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Enhanced current generation using mutualistic interaction of yeast-bacterial co-culture in dual chamber microbial fuel cell M Amirul Islam, Baranitharan Ethiraj, Chin Kui Cheng, Abu Yousuf, Selvakumar Thiruvenkadam, Reddy Prasad, and Md. Maksudur Rahman Khan Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b01855 • Publication Date (Web): 21 Dec 2017 Downloaded from http://pubs.acs.org on December 22, 2017

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Industrial & Engineering Chemistry Research

Enhanced current generation using mutualistic interaction of yeastbacterial co-culture in dual chamber microbial fuel cell

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M Amirul Islam , Baranitharan Ethiraj , Chin Kui Cheng , Abu Yousuf , Selvakumar Thiruvenkadam , † ¥£ Reddy Prasad , Md. Maksudur Rahman Khan *

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Although exogenous mediators can distinctly enhance the performance of yeast driven microbial fuel cell (MFC), the possibility of mediator's toxicity, environmental risk and cost are the main challenges facing towards its application in MFCs. Therefore, the use of naturally produced electron shuttles for unmediated yeast would be of great interest since it can solve most of the above-mentioned problems. The present study is to investigate the possibility of the use of electron shuttle producing bacteria Klebsiella pneumonia (K. pneumonia) to boost up the performance of yeast Lipomyces starkeyi (L. starkeyi) driven MFC. The MFCs inoculated with L. starkeyi and K. pneumoniae co-culture achieved a maximum power 3 density of 12.87 W/m which is about 3 and 6 times higher than that of MFC solely inoculated with pure yeast and bacteria, respectively, demonstrating that the yeast cells have successfully utilized the reduced electron shuttles excreted by the bacteria. The occurrence of the mutualistic interactions was further supported by the CV and EIS results. The findings of this work suggest that the use of mutualistic interaction of yeast and bacteria could be a new way to increase the performance of the MFCs.

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Introduction

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The mutualistic interaction between microbes in microbial communities usually have closely coupled cell1 cell associated interactions and plays a crucial role in bio-electrochemical systems . The potential, created by the anodic catalyst at the anode controls the mutualistic interaction which influences the electron discharging capacity from the biocatalyst as well as the electron transport from organic substrate 2 to anode during its metabolism . In the past decades, several research initiatives have been created for 3 the development of models to demonstrate mutualistic interactions in microbial communities . Some research results showed that the combined output through mutualistic interactions is usually higher than 4, 5 that of each of the monoculture systems . For instance, the binary culture containing fermentative bacteria, Clostridium cellulolyticum, and the electrogenic bacteria, Geobacter sulfurreducens respires on 6 4 an external electrode to convert the cellulosic biomass to useful energy . Moreover, in another study, it

Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia Pahang, 26300, Pahang, Malaysia. ₭ Department of Biotechnology, Bannari Amman Institute of Technology, Sathyamangalam, Erode District, Tamil Nadu, 638401, India §

Faculty of Engineering Technology, Universiti Malaysia Pahang, 26300, Pahang, Malaysia

£

Centre of Excellence for advancement Research Fluid Flow (CARIFF), Universiti Malaysia Pahang, 26300 Pahang, Malaysia € Department of Chemical and Environmental Engineering, Universiti Putra Malaysia, 43400, Serdang, Malaysia. † Department of Petroleum and Chemical Engineering, Institut Teknologi Gadong, BE1410, Brunei. *Corresponding Author: Email: [email protected], [email protected], Tel: +6095492872 Abstract

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was found that the mutualistic interactions between Enterobacter aerogenes and Pseudomonas aeruginosa PA14 through metabolites have significantly enhanced the power generation in bioelectrochemical systems.

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Generally, the bacterial species are employed as anodic biocatalysts in microbial fuel cells 7-9. (MFCs) since it offers some satisfactory results The bacterial species such as Shewanella, Geobacter, Aeromonas, Rhodoferax, Escherichia, and Klebsiella have been proffered because of its high catalytic 10-12 activity in MFCs . However, in some cases, it is not feasible as they require specific substrates and growth conditions. On the contrary, eukaryotic species (i. e yeast) have recently received renewed attention due to their faster growth, high tolerance, capable to withstand a wide range of stressful environmental conditions, mostly non-pathogenic, easy to handle and some species can even utilize 13 broad range of substrates . However, in most cases, yeast driven fuel cells yield lower power than bacteria driven fuel cells due to less extracellular electron transfer efficiency.

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In addition to that, the electron transfer mechanism of biocatalysts plays a major role in the 14 performance of MFCs . The two primary electron transfer mechanisms that have been reported so far in MFCs are the direct and mediated electron transfer. The direct electron transfer (DET) occurs through 15 nanowires, cytochrome or trans-membrane electron transport proteins . Whereas, the mediated electron transfer (MET) occurs through electron shuttle mediator, in such a case the efficiency of electron transfer 11, 16 significantly enhanced . Usually, in an anaerobic condition, microbes shift to fermentation reaction where pyruvate produced from a glucose molecule converted into alcohol or organic acid through + 7 NADH/NAD recycling process which is an essential step to continue glycolysis . Moreover, the NADH can be easily available for the mediator located in the cell membrane of the yeast since the glycolysis reactions occur in the cytosol of the microbial cell. Besides that, the energy extraction process of MFC not + affected the glycolysis since NADH is oxidized back to NAD while the mediator molecule gets reduced. These features make yeast operated fuel cells to be used directly in fermenters for the power 17 generation . Moreover, the yeast cells have natural electron shuttlers such as ferredoxin, azurin and cytochromes for exchanging electrons with electrodes and play a key role in the electrochemical behavior 18 of the biocatalyst . In addition, the yeast cell membranes usually have a higher protein content which is 19 considered as one of the key characteristics of electrogenic species .

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Previous studies reported that only a few species of yeasts such as Candida melibiosica, Arxula adeninivorans possesses the ability to transfer electron through both the membrane bound cytochrome 10 and the endogenous redox mediator(s) . So, most of the yeast species are unable to produce mediators and achieved lower performance in yeast inoculated MFCs. Thus, there are many concerns for studying and enhancing the electron transfer in a mediator less yeast operated MFCs. Generally, the 15, 20 exogenous artificial mediators are usually added to enhance the electron transfer in yeast MFCs . Several studies reported that the performance of MFC enhanced with the addition of exogenous 13, 15 20 mediators in MFCs . For instance, Rahimnejad, et al. reported that addition of thionine mediators in yeast (Saccharomyces cerevisiae) MFCs enhanced the power generation by 20 fold. However, the use of artificial mediators is not so advantageous because they are toxic to microbes at high concentrations, 16 expensive and environmentally unacceptable. . Therefore, harmless, inexpensive, and efficient exogenous mediators would be necessary from the technical as well as in practical point of view. Instead of using exogenous mediators, if yeast could be supported by the bacterial mediators for transferring the electrons to the electrode, it may effectively improve the power generation in the yeast driven MFC.

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Although yeasts and bacteria have antagonistic effects upon one another , some yeasts and 22 bacteria have the ability to live together for their mutual benefits. Zhang, et al. reported that yeastbacterial co-culture consisting three bacterial strains and a yeast strain degraded more total petroleum hydrocarbon (56%) using their synergistic interactions in bioremediation process than yeast (37.7%) and

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bacterial (40.1%) monocultures. But, as far as the authors are concerned, no one has reported the synergistic relationship of yeast-bacterial co-culture in MFCs. Therefore, in this study, the synergistic relationship between electron shuttle producing bacterial spp. Klebsiella pneumonia (K. pneumoniae) and yeast spp. Lipomyces starkeyi (L. starkeyi) was investigated to improve the electron transfer efficiency in palm oil wastewater operated MFCs. K. pneumoniae and L. starkeyi were chosen based on the fact that 23-25 both microbes were reported to utilize POME as substrate .The influence of artificial mediator (pyocyanine) and naturally produced mediators by K. pneumoniae on the electrochemical behavior of L. starkeyi driven MFCs was analyzed. Furthermore, the possible electron transfer mechanism between yeast and bacteria has been discussed.

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2. Materials and Methods

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2.1 Sample collection and characterization

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The palm oil mill effluent (POME) was collected from LKPP Corporation Sdn. Bhd., Gambang, Pahang and stored at 4 °C to prevent the deterioration of ingredients. The solids and debris were removed through a Whatman No.1 filter paper and subsequently, the wastewater was sterilized by autoclaving at 121 °C for 15 min before it is used as the culture medium. All the experiments were conducted using POME as the substrate without adding additional nutrients. POME samples were prepared by diluting the raw POME as described in Table 1.

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Table 1 Description of POME samples Sample

Description

100% POME

Raw (undiluted) POME after removal of solid particles

50% POME

Raw POME and de-ionized water in the ratio of 1:1

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2.2 Culture conditions and inoculum preparation

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The pure culture of yeast strain L. starkeyi was collected from the laboratory of Biochemical Engineering, University of Naples Federico II, Italy. While the bacterial strain K. pneumoniae was isolated from the municipal wastewater, Kuantan, Pahang, Malaysia. The strain isolation and identification by PCR and 23 sequencing have been described by Islam, et al. . The pure cultures were enriched by preparing an overnight culture in Luria Bertani broth (10% v/v) and incubated at 35°C in a shaker incubator (150 rpm). The 1mL of broth culture was used as inoculum in the MFC.

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2.3 MFC construction

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The MFCs (MFC1, MFC2, MFC3, MFC4, MFC5 and MFC6) were fabricated (5 cm x 5 cm x 5 cm) using a cubic plexi glass (Shanghai, Sunny Scientific, China) with a working volume of 20 mL. Stainless steel (3 x 0.4 x 3 cm) was used as both anode and cathode electrode in all the experiments. In this study, stainless steel electrode (SS electrode) was chosen due to its biocompatibility, corrosion resistivity, high chemical 26, 27 stability and conductivity . 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 placed in between the anode and cathode compartments of MFC. Prior to use, the Nafion membrane was pre-treated using dilute 0.1 M H2SO4 for 1 hour followed



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by washing with de-ionized water several times. After that, the entire set up was tightened up with the screws. The anode compartments were filled with 20 mL of sterilized 50% POME and subsequently, the 1 -6 -6 mL of pure culture bacteria (128 CFU x 10 /mL) and the 1 mL of pure culture yeast (116 x 10 CFU/mL) were inoculated into it while the cathode chamber was filled with KMnO4 solution as an oxidizing agent and then operated for 15 days. The descriptions of MFCs used in this study were shown in Table 2.

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Table 2 Descriptions of MFCs MFCs

Inoculums

Working volume (mL)

MFC1

L. starkeyi

20

MFC2

K. pneumoniae

20

MFC3

L. Starkeyi + K. pneumoniae

20

MFC4

L. starkeyi + Artificial mediator (pyocyanin, added th at 7 day) th L. starkeyi + K. pneumonia (added at 7 day)

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MFC5

20

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2.4 Measurement and analyses

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The voltage across an external resistor (1 kΩ) was monitored every 15 minutes using a digital multimeter (Fluke 289 True RMS Multimeter, USA) with data logger. Polarization curves were obtained by changing the external resistance from 50 to 20,000Ω at various time intervals using the Crotech DRB-9 rheostat, UK. Power density was normalized by the volume of the MFC. 3 Volumetric power densities (ܲ௩ , W/m ) were calculated using the Eqs. (1) – (2).

ܲ = ܷ‫ܫ‬

(1)

2

(2)

ܲ௩ = 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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Moreover, the number of bacteria (cell growth) in the liquid phase of anodic chamber was quantified to monitor the planktonic population via spread plate technique where 1 mL of sample was taken out from -6 the anodic chamber and serially diluted up to 10 and 1 mL of each diluted sample was spread on the agar plate. The cultured colonies were counted every 24 hours to follow the growth profile.

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2.5 FESEM analysis

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Biofilm formation on the anode electrode was visualized using FESEM (5 kV) at various time intervals of


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the MFC experiments. The small sections of the anodes were cut off from the anode compartment and rinsed using sterile water. Subsequently, it was soaked in a beaker containing anaerobic solution of glutaraldehyde (3%). The samples were then washed twice with 0.1 M phosphate buffer and dehydrated successively at various ethanol concentrations for about 10 minutes. After that, the samples were dried and coated with platinum to a thickness of 10 nm. Finally, the specimens were observed by FESEM.

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2.6 Mediator analysis

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The excreted mediators were measured and quantified using Gas Chromatography-Mass Spectrometry (GC-MS, Agilent Corporation, USA). The required amounts of samples were taken out from the anode chamber and were pretreated with an equal volume of n-hexane (chromatogram pure grade, Sigma Chemicals, USA) to extract the organic compounds. A 1 µL of extract was analyzed using 6890N/5973 GC–MS system. 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 compartment was controlled as follows: 40 °C for o o 5 min; then increased to 280 C at a rate of 3 C /min.

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2.6 Cyclic voltammetry analysis

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Cyclic voltammetry (CV) was used to examine 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 helps to reveal the precise role of redox mediators, cytochromes and pili in the electrochemical reactions. The CV was performed using a electrochemical system, PARSTAT 2273, USA. A three electrode system was applied to collect the CV data where anode and cathode were used as working and counter electrodes respectively. The Silver/Silver chloride (1.0 M KCl) electrode was disinfected using 70% ethanol (Sigma) and used as a reference electrode. Moreover, the reference electrode was placed closer to the anode surface during CV operation. The CV was performed at a scan rate of 30 mV/s in the potential range of +1.0 to -1.0 V and before each measurement; nitrogen gas was purged for about 15 min.

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2.7 Electrochemical impedance spectroscopy

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Electrochemical impedance spectroscopy (EIS) measurements were performed using a potentiostat, PARSTAT 2273, USA. In order to analyze the anodic internal resistances of MFC, a three electrode system was used where the anode and cathode electrode was 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 (10 mV, 100 kHz – 5 mHz) was applied to prevent the biofilm detachment as well as to minimize the disturbance on systematic stability. The EIS data were fitted with the 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 Performance of MFC

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The performance of MFCs (MFC1, MFC2 and MFC3) using different inoculums have been presented in Figure 1. As can be seen in Figure 1a, all the MFCs showed a similar trend in their performance and the th maximum power generation of MFCs was obtained on the 11 day of operation. Therefore, the th polarization data of the MFCs on 11 day of operation was plotted and shown in Figure 1b. It can be 3 observed that the maximum power density of about 2.67 W/m was achieved for yeast driven MFC



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(MFC1) which is comparatively lower than that of other MFCs. On the other hand, bacteria driven MFC 3 (MFC2) attained a maximum power density of 4.36 W/m which is about 1.8 times higher than that of MFC1. However, interestingly, the yeast-bacterial co-culture driven MFC (MFC3) obtained maximum 3 power generation of 12.87 W/m which is about 6 and 3 times higher than that of MFC1 and MFC2, respectively, might be due to the synergistic relationship between L. starkeyi and K. pneumoniae.

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Figure 1 Electrochemical activity and performance of MFCs, a) Maximum power densities of MFCs using 50% of initial COD vs. Time b) Polarization curves of MFC using different inoculums, c) CV for the anode using different MFCs, d) Fitting results of Nyquist plots for different MFCs, magnified high frequency region of Nyquist plots (inset)

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CV was conducted to investigate the electron transfer mechanism as well as catalytic efficiency in the anode of MFCs. The characteristics of CV rely on various factors, such as the presence of chemical and biological species, the rate of the electron transfer reactions and the rates of diffusion of electroactive 28, 29 species . Besides that, it helps to reveal the precise function of redox mediators in the 30 31 electrochemical reactions . Rabaey, et al. employed CV for reporting the involvement of excreted redox mediators by Pseudomonas aeruginosa in MFC for the transfer of electrons to the anodes. 32 Moreover, on the basis of CV results, Marsili, et al. also reported that Shewanella oneidensis strains capable to secrete soluble mediators which help for the extracellular electron transfer between the biofilm th and the anodes. CV was performed before and after inoculation of MFCs (11 day) as shown in Figure


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1c. Before inoculation (virgin), no redox peak was noticed either during the forward or reverse scan in the controlled electrochemical cell. The MFC1 inoculated with L. starkeyi showed the absence of redox peak which demonstrated that the electrochemical activity was mainly due to the membrane bound 15 cytochromes. Sayed, et al. also observed similar properties (without redox peak) of CV data while MFC inoculated with baker’s yeast (Saccharomyces cerevisiae). However, a redox peak was observed from the MFC inoculated with K. pneumoniae suggest that the formation of reversible redox couples, due to 33 34 the presence of mediators that are reversibly oxidized and reduced during CV tests . Deng, et al. also reported the similar range CV peak for K. pneumonia L17; therefore, K. pneumoniae strain of the present 35 work might also use the similar electron shuttle mechanisms and these redox reactions mostly affect the formal potential of the cell during turnover CV. The more intense redox peak observed in MFC3 compared to other MFCs represents the higher catalytic efficiency which may possibly be due to the production of more electron shuttles in MFC3. The synergistic interactions between the yeast and bacteria might have reduced the cell growth inhibiting compounds which would have consecutively increased the cell growth 22 in MFC3 . Thus, the number of electrogens (K. pneumoniae) also increased which might have produced more redox compounds and that would have resulted higher performance in MFC3. The strong redox peak compared to other MFCs clearly indicates that higher and rapid charge transfer occurred in MFC3. Moreover, it suggest the development of matured and conductive biofilm on the surface of the anode which would have improved the electron transfer efficiency between the microorganisms and the anode of 36 MFC . This finding reveals that the synergistic interaction between yeast and bacteria and efficient use of electron shuttles by both microorganisms improved the power generation in MFC3 by improving the 37 kinetics of the electrochemical reactions since it reduced the anode activation losses .

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The measurement of EIS spectra of the anode shows a key information which helps to analyze the 38 electrochemical reactions on the electrode and bacterial metabolism. . In order to obtain the quantitative values of the resistances, the impedance data were fitted to the equivalent circuit: [R(RQ)(R[QT])], where Rct, Rohm, Q and T represent the charge transfer resistance, ohmic resistance, constant phase element 39 (CPE) and mass transfer resistance respectively . The low frequency regions of the Nyquist plots were dominated by the finite Warburg diffusion element. Nyquist plots for the anode configuration of different th MFCs were recorded on 11 day as shown in Figure 1d. The anode charge transfer resistance (Rct) values were found to be 29.9 Ω, 17.3 Ω and 7.7 Ω for MFC1, MFC2 and MFC3 respectively (Table 3). In MFC1, the higher Rct (29.9 Ω) was obtained which might be due to the absence of electron shuttle 20 compounds (Figure 1c). Rahimnejad, et al. reported that the soluble redox mediators significantly reduced the charge transfer resistance thus the absence of mediators lowered the performance in MFC1. In MFC2, lower Rct (17.3 Ω) was observed compared to the MFC1 (29.9 Ω) demonstrating that the effective biofilm was formed by the electrogenic bacteria hence produced more electron shuttle compounds 29 (Figure 1c) in the anode of MFC . Thus, the activation losses were decreased by improving the kinetics 11, 40 of the electrochemical reactions .

242 Type of MFCs

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Table 3 The contribution of internal resistance of anode at different MFCs Rohm (Ω) Rct (Ω) Rdif(Ω) CPE Total resistance(Ω)

MFC1

1.8

29.9

47.8

5.4

79.5

MFC2

1.9

17.3

27.2

8.9

46.4

MFC3

1.6

7.7

8.4

23.3

17.7

The higher diffusion resistance of MFC1 (47.8 Ω, Table 3) might be due to the inefficient electron transfer between microbes and electrode. The reason could be that the electrons produced from the



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microorganisms not able to travel a long distance to reach the electrode surface without electron shuttle compounds. However, in MFC2, the diffusion resistance was significantly reduced by 43% (27.2 Ω) might be due to the presence of electro-active biofilm on the surface (interface) resulting in a decrease in diffusion path of electron shuttles. In MFC3, the diffusion resistance was drastically reduced compared to other MFCs (8.4 Ω) suggesting that the synergistic interactions between yeast and bacteria would have increased the electron shuttles as well as formed the conductive biofilm thereby decreased the diffusion resistance. In addition to that, the MFC3 achieved a maximum capacitance of 23.3 µF (which is inversely proportional to the Rct) and that corresponds to the enhanced charge holding capacity of MFC. These results showed that the co-culture of L. starkeyi and K. pneumoniae inoculum were useful to achieve higher bio-capacitance with lower charge transfer resistance thereby improved the power output, due to the decrease in overall internal resistance of the cell. These results are consistent with the polarization data and the CV.

Figure 2 Concentrations of mediator on 11

th

day using different MFCs

th

The mediators produced by microbes in MFCs on the 11 day of operation have been detected and analyzed as shown in Figure 2. The absence of mediators in MFC1 inoculated with yeast (Figure 2 and Figure S1) suggesting that yeast cells (L. starkeyi) are unable to produce electron shuttling compounds. Generally, yeast cell produces lower power since it predominantly uses cytochrome mediated electron transfer mechanism which is comparatively less efficient than the other mechanisms ( i.e electron 41 shuttling, electron conducting pili) . Therefore, lower performance was obtained in MFC1 (inoculated with L. starkeyi) compared to other MFCs, possibly due to the inefficient electron transfer between yeast and electrode. Previous studies showed that yeast cells (i.e Hansenula anomala, Saccharomyces cerevisiae, and Candida melibiosica) produced lower electrical power without the addition of electron transfer 42 mediators in MFC . This is because, the wall of a yeast cell is usually composed of thick (100 to 200 nm) polysaccharides and proteins. Moreover, the cytochromes of the yeast are found in the mitochondrial


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region and the trans-plasma membrane electron transport systems are found in the cell membrane, which are surrounded by the cell wall. Therefore, to get an electrochemical response from yeast, it is believed that the mediator compounds should pass through the cell wall and interact with the components of the membrane and/or internal redox centres or that the response comes from soluble electroactive 43 42 compounds released from the cell . Lee, et al. reported that the redox compounds, changing between an oxidized state (Medox) and a reduced state (Medred) are necessary for the yeast to reach the electron transport chain (ETC) located in the mitochondria of the cell.

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However, in MFC2, it can be observed that K. pneumoniae produced electron shuttling mediators of 2, 44 6 Di-tert-butyl benzoquinone (Figure 2 and Figure S2-4). Li, et al. reported that K. pneumonia could produce electron shuttling compounds by utilizing glycerol as a substrate, which was confirmed by 34 electrochemical analysis. In another study Deng, et al. observed that K. pneumonia produced electron shuttling compound of 2,6-di-tert-butyl-p-benzoquinon in glucose fed MFC at neutral pH (pH=7.0) and at o 11 30±1 C. Our earlier study also demonstrated that K. pneumoniae possesses the ability to produce mediator compound which can significantly improve the kinetics of the electrochemical reactions and thereby reduce the charge transfer resistance of MFCs. In the present study, MFC2 exhibited higher performance than MFC1 which was due to the higher catalytic efficiency of K. pneumoniae. In MFC3 the mediator concentration was found to be increased by ~2 times suggesting the presence of synergistic interactions between K. pneumoniae and L. starkeyi. The results shown in Figure 2 are in agreement with the polarization, CV and EIS results (Figure 1b-d). Additionally, the electron shuttle compound produced by K. pneumoniae might have helped both the yeast and bacteria to transfer electrons to the electrode and consequently achieved higher performance in MFC3. The findings of this study scientifically emphasize the positive effect of yeast and bacterial co-culture systems in MFCs.

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3.2 FESEM analysis of biofilm

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Figure 3 Biofilm visualization of MFCs for different inoculums, a) L. starkeyi, b) K. pneumonia, c) K.



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298

pneumonia and L. starkeyi co-culture

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The FESEM images of the biofilm using different inoculums were analyzed on 11 and presented in Figure 3. It can be seen from the Figure 3a (MFC1), the pure yeast cells (L. starkeyi) stick to each other and embedded in a self-produced matrix of extracellular polymeric substance (EPS). On the other hand, colonization of pure bacterial cells has been observed in the MFC2 anode as shown in Figure 3b. However, as expected, in MFC3, both yeast and bacterial cells are observed in the biofilm (Figure 3c). In MFCs, obtaining maximum power generation and a decline in performance not only depends on 23 electrogenesis capability of microorganisms but rather depends on effective biofilm formation . Therefore, the higher power production (Figure 1b) by co-culture of L. starkeyi and K. pneumoniae compared to other MFCs might be due to the formation of effective biofilm which uses both membrane bound cytochromes (DET) and electron shuttles for transferring the electrons to the electrode.

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3.3 Influence of mediators on the yeast performance

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In order to observe the effect of mediators, MFC4 and MFC5 were operated under similar conditions of previous MFCs. The Influence of artificial mediators and the mediators produced from the microorganisms (K. pneumoniae) on the performance of yeast (L. starkeyi) has been investigated and presented in Figure th 4. In MFC4, on 7 day while the current generation reached a stable value, 500 μM of artificial mediators (pyocyanin) was added to observe the changes in the MFC performance. As can be seen from the Figure 4a, the current generation increased sharply at the rate of 52.29 µA/day (Figure S5) and reached to th 224±10 µA (Figure 4a) on 8 day of operation. It shows that the addition of artificial mediator (pyocyanine) significantly improved the current generation by increasing the electron transfer rate between bacteria and electrode. Previous literatures reported that the use of exogenous electron shuttling mediators such as methyl viologen, methylene blue, neutral red, and thionine have significantly enhanced 20 the performance of yeast operated MFCs . However, after 8 days of operation, the power generation of 13 MFC4 sharply reduced might be due to the cytotoxicity of artificial mediators (pyocyanin) . The pyocyanin also plays a significant role in the pathogenesis of pseudomonal infections thus it shows toxic 45 effects in eukaryotic cells . Moreover, from Figure S6, it can be seen that the yeast cell growth started declining after 24 h of the addition of artificial mediator suggesting the antagonistic relationship between yeast cell and artificial mediators for longer time of operation. On the other hand, in MFC5, after the th addition of 1 mL of K. pneumoniae broth culture (at 7 day), the current generation sharply increased at the rate 71.23 µA/day (Figure S5) and reached to the value of 247±10µA which is significantly higher than the maximum current generation obtained from MFC4. Generally, the electron shuttle produced by + bacteria has a redox potential close to that of NADH/NAD which can facilitate electron shuttling between the reaction center inside of the cell and terminal electron acceptor (anode electrode) thereby achieved 20 higher performance in MFC . To further verify the effect of naturally produced mediator (secreted by K. pneumoniae) on performance of yeast driven MFC, an MFC inoculated with K. pneumoniae was run for 7 days and the anolyte was filtered to screen out the microbes. The COD of the filtrate was adjusted by fresh sterilized POME and was used to run another MFC inoculated with L. starkeyi (MFC6, Figure S7). It can be seen that the current generation in MFC6 (350±20 μA) was significantly higher compared to the th th MFC1 (190±20 μA, Figure S7) on the 11 day of operation. The polarization data of MFC4 (on 8 and th th 11 day) and MFC5 (11 day) were plotted as shown in Figure 4b. It can be observed that the MFC4 3 th achieved maximum power density of 5.1 W/m (Figure 4b) on the 8 day (after 24 h of adding th payocyanine); however, on the 11 day the performance of MFC drastically dropped and reached to the 3 1.6 W/m which might be due to the adverse effect of artificial mediator on MFC. On the other hand, the MFC5 achieved 4 and 6.5 times higher power density compared to MFC1 and MFC4, respectively on the th 11 day.

th


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Figure 4 Electrochemical behaviour of MFC4 and MFC5 (a) current generation trend, (b) polarization curves (c) cyclic voltammograms, magnified CV peak of MFC4 (inset) d) Fitting results of MFC Nyquist plots

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The CV and EIS data were recorded for MFC4 (on 8 and 11 day) and MFC5 (11 day) as presented in Figure 4c and 4d respectively. From Figure 4c, a noticeable redox peak was observed for th MFC4 on 8 day and after the addition of pyocyanine and the redox peak was not significantly changed th th on the 11 day of operation. However, the prominent redox peak obtained by MFC5 on 11 day suggesting the presence of adequate amount of bacterial redox compounds performing as natural mediator. From the Figure 4d and Table S1, it can be seen that the Rct and Rdif was increased from 29.21 Ω and 43.91 Ω to 52.67 Ω and 63.89 Ω form day 8 to day 11 respectively for MFC4 where the power generation was dropped concomitantly. In contrast, the more intense reversible redox peak as well as less charge transfer resistance (reduced by 69%, Table S1) observed in MFC5 compare to MFC4 (on the th 11 day) indicates that an effective and conductive biofilm was formed which helped to transfer the electrons from microbes to the anode with minimum diffusion path. The main reason for the drastic th decrease in the performance of MFC4 on the 11 day (Figure 4b) was ineffective biofilm formation which was related to the inhibition of the yeast cell growth by artificial mediators, as proven in Figure S8. These results suggest that the presence of the compounds secreted by K. pneumoniae positively influenced the performance of the L. starkeyi driven MFC.

th


th

th


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Figure 5 Synergistic electron transfer mechanism of yeast- bacterial co-culture (schematic diagram)

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Based on these results, the proposed mechanism for the synergistic effect between L. starkeyi and K. pneumoniae has been presented in Figure 5. It illustrates that L. starkeyi could transfer the electron using cytochromes resulting in unutilization of the electrons produced by the yeast cells in bulk which leads to a low power generation in the yeast driven MFC. In contrast, K. pneumoniae could produce electron shuttle compounds as suggested by our CV results that enables the long distance electron transfer in MFC. In presence of mediator (artificial or natural), the electron transfer for yeast driven MFC can be occurred by both cytochrome based and electron shuttle mediated paths (Figure 5). The naturally produced electron shuttle compounds by K. pneumoniae enhanced the electrogenesis capability of a mixed culture of yeast and bacteria.

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4. Conclusions

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The results of this study demonstrated that the co-culture of L. starkeyi (cytochrome mediated DET) and K. pneumoniae (electron shuttle producing microorganisms) significantly enhanced the power generation 3 in MFC. The MFC with co-culture showed the maximum volumetric power density of 12.87 W/m which is about 3 and 6 times higher than that of MFC with monoculture of L. starkeyi and K. pneumoniae respectively. The co-culture achieved more intense redox peak and lower charge transfer resistance compared to other MFCs which clearly indicates that efficient electron transfer occurs between microorganisms and electrode. Furthermore, a significant decrease in current generation after addition of artificial mediators can be attributed to the antagonistic effect (cytotoxicity) of mediators on yeast. These findings suggest that selected natural mediator producing microorganisms can be exploited to enhance the electron transfer efficiency of unmediated yeast for maximizing the power generation in MFC.

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Associated Content

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Supporting information


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th

th

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GC spectrum of MFC1 on the 11 day of operation (Figure S1), GC spectrum of MFC2 on the 11 day of th operation (Figure S2), GC spectrum of MFC3 on the 11 day of operation (Figure S3). The mass spectrum for 2 6 Di-tert-butyl benzoquinone, a) analytical standard, b) sample taken from the anode of MFC2 (Figure S4), Current generation rate of (a) MFC4 and (b) MFC5 (Figure S5), Cell growth profile of MFC4 and MFC5 (Figure S6), Current generation profile of different MFCs with time under the fixed th external resistance (Figure S7), Biofilm visualization of different MFCs on 11 day, a) MFC4, b) MFC5 (Figure S8), The contribution of internal resistance of anode for MFC4 and MFC5 (Table S1),

393

Author Information

394

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, Universiti 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 Universiti Malaysia Pahang, Malaysia (RDU 140322 and GRS 150371).

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References

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