Bioresource Technology 104 (2012) 315–323

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Bioresource Technology
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Comparative bioelectricity production from various wastewaters in microbial

fuel cells using mixed cultures and a pure strain of Shewanella oneidensis
Vanita Roshan Nimje a, Chien-Yen Chen b,⇑, Hau-Ren Chen a,⇑, Chien-Cheng Chen c, Yuh Ming Huang d,
Min-Jen Tseng a, Kai-Chien Cheng b, Young-Fo Chang b
a
Department of Life Science, National Chung Cheng University, 168 University Road, Minhsiung, Chiayi 621, Taiwan
b
Department of Earth and Environmental Sciences, National Chung Cheng University, 168 University Road, Minhsiung, Chiayi 621, Taiwan
c
Department of Biotechnology, National Kaohsiung Normal University, No. 62, Shenjhong Rd., Yanchao Township, Kaohsiung County 82444, Taiwan
d
Department of Soil and Environmental Science, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: Current and power density from four wastewaters, agriculture (AWW), domestic (DWW), paper (PWW),
Received 29 May 2011 and food/dairy (FDWW), were comparatively evaluated in combination with three inocula: wastewater
Received in revised form 27 September 2011 endogenous microbes (MFC1), Shewanella oneidensis MR-1 (MFC2), and wastewater endogenous microbes
Accepted 29 September 2011
with MR-1 (MFC3) in single chamber microbial fuel cells (MFC). Using AWW (0.011 mA/cm2; 0.0013 mW/
Available online 29 October 2011
cm2) and DWW (0.017 mA/cm2; 0.0036 mW/cm2), MFC2 was the best candidate providing the maximum
current, whereas AWW-MFC1 and DWW-MFC1 were unable to construct a well-established MFC. FDWW
Keywords:
produced a maximum current from MFC3 (0.037 mA/cm2; 0.015 mW/cm2), and confirmed the unsuitabil-
Microbial fuel cell
Wastewater
ity of MFC2 at an alkaline pH. FDWW-MFC3 also performed best with the highest substrate degradation
Endogenous microbe and coulombic efficiency. Mixed culture in MFC3 resulted in higher current generation under the influence
Shewanella oneidensis of MR-1 (except in PWW), indicating the endogenous microbes were not solely responsible for the current
Inocula but the outperformance was significantly attributed to the association of MR-1.
Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction as fuel (Velasquez-Orta et al., 2011) and different microbial sources

Biological fuel cells (microbial and enzymatic) are rapidly grow-
ing alternative energy technologies in addition to the more tradi-
tional environmental energy sources such as wind, solar, and
geothermal power. A microbial fuel cell (MFC) is a system in which
microbes convert chemical energy produced by the oxidation of or-
ganic/inorganic compounds into ATP by sequential reactions in
which electrons are transferred to a terminal electron acceptor to
generate an electrical current (Torres et al., 2009).
Unlike chemical or enzyme-based fuel cells, which are custom-
ized to oxidize particular electron donors, MFCs have an enormous
electron donor adaptability (Ren et al., 2007). Substrates of partic-
ular interest for use in MFCs are soluble by-products of dark fer-
mentation (Kumar et al., 2008), with most of the focus being on
acetic acid and ethanol (Kiely et al., 2011a). Subsequently, the
interest in complex substrates led to tests on starch and cellulose
(Spets et al., 2008). In parallel, the use of different wastewaters
⇑ Corresponding authors. Tel.: +886 5 2720411x66220; fax: +886 5 2720807 (C.-
Y. Chen), tel.: +886 5 2720411x66503; fax: +886 5 2722871 (H.-R. Chen).
E-mail addresses: chien-yen.chen@oriel.oxon.org, yen@eq.ccu.edu.tw (C.-Y. Chen),
biohrc@ccu.edu.tw (H.-R. Chen).
(Logan and Regan, 2006) has been studied in depth. In general, for
all substrates collectively, there were large variations in power,
coulombic efficiency (CE), and the composition of the microbial
community.
To date, discrete entities of bacterial species of genus Geobacter
(Richter et al., 2008), Enterobacter (Rezaei et al., 2009), Shewanella
(Watson and Logan, 2010) and Bacillus (Nimje et al., 2009) have
been tested with respect to their generation or maximization of
the power output in MFCs. Recently, MFCs populated by mixed
microbial communities have garnered much attention owing to
their stability, robustness due to nutrient adaptability, stress resis-
tance and general tendency to produce higher current densities
than those obtained using pure cultures. However, in mixed-cul-
ture MFC systems direct comparison is not possible unless all other
conditions are kept exactly the same. Contrarily, the use of single
strains allows mechanistic and physiological details to be observed
directly. However, there is a compelling need to identify pure cul-
tures that can serve as appropriate models for electron transfer to
the anodes of MFCs, because little is known about the physiology of
this process (Lanthier et al., 2008).
Shewanella species were the first organisms proposed to trans-
fer electrons to the surface of electrodes via electron-transfer

0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2011.09.129
316 V.R. Nimje et al. / Bioresource Technology 104 (2012) 315–323
proteins (Kim et al., 1999). These species that assimilate fermenta-
tion products (Nealson and Scott, 2006) are nonfermenting, motile,
facultatively anaerobic bacteria found in suboxic sediments. It is
also very well known that wastewater contains complex organic
substrates whose degradation occurs through a complex series of
biochemical reactions manifested by a diverse group of selective
bacteria with prerequisite acidogens and methanogens (Mohan
et al., 2007). Under such circumstances, fermentation is the most
likely process that might occur in a MFC. The occurrence of an iso-
lated electrochemically active microbe that can oxidize different
electron donors/fermentative products, without necessitating the
expense of chemical catalysts, in synergy with a mixed culture
(producing fermentation products) would overcome the loss of
electrons to soluble fermentation products. This makes it an abso-
lute equivalent of Shewanella, electrochemically-active bacteria
that can oxidize a variety of fermentation products and utilize an
electrode as an electron acceptor (Kim et al., 1999). The importance
of Shewanella as a potential bioelectrochemical catalyst for MFC
technologies continues to drive research into the adaptability of
Shewanella for various substrates. There have been relatively few
such direct comparisons of pure and mixed cultures under identi-
cal conditions to mutually oxidize wastewater complex organic
substrates with fermentative products or byproducts.
Recently, Shewanella (MR-1) has been studied in MFCs by Kiely
et al. (2010), and its power production has been compared with
that of a mixed culture. The distinctive feature of their study lies
in the fact that MR-1 produced a greater power density than a
mixed culture. In another study, Watson and Logan (2010) com-
pared the power production by MR-1 to that of mixed cultures in
different reactors under constant solution chemistry. Contrary to
Kiely et al. (2010), the data obtained by Watson and Logan
(2010) showed a lower power production by MR-1 as compared
with the mixed cultures. However, it is difficult to ascertain these
differences in power production which might be due to (i) the dif-
ferent electron transfer mechanisms used by MR-1 in comparison
to the mechanisms of mixed cultures, or (ii) different pathways
may be used by the pure culture and mixed cultures in different
reactor configurations (Watson and Logan, 2010).
In order to more fully understand the differences in the current
produced by a Shewanella in pure culture system or in a mixed cul-
ture system, it is important to study their behavior individually or
in combination and analyze their roles in MFC, keeping the reactor
configuration the same. Such comparisons are needed to provide
insight into the effects of pure and mixed cultures on power
production.
The objective of our study was therefore to examine the current
production by MR-1 compared to that of a diverse consortium of
microbes using four different types of wastewaters as the sub-
strate. Wastewater contains high levels of easily degradable organ-
ic material. Moreover, the structure and composition of different
wastewaters strongly affects the power output by the microbial
community in MFCs. Hence, individual wastewaters were used un-
der three circumstances, which were then compared with regards
to the current-producing capability: (i) wastewater endogenous
microflora (MFC1), (ii) MR-1 (MFC2), and (iii) wastewater endoge-
nous microflora with MR-1 (MFC3). The overall performance of the
MFC was reviewed in terms of chemical oxygen demand (COD),
coulombic efficiency (CE), maximum power density from polariza-
tion and power curves, and cyclic voltammetry.
2. Methods
2.1. Wastewater as substrates and their sources
Agricultural wastewater (AWW) was collected from a banana
orchard in Chaiyi (Taiwan); domestic wastewater (DWW) was
collected from the wastewater treatment unit at National Chung
Cheng University, Chia-yi, Taiwan, prior to treatment; paper
industry wastewater (PWW) was collected from the Cheng Loong
Corp., Dali (Taiwan); and food/dairy wastewater (FDWW) was col-
lected from the Kuang Chuan Dairy Co., Minsyong (Taiwan).
2.2. Analysis of various parameters
Although, origin and nature of wastewater are crucial for the
current generation, the characteristics of wastewater also play an
important role in the electricity generation. Organic material and
wastewater components are prerequisites for microbial metabolic
reactions, which are also required for current production. Waste-
water parameters are characterized as chemical oxygen demand
(COD), biochemical oxygen demand (BOD), total dissolved solids
(TDS), volatile fatty acids (VFA), color, temperature, pH, etc. Being
rich in COD, organic material broken down by the microorganisms
could result in the formation of VFAs, however, altering the pH of
the wastewater. Hence, this is the most likely reason to character-
ize the parameters such as pH, temperature, COD, TDS, conductiv-
ity and VFA. The wastewater characteristics of the raw effluents are
presented in Table 1. pH, temperature, COD, TDS, conductivity and
VFA were determined before and during the experimental period.
pH, temperature, TDS and conductivity were measured using an
HI-9828 multiparameter (Code No. HI710046; made in Romania).
COD was measured using a commercially-available kit (COD
Reagent; COD-S; CAS [7664-93-9]). VFA was determined by gas
chromatography (Bogaard Van-Den et al., 1986).
2.3. Microorganisms and culture conditions
2.3.1. MFC1
Wastewater with its endogenous microflora. Mixed cultures
were prepared as follows: 75 mL AWW, DWW, PWW and FDWW
were individually collected in 200-mL conical flasks, then an anode
(surface area – 22.5 cm2) was inserted and acclimatized by the daily
addition of raw wastewater using a syringe placed in a screw cap.
2.3.2. MFC2
Shewanella oneidensis (MR-1) with different wastewaters. MR-1
was initially grown by inoculating 50 mL of LB broth with gentle
shaking (100 rpm) for approximately 70 h. The LB growth media
was dissolved in 50 mM phosphate buffer adjusted to pH 7.
AWW, DWW, PWW, and FDWW were sterilized in autoclave at
121 °C for 20 min for use in MFC2. No significant changes were
measured in the parameters such as pH, COD, TDS, conductivity
and VFA before and after wastewater sterilization. From the LB
broth, 5 mL of MR-1 culture was harvested and subcultured in
75 mL sterilized and autoclaved AWW, DWW, PWW and FDWW
collected in a 200-mL conical flask. An anode (surface area
22.5 cm2) was inserted and acclimatized by the daily addition of
sterilized wastewater using a syringe placed in a screw cap.
2.3.3. MFC3
S. oneidensis (MR-1) with different wastewaters and their endoge-
nous microflora. Following MR-1 growth in LB broth, 5 mL of culture
and 75 mL raw AWW, DWW, PWW and FDWW were collected in 200-
mL conical flasks. An anode (surface area 22.5 cm2) was inserted and
acclimatized by the daily addition of raw wastewater using a syringe
placed in a screw cap.
Wastewater as a substrate was added daily for 5 days for MFC1,
MFC2 and MFC3 and the whole acclimatization period was 10 days.
Before addition, 15 mL of substrate/wastewater was first removed
from each 200-mL conical flasks (75 mL broth) followed by the
addition of 15 mL of sterilized wastewater. During the acclimatiza-
tion period, the microbes evolved rapidly to anaerobic conditions.
 V.R. Nimje et al. / Bioresource Technology 104 (2012) 315–323 317

Table 1
Wastewater characteristics and open circuit potential (OCP), maximum current, maximum COD removal and coulombic efficiency (CE) from different MFCs.

Substrate characteristics OCP (mV) CCP (mV) Current (mA) CODTotal consumed VFA consumed CE (%)
4 day 7 day 12 day 18 day
AWW MFC1 301 140 0.14 361 13 0 ND ND 0.0002
pH – 7.1 MFC2 326 250 0.25 256 0 0 ND ND 0.003
TDS – 98 MFC3 311 250 0.25 269 0 0 ND ND 0.003
EC – 68
COD Total – 397 ± 15
VFA – 17
DWW MFC1 321 320 0.32 539 11 6 ND ND 0.18
pH – 6.8 MFC2 466 430 0.43 496 0 0 ND ND 0.71
TDS – 486 MFC3 457 430 0.43 481 0 0 ND ND 0.64
EC – 103
CODTotal – 671 ± 20
VFA – 19
PWW MFC1 571 430 0.43 996 117 96 124 ND 1.4
pH – 8.6 MFC2 687 320 0.32 435 0 0 ND ND 0.008
TDS – 571 MFC3 542 450 0.45 987 121 132 127 ND 1.5
EC – 76
CODTotal – 1250 ± 15
VFA - 113
FDWW MFC1 647 600 0.6 1202 189 137 96 84 2.5
pH – 5.3 MFC2 622 700 0.7 1296 0 0 0 0 2.7
TDS – 572 MFC3 624 730 0.73 1374 156 123 47 12 2.9
EC – 282
CODTotal – 1562 ± 20
VFA – 147

(1) AWW – agricultural wastewater; DWW – domestic wastewater; PWW – paper wastewater; FDWW – food/dairy wastewater.
(2) TDS – total dissolved solids in mg/L; EC – electrical conductivity in lS/cm; CODTotal – total COD (in mg/L) including VFA; VFA (in mg/L) was obtained separately and added
in the COD value to obtain CODTotal.
(3) CCP (mV) – closed circuit potential obtained at an external resistance of 1 kX.
(4) Current (mA) – maximum current obtained at an external resistance of 1 kX.
(5) VFA was measured separately at the mentioned time (at 4, 7, 12 and 18 day) and added in the COD value to obtain CODTotal consumed.
(6) ND – not detected.

The anodes from these cultures were obtained for direct use in the
MFC experiments. The schematics of the three different MFCs,
MFC1, MFC2 and MFC3, using different MFC inocula are presented
in Fig. 1(A).
2.4. Microbial fuel cell construction and operation
All experiments were carried out using a simplified single-
chamber MFC as depicted in Fig. 1(B). The anode (dimensions:
2.5  4.5 cm; projected area of 22.5 cm2) and cathode electrodes
were made of carbon cloth (internal diameter: 3 cm) and the
cathode was loaded with four PTFE diffusion layers, as de-
scribed by Cheng et al. (2006), without a platinum catalyst.
Copper wire was used to connect the circuit, and the fuel cell
was placed under a constant load by connecting the anode
and cathode to an external resistance of 1 kX. The MFC was
operated in a batch mode over a full cycle, where the duration
for each batch cycle varied depending on the substrate con-
sumption and voltage reduction to a low value of 50 ± 5 mV
from the maximum. All experiments were conducted at room
temperature (22 ± 2 °C).
2.5. Data acquisition and calculations
The potential of the reactor was measured across an external
resistance (Rext) of 1 kX every one minute using a PicoLog recorder
(PicoLog@ v. 5.09.4 recorder software (Pico Technology)) with an
RS232 interface connected to an ADC 20–21 A–D converter (Pico
Technology Ltd., Cambridgeshire, UK). In order to evaluate the
performance of the MFC, in a period of maximum current genera-
tion, polarization and power curves were obtained. To obtain the
polarization curves, different Rext (3900, 2700, 1000, 680, 560, 470,
390, and 330 X) were applied, with each resistance being connected
for 15 min. Potential (V) was monitored in order to calculate the cur-
rent (I), as I = V/R. Power was normalized based on the cross-sec-
tional area (projected area = 22.5 cm2) of the anode in order to
calculate the power density in mW/cm2 as: P = I  V/22.5 cm2. Based
upon the polarization curve, the internal resistance of the MFC was
calculated as: Rint = (Vo-c/IL)  RL, where Vo-c is the open circuit volt-
age, IL is the current under resistance, and RL is the external resis-
tance (Ieropoulos et al., 2008). Coulombic efficiency is calculated
R
as CE = ( I dt/[(DCOD/32  1000)  4  V  96,480])  100%, where
I is current, DCOD (mg/L) is the difference of COD (mg/L) value be-
tween influent and effluent of anode chamber, V (L) is the volume
of wastewater treated, 4 is the number of mol of 1 mol O2 related
electrons, 32 is the molecular weight of O2 (Zhang et al., 2010).
2.6. Cyclic voltammetry
Cyclic voltammetry (CV) was carried out using a potentiostat
(CHI 627C; CH instrument, USA) connected to a personal computer
(CHI627C Electrochemical Analyzer) with a scan rate of 0.1 V s1,
ranging from 0.45 to 0.6 V. A conventional three-electrode set-
up was employed, with the anode as the working electrode, an
Ag/AgCl reference electrode, and platinum wire as the counter
electrode. CV is a standard tool in electrochemistry, and can pro-
vide valuable insight into the electron transfer interactions be-
tween microorganisms or microbial biofilms and microbial fuel
cells. Hence, voltammograms were obtained during a period of
maximum current generation to detect the electrochemical prop-
erties of the MFCs.
318 V.R. Nimje et al. / Bioresource Technology 104 (2012) 315–323

Fig. 1. (A) Pictorial representation of agriculture (AWW), domestic (DWW), paper (PWW) and food/dairy (FDWW) wastewaters used in MFC1, MFC2 and MFC3. (B) Schematic
of single chamber microbial fuel cell.

Table 2
3. Results and discussion Current, power density and internal resistances from polarization curve obtained
against various external resistances from different MFCs.
3.1. Utilization of different wastewaters for electricity generation with Substrate Current (mA) Power density (mW/cm2) Rext (X) Rint (X)
different microbial inocula
AWW
MFC1 0.18 0.0009 680 990
The ability of different bacterial inocula to utilize wastewater as MFC2 0.23 0.0013 560 800
a substrate to produce a current in an MFC was assessed using MFC3 0.22 0.0012 560 800
AWW, DWW, PWW and FDWW. For individual wastewaters, the DWW
current generation under three different conditions of microbial MFC1 0.2 0.0016 560 868
inocula was evaluated (as mentioned in Section 2 and Fig. 1) by MFC2 0.38 0.0036 560 676
MFC3 0.41 0.0042 560 560
comparing the OCP, maximum current generation, COD and VFA
removal, and CE, the values of which are presented in Tables 1 PWW
MFC1 0.51 0.008 680 320
and 2. MFC2 0.3 0.0028 680 653
MFC3 0.52 0.0082 680 320
3.2. Agriculture wastewater FDWW
MFC1 0.65 0.013 680 320
AWW with an initial COD of 397 ± 15 mg/L at pH 7.1 Visible MFC2 0.66 0.013 680 229
improvement in the current generation pattern was observed in MFC3 0.84 0.015 470 244

the order MFC3 > MFC2 > MFC1, as depicted in Fig. 2. The lowest
OCP and current was recorded from MFC1, with steady-state cur-
rent generation followed by progressive reduction. Contrary to
MFC1, MFC2 exhibited a marked improvement in OCP and an
appreciable and rapid current increase and, most probably, com-
plete consumption of the substrate resulted in a current decrease
after the stable plateau in a relatively short period as compared
with MFC1 (Fig. 2(C)). The trend of current generation by MFC1
indicated the presence of non-electroactive microbes that might
have occupied space on the electrode, and metabolizing the sub-
strate to thrive or at least to survive rather than generating current.
Whereas, the transfer of electrons to the anode was favored in the
presence of MR-1 (MFC2), with enhanced current generation.
Alternatively, the presence of MR-1 in MFC3 minimized the effect
of the low current generation from MFC1, producing a similar
maximum current (0.25 mA; 250 mV) within a shorter period than
that of MFC1 and MFC2. These results are in good agreement with
Kiely et al. (2010), who also demonstrated this unpredicted finding
and the electroactive capability of Shewanella to eventually pro-
duce a higher current than a mixed culture.
Further, the COD degradation and CE in all three MFCs were eval-
uated (Fig. 2(B)). The COD decreased in all three MFCs from the
influent initial value of 397 ± 15 mg/L down to a steady state value
of 361 mg/L (MFC1), 269 mg/L (MFC2), and 256 mg/L (MFC3).
Hence, it is interesting to interpret that MFC1 consumed the maxi-
mum substrate/COD for the production of new non-electroactive
biomass and exhibited a quite slow electron discharge, with the
lowest CE of 0.0002%. When MFC2 and MFC3 were operated with
 V.R. Nimje et al. / Bioresource Technology 104 (2012) 315–323 319

0.35 AWW-B
AWW-A
MFC1 400
0.30 MFC2
MFC3

COD (mg/l) -CE (%)

300
Current (mA) 0.25

0.20 200

0.15
100
0.10
0
0.05 MFC1 MFC2 MFC3

0.00 COD 361 269 256

0 2 4 6 8 CE
Time (Days) 0.0002 0.003 0.003

400 0.0015 1.0

AWW-C AWW-D

Power density (mW/cm )

2
0.0012 0.5
Potential (mV)

300

Current (mA)
0.0009 0
200
0.0006 -0.4

100
0.0003
-0.9

0 0
0 0.1 0.2 0.3 -1.4
-0.5 -0.3 -0.2 0 0.1 0.3 0.5 0.6
Current (mA) Potential (V)

Fig. 2. Current output (A); COD removal and CE (B); power and polarization curve (C) [MFC1 (N), MFC2 (d), MFC3 (})]; cyclic voltammogram (D) for AWW.

an attached biofilm on the anode, along with COD removal, an in- 3.3. Domestic wastewater
creased CE was also achieved as compared with MFC1. Alterna-
tively, greater COD removal in MFC3 as compared with MFC2 also DWW was tested with regards to its capacity to produce cur-
raises the prospect of simultaneous substrate utilization (electron rent, with an initial COD of 671 ± 20 mg/L and pH 6.8, as shown
consumption) by AWW endogenous microbes for their growth in Fig. 3(A). In MFC1, four phases of the current–time curve were
and by electroactive MR-1 for electron transfer to the anode. Hence, observed. Following a lag phase (20 h), an exponential increase
the result concluded the existence of a unique versatile electricigen (48 h) and a stable plateau (72 h; 0.32 mA; 320 mV) were
MR-1 with greater performance in MFC2 and MFC3 that has the po- observed, followed by progressive current reduction. This behavior
tential to metabolize AWW more efficiently and in less time than suggested that although a fairly long enrichment period (10 days)
the AWW mixed culture. in closed flasks was provided (see Section 2), the endogenous
Polarization and power density curves were obtained from the microbes in MFC1 were growing entirely in a planktonic phase.
MFCs with AWW, as shown in Fig. 2(C). The variable resistance de- These results also implied that the current generated during the
picts how effectively the bacteria generated the maximum power exponential phase was due to the utilization of easily-assimilated
and current. MFC1 exhibits a steep voltage drop, implying higher compounds, probably for biomass production, followed by the oxi-
polarization/electron transport resistance and hence lower power dation of complex compounds in a stable phase of current genera-
densities (Table 2). In contrast, MFC2 and MFC3 exhibited a maxi- tion, where biomass growth and stability was at a maximum.
mum power density, which produced the highest OCP, maximum Contrarily, an instantaneous increase in the current generation
current, and a constant rate of voltage fall during polarization. (maximum 0.43 mA; 430 mV) with a stable period of 5 days, fol-
Fig. 2(D) elucidate that MFC1 produced flat voltammetry curves lowed by rapid current reduction, was recorded in MFC2, demon-
and no Faradic peaks between 0.45 and 0.6 V (vs. Ag/AgCl). How- strating that the attached bacteria on the anode rather than the
ever, there was the possibility that monolayers of cells might dis- suspended bacteria mainly contributed to electricity generation.
perse across the anode, due to which a sudden current rise was Otherwise, the current output should decrease due to the removal
observed in CV. The voltammogram generated in MFC2 had the of most of the suspended bacteria with anolyte replacement (when
appearance of uniform oxidation peak active near +0.3 V (0.55 the anode with attached bacteria was transferred from the closed
mA), whereas this peak reappeared with a high current intensity flask to the MFC reactor). In parallel, the immediate current gener-
(+0.3 V; 0.85 mA) in MFC3. No oxidation peak in the potential ation (maximum 0.43 mA; 430 mV) by MFC3 was attributed to the
region was observed for MFC1; this discrepancy could be a result direct consequence of the thick biofilm formation by MR-1 and
of differing metabolite and/or electron donor concentrations be- endogenous microbes, which was explained in the research
tween MFC1 (without MR-1), MFC2 and MFC3 (with MR-1) (Fricke conducted by Dumas et al. (2008). According to this research, bac-
et al., 2008). This result also suggests that the mediator of en- teria in a biofilm have comparatively different properties than
hanced electrogenic activity in the wastewater was present at sig- planktonic bacteria. The dense and protected environment of the
nificant concentrations after MR-1 enrichment rather than being a film allows the bacteria to cooperate and interact by various phys-
redox component already present at high concentrations in the ical and chemical means. However, continuous conductance of
wastewater. electron transfer through the biofilm (MR-1 covering the anode
320 V.R. Nimje et al. / Bioresource Technology 104 (2012) 315–323

0.6 MFC1
DWW-A
DWW-B
MFC2
MFC3 600
0.5

COD (mg/l) -CE (%)

500
0.4
Current (mA)

400

0.3 300

200
0.2
100

0.1 0
MFC1 MFC2 MFC3
COD 539 496 481
0.0
0 2 4 6 8 CE 0.18 0.71 0.64
Time (Days)
500 0.0048 3.7
DWW-D
DWW-C

Power density (mW/cm 2 )

400 1.7
0.0036
Potential (mV)

Current (mA)
300
-0.3
0.0024
200
-2.3
0.0012
100
-4.3

0 0
0 0.2 0.4 0.6 -6.3
-0.4 -0.2 0 0.2 0.4 0.6
Current (mA) Potential (V)

Fig. 3. Current output (A); COD removal and CE (B); power and polarization curve (C) [MFC1 (N), MFC2 (d), MFC3 (})]; cyclic voltammogram (D) for DWW.

surface) reorients and selects specific electroactive bacteria from oxidation peak at the midpoint potential (0.0 V; 2.7 mA) might be
the planktonic growth in the biofilm matrices, which might be a related to some electroactive bacterial species from planktonic
probable reason for the thick biofilm formation and high electro- growth in biofilm matrices that might interact with the anode
chemical current observed in MFC3 comparative to MFC1 (which through a different mediator. To be more explicit, the oxidation
can be better observed from the voltammogram (Fig. 3(D)). peaks in MFC2 and MFC3 were presumably related to the continu-
Fig. 3(B) depicts the COD decrease for MFC1, MFC2 and MFC3 ous production or presence of a redox component (by MR-1), which
from the initial 671 ± 20 mg/L down to a steady state value of catalyzed electron transfer to the anode (Velasquez-Orta et al.,
539 mg/L (CE – 0.18%), 496 mg/L (CE – 0.71%) and 481 mg/L (CE 2011).
– 0.64%). From MFC1, it can be inferred that the planktonic bacteria
were utilizing the highest COD while achieving the lowest CE. 3.4. Paper industry wastewater
However, clear observation suggested that MFC2 and MFC3 under
the influence of MR-1 appeared to attain a maximum current in In MFC1 and MFC3, the pattern of current generation was highly
quick succession, with a greater CE than that observed for MFC1. reproducible: the current increased immediately, with a com-
Fig. 3(C) illustrates the polarization and power density curves as pletely different behavior of a bimodal nature with progressive
a function of current. It can be seen that the maximum power den- current increasing and decreasing pattern (Fig. 4(A)). This bimodal
sity increased from MFC1 to MFC3. In MFC1, the voltage dropped current generation behavior was likely due to progressive utiliza-
suddenly in conjunction with an increased current, notably due tion of soluble COD to particulate COD (Huang and Logan, 2008),
to the increased internal resistance (868 X) and most likely due a strategy followed by mixed bacterial communities to progres-
to the corresponding increase in ohmic loss (Hong et al., 2009). Po- sively deplete an organic substrate (Cercado-Quezada et al.,
tential dropped linearly with increasing current, which showed 2010). The first peak could have been due to utilization of easily-
that ohmic losses caused by resistance to the flow of electrons assimilated compounds such as VFAs, while the secondary peak
through the electrodes and through the electrolyte were dominant might be a result of complex organics utilization (Kiely et al.,
in MFC2 and MFC3. 2011b). It was surprising to note that MFC2 achieved the highest
Voltammograms plotted for DWW MFC1 did not show any Fara- OCP (687 mV > MFC1 > MFC3, Fig. 4(C)) but was found to be inop-
dic peaks (Fig. 3(D)). However, the current produced was due to sus- erative and generated the lowest current among all three MFCs
pended bacteria, whereas a biofilm was necessary for the oxidation with PWW. A study conducted by Aelterman et al. (2008) demon-
reaction to occur, which could facilitate electron transfer by achiev- strated that the increasing loading rate (COD) needs to be accom-
ing direct contact with the electrode and may be due to compounds panied by a decreased Rext in order to increase the continuous
bound to the cell membrane (Parot et al., 2008). An oxidation peak current generation. Contrarily, the results from AWW-MFC2 and
in MFC2 at the potential of +0.3 V (3 mA) confirmed the redox DWW-MFC2 showed an efficient performance with respect to cur-
system, which appeared typically and was pertinent to MR-1 in rent generation at the same Rext of 1 kX, although DWW was asso-
our study. The simplicity of the voltammogram with only one oxida- ciated with a high COD content. However, the result of low current
tion peak in MFC2 gives way to more complex behavior in MFC3, generation from PWW-MFC2 (MR-1) was unexplainable by the fact
with multiple oxidation peaks. The MR-1-related oxidation peak that the high resistance leads to less current and a decreased elec-
occurred at a high catalytic current (+0.3 V; 3.7 mA), and an tron transfer rate to the anode, leading to less energy generation
 V.R. Nimje et al. / Bioresource Technology 104 (2012) 315–323 321

0.6 PWW-A PWW-B

MFC1
MFC2 1000
0.5 MFC3

COD (mg/l) -CE (%)

800
0.4
Current (mA)

600
0.3
400
0.2
200

0.1 0
MFC1 MFC2 MFC3
0.0 COD 996 435 987
0 2 4 6 8 10 12 14 16
Time(Days) CE 1.4 0.08 1.5

800 0.0105 3.6

PWW-D

Power density (mW/cm 2)

PWW-C
0.0084 2.4 MFC3
600 MFC2
Potential (mV)

Current (mA)
0.0063 1.2
400 MFC1
0.0042 0

200
0.0021
-1.2

0 0
0 0.15 0.3 0.45 0.6 -2.4
-0.5 -0.3 -0.2 0 0.1 0.3 0.5 0.6
Current (mA)
Potential (V)

Fig. 4. Current output (A); COD removal and CE (B); power and polarization curve (C) [MFC1 (N), MFC2 (d), MFC3 (})]; cyclic voltammogram (D) for PWW.

for an electrogenic microbe. However, this dampening effect of low produced by the mixed cultures (MFC1 and MFC3) with progres-
current generation at pH 8.6 for MR-1 is relatively uncharted. sive current diminution revealed the presence of a well-estab-
In terms of COD removal (Fig. 4(B)), MFC1 and MFC3 exhibited lished acidogenic microbial community in the wastewater, but
good substrate degradation, with similar COD removals of 996 mg/ the similar electrical current attained using MR-1 at an acidic pH
L (CE – 1.4%) and 987 mg/L (CE – 1.5%), while the lowest substrate in MFC2 with rapid reduction was quite noteworthy (Fig. 5(A)).
degradation (435 mg/L COD; CE – 0.008%) was recorded in MFC2. A Most studies emphasize a better electrical activity and maximum
reduced pH of 7.4 ± 1 with higher VFA generation were also regis- current generation by microbes at neutral to basic pHs, whereas
tered in MFC1 and MFC3, which remained non-assimilated until an acidic pH has been found to have a negative effect on microbial
the end of cycle (Table 1). physiology. Gaboriaud et al. (2006) very precisely demonstrated
Fig. 4(C) compares the polarization curves as a function of the how experimental conditions, specifically pH, can drastically affect
measured steady-state currents. For MFC1 and MFC3, a compara- bacterial adhesion and physiology. Changes in the current-produc-
tively higher potential drop with effective electron discharge was ing capability of Shewanella are predominantly related to pH:
observed at lower Rext, which indicates increased power output. Shewanella’s polymeric outer membrane swells and softens in
A consistent increase in current was also observed with a decrease response to an increase in pH from 4 to 10, changing the electro-
in Rext. On the contrary, the highest OCP (687 mV) for MFC2 sug- phoretic transport rates of particles through the membrane and
gests that although Shewanella can accumulate charge, the release affecting the electron transfer rate through the membrane, which
of electrons is less efficient than in the mixed culture, as the de- ultimately decreases power production at higher pHs (Dague et
crease in working voltage in the low current region indicates larger al., 2006; Gaboriaud et al., 2006). This could be why we observed
activation losses and relatively high Rint (653 O) could result in better electrical activity in MFC2 (MR-1) with an acidic pH as com-
higher anode overpotentials, limiting the power generation in pared with PWW-MFC2 (MR-1) with an alkaline pH. Considering
MFC2 (Watson and Logan, 2010). that Shewanella may synthesize mediators for enhanced electron
CV depicted no redox peaks for MFC1 and MFC3 (Fig. 4(D)), transfer, the role acidity plays on autologous mediators could be
although bacterial attachment in the form of a biofilm was clearly quite pronounced (Biffinger et al., 2008). Maximum current in
visualized on the anode possibly because of increased fermentable MFC3 with MR-1 might be attributed to the increased electro-
compounds that favored fermentative microbial metabolism over chemical activity of the mediators synthesized by Shewanella at
electron transfer to the anode (Freguia et al., 2007). Similarly, no pH 5.3, which influenced the process performance and accounted
redox activity was noted from MFC2, which is still a matter for for the increased current generation in MFC3.
investigation. Finally, the rapid and progressive behavior of current reduction
was explained suitably by Liu et al. (2010). The trend of progressive
3.5. Food/dairy processing wastewater current diminution is a biological phenomenon that might be a
consequence of the oxidation of fermentation products rather than
Studies on FDWW with a COD of 1562 ± 20 mg/L and pH 5.3 a chemical reaction; this was clearly observed in MFC1 and MFC3
shows a significant current generation from MFC1 and MFC2 with FDWW endogenous microbes. Contrarily, current generation
(0.65 ± 0.05 mA; 650 ± 50 mV). The effective current at pH 5.3 from MFC2 by MR-1 could be a chemical phenomenon, as a rapid
322 V.R. Nimje et al. / Bioresource Technology 104 (2012) 315–323

0.9 FDWW-A
MFC1 FDWW-B
0.8 MFC2
1500
MFC3

COD (mg/l) -CE (%)

0.7
1200
0.6
Current (mA)

900
0.5

0.4 600
0.3 300
0.2
0
0.1 MFC1 MFC2 MFC3
COD 1202 1296 1374
0.0
0 2 4 6 8 10 12 14 16 18 20 CE 2.5 2.7 2.9
Time (Days)
1.1
800 0.016
FDWW-C FDWW-D
0.8

Power density (mW/cm 2)

600 0.012
Potential (mV)

Current (mA)
0.4

400 0.008
0.1

200 0.004
-0.3

0 0 -0.6
0 0.2 0.4 0.6 0.8 1 -0.4 -0.2 0 0.2 0.4 0.6
Current (mA) Potential (V)

Fig. 5. Current output (A); COD removal and CE (B); power and polarization curve (C) [MFC1 (N), MFC2 (d), MFC3 (})]; cyclic voltammogram (D) for FDWW.

current reduction was always observed (with AWW, DWW, PWW, The best performance was obtained from MFC3 with a maximum
and FDWW). The progressive current reduction in MFC3 (after power density of 0.015 mW/cm2 and a current of 0.84 mA (840
18 days) might be due to slow and steady utilization of the few mV) at 470 X Rext. Whereas, MFC3 and MFC2 documented the
remaining fermentation products by MR-1/electrogenic microbes. lowest Rint in comparison with MFC1. Hence, mediator production
High COD and greater community diversity of either fermenta- (by MR-1) might be the foremost reason for the high power density
tive/methanogenic and/or exoelectrogenic bacteria present in the and low Rint (Logan and Regan, 2006).
FDWW are of greater values because, fermentative/methanogenic The voltammogram shown in Fig. 5(D) reveals the electrochem-
microbes could oxidize complex organics into breakdown products ical activity to be in the order of MFC3 and/or MFC2 > MFC1. MFC1
like amino acids and VFA’s while, exoelectrogenic bacterial popula- produced a flat curve with no redox peaks, which might be due to
tion like MR-1 reflect the capability to assimilate the byproducts of the rise in fermentable compounds (Freguia et al., 2007), whereas
fermentation/respiration. However, the COD and VFA concentra- an oxidation peak pertinent to MR-1 (0.3 V; 0.5 mA) was obtained
tions were monitored to estimate substrate removal and the in MFC2, with its reappearance in MFC3 (0.3 V; 0.52 mA). It is be-
changes in fermentable compounds. Maximum COD degradation lieved that sugar-fermenting bacteria in dairy wastewater can
and maximum CE followed the order MFC3 > MFC2 > MFC1 interact with current-producing microbes in a fashion that is either
(Fig. 5(B)). While MFC1 and MFC3 exhibited a transient increase neutral, positively-enhancing, or even negatively-affecting (Gupta
in VFA followed by its assimilation at different concentrations et al., 2011). Using (electro)-chemical analysis and transcription
(Table 1), MFC2 exhibited no VFA accumulation, with moderate profiling, Rosenbaum et al. (2011) found that the bioelectrochem-
COD removal and a moderate CE was established. Although VFA ical performance and S. oneidensis physiology did not differ signif-
accumulation was observed in MFC3, it was followed by maximum icantly whether it was grown as a pure culture or a co-culture.
consumption corresponding to that of MFC1 until the end of the Thus, the microbes worked together in a purely substrate-based
cycle. With the knowledge that fermentation side products were (neutral) relationship. From our results, it could be postulated that
possibly produced in MFC1, and MR-1 does not produce VFA MR-1 was positively interacting with the inoculum/substrate; this
(MFC2), it was confirmed that Shewanella is a non-fermenting spe- physiology of MR-1 appears to be true in MFC3. Although FWW
cies, whereas it can also assimilate fermentative products (Nealson endogenous microbes generated fermentative products, MR-1
and Scott, 2006) and redirect the electrons from VFA oxidation to worked in the same system purely in a substrate-based relation-
produce more electrons in MFC3. This increased versatility allowed ship, with the same bioelectrochemical activity and same physiol-
the metabolism of VFA in MFC3. ogy of mediator production, as confirmed by the reappearance of
The polarization performance of all three MFCs demonstrates an an oxidation peak in MFC3 along with an identical and intense cur-
almost equal performance in an acidogenic environment (Fig. 5(C)). rent production.
The values for maximum current, maximum power density and The overall results from AWW, DWW, PWW, and FDWW reveal
internal resistance are presented in Table 2. Rapid stabilization of MR-1 as a versatile microbe that can metabolize multiple electron
OCP was also observed at higher Rext and a relatively high potential donors for efficient electricity generation in conjunction with a
drop and slow stabilization of the voltage was observed at lower mixed culture, whereas this interaction of MR-1 with a mixed
Rext, which might be due to effective electron discharge at lower Rext. culture could be positively or negatively dominated under certain
 V.R. Nimje et al. / Bioresource Technology 104 (2012) 315–323
circumstances: positively dominated in (1) surrounding microenvi-
ronment with nonelectrogenic microbes (AWW) and (2) where mi-
crobes in the nearby environment follow a typical bacterial growth
behavior, whereas the anode with attached MR-1 functions well and
initiates immediate electron transfer (DWW); (3) this interaction is
negatively affected/dominated by high alkaline conditions (PWW);
(4) positively interacted by producing mediators of high electro-
chemical activity under acidic conditions (FDWW). In accordance
with Biffinger et al. (2008), pH is a very important aspect of Shewa-
nella’s physiology, and is linked to the potential secretion of media-
tors, which could impart a negative or positive effect on current
generation by Shewanella. Experiments performed by Biffinger
et al. (2008) demonstrated the current production ability of Shewa-
nella, where at an acidic pH of 5 mediator production accounted for
>70% of the electrochemical activity at pH 7. Hence, this could be a
likely reason why FDWW (pH 5.3) exhibited the best electrical cur-
rent and electrochemical activity with MR-1 in contrast to PWW
(pH 8.6) where, lowest electrical current and activity was achieved
and maximum substrate remains quite recalcitrant.
4. Conclusion
Electricity generation was compared with COD removal from
different wastewaters in combination with three inoculum
sources. AWW not well-suitable unless accompanied by electro-
genic microbes; DWW endogenous microbes with planktonic
growth phase took longer period to attain maximum current gen-
eration comparative to MR-1; PWW endogenous microbes were
able to produce electricity at an alkaline pH, but MR-1 performance
gets drastically affetcted with lower electron discharge; while
FDWW was a suitable substrate to exploit in a MFC with its endog-
enous microbes and specifically with electrogeneic bacterial spe-
cies such as Shewanella, which has the potential to utilize
fermentation products from FDWW.
Acknowledgement
The authors are very grateful to the Science Council of Taiwan
for sponsoring this research (NSC 99-2116-M-194-004).
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