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Correlation of power generation with time-course biofilm architecture using Klebsiella variicola in dual chamber microbial fuel cell
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 2 ( 2 0 1 7 ) 2 5 9 3 3 e2 5 9 4 1
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Correlation of power generation with time-course biofilm architecture using Klebsiella variicola in dual chamber microbial fuel cell M. Amirul Islam a, Chee Wai Woon a, Baranitharan Ethiraj a, Chin Kui Cheng a,c, Abu Yousuf b, Md. Maksudur Rahman Khan a,c,* a
Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang, Malaysia b Faculty of Engineering Technology, Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang, Malaysia c Centre of Excellence for Advanced Research in Fluid Flow (CARIFF), University Malaysia Pahang, 26300 Kuantan, Pahang, Malaysia
article info
abstract
Article history:
In the present work, the wild type Klebsiella variicola was investigated in double chamber
Received 11 June 2017
microbial fuel cell (MFC) using palm oil mill effluent as substrate which achieved high
Received in revised form
power density (4.5 W/m3) and coulombic efficiency (63%) while maintaining the moderate
4 August 2017
chemical oxygen demand (COD) removal efficiency (58%). The effect of biofilm formation
Accepted 28 August 2017
on power generation over time was also evaluated and found that an effective biofilm with
Available online 19 September 2017
the discrete distribution of single layer microorganisms can produce high power corresponding to low charge transfer resistance. The growth of biofilm in multilayers consisting
Keywords:
of outnumbered dead cells in the vicinity of the electrode surface caused the polarization
Microbial fuel cell
resistance and diffusion resistance resulting in a sharp drop in the current generation. The
Klebsiella variicola
removal of multilayer biofilm from the anode surface positively influenced the cell per-
Biofilm architecture
formance which led to a rapid increase in current generation and thus revealed that
Palm oil mill effluent
effective biofilm predominated by live cells can be an emergent factor for achieving
Cell viability
maximum performance in MFC. © 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction In bio-electrochemical systems (BES), especially in microbial fuel cells (MFCs), the bio-catalytic activity of microorganisms plays a crucial role in enhancing the power generation of system [1,2]. In the past decade, a number of new strains of microorganisms have been reported which
can generate electrical current through MFCs [3]. However, only a few strains are capable of producing significant power using complex wastewater as a substrate [4,5]. MFCs are being considered to be a promising sustainable technology to combat the energy crisis, especially using wastewaters as substrates, because it can simultaneously generate electricity and accomplish wastewater treatment [5,6]. Moreover, it is reported that the energy efficiencies of
* Corresponding author. Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang, Malaysia. E-mail addresses: [email protected], [email protected] (Md.M.R. Khan). http://dx.doi.org/10.1016/j.ijhydene.2017.08.193 0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
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common renewable energy sources such as wind turbine (15e40%) [7] and biogas plant (5e20.7%) [8] are lower compare to the MFC (77.6%) [9]. Besides, MFCs can also be able to utilize a variety of substrates ranging from simple to complex. Wide varieties of simple substrates such as glucose, acetate etc. [10] and complex organic substrates such as industrial and domestic [11,12] wastewater have been tested so far in MFC. Anaerobic sludge (mixed culture) is one of the most widely used inoculum in the MFC for electricity generation and treatment of wastewater [13]. However, the sludge, which is not confined to a specific location, raises the difficulty to reproduce the electricity generation. In addition, when using mixed microbial cultures, it becomes hard to determine the mechanisms and roles of the individual microorganisms contributing to power generation. MFCs operated with the complex substrates using mixed microbial cultures usually showed high power densities compared to pure cultures [14] because, most of the pure cultures are not able to utilize the wide range of substrates [15]. So far, only a few pure cultures have been used in MFC operated with wastewater. Li et al. [16] and Nor et al. [17] have reported that pure cultures of Pseudomonas aeruginosa ZH1 and Shewanella oneidensis produced 4.45 W/m3 and 0.784 W/m3 using POME and municipal wastewater respectively. Very recently, Klebsiella variicola (K. varricola) was described as a new bacterial species and found to be more closely related to an electrogen Klebsiella pneumoniae [18] which can able to produce 2,6 DTBBQ electron shuttle in MFC [19]. Since the phenotypic and biochemical characteristics of K. varricola are identical to K. pneumoniae, it might also have electrogenic properties. Moreover, K. varricola can able to consume various kinds of substrates ranging from simple to complex [18]. Palm Oil Mill Effluent (POME) is one of the major wastewater sources in Malaysia comprising with amino acids, short fibres, nitrogenous constituents, free organic acids and a mixture of carbohydrates ranging from hemicelluloses to simple sugars [20,21]. Since K. varricola can utilize a diverse range of substrates; it might also be able to utilize POME efficiently, which has not been investigated so far in MFC. Biofilm is considered as a power house [22] that inevitably formed on the anode surface during MFC operation. In MFC, it is imperative if electroactive biofilms are produced that have the ability to respire the terminal electrons from metabolism onto electrode surfaces [23]. However, the thickness of biofilm is a crucial factor for MFC performance [24]. Thick biofilm with predominant dead cells accumulation at the vicinity of the electrode surface could negatively influence the power density by increasing the charge transfer and diffusion resistances [25]. Therefore, an optimum thickness is favourable for achieving substantial current densities [23]. In this research, the performance of wild type pure K. varricola was investigated in double chamber MFC operated with POME and the electricity production was interpreted with biofilm architecture comprising with live and dead cells. The power generation as a function of time was correlated with the electrochemical impedance data to elucidate the contribution of the resistances involved in the MFC.
Materials and methods Sample collection and characterization POME samples were collected from Panching palm oil mill (FELDA) located in Kuantan, Pahang, Malaysia. The samples were collected before the effluent discharge into the mixing pond at about 80e90 C. The collected samples were filtered using Whatman no.1 filter paper (2.5 mm) and the filtrate was autoclaved at 121 C, 15 psi for 15 min. Municipal wastewater (MWW) was collected from drainage discharge point of Kuantan city, Malaysia and filtered using Whatman filter paper and the filtrate was used as primary inoculum.
Isolation and characterization of microorganism The filtered MWW was serially diluted (101e106) and the pure culture bacteria were obtained from 106 dilution using the spread plate technique. The enrichment of the cultures was carried out by preparing an overnight culture in LB (Luria Bertani) broth (10% v/v) and incubated at 37 C with shaking at 150 rpm. Several pure culture strains were identified using Biolog gen III analysis but only Klebsiella spp. was selected for further identification and used as inoculum in MFCs. The strain characterization by PCR and sequencing has been described by Islam et al. [18].
Fabrication and operation of MFC The dual chamber MFCA and MFCB were fabricated using a cubic plexi glass (5 cm 5 cm x 5 cm) with the working volume of 20 mL (Shanghai, Sunny Scientific, China). A small brush with 2.5 cm in outer diameter and 2.5 cm long was used as anode and cathode electrode for all the experiments. The electrodes were cleaned with 1.0 M NaOH and subsequently with 1.0 M HCl after each experiment and stored in distilled water before use. The anode and cathode compartments were separated using a cation exchange membrane (Nafion 117, Dupont Co., USA). Prior to use, the cation exchange membrane was pretreated using dilute HCl for 1 h and thereafter it was washed with DI (De-ionized) water several times. After that, the whole set up was tightened with the screws. The 20 mL of sterilized POME (POME/water volume ratio 1:1) was poured into the anode compartment and subsequently inoculated with pure culture bacteria (1 mL) while the cathode chamber was filled with KMnO4 solution (20 mL), as an oxidizing agent.
Measurement and analyses Polarization measurements were performed at regular intervals (15 min) to estimate the power production of MFC at various external resistances ranging from 50 to 20,000U using an external resistor. The voltage data were taken using a digital multimeter with built in data logger (Fluke 289 true RMS industrial logging digital multimeter, USA) after it reached the stable value for obtaining the polarization curves. Power density normalized by volume (PV, W/m3) was calculated using the following equation.
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Cyclic voltammetry analysis V2 Pv ¼ vR
(1)
where, V is the output voltage, v is the volume and R is the resistance. The COD removal efficiency and columbic efficiency of dual chamber MFC was calculated as described by Baranitharan et al. [26]. The COD was determined using a COD reactor (HACH DRB 200, USA). The COD removal efficiency was calculated using Eq. (2) COD removal efficiency ¼
CODi CODt 100% CODi
(2)
where, CODi and CODt represent the initial COD (mg/L) of the anode chamber and the COD of the anode chamber at a particular time. The columbic efficiency (CE) was calculated using Eq. (3) Z 8 CE ¼
Idt
F$Van DCOD
(3)
where, I is the current (A), F is the Faraday's constant (96,485 C/mol), and Van is the anode volume (L). D 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 oxygen (32 g/mol) and assuming that 4 electrons exchanged per mole of oxygen.
Cell viability count of biofilm Fluorescence microscope (Olympus BX53, Germany) with 20X objective lens was employed to determine the cell viability with the help of LIVE/DEAD Bacterial Viability Kit (BD™ Cell Viability Kit). In brief, cell viability of biofilm was performed by detruncating small part (1 cm) of the anode using a sterilized scissor and then immediately immersed in the 50 mM phosphate buffer. In order to separate the microorganisms from carbon brush, it was centrifuged at 5000 rpm for 1 min. Thereafter, the cell suspension was serially diluted and stained using a viability staining kit. Finally, a 0.4 mm membrane filter was used to filter the stained cells and the cells were then counted using a microscope. The cell density was calculated per anode geometric area using the dilution factor, the filtered volume and the ratio of total filtered area to image area [27].
FESEM analysis of the biofilm Field emission scanning electron microscopy (FESEM) was used to visualize the anode biofilm at different time intervals of the anode. Small parts of the anode with biofilm was cut off and rinsed with sterile medium followed by immediate soaking. After that, the samples were immediately soaked into anaerobic solution (3% glutaraldehyde). The sample was then washed twice using 0.1 M phosphate buffer and dehydrated by successive 10 min incubations with different dilution of ethanol concentrations (40%, 60%, 80%, 90% and 100%). The samples were dried at room temperature (27 ± 2 C) and coated with platinum using an ion-sputter to a thickness of 10 nm. Finally, the samples were visualized under the FESEM (Model: JEOL JSM7800F) at 5 kV.
The catalytic behavior of the MFC was determined using cyclic voltammetry (CV). The electrocatalytic behavior and electron transfer interactions between the biofilm (biocatalyst) and the anode of the MFC can be characterized by CV analysis. Besides, it helps to elucidate the role of membrane bound cytochromes, mediators and pili in the electrochemical reactions [28]. The CV was conducted using AUTOLAB electrochemical station (USA). The three electrode system was applied in order to collect the CV data where the anode and cathode were used as working and counter electrodes respectively. The (Ag/AgCl, 1.0 M KCl) was used as reference electrode and before plugging in the anode chamber, it was disinfected by 75% ethanol (Sigma). CV was carried out at a scan rate of 30 mV/s between the potential ranges of þ1.0 to 1.0 V (vs Ag/AgCl). Prior to electrochemical measurements, the solution was purged with nitrogen gas for 15 min.
Electrochemical impedance spectroscopy Electrochemical impedance spectroscopy (EIS) experiments were performed using AUTOLAB electrochemical station (USA) in order to evaluate the contribution of internal resistances in MFC. A three electrode system was used to examine the anode electrode where the anode and cathode electrode was used as working and counter electrode respectively. The saturated Ag/AgCl electrode (1.0 M KCl) was used as the reference electrode and it was positioned near to the anode. EIS was measured by applying AC potential (maximum amplitude of 10 mV) in the frequency range of 100 kHze5 mHz to prevent the biofilm detachment as well as to minimize the disturbance on systematic stability. The EIS data were plotted in the form of a Nyquist curve in which the charge transfer resistance (Rct) and ohmic resistance (RU) were calculated by fitting the measured impedance data to an equivalent circuit (EC): R(Q[RW]) using Z view software.
Results and discussion Isolation and characterization of the microorganism The predominant microorganism present in the biofilm was isolated and identified as K. variicola. K. variicola is a facultative non-spore forming, non-motile and fermentative which can be readily isolated from water and soil environments [29,30].
Performance of K. variicola in MFC The electrochemical performance of wild type K. variicola was investigated using MFCA fed with POME (COD ¼ 28,180 mg/L) and inoculated with 1 mL of K. variicola liquid culture. The current generation data was recorded for 26 days as shown in Fig. 1. Generally, the pure cultures takes longer time to attain stable current than that of anaerobic sludge (AS) in MFC [17]. But, in this study, the stable current generation was achieved on 3rd day which might be due to the utilization of wide range substrates. Stable average current generation was observed from 3rd day to 16th day, where maximum average current
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Thus, it can be one of the potential microorganisms in the preparation of effective inoculum for the treatment of wastewater in the MFCs.
Role of time-course biofilm on power generation
Fig. 1 e a) Profile of current generation of MFCA using K. variicola with time under fixed external resistance (1000 U), b) average current value with standard error (inset).
generation was obtained on 11th day (172 mA). However, after 16 days of operation, the current generation started decreasing sharply and after 20 days of operation, it reached to 74 mA and thereafter no significant change was observed. The polarization and power density data at different days of operation were recorded and presented in Fig. 2. On the 3rd day, the maximum power density of 2924 mW/m3 was achieved and after 6 days of operation it reached to 3239 mW/m3. On the 11th day, the maximum power generation achieved was about 4426 mW/m3. But, after 20 days of operation, the maximum power density was drastically dropped to 2475 mW/m3 which was even lower than the 3rd day performance. Besides that, after 26 days of operation, the COD removal efficiency and CE was determined which was about 58% and 63%, respectively. The higher COD removal efficiency (58%) indicating the effective utilization of POME substrates by K. variicola during MFC operation. Moreover, the higher CE (63%) observed in MFCA indicates that a major proportion of the substrates were being used by K. variicola. These results clearly show that K. varricola possess the ability to utilize the POME efficiently and generate substantial power in MFC.
Fig. 2 e Polarization curves of MFCA on different days of operation using K. variicola.
To evaluate the role of biofilm, another cell (MFCB) was operated with similar conditions where similar current generation was observed until 21 days of operation as shown in Fig. 3. On the 22nd day, the carbon brush was taken out from the anode compartment of the MFCB and the biofilm from the surface of the carbon brush was completely removed using the sterile brush in laminar air condition, and finally it was autoclaved for 2 h. The carbon brush was again placed in the anode chamber and continued to run the MFCB. Surprisingly, the current generation went up immediately and on the 22nd day, where the average current generation reached 137 mA which is about 2 times higher than the 1st day (60 mA). The higher current on the 22nd day might be due to the existence of a large number of microorganisms in the bulk solution compared to the 1st day of operation which was evaluated by measuring the absorbance at 600 nm (OD600, data not shown). Several reports have presented a similar trend in the current generation of MFC [31,32]. However, in these studies, the major reason for the decrease in current generation was due to the depletion of substrates, in particular, volatile fatty acids (VFA) whereas in our case the current generation was declined while sufficient substrate (13,526 mg/L) was available in the anode compartment for the microorganisms. Therefore, in order to rule out the possible effect of the substrate as well as VFA on MFC performance, an MFC was operated where the old medium was replaced by the fresh medium (Fig. S1). It was observed that the current generation did not increase after replacing the medium (Fig. S1). Thus, it was hypothesized that the decrease in current generation was due to the thickness of the biofilm because it could have affected the diffusion of substrates into the biofilm resulted in the formation of two layers (outer and inner) in the biofilm [25,33]. The time-course biofilm formation was analyzed using FESEM images of the
Fig. 3 e a) Profile of current generation of MFCB using K. variicola with time under fixed external resistance (1000 U), b) average current value with standard error (inset).
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anode presented in Fig. 4. On the 3rd day, small quantities of loosely attached microorganisms were observed on the electrode surface as shown in Fig. 4b might be enough to achieve stable current generation. It can be seen that, during the initial period, bacteria started to colonize on the electrode surface and consequently, adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substances (EPS) that can be transformed to multilayer biofilm. On the 21st day, multilayer thick biofilm was noticed as shown in Fig. 4c, which caused low power generation. On the 22nd day, the biofilm was removed and after 25 days of operation, bacteria again started to colonize on anode surface (Fig. 4d) resulting in higher power generation. The bacterial viability of the biofilm was analyzed on different days of MFCB operation and presented in Fig. 5. The number of live cells increased with the time until 15th day of operation where live cells outnumbered the dead cells until 11th day. The number of dead cells started rising from 7th day of operation and eventually exceeded the number of live cells by many folds on 21st day while thick biofilm was observed (Fig. 4c). Although we could not analyze the localization of live and dead cells in multilayer biofilm, the previous literature [25] reported that the live cells can exist only at the outer layer of thick biofilm which might be due to the availability of nutrients as shown in schematic diagram (Fig. 6). The biofilm architecture and the electron transfer mechanism have been illustrated in Fig. 6. The initial biofilm comprising with live cells could easily transfer the electron whereas after forming the thick biofilm, electron transfer process of the top layer cells could be interrupted by the dead cell layer thus reducing the electron transfer to the anode. The outer layer with live K. variicola might not have nanowires (not seen in FESEM) or pili because K. variicola has similar physical and biochemical
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Fig. 5 e Cell viability count of anode biofilm on different days of operations.
characteristics as that of K. pneumonia [29] which lacks pili and uses electron shuttle mechanism. Therefore, K. variicola might also be using electron shuttle mechanism for transferring electrons to the anode. But this dead inner layer hindered the electron transfer from the live K. variicola present in the outer layer of the biofilm and bulk solution which caused current densities to be reduced and low power was obtained after 21 days of operation. These results suggest that the thick biofilm is not imperative to get optimum current generation for K. variicola. However, it varies for other microorganisms such as Geobacter sulfurreducens because it usually forms thick biofilm but generates higher current using conductive pili [34]. These results also suggest that, biofilm is a crucial component of MFC that allows considerable conversion
Fig. 4 e FESEM images on anode carbon brush at different days of operation (MFCB), (a) virgin, (b) 3rd day, (c) 21st day and (d) 25th day.
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Fig. 6 e Schematic diagram of electron transfer mechanism in monolayer and multilayer biofilm.
capacity and opportunities for extracellular electron transfer [35]. However, the increase in the thick inactive biofilm layer over time could reduce the electricity generation which might be due to the less electrical conductivity of the dead biofilm compared to the viable biofilm, the need of greater diffusion path for the transport of electrons from live cells to the surface and the depletion of the substrates at the electrode surface [25,36].
Electrochemical characterization of biofilm CV analysis CV was conducted to investigate the electron transfer mechanism in the biofilm. The CV was performed before inoculation and after inoculation (3rd, 7th, 11th and 21st day of operation) as presented in Fig. 7. It can be seen that before the inoculation (virgin), no redox peak was noticed during either the forward or reverse scan in the controlled electrochemical cell whereas, on the 3rd day, small redox peak was observed which was not initially detected in the CV curve. Thus it is indicating that the electrochemical activity was primarily due to the redox mediators excreted from the microorganisms. Moreover, the formation of reversible redox couples confirmed the presence of mediators that reversibly oxidized and reduced during CV analysis [14]. Zhang et al. [37] also observed similar CV peak for K. pneumonia L17 suggesting that K. variicola could also have transferred the electrons through the similar redox mechanisms. The electrochemical activity of K. varricola behaved differently during different stages of operation, indicating the growth of suspension cells or adherence of suspension cells onto the anode to form the biofilm in the MFC [38]. Therefore, on the 7th day, intense redox peak was observed which can be attributed to the higher active biomass (live cells, Fig. 5) within the biofilm.
While on the 11th day, a more intense redox peak was detected, indicating that the mature and effective biofilm was formed hence it facilitated the extracellular electron transfer (EET) between microbes and anode with minimum diffusion path [18]. In addition, effective biofilm comprising with predominant active microorganisms (Fig. 5) produced more redox compounds which enhanced the electron transfer efficiency thereby increased the current generation. However, on the 21st day, redox peak intensity was reduced compared to day 11, which might be due to the formation of thick ineffective biofilm (Fig. 4c) comprising a higher number of dead cells than live cells. Consequently, the quantity of redox mediators reduced which in turn affected the performance of MFC. Besides that, the thick ineffective biofilm (Fig. 4c) caused a
Fig. 7 e Cyclic voltammograms for the anode on different days of operation.
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spatial obstruction which resulted in diffusion limitation between the solution and the anode [39] thus achieved lower performance on the 21st day.
Electrochemical impedance spectroscopy Nyquist plots for the anode configuration on different days (day 3, 7, 11, 21 and 25) of MFC operation are shown in Fig. 8. The equivalent circuit EC: R(Q[RW]) was used to fit the impedance spectra of anode MFC where Rohm, Rct, Q and W represent the ohmic resistance, charge transfer resistance, constant phase element and diffusion resistance, respectively [40]. It can be observed that the EIS data were fitted well with the afore-mentioned equivalent circuit model. The higher Rct (148 U) on the 3rd day might be due to the presence of fewer microbes on the anode surface whereas on 7th day 31% reduction in Rct was achieved which could be due to the presence of live bacteria in the form of initial biofilm. On the 11th day, the Rct dropped drastically by ~89% compared to the 3rd day, suggesting the formation of effective biofilm. The effective biofilm was meant by the high coverage of the anode surface with a predominant number (~2.5 times) of live cells (Fig. 5) that enhanced the kinetics of the bio-electrochemical reaction by decreasing the anode activation losses [41]. But, a drastic increase in Rct (~422%) on 21st day revealed the formation of multilayer biofilm on anode by predominant dead cells as illustrated in Fig. 6. The dead cells in the thick biofilm might have hindered the EET process which resulted in increased charge transfer resistance [42]. It can be seen that on the 25th day, lower Rct (49.83) was obtained compared to the 21st day which could be due to the removal of nonconductive thick biofilm layer after 21 days of operation. These results suggest that the Rct is proportional to the number of live cells directly attached to the anode surface (thin effective biofilm). The anode diffusion resistances on 3rd, 7th, 11th, 21st and 25th were found to be 0.0048 U, 1.30 U, 116.7 U, 141 U and 117 U respectively. Initially (3rd day), the diffusion resistance (0.0048U) found very less possibly owing to the mediators (electron shuttles) produced by the microorganisms in the bulk because these mediators could easily travel to the surface
Fig. 8 e a) Fitting results of MFC Nyquist plots at different days of operation, b) magnified high frequency region of Nyquist plots (inset).
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of the electrode without any hindrance due to the absence of biofilm. Diffusion resistance increased until 21st day due to the thickness of biofilm increased with the time which affected the diffusion of substrates into the biofilm thus significantly increased the diffusion resistance [43]. Moreover, the electron needed greater diffusion path to reach electrode due to the formation of thick biofilm [5]. Interestingly, after removal of thick biofilm (on 22nd day), on the 25th day, it reduced by 17% compared to the 21st day. Total internal resistance accounted on MFC at different days of operations is shown in Fig. 9. On the 3rd day, the total internal resistance was observed as 150.83 U and it decreased to 110.01 U until 7 days of operation, thereafter no significant changes were observed until 11th day. However, on the 21st day, very high internal resistance (916.57) was found which was mainly due to the very high Rct (773.3 U). Interestingly, after the removal of biofilm from the anode, the total resistance was decreased again by 81.39%. It can be seen that, the total internal resistance trend is completely consistent with the power generation trend. Besides, in MFCs, among the different types of electron transfer mechanisms, the direct and mediated electron transfer mechanisms are reported as primary mechanisms. The direct electron transfer (DET) takes place through the outer membrane cytochrome, nanowires, or trans-membrane electron transport proteins [44]. Whereas, the mediated electron transfer (MET) takes place with the help of an electron shuttle mediator, in such a case the efficiency of electron transfer significantly enhanced [18,45]. Generally, identical bacterial genus accustomed similar electron transfer mechanism. Although Shewanella sp. could transfer electron through the soluble electron shuttles, electron conducting pili and other mechanisms, most of the reports have shown that predominant electron transfer occurs trough the nanowire (pili) for this genus. For example, Peng et al. [46] and Liu et al. [47] reported that Shewanella oneidensis and Shewanella loihica uses direct electron transfer mechanism (through the nanowire). Whereas, Pseudomonas spp., such as Pseudomonas aeruginosa [23] and Pseudomonas alcaliphila [48] predominantly uses the similar electron shuttle mechanism. Therefore, since
Fig. 9 e Total internal resistance of anode at different days of operation.
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K. variicola is very similar to K. pneumoniae in both phenotypic and biochemical characteristics [19] that use electron shuttle (2,6 DTBBQ) mechanism for electron transfer, it might also use similar electron shuttle mechanism. However, further study is needed to elucidate the electron transfer mechanism of K. variicola and the redox compound produced by it.
Conclusions In this study, a wild type K. variicola was employed to enhance the power generation in dual chamber MFC operated with POME. The performance of MFC was analyzed using polarization measurements. It was found that the MFC operated with K. variicola can exhibit maximum power density of about 4.5 W/m3 which is potentially higher than the commonly used anaerobic sludge as inoculum. The time-course architecture of biofilm was analyzed and correlated with power generation, CV and EIS which revealed that thick biofilm could negatively impact on MFC performance due to the accumulation of outnumbering dead cells at the vicinity of electrode surface that increased the Rct as well as diffusion path of the electron. Moreover, the CV peak (reversible) confirmed that K. variicola produced mediator compounds that reduced the Rct thereby enhanced the electron transfer efficiency in the anode of MFCs. The maximum COD removal efficiency and coulombic efficiency were achieved as 58% and 63% respectively which ascribed that it can utilize various fuels ranging from simple to complex substrates. Therefore, it can be one of the potential microorganisms in the preparation of effective inoculum for the treatment of wastewater in the MFCs. However, further studies are needed to elucidate the electron transfer mechanism and substrate utilization kinetics of K. variicola. It can be concluded that electroactive and metabolic biofilm with outnumbered live cells is indispensable to achieve higher performance in MFCs.
Acknowledgements This work was supported by the University Malaysia Pahang, Malaysia (RDU 140322 and GRS 150371).
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ijhydene.2017.08.193.
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[21] Mishra P, Thakur S, Singh L, Krishnan S, Sakinah M, Ab Wahid Z. Fermentative hydrogen production from indigenous mesophilic strain Bacillus anthracis PUNAJAN 1 newly isolated from palm oil mill effluent. Int J Hydrogen Energy 2017;42:16054e63. [22] Franks AE, Malvankar N, Nevin KP. Bacterial biofilms: the powerhouse of a microbial fuel cell. Biofuels 2010;1:589e604. [23] Kumar R, Singh L, Zularisam A. Exoelectrogens: recent advances in molecular drivers involved in extracellular electron transfer and strategies used to improve it for microbial fuel cell applications. Renew Sustain Energy Rev 2016;56:1322e36. [24] Sun D, Chen J, Huang H, Liu W, Ye Y, Cheng S. The effect of biofilm thickness on electrochemical activity of Geobacter sulfurreducens. Int J Hydrogen Energy 2016;41:16523e8. [25] Sun D, Cheng S, Wang A, Li F, Logan BE, Cen K. Temporalspatial changes in viabilities and electrochemical properties of anode biofilms. Environ Sci Technol 2015;49:5227e35. [26] Baranitharan E, Khan MR, Prasad D, Salihon JB. Bioelectricity generation from palm oil mill effluent in microbial fuel cell using polyacrylonitrile carbon felt as electrode. Water Air Soil Pollut 2013;224:1e11. [27] Ren Z, Ramasamy RP, Cloud-Owen SR, Yan H, Mench MM, Regan JM. Time-course correlation of biofilm properties and electrochemical performance in single-chamber microbial fuel cells. Bioresour Technol 2011;102:416e21. [28] Varanasi JL, Nayak AK, Sohn Y, Pradhan D, Das D. Improvement of power generation of microbial fuel cell by integrating tungsten oxide electrocatalyst with pure or mixed culture biocatalysts. Electrochim Acta 2016;199:154e63. nchez J, Martı´nez-Romero E, [29] Garza-Ramos U, Silva-Sa Tinoco P, Pina-Gonzales M, Barrios H, et al. Development of a multiplex-PCR probe system for the proper identification of Klebsiella variicola. BMC Microbiol 2015;15:1. [30] Suzuki T, Nishikawa C, Seta K, Shigeno T, NakajimaKambe T. Ethanol production from glycerol-containing biodiesel waste by Klebsiella variicola shows maximum productivity under alkaline conditions. New Biotechnol 2014;31:246e53. [31] Friman H, Schechter A, Ioffe Y, Nitzan Y, Cahan R. Current production in a microbial fuel cell using a pure culture of Cupriavidus basilensis growing in acetate or phenol as a carbon source. Microb Biotechnol 2013;6:425e34. [32] Wang Z, Lee T, Lim B, Choi C, Park J. Microbial community structures differentiated in a single-chamber air-cathode microbial fuel cell fueled with rice straw hydrolysate. Biotechnol Biofuels 2014;7:1. [33] Yates MD, Siegert M, Logan BE. Hydrogen evolution catalyzed by viable and non-viable cells on biocathodes. Int J Hydrogen Energy 2014;39:16841e51.
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[34] Malvankar NS, Vargas M, Nevin KP, Franks AE, Leang C, Kim BC, et al. Tunable metallic-like conductivity in microbial nanowire networks. Nat Nanotechnol 2011;6:573e9. [35] Read ST, Dutta P, Bond PL, Keller J, Rabaey K. Initial development and structure of biofilms on microbial fuel cell anodes. BMC Microbiol 2010;10:1. [36] Liu Y, Kim H, Franklin R, Bond DR. Gold line array electrodes increase substrate affinity and current density of electricityproducing G. sulfurreducens biofilms. Energy Environ Sci 2010;3:1782e8. [37] Zhang L, Zhou S, Zhuang L, Li W, Zhang J, Lu N, et al. Microbial fuel cell based on Klebsiella pneumoniae biofilm. Electrochem Commun 2008;10:1641e3. [38] Rabaey K, Boon N, Siciliano SD, Verhaege M, Verstraete W. Biofuel cells select for microbial consortia that self-mediate electron transfer. Appl Environ Microbiol 2004;70:5373e82. [39] Nimje VR, Chen C-Y, Chen C-C, Jean J-S, Reddy AS, Fan C-W, et al. Stable and high energy generation by a strain of Bacillus subtilis in a microbial fuel cell. J Power Sources 2009;190:258e63. [40] He Z, Wagner N, Minteer SD, Angenent LT. An upflow microbial fuel cell with an interior cathode: assessment of the internal resistance by impedance spectroscopy. Environ Sci Technol 2006;40:5212e7. [41] Ramasamy RP, Ren Z, Mench MM, Regan JM. Impact of initial biofilm growth on the anode impedance of microbial fuel cells. Biotechnol Bioeng 2008;101:101e8. [42] Islam MA, Woon CW, Ethiraj B, Cheng CK, Yousuf A, Khan MMR. Ultrasound driven biofilm removal for stable power generation in microbial fuel cell. Energy Fuels 2016;31:968e76. [43] Picioreanu C, Head IM, Katuri KP, van Loosdrecht MC, Scott K. A computational model for biofilm-based microbial fuel cells. Water Res 2007;41:2921e40. [44] Sayed ET, Barakat NA, Abdelkareem MA, Fouad H, Nakagawa N. Yeast extract as an effective and safe mediator for the Baker's-yeast-based microbial fuel cell. Ind Eng Chem Res 2015;54:3116e22. [45] Hubenova YV, Rashkov RS, Buchvarov VD, Arnaudova MH, Babanova SM, Mitov MY. Improvement of yeast biofuel cell output by electrode modifications. Ind Eng Chem Res 2010;50:557e64. [46] Peng L, You S-J, Wang J-Y. Carbon nanotubes as electrode modifier promoting direct electron transfer from Shewanella oneidensis. Biosens Bioelectron 2010;25:1248e51. [47] Liu X, Wu W, Gu Z. Poly (3, 4-ethylenedioxythiophene) promotes direct electron transfer at the interface between Shewanella loihica and the anode in a microbial fuel cell. J Power Sources 2015;277:110e5. [48] Zhang T, Zhang L, Su W, Gao P, Li D, He X, et al. The direct electrocatalysis of phenazine-1-carboxylic acid excreted by Pseudomonas alcaliphila under alkaline condition in microbial fuel cells. Bioresour Technol 2011;102:7099e102.
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Correlation of power generation with time-course biofilm architecture using Klebsiella variicola in dual chamber microbial fuel cell M. Amirul Islam a, Chee Wai Woon a, Baranitharan Ethiraj a, Chin Kui Cheng a,c, Abu Yousuf b, Md. Maksudur Rahman Khan a,c,* a
Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang, Malaysia b Faculty of Engineering Technology, Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang, Malaysia c Centre of Excellence for Advanced Research in Fluid Flow (CARIFF), University Malaysia Pahang, 26300 Kuantan, Pahang, Malaysia
article info
abstract
Article history:
In the present work, the wild type Klebsiella variicola was investigated in double chamber
Received 11 June 2017
microbial fuel cell (MFC) using palm oil mill effluent as substrate which achieved high
Received in revised form
power density (4.5 W/m3) and coulombic efficiency (63%) while maintaining the moderate
4 August 2017
chemical oxygen demand (COD) removal efficiency (58%). The effect of biofilm formation
Accepted 28 August 2017
on power generation over time was also evaluated and found that an effective biofilm with
Available online 19 September 2017
the discrete distribution of single layer microorganisms can produce high power corresponding to low charge transfer resistance. The growth of biofilm in multilayers consisting
Keywords:
of outnumbered dead cells in the vicinity of the electrode surface caused the polarization
Microbial fuel cell
resistance and diffusion resistance resulting in a sharp drop in the current generation. The
Klebsiella variicola
removal of multilayer biofilm from the anode surface positively influenced the cell per-
Biofilm architecture
formance which led to a rapid increase in current generation and thus revealed that
Palm oil mill effluent
effective biofilm predominated by live cells can be an emergent factor for achieving
Cell viability
maximum performance in MFC. © 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction In bio-electrochemical systems (BES), especially in microbial fuel cells (MFCs), the bio-catalytic activity of microorganisms plays a crucial role in enhancing the power generation of system [1,2]. In the past decade, a number of new strains of microorganisms have been reported which
can generate electrical current through MFCs [3]. However, only a few strains are capable of producing significant power using complex wastewater as a substrate [4,5]. MFCs are being considered to be a promising sustainable technology to combat the energy crisis, especially using wastewaters as substrates, because it can simultaneously generate electricity and accomplish wastewater treatment [5,6]. Moreover, it is reported that the energy efficiencies of
* Corresponding author. Faculty of Chemical and Natural Resources Engineering, Universiti Malaysia Pahang, Lebuhraya Tun Razak, 26300 Gambang, Kuantan, Pahang, Malaysia. E-mail addresses: [email protected], [email protected] (Md.M.R. Khan). http://dx.doi.org/10.1016/j.ijhydene.2017.08.193 0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
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common renewable energy sources such as wind turbine (15e40%) [7] and biogas plant (5e20.7%) [8] are lower compare to the MFC (77.6%) [9]. Besides, MFCs can also be able to utilize a variety of substrates ranging from simple to complex. Wide varieties of simple substrates such as glucose, acetate etc. [10] and complex organic substrates such as industrial and domestic [11,12] wastewater have been tested so far in MFC. Anaerobic sludge (mixed culture) is one of the most widely used inoculum in the MFC for electricity generation and treatment of wastewater [13]. However, the sludge, which is not confined to a specific location, raises the difficulty to reproduce the electricity generation. In addition, when using mixed microbial cultures, it becomes hard to determine the mechanisms and roles of the individual microorganisms contributing to power generation. MFCs operated with the complex substrates using mixed microbial cultures usually showed high power densities compared to pure cultures [14] because, most of the pure cultures are not able to utilize the wide range of substrates [15]. So far, only a few pure cultures have been used in MFC operated with wastewater. Li et al. [16] and Nor et al. [17] have reported that pure cultures of Pseudomonas aeruginosa ZH1 and Shewanella oneidensis produced 4.45 W/m3 and 0.784 W/m3 using POME and municipal wastewater respectively. Very recently, Klebsiella variicola (K. varricola) was described as a new bacterial species and found to be more closely related to an electrogen Klebsiella pneumoniae [18] which can able to produce 2,6 DTBBQ electron shuttle in MFC [19]. Since the phenotypic and biochemical characteristics of K. varricola are identical to K. pneumoniae, it might also have electrogenic properties. Moreover, K. varricola can able to consume various kinds of substrates ranging from simple to complex [18]. Palm Oil Mill Effluent (POME) is one of the major wastewater sources in Malaysia comprising with amino acids, short fibres, nitrogenous constituents, free organic acids and a mixture of carbohydrates ranging from hemicelluloses to simple sugars [20,21]. Since K. varricola can utilize a diverse range of substrates; it might also be able to utilize POME efficiently, which has not been investigated so far in MFC. Biofilm is considered as a power house [22] that inevitably formed on the anode surface during MFC operation. In MFC, it is imperative if electroactive biofilms are produced that have the ability to respire the terminal electrons from metabolism onto electrode surfaces [23]. However, the thickness of biofilm is a crucial factor for MFC performance [24]. Thick biofilm with predominant dead cells accumulation at the vicinity of the electrode surface could negatively influence the power density by increasing the charge transfer and diffusion resistances [25]. Therefore, an optimum thickness is favourable for achieving substantial current densities [23]. In this research, the performance of wild type pure K. varricola was investigated in double chamber MFC operated with POME and the electricity production was interpreted with biofilm architecture comprising with live and dead cells. The power generation as a function of time was correlated with the electrochemical impedance data to elucidate the contribution of the resistances involved in the MFC.
Materials and methods Sample collection and characterization POME samples were collected from Panching palm oil mill (FELDA) located in Kuantan, Pahang, Malaysia. The samples were collected before the effluent discharge into the mixing pond at about 80e90 C. The collected samples were filtered using Whatman no.1 filter paper (2.5 mm) and the filtrate was autoclaved at 121 C, 15 psi for 15 min. Municipal wastewater (MWW) was collected from drainage discharge point of Kuantan city, Malaysia and filtered using Whatman filter paper and the filtrate was used as primary inoculum.
Isolation and characterization of microorganism The filtered MWW was serially diluted (101e106) and the pure culture bacteria were obtained from 106 dilution using the spread plate technique. The enrichment of the cultures was carried out by preparing an overnight culture in LB (Luria Bertani) broth (10% v/v) and incubated at 37 C with shaking at 150 rpm. Several pure culture strains were identified using Biolog gen III analysis but only Klebsiella spp. was selected for further identification and used as inoculum in MFCs. The strain characterization by PCR and sequencing has been described by Islam et al. [18].
Fabrication and operation of MFC The dual chamber MFCA and MFCB were fabricated using a cubic plexi glass (5 cm 5 cm x 5 cm) with the working volume of 20 mL (Shanghai, Sunny Scientific, China). A small brush with 2.5 cm in outer diameter and 2.5 cm long was used as anode and cathode electrode for all the experiments. The electrodes were cleaned with 1.0 M NaOH and subsequently with 1.0 M HCl after each experiment and stored in distilled water before use. The anode and cathode compartments were separated using a cation exchange membrane (Nafion 117, Dupont Co., USA). Prior to use, the cation exchange membrane was pretreated using dilute HCl for 1 h and thereafter it was washed with DI (De-ionized) water several times. After that, the whole set up was tightened with the screws. The 20 mL of sterilized POME (POME/water volume ratio 1:1) was poured into the anode compartment and subsequently inoculated with pure culture bacteria (1 mL) while the cathode chamber was filled with KMnO4 solution (20 mL), as an oxidizing agent.
Measurement and analyses Polarization measurements were performed at regular intervals (15 min) to estimate the power production of MFC at various external resistances ranging from 50 to 20,000U using an external resistor. The voltage data were taken using a digital multimeter with built in data logger (Fluke 289 true RMS industrial logging digital multimeter, USA) after it reached the stable value for obtaining the polarization curves. Power density normalized by volume (PV, W/m3) was calculated using the following equation.
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Cyclic voltammetry analysis V2 Pv ¼ vR
(1)
where, V is the output voltage, v is the volume and R is the resistance. The COD removal efficiency and columbic efficiency of dual chamber MFC was calculated as described by Baranitharan et al. [26]. The COD was determined using a COD reactor (HACH DRB 200, USA). The COD removal efficiency was calculated using Eq. (2) COD removal efficiency ¼
CODi CODt 100% CODi
(2)
where, CODi and CODt represent the initial COD (mg/L) of the anode chamber and the COD of the anode chamber at a particular time. The columbic efficiency (CE) was calculated using Eq. (3) Z 8 CE ¼
Idt
F$Van DCOD
(3)
where, I is the current (A), F is the Faraday's constant (96,485 C/mol), and Van is the anode volume (L). D 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 oxygen (32 g/mol) and assuming that 4 electrons exchanged per mole of oxygen.
Cell viability count of biofilm Fluorescence microscope (Olympus BX53, Germany) with 20X objective lens was employed to determine the cell viability with the help of LIVE/DEAD Bacterial Viability Kit (BD™ Cell Viability Kit). In brief, cell viability of biofilm was performed by detruncating small part (1 cm) of the anode using a sterilized scissor and then immediately immersed in the 50 mM phosphate buffer. In order to separate the microorganisms from carbon brush, it was centrifuged at 5000 rpm for 1 min. Thereafter, the cell suspension was serially diluted and stained using a viability staining kit. Finally, a 0.4 mm membrane filter was used to filter the stained cells and the cells were then counted using a microscope. The cell density was calculated per anode geometric area using the dilution factor, the filtered volume and the ratio of total filtered area to image area [27].
FESEM analysis of the biofilm Field emission scanning electron microscopy (FESEM) was used to visualize the anode biofilm at different time intervals of the anode. Small parts of the anode with biofilm was cut off and rinsed with sterile medium followed by immediate soaking. After that, the samples were immediately soaked into anaerobic solution (3% glutaraldehyde). The sample was then washed twice using 0.1 M phosphate buffer and dehydrated by successive 10 min incubations with different dilution of ethanol concentrations (40%, 60%, 80%, 90% and 100%). The samples were dried at room temperature (27 ± 2 C) and coated with platinum using an ion-sputter to a thickness of 10 nm. Finally, the samples were visualized under the FESEM (Model: JEOL JSM7800F) at 5 kV.
The catalytic behavior of the MFC was determined using cyclic voltammetry (CV). The electrocatalytic behavior and electron transfer interactions between the biofilm (biocatalyst) and the anode of the MFC can be characterized by CV analysis. Besides, it helps to elucidate the role of membrane bound cytochromes, mediators and pili in the electrochemical reactions [28]. The CV was conducted using AUTOLAB electrochemical station (USA). The three electrode system was applied in order to collect the CV data where the anode and cathode were used as working and counter electrodes respectively. The (Ag/AgCl, 1.0 M KCl) was used as reference electrode and before plugging in the anode chamber, it was disinfected by 75% ethanol (Sigma). CV was carried out at a scan rate of 30 mV/s between the potential ranges of þ1.0 to 1.0 V (vs Ag/AgCl). Prior to electrochemical measurements, the solution was purged with nitrogen gas for 15 min.
Electrochemical impedance spectroscopy Electrochemical impedance spectroscopy (EIS) experiments were performed using AUTOLAB electrochemical station (USA) in order to evaluate the contribution of internal resistances in MFC. A three electrode system was used to examine the anode electrode where the anode and cathode electrode was used as working and counter electrode respectively. The saturated Ag/AgCl electrode (1.0 M KCl) was used as the reference electrode and it was positioned near to the anode. EIS was measured by applying AC potential (maximum amplitude of 10 mV) in the frequency range of 100 kHze5 mHz to prevent the biofilm detachment as well as to minimize the disturbance on systematic stability. The EIS data were plotted in the form of a Nyquist curve in which the charge transfer resistance (Rct) and ohmic resistance (RU) were calculated by fitting the measured impedance data to an equivalent circuit (EC): R(Q[RW]) using Z view software.
Results and discussion Isolation and characterization of the microorganism The predominant microorganism present in the biofilm was isolated and identified as K. variicola. K. variicola is a facultative non-spore forming, non-motile and fermentative which can be readily isolated from water and soil environments [29,30].
Performance of K. variicola in MFC The electrochemical performance of wild type K. variicola was investigated using MFCA fed with POME (COD ¼ 28,180 mg/L) and inoculated with 1 mL of K. variicola liquid culture. The current generation data was recorded for 26 days as shown in Fig. 1. Generally, the pure cultures takes longer time to attain stable current than that of anaerobic sludge (AS) in MFC [17]. But, in this study, the stable current generation was achieved on 3rd day which might be due to the utilization of wide range substrates. Stable average current generation was observed from 3rd day to 16th day, where maximum average current
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Thus, it can be one of the potential microorganisms in the preparation of effective inoculum for the treatment of wastewater in the MFCs.
Role of time-course biofilm on power generation
Fig. 1 e a) Profile of current generation of MFCA using K. variicola with time under fixed external resistance (1000 U), b) average current value with standard error (inset).
generation was obtained on 11th day (172 mA). However, after 16 days of operation, the current generation started decreasing sharply and after 20 days of operation, it reached to 74 mA and thereafter no significant change was observed. The polarization and power density data at different days of operation were recorded and presented in Fig. 2. On the 3rd day, the maximum power density of 2924 mW/m3 was achieved and after 6 days of operation it reached to 3239 mW/m3. On the 11th day, the maximum power generation achieved was about 4426 mW/m3. But, after 20 days of operation, the maximum power density was drastically dropped to 2475 mW/m3 which was even lower than the 3rd day performance. Besides that, after 26 days of operation, the COD removal efficiency and CE was determined which was about 58% and 63%, respectively. The higher COD removal efficiency (58%) indicating the effective utilization of POME substrates by K. variicola during MFC operation. Moreover, the higher CE (63%) observed in MFCA indicates that a major proportion of the substrates were being used by K. variicola. These results clearly show that K. varricola possess the ability to utilize the POME efficiently and generate substantial power in MFC.
Fig. 2 e Polarization curves of MFCA on different days of operation using K. variicola.
To evaluate the role of biofilm, another cell (MFCB) was operated with similar conditions where similar current generation was observed until 21 days of operation as shown in Fig. 3. On the 22nd day, the carbon brush was taken out from the anode compartment of the MFCB and the biofilm from the surface of the carbon brush was completely removed using the sterile brush in laminar air condition, and finally it was autoclaved for 2 h. The carbon brush was again placed in the anode chamber and continued to run the MFCB. Surprisingly, the current generation went up immediately and on the 22nd day, where the average current generation reached 137 mA which is about 2 times higher than the 1st day (60 mA). The higher current on the 22nd day might be due to the existence of a large number of microorganisms in the bulk solution compared to the 1st day of operation which was evaluated by measuring the absorbance at 600 nm (OD600, data not shown). Several reports have presented a similar trend in the current generation of MFC [31,32]. However, in these studies, the major reason for the decrease in current generation was due to the depletion of substrates, in particular, volatile fatty acids (VFA) whereas in our case the current generation was declined while sufficient substrate (13,526 mg/L) was available in the anode compartment for the microorganisms. Therefore, in order to rule out the possible effect of the substrate as well as VFA on MFC performance, an MFC was operated where the old medium was replaced by the fresh medium (Fig. S1). It was observed that the current generation did not increase after replacing the medium (Fig. S1). Thus, it was hypothesized that the decrease in current generation was due to the thickness of the biofilm because it could have affected the diffusion of substrates into the biofilm resulted in the formation of two layers (outer and inner) in the biofilm [25,33]. The time-course biofilm formation was analyzed using FESEM images of the
Fig. 3 e a) Profile of current generation of MFCB using K. variicola with time under fixed external resistance (1000 U), b) average current value with standard error (inset).
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anode presented in Fig. 4. On the 3rd day, small quantities of loosely attached microorganisms were observed on the electrode surface as shown in Fig. 4b might be enough to achieve stable current generation. It can be seen that, during the initial period, bacteria started to colonize on the electrode surface and consequently, adherent cells are frequently embedded within a self-produced matrix of extracellular polymeric substances (EPS) that can be transformed to multilayer biofilm. On the 21st day, multilayer thick biofilm was noticed as shown in Fig. 4c, which caused low power generation. On the 22nd day, the biofilm was removed and after 25 days of operation, bacteria again started to colonize on anode surface (Fig. 4d) resulting in higher power generation. The bacterial viability of the biofilm was analyzed on different days of MFCB operation and presented in Fig. 5. The number of live cells increased with the time until 15th day of operation where live cells outnumbered the dead cells until 11th day. The number of dead cells started rising from 7th day of operation and eventually exceeded the number of live cells by many folds on 21st day while thick biofilm was observed (Fig. 4c). Although we could not analyze the localization of live and dead cells in multilayer biofilm, the previous literature [25] reported that the live cells can exist only at the outer layer of thick biofilm which might be due to the availability of nutrients as shown in schematic diagram (Fig. 6). The biofilm architecture and the electron transfer mechanism have been illustrated in Fig. 6. The initial biofilm comprising with live cells could easily transfer the electron whereas after forming the thick biofilm, electron transfer process of the top layer cells could be interrupted by the dead cell layer thus reducing the electron transfer to the anode. The outer layer with live K. variicola might not have nanowires (not seen in FESEM) or pili because K. variicola has similar physical and biochemical
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Fig. 5 e Cell viability count of anode biofilm on different days of operations.
characteristics as that of K. pneumonia [29] which lacks pili and uses electron shuttle mechanism. Therefore, K. variicola might also be using electron shuttle mechanism for transferring electrons to the anode. But this dead inner layer hindered the electron transfer from the live K. variicola present in the outer layer of the biofilm and bulk solution which caused current densities to be reduced and low power was obtained after 21 days of operation. These results suggest that the thick biofilm is not imperative to get optimum current generation for K. variicola. However, it varies for other microorganisms such as Geobacter sulfurreducens because it usually forms thick biofilm but generates higher current using conductive pili [34]. These results also suggest that, biofilm is a crucial component of MFC that allows considerable conversion
Fig. 4 e FESEM images on anode carbon brush at different days of operation (MFCB), (a) virgin, (b) 3rd day, (c) 21st day and (d) 25th day.
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Fig. 6 e Schematic diagram of electron transfer mechanism in monolayer and multilayer biofilm.
capacity and opportunities for extracellular electron transfer [35]. However, the increase in the thick inactive biofilm layer over time could reduce the electricity generation which might be due to the less electrical conductivity of the dead biofilm compared to the viable biofilm, the need of greater diffusion path for the transport of electrons from live cells to the surface and the depletion of the substrates at the electrode surface [25,36].
Electrochemical characterization of biofilm CV analysis CV was conducted to investigate the electron transfer mechanism in the biofilm. The CV was performed before inoculation and after inoculation (3rd, 7th, 11th and 21st day of operation) as presented in Fig. 7. It can be seen that before the inoculation (virgin), no redox peak was noticed during either the forward or reverse scan in the controlled electrochemical cell whereas, on the 3rd day, small redox peak was observed which was not initially detected in the CV curve. Thus it is indicating that the electrochemical activity was primarily due to the redox mediators excreted from the microorganisms. Moreover, the formation of reversible redox couples confirmed the presence of mediators that reversibly oxidized and reduced during CV analysis [14]. Zhang et al. [37] also observed similar CV peak for K. pneumonia L17 suggesting that K. variicola could also have transferred the electrons through the similar redox mechanisms. The electrochemical activity of K. varricola behaved differently during different stages of operation, indicating the growth of suspension cells or adherence of suspension cells onto the anode to form the biofilm in the MFC [38]. Therefore, on the 7th day, intense redox peak was observed which can be attributed to the higher active biomass (live cells, Fig. 5) within the biofilm.
While on the 11th day, a more intense redox peak was detected, indicating that the mature and effective biofilm was formed hence it facilitated the extracellular electron transfer (EET) between microbes and anode with minimum diffusion path [18]. In addition, effective biofilm comprising with predominant active microorganisms (Fig. 5) produced more redox compounds which enhanced the electron transfer efficiency thereby increased the current generation. However, on the 21st day, redox peak intensity was reduced compared to day 11, which might be due to the formation of thick ineffective biofilm (Fig. 4c) comprising a higher number of dead cells than live cells. Consequently, the quantity of redox mediators reduced which in turn affected the performance of MFC. Besides that, the thick ineffective biofilm (Fig. 4c) caused a
Fig. 7 e Cyclic voltammograms for the anode on different days of operation.
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spatial obstruction which resulted in diffusion limitation between the solution and the anode [39] thus achieved lower performance on the 21st day.
Electrochemical impedance spectroscopy Nyquist plots for the anode configuration on different days (day 3, 7, 11, 21 and 25) of MFC operation are shown in Fig. 8. The equivalent circuit EC: R(Q[RW]) was used to fit the impedance spectra of anode MFC where Rohm, Rct, Q and W represent the ohmic resistance, charge transfer resistance, constant phase element and diffusion resistance, respectively [40]. It can be observed that the EIS data were fitted well with the afore-mentioned equivalent circuit model. The higher Rct (148 U) on the 3rd day might be due to the presence of fewer microbes on the anode surface whereas on 7th day 31% reduction in Rct was achieved which could be due to the presence of live bacteria in the form of initial biofilm. On the 11th day, the Rct dropped drastically by ~89% compared to the 3rd day, suggesting the formation of effective biofilm. The effective biofilm was meant by the high coverage of the anode surface with a predominant number (~2.5 times) of live cells (Fig. 5) that enhanced the kinetics of the bio-electrochemical reaction by decreasing the anode activation losses [41]. But, a drastic increase in Rct (~422%) on 21st day revealed the formation of multilayer biofilm on anode by predominant dead cells as illustrated in Fig. 6. The dead cells in the thick biofilm might have hindered the EET process which resulted in increased charge transfer resistance [42]. It can be seen that on the 25th day, lower Rct (49.83) was obtained compared to the 21st day which could be due to the removal of nonconductive thick biofilm layer after 21 days of operation. These results suggest that the Rct is proportional to the number of live cells directly attached to the anode surface (thin effective biofilm). The anode diffusion resistances on 3rd, 7th, 11th, 21st and 25th were found to be 0.0048 U, 1.30 U, 116.7 U, 141 U and 117 U respectively. Initially (3rd day), the diffusion resistance (0.0048U) found very less possibly owing to the mediators (electron shuttles) produced by the microorganisms in the bulk because these mediators could easily travel to the surface
Fig. 8 e a) Fitting results of MFC Nyquist plots at different days of operation, b) magnified high frequency region of Nyquist plots (inset).
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of the electrode without any hindrance due to the absence of biofilm. Diffusion resistance increased until 21st day due to the thickness of biofilm increased with the time which affected the diffusion of substrates into the biofilm thus significantly increased the diffusion resistance [43]. Moreover, the electron needed greater diffusion path to reach electrode due to the formation of thick biofilm [5]. Interestingly, after removal of thick biofilm (on 22nd day), on the 25th day, it reduced by 17% compared to the 21st day. Total internal resistance accounted on MFC at different days of operations is shown in Fig. 9. On the 3rd day, the total internal resistance was observed as 150.83 U and it decreased to 110.01 U until 7 days of operation, thereafter no significant changes were observed until 11th day. However, on the 21st day, very high internal resistance (916.57) was found which was mainly due to the very high Rct (773.3 U). Interestingly, after the removal of biofilm from the anode, the total resistance was decreased again by 81.39%. It can be seen that, the total internal resistance trend is completely consistent with the power generation trend. Besides, in MFCs, among the different types of electron transfer mechanisms, the direct and mediated electron transfer mechanisms are reported as primary mechanisms. The direct electron transfer (DET) takes place through the outer membrane cytochrome, nanowires, or trans-membrane electron transport proteins [44]. Whereas, the mediated electron transfer (MET) takes place with the help of an electron shuttle mediator, in such a case the efficiency of electron transfer significantly enhanced [18,45]. Generally, identical bacterial genus accustomed similar electron transfer mechanism. Although Shewanella sp. could transfer electron through the soluble electron shuttles, electron conducting pili and other mechanisms, most of the reports have shown that predominant electron transfer occurs trough the nanowire (pili) for this genus. For example, Peng et al. [46] and Liu et al. [47] reported that Shewanella oneidensis and Shewanella loihica uses direct electron transfer mechanism (through the nanowire). Whereas, Pseudomonas spp., such as Pseudomonas aeruginosa [23] and Pseudomonas alcaliphila [48] predominantly uses the similar electron shuttle mechanism. Therefore, since
Fig. 9 e Total internal resistance of anode at different days of operation.
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K. variicola is very similar to K. pneumoniae in both phenotypic and biochemical characteristics [19] that use electron shuttle (2,6 DTBBQ) mechanism for electron transfer, it might also use similar electron shuttle mechanism. However, further study is needed to elucidate the electron transfer mechanism of K. variicola and the redox compound produced by it.
Conclusions In this study, a wild type K. variicola was employed to enhance the power generation in dual chamber MFC operated with POME. The performance of MFC was analyzed using polarization measurements. It was found that the MFC operated with K. variicola can exhibit maximum power density of about 4.5 W/m3 which is potentially higher than the commonly used anaerobic sludge as inoculum. The time-course architecture of biofilm was analyzed and correlated with power generation, CV and EIS which revealed that thick biofilm could negatively impact on MFC performance due to the accumulation of outnumbering dead cells at the vicinity of electrode surface that increased the Rct as well as diffusion path of the electron. Moreover, the CV peak (reversible) confirmed that K. variicola produced mediator compounds that reduced the Rct thereby enhanced the electron transfer efficiency in the anode of MFCs. The maximum COD removal efficiency and coulombic efficiency were achieved as 58% and 63% respectively which ascribed that it can utilize various fuels ranging from simple to complex substrates. Therefore, it can be one of the potential microorganisms in the preparation of effective inoculum for the treatment of wastewater in the MFCs. However, further studies are needed to elucidate the electron transfer mechanism and substrate utilization kinetics of K. variicola. It can be concluded that electroactive and metabolic biofilm with outnumbered live cells is indispensable to achieve higher performance in MFCs.
Acknowledgements This work was supported by the University Malaysia Pahang, Malaysia (RDU 140322 and GRS 150371).
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ijhydene.2017.08.193.
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