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Effect of isolated bacterial strains from distillery wastewater on power generation in microbial fuel cell
Accepted Manuscript Title: Effect of isolated bacterial strains from distillery wastewater on power generation in microbial fuel cell Author: N. Samsudeen T.K. Radhakrishnan M. Matheswaran PII: DOI: Reference:
S1359-5113(16)30185-4 http://dx.doi.org/doi:10.1016/j.procbio.2016.06.007 PRBI 10711
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
10-8-2015 2-6-2016 8-6-2016
Please cite this article as: N.Samsudeen, T.K.Radhakrishnan, M.Matheswaran.Effect of isolated bacterial strains from distillery wastewater on power generation in microbial fuel cell.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.06.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of isolated bacterial strains from distillery wastewater on power generation in microbial fuel cell
N.Samsudeen, T.K.Radhakrishnan, M.Matheswaran
Department of Chemical Engineering, National Institute of Technology, Tiruchirappalli, India.
Corresponding
author: Tel: +91-4312503120; Fax. +91-4312503102
Email: [email protected] (Manickam Matheswaran)
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Graphical abstract
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Voltage (mV)
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MFC-1 MFC-2 MFC-3 MFC-4
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HIGHLIGHTS Effect of isolated bacterial strains on power generation in MFC was investigated. Three strains isolated from distillery wastewater used as biocatalyst in the MFC. Isolated strains were identified using 16S rRNA gene sequences. L. sphaericus SN-2 produced highest power density at pH 8. L. sphaericus SN-2 was considered as potential bacteria for power generation in MFC.
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ABSTRACT The effect of isolated bacterial strains on power generation in microbial fuel cell (MFC) using distillery wastewater as a substrate was investigated. The strains were isolated from wastewater and identified using 16S rRNA gene sequences. The isolated species was found to be of Bacillus types and the strains were designated as L. sphaericus SN-1, L. sphaericus SN-2 and B. safensis SN-3, respectively. The strains were used as biocatalyst for the generation of electricity and treatment of wastewater in the MFC. The result showed that each strain has different current generation capacity and wastewater treatment efficiency. The MFC inoculated with L. sphaericus SN-2 produced a maximum open-circuit voltage (OCV) of 646 ± 5 mV and peak power density of 104 ± 3 mW/m2 with higher treatment efficiency of 63.4 ± 0.5 %. The effect of wastewater pH (6, 7 and 8), COD concentration (3200, 4800 and 6400 mg/L) and buffer on power generation in the MFC were investigated. Based on the performance of the MFC, L. sphaericus SN-2 can be used as a biocatalyst for electricity generation from the distillery wastewater. Keywords: Chemical oxygen demand, Distillery wastewater, L. sphaericus, Microbial fuel cell, Power density, ,
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1. Introduction Alcoholic distillery industries release about 8−15 liters of wastewater for the production of every liter of alcohol during the process. The distillery wastewater is the most complex substrate which leads to pollution in nearby water bodies when they are discharged into the environment [1, 2]. The color of wastewater does not allow penetration of sunlight and thus causes deleterious effects on aquatic life. The treatment of distillery wastewater by various methods such as biodegradation, flocculation, electrochemical and membrane filtration has been investigated [3]. Most of the conventional treatment processes need energy which is not economical for attaining sustainability. The MFC is a novel green technology to generate electricity during the wastewater treatment which supports the combined issues of energy crisis and environmental safety [4, 5]. The MFC is a hybrid bioelectrochemical system that directly converts the chemical energy into electricity by the oxidation of organic matter in the presence of microorganisms under ambient conditions. The voltage generation due to the bacterial metabolic activity in the anode and the electron acceptor conditions in the cathode results in bioelectricity [6-8]. The distillery wastewater contains high organic matter that can provide a good source for electricity production in the MFCs and easily biodegradable [9]. The distillery wastewater has been widely examined as a substrate for electricity generation in MFC using mixed microbial community, simultaneously leading to the treatment of the wastewater pollutant [10, 11]. Zhang et al. [12] and Huang et al. [13] have developed a coupled system of MFC technology with conventional anaerobic/aerobic processes such as up-flow anaerobic sludge blanket reactor– microbial fuel cell–biological aerated filter (UASB−MFC−BAF) and anaerobic fluidized bed−microbial fuel cell (AFB−MFC). This system showed the feasibility of electricity generation with the simultaneous removal of pollutants from the molasses wastewater. Mohanakrishna et al.
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[14] reported that the MFC could effectively reduce chemical oxygen demand (COD), color and dissolved oxygen content of the distillery wastewater. Various unit operations pertaining to wastewater treatment such as biological treatment (anaerobic), electrolytic dissociation and electrochemical oxidation can be performed in the MFC. The performance of MFC was affected by various intrinsic and extrinsic factors such as types of substrate and its concentration, solution pH, electrode materials, system architecture, temperature and types of microorganism during wastewater treatment [15-17]. The presence of a microorganism as a biocatalyst either in pure strain or mixed community plays a crucial role in the generation of electricity through metabolic reaction during the removal of pollutants from the wastewater. Many of the researchers evaluated the performance of MFC in terms of electricity generation and treatment of wastewater by inoculating with the mixed microbial communities [18, 19]. Nevertheless, roles of the individual microorganism and the mechanisms involved contribute to power generation, and understanding the treatment efficiency becomes difficult using mixed microbial community as a biocatalyst. The pure bacterial strains including gram-positive Corynebacterium sp., B. subtillis, E. cloacae etc. have been used to investigate the extent of power generation and utilization of wastewater from various sources in the MFC [20-22]. The exoelectrogenic activities of bacterial strains were altered with pH conditions in the MFC. Liu et al. [20] reported that the higher electrochemical activity of strain gram-positive Corynebacterium sp. MFC03 at pH 9 as compared with pH 8. Kim et al. [23] studied the performance of MFC using low-pH distillery wastewater as a substrate without pH control under continuous mode. They reported Caldiserica might significantly contribute to electricity generation of single chamber MFC using low-pH wastewater and high-Rext. Ganapathy et al. [24] reported that the isolated strain of Nostoc muscorum, belonging to the phylum Cyanobacteria was found in the distillery
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wastewater which could be potentially used for the biological treatment. Ha et al. [25] used uncultured Bacteroidetes bacterium obtained from thermophilic anaerobic digestion sludge for the treatment of distillery wastewater in the MFC. In this study, the feasibility of using inherent microbes isolated from the distillery wastewater was tested in a dual chamber MFC for power generation along with treatment. The isolated strains were identified by 16S rRNA gene sequencing based on nucleotide homology and phylogenetic analysis. The electrochemical activities of each strain were investigated by cyclic voltammetry (CV) and compared with those of mixed culture. The effect of wastewater inoculated with isolated strains on power generation, COD removal and columbic efficiency in the MFC was studied. The performance of MFC was investigated under different pH conditions, wastewater COD concentration and also compared in the presence and absence of phosphate buffer. 2. Materials and Methods 2.1 Wastewater characteristics The wastewater was used as a substrate that collected nearby distillery industries Trichy, India. The important characteristics of the distillery wastewater were: pH: 4.9 ± 0.05, COD: 80,000– 90,000 mg/L, TDS: 18460−20200 mg/L, conductivity: 33.30 ±3 mS/cm and color – dark brown, odor – burnt sugar. The pH, conductivity and TDS of wastewater were determined using multiparameter analyzer (CyberScan PC650, Eutech’s). The collected wastewater was stored in refrigerator at 4 ± 1oC prior to use. 2.2 Isolation of Bacteria Organisms present in the distillery wastewater were isolated using a liquid nutrient medium. The raw distillery wastewater (DW) was diluted into 25 times using deionized water for isolating the bacteria. The bacterial strains were isolated from the diluted DW using serial dilution followed
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by pour plate and streak plate techniques. The nutrient agar was prepared using peptic digest (5 g/L), Beef extract (1.5 g/L), sodium chloride (5 g/L), yeast extract (1.5 g/L) and 15 g agar for 1000 mL medium. The prepared agar media was sterilized in an autoclave at 121 ± 2 °C for 15 mins. The distillery wastewater of 0.1 mL was spread on the nutrient agar plates and incubated at 37 C for 24 h. The isolated strain was repeatedly subcultured for obtaining a pure culture. After incubation, bacterial colonies were selected and subjected to preliminary screening, phenotypic and genotypic characterization. 2.3. Identification of Bacteria For identification of bacteria, overnight cultures of each isolate in nutrient broth were considered. All isolates were initially tested for gram reaction and subjected to biochemical test. The genomic DNA was extracted using a modified phenol−chloroform−isoamyl alcohol method. For genotypic identification, the bacterial 16S rDNA fragments in genomic DNA were amplified using universal 16S rDNA primers, forward primer 8F (5’AGA GTT TGA TCC TGG CTC AG 3’) and reverse primer 1492R (5’ CGG TTA CCT TGT TAC GAC TT-3’). PCR amplification was conducted in an automated thermal cycler (BIORAD T100TM) using the following protocol. The final reaction PCR mixture contained 1 µL of DNA, 400 ng of reverse primer, 4 µL of deoxynucleotide triphosphate mixture, 10 µL of Taq polymerase buffer and 1 µL of Taq polymerase. The amplification condition was 94 °C for 5 min, followed by 35 cycles of denaturation at 92 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 °C for 2 min. A final extension step was carried out at 72 °C for 5 min. The PCR products were analyzed by 1.4% (W/V) agarose gel electrophoresis with ethidium bromide (0.5 g/mL) for 1 h and visualized on a UV transilluminator. The PCR products were purified and quantified photo metrically (Model UV-1700 Shimadzu). The 16S rDNA sequence was aligned and identified by GenBank using the
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BLAST program. The closest known relatives of the isolates were determined by performing a sequence database search. Distance matrix was generated using the Ribosomal Database Project RDP database and the phylogenetic tree was constructed using MEGA 4 [26-29]. 2.5 MFC configuration and operation Four identical MFCs were constructed using a non-conducting polymeric material (Plexi glass) with working volume of 210 mL in 230 mL of the total volume. The MFC consists of an anode and a cathode compartment separated by a proton exchange membrane (PEM) (NafionTM 117, Dupont Co,). The membrane was pretreated using 0.5 M H2SO4 followed by 5 % H2O2 at 70−80 o
C for 1 h before used. The anode and cathode electrodes were made of graphite plates with the
dimension of 4 cm x 6 cm x 0.3 cm. The electrodes were placed at a distance of 1 cm on either sides of the membrane. Prior to use, the electrodes were soaked in deionized water for 24 h. Copper wires were used to provide connection between the anode and cathode electrodes. The anode and cathode compartments were filled with sterilized distillery wastewater and potassium ferricyanide (50 mM) in phosphate buffer (50 mM) respectively. The isolated liquid broth of 5 mL was added in the anode compartment. The anodic solution pH was adjusted using orthophosphoric acid (88%) and 3 N NaOH with respect to the experimental conditions. The cathodic solution pH was adjusted to 7.5. The sterilized wastewater was replaced once the cell voltage started to drop under the open-circuit mode. In order to maintain the anaerobic environment, the anode compartment was completely sealed and then purged with N2 gas for 10 min at every feeding event. All the experiments were carried out inside the laminar flow cabinet to avoid the cross-contamination of microorganisms. The system was operated in batch mode under ambient conditions (31 ± 2 oC and 1 atm). The MFC was operated under OCV mode for several cycles (8 days per cycle) to obtain the stable potential generation. The polarization,
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coulombic efficiency and COD removal efficiency were measured after attaining the stable potential. The effect of MFC performance was investigated by varying COD concentration (3200, 4800 and 6400 mg/L) and pH (6, 7 and 8) of wastewater. The schematic representation of microbial fuel cell was shown in Fig. 1. 2.6 Electrochemical parameter Analysis The voltage generation in the MFC was recorded using a digital multimeter. The current (mA) and power (mW) were calculated by Ohm’s law I = E/R, and P = I2/R, where, I is the current (mA), E is the measured voltage (mV) and R is the external resistance (Ω). For polarization curve, the current was recorded at various external resistances (15,000 Ω to 100 Ω) connected and readings were noted after stabilization. The power density (mW/m2) and current density (mA/m2) were calculated based on the anode surface area (m2). The pH of the anolyte and catholyte was monitored continuously during the experiment. The COD was determined using closed reflux (potassium dichromate titration) method. The COD removal efficiency (ηCOD) was calculated using the following equation. ηCOD,R
Ci Cf x100 Ci
(1)
where Ci is the initial COD concentration (mg/L) and Cf is the final COD concentration (mg/L). The conversion of chemical energy into electrical energy was determined as columbic efficiency (CE) by the following equation. t
CE
M Idt
(2)
0
FbVan COD
Where F is Faradays constant (1 Faradays = 96,486 coulomb/mol), b = 4 the number of electrons (e−) exchanged per mole of oxygen, Van is the anolyte volume (mL) and ∆COD is the change in COD (mg/L) with respect to time t. 9
In order to ascertain the electrochemical activity of the individual microorganism, CV analysis was performed using classical three-electrode system in the Galvanostat−Potentiostat PGSTAT101 (Metrohm Autolab, Netherlands). The CV of the mixed culture and individual bacterial strain was performed using a graphite plate as the working electrode, Ag/AgCl and platinum rod as the reference and counter electrode respectively. Before the start of the CV experiments, the cells were purged with N2 gas for 10 min. The CV experiment was carried out with a scan range of −1 V to 1 V at a scan rate of 100 mV/s. 3. Results and Discussions 3.1 Characterization of bacteria The biochemical characterizations of three isolated strains form distillery wastewater were found to be Gram-positive and possessed a rod shaped-structure (supplementary material Fig. S1 A, B and C). The substrate utilization of individual strains was given in Table S1. The PCRamplified products were analyzed for 16S rDNA sequence to identify the strain. The consensus sequence of 1168-, 1184- and 1183-bp was determined for isolates 1, 2 and 3 respectively. Gene analysis using the online BLAST tool showed that the isolate -1 & 2 were similar to L. sphaericus and isolate- 3 was B. safensis respectively. 16S rDNA gene analysis showed that isolate-1 was closest match to L. sphaericus strain IV (4)13 with a homology of 100 %. The isolate-2 was closely related to 11 strains of L. sphaericus and maximum of 99% similarity with L. sphaericus strain IV (4)10. Finally, isolate-3 showed a closer relatedness to the strains of B. safensis, B. pumilus and Bacillus sp. Among them B. safensis CCMM B629 showed 100% homology. In addition, the phylogenic tree was constructed to characterize the relationship between the isolates and the types of strain sequences were shown in Fig. 2. A, B and C. The gene sequences have been deposited in the NCBI database with an accession number of isolate-1 (GenBank Accession Number:
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KT003214), isolate-2 (GenBank Accession Number: KR996141) and isolate-3 (GenBank Accession Number: KT003213). The identified strains from isolates 1, 2 and 3 were designated as, L. sphaericus SN-1, L. sphaericus SN-2 and B. safensis SN-3, respectively. The numbers at the nodes indicated the levels of bootstrap support based on the neighbor-joining tree method. 3.2 Electricity generation using isolated strains The isolated bacterial strains were used to investigate the electricity generation capacity and COD removal efficiency during the treatment of distillery wastewater in the MFC. The anode compartment was filled with sterilized distillery wastewater with COD concentration of 3200 mg/L and 5 mL of each isolated liquid broth strain. The pH of the anolyte and catholyte solutions was adjusted to 8 and 7.5 respectively. Based on the inoculation of L. sphaericus SN-1, L. sphaericus SN-2, and B. safensis SN-3 in the anode compartment, the system was represented as MFC-1, MFC-2 and MFC-3, respectively. The raw distillery wastewater (in diluted form) containing various strains including the isolated strains was used as inoculum for the mixed culture experiment (MFC-4). The MFC was operated for several cycles (8 days/cycle). The start-up time required for the MFC operation around 4 hrs for all isolated strains and mixed culture. During the cycle, the voltage generation increased with increasing the bacterial activity and attained peak voltage under the stable condition. Under OCV conditions, the cell voltage was measured for each strain and mixed culture as shown in Fig. 3. The highest OCV values of 632 ± 5, 646 ± 5, 615 ± 7 and 642 ± 5 mV were recorded in the MFC-1, MFC-2, MFC-3 and MFC-4 respectively. The cell voltage decreased gradually with time and reached 545 ± 5, 522 ± 5, 510 ± 7 and 540 ± 5 mV on the 8th day in the respective MFCs. Under closed-circuit conditions, a significant power generation was observed with respect to the bacterial strains used in the MFC. From the polarization and power density curve, the peak
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power densities of 77 ± 2, 88.8 ± 5, 76 ± 2 and 70 ± 2 mW/m2 with a corresponding current densities of 276 ± 8, 287 ± 12, 277 ± 7 and 265 ± 8 mA/m2 were observed at 100 Ω in the MFC-1, MFC-2, MFC-3 and MFC-4, respectively. These results showed that the current and power density in the MFC-2 was higher than the other MFCs as shown in Fig. 4. However, the power density of the mixed culture (MFC-4) was slightly lower than the individual strains. This might be due to the presence of non-electroactive microbes in the mixed culture occupying the electrode surface and metabolizing the substrate to survive rather than generating current. Xing et al. [30] and Kiely et al. [31] reported that the mixed culture microorganism showed a poor performance as compared to the pure strains used in the MFC. Under OCV conditions, the cell voltage exhibits smaller variations between the different microbes used. However, the power density significantly varied with respect to the isolated strains which might be due to different substrate degradation pathways of the strains. The distillery wastewater contains various organic matter components including, starch, acetate, glucose, fructose, volatile fatty acids (VFA) etc. that are utilized by the diverse bacterial strains for electrons generation and their growth. The presence of different organic sources in the distillery wastewater influences the different degradation behaviors of each strain. Rezaei et al. [32] reported that E. cloacae ACTT and E. cloacae FR showed the different chemical activities for the carbon source and electron donors used in the MFCs. The organic matter utilization of strains indicates its potential relevance for use with wastewater having variable compositions. Thus, the current generation varied based on the strains used in the MFCs. Zuo et al. [33] reported that the different current production was observed for various substrates such as acetate, glucose and cellobiose using Ochrobactrum anthropic YZ-1. Kim et al. [34] observed that the current generation varied with different strains of S. putrefaciens (MR-1, IR-1, and SR-21) in an anaerobic environment using lactate as a substrate.
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CV experiment was conducted to investigate the effect of electrochemical activity of the individual strains and also compared with control wastewater. Fig. 5 of voltammogram showed that flat curve for control wastewater without any Faradic peaks between − 1 V to +1 V. However, the peaks could be observed after the development of biofilm enriched by exoelectrogenic activity of isolated strains on the electrode surface. Nimje et al. [35] reported that the biofilm is required to catalyze the oxidation reaction, which could facilitate electron transfer by achieving direct contact with the electrode and may also be due to compounds bound to the cell membrane. The peak and increasing current generation observed during sweep in the CV of individual strain were due to the presence of electroactive species in the biofilm. From Fig 5, the anodic peak potential at −0.25 V (0.93 mA) and cathodic peak potential at 0.25 V (1.1 mA) were observed for L. sphaericus SN-1. Similarly, the anodic peak potential at −0.08 V (0.7 mA) and cathodic peak potential at −0.27 V (0.46 mA) was obtained for L. sphaericus SN-2. B. safensis SN-3 and the mixed culture showed an anodic peak potential of 0.35 V (0.8 mA) and −0.25 V (0.45 mA) and a cathodic peak potential of 0.046 V (0.4 mA) and 0.3V (0.54 mA), respectively. The results indicate that the exoelectrogenic activity of the individual strains may be involved in extracellular electron transfer. The COD removal rate of the each strain varied from 0.18 to 0.24 kg/ m 3.d in the MFC operating under batch mode. The COD removal efficiency of 51.9 ± 0.5 % (0.21 kg/m3.d), 57.4 ± 0.4% (0.24 kg/m3.d), 46.2 ± 0.2% (0.18 kg/m3.d) and 54.5 ± 0.5 % (0.218 kg/m3.d) for the isolate1, 2, 3 and mixed culture respectively. The coulombic efficiency of the system was found to be maximum of 22.4, 27, 15.8, and 13.2 % for the MFC -1, 2, 3 and MFC-4 respectively. The use of L. sphaericus SN-2 in MFC-2 might have a higher electron generation and transferring capability to anode surface as compared with other strains in MFC. The performance in terms of power
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density and COD removal efficiency for the MFC was compared between the isolated strain and the various strains reported by researchers in the literature as given in the Table. 1. 3.3 Influence of anodic pH The wastewater pH is the most important factor which affects the microbial activity for the metabolic reaction and proton and electron generation mechanism. The effect of anolyte pH was investigated by adjusting the electrolyte pH to 6, 7 and 8. The MFC-2 was filled with wastewater COD concentration of 3200 mg/L containing 5 mL of isolated broth of L. sphaericus SN-2 in the anodic chamber. During the experiment, the anolyte solution pH was changed with respect to the feed pH due to electrochemical and microbial metabolic activity. A significant voltage difference was observed under different pH, when no current was flowing through an external circuit. Under OCV conditions, the maximum voltage of 561 ± 10 mV, 605 ± 5 mV and 643 ± 5 mV was observed at pH 6, 7 and 8 respectively. Liu et al. [20] reported that the anodic over potential can be reduced thermodynamically by increasing pH which will lead to an increase in the overall cell potential. The polarization and power density behavior of MFC-2 during operation were obtained at various resistances (15,000−100 Ω) under different pHs as shown in Fig. 6 A. The power density of 88.8 ± 5 mW/m2 with the corresponding current density of 287 ± 12 mA/m2 was higher at pH 8 as compared to pH 6 and 7 at 100 Ω. The COD removal efficiency and CE during MFC operation with respect to the pH conditions was shown in Fig. 6 B. The maximum COD removal efficiency and CE at pH 8 was observed to be 57.4 ± 0.4% (0.24 kg/m3.d) and 27 %, respectively. The results show that the highest performance was achieved at alkaline conditions rather than acidic and neutral. At pH 8, the electrochemical activity of the particular strain was increased due to that the charge transfer, and diffusion resistance may be decreased which might enhance power generation.
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The performance in MFC-2 was lower in the acidic conditions (pH 6) which may be due to the slower microbial activity in the anolyte solution [38]. 3.4 Effect of buffer on power generation The presence of buffer in the anode compartment is essential not only for optimum microbial activities, but also to compensate the slow proton transport rate through the membrane. The experiments were conducted to investigate the effect of buffer on the power generation and wastewater treatment efficiency. The MFC was filled with the sterilized wastewater COD concentration of 3200 mg/L containing 5 mL of L. sphaericus SN-2. The phosphate buffer was added to maintain pH 8 in the anode compartment. The maximum OCV values of 646 ± 5 mV and 633 ± 5 mV were recorded in the presence and absence of buffer, respectively. However, a different performance of MFC was observed in polarization and power density curve as shown in the Fig 7. At 200 Ω, the peak power density observed in MFC was higher in the presence of buffer than the absence of buffer. At current density of 310.2 ± 9 mA/m2, the maximum power density of 104 ± 3 mW/m2 was produced with buffer in the anolyte. The COD removal efficiency of 63.4 ± 0.5 % (0.26 kg/m3.d) was achieved in the system. Higher performance obtained in the MFC with buffer might be due to the presence of phosphate which increases the solution conductivity and in turn reduces the ohmic resistance, thus improving the proton transfer between the electrodes and the solution [39]. The phosphate buffer system significantly increased the power generation by affecting the electrochemical reactions. However, there no significant change was found in the COD removal efficiency of MFC. 3.5
Effect of wastewater COD concentration The anode chamber was filled with different COD concentration (3200, 4800 and 6400 mg/L)
of wastewater to investigate the performance of MFC using L. sphaericus SN-2 as inoculum.
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Under OCV conditions, the maximum voltage of 645 ± 7, 685 ± 5 and 715 ± 5 mV were recorded for the COD concentrations of 3200, 4800 and 6400 mg/L respectively. The power generation significantly increased with the increasing COD concentration during the polarization as shown in Fig 8. The highest power density of 123.5 ± 3 mW/m2 (323.4 ± 4 mA/m2) was observed for the concentration of 6400 mg/L. When the MFC operating at lower COD concentration of 3200 and 4800 mg/L, the peak power densities of 88.5 ± 5 mW/m2 (286 ± 2 mA/m2) and 110 ± 2 mW/m2 (304.5 ± 5 mA/m2) respectively was achieved. The COD removal efficiency was found to be maximum of 64.8 ± 0.3 % (0.52 kg/m3.d), 62.2 ± 0.5 % (0.37 kg/m3.d) and 57.4 ± 0.4 % (0.24 kg/m3.d) for the concentration of 6400, 4800 and 3200 mg/L respectively in the MFC. Haung and Logan reported that the power production was dependent on the initial COD concentration of wastewater and this was rapidly converted into electricity by anodic biofilm [39]. When the substrate concentration increased the reaction rates were accelerated, due to which higher power generation was observed. 4. Conclusion The performance of MFC was investigated using an isolated bacteria and mixed culture from distillery wastewater as a biocatalyst. 16S rRNA Gene analysis by online BLAST tool showed that the isolated culture was similar to L. sphaericus and B. safensis. The isolated strains were designated as L. sphaericus SN-1, L. sphaericus SN-2 and B. safensis SN-3, respectively. All isolated strains have exhibited significant power generation capacity and wastewater treatment efficiency. The MFC operated using an isolated strain of L. sphaericus SN-2 produced a maximum power density of 88.8 ± 5 (287 ± 12 mA/m2) and 57.4 ± 0.4 % COD removal efficiency was achieved at pH 8. The wastewater pH, COD concentration and buffer usage significantly affected the power production using L. sphaericus SN-2 as inoculum in the MFC. The highest power
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density of 123.5 ± 3 mW/m2 (323.4 ± 4 mA/m2) was achieved at the COD concentration of 6400 mg/L under pH 8. Hence, L. sphaericus SN-2 obtained from the distillery wastewater can be used as a biocatalyst in the MFC for power generation and removal of COD from the wastewater. Acknowledgement The authors acknowledge the Department of Biotechnology, Government of India under the scheme of Rapid Grant for Young Investigators (No.BT/PR6080/GBD/27/503/2013) for providing financial support to carry out the research work. The authors are very grateful to Ms. Harshini Muthukumar (PhD scholar, NIT Trichy) for helping to carry out the gram staining and biochemical characterization studies.
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5. References [1] Mohana S, Acharya B. K, Madamwar D. Distillery spent wash: Treatment technologies and potential applications. J Hazard Mater 2009;163:12-25. [2] Tewari P. K, Batra VS, Balakrishnan M. Water management initiatives in sugarcane molasses based distilleries in India. Resour Conserv Recycl 2007;52:351-367. [3] Zhang W, Xiong R, Wei G. Biological flocculation treatment on distillery wastewater and recirculation of wastewater. J Hazard Mater 2009;172:1252-1257. [4] Venkata Mohan S, Mohanakrishna G, Reddy B.P, Saravanan R, Sarma PN. Bioelectricity generation from chemical wastewater treatment in mediatorless (anode) microbial fuel cell (MFC) using selectively enriched hydrogen producing mixed culture under acidophilic microenvironment. Biochem Eng J 2008;39:121-130. [5] Logan B. E, Hamelers B, Rozendal R, Schröder U, Keller J, Freguia S, Aelterman P, Verstraete W, Rabaey K. Microbial Fuel Cells: Methodology and Technology. Environ Sci Technology 2006;40:5181-5192. [6] E.Logan B. Microbial Fuel Cells. United States of America: A John Wiley & Sons, Inc.,; 2008. [7] Venkata Mohan S, Veer Raghavulu S, Sarma PN. Influence of anodic biofilm growth on bioelectricity production in single chambered mediatorless microbial fuel cell using mixed anaerobic consortia. Biosens Bioelectron 2008;24:41-47. [8] Jang JK, Pham TH, Chang IS, Kang KH, Moon H, Cho KS, Kim BH. Construction and operation of a novel mediator- and membrane-less microbial fuel cell. Process Biochem 2004;39:1007-1012. [9] Samsudeen N, Radhakrishnan TK, Matheswaran M. Performance comparison of triple and dual chamber microbial fuel cell using distillery wastewater as a substrate. Environ Prog Sustainable Energy 2015;34:589-594. [10] Kaushik A, Chetal A. Power generation in microbial fuel cell fed with post methanation distillery effluent as a function of pH microenvironment. Bioresour Technol 2013;147:7783. [11] Sevda S, Dominguez-Benetton X, Vanbroekhoven K, De Wever H, Sreekrishnan TR, Pant D. High strength wastewater treatment accompanied by power generation using air cathode microbial fuel cell. Appl Energy 2013;105:194-206.
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[12] Zhang B, Zhao H, Zhou S, Shi C, Wang C, Ni J. A novel UASB–MFC–BAF integrated system for high strength molasses wastewater treatment and bioelectricity generation. Bioresour Technol 2009;100:5687-5693. [13] Huang J, Yang P, Guo Y, Zhang K. Electricity generation during wastewater treatment: An approach using an AFB-MFC for alcohol distillery wastewater. Desalination 2011;276:373-378. [14] Mohanakrishna G, Venkata Mohan S, Sarma PN. Bio-electrochemical treatment of distillery wastewater in microbial fuel cell facilitating decolorization and desalination along with power generation. J Hazard Mater 2010;177:487-494. [15] Li Z, Zhang X, Lei L. Electricity production during the treatment of real electroplating wastewater containing Cr6+ using microbial fuel cell. Process Biochem 2008;43:13521358. [16] Du Z, Li H, Gu T. A state of the art review on microbial fuel cells: A promising technology for wastewater treatment and bioenergy. Biotechnol Adv 2007;25:464-482. [17] Li XM, Cheng KY, Selvam A, Wong JWC. Bioelectricity production from acidic food waste leachate using microbial fuel cells: Effect of microbial inocula. Process Biochem 2013;48:283-288. [18] Mohan SV, Raghavulu SV, Peri D, Sarma PN. Integrated function of microbial fuel cell (MFC) as bio-electrochemical treatment system associated with bioelectricity generation under higher substrate load. Biosens Bioelectron 2009;24:2021-2027. [19] Sangeetha T, Muthukumar M. Influence of electrode material and electrode distance on bioelectricity production from sago-processing wastewater using microbial fuel cell. Environ Prog Sustain Energy 2013;32:390-395. [20] Liu M, Yuan Y, Zhang L-x, Zhuang L, Zhou S-g, Ni J-r. Bioelectricity generation by a Grampositive Corynebacterium sp. strain MFC03 under alkaline condition in microbial fuel cells. Bioresour Technol 2010;101:1807-1811. [21] Nimje VR, Chen C-Y, Chen C-C, Jean J-S, Reddy AS, Fan C-W, Pan K-Y, Liu H-T, Chen JL. Stable and high energy generation by a strain of Bacillus subtilis in a microbial fuel cell. J Power Sources 2009;190:258-263. [22] Nimje VR, Chen C-Y, Chen C-C, Tsai J-Y, Chen H-R, Huang YM, Jean J-S, Chang Y-F, Shih R-C. Microbial fuel cell of Enterobacter cloacae: Effect of anodic pH 19
microenvironment on current, power density, internal resistance and electrochemical losses. Int J Hydrogen Energy 2011;36:11093-11101. [23] Kim H, Kim B, Kim J, Lee T, Yu J. Electricity generation and microbial community in microbial fuel cell using low-pH distillery wastewater at different external resistances. J. Biotechnol 2014;186:175-180. [24] Ganapathy Selvam G BRaMP. Microbial diversity and bioremediation of distilleries effluent. J Research Biol 2011;1:153-162. [25] Ha PT, Lee TK, Rittmann BE, Park J, Chang IS. Treatment of alcohol distillery wastewater using a Bacteroidetes-dominant thermophilic microbial fuel cell. Environ Sci Technol 2012;46:3022-3030. [26] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-1599. [27] Felsenstein J. Confidence Limits on Phylogenies: An Approach Using the Bootstrap. 1985;39:783-791. [28] Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16:111-120. [29] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-425. [30] Xing D, Zuo Y, Cheng S, Regan JM, Logan BE. Electricity Generation by Rhodopseudomonas palustris DX-1. Environ Sci Technol 2008;42:4146-4151. [31] Kiely P, Call D, Yates M, Regan J, Logan B. Anodic biofilms in microbial fuel cells harbor low numbers of higher-power-producing bacteria than abundant genera. Appl Microbiol Biotechnol 2010;88:371-380. [32] Rezaei F, Xing D, Wagner R, Regan JM, Richard TL, Logan BE. Simultaneous cellulose degradation and electricity production by Enterobacter cloacae in a microbial fuel cell. Appl Environ Microbiol 2009;75:3673-3678. [33] Zuo Y, Xing D, Regan JM, Logan BE. Isolation of the exoelectrogenic bacterium Ochrobactrum anthropi YZ-1 by using a U-tube microbial fuel cell. Appl Environ Microbiol 2008;74:3130-3137.
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[34] Kim HJ, Park HS, Hyun MS, Chang IS, Kim M, Kim BH. A mediator-less microbial fuel cell using a metal reducing bacterium, Shewanella putrefaciens. Enzyme Microb Technol 2002;30:145-152. [35] Nimje VR, Chen C-Y, Chen H-R, Chen C-C, Huang YM, Tseng M-J, Cheng K-C, Chang YF. Comparative bioelectricity production from various wastewaters in microbial fuel cells using mixed cultures and a pure strain of Shewanella oneidensis. Bioresour Technol 2012;104:315-323. [36] Han J-L, Wang C-T, Hu Y-C, Liu Y, Chen W-M, Chang C-T, Xu H-Z, Chen B-Y. Exploring power generation of single-chamber microbial fuel cell using mixed and pure cultures. J Taiwan Inst Chem Eng 2010;41:606-611. [37] Bond DR, Lovley DR. Electricity production by Geobacter sulfurreducens attached to electrodes. Appl Environ Microbiol 2003;69:1548-1555. [38] Gil GC, Chang IS, Kim BH, Kim M, Jang JK, Park HS, Kim HJ. Operational parameters affecting the performannce of a mediator-less microbial fuel cell. Biosens Bioelectron 2003;18:327-334. [39] Huang L, Logan BE. Electricity generation and treatment of paper recycling wastewater using a microbial fuel cell. Appl Microbiol Biotechnol 2008;80:349-355.
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FIGURE CAPTION Fig 1. Schematic representation of Microbial fuel cell. Fig 2. A, B and C. Neighbor-joining tree showing the phylogenetic relationship of bacterial 16S rRNA obtained from the isolate-1 (L. sphaericus SN-1), isolate-2 (L. sphaericus SN-2) and isolate-3 (B. safensis SN-3) respectively. Fig 3. Open circuit voltage generation during the MFC operation for isolated strains and mixed culture. Fig 4. Polarization behavior and power densities curve for isolated strains and mixed culture used in the MFC. Fig 5. Cyclic voltammograms curve for isolated strains and mixed culture using distillery wastewater as a substrate. Fig 6.A Polarization curve of MFC operating under different wastewater feed pH. Fig 6.B. Effect of wastewater pH on COD removal and coulombic efficiency in the MFC. Fig 7. Polarization behavior and power density curve for the presence and absence of buffer in the anodic chamber. Fig 8. Power density curve for different the wastewater COD concentration in the MFC.
22
FIGURES
Fig. 1
23
Fig. 2.A
Fig. 2.B
24
Fig. 2.C
25
700
Voltage (mV)
625
550 MFC-1 MFC-2 475
MFC-3 MFC-4
400 0
2
4
6
Time (Days)
Fig. 3
26
8
750
100
600
450 50 300
MFC-1 MFC-2 25
MFC-3
150
MFC-4 0
0
0
100
200 Current Density (mA/m2)
300
Fig. 4
27
400
Power Density (mW/m2)
Voltage (mV)
75
0.003
Current (A)
0.002
B. safensis SN-3 L. sphaericus SN-2 L. sphaericus SN-1 Mixed culture Control WW
0.001
0.000
-0.001
-0.002 -1.2
-0.9
-0.6
-0.3
0.0
0.3
Applied Potential (V)
Fig. 5
28
0.6
0.9
1.2
100
600
Voltage (mV)
450 60
300
40 pH 6
Power Density (mW/m2)
80
pH 7 pH 8
150
20
0
0 0
100
200 Current Density (mA/m2)
300
400
60
32
45
24
30
16
15
8
0
0
pH 6 pH 7 COD Removal Efficiency
pH 8 Coulombic Efficiency
Fig. 6.B
29
Coulombic Efficiency (%)
COD removal Efficiency (%)
Fig. 6.A
120
600
Voltage (mV)
90 450 60 300
Presence of buffer Absence of buffer
150
30
0
0 0
100
200
300
Current Density (mA/m2)
Fig. 7
30
400
Power Density (mW/m2)
750
140
Power Density (mW/m2)
105
70
3200 mg/l
4800 mg/l
35
6400 mg/l
0
0
125
250
Current Density
375
(mA/m2)
Fig. 8
31
500
Table caption: Table 1. Performance comparison of MFC operated under the isolated strains and pure cultures from the wastewater.
S.No.
Substrate / wastewater
1.
Cellulose
2. 3. 4. 5.
Glucose Food/dairy Acetate Distillery
6.
Distillery
Microorganism Gram-positive Corynebacterium sp.strain Aeromonas punctata Shewanella oneidensis Geobacter sulfurreducens Mixed culture Lysinibacillus sphaericus SN-2
32
Power density (mW/m2)
COD removal efficiency
References
7.3
-
[20]
68.5 150 13.1 93.99
13.1 56.7
[36] [35] [37] [14]
88.8 ± 3
57.4 ± 0.4
This study
S1359-5113(16)30185-4 http://dx.doi.org/doi:10.1016/j.procbio.2016.06.007 PRBI 10711
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
10-8-2015 2-6-2016 8-6-2016
Please cite this article as: N.Samsudeen, T.K.Radhakrishnan, M.Matheswaran.Effect of isolated bacterial strains from distillery wastewater on power generation in microbial fuel cell.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.06.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Effect of isolated bacterial strains from distillery wastewater on power generation in microbial fuel cell
N.Samsudeen, T.K.Radhakrishnan, M.Matheswaran
Department of Chemical Engineering, National Institute of Technology, Tiruchirappalli, India.
Corresponding
author: Tel: +91-4312503120; Fax. +91-4312503102
Email: [email protected] (Manickam Matheswaran)
1
Graphical abstract
100
Voltage (mV)
600
75
450 50 300
MFC-1 MFC-2 MFC-3 MFC-4
150
25
0
Power Density (mW/m2)
750
0 0
100 200 300 Current Density (mA/m2)
400
HIGHLIGHTS Effect of isolated bacterial strains on power generation in MFC was investigated. Three strains isolated from distillery wastewater used as biocatalyst in the MFC. Isolated strains were identified using 16S rRNA gene sequences. L. sphaericus SN-2 produced highest power density at pH 8. L. sphaericus SN-2 was considered as potential bacteria for power generation in MFC.
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ABSTRACT The effect of isolated bacterial strains on power generation in microbial fuel cell (MFC) using distillery wastewater as a substrate was investigated. The strains were isolated from wastewater and identified using 16S rRNA gene sequences. The isolated species was found to be of Bacillus types and the strains were designated as L. sphaericus SN-1, L. sphaericus SN-2 and B. safensis SN-3, respectively. The strains were used as biocatalyst for the generation of electricity and treatment of wastewater in the MFC. The result showed that each strain has different current generation capacity and wastewater treatment efficiency. The MFC inoculated with L. sphaericus SN-2 produced a maximum open-circuit voltage (OCV) of 646 ± 5 mV and peak power density of 104 ± 3 mW/m2 with higher treatment efficiency of 63.4 ± 0.5 %. The effect of wastewater pH (6, 7 and 8), COD concentration (3200, 4800 and 6400 mg/L) and buffer on power generation in the MFC were investigated. Based on the performance of the MFC, L. sphaericus SN-2 can be used as a biocatalyst for electricity generation from the distillery wastewater. Keywords: Chemical oxygen demand, Distillery wastewater, L. sphaericus, Microbial fuel cell, Power density, ,
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1. Introduction Alcoholic distillery industries release about 8−15 liters of wastewater for the production of every liter of alcohol during the process. The distillery wastewater is the most complex substrate which leads to pollution in nearby water bodies when they are discharged into the environment [1, 2]. The color of wastewater does not allow penetration of sunlight and thus causes deleterious effects on aquatic life. The treatment of distillery wastewater by various methods such as biodegradation, flocculation, electrochemical and membrane filtration has been investigated [3]. Most of the conventional treatment processes need energy which is not economical for attaining sustainability. The MFC is a novel green technology to generate electricity during the wastewater treatment which supports the combined issues of energy crisis and environmental safety [4, 5]. The MFC is a hybrid bioelectrochemical system that directly converts the chemical energy into electricity by the oxidation of organic matter in the presence of microorganisms under ambient conditions. The voltage generation due to the bacterial metabolic activity in the anode and the electron acceptor conditions in the cathode results in bioelectricity [6-8]. The distillery wastewater contains high organic matter that can provide a good source for electricity production in the MFCs and easily biodegradable [9]. The distillery wastewater has been widely examined as a substrate for electricity generation in MFC using mixed microbial community, simultaneously leading to the treatment of the wastewater pollutant [10, 11]. Zhang et al. [12] and Huang et al. [13] have developed a coupled system of MFC technology with conventional anaerobic/aerobic processes such as up-flow anaerobic sludge blanket reactor– microbial fuel cell–biological aerated filter (UASB−MFC−BAF) and anaerobic fluidized bed−microbial fuel cell (AFB−MFC). This system showed the feasibility of electricity generation with the simultaneous removal of pollutants from the molasses wastewater. Mohanakrishna et al.
4
[14] reported that the MFC could effectively reduce chemical oxygen demand (COD), color and dissolved oxygen content of the distillery wastewater. Various unit operations pertaining to wastewater treatment such as biological treatment (anaerobic), electrolytic dissociation and electrochemical oxidation can be performed in the MFC. The performance of MFC was affected by various intrinsic and extrinsic factors such as types of substrate and its concentration, solution pH, electrode materials, system architecture, temperature and types of microorganism during wastewater treatment [15-17]. The presence of a microorganism as a biocatalyst either in pure strain or mixed community plays a crucial role in the generation of electricity through metabolic reaction during the removal of pollutants from the wastewater. Many of the researchers evaluated the performance of MFC in terms of electricity generation and treatment of wastewater by inoculating with the mixed microbial communities [18, 19]. Nevertheless, roles of the individual microorganism and the mechanisms involved contribute to power generation, and understanding the treatment efficiency becomes difficult using mixed microbial community as a biocatalyst. The pure bacterial strains including gram-positive Corynebacterium sp., B. subtillis, E. cloacae etc. have been used to investigate the extent of power generation and utilization of wastewater from various sources in the MFC [20-22]. The exoelectrogenic activities of bacterial strains were altered with pH conditions in the MFC. Liu et al. [20] reported that the higher electrochemical activity of strain gram-positive Corynebacterium sp. MFC03 at pH 9 as compared with pH 8. Kim et al. [23] studied the performance of MFC using low-pH distillery wastewater as a substrate without pH control under continuous mode. They reported Caldiserica might significantly contribute to electricity generation of single chamber MFC using low-pH wastewater and high-Rext. Ganapathy et al. [24] reported that the isolated strain of Nostoc muscorum, belonging to the phylum Cyanobacteria was found in the distillery
5
wastewater which could be potentially used for the biological treatment. Ha et al. [25] used uncultured Bacteroidetes bacterium obtained from thermophilic anaerobic digestion sludge for the treatment of distillery wastewater in the MFC. In this study, the feasibility of using inherent microbes isolated from the distillery wastewater was tested in a dual chamber MFC for power generation along with treatment. The isolated strains were identified by 16S rRNA gene sequencing based on nucleotide homology and phylogenetic analysis. The electrochemical activities of each strain were investigated by cyclic voltammetry (CV) and compared with those of mixed culture. The effect of wastewater inoculated with isolated strains on power generation, COD removal and columbic efficiency in the MFC was studied. The performance of MFC was investigated under different pH conditions, wastewater COD concentration and also compared in the presence and absence of phosphate buffer. 2. Materials and Methods 2.1 Wastewater characteristics The wastewater was used as a substrate that collected nearby distillery industries Trichy, India. The important characteristics of the distillery wastewater were: pH: 4.9 ± 0.05, COD: 80,000– 90,000 mg/L, TDS: 18460−20200 mg/L, conductivity: 33.30 ±3 mS/cm and color – dark brown, odor – burnt sugar. The pH, conductivity and TDS of wastewater were determined using multiparameter analyzer (CyberScan PC650, Eutech’s). The collected wastewater was stored in refrigerator at 4 ± 1oC prior to use. 2.2 Isolation of Bacteria Organisms present in the distillery wastewater were isolated using a liquid nutrient medium. The raw distillery wastewater (DW) was diluted into 25 times using deionized water for isolating the bacteria. The bacterial strains were isolated from the diluted DW using serial dilution followed
6
by pour plate and streak plate techniques. The nutrient agar was prepared using peptic digest (5 g/L), Beef extract (1.5 g/L), sodium chloride (5 g/L), yeast extract (1.5 g/L) and 15 g agar for 1000 mL medium. The prepared agar media was sterilized in an autoclave at 121 ± 2 °C for 15 mins. The distillery wastewater of 0.1 mL was spread on the nutrient agar plates and incubated at 37 C for 24 h. The isolated strain was repeatedly subcultured for obtaining a pure culture. After incubation, bacterial colonies were selected and subjected to preliminary screening, phenotypic and genotypic characterization. 2.3. Identification of Bacteria For identification of bacteria, overnight cultures of each isolate in nutrient broth were considered. All isolates were initially tested for gram reaction and subjected to biochemical test. The genomic DNA was extracted using a modified phenol−chloroform−isoamyl alcohol method. For genotypic identification, the bacterial 16S rDNA fragments in genomic DNA were amplified using universal 16S rDNA primers, forward primer 8F (5’AGA GTT TGA TCC TGG CTC AG 3’) and reverse primer 1492R (5’ CGG TTA CCT TGT TAC GAC TT-3’). PCR amplification was conducted in an automated thermal cycler (BIORAD T100TM) using the following protocol. The final reaction PCR mixture contained 1 µL of DNA, 400 ng of reverse primer, 4 µL of deoxynucleotide triphosphate mixture, 10 µL of Taq polymerase buffer and 1 µL of Taq polymerase. The amplification condition was 94 °C for 5 min, followed by 35 cycles of denaturation at 92 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 °C for 2 min. A final extension step was carried out at 72 °C for 5 min. The PCR products were analyzed by 1.4% (W/V) agarose gel electrophoresis with ethidium bromide (0.5 g/mL) for 1 h and visualized on a UV transilluminator. The PCR products were purified and quantified photo metrically (Model UV-1700 Shimadzu). The 16S rDNA sequence was aligned and identified by GenBank using the
7
BLAST program. The closest known relatives of the isolates were determined by performing a sequence database search. Distance matrix was generated using the Ribosomal Database Project RDP database and the phylogenetic tree was constructed using MEGA 4 [26-29]. 2.5 MFC configuration and operation Four identical MFCs were constructed using a non-conducting polymeric material (Plexi glass) with working volume of 210 mL in 230 mL of the total volume. The MFC consists of an anode and a cathode compartment separated by a proton exchange membrane (PEM) (NafionTM 117, Dupont Co,). The membrane was pretreated using 0.5 M H2SO4 followed by 5 % H2O2 at 70−80 o
C for 1 h before used. The anode and cathode electrodes were made of graphite plates with the
dimension of 4 cm x 6 cm x 0.3 cm. The electrodes were placed at a distance of 1 cm on either sides of the membrane. Prior to use, the electrodes were soaked in deionized water for 24 h. Copper wires were used to provide connection between the anode and cathode electrodes. The anode and cathode compartments were filled with sterilized distillery wastewater and potassium ferricyanide (50 mM) in phosphate buffer (50 mM) respectively. The isolated liquid broth of 5 mL was added in the anode compartment. The anodic solution pH was adjusted using orthophosphoric acid (88%) and 3 N NaOH with respect to the experimental conditions. The cathodic solution pH was adjusted to 7.5. The sterilized wastewater was replaced once the cell voltage started to drop under the open-circuit mode. In order to maintain the anaerobic environment, the anode compartment was completely sealed and then purged with N2 gas for 10 min at every feeding event. All the experiments were carried out inside the laminar flow cabinet to avoid the cross-contamination of microorganisms. The system was operated in batch mode under ambient conditions (31 ± 2 oC and 1 atm). The MFC was operated under OCV mode for several cycles (8 days per cycle) to obtain the stable potential generation. The polarization,
8
coulombic efficiency and COD removal efficiency were measured after attaining the stable potential. The effect of MFC performance was investigated by varying COD concentration (3200, 4800 and 6400 mg/L) and pH (6, 7 and 8) of wastewater. The schematic representation of microbial fuel cell was shown in Fig. 1. 2.6 Electrochemical parameter Analysis The voltage generation in the MFC was recorded using a digital multimeter. The current (mA) and power (mW) were calculated by Ohm’s law I = E/R, and P = I2/R, where, I is the current (mA), E is the measured voltage (mV) and R is the external resistance (Ω). For polarization curve, the current was recorded at various external resistances (15,000 Ω to 100 Ω) connected and readings were noted after stabilization. The power density (mW/m2) and current density (mA/m2) were calculated based on the anode surface area (m2). The pH of the anolyte and catholyte was monitored continuously during the experiment. The COD was determined using closed reflux (potassium dichromate titration) method. The COD removal efficiency (ηCOD) was calculated using the following equation. ηCOD,R
Ci Cf x100 Ci
(1)
where Ci is the initial COD concentration (mg/L) and Cf is the final COD concentration (mg/L). The conversion of chemical energy into electrical energy was determined as columbic efficiency (CE) by the following equation. t
CE
M Idt
(2)
0
FbVan COD
Where F is Faradays constant (1 Faradays = 96,486 coulomb/mol), b = 4 the number of electrons (e−) exchanged per mole of oxygen, Van is the anolyte volume (mL) and ∆COD is the change in COD (mg/L) with respect to time t. 9
In order to ascertain the electrochemical activity of the individual microorganism, CV analysis was performed using classical three-electrode system in the Galvanostat−Potentiostat PGSTAT101 (Metrohm Autolab, Netherlands). The CV of the mixed culture and individual bacterial strain was performed using a graphite plate as the working electrode, Ag/AgCl and platinum rod as the reference and counter electrode respectively. Before the start of the CV experiments, the cells were purged with N2 gas for 10 min. The CV experiment was carried out with a scan range of −1 V to 1 V at a scan rate of 100 mV/s. 3. Results and Discussions 3.1 Characterization of bacteria The biochemical characterizations of three isolated strains form distillery wastewater were found to be Gram-positive and possessed a rod shaped-structure (supplementary material Fig. S1 A, B and C). The substrate utilization of individual strains was given in Table S1. The PCRamplified products were analyzed for 16S rDNA sequence to identify the strain. The consensus sequence of 1168-, 1184- and 1183-bp was determined for isolates 1, 2 and 3 respectively. Gene analysis using the online BLAST tool showed that the isolate -1 & 2 were similar to L. sphaericus and isolate- 3 was B. safensis respectively. 16S rDNA gene analysis showed that isolate-1 was closest match to L. sphaericus strain IV (4)13 with a homology of 100 %. The isolate-2 was closely related to 11 strains of L. sphaericus and maximum of 99% similarity with L. sphaericus strain IV (4)10. Finally, isolate-3 showed a closer relatedness to the strains of B. safensis, B. pumilus and Bacillus sp. Among them B. safensis CCMM B629 showed 100% homology. In addition, the phylogenic tree was constructed to characterize the relationship between the isolates and the types of strain sequences were shown in Fig. 2. A, B and C. The gene sequences have been deposited in the NCBI database with an accession number of isolate-1 (GenBank Accession Number:
10
KT003214), isolate-2 (GenBank Accession Number: KR996141) and isolate-3 (GenBank Accession Number: KT003213). The identified strains from isolates 1, 2 and 3 were designated as, L. sphaericus SN-1, L. sphaericus SN-2 and B. safensis SN-3, respectively. The numbers at the nodes indicated the levels of bootstrap support based on the neighbor-joining tree method. 3.2 Electricity generation using isolated strains The isolated bacterial strains were used to investigate the electricity generation capacity and COD removal efficiency during the treatment of distillery wastewater in the MFC. The anode compartment was filled with sterilized distillery wastewater with COD concentration of 3200 mg/L and 5 mL of each isolated liquid broth strain. The pH of the anolyte and catholyte solutions was adjusted to 8 and 7.5 respectively. Based on the inoculation of L. sphaericus SN-1, L. sphaericus SN-2, and B. safensis SN-3 in the anode compartment, the system was represented as MFC-1, MFC-2 and MFC-3, respectively. The raw distillery wastewater (in diluted form) containing various strains including the isolated strains was used as inoculum for the mixed culture experiment (MFC-4). The MFC was operated for several cycles (8 days/cycle). The start-up time required for the MFC operation around 4 hrs for all isolated strains and mixed culture. During the cycle, the voltage generation increased with increasing the bacterial activity and attained peak voltage under the stable condition. Under OCV conditions, the cell voltage was measured for each strain and mixed culture as shown in Fig. 3. The highest OCV values of 632 ± 5, 646 ± 5, 615 ± 7 and 642 ± 5 mV were recorded in the MFC-1, MFC-2, MFC-3 and MFC-4 respectively. The cell voltage decreased gradually with time and reached 545 ± 5, 522 ± 5, 510 ± 7 and 540 ± 5 mV on the 8th day in the respective MFCs. Under closed-circuit conditions, a significant power generation was observed with respect to the bacterial strains used in the MFC. From the polarization and power density curve, the peak
11
power densities of 77 ± 2, 88.8 ± 5, 76 ± 2 and 70 ± 2 mW/m2 with a corresponding current densities of 276 ± 8, 287 ± 12, 277 ± 7 and 265 ± 8 mA/m2 were observed at 100 Ω in the MFC-1, MFC-2, MFC-3 and MFC-4, respectively. These results showed that the current and power density in the MFC-2 was higher than the other MFCs as shown in Fig. 4. However, the power density of the mixed culture (MFC-4) was slightly lower than the individual strains. This might be due to the presence of non-electroactive microbes in the mixed culture occupying the electrode surface and metabolizing the substrate to survive rather than generating current. Xing et al. [30] and Kiely et al. [31] reported that the mixed culture microorganism showed a poor performance as compared to the pure strains used in the MFC. Under OCV conditions, the cell voltage exhibits smaller variations between the different microbes used. However, the power density significantly varied with respect to the isolated strains which might be due to different substrate degradation pathways of the strains. The distillery wastewater contains various organic matter components including, starch, acetate, glucose, fructose, volatile fatty acids (VFA) etc. that are utilized by the diverse bacterial strains for electrons generation and their growth. The presence of different organic sources in the distillery wastewater influences the different degradation behaviors of each strain. Rezaei et al. [32] reported that E. cloacae ACTT and E. cloacae FR showed the different chemical activities for the carbon source and electron donors used in the MFCs. The organic matter utilization of strains indicates its potential relevance for use with wastewater having variable compositions. Thus, the current generation varied based on the strains used in the MFCs. Zuo et al. [33] reported that the different current production was observed for various substrates such as acetate, glucose and cellobiose using Ochrobactrum anthropic YZ-1. Kim et al. [34] observed that the current generation varied with different strains of S. putrefaciens (MR-1, IR-1, and SR-21) in an anaerobic environment using lactate as a substrate.
12
CV experiment was conducted to investigate the effect of electrochemical activity of the individual strains and also compared with control wastewater. Fig. 5 of voltammogram showed that flat curve for control wastewater without any Faradic peaks between − 1 V to +1 V. However, the peaks could be observed after the development of biofilm enriched by exoelectrogenic activity of isolated strains on the electrode surface. Nimje et al. [35] reported that the biofilm is required to catalyze the oxidation reaction, which could facilitate electron transfer by achieving direct contact with the electrode and may also be due to compounds bound to the cell membrane. The peak and increasing current generation observed during sweep in the CV of individual strain were due to the presence of electroactive species in the biofilm. From Fig 5, the anodic peak potential at −0.25 V (0.93 mA) and cathodic peak potential at 0.25 V (1.1 mA) were observed for L. sphaericus SN-1. Similarly, the anodic peak potential at −0.08 V (0.7 mA) and cathodic peak potential at −0.27 V (0.46 mA) was obtained for L. sphaericus SN-2. B. safensis SN-3 and the mixed culture showed an anodic peak potential of 0.35 V (0.8 mA) and −0.25 V (0.45 mA) and a cathodic peak potential of 0.046 V (0.4 mA) and 0.3V (0.54 mA), respectively. The results indicate that the exoelectrogenic activity of the individual strains may be involved in extracellular electron transfer. The COD removal rate of the each strain varied from 0.18 to 0.24 kg/ m 3.d in the MFC operating under batch mode. The COD removal efficiency of 51.9 ± 0.5 % (0.21 kg/m3.d), 57.4 ± 0.4% (0.24 kg/m3.d), 46.2 ± 0.2% (0.18 kg/m3.d) and 54.5 ± 0.5 % (0.218 kg/m3.d) for the isolate1, 2, 3 and mixed culture respectively. The coulombic efficiency of the system was found to be maximum of 22.4, 27, 15.8, and 13.2 % for the MFC -1, 2, 3 and MFC-4 respectively. The use of L. sphaericus SN-2 in MFC-2 might have a higher electron generation and transferring capability to anode surface as compared with other strains in MFC. The performance in terms of power
13
density and COD removal efficiency for the MFC was compared between the isolated strain and the various strains reported by researchers in the literature as given in the Table. 1. 3.3 Influence of anodic pH The wastewater pH is the most important factor which affects the microbial activity for the metabolic reaction and proton and electron generation mechanism. The effect of anolyte pH was investigated by adjusting the electrolyte pH to 6, 7 and 8. The MFC-2 was filled with wastewater COD concentration of 3200 mg/L containing 5 mL of isolated broth of L. sphaericus SN-2 in the anodic chamber. During the experiment, the anolyte solution pH was changed with respect to the feed pH due to electrochemical and microbial metabolic activity. A significant voltage difference was observed under different pH, when no current was flowing through an external circuit. Under OCV conditions, the maximum voltage of 561 ± 10 mV, 605 ± 5 mV and 643 ± 5 mV was observed at pH 6, 7 and 8 respectively. Liu et al. [20] reported that the anodic over potential can be reduced thermodynamically by increasing pH which will lead to an increase in the overall cell potential. The polarization and power density behavior of MFC-2 during operation were obtained at various resistances (15,000−100 Ω) under different pHs as shown in Fig. 6 A. The power density of 88.8 ± 5 mW/m2 with the corresponding current density of 287 ± 12 mA/m2 was higher at pH 8 as compared to pH 6 and 7 at 100 Ω. The COD removal efficiency and CE during MFC operation with respect to the pH conditions was shown in Fig. 6 B. The maximum COD removal efficiency and CE at pH 8 was observed to be 57.4 ± 0.4% (0.24 kg/m3.d) and 27 %, respectively. The results show that the highest performance was achieved at alkaline conditions rather than acidic and neutral. At pH 8, the electrochemical activity of the particular strain was increased due to that the charge transfer, and diffusion resistance may be decreased which might enhance power generation.
14
The performance in MFC-2 was lower in the acidic conditions (pH 6) which may be due to the slower microbial activity in the anolyte solution [38]. 3.4 Effect of buffer on power generation The presence of buffer in the anode compartment is essential not only for optimum microbial activities, but also to compensate the slow proton transport rate through the membrane. The experiments were conducted to investigate the effect of buffer on the power generation and wastewater treatment efficiency. The MFC was filled with the sterilized wastewater COD concentration of 3200 mg/L containing 5 mL of L. sphaericus SN-2. The phosphate buffer was added to maintain pH 8 in the anode compartment. The maximum OCV values of 646 ± 5 mV and 633 ± 5 mV were recorded in the presence and absence of buffer, respectively. However, a different performance of MFC was observed in polarization and power density curve as shown in the Fig 7. At 200 Ω, the peak power density observed in MFC was higher in the presence of buffer than the absence of buffer. At current density of 310.2 ± 9 mA/m2, the maximum power density of 104 ± 3 mW/m2 was produced with buffer in the anolyte. The COD removal efficiency of 63.4 ± 0.5 % (0.26 kg/m3.d) was achieved in the system. Higher performance obtained in the MFC with buffer might be due to the presence of phosphate which increases the solution conductivity and in turn reduces the ohmic resistance, thus improving the proton transfer between the electrodes and the solution [39]. The phosphate buffer system significantly increased the power generation by affecting the electrochemical reactions. However, there no significant change was found in the COD removal efficiency of MFC. 3.5
Effect of wastewater COD concentration The anode chamber was filled with different COD concentration (3200, 4800 and 6400 mg/L)
of wastewater to investigate the performance of MFC using L. sphaericus SN-2 as inoculum.
15
Under OCV conditions, the maximum voltage of 645 ± 7, 685 ± 5 and 715 ± 5 mV were recorded for the COD concentrations of 3200, 4800 and 6400 mg/L respectively. The power generation significantly increased with the increasing COD concentration during the polarization as shown in Fig 8. The highest power density of 123.5 ± 3 mW/m2 (323.4 ± 4 mA/m2) was observed for the concentration of 6400 mg/L. When the MFC operating at lower COD concentration of 3200 and 4800 mg/L, the peak power densities of 88.5 ± 5 mW/m2 (286 ± 2 mA/m2) and 110 ± 2 mW/m2 (304.5 ± 5 mA/m2) respectively was achieved. The COD removal efficiency was found to be maximum of 64.8 ± 0.3 % (0.52 kg/m3.d), 62.2 ± 0.5 % (0.37 kg/m3.d) and 57.4 ± 0.4 % (0.24 kg/m3.d) for the concentration of 6400, 4800 and 3200 mg/L respectively in the MFC. Haung and Logan reported that the power production was dependent on the initial COD concentration of wastewater and this was rapidly converted into electricity by anodic biofilm [39]. When the substrate concentration increased the reaction rates were accelerated, due to which higher power generation was observed. 4. Conclusion The performance of MFC was investigated using an isolated bacteria and mixed culture from distillery wastewater as a biocatalyst. 16S rRNA Gene analysis by online BLAST tool showed that the isolated culture was similar to L. sphaericus and B. safensis. The isolated strains were designated as L. sphaericus SN-1, L. sphaericus SN-2 and B. safensis SN-3, respectively. All isolated strains have exhibited significant power generation capacity and wastewater treatment efficiency. The MFC operated using an isolated strain of L. sphaericus SN-2 produced a maximum power density of 88.8 ± 5 (287 ± 12 mA/m2) and 57.4 ± 0.4 % COD removal efficiency was achieved at pH 8. The wastewater pH, COD concentration and buffer usage significantly affected the power production using L. sphaericus SN-2 as inoculum in the MFC. The highest power
16
density of 123.5 ± 3 mW/m2 (323.4 ± 4 mA/m2) was achieved at the COD concentration of 6400 mg/L under pH 8. Hence, L. sphaericus SN-2 obtained from the distillery wastewater can be used as a biocatalyst in the MFC for power generation and removal of COD from the wastewater. Acknowledgement The authors acknowledge the Department of Biotechnology, Government of India under the scheme of Rapid Grant for Young Investigators (No.BT/PR6080/GBD/27/503/2013) for providing financial support to carry out the research work. The authors are very grateful to Ms. Harshini Muthukumar (PhD scholar, NIT Trichy) for helping to carry out the gram staining and biochemical characterization studies.
17
5. References [1] Mohana S, Acharya B. K, Madamwar D. Distillery spent wash: Treatment technologies and potential applications. J Hazard Mater 2009;163:12-25. [2] Tewari P. K, Batra VS, Balakrishnan M. Water management initiatives in sugarcane molasses based distilleries in India. Resour Conserv Recycl 2007;52:351-367. [3] Zhang W, Xiong R, Wei G. Biological flocculation treatment on distillery wastewater and recirculation of wastewater. J Hazard Mater 2009;172:1252-1257. [4] Venkata Mohan S, Mohanakrishna G, Reddy B.P, Saravanan R, Sarma PN. Bioelectricity generation from chemical wastewater treatment in mediatorless (anode) microbial fuel cell (MFC) using selectively enriched hydrogen producing mixed culture under acidophilic microenvironment. Biochem Eng J 2008;39:121-130. [5] Logan B. E, Hamelers B, Rozendal R, Schröder U, Keller J, Freguia S, Aelterman P, Verstraete W, Rabaey K. Microbial Fuel Cells: Methodology and Technology. Environ Sci Technology 2006;40:5181-5192. [6] E.Logan B. Microbial Fuel Cells. United States of America: A John Wiley & Sons, Inc.,; 2008. [7] Venkata Mohan S, Veer Raghavulu S, Sarma PN. Influence of anodic biofilm growth on bioelectricity production in single chambered mediatorless microbial fuel cell using mixed anaerobic consortia. Biosens Bioelectron 2008;24:41-47. [8] Jang JK, Pham TH, Chang IS, Kang KH, Moon H, Cho KS, Kim BH. Construction and operation of a novel mediator- and membrane-less microbial fuel cell. Process Biochem 2004;39:1007-1012. [9] Samsudeen N, Radhakrishnan TK, Matheswaran M. Performance comparison of triple and dual chamber microbial fuel cell using distillery wastewater as a substrate. Environ Prog Sustainable Energy 2015;34:589-594. [10] Kaushik A, Chetal A. Power generation in microbial fuel cell fed with post methanation distillery effluent as a function of pH microenvironment. Bioresour Technol 2013;147:7783. [11] Sevda S, Dominguez-Benetton X, Vanbroekhoven K, De Wever H, Sreekrishnan TR, Pant D. High strength wastewater treatment accompanied by power generation using air cathode microbial fuel cell. Appl Energy 2013;105:194-206.
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[12] Zhang B, Zhao H, Zhou S, Shi C, Wang C, Ni J. A novel UASB–MFC–BAF integrated system for high strength molasses wastewater treatment and bioelectricity generation. Bioresour Technol 2009;100:5687-5693. [13] Huang J, Yang P, Guo Y, Zhang K. Electricity generation during wastewater treatment: An approach using an AFB-MFC for alcohol distillery wastewater. Desalination 2011;276:373-378. [14] Mohanakrishna G, Venkata Mohan S, Sarma PN. Bio-electrochemical treatment of distillery wastewater in microbial fuel cell facilitating decolorization and desalination along with power generation. J Hazard Mater 2010;177:487-494. [15] Li Z, Zhang X, Lei L. Electricity production during the treatment of real electroplating wastewater containing Cr6+ using microbial fuel cell. Process Biochem 2008;43:13521358. [16] Du Z, Li H, Gu T. A state of the art review on microbial fuel cells: A promising technology for wastewater treatment and bioenergy. Biotechnol Adv 2007;25:464-482. [17] Li XM, Cheng KY, Selvam A, Wong JWC. Bioelectricity production from acidic food waste leachate using microbial fuel cells: Effect of microbial inocula. Process Biochem 2013;48:283-288. [18] Mohan SV, Raghavulu SV, Peri D, Sarma PN. Integrated function of microbial fuel cell (MFC) as bio-electrochemical treatment system associated with bioelectricity generation under higher substrate load. Biosens Bioelectron 2009;24:2021-2027. [19] Sangeetha T, Muthukumar M. Influence of electrode material and electrode distance on bioelectricity production from sago-processing wastewater using microbial fuel cell. Environ Prog Sustain Energy 2013;32:390-395. [20] Liu M, Yuan Y, Zhang L-x, Zhuang L, Zhou S-g, Ni J-r. Bioelectricity generation by a Grampositive Corynebacterium sp. strain MFC03 under alkaline condition in microbial fuel cells. Bioresour Technol 2010;101:1807-1811. [21] Nimje VR, Chen C-Y, Chen C-C, Jean J-S, Reddy AS, Fan C-W, Pan K-Y, Liu H-T, Chen JL. Stable and high energy generation by a strain of Bacillus subtilis in a microbial fuel cell. J Power Sources 2009;190:258-263. [22] Nimje VR, Chen C-Y, Chen C-C, Tsai J-Y, Chen H-R, Huang YM, Jean J-S, Chang Y-F, Shih R-C. Microbial fuel cell of Enterobacter cloacae: Effect of anodic pH 19
microenvironment on current, power density, internal resistance and electrochemical losses. Int J Hydrogen Energy 2011;36:11093-11101. [23] Kim H, Kim B, Kim J, Lee T, Yu J. Electricity generation and microbial community in microbial fuel cell using low-pH distillery wastewater at different external resistances. J. Biotechnol 2014;186:175-180. [24] Ganapathy Selvam G BRaMP. Microbial diversity and bioremediation of distilleries effluent. J Research Biol 2011;1:153-162. [25] Ha PT, Lee TK, Rittmann BE, Park J, Chang IS. Treatment of alcohol distillery wastewater using a Bacteroidetes-dominant thermophilic microbial fuel cell. Environ Sci Technol 2012;46:3022-3030. [26] Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 2007;24:1596-1599. [27] Felsenstein J. Confidence Limits on Phylogenies: An Approach Using the Bootstrap. 1985;39:783-791. [28] Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980;16:111-120. [29] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 1987;4:406-425. [30] Xing D, Zuo Y, Cheng S, Regan JM, Logan BE. Electricity Generation by Rhodopseudomonas palustris DX-1. Environ Sci Technol 2008;42:4146-4151. [31] Kiely P, Call D, Yates M, Regan J, Logan B. Anodic biofilms in microbial fuel cells harbor low numbers of higher-power-producing bacteria than abundant genera. Appl Microbiol Biotechnol 2010;88:371-380. [32] Rezaei F, Xing D, Wagner R, Regan JM, Richard TL, Logan BE. Simultaneous cellulose degradation and electricity production by Enterobacter cloacae in a microbial fuel cell. Appl Environ Microbiol 2009;75:3673-3678. [33] Zuo Y, Xing D, Regan JM, Logan BE. Isolation of the exoelectrogenic bacterium Ochrobactrum anthropi YZ-1 by using a U-tube microbial fuel cell. Appl Environ Microbiol 2008;74:3130-3137.
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[34] Kim HJ, Park HS, Hyun MS, Chang IS, Kim M, Kim BH. A mediator-less microbial fuel cell using a metal reducing bacterium, Shewanella putrefaciens. Enzyme Microb Technol 2002;30:145-152. [35] Nimje VR, Chen C-Y, Chen H-R, Chen C-C, Huang YM, Tseng M-J, Cheng K-C, Chang YF. Comparative bioelectricity production from various wastewaters in microbial fuel cells using mixed cultures and a pure strain of Shewanella oneidensis. Bioresour Technol 2012;104:315-323. [36] Han J-L, Wang C-T, Hu Y-C, Liu Y, Chen W-M, Chang C-T, Xu H-Z, Chen B-Y. Exploring power generation of single-chamber microbial fuel cell using mixed and pure cultures. J Taiwan Inst Chem Eng 2010;41:606-611. [37] Bond DR, Lovley DR. Electricity production by Geobacter sulfurreducens attached to electrodes. Appl Environ Microbiol 2003;69:1548-1555. [38] Gil GC, Chang IS, Kim BH, Kim M, Jang JK, Park HS, Kim HJ. Operational parameters affecting the performannce of a mediator-less microbial fuel cell. Biosens Bioelectron 2003;18:327-334. [39] Huang L, Logan BE. Electricity generation and treatment of paper recycling wastewater using a microbial fuel cell. Appl Microbiol Biotechnol 2008;80:349-355.
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FIGURE CAPTION Fig 1. Schematic representation of Microbial fuel cell. Fig 2. A, B and C. Neighbor-joining tree showing the phylogenetic relationship of bacterial 16S rRNA obtained from the isolate-1 (L. sphaericus SN-1), isolate-2 (L. sphaericus SN-2) and isolate-3 (B. safensis SN-3) respectively. Fig 3. Open circuit voltage generation during the MFC operation for isolated strains and mixed culture. Fig 4. Polarization behavior and power densities curve for isolated strains and mixed culture used in the MFC. Fig 5. Cyclic voltammograms curve for isolated strains and mixed culture using distillery wastewater as a substrate. Fig 6.A Polarization curve of MFC operating under different wastewater feed pH. Fig 6.B. Effect of wastewater pH on COD removal and coulombic efficiency in the MFC. Fig 7. Polarization behavior and power density curve for the presence and absence of buffer in the anodic chamber. Fig 8. Power density curve for different the wastewater COD concentration in the MFC.
22
FIGURES
Fig. 1
23
Fig. 2.A
Fig. 2.B
24
Fig. 2.C
25
700
Voltage (mV)
625
550 MFC-1 MFC-2 475
MFC-3 MFC-4
400 0
2
4
6
Time (Days)
Fig. 3
26
8
750
100
600
450 50 300
MFC-1 MFC-2 25
MFC-3
150
MFC-4 0
0
0
100
200 Current Density (mA/m2)
300
Fig. 4
27
400
Power Density (mW/m2)
Voltage (mV)
75
0.003
Current (A)
0.002
B. safensis SN-3 L. sphaericus SN-2 L. sphaericus SN-1 Mixed culture Control WW
0.001
0.000
-0.001
-0.002 -1.2
-0.9
-0.6
-0.3
0.0
0.3
Applied Potential (V)
Fig. 5
28
0.6
0.9
1.2
100
600
Voltage (mV)
450 60
300
40 pH 6
Power Density (mW/m2)
80
pH 7 pH 8
150
20
0
0 0
100
200 Current Density (mA/m2)
300
400
60
32
45
24
30
16
15
8
0
0
pH 6 pH 7 COD Removal Efficiency
pH 8 Coulombic Efficiency
Fig. 6.B
29
Coulombic Efficiency (%)
COD removal Efficiency (%)
Fig. 6.A
120
600
Voltage (mV)
90 450 60 300
Presence of buffer Absence of buffer
150
30
0
0 0
100
200
300
Current Density (mA/m2)
Fig. 7
30
400
Power Density (mW/m2)
750
140
Power Density (mW/m2)
105
70
3200 mg/l
4800 mg/l
35
6400 mg/l
0
0
125
250
Current Density
375
(mA/m2)
Fig. 8
31
500
Table caption: Table 1. Performance comparison of MFC operated under the isolated strains and pure cultures from the wastewater.
S.No.
Substrate / wastewater
1.
Cellulose
2. 3. 4. 5.
Glucose Food/dairy Acetate Distillery
6.
Distillery
Microorganism Gram-positive Corynebacterium sp.strain Aeromonas punctata Shewanella oneidensis Geobacter sulfurreducens Mixed culture Lysinibacillus sphaericus SN-2
32
Power density (mW/m2)
COD removal efficiency
References
7.3
-
[20]
68.5 150 13.1 93.99
13.1 56.7
[36] [35] [37] [14]
88.8 ± 3
57.4 ± 0.4
This study