (V)

Current (mA)

Voltage (V)

Current (mA)

Voltage (V)

Current (mA)

Voltage (V)

Current (mA)

Voltage (V)

Current (mA)

Day 1

1.08

0.001

3.48

0.042

1.09

0.001

3.18

0.030

1.13

0.001

4.21

0.008

1.18

0.001

5.02

0.026

0.88

0.001

3.27

0.005

Day 2

1.06

0.001

3.34

0.050

1.09

0.001

3.51

0.011

1.12

0.001

4.08

0.007

1.17

0.001

5.03

0.037

0.85

0.001

3.13

0.030

Day 3

1.04

0.001

3.26

0.011

1.07

0.001

3.41

0.006

1.11

0.001

3.82

0.009

1.16

0.001

4.35

0.025

0.85

0.001

2.82

0.006

Day 4

1.03

0.001

2.59

0.004

1.06

0.001

3.27

0.054

1.09

0.001

3.58

0.006

1.14

0.001

4.01

0.012

0.83

0.001

2.65

0.013

Day 5

0.99

0.001

2.22

0.003

1.04

0.001

2.66

0.006

1.07

0.001

3.15

0.006

1.13

0.001

3.69

0.006

0.82

0.001

2.29

0.028

Table (02) - Effect of operating pH on electricity production for five consequent days by isolate WRS – 6

Time (days)

pH range

pH 5

pH 6

pH 7

pH 8

pH 9

Voltage (V)

Current (mA)

Voltage (V)

Current (mA)

Voltage (V)

Current (mA)

Voltage (V)

Current (mA)

Voltage (V)

Current (mA)

Day 1

1.15

0.001

3.21

0.008

1.19

0.001

4.73

0.009

1.18

0.001

4.58

0.029

1.03

0.001

2.98

0.004

0.94

0.001

2.11

0.008

Day 2

1.13

0.001

3.74

0.007

1.20

0.001

5.59

0.005

1.17

0.001

4.67

0.008

1.01

0.001

3.18

0.006

0.90

0.001

1.98

0.003

Day 3

1.12

0.001

3.36

0.009

1.19

0.001

5.09

0.007

1.16

0.001

4.41

0.005

0.97

0.001

2.95

0.009

0.83

0.001

1.85

0.003

Day 4

1.08

0.001

3.24

0.006

1.18

0.001

4.93

0.009

1.16

0.001

4.18

0.006

0.95

0.001

2.69

0.004

0.77

0.001

1.65

0.001

Day 5

1.06

0.001

3.05

0.005

1.18

0.001

4.44

0.013

1.14

0.001

3.85

0.014

0.89

0.001

2.39

0.009

0.79

0.001

1.45

0.001

Alteration of pH characteristics of catholyte indicates that, there is good transferred of proton as compared to electrons which led to decrease in pH (initial pH 7.103±0.032 to 6.720±0.140) of the catholyte. The performance in this case could be limited by the availability of the electrons resulted from microbial action in anode chamber which is transferred through anodic electrode to cathode chamber to final reduction reaction of the oxygen (Jadhav and Ghangrekar, 2009). The condition suggests that there is need for remedies like equipping the cathode with stirring conditions or the air sparger for the enhanced cathodic reactions.

3.4. Electrochemical Activity of native Bacillus sp. (WRS-6):

In present study rice bran oil refinery wastewater is used as substrate. The wastewater from these industries are generated from washing of extraction units; thus, may contain some complex components like mono-saturated fats, poly-unsaturated fats, saturated fats, great amount of essential fatty acids, Linolenic acids, Linoleic fatty acid, and trace amount of other nutrients which are recommended for edible oils (Ghosh, 2007). Therefore, the wastewater from the industry might contain sufficient values of nutrition as well as high organic load to support bacterial growth (Akhter et al., 2014). The members of Bacillus sp. are known to found widely in environment and have strong survivability even in hostile conditions probably due ecological adaptation in different environments which provide them metabolic diversity (Ivanova et al., 2003; Bottone, 2010). Member of Bacillus sp. are also reported to use organic waste compounds as carbon source at moderate temperatures. This makes them suitable for MFC experiments which are operated under moderate temperature. Also, the multiplicity of carbohydrate catabolic pathways possess by the members of Bacillus sp. help them to utilize variety of carbohydrates, peptides, amino- acids and vitamins as nutrient source majorly from plant-derived materials present in the soil (Ivanova et al., 2003).

Demonstration of useful energy from newly isolated Bacillus sp. (designated as WRS-6):

Individual set of MFCs (each contained 3 units) fed with only sterile oil refinery wastewater (SORW), sterile wastewater + starch from cooked rice (SORW+S), sterile oil refinery wastewater + rotten fruit sap (SORW+FS), and sterile oil refinery wastewater + sap of vegetable waste from kitchen (SORW+VWS), were combined in series to glowing of LED of 4V. These demonstrations were intended to check the efficiency of isolate WRS-6 in MFC for practical use. All the individual units were able to produce near about 1.1 V of OCV and current of 3.5 mA on an average. Final output from series arrangement produced OCV of 3.05 V and current of 2.67 mA, 2.47 V and current of 1.05 mA. 3.08 V and current of 4.40 mA, and 2.68 V and current of 2.15 mA from MFC fed with SORW, SORW+VWS, SORW+S, and SORW+FS, respectively; which was high enough to glow an LED of 4V (Figure -05). The intensity of light was quite good for 11 days, for all the MFCs except SORW+VWS (intensity dropped after 5 days); after that decline in intensity has been observed. Although, the electricity generated was quite stable from day 3 to 15 day in terms of voltage. A gradual decrease in current along with drop in voltage has been noticed. In present study, prominent limitation could be the transfer of electrode mass from anode to cathode compartment as deposition is seen on cathodic electrode leading to remarkable electrode fouling in anode side. On an average, 28.71% and 1.51% loss of weight of anode and cathode has been recorded. Although for both the electrode negligible loss in activity has been seen. Huang et al. (2011) also reported that mass transfer of anode to the cathode and cathode limitations are the factors limiting performance of MFC. It is shown in many reports that oxidation of fuel in anode can cause acidification due to production of cations in higher concentrations rather than protons. This can hinder the performance of MFC. In cathode compartment, the nonspecific transport of cations other than protons through the salt bridge and production of alkaline by oxygen reduction can also limits the performance of MFC (Rismani-Yazdi et al., 2008; Zhao et al. 2005). So, recommendations from present study include minimizing the cathode limitations by employing oxygen sparger to enhance the reaction of oxygen reduction. Also, preventing the loss of electrode mass by coating them with suitable aid prior to use especially in long term experiments which may also help in enhancing the electron transfer (Xie et al., 2012). In real wastewater, the varying amount of degradation of organic component cause the variation in concentration of proton generated over a long period of time. This can also significantly change the stable performance of MFC; hence affect the predictions of electrochemical measure for these real MFCs (Liu et al., 2013).

Figure (05) –Snap of Glowing of 4V LED using MFCs fed with (A) SORW+S, (B) SORW+FS, (C) SORW+VWS, and (D) SORW using isolate WRS -6 as inoculum

Monitoring of wastewater treatment progress:

Progress of wastewater treatment was determined experimentally in terms of various parameters of typical wastewater characteristics viz., pH, DO, BOD, total dissolved solids, and electrical conductivity, before and after operation using standard methods. The color and odor were found to be minimized after the experimental run (Table-03). The presence of inorganic ions due to the electrochemical activity of bacterial isolate resulted in increased value of in electrical conductivity of anolyte. Organic substances present in the wastewater is degraded during the metabolic process and electricity generation producing simpler compounds and other successive metabolized products which may have caused increase in TDS (Uwidia and Ukulu, 2013). Alteration in catholyte characteristics was observed. Decrease in pH from7.53±0.032 to 6.84±0.056 was noted which could be due to transfer of more amount of protons from salt bridge as compared to electrons or cathodic limitations. Increase in EC (from 6.801±0.013 to 9.923±0.352 mS/cm) is the result from charge transfer from the circuitry. Increase in TDS (2.023±0.011 to 8.138±0.532 ppt) is resulted from mass transfer from anode to cathode and fouling of salt bridge.

Table (03) - Showing characteristics of wastewater before and after experimental run under optimized conditions

Parameter	Before experimental run	After experimental run
Odor	Pungent	Less odorous
Color	Buff	Translucent
pH	6.05± 0.05	5.78± 0.45
Total dissolve solutes (TDS) (ppt)	3.558± 3.11	6.615± 3.32
Electrical Conductivity (mS/cm)	7.868±0.42	9.56 ±0.92
Dissolve Oxygen (mg/L)	1.9 ± 1.5	0.08± 0.07
Biological oxygen demand (BOD) (mg/L)	43.43± 2.5	21.53± 0.94
COD (mg/L)	5390± 462	2918± 320

In about present study, about 56.99% and 49.55% of removal of COD and BOD has been observed. Previous reports also suggest that MFCs are efficient in lowering the organic loads (ranging from 27 to 98%) from various real wastewaters. 27±2% to 76±4% removal of TCOD from paper and pulp wastewater in an open-circuit control (Huang and Logan, 2008), 98.0% COD removal from starch processing wastewater (Lu et al., 2009), COD and BOD removal efficiency of 45.21% and 45% from palm oil mill wastewater, respectively (Baranitharan et al., 2013), COD removal efficiency around 90% of dairy industry wastewater (Elakkiya and Matheswaran, 2013), approximately 80% removal of COD from municipal wastewater (Buitrón and Cervantes-Astorga, 2013), 91% removal of COD at 40 days hydraulic retention time while using coconut husk retting wastewater as substrate (Jayashree et al., 2014), and 30% COD removal of oil refinery wastewater (Majumder et al., 2014). These MFCs can further scale up for enhancing the power output as well as wastewater treatment efficiency using co-cultures, combinations of series and parallel connection, addition of mediators or can be targed towards remediation of specific compound etc. (Behera et al., 2010). It is also reported that MFCs can be coupled with the process with recovery of different end products based on the nature and components of substrate which could lead to increase the efficiency of wastewater treatment as well as removal of desired organics from the substrate with major goal of concurrent electricity generation (Behera et al., 2010; Kaushik and Jadhav, 2017; Patil et al., 2009).

CONCLUSIONS:

The results elucidate that newly isolated Bacillus sp. (designated as WRS-6) can be utilize as electrogen in mediator-less MFCs fed with various of organic substrates under optimized operating conditions for efficient electricity production along with its treatment using inexpensive metal electrodes as sole electron acceptors.

ACKNOWLEDGEMENT:

The authors thank University Grants Commission, New Delhi to support the research scholar under National Eligibility Test- Senior Research Fellowship [F.15-6(DEC.2013)/2014(NET), UGC-Ref. No: 3674/(NET-DEC.2013)] and Department of Science and Technology, New Delhi for providing financial support through DST-FIST scheme to School of Studies in Biotechnology, Pt. Ravishankar Shukla University, Raipur.

Declaration of interest:

Authors have no conflicts of interest to disclose regarding any financial and personal relationships with other people or organizations that could inappropriately influence (bias) our work which includes employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/ registrations, and grants or other funding.

REFERENCES:

1. Ahn, Y. and Logan, B. E. (2010). Effectiveness of domestic wastewater treatment using microbial fuel cells at ambient and mesophilic temperatures. Bioresource Technology, 101:469-475.

2. Akhter, M., M. A. Ali, Z. Haider, and S. Muzammil. (2014). Efficacy of parboiling on physico-chemical properties of some promising lines/varieties of rice (Oryza sativa L.). Sci Tech and Dev 33 (3): 115-122.

3. Aldrovandi, A., Marsili, E., and Stante, L. (2009). Sustainable power production in a membrane-less and mediator-less synthetic wastewater microbial fuel cell. Bioresour Technol 100(13):3252–3260.

4. APHA American Water Works Association (1998). Water Pollution Control Federation. Standard methods for the examination of water and wastewater. American Public Health Association, Washington.

5. Baranitharan, E., Khan, M. R., Prasad, D. M. R. and Salihon, J. B. (2013) Bioelectricity generation from palm oil mill effluent in microbial fuel cell using polacrylonitrile carbon felt as electrode. Water Air Soil Pollut., 224:1533-1544.

6. Behera, M., Jana, P. S., More, T. T., and Ghangrekar, M. M. (2010). Rice mill wastewater treatment in microbial fuel cells fabricated using proton exchange membrane and earthen pot at different pH. Bioelectrochemistry 79:228-233.

7. Bond, D. R. and Lovley, D. R. (2003). Electricity production by Geobacter sulfurreducens attached to electrodes. Applied and Environment Microbiology, 69:1548- 1555.

8. Bottone, E. J. (2010). Bacillus cereus, a Volatile Human Pathogen. Clinical, Microbiology Reviews 23(2): 382–398.

9. Brown, M. A. (2001). Market failures and barriers as a basis for clean energy policies. Energy Policy, 29:1197–1207.

10. Buitrón, G., and Cervantes-Astorga, C. (2013) Performance evaluation of a low-cost microbial fuel cell using municipal wastewater, Water Air Soil Pollut, 224:1470-1478.

11. Chaudhuri, S. K., and Lovley, D. R. (2003) Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells. Nat. Biotechnol., 21:1229–1232.

12. Choi, J., and Ahn, Y. (2013) Continuous electricity generation in stacked air cathode microbial fuel cell treating domestic wastewater. Journal of Environmental Management, 130: 146-152.

13. Daniel, D. K., Mankidy, B. D., Ambarish, K. and Manogari, R. (2009) Construction and operation of a microbial fuel cell for electricity generation from wastewater. International Journal of Hydrogen Energy, 34: 7555–7560.

14. Das, S. and Mangwani, N. (2010). Recent development in microbial fuel cells: a review. Journal of Scientific and Industrial Research 69: 727-731.

15. Elakkiya, E. and Matheswaran, M. 2013 Comparison of anodic metabolisms in bioelectricity production during treatment of dairy wastewater in Microbial Fuel Cell. Bioresource Technology, 136: 407–412.

16. Feng, Y., X. Wang, B. E. Logan, and H. Lee. (2008) Brewery wastewater treatment using air-cathode microbial fuel cells. Applied Microbiology and Biotechnology, 78:873-880.

17. Fernando, E., Keshavarz, T. and Kyazze, G. (2013). Simultaneous co-metabolic decolourisation of azo dye mixtures and bio-electricity generation under thermophillic (50ºC) and saline conditions by an adapted anaerobic mixed culture in microbial fuel cells. Bioresource Technology, 127: 1–8.

18. Gajda, I., Greenman, J., Melhuish, C., and Ieropoulos, I. (2015). Simultaneous electricity generation and microbially-assisted electrosynthesis in ceramic MFCs. Bioelectrochemistry, 104: 58–64.

19. Ghosh, M. (2007) Review on recent trends in rice bran oil processing, J Amer Oil Chem Soc, 84:315–324.

20. Gil, G-C., Chang, I., Kim, B. H., Kim, M., Jang, J-K., Park, H. S., and Kim, H. J. (2003). Operational parameters affecting the performance of a mediator-less microbial fuel cell. Biosensors and Bioelectronics, 18: 327- 334.

21. Gregory, K. B., Bond, D. R., and Lovley, D. R. (2004). Graphite electrodes as electron donors for anaerobic respiration. Environment Microbiology, 6: 596- 604.

22. Huang, L., Logan, B. E. (2008) Electricity generation and treatment of paper recycling wastewater using a microbial fuel cell. Applied Microbiology and Biotechnology, 80:349- 355.

23. Huang, L., Regan, J. M., Quan, X. (2011) Electron transfer mechanisms, new applications and performance of biocathode microbial fuel cells. Bioresource Technology, 102:316–323.

24. Ivanova, N., Sorokin, A., Anderson, I., Galleron, N., Candelon, B., Kapatral, V., Bhattacharyya, A., Reznik, G., Mikhailova, N., Lapidus, A., Chu, L., Mazur, M., Goltsman, E., Larsen, N., D’Souza, M., Walunas, T., Grechkin, Y., Pusch, G., Haselkorn, R., Fonstein, M., S. D. Ehrlich, R. Overbeek, and N. Kyrpides. (2003). Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423(6935):87-91.

25. Jadhav, G.S., and Ghangrekar, M.M. (2009) Performance of microbial fuel cell subjected to variation in pH, temperature, external load and substrate concentration, Bioresource Technology 100:717–723.

26. Jayashree, C., Arulazhagan, P., Kumar, A. S., Kaliappan, S., Yeom, I. T., Banu, J. R. (2014) Bioelectricity generation from coconut husk retting wastewater in fed batch operating microbial fuel cell by phenol degrading microorganism, Biomass and Bioenergy, 69:249-254.

27. Jiang, H., Luo, S., Shi, X., Dai, M. and Guo, R. (2012). A novel microbial fuel cell and photobioreactor system for continuous domestic wastewater treatment and bioelectricity generation. Biotechnology Letters, 34: 1269–1274.

28. Jung, S., and Regan, J. M. (2007). Comparison of anode bacterial communities and performance in microbial fuel cells with different electron donors. Appl Microbiol Biotechnol 77:393–402.

29. Kalathil, S., Lee, J., and Cho, M. H. (2012). Efficient decolorization of real dye wastewater and bioelectricity generation using a novel single chamber biocathode-microbial fuel cell. Bioresource Technology 119:22-27.

30. Kaushik, A., and Jadhav, S. K. (2017). Conversion of waste to electricity in a microbial fuel cell using newly identified bacteria: Pseudomonas fluorescens. International Journal of Environmental Science and Technology 14: 1771–1780.

31. Kim, J. E., Dec, J., Bruns, M. E., Logan, B. E. (2008) Reduction of Odors from Swine Wastewater by Using Microbial Fuel Cells. Applied and Environmental Microbiology, 74(8): 2540- 2543.

32. Krishna, K.V., Sarkar, O., and Mohan, V. S. (2014) Bioelectrochemical treatment of paper and pulp wastewater in comparison with anaerobic process: integrating chemical coagulation with simultaneous power production. Bioresource Technology, 174:142–151.

33. Kumar, S., Kumar, H. D., and Babu, G. K. (2012). A study on the electricity generation from the cow dung using microbial fuel cell. Journal of Biochemical Technology, 3:442- 447.

34. Liu, H., Ramnarayanan, R., and Logan, B. E. (2004). Production of electricity during wastewater treatment using a single chamber microbial fuel cell. Environ. Sci. Technol., 38: 2281-2285.

35. Liu, M., Yuan, Y., Zhang, L., Zhuang, L., Zhou, S., and Ni, J. (2010). Bioelectricity generation by a Gram-positive Corynebacterium sp. strain MFC03 under alkaline condition in microbial fuel cells. Bioresource Technology, 101: 1807–1811.

36. Liu, Y., Liu, H., Wang, C., Hou, S., and Yang, N. (2013). Sustainable energy recovery in wastewater treatment by microbial fuel cells: stable power generation with nitrogen- doped graphene cathode. Environmental Science & Technology 47:13889-13895.

37. Logan B. E. (2009). Exoelectrogenic bacteria that power microbial fuel cells. Nature Reviews Microbiology, 7: 375–381.

38. Logan, B. E., and Regan, J. M. (2006). Microbial challenges and applications. Environmental Science & Technology, 40 (17): 5172-5180.

39. Lovley, D. R. (2006). Microbial energizers: fuel cells that keep on going microbes that produce electricity by oxidizing organic compounds in biomass may someday power useful electronic devices. Microbe, 1(7):323-329.

40. Lovley, D. R., Giovannoni, S. J., White, D. C., Champine, J. E., Phillips, E. J., Gorby, Y. A., and Goodwin, S. (1993). Geobacter metallireducens gen. nov. sp. nov., a microorganism capable of coupling the complete oxidation of organic compounds to the reduction of iron and other metals. Arch Microbiol, 159(4):336-44.

41. Lu, N., Zhou, S., Zhuang, L., Zhang, J., Ni, J. (2009) Electricity generation from starch processing wastewater using microbial fuel cell technology, Biochemical Engineering Journal, 43:246–251.

42. Luo, H., Liu, G., Zhang, R., Jin, S. (2009) Phenol degradation in microbial fuel cells. Chemical Engineering Journal, 147: 259- 264.

43. Majumder, D., Maity, J. P., Tseng, M., Nimje, V. R., Chen, H., Chen, C., Chang, Y., Yang, T. and Chen, C (2014) Electricity generation and wastewater treatment of oil refinery in microbial fuel cells using Pseudomonas putida. Int. J. Mol. Sci., 15:16772-16786.

44. Merino-Jimenez, I., Greenman, J., Ieropoulos, I. (2017). Electricity and catholyte production from ceramic MFCs treating urine. International Journal of Hydrogen Energy, 42:1791 – 1799.

45. Min, B., and Logan. B. E. (2004). Continuous electricity generation from domestic wastewater and organic substrates in a flat plate microbial fuel cell. Environ. Sci. Technol., 38: 5809-5814.

46. Min, B., Cheng, S. and Logan, B. E. (2005). Electricity generation using membrane and salt bridge microbial fuel cells. Water Research, 39(9): 1675-1686.

47. Min, B., Roman, O. B. and Angelidaki, I. (2008) Importance of temperature and anodic medium composition on microbial fuel cell (MFC) performance. Biotechnol. Lett., 30:1213–1218.

48. Nimje, V. R., Labrath, Y. P. and Gaikar, V. G. (2013) Development of Microbial Fuel Cell Using Distillery Spent Wash: Evaluation of Current Generation and COD Removal with Respect to pH. Iranica Journal of Energy & Environment, 4(4): 348-356.

49. Pangare, G., Pangare, V., Das, B., (2006) Springs of Life: India's Water Resource, Academic foundation, World Water Institute, New Delhi India.

50. Park, D. H., and Zeikus, J. G. (2000). Electricity Generation in Microbial Fuel Cells Using Neutral Red as an Electronophore. Applied and Environmental Microbiology, 66(4): 1292-1297.

51. Patil S. A., Venkata, P. S., Koul, S., Ijmulwar, S., Amar, V., Shouche, Y. S., and Kapadnis, B. P. (2009). Electricity generation using chocolate industry wastewater and its treatment in activated sludge based microbial fuel cell and analysis of developed microbial community in the anode chamber. Bioresource Technology, 100: 5132–5139.

52. Pham, C.A., Jung, S.J., Phung, N.T., Lee, J., Chang, I.S., Kim, B.H., Yi, H. and Chun, J. (2003) A novel electrochemically active and Fe (III)- reducing bacterium phylogenetically related to Aeromonas hydrophila, isolated from a microbial fuel cell. FEMS Microbiology Letters, 223: 129–134.

53. Pham, T. H., Boon, N., Aelterman, P., Clauwaert, P., Schamphelaire, L., Vanhaecke, L., Maeyer. K., Höfte, M., Verstraete, W., Rabaey, K. (2008) Metabolites produced by Pseudomonas sp. enable a Gram positive bacterium to achieve extracellular electron transfer, Appl Microbiol Biotechnol, 77:1119–1129.

54. Potter, M. C. (1911). Effects accompanying the decomposition of organic compounds. Proceedings of the royal society of London. Series b, containing papers of a biological character, 84(571):260-276.

55. Prescott, L. M., and Harley, J. P. (2002). Laboratory exercises in microbiology. McGraw Hill, New York.

56. Rabaey, K., Clauwaert, P., Aelterman, P., and Verstraete, W. (2005). Tubular Microbial Fuel Cells for Efficient Electricity Generation, Environ. Sci. Technol., 39: 8077-8082.

57. Raghavulu, S.V., Mohan, V. S., Goud, R.K. and Sarma, P.N. (2009) Effect of anodic pH microenvironment on microbial fuel cell (MFC) performance in concurrence with aerated and ferricyanide catholytes. Electrochemistry Communications, 11: 371–375.

58. Ringeisen, B. R., Ray, R., and Little, B. (2007). A miniature microbial fuel cell operating with an aerobic anode chamber. Journal of Power Sources, 165:591–597.

59. Rismani-Yazdi, H., Carver, S. M., Christya, A. D., Tuovinen, O. H. (2008) Cathodic limitations in microbial fuel cells: An overview. Journal of Power Sources, 180: 683–694.

60. Shukla, A.K., Suresh, P., Berchmans, S. and Rajendran, A. (2004) . Biological Fuel Cells and Their Applications. Current Science, 87(4): 455-468.

61. Sund, C. J., McMasters, S., Crittenden, S. R., Harrell, L. E., and Sumner, J. J. (2007). Effect of electron mediators on current generation and fermentation in a microbial fuel cell. Appl Microbiol Biotechnol 76:561–568.

62. Uwidia, I.E. and Ukulu, H.S. (2013) Studies on electrical conductivity and total dissolved solids concentration in raw domestic wastewater obtained from an estate in Warri, Nigeria. Greener Journal of Physical Sciences, 3(3): 110-114.

63. Water for People, Water for Life - UN World Water Development Report (WWDR) March 2003, Retrieved on 12-11-2014.

64. Water for People, Water for Life - UN World Water Development Report (WWDR) March 2003, Retrieved on 12-11-2014.

65. Wei, L., Han, H. and Shen, J. (2013) Effects of temperature and ferrous sulfate concentrations on the performance of microbial fuel cell. International Journal of Hydrogen Energy, 38: 11110–11116.

66. Wei, M., Patadia, S., Kammen, D. M. (2010). Putting renewable and energy efficiency to work: How many jobs can the clean energy industry generate in the US? Energy Policy, 38: 919–931.

67. Xie, X., Ye, M., Hu, L., Liu, N., McDonough, J. R., Chen, W., Alshareef, H. N., Criddle, C. S., Cui, Y., (2012) Carbon nanotube-coated macroporous sponge for microbial fuel cell electrodes. Energy & Environmental Science, 5:5265–5270.

68. Zhao, F., Harnisch, F., Schroder, U., Scholz, F., Bogdanoff, P., Herrmann, I., (2005) Challenges and Constraints of Using Oxygen Cathodes in Microbial Fuel Cells. Electrochemistry Communications, 7: 1405–1410.

Received on 28.05.2018 Modified on 19.06.2018

Research J. Pharm. and Tech. 2019; 12(5):2213-2221.

DOI: 10.5958/0974-360X.2019.00369.X