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Electrogenic and anti-methanogenic properties of Bacillus cereus for enhanced power generation in anaerobic sludge driven microbial fuel cell M Amirul Islam, Baranitharan Ethiraj, Chin Kui Cheng, Abu Yousuf, and Md. Maksudur Rahman Khan Energy Fuels, Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 23, 2017

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Electrogenic and anti-methanogenic properties of Bacillus cereus for

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enhanced power generation in anaerobic sludge driven microbial fuel

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cell

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¥

¥£

§

¥£

M Amirul Islam , Baranitharan Ethiraj , Chin Kui Cheng , Abu Yousuf , Md Maksudur Rahman Khan * ¥

Faculty of Chemical and Natural Resources Engineering, University Malaysia Pahang, 26300 Kuantan, Malaysia § Faculty of Engineering Technology, University Malaysia Pahang, 26300 Kuantan, Malaysia £ Centre of Excellence for advancement Research Fluid Flow (CARIFF), University Malaysia Pahang, 26300, Kuantan, Malaysia. *Corresponding Author: Email: [email protected], [email protected], Tel: +6095492872

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Abstract

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Mutualistic interaction between microorganisms plays a vital role in the formation of electroactive biofilms which is a key element to longevity and success in bioelectrochemical systems. The present study is intended to examine both the electrogenic property of B. cereus and its ability to inhibit the methanogenesis in microbial fuel cell (MFC). The potential influence of the incorporation of B. cereus in anaerobic sludge (AS) on the electrochemical activity was assessed using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) analysis. The CV of MFC with B. cereus showed strong redox peak suggesting that B. cereus possesses electrogenetic properties. Moreover, the incorporation of 3 B. cereus in AS showed the enhancement in power generation (4.83 W/m ) and CE (22%) of MFC 3 compared to the MFC solely inoculated with AS (1.82 W/m , 12%). The increase in power generation could be due to the anti-methanogenic property of B. cereus which would be evident from the 54% reduction in methane production. The results of this study suggest that the incorporation of microorganisms possessing electrogenic and anti-methanogenic properties in AS promotes the formation of electroactive biofilm and maximizes the power generation of MFC by suppressing the methanogenesis.

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1. Introduction

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Microbial fuel cells (MFCs) are electrochemical devices that directly converts chemical energy of wastes 1 into electricity by the metabolic activity of microorganisms . In the past ten years, various efforts have been made to enhance the power generation of MFCs such as optimizing electrode composition, reactor 2 design and other engineering aspects in both the anode and cathode chambers . However, relatively less effort has been devoted to identify the efficient synergistic combination of microorganisms in MFCs. Generally, pure and mixed cultures of microorganisms are used as inoculum in MFCs, but pure microorganisms may not be suitable for the practical operation such as treatment of industrial effluents due to high costs and its inefficiency to utilize the large varieties of complex substrates. On the other hand, mixed culture of microbes from natural sources such as soil, wastewater and anaerobic sludge (AS) have been widely used as inoculum in MFCs since they are more easily obtainable in large amounts, more tolerant to environmental fluctuations, and more accommodating to broad range of

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substrates . However, AS could not achieve significant performance because of the presence of 4, 5 nonelectrogenic bacteria as well as methanogenic bacteria .

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As methanogens are prevalent in AS , attaining a high performance from MFCs inoculated with AS is cumbersome because methanogenesis cannot be easily evaded since the growth conditions of 7 methanogenic bacteria is similar to that of electrogenic bacteria . Methanogenesis diverts energy away 7 from electrogenesis, and thus affects the coulombic efficiency of MFCs. Chae et al. 2010 reported that 8 MFC inoculated with anaerobic digester sludge showed 66% of electron loss due to methanogenesis . Therefore, enhancing the electrogenesis by suppressing the methanogenesis in the anode biofilm is exigent to obtain higher performance in MFCs. In last ten years, several attempts have been made by 7, 9 researchers to suppress the methanogenesis in order to improve the performance of MFC . Recently, 6 Rajesh et al. 2015 used marine algae (Chaetoceros powder) as a methanogen inhibitor to enhance the performance of AS operated MFC. But, this marine alga is not readily available in the environment. Moreover, significant power generation was not achieved from their study might be due to the incomplete 10 synergy between AS and marine algae (Chaetoceros). In another study, Zhuang et al. 2012 used 2Bromoethanesulfonate to control the methanogenesis. But, though it effectively suppressed the methanogenesis, it is not considered as a cost effective method for large scale MFC systems.

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Besides that, some recent studies have reported that long chain saturated fatty acid (SFA), 11, 12 hexadecanoic acid and octadecanoic acids possesses methanogen inhibition properties . In particular, the SFAs are believed to be responsible for the inhibition of the growth of methanogens through 13 14 adsorption and disruption of cell membranes . Zhou et al. 2013 found that the addition of SFAs to the substrate can disrupt the cell membrane and trigger potassium ions efflux in methanogenic bacteria during rumen fermentation process under anaerobic condition. Even the dominant methanogen, M. 14 ruminantium was found to be inhibited by the addition of SFAs .

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Moreover, few studies showed that gram-positive bacteria especially Bacillus spp. have the 15 capability to degrade organic waste compounds as well as use it as a carbon source . At moderate 16 temperatures, B. cereus is capable to synthesize large amounts of fatty acids . In vitro transcription and binding studies on Bacillus subtilis FapR demonstrated that malonyl CoA (Coenzyme A) is a specific inducer and, a conserved transcriptional repressor that regulates the expression of several genes 17 engaged in bacterial fatty acid synthesis . Therefore, Bacillus cereus might also be used as an effective modulator to suppress the methanogenic electron loss in MFC. Although, indirect evidence shows that B. cereus capable to suppress the methanogenesis but until now no one has conducted experiments especially in MFC.

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Besides that, bacteria generally form thick multilayer biofilm on the anode surface during long 5 term operation . The thick biofilm severely affects the diffusion of nutrients into the biofilm and causes dead cells to accumulate in the vicinity of the surface which leads to deterioration in the MFC 18 performance . Moreover, the biofilm usually consists of both nonelectrogenic (especially, methanogenic) and electrogenic microorganisms. The presence of methanogenic microorganisms mainly hinders the 2 19 electron transfer from the biofilm to the electrode surface . . Therefore, the formation of efficient biofilm by avoiding nonelectrogens especially methanogenic bacteria is necessary to allow efficient EET in 20 MFCs . It is thus highly desirable and important to develop simple and efficient strategies for large amounts of electrogenic bacteria to adhere onto the anode surface. So far, several initiatives such as controlling enivironmental stress (low temperature, low pH, inhibitor and variations in external 7 6 9 resistance) , utilizing pretreated inoculum and other different operating strategies have been developed to overcome the methanogenesis, but utilizing the synergistic effects of microorganisms in inoculum for the suppression of methanogenesis has never been studied in MFCs.

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Therefore, in the present study, the electrogenetic property of B. cereus and its inclusion in anaerobic sludge (AS) was investigated to enhance the power generation of MFC. Moreover, the effect of B. cereus on the suppression of methanogenesis was correlated with the current generation, coulombic efficiency, EIS and CV analysis to elucidate its effect on MFC performance.

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2. Experimental methods

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2.1 Sample collection and characterization

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Palm oil mill effluent (POME) was collected from Panching Palm Oil Mill (FELDA), Kuantan, Pahang, Malaysia. The Raw POME was collected before the effluent discharge into the mixing pond at about 8090 °C. AS was collected from currently running anaerobic digester of palm oil mill and municipal wastewater (MWW) was collected from drainage discharge point of Kuantan city, Malaysia. All the samples were stored in sterilized glass bottles at 4 °C.

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2.2 Isolation and characterization of B. cereus

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The collected MWW was filtered using Whatman no. 1 filter paper to remove suspended solid particles. -1 -6 Subsequently, the filtrate was serially diluted (10 to 10 ) and the pure culture bacteria were obtained -6 from 10 dilution using the spread plate technique. Enrichment of the cultures were carried out by preparing an overnight culture in Luria Bertani (LB) broth (10% v/v) and incubated at 37 °C with shaking at 150 rpm. Biolog gen III analysis was done for the primary identification of isolated bacteria. The molecular analysis of bacteria was then performed and presented in Supporting Information (Figure S1).

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2.3 MFC assembly and operation

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The MFCs (MFC1, MFC2, MFC3, MFC4) were fabricated with a cubic plexi glass (Shanghai, Sunny Scientific, China) which has a dimension of 5 cm x 5 cm x 5 cm and a total working volume of 20 mL. Carbon felt (3 x 0.8 x 3 cm) was used as both anode and cathode electrode in all the experiments. The electrodes were cleaned with 1.0 M sodium hydroxide followed by 1.0 M hydrochloric acid after each experiment and stored in distilled water before use. A cation exchange membrane (Nafion 117, Dupont Co., USA) was used to separate the anode and cathode compartments of MFC. Prior to use, the Nafion membrane was pre-treated using dilute HCl for 1 hour followed by washing with Deionized water several times. Thereafter, the whole MFC set up was tighten up with screws, the anode compartments were filled with 20 mL of sterilized 50% POME and subsequently the pure culture bacteria and AS (1mL) were inoculated into it while the cathode chamber was filled with KMnO4 solution, as oxidizing agent and then operated for 15 days. In order to measure the CH4 gas production, another three sets of 450 ml (working 21 volume) MFCs (MFC5, MFC6, MFC7) were fabricated as described by Baranitharan et al. 2015 and operated for 15 days using previously mentioned conditions. The descriptions of MFCs used in this study are shown in Table 1. The gas from MFC was measured by liquid displacement system using a flask containing 10% sodium hydroxide (w/v) solution. The thymol blue indicator was added into sodium hydroxide solution so that when CO2 absorption capacity of the solution was exhausted, the blue color of 22 the indicator was discharged . Methane gas was analyzed using a Hewlett-Packard 5890 Series II gas chromatograph with a thermal conductivity detector equipped using HP-624 capillary column. The schematic diagram of gas collection from MFC reactors has been presented in Supporting Information (Figure S2).

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Table 1. Description of MFCs


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MFCs

Inoculum

Working volume (mL)

MFC1

B. cereus (for continuous current generation)

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MFC2

AS

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MFC3

B. cereus

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MFC4

AS + B. cereus

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MFC5

AS

450

MFC6

B. cereus

450

MFC7

AS + B. cereus

450

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2.4 Analyses and calculations

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The voltage across an external resistance (1 kΩ) in the MFC circuit was monitored at regular intervals (15 min) using a digital multimeter (Fluke 289 True RMS Multimeter, USA) with data logger. Polarization curves at different time intervals were obtained by varying the external resistance from 50 to 20,000Ω using the rheostat (Crotech DRB-9, UK). Power density was 3 normalized by volume of the MFC. Volumetric power densities ( , W/m ) were calculated using the Eqs. (1) – (2). =

(1)

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(2)

= RVI /V 3

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where, V is the volume of the MFC (m ), U is the voltage of the cell (V) and Rv is the external resistance (Ω), and I is the current (A).

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The COD removal efficiency and columbic efficiency of dual chamber MFC was calculated as described 23 by Baranitharan et al. 2015 . The COD was determined using digestive solution and measured using a COD reactor (HACH DRB 200, USA). The COD removal efficiency was calculated using the equation (2)

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COD removal efficiency =

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where, CODi is the initial COD (mg/L) of the anode chamber and CODt is the COD of the anode chamber at any time. The columbic efficiency (CE) was calculated using equation (3)

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CE =

! " #$% &.()*∆,-.

× 100%

(2)

(3)


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where, I is the current (A), F is the Faraday’ constant (96485 C/mol), and Van is the anode volume (L). ∆COD is the difference between CODin and CODt (values in g/L). In Eq. 3, the constant (8) is calculated based on the molecular weight of O2 (32 g/mol) and assuming that 4 electrons exchanged per mole of oxygen.

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2.5 Gas Chromatography-Mass Spectrometry analysis

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2.6 FESEM analysis of the biofilm

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Biofilm formation was visualized at different time intervals of anode electrode using FESEM (Model: JEOL JSM7800F) at 5 kV. Small portion (1 cm) of anode electrodes with bacteria on the surface were cut off from the anode compartment and rinsed with sterile medium followed by immediate soaking into anaerobic solution of 3% glutaraldehyde. The electrode samples were then washed two times with 0.1 M phosphate buffer and dehydrated by successive 10 minutes incubations in 30%, 70%, 90% and 100% ethanol. The samples were then dried and coated with platinum using an ion-sputter to a thickness of 10 nm. After this procedure, specimens were examined by FESEM.

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2.7 Electrochemical analysis

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2.7.1 Cyclic voltammetry analysis

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Cyclic voltammetry (CV) was used to analyze the catalytic behaviour of the MFCs. The CV analysis aids in characterizing the electron transfer interactions between the biofilm (biocatalyst) and the anode electrode of the MFC. Moreover, it aids to elucidate the explicit role of redox mediators, membrane bound 24 cytochrome and electron conducting pili in the electrochemical reactions . The CV was conducted using a PARSTAT 2273 electrochemical system (USA). In order to collect the CV data, three electrode systems were applied where anode and cathode were used as working and counter electrodes respectively. The Silver/Silver chloride (1.0 M KCl) electrode was used as reference electrode and before plugging the reference electrode in the anode chamber was disinfected by 75% ethanol (Sigma). Besides that, the reference electrode was placed in the vicinity of the anode electrode surface during CV operation. The CV was performed using 30 mV/s scan rate at potential range from +1.0 to -1.0 V. Nitrogen gas was purged for 15 min before the electrochemical measurements.

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2.7.2 Electrochemical impedance spectroscopy

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Electrochemical impedance spectroscopy (EIS) was used to measure the MFC anode internal resistances, and the measurement was conducted using a potentiostat (PARSTAT 2273, USA). A three electrode system was used to examine the anode electrode where the anode and cathode electrode were used as working and counter electrode respectively. The saturated Silver/Silver chloride electrode (1.0 M KCl) was used as the reference electrode. An AC signal with amplitude of 10 mV and a frequency range of 100 kHz – 5 mHz was applied in order to prevent the biofilm detachment and to minimize the

Gas Chromatography-Mass Spectrometry analysis (GC–MS) was employed for the analysis of organic compounds which was extracted through liquid–liquid extraction pretreatment using n-hexane (chromatogram pure grade, Sigma Chemicals, USA). The 1 µL of pretreated sample was analyzed by using 6890N/5973 GC–MS system (Agilent Corporation, USA). Highly pure Helium (99.999%) was used as carrier gas with a flow rate of 1 mL/min. A DB-35MS capillary column with inner diameter of 0.25 mm and length of 30 m was adopted in the separation system. The temperature of the gasification o o compartment was controlled as follows: 40 °C for 5 min; then increased to 280 C at a rate of 3 C /min.


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disturbance on systematic stability. The EIS data were plotted in the form of Nyquist curve in which the charge-transfer resistance (Rct) and ohmic resistance (RΩ) were determined by fitting the measured impedance data to an equivalent circuit (EC): [R(RQ)(R[QT])] using Nova 1.11 software.

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3. Results and discussion

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3.1 Electrochemical activity of B. cereus

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The electrochemical activity of B. cereus (MFC1) was analyzed using polarization curve, CV and EIS analysis. The current generation curve of MFC with time is presented in Figure 1a. As can be seen from th the Figure 1a, the current generation gradually increased until 5 day of operation; thereafter it showed a plateau and finally decreased after 11 days of operation. The polarization and power density curves of rd th MFC on 3 and 11 day of operation are presented in Figure 1b. Moreover, the biofilm formation capability of B. cereus was visualized by FESEM analysis as shown in Figure 2. From Figure 2a, it can be observed that after 2 days of operation, incomplete biofilm was formed because during the initial period, bacteria started to colonize on the electrode surface thereby achieved lower performance. But, thereafter, these cells adhere to each other and frequently embedded inside a self-produced matrix of extracellular polymeric substance (EPS) that can be transformed to effective biofilm and exhibited higher performance after 10 days of operation as shown in Figure 2b.

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Figure 1. Performance trend (a) and polarization and power density curves (b) of MFC1 after different days of operation using B. cereus. The CV was performed to characterize the electron transfer interactions between the biofilm (biocatalyst) and the anode of the MFC. The characteristics of CV rely on various factors, such as the chemical and biological species present, the rate of the electron transfer reactions and the rates of 25 diffusion of electroactive species . Moreover, it helps to elucidate the explicit role of redox mediators in 24 26 the electrochemical reactions . Rabaey et al. 2004 employed CV for reporting the involvement of excreted redox mediators by Pseudomonas aeruginosa in MFC for the transfer of electrons to the 27 anodes. Moreover, on the basis of CV results, Marsili et al. 2008 also reported that Shewanella


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oneidensis strains capable to secrete soluble mediators which help for the extracellular electron transfer between the biofilm and the anodes. As can be seen from the Figure3a, after 3 days of operation, less intense reversible redox peak was obtained which demonstrated that the initial electrochemical activity 28 was mainly due to the excreted redox mediators. Patil et al. 2012 reported the formation of reversible redox couples, due to the presence of mediators that are reversibly oxidized and reduced during CV 29 tests. Besides that Nimje et al. 2009 reported the similar range CV peak for B. subtilis; therefore, B. rd cereus might likely contains similar electron shuttle mechanisms. On 3 day of operation, less intense redox peak (Figure 3a) was obtained which indicated that lesser number of cells was attached on the th anode surface. However, on 11 day of operation, strong redox peak (increased by 98%, Figure 3a) was obtained because of the presence of effective biofilm as shown in Figure 2b.

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th

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Figure 2. FESEM images on anode carbon felt at different days of operation (MFC1), (a) 3 day, (b) 11 day.

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Nyquist plots for the anode configuration on different days of MFC operation are shown in Figure 3b. The measurement of EIS spectra of the anode demonstrates key information which helps to analyze the electrochemical reactions on electrode and bacterial metabolism. The electrochemical polarization resistance of the working electrode in the anode configuration can be qualitatively determined from the 30 magnitude of Nyquist arc whereas the quantitative values can be obtained by an EC fitting of the data . The impedance spectra of the anode MFC were analyzed by fitting to the equivalent circuit: [R(RQ)(R[QT])], where Rct, Rohm, Q and T represent the charge transfer resistance, ohmic resistance, 31 constant phase element (CPE) and mass transfer resistance respectively . CPE was used rather than capacitor in order to model the double layer capacitor; because the surface roughness or a distribution of 31 reactions across the surface can affect the overall kinetics . The low frequency regions of the Nyquist plots were dominated by the finite Warburg diffusion element. The anode (Rct) is the major contribution to


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Figure 3. a) Cyclic voltammograms for the anode on different days of operation, b) Fitting results of MFC Nyquist plots at different days of operation.

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3.2. Effect of B. cereus on AS

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The polarization data for MFCs (MFC2, MFC3, MFC4) were recorded and plotted as shown in Figure 4a. In 3 Figure 4a, MFC2 achieved maximum power density of 1.828 W/m which is comparatively lower than that of other MFCs. Moreover, the microbes in the mixed culture which are not electrochemically active species might have suppressed the growth of electrogenic microorganisms in the anode biofilm resulted 3 in lower power generation in MFC2 . On the other hand, MFC3 achieved maximum power density of 2.87

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the total internal resistance which plays an important role in the performance MFC . Usually, a lower Rct 30 can be obtained while conductive efficient microbial biofilm is formed in MFC . The anode Rct values rd th were found to be 87.4 Ω and 13.45 Ω on 3 and 11 day of operation respectively. Moreover, the peak intensity of voltammogram usually increased with time due to an increase in bacterial density, since it 26 increases the amount of metabolites in solution and that in turn influences the conductivity and capacity . However, in most cases, redox compounds exhibit irreversible electrochemical behaviour in MFCs due to 25 very slow heterogeneous electron transfer that occurs at the surface of the electrode . But in this study, the reversible CV peak was observed at +0.140 V to -0.160 V, suggesting the occurrence of rapid electron transfer reactions between microbes and electrodes. Moreover, when scanning from negative to positive potential and positive to negative potential, a peak centered at +0.140 V and -0.160 V was rd th th detected on both 3 and 11 day of MFC operation, and the height of the 11 day peak centered at 0.140 rd V increased by 35% (0.0030 A for 3 day and 0.0046 A for 11th day), indicating the higher 27 electrochemical activity of the microorganisms in the biofilm . Besides that, the EIS results (Figure 3b) th rd demonstrated that the Rct was significantly reduced by 84.6% on 11 day (13.45) compared to the 3 day (87.4) might be due to the formation of effective biofilm. Thus, it is clear that the effective biofilm 33 increased the mediator concentration which would have reduced the anode activation losses and facilitated the kinetics of the electrochemical reactions.


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W/m which is about 1.5 times higher than that of MFC2. The higher power in MFC3 might be due to the presence of electrogenic properties in B. cereus. Moreover, usually gram positive bacteria develop viable biofilm compared to gram negative bacteria and that could also be a reason for achieving higher 34 3 performance in MFC3 . Interestingly, in the case of MFC4 maximum power generation of 4.87 W/m was obtained which is about 1.8 and 2.2 times higher than MFC2 and MFC3 respectively. Higher power generation was obtained from MFC4 might be due to the synergistic relationship between B. cereus and 34 AS. Read et al. 2010 reported that when gram positive and gram negative bacteria (AS contains predominantly gram negative bacteria) grown together current generation increased significantly in MFC.

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Figure 4. a) Polarization curves of MFC using different inoculums, b) Cyclic voltammograms for the anode using different MFCs, c) Fitting results Nyquist plots and polarization curve using different MFCs, d) Comparison of COD removal efficiency of MFCs using different inoculums.

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In MFCs, obtaining maximum power generation and decline in performance may not completely depend on electrogenesis capability of microorganisms but rather depends on effective biofilm formation by inhibiting nonelectrogenic bacteria. In this study, B. cereus incorporated AS showed significant power 34 production and that might be due to the formation of effective biofilm with electrogens . Furthermore, B. cereus has the capability of producing a wide variety of enzymes (i.e lipase, protease, amylase, cellulase 35 etc.) that can efficiently hydrolyze the complex wastewater (i.e POME) . Thus, B. cereus can convert the complex to simple substrates and which can be effectively utilized by other electrogenic bacteria.

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CV analysis was used to study the effect of MFC on the catalytic behavior of microorganisms in the anode chamber; the CV was conducted for MFC2, MFC3 and MFC4 after 10 days of operation as shown in Figure 4b. In MFC2, the presence of several less intense irreversible redox peaks (at 0.0014 A and 0.00058 A) indicating that some electrochemically active bacteria were present in AS and which could have produced electron shuttle compounds in MFC. However, significant power generation was not achieved possibly due to the suppression of electrochemically active bacteria by methanogens in the AS.


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Varanasi et 2016 reported that the lower catalytic behavior of mixed culture systems was due to the multiple possible side reactions which triggered on MFCs. The intense reversible redox peak (at 0.0020 A) at higher applied voltage region in MFC3 compared to MFC2 represents higher catalytic efficiency which may possibly due to the production of higher electron shuttles compared to mixed culture and that in turn 5, 33 enhanced the kinetics of the electrochemical reactions since it reduced the anode activation losses . The MFC4 showed strong redox peak (0.0062 A) compared to other MFCs which clearly indicates that rapid charge transfer occurred with lower methanogenic electron loss in MFC4. During both forward and reverse scan, the peaks amplitude showed the presence of redox components in the anodic chamber that 6 get oxidized and reduced . These redox reactions mostly affect the formal potential of the cell during turnover CV. The prominent redox peak suggested that the formation of matured and conductive biofilm on the anode surface would have enhanced the electron transfer efficiency between the microorganisms 36 and the anode of MFC . The conductive biofilm formation comprising with predominant electrogens might have produced more redox compounds and that would have resulted in maximum performance in MFC4. This finding also suggests that elevated current generation in MFC4 was due to enhanced electrochemical activity of electrogens while controlling methanogenesis. The results revealed that the CV data were consistent with the MFC performance. Nyquist plots for the anode configuration on different MFCs are shown in Figure 4c. The anode charge transfer resistance (Rct) values were found to be 174.4 Ω, 60.7 Ω and 27.7 Ω for MFC2, MFC3 and MFC4 respectively as shown in Table S1. In MFC2, the higher Rct (174.4 Ω) was obtained which might be due to the formation of ineffective thick biofilm on electrode surface. Moreover, the presence of unwanted methanogenic microorganisms would have dominated the electrogenic microorganisms on the anode biofilm thus resulted in the reduction of electron transfer efficiency of biofilm in MFC2. In MFC4, lower Rct (27.7 Ω) was observed compared to the MFC2 (174.4 Ω) and MFC3 (60.7 Ω) demonstrating that the effective biofilm formed by electrogenic bacteria while methanogens were inhibited on the anode and thus would have decreased the anode activation losses by enhancing the kinetics of the bio-electrochemical 37 reactions . Moreover, it showed that the growth of effective biofilm on the anode was found to decrease the anode charge resistance and facilitate the kinetics of the bioelectrochemical reactions. The lower diffusion resistance (12.2 Ω) obtained from MFC4 compared to MFC2 (34.8 Ω) showed that 38 the diffusion resistance increased mainly due to the formation of thick ineffective biofilm . In addition, MFC4 showed the maximum capacitance of 37.5µF (which is inversely proportional to the Rct) and that corresponds to the enhanced charge holding capacity of MFC. These results showed that the B. cereus incorporated AS inoculum were useful to achieve higher biocapacitance with lower charge transfer resistance thereby improved the power output, due to the decrease in overall internal resistance of the cell.


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Figure 5. CE and methane production on different MFCs. The COD removals after 11 days of operation in MFCs have been presented in Figure 4d. It can be seen from the Figure 4d, the MFC2 obtained higher COD removal efficiency (68%) though less electricity was produced. The higher COD removal efficiency were achieved probably due to the presence of diverse range of bacteria as well as methanogenic archaea in the inoculum that developed into a 5, 39 community which then efficiently utilized the substrates in the complex wastewater . Besides that, 7 Chae et al. 2010 reported that in AS operated MFC, after certain time of operation, the methanogenic activity increased and therefore it could also be the reason for achieving high COD removal efficiency in MFC2. But, MFC3 achieved lower COD removal efficiency (42%) compared to MFC2 because pure culture of B. cereus could not be able to utilize wide variety of substrates present in the wastewater whereas MFC4 achieved slightly higher COD removal efficiency than MFC3 but lower than MFC2, which might be due to the less concentration of wide range of bacteria in the inoculum compared to MFC2. In order to observe how much of the available substrates were converted into electrical current in the MFC, another set of 450 ml MFCs (MFC5, MFC6, MFC7) were operated at similar conditions. The CE and methane production were analyzed after 11 days of operation to observe the methanogenic effects on the performance of MFCs as shown in Figure 5. The MFC5 obtained maximum CE of 12% with higher CH4 generation (21.33 mmol) whereas MFC6 attained CE of about 32% with very low CH4 generation (3.2 mmol). The CE and methane production were around 2.7 times higher and 7 times lower than that of MFC5 respectively. The lower CE observed in MFC5 indicates that major proportion of the substrates were 6 being used by methanogenic bacteria . However, the inclusion of B. cereus in AS (MFC7) achieved higher CE (22%) with lower CH4 (12.2 mmol) production compared to the MFC which solely inoculated with AS (MFC5). This result clearly indicates the dominance of electrogens in the inoculum which efficiently used major part of the substrates for generating the current during MFC operation. Furthermore, the increase in CE of MFC7 suggests that more electrons are used for bioelectrochemical reactions, while inhibiting 40 methane formation . 4. Implications In microbial fuel cells, researchers have limited options to enhance the performance of MFC. Currently much emphasis has been given to the development of materials and identification of new microorganisms to achieve a high performance in MFC but the investigation on synergistic interactions between


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microorganisms in AS to improve the MFC performance is still lagging. Moreover, it is well known that electrogenic microorganisms are required for the formation of effective biofilm in MFCs but the presence of methanogenic microorganisms in AS suppresses the growth of electrogens and causes the major electron loss in AS operated MFCs. Therefore, our work forms the foundation to investigate the effect of incorporation of microorganisms possessing electrogenic and anti-methanogenic properties in AS operated MFCs.

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In this study, we have introduced an electrogen (B. cereus) with methanogen inhibition properties that can simply be incorporated into AS to achieve higher performance in AS operated MFC. The CV results (Figure 3a) demonstrated that B. cereus can able to produce mediators that can effectively transfer the electrons to anodes which clearly indicated that B. cereus possesses electrogenic properties. 41 Besides that, literatures showed that the Bacillus sp capable to produce fatty acids which can suppress the growth of methanogenesis. Similarly, the GC-MS data (Table S2) of our study also showed the higher existence of fatty acid compounds in MFC4 (with B. cereus incorporated AS) compared to MFC2 (with AS), indicating that the B. cereus possesses the ability to produce fatty acid compounds which might have inhibited the growth of methanogenic bacteria and that in turn improved the power generation in MFC4. Based on these results, the proposed mechanism of methanogenesis inhibition by B. cereus is illustrated in Figure 6. It shows that B. cereus might have excreted lipase enzymes which catalyzed the lipids and proteins in the POME thereby produced fatty acids such as palmitic acid and stearic acid (Table S2). These fatty acids adsorbed by methanogenic bacterial cell membranes [20] that could have damaged the cell membrane as determined via loss of potassium ions, proteins and ATP and might have played a role in cell death.

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Figure 6. Schematic diagram of inhibition of methanogenic bacteria. 42

Moreover, Zeitz et al. 2013 also reported that methane production in the rumen fermentation process was successfully inhibited by supplementing the medium with long chain saturated fatty acids (SFAs). SFA hinders the growth of methanogenic bacteria through adsorption and disruption of cell membranes thereby decreased the CH4 formation rate by 90% for specific species of methanogenic 10 43 archaea . In another study , medium chain fatty acid (lauric acid) was used to suppress the substrate


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loss caused by methanogenic microorganisms. As discussed above, these findings greatly expand the potential of MFC which uses B. cereus incorporated AS to suppress the methanogenesis because previously described methods either did not effectively suppress the methanogenesis or incurred higher costs. Moreover, most of the previous methods required the addition of external compounds such as 6 10 Chaetoceros , 2-Bromoethanesulfonate etc. In future, in depth analysis on microbial community structure of B. cereus incorporated AS will be carried out to observe the inhibiting effect of B. cereus on methanogens in the AS operated MFCs.

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Associated Content

390

Supporting information

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Molecular analysis of the microorganism (Figure S1), the schematic diagram of gas collection and analysis from MFC reactors (Figure S2), electrochemical parameters of different MFCs obtained by fitting nyquist plots (Table S1), qualitative analysis of main organic compound by GC-MS analysis (Table S2).

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Author Information

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Corresponding Author

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*E-mail: [email protected], [email protected]. Tel: +6095492872.

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ORCID: Md. Maksudur Rahman Khan: 0000-0001-6594-5361

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Present Address

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Faculty of Chemical and Natural Resources Engineering, University Malaysia Pahang, 26300 Kuantan, Malaysia.

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Notes

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The authors declare no competing financial interest.

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Acknowledgements

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This work was supported by the University Malaysia Pahang, Malaysia (RDU 140322 and GRS 150371)

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References

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