Ultrasound Driven Biofilm Removal for Stable Power Generation in

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Ultrasound driven biofilm removal for stable power generation in microbial fuel cell M Amirul Islam, Chee Wai Woon, Baranitharan Ethiraj, Chin Kui Cheng, Abu Yousuf, and Md. Maksudur Rahman Khan Energy Fuels, Just Accepted Manuscript • DOI: 10.1021/acs.energyfuels.6b02294 • Publication Date (Web): 01 Dec 2016 Downloaded from http://pubs.acs.org on December 6, 2016

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Ultrasound driven biofilm removal for stable power generation in microbial fuel cell

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M Amirul Islam , Chee Wai Woon , Baranitharan Ethiraj , Chin Kui Cheng ¥£ Rahman Khan *

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, Abu Yousuf , Md Maksudur

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Anodic biofilm plays a crucial role in bioelectrochemical system to make it sustain for long term performance. However, the accumulation of dead cells over time within the anode biofilm can be particularly detrimental for current generation. In this study, the effect of ultrasound on anode biofilm thickness was investigated in microbial fuel cell (MFC). Ultrasonic treatment was employed for different duration to evaluate its ability to control the thickness of biofilm for maintaining the stable power generation. Cell viability count and field emission scanning electron microscopy (FESEM) analysis of the biofilms over time showed that the number of dead cells increased with the increase of biofilm thickness, and eventually exceeded the number of live cells by many folds. Electrochemical impedance spectroscopy (EIS) analysis indicated that the high polarization resistance was appeared due to the dead layer formation, thus the catalytic efficiency was reduced in MFCs. The stable power generation was achieved by employing ultrasonic treatment for 30 min on every 6 days with some initial exception. The low frequency ultrasound treatment successfully dislodged the ineffective biofilm from the surface of the anode. Moreover, the ultrasound could increase the mass transfer rate of the nutrients and cellular wastes through the biofilm leading to the increase in the cell growth. Therefore, ultrasonic treatment is verified as an efficient method to control the thickness of biofilm as well as to enhance the cell viability in biofilm thereby maintain the stable power generation in the MFC.

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1. Introduction

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Microbial fuel cell (MFC) is a type of bioelectrochemical system (BES) that can convert the energy 1 available in a substrate directly into electricity through the catalytic activity of electroactive bacteria . MFCs have been one of the most studied systems for applications in electricity generation as well as in 2 wastewater treatment compared to other bioelectrochemical systems, because it can simultaneously accomplish both energy and environmental issues. In MFCs, electrogenic bacteria evolved with the ability to completely oxidize the organic compounds to carbon dioxide with an electrode serving as the sole 3 electron acceptor . However, the requirement for most of the electrogens to establish contact with the fuel cell anode to transfer electrons for the production of electricity could potentially be a limiting factor in 4 power production for MFCs . Although research on MFCs has experienced a significant development in recent years, but still numerous studies are reporting unstable and deteriorated performance of MFCs 5 during long-term operation, especially those fuelled with real wastes . Generally, the biofilm formation is detrimental since it causes biofouling on membrane bioreactors (MBR) as well as in other biological wastewater treatment systems. However, unlike other process, in MFCs, biofilm is considered as the power house due to the presence of high cell density through which

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

Faculty of Engineering Technology, University Malaysia Pahang, 26300 Kuantan, Malaysia

£

Centre of Excellence for advancement Research Fluid Flow (CARIFF), University Malaysia Pahang, 26300 Kuantan, Malaysia *Corresponding Author: Email: [email protected], [email protected], Tel: +6095492872

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predominant electron transfer occurs with minimum diffusion path . In presence of nutrients, microorganisms naturally can able to grow perpendicularly and horizontally through the extracellular polymorphic substances and over the time they form a thick layer of biofilm. However, in case of thick biofilm formation, the inner layer might experience the lack of nutrient supply and eventually it forms a 7 dead layer at the proximity of the surface, can be called as electrode fouling in MFC which may 8 negatively influence the performance of MFC. Sun, et al. reported that during biofilm development, the live cells solely responsible for current generation tend to accumulate in the outer layer, whereas dead cells continued to accumulate in the vicinity of the electrode surface and in the long run it eventually forms a thick biofilm. Moreover, the release of protons within the biofilm caused low pH generation near the anode surface which would have limited the metabolism in this zone, thus facilitated the dead cell 9 accumulation and it in turn reduced the power generation . Therefore, the optimal biofilm thickness by removing cell clogging is exigent to vitalize the biofilm for allowing efficient electron transfer and substrate 10 access . Recent studies reported that, only few microorganisms are capable of long distance electron transfer 11-13 through the biofilms . Long range extracellular electron transfer (EET) can be occurred through the 8 thick biofilm at distances of tens or even hundreds of microns distant from the electrode surface . As of now, two competing models have been reported for long-distance EET, such as electron hopping and metallic-like conductivity through the electrically conductive biofilms. According to the electron hopping model, electrons are relayed through the biofilm by electron tunneling using mediators positioned in the exopolymer matrix and also on pili (nanowire). In contrast, the metallic-like conduction model electron 12 transport occurs by delocalized electrons in the pili network within the biofilm . However, evidence in the literature suggests that some biofilms, especially Shewanella oneidensis produce the requisite 13 components for both mechanisms . Microorganisms even with electron conducting pili could not be able to achieve high performance while operated with real wastewater which might be due to the inability of 14 those microorganisms to utilize complex substrates .

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Figure 1 Schematic diagram of the effects of ultrasonic treatment on monolayer and multilayer biofilms

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The detrimental effects of thick biofilm on MFC performance can be alleviated by retrenching the 7 thickness of biofilm and through the removal of excess inert biomass . The thickness of biofilm can be 7 controlled by using a turbulent environment and that may possibly maximize the performance of MFCs . In membrane bioreactor (MBR) system, different methods such as ultrasonic treatment, oxygenation, rotation, chemical (antibiotic), mechanical cleaning etc. have been implemented to remove the inactive biomass (dead cells) from the surface of the membrane. Unlike the MBR system, the MFC requires efficient biofilm comprising with more viable cells for stable power generation. Therefore, an appropriate


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method is needed to considerably reduce the thickness of the biofilm as well as to increase the cell viability in the biofilm.

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Therefore, the current research addressed the possibility of the use of low frequency ultrasound effect on anode biofilm and its impact on power generation of MFC. The effect of ultrasound on anode biofilm was visualized and correlated with the current generation, EIS and CV analysis to elucidate its effect on MFC performance.

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2. Experimental Methods

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2.1 Sample collection and characterization

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Palm oil mill effluent (POME) was collected from Panching palm oil mill (FELDA) located in Kuantan, Pahang, Malaysia. The sample was collected before the effluent get discharge into the mixing pond at about 80-90°C. POME was filtered using Whatman no.1 filter paper (2.5 µm) and the filtrate was autoclaved at 121°C, 15 psi for 15 minutes. The municipal wastewater (MWW) was collected from drainage discharge point of Kuantan city, Malaysia. All samples were stored in sterilized glass bottles at 4°C.

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2.2 Isolation and characterization of bacteria

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MWW was filtered using Whatman no. 1 filter paper and filtrate was serially diluted (10 to 10 ) and -6 the pure culture bacteria were obtained from 10 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) incubated at 37°C with shaking at 150 rpm. Several pure culture strains were identified using Biolog gen III analysis but only electroactive Klebsiella spp. was selected for further identification and used as inoculum in MFCs. The molecular characterization of bacteria has been presented in supplementary section (Fig. S1).

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2.3 MFC assembly and operation

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The MFC experiments were operated at room temperature (22 ± 2 °C) in fed batch mode. Six dual chamber MFCs (MFC1, MFC2, MFC3, MFC4, MFC5 and MFC6) were fabricated with a cubic plexi glass which has a dimension of 5 cm x 5 cm x 5 cm (Shanghai, Sunny Scientific, China) and a total working

Among the available methods, ultrasound treatment is one of the most potential in-vitro methods since 15 it can successfully remove the thick biofilm within a short period of time . Ultrasonic treatment causes detachment of bacteria by using strong shear forces and thus prevents the deposition of a dense bacterial layer on the surface as shown in Fig 1 (schematic diagram). The figure illustrates that ultrasound can create cavitation bubbles in the liquid adjacent to the surface, or in the narrow volume between the surface and loosely attached inert biomass thereby multilayer biofilm can easily be dispersed from the 16 surface. Moreover, Dror et al. (2009) reported that the ultrasonic treatment with low frequency (20 kHz) 17 efficiently removed 87.5% of the biofilm from the glass surface. In addition to that, Pitt et al. (2003) reported that the low frequency ultrasound enhanced the cell growth due to the following properties of ultrasound: 1) its ability to increase the transport of small molecules in solution, and 2) its inability to completely remove cells from surfaces. Besides that, the ultrasound treatment can increase the mass 18 transfer of nutrients to the cells as well as increases the transport of cellular waste products away from 17 8 the cells thus it can increase the cell viability . Sun, et al. reported that biofilm comprised with more viable cells enhanced the power generation in MFC. But, as long as the authors are concerned, the effect of ultrasonic treatment has never been studied on MFCs.

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volume of 25 mL. Carbon brush was used as anode and cathode electrodes in all the experiments. The electrodes were cleaned with 1.0 M NaOH followed by 1.0 M HCL after each experiment and stored in distilled water before use. A cation exchange membrane (Nafion 117, Dupont Co., USA) was used to separate the anode and cathode compartments of MFC. Prior to use, the Nafion membrane was pretreated using dilute HCL for 1 hour followed by washing with DI (Deionized) water several times. After that, the whole assembly was held in place and tightened with the screws. The anode compartment was filled with 20mL of sterilized POME (POME/water volume ratio 1:1) and the pure culture bacteria (1mL) were subsequently inoculated into it while the cathode chamber was filled with KMnO4 solution, as oxidizing agent. The anode chamber was continuously flushed with N2/CO2 (80:20) to maintain anaerobic conditions and maintain the pH balance of the POME. The anode and cathode electrodes were connected by using titanium wires with a rheostat (Crotech DRB-9, UK) to form a circuit. The MFC1 was operated to observe the effect of time on the removal of COD from POME. In order to measure the COD, 1ml of POME solution was pippetted out after every 6 days of operation. Besides that, all the MFCs (MFC1 to MFC6) were placed in ultrasonic bath (CREST ULTRASONICS; Model 690DAE; 20 KHz) in switched off mode and operated under the analogous conditions. In addition, whenever the ultrasound treatment was employed, the bath was “switched on” for desired time period (15 min, 30 min and 60 min). Furthermore, the temperature was maintained at 26±2°C so that the real time power measurement cannot be interrupted.

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2.4 Cell viability count of biofilm

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The 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 into the 50mM phosphate buffer. In order to separate the microorganisms from carbon brush, it was centrifuged at 5000rpm for 1 min . Thereafter, the cell suspension was serially diluted and stained using a viability staining kit. Finally, stained cells were filtered through 0.4 µm membrane filter and counted using microscope. The cell density per anode geometric area was calculated using the dilution factor, the filtered volume and the ratio of total filtered area to 19 image area . The method was replicated using 4-6-diamidino-2-phenyl indole (DAPI) for comparison.

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2.5 FESEM analysis of the biofilm

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Biofilm formation was visualized at different time intervals of anode electrode using field emission electron microscopy (FESEM) (Model: JEOL JSM7800F) at 5 kV. Small portions (1 cm) of the anode electrodes with bacteria on the surface were cut off from the anode chamber and rinsed with sterile medium followed by immediate soaking into anaerobic solution of 3% glutaraldehyde. The samples were then washed twice with 0.1 M phosphate buffer and dehydrated by successive 10 minutes incubations in 40%, 60%, 80%, 90% and 100% ethanol. The samples were then dried with a critical-point drier and coated with platinum using an ion-sputter to a thickness of 10 nm. After this procedure, specimens were examined by FESEM.

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2.6 Analyses

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

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Pv =

(1)


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where, V is the output voltage, v is the volume and R is the resistance.

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The COD removal efficiency and columbic efficiency of dual chamber MFC was calculated as described 20 by Baranitharan et al., . The COD was determined using digestive solution (0- 1500 mg/L range; Hach, USA) and measured using a COD reactor (HACH DRB 200, USA). The COD removal efficiency was calculated using equation (2)

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COD removal efficiency =

× 100%

(2)

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

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CE =

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where, I is the current (A), F is the Faraday’s constant (96485 C/mol), and Van is the anode volume (L). COD is the difference between CODin and CODt (values in g/L). In Eq. 3, the constant (8) is calculated based on the molecular weight of oxygen (32 g/mol) and assuming that 4 electrons exchanged per mole of oxygen. Moreover, optical density (OD) of the anolyte was measured using UV-spectrophotometer (Shimadzu model UV -160A) at 600 nm for monitoring the planktonic population growth during the initial 21 days of experiments .

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2.7 Electrochemical analysis

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2.7.1 Electrochemical impedance spectroscopy

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EIS is a well known nondestructive electrochemical technique for analyzing bio-electrochemical reactions on electrodes, internal resistances, biofilm development and studying the mass transfer 22 resistances due to the diffusion limitations of the reactants . EIS was employed to measure the MFC anode internal resistances, and the measurement was conducted using a potentiostat (PARSTAT 2273, USA) at the open circuit potential in a frequency of 100 kHz to 50 mHz with the anode as the working electrode, the cathode as the counter electrode, and saturated Ag/AgCl (1.0 M KCl) electrode as the reference electrode. An AC signal with amplitude of 10 mV and a frequency range of 100 kHz – 5 mHz was applied to prevent the biofilm detachment and to minimize the disturbance on systematic stability. The EIS data were plotted in the form of Nyquist curve were analyzed using Zview software. The ohmic resistance (RΩ) and charge-transfer resistance (Rct) were determined by fitting the measured impedance data to an equivalent circuit: R(Q[RW]).

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2.7.2 Cyclic voltammetry

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Cyclic voltammetry (CV) was used to study the catalytic behavior of the MFCs. It helps in characterizing the electron transfer interactions and mechanisms between the biofilm (biocatalyst) and the anode of the MFC. Besides that, It helps in understanding the specific role of redox mediators taking 23 part in the bioelectrochemical reactions . The CV measurements were performed with a PARSTAT 2273 electrochemical system (USA). The anode and cathode were used as working and counter electrodes respectively. Ag/AgCl (sat. KCl, 222 mV vs. SHE) electrode was used as reference electrode and

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disinfected with 75% alcohol before plugging into the anode- chamber filled with POME. CV was conducted at a scan rate of 30 mV/s, in the potential range from +1 to -1 V vs reference electrode. All solutions were purged with the N2 for 15 min before electrochemical measurements.

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3. Results and discussion

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3.1. Performance of K. pneumoniae in MFC

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The performance of wild type K. pneumoniae was investigated using MFC1 fed with POME (COD=23,258 mg/L). From the polarization data, power at 0.4 V was calculated and plotted vs time as shown in Fig. 2. 3 On day 1(after 18 hours of operation), MFC1 achieved maximum power density of 870 mW/m and thereafter it sharply increased until 5 days of operation. After 5 days of operation, power density 3 increased gradually and it reached to a stable value of 3907±136mW/m , thereafter stable power th 3 generation continued until 15 day and then, started decreasing and finally it reached to 1136 mW/m st after 27 days of operation which is near to the 1 day performance. 25000 4000

3200 15000

3

2400

P (mW/m ) at 0.4 V

20000

COD value (mg/L)

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10000 1600

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0

0 0

5

10

15

20

25

Time (day)

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Figure 2 Profile of maximum power densities (at 0.4 V) and COD removal of MFC1 with time

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Although the number of microorganisms reached to maximum on day 2 (Fig S2) itself, the MFC showed maximum power density only after 11 days of operation. This phenomenon suggests that effective biofilm formation is necessary for the maximum power generation rather than the number of microorganisms. Moreover, the performance was not dropped even though the COD content was reduced in the anode compartment. 3 The maximum power generation of 5061 mW/m (Fig S3) at 0.8 V in the present study has been compared with the literature and presented in Table 1. Several reports showed that pure culture bacteria produces lower power compared to mixed culture bacteria while operated using complex wastewater as a 24 substrate . In this study, K. pneumoniae showed


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Table 1 Performance comparison to literature on dual chamber MFC systems

Substrate (Wastewat er)

COD (mg/L)

Inoculum

Electrode material

Power Density 3 (W/ m )

POME

28,230

K. pneumoniae

Carbon brush

Dairy Municipal

-

L. pentosus S. oneidensis

Oil refinery

2,213

POME

1,000

CE (%)

Reference

5.1

COD removal efficiency (%) 62

72

This study (MFC1)

Graphite Graphite

0.02* 0.784

56 89

-

Boas, et al. 14 Li, et al.

P. putida

Carbon cloth

0.09*

-

0.06

Majumder, et al.

AS

PACF

0.472

32

74

Baranitharan, et al.

25

26 27

28

Dairy

12,400

AS

Graphite

0.715

54

9

Jayashree, et al.

POME Dairy

60,600 1,600

AS AS

PACF Graphite

0.304 2.7

45 91

0.8 17

Food waste

1,000

AS

Carbon felt

0.432

92

20

Baranitharan, et al. Elakkiya and 29 Matheswaran 30 Li, et al.

Municipal

-

AS

Graphite

1.43

94

-

Wang, et al.

Swine

3,250

AS

20

31

237

Stainless 1.41 1.7 steel 3 2 *the values were recalculated to show in W/m but originally it was reported in W/m

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significant power production which is comparatively higher than other reported literatures. It might be due 33 to the ability of K. pneumoniae to utilize simple to complex range of substrates . The heterologous expression of the NADH oxidase, and the production of 2,3-butanediol by K. pneumoniae largely + decreases the intracellular NADH /NAD ratio (2.0 fold) which activates the extracellular electron excretion 34 pathway and consecutively it enhances the power generation in MFCs . However, in MFCs, the maximum power generation and its subsequent decline may not completely depend on electrogenesis 35 9 capability of microorganisms rather depends on effective (conductive) biofilm formation . Franks, et al. reported that the different layers of the biofilm may have substantially different activities, however which 11 layer to be the most electroactive, is still an obscure in MFC research . The genes treC and sugE in K. pneumoniae stimulates the biofilm formation by modulating the capsular polysaccharide production 36 consequently it forms thick biofilm with time . Generally, bacteria can form thin biofilm within 3 days of 37 MFC operation and the cells then increase continuously and forms an ineffective multilayer biofilm which deteriorate the power generation of MFC. It can be seen from the Fig. 2, the power generation commenced reducing after around 16 days of operation even though higher COD was observed (9,244 mg/L) in anode compartment. Therefore, it was hypothesized that the formation of thick biofilm on anode surface reduced the power generation after 15 days of operation.

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3.2 Effect of biofilm removal on power generation

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In order to prove the hypothesis, another cell (MFC2) was operated where the similar trend was observed as in MFC1. The current generation as a function of time is presented in Fig. 3. After 21 days of operation when the current generation reached the minimum, the carbon brush was taken out from the anode chamber and the biofilm from the surface of the carbon brush was completely removed using sterile brush in laminar


Ryu, et al.

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Figure 3 Profile of current generation of MFC2 (under fixed external resistance, 1000 Ω) using K. pneumoniae with time (the fresh medium injections are indicated by small arrows) air condition, and finally it was autoclaved for 2 hours. Thereafter, the carbon brush was placed again in anode compartment and continued to run in the MFC2.The current generation went up sharply and on th 24 day, it reached the stable value. In MFC2, the biofilm formation was analyzed using FESEM images of the anode (before and after removal) which are presented in Fig. 4a and 4b. In Fig 4a, after 20 days of operation, very thick layer of nd biofilm was observed whereas after the removal of biofilm on 22 day, only few microorganisms scattered around the electrode surface were observed as shown in Fig. 4b. Bacterial cell viability of the biofilm was analyzed on different days of MFC2 operation and presented in Fig 4c. After 10 days of operation, the number of live cells increased significantly and outnumbered the dead cells by 4.5 fold. Thereafter, the number of dead cells increased with time until 21 days of operation, and eventually exceeded the number of the live cells by many folds within 22-25 days of operation which might be due to the formation of multilayer biofilm as shown in Fig.4a. Generally, live bacteria prefer to localize and exist on the outer layer 38 of the biofilm due to the easier availability of substrates therefore deposition of bacteria with time led to the accumulation of more dead cells on anode surface. After 24 days of operation, very less number of nd dead cells were observed due to the immediate removal of thick biofilm (On 22 day) comprising with inert biomass (dead cell).


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Nyquist plots and CV for the anode configuration on day 11, 21 and 25 have been presented in Fig. 4d and 4e respectively. In Fig. 4d, after 10 days of operation, polarization resistance was observed as 5.43Ω. However, on day 21 it increased to 233.48Ω, suggesting the accumulation of more dead cells on the surface of the anode (Fig. 4c). But, after the removal of biofilm and subsequent operation the polarization th resistance was reduced to 20.33Ω (25 day). Fig. 4e, shows the CV data of anode on different days of operation. The reversible redox peak was observed at +0.2 V to –0.2 V, which reduced from 0.068 mA (day 11) to 0.038 mA on day 21. After 24 days of operation, the catalytic efficiency again increased and reached to 0.072 mA. The reversible redox peak suggested that electron transfer occurs through the 39 mediator .

Figure 4 Performance of MFC2 using K. pneumoniae under fixed external resistance (1000 Ω). FESEM st nd images on anode carbon brush (a) before (21 day) and (b) after (22 day) the removal of biofilm, (c) Cell viability count, (d) Nyquist plots, (e) Cyclic voltammograms On day 11, lower charge transfer resistance (5.43 Ω) and a strong redox peak were observed, and it is due to the presence of higher number of live cells in the biofilm (Fig. 4d) which would have increased the mediator (2,6 – DTBBQ) content and thus facilitated the kinetics of the electrochemical reactions since it 40 reduced the anode activation losses . Whereas, after 20 days of operation, higher charge transfer resistance (increased by 92%) and a small redox peak (reduced by 51%) were achieved owing to the formation of multilayer biofilm comprising with more dead cells and this dead inner layer hindered the electron transfer from the live bacteria present in the outer layer of the biofilm and bulk solution. Moreover, the electrons in the biofilm needed to travel over substantial distances to reach the anode and the resistance associated with long-distance electron transport is substantially higher than the resistance associated with the single electron transfer step from the biofilm to the anode at the biofilm/anode 41 th interface . On 25 day, again lower Rct and prominent redox peak were observed. The higher power on th st 25 day compared to 21 day suggesting that the removal of dead cells and inert biomass (Fig. 4d) increased the electron flow between the microorganisms and electrode through electron shuttles. These results revealed that the effective biofilm formation can directly influence the electrochemical transport processes and play a crucial role in MFC performance.


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3.3 Ultrasound effect on biofilm and power generation

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To observe the effect of ultrasound on biofilm dispersal and current generation in microbial fuel cell, the MFC3, MFC4, MFC5 and MFC6 were operated under similar conditions as that of MFC1 and MFC2.

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3.3.1 15 minutes impact

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In MFC3, after 21 days of operation, while current generation was dropped, ultrasonic treatment (20 2 kHz, 64 W/cm ) was employed for about 15 minutes, it was observed that the current generation boosted up (26.20 µA/day) immediately and showed the stable value of about 175±10 µA (Fig 5a), which possibly due to the removal of inert biomass from the electrode surface and thereafter no significant change was observed until 27 days of operation. Under post-ultrasonic treatment, the current generation reached the stable value very fast, but it was significantly lower than the maximum stable value that was achieved before the ultrasonic treatment.

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Figure 5 Effect of ultrasonic treatment (15 min) under fixed external resistance (1000 Ω) on MFC3 (a) current generation (the fresh medium injections are indicated by small arrows), (b) biofilm visualization (c) Cyclic voltammograms, (d) Nyquist plots

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On 22 day of operation, the anode biofilm was visualized by FESEM to observe the effect of ultrasonic treatment as shown in the Fig. 5b. It can be seen that loosely attached microbial clumps sticking with biomass were scattered around the electrode surface. After 24 days of operation, the current generation reached the stable value and then polarization data, CV and EIS were measured as shown in Fig S4, 5c 3 and 5d. It can be seen that, higher Rct (43 Ω), lower power (2570 mW/m ) and small redox peak (maximum current 0.056 mA) were achieved compared to MFC2 suggests that the multilayer biofilm rapidly formed on anode surface due to the scrappy removal of inactive biomass as well as higher number of microorganisms in the bulk. These results revealed that 15 min ultrasonic treatment is not effective for controlling the anode biofilm thickness.

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In MFC4, after 21 days of operation, ultrasonic treatment was employed for about 30 minutes. It was observed that the current generation increased sharply (34.47 µA/day) and reached the stable value of about 230±10 µA (after 24 days of operation), which is almost near to the maximum value in this cycle as shown in Fig 6a. The reason could be that the 30 min ultrasonic treatment might have almost completely removed the ineffective biofilm from the surface. After 21days of operation, the anode biofilm was visualized under FESEM to observe the effect of ultrasonic treatment as shown in Fig. 6b.

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Figure 6 Effect of ultrasonic treatment (30 min) under fixed external resistance (1000 Ω) on MFC4 (the fresh medium injections are indicated by small arrows), (a) current generation, (b) biofilm visualization, (c) Cyclic voltammograms, (d) Nyquist plots

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It can be seen that, microorganisms were scattered around the electrode but most of the electrode surface was not covered therefore the electron shuttle from bulk solution can easily travel between the microorganisms and electrode thus resulted in stable high current generation. After 24 days of operation, the current generation reached the stable value at a rate of 34.47 µA/day; thereafter the polarization data, EIS and CV were measured as shown in Fig. S4, 6c and 6d. Maximum power density of 3920 3 mW/m was achieved which is significantly higher than that of MFC3 due to the complete removal of inert biomass from the anode surface consecutively it reduced the Rct (20.02 Ω). Moreover, a strong redox peak (0.068 mA) compared to MFC3 indicating the salient electrochemical activities of bacteria and the presence of more redox active compounds in the anode biofilm and thus would have enhanced the 42 extracellular electron transfer process . These results suggest that 30 min ultrasonic treatment played an imperative role in removing the thickness of biofilm and also in increasing the cell growth rate thereby enhanced the power generation.

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In MFC5, after 21 days of operation, ultrasonic treatment was employed for about 60 minutes, it was th observed that the current generation increased (10.35 µA/day) very slowly until 27 day of operation. The reason could be that the long term ultrasound treatment initiated porosity in the cell wall that led to water influx

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Figure 7 Effect of ultrasonic treatment (60 min) under fixed external resistance (1000 Ω) on MFC5 (the fresh medium injections are indicated by small arrows), (a) current generation, (b) biofilm visualization, (c) Cyclic voltammograms, (d) Nyquist plots

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into the cells resulting in swelling and subsequent rupture of cells . Moreover, long term ultrasonication can create two major effects such as bacterial declumping and bacterial killing and which are detrimental 44 st for power generation in MFCs . On 21 day of operation after the ultrasonic treatment, the anode biofilm was visualized under FESEM, to observe the effect of ultrasonic treatment as shown in Fig. 7b. It can be seen that, very less microorganisms were found around the electrode and most of the electrode surfaces were not covered due to the complete detachment of biofilm. After 24 days of operation, polarization data, 3 CV and EIS were measured as shown in Fig. S4, 7c and 7d. The maximum power density of 3083 mW/m was achieved which is significantly lower than that of MFC3 and MFC4 due to the presence of less number of live microorganisms in the bulk as well as on anode surface. Besides that, the Rct (74.20 Ω) was significantly higher compared to the MFC3 and MFC4. Moreover, CV results showed poor bioelectrocatalytic activity (maximum current, 0.043 mA) due to the presence of less redox active compounds in the anode and which would have reduced the extracellular electron transfer process (Fig. 7c). Thus, it clearly shows that the long term ultrasonic treatment is detrimental to cell viability as well as to MFC performance.

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These results revealed that the excessive bacterial colonization on the anode over the time brings nonconductive debris and inert biomass onto the electrode surface thus increases the Rct as well as reduce the electrocatalytic activity due to limited substrate diffusion into the biofilm thereby reduced the MFC performance and which can be eliminated by using ultrasonic treatment.

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However, the long exposure of ultrasound (60 min) almost cleaned the surface as seen from the FESEM image (Fig. 7b) whereas, the 15 and 30 min exposure resulted in only partial removal of the biofilm (Fig. 5b, 6b). Besides that, in case of 60 min exposure, the reformation of the biofilm took longer time which can be seen from the current vs time plot (Fig. 7a). But, the 15 and 30 min exposure (Fig. 5a and 6a) required only around 72 hours for achieving the stable power generation indicating that the reformation of efficient biofilm occurred in a relatively short time compared to the 60 min exposure. So it is clear that the ultrasonic treatment is solely responsible for stabilizing the performance of MFC as injection of the fresh medium did not show any significant fluctuations on power generation. Furthermore, it is important to note that the rate of current generation (during the post-ultrasound treatment) achieved for 30 min exposure (37.47 µA/day) is higher compared to that of 15 (26.20 µA/day) and 60 (10.35 µA/day) 44 min respectively indicating the faster reformation of viable biofilm. Ewe, et al. reported that the low frequency ultrasound treatment for short term affected the fatty acids chain of the cellular membrane lipid bilayer, thus increased the lipid peroxidation. This led to increased membrane fluidity and subsequently, membrane permeability. The permeabilized cellular membranes had facilitated nutrient internalization and subsequent growth enrichment of microorganisms. Thus, it helped to achieve faster and viable biofilm formation. However, the long term (60 min) ultrasound treatment is detrimental to the cell growth since it 43, 45 creates severe shear forces that might rupture the membrane of bacterial cells .

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3.3.4 Effect of periodic ultrasonic treatment

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As can be seen from the current generation trend of MFC2 to MFC5, the current generation decreased th sharply on 18 day of operation. Moreover, the 30 min ultrasonic treatment in MFC4 showed that it can effectively revitalize the biofilm. Therefore, in order to achieve an uninterrupted stable MFC performance another MFC (MFC6) was operated where intermittent ultrasound treatment was applied for about 30 minutes (Fig.8). The intermittent ultrasound treatment was provided after 18 days of operation and thereafter, every 6 days of interval the same ultrasonic treatment was repeated. From Fig 4c it can be seen that the complete aging of the biofilm occurred within 10 days of operation (day 11 to day 21). The intervention in that process is required within that time interval. Therefore, the intermittent ultrasonic treatment was conducted at an interval of 6 days. It can be seen that the current generation was not th dropped and maintained the stable performance until 40 day of operation. These results suggest that viable biofilm can be maintained by intermittent ultrasonic treatment leading to a stable power generation. However, further research is needed to optimize the interval time as well as the duration of the treatment for reducing the energy consumptions in the MFCs. Besides that, under continuous mode of operation there are concerns that due to the ultrasonic effect, the viable cells from the biofilm will also be carried away from the system along with the dead cells and debris. Therefore, for continuous mode of operation a systematic study will be required for the optimization of the ultrasonic treatment.

Hence, it can be concluded that the low frequency ultrasound for 30 min not only removed the ineffective biofilm but also revitalized the biofilm by enhancing the live cells in the anode biofilm thus maintained the stable power generation in MFCs.


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Figure 8 Profile of current generation of MFC6 (under fixed external resistance, 1000 Ω) with intermittent 30 min ultrasonic treatment (the fresh medium injections are indicated by small arrows)

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Conclusions

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In this study, we have successfully used the ultrasonic treatment for retrenching the ineffective biofilm in order to maintain the stable power generation in MFC. After prolonged operation, microbes in the MFC usually form thick multilayer biofilm on the anode surface which deteriorates the performance of MFC. It can be observed from the FESEM, CV and EIS results, the multilayer thick biofilm comprising with outnumber of dead cells reduced the electron transfer efficiency and thus increased the charge transfer resistance. To dispel the ineffective biofilm, ultrasonic treatment was employed on MFC for different duration and among them 30 minutes of ultrasound (20 kHz) treatment restored the power generation. The ultrasonic treatment for about 30 minutes was employed at regular intervals throughout the operation that resulted in an uninterrupted stable power generation in MFC. Moreover, it is important to note that, even though, the ultrasound treatment removes both live and dead cells, it increased the cell viability and thus helped to achieve faster and viable biofilm formation. Therefore, long term stable power output can be obtained by employing low frequency ultrasound treatment for short period of time. The enhanced performance by the ultrasonic treatment clearly showed its promising potential in stabilizing the power generation in MFC. Further study will be carried out to observe the effect of the ultrasonic treatment in the continuous mode MFCs.

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Supporting information

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Molecular analysis and phylogenetic relationship of K. pneumonia (Figure S1), Optical density measurements of K. pneumonia (Figure S2), Polarization curves of MFC1 after 10 days operation (Figure th S3), Polarization curves of MFC3, MFC4, and MFC5 on 25 day (Figure S4).

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Author information

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

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Corresponding author *Phone: +609-5492872, e-mail: [email protected]

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


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