APPLICATION OF DIGITAL CELLULAR RADIO FOR MOBILE LOCATION ESTIMATION

IIUM Engineering Journal, Vol. 18, No. 2, 2017 Islam et al.

BIOELECTROCHEMICAL BEHAVIOR OF WILD

TYPE BACILLUS CEREUS IN DUAL CHAMBER

MICROBIAL FUEL CELL

MOHAMMED AMIRUL ISLAM
1
, CHEE WAI WOON

1
, BARANITHARAN ETHIRAJ

2
,

ABU YOUSUF
3
, HUEI RUEY ONG

1
AND MD MAKSUDUR RAHMAN KHAN

1
Faculty of Chemical Engineering & Natural Resources, Universiti Malaysia Pahang,

Lebuhraya Tun Razak, 26300 Gambang, Pahang, Malaysia.
2
Department of Biotechnology, Bannari Amman Institute of Technology,

Sathyamangalam, Erode District, India.
3
Faculty of Engineering Technology, Universiti Malaysia Pahang,

Lebuhraya Tun Razak, 26300 Gambang, Pahang, Malaysia.

*Corresponding author: mrkhancep@yahoo.com

(Received: 27
th

Feb. 2017; Accepted: 30
th

Oct. 2017; Published on-line: 1
st
Dec. 2017)

ABSTRACT: A microbial fuel cell (MFC) is a bioelectrochemical system that uses

living microbes as biocatalyst to oxidize organic substrates as well as release electrons

that can be harvested in an external circuit to produce electrical energy. In this study, a

proteolytic biocatalyst, Bacillus cereus, has been employed for the first time in a

microbial fuel cell (MFC). The wild type pure culture was isolated from municipal

wastewater and identified using Biolog Gen III analysis. The MFCs were fueled with

palm oil mill effluent (POME) and attained a maximum power density of about 3.88

W/m
3
. The electrochemical behavior of the MFC was evaluated using a polarization

curve, electrochemical impedance spectroscopy (EIS), and cyclic voltammetery (CV)

analysis. The CV and EIS results suggest that the predominant electron transfer occurred

through the electron shuttle mechanism. The electron shuttle mediators excreted by B.

cereus significantly reduced the anode charge transfer resistance (52.95%). The FESEM

result shows that B. cereus has the capability to form an effective biofilm on the anode

electrode surface. These results revealed the electrocatalytic potentiality of B. cereus,

making it a promising candidate to be used in MFCs. Therefore, this biocatalyst can be

used to generate electricity through wastewater valorization.

ABSTRAK: Sel bahan api mikrob (MFC) adalah sistem bioelektrokimia yang

menggunakan mikrob hidup sebagai bio pemangkin untuk mengoksida substrat organik

bagi menghasilkan tenaga elektrik dengan membebaskan elektron mengunakan litar

tertentu. Kajian ini telah menggunakan bio pemangkin proteolitik Bacillus cereus buat

pertama kali dalam sel bahan api mikrob (MFC). Kultur asli jenis liar telah diasingkan

daripada sisa air kumbahan dan dikenal pasti dengan menggunakan analisis Biolog Gen

III. MFC ini telah diberi bahan bakar bersama efluen kilang minyak sawit (POME) dan

didapati ketumpatan kuasa maksimum adalah sebanyak 3.88 W/m
3
. Tindak balas

elektrokimia MFC ini dinilai menggunakan analisis lengkung polarisasi, Elektrokimia

Impedan Spektroskopi (EIS) dan kitaran voltammetri (CV). Keputusan CV dan EIS

menunjukkan dominasi pemindahan elektron telah berlaku menerusi mekanisme

pemindahan elektron. Pengantara pemindahan elektron ini yang terhasil daripada B.

cereus telah menurunkan rintangan pemindahan cas anod dengan ketara (52.95%). Hasil

FESEM menunjukkan bahawa B. cereus mempunyai keupayaan membentuk biofilem

yang berkesan pada permukaan elektrod anod. Keputusan ini menunjukkan potensi

IIUM Engineering Journal, Vol. 18, No. 2, 2017 Islam et al.

elektrokatalitik B. cereus sebagai calon terbaik dalam MFC. Ini kerana bio pemangkin

ini boleh digunakan untuk menjana tenaga elektrik melalui volarisasi air kumbahan.

KEYWORDS: microbial fuel cell; Bacillus cereus; biofilm; palm oil mill effluent; cyclic

voltammetry; electron transfer mechanism

1. INTRODUCTION

The microbial fuel cell (MFC) is an important type of bioenergy source for the future

because it can use a wide variety of substrates, including soluble or dissolved complex

organic wastes and various types of artificial or real wastewater [1]. In MFCs, the use of

pure cultures may not be suitable due to high costs and its inefficiency to utilize the

complex substrates; therefore, it is not efficient for practical operation such as treatment of

industrial effluents. On the other hand, mixed cultures from natural sources such as soil,

wastewater, and anaerobic sludge (AS) have been widely used as inoculum in MFCs since

they are more readily obtainable in large quantities, more tolerant to environmental

fluctuations, and more accommodating to a variety of substrates [2, 3]. However, AS

could not achieve a significant performance because of the presence of non-electrogenic

bacteria (i.e., methanogenic bacteria and denitrifying bacteria) in mixed cultures that

consume organic substrates without generating electricity [4]. Therefore, this problem can

be solved using wild type pure culture bacteria that can utilize a wide variety of substrates

as inoculum in MFCs.

In the past decade, a number of new strains of bacteria have been reported to be

capable of generating electrical current through MFCs. Although many new strains have

been identified, only a few strains are capable of producing significant power using

complex wastewater as a substrate [5, 6]. One of them is Bacillus cereus. It is a Gram-

positive, soil-native bacteria, widespread in different habitats around the world. Some

studies showed that Gram-positive bacteria especially Bacillus spp. increased current

generation through the redox shuttles in MFC [7, 8]. Besides that, it has the capability to

degrade toxic waste compounds and efficiently utilize them as a carbon sources [9, 10].

These degrading skills shown by B. cereus could be improved if it grows in adverse

environmental conditions [11, 12]. Therefore, Bacillus cereus can be considered as a

potential biocatalysts to be used in MFC for simultaneous power generation and

wastewater treatment.

Bacteria are, so far, known to transfer electrons to anode surfaces via two

mechanisms. The first is a biofilm mechanism, where exoelectrogens form a biofilm on

the anode surface. Some microorganisms can even produce nanowires carrying electrons

from the cell to anode surface. The second is the electron-shuttle mechanism, where the

microorganism itself can produce and use low molecular weight compounds, soluble

mediators (e.g. phenazine, quinones) that eliminate the need for direct contact between the

cell, and electron acceptors [13]. The understanding of electron shuttles in these electron-

generating processes is emergent to effectively harvest the electricity from MFCs. A pure

exoelectrogen strain is required for studying the mechanism of electricity production in

MFC. Currently, quite a number of pure exoelectrogens are demonstrated to have

electrochemical activity, such as Geobacter and Shewane1la [14]. However, only a few

strains were capable of producing electron shuttles, such as pyocyanin by Pseudomonas

aeruginosa and riboflavin by S. oneidensis MR-1 [15].

The purpose of this study is to investigate the electrochemical activity as well as

electron transfer mechanism of wild type B. cereus in microbial fuel cells. The

IIUM Engineering Journal, Vol. 18, No. 2, 2017 Islam et al.

electrochemical activity was evaluated using the polarization measurement, biofilm

formation capability, electrochemical impedance spectroscopy and cyclic voltammetry

analysis.

2. MATERIALS AND METHODS

2.1 Source of Microorganism and Sample Collection

Palm oil mill effluent (POME) samples were obtained from a local palm oil mill

industry located in Pahang, Malaysia. Municipal wastewater was collected in a sterilized

bottle from a drainage discharge point in Kuantan, Pahang, Malaysia. All samples were

stored at 4
o
C to minimize the degradation of samples by indigenous microbial activities,

thus preserving the quality of the samples.

2.2 Isolation of Microorganism

The source of microorganisms was municipal wastewater. Enrichment of the cultures

was carried out by preparing an overnight culture in LB broth (10% v/v) incubated at 35
o

C with shaking at 150 rpm. The overnight culture (10% v/v) was used as primary

inoculum in the anode compartment. After 13 days of MFC batch operation, the anode

was taken out and placed in 0.1 M phosphate buffer and shaken vigorously to detach

bacteria that grew as biofilm on the anode. The side of the anode was also carefully

scraped and resuspended in phosphate buffer. The bacterial suspension was serially diluted

and the pure culture bacteria were obtained using the spread plate technique. The

predominant colony was identified using Biolog GEN III, specifically B. cereus.

2.3 MFC Configuration

The dual chamber MFC was fabricated with a Plexiglas cube with a dimension of 5

cm x 5 cm x 5 cm (Shanghai, Sunny Scientific, China) and a total working volume of 25

mL. A carbon brush was used as an electrode in all experiments. Nafion 117 membrane

was used to separate the anode and cathode compartments of MFC (Dupont Co., USA).

Prior to use, the Nafion membrane was pretreated using diluted H2SO4 for 1 hour followed

by washing with DI (de-ionized) water several times. After the whole MFC set up was

tightened up with screws, the anode compartment was filled with sterilized 50% POME

(20mL) and subsequently the pure culture bacteria (1mL) were inoculated into it while the

cathode chamber was filled with KMnO4 solution. The anode chamber was purged with N2

for 30 min and then tightly closed to maintain the anaerobic condition. The whole

experiment was conducted in batch mode. The anode and cathode electrodes were

connected using titanium wires with a rheostat (Crotech DRB-9, UK) to form a circuit.

2.4 FESEM Analysis

Field emission scanning electron microscopy (FESEM) was used to observe the

microorganisms grown on the electrode material. A small portion of the electrodes was cut

off for FESEM observation. The electrode sample was immediately immersed in 3%

glutaraldehyde and 0.1 M phosphate buffer solution and then dehydrated with increasing

ethanol concentration (30–100%) [16]. Thereafter, samples were freeze dried and sputter

coated with a thin layer of platinum under vacuum to neutralize the charging effects prior

to scanning in the FESEM. The morphology was observed under a scanning electron

microscope (Hitachi, Japan, Model: 3400N) with an acceleration voltage of 5 kV during

the scanning.

IIUM Engineering Journal, Vol. 18, No. 2, 2017 Islam et al.

2.5 Measurement and Analyses

The voltage (V) and current (I) across an external resistor (1 kX) in the MFC circuit

was monitored using a digital multimeter with data logger (Fluke 289 True RMS

Multimeter, USA) connected to a computer through a USB cable adapter. To obtain

polarization data, the external resistance was varied from 50 to 20,000 X. Power density

normalized by surface area (PA, W/m
3
) was measured and calculated using the following

equations.

PA = UI (1)

Pv = RvI
2
/V (2)

where V is the volume of the MFC (m
3
), U is the voltage of the cell (V) and Rv is the

external resistance (Ω), and I is the current (A).

2.6 EIS and CV Analysis

Electrochemical impedance spectroscopy (EIS) was carried out using an

electrochemical workstation (AUTOLAB 2273, PAR, USA). A conventional three-

electrode system was employed with the anode as the working electrode, the cathode as

the counter electrode, and an Ag/AgCl reference electrode that was placed as close as

possible to anode electrode [17]. The impedance data were fitted to the equivalent

electrical circuit to obtain key parameters such as the electrochemical charge transfer

resistance for the anode. The Nyquist plots of the impedance spectra were analyzed using

NOVA 1.9 (NOVA Software). The CV was run at a scan rate of 30 mV/s with a scan

range of -1.0 to +1.0 V versus Ag/Agl and the voltammogram was compared for different

days in identical configuration and operational conditions.

3. RESULTS AND DISCUSSIONS

3.1 Performance of MFC

The performance of MFC fed with 50% of POME (COD=18,260 mg/l) and

inoculated with 1 ml of liquid culture was investigated. The MFC performance was

recorded for 13 days, as shown in Fig 1.

Fig. 1: Maximum power densities of

the MFC versus time.

Fig. 2: Polarization curves of the MFC on

the 3
rd

and 11
th

day using B. cereus.

IIUM Engineering Journal, Vol. 18, No. 2, 2017 Islam et al.

The performance of the MFC gradually increased until 7 days of operation and reached

maximum power density of 3.52 mW/m
3
. After 7 days of operation, it showed that stable

power generation was achieved for a few days. Maximum performance of 3,88 mW/m
3

achieved on the 11
th

day and started decreasing after 11 days of operation. Our previous

study [12] showed that B. cereus possesses electrogenic properties and could form an

effective biofilm. Moreover, usually Gram-positive bacteria develop viable biofilm

compared to gram negative bacteria and that could also be a reason for achieving a higher

performance in MFC3 [7]. However, the drastic decrease in power generation could be due

to the excessive bacterial colonization on the anode surface, which might have limited the

substrate access to the inner layer of the biofilm [18]. Hence, it reduced the number of

viable cells in the inner layer which in turn declined the power generation. The

polarization curve for the 3
rd

day and 11
th

day has been shown in Fig. 2. As can be seen

from the Fig. 2, the power generation on 11
th

day was around 3 times higher than the 3
rd

day. The higher performance on 11
th

day was attributed to the formation of an effective

biofilm on the anode surface that resulted in higher kinetics of the bio-electrochemical

reaction on the surface of the MFC anode [19].

3.2 Biofilm Analysis

The formation of a biofilm is vital for the MFC because it significantly influences the

electron transfer through the extracellular polymeric substances [20]. FESEM analysis was

done to visualize the biofilm on the anode electrode surface before and after operation (on

the 3
rd

and 11
th

days) of the MFC, and the results have been presented in Fig. 3a and 3b.

On day 3, as shown in Fig 3a, less bacterial colonization was scattered around the

electrode and loosely associated microbial clumps were observed in some locations on the

electrode surface. However, on day 11, it can be seen that the significant bacterial cells,

that colonized the electrode surface and formed the mature biofilm, are embedded in a

self-produced matrix of extracellular polymeric substance (EPS) (Fig. 3b). Hence

maximum performance was achieved by the MFC. Read et al. [7] reported that a biofilm is

a crucial component of the MFC that allows considerable conversion capacity and

opportunities for extracellular electron transfer. Moreover, the cells attached on the

electrode surface need less of a diffusion path for the transport of electrons from the

microbes to the electrode [21]. The results suggest that an effective biofilm, comprising of

higher active microorganisms might have produced more redox compounds that enhanced

the electron transfer efficiency as well as the performance of the MFC.

Fig. 3: FESEM images on anode carbon brush in the MFC fed with

POME as substrate, (a) day 3 and (b) day 11.

IIUM Engineering Journal, Vol. 18, No. 2, 2017 Islam et al.

3.3 Electrochemical Impedance Analysis

Nyquist plots for the anode configuration on different days of operation are shown in

Fig. 4. The measurement of EIS spectra for the anode demonstrates the key information of

resistance created by electrode and bacterial metabolism. The impedance at the high

frequency region is attributed to the ohmic resistance and the diameter of the semicircle

represents the polarization resistance (or charge transfer resistance), which is affected by

the kinetics of electrode reactions [22]. The charge transfer resistance (Rct) values using B.

cereus on the 3
rd

and 11
th

day were found to be 89.7 Ω, and 42.37 Ω, respectively. The

higher Rct using B. cereus (89.7Ω) on the 3
rd

day might be due to the absence of mature

biofilm on electrode surface (Fig. 3a). However, on the 11
th

day, the Rct was reduced by

52.95% (42.37 Ω) indicating that the B. cereus formed an effective biofilm on the anode.

Thus, it decreased the anode activation losses by enhancing the kinetics of the bio-

electrochemical reactions [19]. These results revealed that the effective biofilm is

necessary to facilitate the anodic electron transfer as well as power generation in the

MFCs.

Fig. 4: Nyquist plots of B. cereus on the

3
rd

and 11
th

day of operations.

Fig. 5: Cyclic voltammetry curves of B.

cereus on the 3
rd

day and 11
th

day of

operations.

3.4 Cyclic Voltammetry Analysis

CV analysis has been extensively used to evaluate the bioelectrocatalytic activity of

anodic biofilms and to determine the electron transfer mechanisms [23]. The CV was

conducted before inoculation and after inoculation on day 3 and 11, as shown in Fig 5.

Marked variations of catalytic behavior between the 3
rd

and 11
th

day are clearly observed

by CV analysis, as shown in Fig. 5. The observation that the maximum current reached up

to 0.23 A on day 11, which is about 2 times higher than that of 3
rd

day (0.01 A), might be

due to the excretion of large amounts of electron shuttle compounds in the anode [24].

Moreover, a reversible CV peak apparently demonstrates the redox transformation of

electro chemically active species [21]. The abundance of electron shuttle compounds

decreased the charge transfer resistance in the MFC by around 52%. The catalytic

measurements demonstrated that the presence of a recyclable electrochemical active

compounds produced by B. cereus would have mainly contributed to the electricity

generation in the MFC [12]. Moreover, the electrochemical activity of B. cereus behaved

distinctly different during different stages, suggesting that suspension cells were growing

or adhering onto the anode to form a biofilm in the MFC [25]. However, further study is

0 80 160 240 320 400
0

160

240

320

400

(B )

-Z
"

(o
h

m
)

Z' (ohm)

Day 3

Day 11

IIUM Engineering Journal, Vol. 18, No. 2, 2017 Islam et al.

needed in order to identify the redox compound produced by B. cereus.

4. CONCLUSION

The outcome of this study suggests that B. cereus is an efficient microorganism to be

used as inoculum in an MFC. B. cereus showed significant power production of about 3.9

W/m
3

using POME as substrate. The reversible CV peak confirmed that B. cereus

produced electron shuttle compounds that enhanced the electron transfer efficiency. The

anode charge transfer resistance decreased by 52%, indicating that the presence of an

efficient biofilm formation. The visualization of the biofilm by FESEM further confirms

the formation of effective biofilm after 10 days of operation. Further developments can be

made by mixing different kinds of microorganisms with B. cereus in order to possibly

improve the power generation of the MFC.

ACKNOWLEDGEMENTS

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

GRS 150371).

REFERENCES

[1] Pant D, Van Bogaert G, Diels L, Vanbroekhoven K. (2010) A review of the substrates used

in microbial fuel cells (MFCs) for sustainable energy production. Bioresource Technology,

101:1533-1543.

[2] Ismail ZZ, Jaeel AJ. (2013) Sustainable Power Generation in Continuous Flow Microbial

Fuel Cell Treating Actual Wastewater: Influence of Biocatalyst Type on Electricity

Production. The Scientific World Journal, 2013:13515-13522.

[3] Islam MA, Khan MR, Yousuf A, Wai WC, Cheng CK. (2016) Electricity generation form

pretreated palm oil mill effluent using Klebsiella variicola as an inoculum in Microbial fuel

cell. 4th International Conference on the Development in the in Renewable Energy

Technology (ICDRET): IEEE; p.1-4.

[4] Logan BE, Regan JM. (2006) Microbial fuel cells-challenges and applications.

Environmental Science & Technology,40:5172-5180.

[5] Majumder D, Maity JP, Tseng M-J, Nimje VR, Chen H-R, Chen C-C, et al. (2014)

Electricity generation and wastewater treatment of oil refinery in microbial fuel cells using

Pseudomonas putida. International Journal of Molecular Sciences,15:16772-16786.

[6] Li N, Liu L, Yang F. (2014) Power generation enhanced by a polyaniline–phytic acid

modified filter electrode integrating microbial fuel cell with membrane bioreactor.

Separation and Purification Technology,132:213-217.

[7] Read ST, Dutta P, Bond PL, Keller J, Rabaey K. (2010) Initial development and structure of

biofilms on microbial fuel cell anodes. BMC Microbiology,10:1-10.

[8] Nimje VR, Chen C-Y, Chen C-C, Jean J-S, Reddy AS, Fan C-W, et al. (2009) Stable and

high energy generation by a strain of Bacillus subtilis in a microbial fuel cell. Journal of

Power Sources,190:258-263.

[9] Vélez-Lee AE, Cordova-Lozano F, Bandala ER, Sanchez-Salas JL. (2015) Cloning and

expression of vgb gene in Bacillus cereus, improve phenol and p-nitrophenol

biodegradation. Physics and Chemistry of the Earth,91:38-45.

[10] Islam MA, Chee WW, Baranitharan E, Abu Y, Chin KC, Khan MR. (2016) Evaluation of

Electricity Generation and Wastewater Treatment from Palm Oil Mill Effluent Using Single

and Dual Chamber microbial fuel cell.

[11] Banerjee A, Ghoshal AK. (2010) Phenol degradation by Bacillus cereus: pathway and kinetic

modeling. Bioresource Technology,101:5501-5507.

IIUM Engineering Journal, Vol. 18, No. 2, 2017 Islam et al.

[12] Islam MA, Ethiraj B, Cheng CK, Yousuf A, Khan MMR. (2017) Electrogenic and anti-

methanogenic properties of Bacillus cereus for enhanced power generation in anaerobic

sludge driven microbial fuel cell. Energy & Fuels, 31(6):613-6139.

[13] Deng L, Li F, Zhou S, Huang D, Ni J. (2010) A study of electron-shuttle mechanism in

Klebsiella pneumoniae based-microbial fuel cells. Chinese Science Bulletin,55:99-104.

[14] Wrighton KC, Agbo P, Warnecke F, Weber KA, Brodie EL, DeSantis TZ, et al. (2008) A

novel ecological role of the Firmicutes identified in thermophilic microbial fuel cells. The

ISME journal,2:1146-1156.

[15] Marsili E, Baron DB, Shikhare ID, Coursolle D, Gralnick JA, Bond DR. (2008) Shewanella

secretes flavins that mediate extracellular electron transfer. Proceedings of the National

Academy of Sciences,105:3968-3973.

[16] Nandy A, Kumar V, Kundu PP. (2013) Utilization of proteinaceous materials for power

generation in a mediatorless microbial fuel cell by a new electrogenic bacteria Lysinibacillus

sphaericus VA5. Enzyme and Microbial Technology,53:339-344.

[17] Baranitharan E, Khan MR, Yousuf A, Teo WFA, Tan GYA, Cheng CK. (2015) Enhanced

power generation using controlled inoculum from palm oil mill effluent fed microbial fuel

cell. Fuel,143:72-79.

[18] Islam MA, Rahman M, Yousuf A, Cheng CK, Woon CW. (2016) Performance of Klebsiella

oxytoca to generate electricity from POME in microbial fuel cell. MATEC Web of

Conferences: EDP Sciences, 38:1-6.

[19] Ramasamy RP, Ren Z, Mench MM, Regan JM. (2008) Impact of initial biofilm growth on

the anode impedance of microbial fuel cells. Biotechnology and Bioengineering,101:101-

108.

[20] Marsili E, Rollefson JB, Baron DB, Hozalski RM, Bond DR. (2008) Microbial biofilm

voltammetry: direct electrochemical characterization of catalytic electrode-attached

biofilms. Applied and Environmental Microbiology,74:7329-7337.

[21] Islam MA, Woon CW, Ethiraj B, Cheng CK, Yousuf A, Khan MMR. (2017) Correlation of

power generation with time-course biofilm architecture using Klebsiella variicola in dual

chamber microbial fuel cell. International Journal of Hydrogen Energy, 41(41):25933-

25941.

[22] He Z, Mansfeld F. (2009) Exploring the use of electrochemical impedance spectroscopy

(EIS) in microbial fuel cell studies. Energy & Environmental Science,2:215-219.

[23] Samsudeen N, Radhakrishnan T, Matheswaran M. (2015) Bioelectricity production from

microbial fuel cell using mixed bacterial culture isolated from distillery wastewater.

Bioresource Technology,195:242-247.

[24] Islam MA, Woon CW, Ethiraj B, Cheng CK, Yousuf A, Khan MMR. (2016) Ultrasound

driven biofilm removal for stable power generation in microbial fuel cell. Energy & Fuels,

38(1):968-976.

[25] Islam MA, Karim A, Woon CW, Ethiraj B, Cheng CK, Yousuf A, et al. (2017)

Augmentation of air cathode microbial fuel cell performance using wild type Klebsiella

variicola. RSC Advances,7:4798-4805.