Membraneless Microbial Fuel Cell: Characterization of

Electrogenic Bacteria and Kinetic Growth Model

Muaz Mohd Zaini Makhtar 1 and Vel Murugan Vadivelu 2

Abstract: The generation of electricity in a membraneless microbial fuel cell (ML-MFC) was studied using dewatered sludge containing a
mixed culture of electrogenic bacteria (EB). The EB acted as a biocatalyst to enhance the degradation of chemical oxygen demand (COD).
Scanning electron microscope (SEM) observations revealed the formation of a biofilm at the anode surface. Phylogenetic analysis proved the
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presence of Pseudomonas species and Bacillus subtilis, which actively boosted the electron transfer, in the biofilm. Moreover, three un-
structured kinetic models for EB growth, namely the logistic, Kono and Asai (KA), and combined continuous logistic and Fermi (CCLF)
models, were proposed and validated. The logistic and KA growth models had high R2 (>0.91) and low root-mean-square error (RMSE)
(<1.082) values, while the CCLF model showed low values for both R2 (0.48) and RMSE (5.431). The experimental data showed that the
logistic model could best describe the growth of EB in the ML-MFC. DOI: 10.1061/(ASCE)EE.1943-7870.0001522. © 2019 American
Society of Civil Engineers.
Author keywords: Bacteria growth; Membraneless microbial-fuel cell; Electrogenic bacteria; Dewatered sludge; Unstructured model.

Introduction a membraneless MFC (ML-MFC) was used. Dewatered sludge

A microbial fuel cell (MFC) is a bioelectrical device that connects
the natural metabolism of microbes to generate electricity (Gude
2016). Several studies found that MFCs contain diverse microbial
communities and confirmed that many different electrogenic bac-
teria (EB) in anodic biofilms can generate power. EB are capable
of performing exocellular electron transfers (Zhang et al. 2017).
They have also been described using various other terms, such
as electrochemically active bacteria, anode-respiring bacteria,
and electricigens (Li et al. 2014). There have been several studies
on EB, such as those comparing different strains of EB species in
terms of electricity generation (Rezaei et al. 2009), applying EB to
different types of substrate (Chae et al. 2009), using EB for bio-
remediation (Morris and Jin 2007), and focusing on several appli-
cations in renewable energy research. EB are distinguished from
other microbes by their ability to directly transport electrons out-
side the cell, which permits them to function in MFCs. Typically, a
MFC uses two individual chambers that are separated by special
membranes such as proton exchange membranes (PEM) and cation
exchange membranes or anion membrane. The role of the mem-
brane is to separate two different environments, namely oxic and
anoxic, that serve as the cathode and anode environment, respec-
tively (De Schamphelaire et al. 2008). At the same time, the mem-
brane also helps to transmit Hþ to complete the electrical circuit in
the MFC. However, these membranes are high in cost, so an alter-
native solution is needed to overcome the problem. In this study,
1
Researcher, School of Chemical Engineering, Engineering Campus,
Universiti Sains Malaysia, Nibong Tebal 14300, Malaysia. Email: muaz
.mikl@gmail.com
2
Associate Professor, School of Chemical Engineering, Engineering
Campus, Universiti Sains Malaysia, Nibong Tebal 14300, Malaysia
(corresponding author). Email: chvel@usm.uy
Note. This manuscript was submitted on April 25, 2018; approved on
October 24, 2018; published online on March 1, 2019. Discussion period
open until August 1, 2019; separate discussions must be submitted for in-
dividual papers. This paper is part of the Journal of Environmental En-
gineering, © ASCE, ISSN 0733-9372.
played a dual role, acting as both the nutrient-rich anodic and as
a pseudomembrane to separate the anode and cathode. This ap-
proach could make this ML-MFC affordable to construct and also
reduce the its cost of operation.
The growth of EB in ML-MFCs has a significant effect on their
voltage generation and chemical oxygen demand (COD) removal
capabilities. There is a need to determine models that provide re-
liable growth kinetics for EB. Normally, kinetic modeling allows
researchers to determine important parameters such as specific
growth rate, process yield, process productivity, process control
criteria, and strategies for the production of products and scale-
up consideration (Thomas et al. 2013). Many researchers studying
microbes in other bioprocess fields have gradually moved away
from the traditional, largely empirical, approaches, toward sim-
pler and better controlled processes. For example, a simple model
was utilized to investigate the effect of specific growth rate on
the biosynthesis of erythromycin by Streptomyces ertyhraeus in
phosphate-limited chemostat culture. Moreover, the correlation
between initial biomass over time of incubation was shown by
the result of computer simulation of fed-batch culture of Penicil-
lium chrysogenum producing penicillin G (Trilli et al. 1987). The
same methods could be applied to a MFC system. Several kinetic
models have been suggested by researchers working on MFCs,
but these tended to focus on the generation of electricity (Luo
et al. 2016; Wen et al. 2009) instead of the growth of EB, which
was the core element of recovering energy using MFCs. In this
study, simple and easily understandable mathematical descrip-
tions of the kinetic model of microbial growth in a ML-MFC were
explored to predict the behavior of EB in a ML-MFC system. This
approach can facilitate the rapid evaluation of the behavior of EB
in the system compared with laboratory experiments, which can
be time consuming. In addition, there are fewer reports on the
kinetic growth models of EB that generate electricity from the
dewatered sludge in ML-MFCs. Thus, this study focused on
selection of a kinetic model to represent the growth of EB in a
ML-MFC. The unstructured logistic, Kono and Asai, and com-
bined continuous logistic Fermi models were evaluated and com-
pared for this purpose.

© ASCE 04019015-1 J. Environ. Eng.

J. Environ. Eng., 2019, 145(5): 04019015


Resistor
Cathode
cm
Dewatered sludge cm
Anode
cm
cm
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Fig. 1. Schematic of membraneless air-cathode MFC used in this
study.
Materials and Methods
Sample Collection
Dewatered sludge was collected from Kerian Indah Water Treat-
ment Plant Parit Buntar, Malaysia. The influent of this secondary
treatment plant mainly comes from the septic tanks of the residen-
tial area (12,000 population equivalent) around the plant, which is
transported using desludging lorries. The collected sample was kept
at 4°C and thawed to room temperature (27°C) before being used in
the study.
Construction of a Membraneless Microbial Fuel Cell
A ML-MFC was built using cylindrical PVC reactors (diameter:
10 cm; height: 10 cm). Fig. 1 shows the schematic of the ML-MFC.
Up to 1 cm of the dewatered sludge was placed in it, and the anode
was then placed on top of it. Then the anode was covered with more
dewatered sludge, to a depth of 6 cm, and the cathode was placed
on top of it, with its upper surface exposed to air. The graphite felt
electrodes (anode and cathode) had radiuses, thicknesses, and sur-
face areas of 4.6 cm, 0.65 cm, and 0.0064 m2 , respectively. The
chamber was then closed with a lid and set at room temperature
(27°C). Generation of electricity was measured using a digital mul-
timeter (UT33D, UNI-T, Hong Kong) that connected the probe to
the anode and cathode wires in the ML-MFC. No other organic
substrate was added to the anolyte.
Operation of the ML-MFC
The effect of the pH, distance between electrodes, moisture content,
and temperature were tested on the voltage generation and the COD
removal. The pH of the dewatered sludge was set to 6.0 by adjust-
ing the ratio of sodium hydroxide (NaOH 1M) and hydrochloric
acid (HCl 1M). The pH was measured using a benchtop pH meter
(UB-10, Denver Instrument, Denver). The electrode distance was
manually adjusted to 3 cm by adding dewatered sludge between the
electrodes. The moisture content of the dewatered sludge was set at
30% (v/w) by increasing the percentage of sterile deionized water
in the mass of dewatered sludge in the ML-MFC. The temperature
of the ML-MFC was set to 35°C by adjusting the temperature
setting of the incubator. The aforementioned operating conditions
were chosen on the basis of the ML-MFC optimization study car-
ried out earlier (Muaz et al. 2018). The ML-MFC was incubated
© ASCE
J. Environ. Eng., 2019, 145(5): 04019015
for 10 d, and voltage and COD removal measurements were re-
corded every 6 h during the entire period that the ML-MFC was
operated, using a multimeter and standard methods (APHA 2015),
respectively.
Determination of Biomass through Volatile Testing
The biomass of the EB was represented by the mass in the volatile
solid (VS) method (APHA 2015). The EB attached to the anode
and formed a biofilm. Dewatered sludge samples were taken from
the surface of the anode and subjected to VS testing. Samples were
taken every 6 h for 10 d
ðA − BÞ × 1,000
Volatile solidðmg=gÞ ¼ ð1Þ
Sample dewatered sludge ðgÞ
where A = weight of residue + dish before drying (mg); and
B = weight of residue + dish after drying (mg).
Determination of Biomass through Colony-Forming
Units
The biomass of EB were also determined using the colony-forming
unit (CFU) method (Blodgett 2008) by taking a sample from the
biofilm on the anode. One gram of the biofilm sample was diluted
102 times by adding 99 mL of sterile water. The sample was stirred
well and the tubes were capped to prevent the introduction of air-
borne bacteria. By diluting 1 mL of the 102 solution with another
99 mL of sterile water and mixing thoroughly, a dilution of 104 was
obtained. The biofilm sample was prepared for 106 dilution, mean-
ing that 1 mL of the 104 dilution was diluted further into 99 mL
of sterile water. Then 0.1 mL of the 106 dilution was aseptically
pipetted onto agar and spread over the surface of the nutrient using
a disinfected rod. The plates were then incubated for 24–48 h. The
numbers of bacterial colonies in 1 g of biofilm sample were calcu-
lated as follows:
Number of colonies
× 106
0.1
¼ number of electrogenic bacteria=gram biofilm ð2Þ
Determination of Chemical Oxygen Demand
One gram of dewatered sludge was diluted in 10 mL of distilled
water in the centrifuge, then the sample was vortexed for 3 min
and centrifuged at 4k rpm for 5 min. Then, the sample was filtered
using syringe filter (MF—Millipore Millex GS syringe filter with
pore size 0.22 mm). Two milliliters of the filtrate was added into the
COD vials that consisted of premixed chemicals (K2Cr2O7,
AgNO3, HgSO4, Potassium hydrogen phthalate, H2SO4). A blank
sample was prepared by adding 2 mL of distilled water into the
vials. The COD vials were digested at 150°C for 2 h. After 2 h,
the content of the vials was cooled down to room temperature.
The COD kit (Checkit Direct, Lovibond) was used to measure the
COD value (APHA 2015).
Analysis of Biofilm
The surface morphology of the anode electrode after being attached
to by the EB was observed using a scanning electron microscope
(SEM) (TM3000, Hitachi, Japan). At the end of the experiment, the
anode was collected and immersed overnight at 4°C in a buffer sol-
ution of paraformaldehyde and glutaraldehyde, with a pH value of
5.4. After this, the anode was coated and observed with a SEM.
04019015-2 J. Environ. Eng.
 Determination of Maximum Power Using a Polarization
Curve
The polarization curve is a conventional method to evaluate
the performance of a MFC. The ML-MFC was connected to a
multimeter to record cell voltage at different external resistance
(47, 100, 220, 470, and 1,000 Ω), and its power was determined
based on Ohm’s law (R ¼ V=I, P ¼ IV). The polarization curve
was plotted throughout the voltage and current measurements. The
peak of the power curve was the maximum power of the ML-MFC
(Logan 2012).
Determination of Cell Phenotype
The EB present in the dewatered sludge were isolated using a
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method described by Richards and Ausubel (1988). The dewatered
sludge was dissolved in a stock deionized water solution at a con-
centration of 0.05 g=mL. A series of dilutions (103 dilutions) were
performed on the stock mixture to reduce its concentration. A few
agar plates were also prepared to culture the bacteria; 25 g of agar
was dissolved in 1 L of deionized water, and this was autoclaved at
120°C for 30 min. The autoclaved agar solution was then distrib-
uted equally in 50 petri dishes. One milliliter of the diluted stock
mixture was added to 20 of those dishes and then stirred gently to
allow equal distribution of EB growths. These 20 petri dishes were
placed in an incubator at 30°C for 24 h. After 24 h, individual col-
onies of EB that had grown in the agar were isolated and cultured
separately in the remaining 30 petri dishes. These were left in the
incubator for another 24 h to allow the EB to grow, after which the
three major populations of isolated EB were picked using an inoc-
ulation loop and streaked in an agar slant. After incubation for 24 h
at 30°C, the slants were stored in a refrigerator for further use.
Cell Molecular and Phylogenic Analysis
The isolated samples (agar slants) were sent to Macrogen, South
Korea, for microbial identification. The primers used for the
polymerase chain reaction analysis were 27F 5′(AGA GTT TGA
TCM TGG CTC AG)3′ and 1492R 5′(TAC GGY TAC CTT
GTT ACG ACT T)3′. The sequences obtained were analyzed
using the National Center for Biotechnology Information (NCBI)
online nucleotide BLAST tool and ribosomal database-II to iden-
tify the taxonomic hierarchy of the sequences. Taxonomically re-
lated 16S rRNA gene sequences were obtained from the NCBI
nucleotide database. The sequences collected were aligned using
the MUSCLE multiple sequence alignment algorithm. The phylo-
genetic tree was constructed and inferred using the neighbor-
joining method and validated using the bootstrap method (1,000
replications). The evolutionary distances were computed using
the maximum composite likelihood method and are presented as
the number of base substitutions per site. All positions containing
gaps and missing data were eliminated. All analyses were per-
formed using MEGA6 (Gobi and Vadivelu 2015).
Kinetic Models
Logistic Model
In many fermentation systems, including the breakdown of organic
matter by the bacteria, cell growth has been characterized by the
logistic equation (Mitchell et al. 2004). To use the logistic model
here, some derivations have been made to make it applicable for use
with Polymath software. Eq. (3) shows how the model represents
both the exponential and stationary phase (Elibol and Mavituna
1999; Rajendran and Thangavelu 2008)
© ASCE 04019015-3
J. Environ. Eng., 2019, 145(5): 04019015
X eμ m t
XðtÞ ¼
Xo o ð3Þ
1 − Xm ð1 − eμm t Þ
where X = biomass concentration (mg=g substrate) at time t (h);
μm = maximum specific growth rate (1=h); X o = initial biomass
concentration; and X m = maximum biomass concentration.
Kono and Asai Model
Rajendra and Thangavelu (2008) reported that the Kono and Asai
model described the exponential and declining growth phase. The
cell multiplication rate was exponential till the critical cell concen-
tration (X c ) was reached, beyond which exponential growth was
not possible. Equations for both exponential and declining growth
are expressed as in Eq. (4)
  
Xc
XðtÞ ¼ X m − ðX m − X c Þ exp μ ðt − tc Þ ð4Þ
Xm − Xc
where tc is the time when critical cell concentration is reached.
The kinetic parameters were then determined by plotting the mi-
crobial growth rates (dX=dt) against cell concentration (X). The
induction and transient phases of growth were followed by an
exponential phase in which dX=dt was found to increase linearly
with cell concentration (Elibol and Mavituna 1999). The slope of
the straight line gave the value of μ. At the end of the exponential
phase, the growth curve ended abruptly, and the growth rate de-
creased as a function of cell concentration. The cell mass at which
the break occurred was equal to the critical cell mass (X c ) and the
critical time (tc ).
Combined Continuous Logistic and Fermi Model
Most standard models have been developed for a specific growth
or decline regime and consequently cannot count for both. There-
fore, a new model, the combined continuous logistic and Fermi
(CCLF) model was developed and modified to describe the entire
growth cycle. To fully describe both the growth and decline phase,
two equations—the continuous logistic equation and Fermi’s
function—were combined. The continuous logistic equation can
be written in the following form (Peleg 1996):
Xm
X L ðtÞ ¼ ð5Þ
1 þ expðμL ðt − tc ÞÞ
where XðtÞ = number of microorganisms at time t; X m = maximum
growth that can be achieved; μL = growth rate constant; and tc in-
dicates the time needed to reach half the environmental capacity
which was Xðtc Þ=X m ¼ 1=2. Fermi’s function was a mirror image
of the logistic equation that showed the decline phase in any micro-
bial growth rate (Du et al. 2007), and is described in Eq. (6)
1
X F ðtÞ ¼ ð6Þ
1 þ expðμF ðt − tc Þ
where μF = constant rate of decline (h−1 ); and tc = time taken to
reach 50% survival. By combining the continuous logistic equation
[Eq. (5)] and Fermi’s function [Eq. (6)], the following model was
obtained (Peleg 1996):
X m ½1 þ expðμF ðt − tc ÞÞ
X T ðtÞ ¼ ð7Þ
1 þ expðμL ðt − tc Þ
where X = biomass (mg=g substrate); Xm = maximum biomass
(mg=g substrate); μL (h−1 ) = constant rate of growth; μF = constant
J. Environ. Eng.
 rate of decline (h−1 ); and tc = time to reach 50% survival in 550 1000 35
900
which Xðtc Þ=X m ¼ 1=2. 800
30
510
All the models were fitted to the experimental data using non- 700 25

Biomass (mg/g)
Voltage (mV)
linear regression. These models are also used by Samsudin and Don

COD (mg/l)
470 600 20
(2015), Elibol and Mavituna (1999), and Pazouki et al. (2008) re- 500
search groups to describe similar processes. The goal is to make use 430 400 15
of mathematical modeling to reduce a complex biological system 300 10
into a simpler mathematical one that can be analyzed in far more 390 200
5
detail, and from which key properties can be identified. Lineariza- 100
tion was done for all selected models to estimate the initial condi- 350 0 0
-50 0 50 100 150 200 250
tion value. The experimental results were analyzed using linearized Time (h)
equations of the selected kinetic models. Voltage (mV) Cod (mg/l) Biomass (mg/g)

Fig. 2. Profile of chemical oxygen demand, electricity voltage gener-

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Results and Discussion
Voltage Generation and EB Biomass in the ML-MFC
Fig. 2 presents the bacterial growth and the voltage generated in
the ML-MFC under the following conditions: a pH value of 6.0,
electrode distance of 3 cm, moisture content of 30% (v/w) and tem-
perature of 35°C. The EB biomass profile is in a lag phase at the
early stage, from 0 to 35 h, due to the EB adapting to the new envi-
ronment in the ML-MFC. The biomass then kept increasing until
168 h, when the voltage and the biomass were 914.1  8.48 mV
and 28.4  0.35 mg=g, respectively. The biomass then stabilized
because it had reached the maximum value. The same trend was
recorded for the voltage generation. The incremental increase in
EB biomass also increased the voltage in the ML-MFC, proving
that EB biomass and voltage were associated with growth. The
oxidation of organic compounds by EB at the anode released elec-
trons, while the reduction of oxygen at the cathode created a high
redox potential, thus recording a high voltage. The process began
with the donated compounds (organics in the dewatered sludge)
being subjected to a series of metabolic pathways: glycolysis, the
citric acid cycle, and oxidative phosphorylation. During the occur-
rence of these natural metabolic pathways, the organics were bro-
ken down and highly reduced biomolecules that act as electron
carriers, such as nicotinamide adenine dinucleotide (NADH), were
generated (Rasmusson et al. 2008). These processes took place at
the anode, to which the electrons from NADH (or any complex
protein) were passed. The electrons reacted with oxygen and pro-
tons at the cathode and electricity was generated.
Crittenden et al. (2006) stated that higher populations of EB
would result in more electrons being transferred to the anode,
and a better performance of the MFC. The increase in biomass con-
centration indicated that the population of bacteria formed colonies
at the anode, thus lowering the COD level (Fig. 2). At the beginning
of the experiment (t ¼ 0 h), the initial COD (CODi ), initial bio-
mass (X i ), and initial number of bacteria (cfui ) were 535 mg=L,
0.021 mg=g, and 8 × 106 EB=g biofilm, respectively. During the
lag phase from 0 to 35 h, there was a slight increase in biomass
value (VS∶2.4 mg=g, cfu: 24 × 106 ) and the COD value was con-
sequently reduced to about 13.2 mg=L. The strong relationship
between both parameters (cfu and VS values) was also shown when
an exponential increase in bacterial biomass took place from 36 h to
168 h: the VS value rose from 2.4 to 28.6 mg=g (an approximately
10-fold increase), the number of colony forming units or cfu (cfu is
the unit used to estimate the number of viable bacteria in a sample
of biofilm at the anode in a ML-MFC) increased from 24 × 106 to
118 × 106 (an approximately sixfold increase), and the COD value
of the dewatered sludge also fell rapidly from 521.8 to 366.7 mg=L
(a decline of up to 30%). The appearance of a colony on the agar
plate indicates the attachment of EB to the anode.
ated, and electrogenic bacteria biomass in membraneless microbial
fuel cell.
Power Generation Using the ML-MFC
The power output of the present study was fairly similar to
other MFC studies that used organic-rich soils and sediments
(De Schamphelaire et al. 2008) and paddy plantation soil
(26 mW · m−2 ) and other sediments (30 mW · m−2 ) (Logan and
Regan 2006). This had proved that the MFC without membrane
also could achieve a high generation of power. The ML-MFC
was subjected to polarization losses via activation potential, ohmic
losses, and mass transfer. The losses can be clearly seen in the
polarization curve through (1) a rapid voltage drop as the current
flowed through the circuit at a high external voltage; (2) a nearly
linear decrease in voltage; and (3) a second rapid voltage decrease
at high-current densities, respectively. The activation potential of
the ML-MFC was reached when the current went from 0 to
0.56 mA, and the voltage dropped rapidly from 927 to 567 mV
(Fig. 3). This could be due to the energy lost as heat while initiating
oxidation or reduction reactions, or that lost through the transfer
of electrons from the terminal proteins in bacterial cells to the
surface of the anode (Logan and Regan 2006). To overcome this
loss of activation potential, the temperature must be raised. When
the current increased from 1.00 to 2.21 mA, a constant voltage drop
(472–221 mV) was observed. This is called ohmic overpotential
and was caused by the electrical resistance of the electrodes. This
was proved by the observation that the voltage improved when the
electrode distance was 3 cm instead of 5 cm (Muaz et al. 2018),
which was due to the energy loss during the transmission of protons
to the cathode.
When the current increased (2.21–2.38 mA), the voltage once
again dropped rapidly, from 221 to 112 mV; this was due to the
(1) (2) (3)
Fig. 3. Polarization curve for the microbial fuel cell.

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J. Environ. Eng., 2019, 145(5): 04019015

Downloaded from ascelibrary.org by Lund University on 03/05/19. Copyright ASCE. For personal use only; all rights reserved.
Fig. 4. Electrogenic bacteria on the surface of an anode: (a) at optimum condition in membraneless microbial fuel cell; and (b) at control
conditions.
strong oxidative forces at the anode. The organic compounds were
being oxidized faster at the anode than they could be transported to
the surface in a phenomenon called concentration or mass transfer
loss (Gude 2016). Each type of polarization loss should be mini-
mized in future studies, to decrease the overall loss of potential, this
would increase power generation.
EB Attachment on the Anode
Scanning electron microscopy observations were conducted to
characterize the biofilm that formed on the surface of the anode
at optimum conditions [pH 6.0, electrode distance 3 cm, moisture
content 30% (v/w), temperature 35°C]. The images obtained
showed an abundant microbial presence on the graphite felt. It
is evident from Fig. 4(a) that EB attach themselves to and colonize
the surface of the anode, forming a living matrix called biofilm. The
EB interact with a conductive solid surface (i.e., the anode), form
electroactive microbial biofilm, and transfer electrons to generate
electricity. In the ML-MFC under control conditions (one that used
natural dewatered sludge), only a thin biofilm formed and few
colonies of EB were present [Fig. 4(b)]. As more bacteria grew
and attached to the anode, the internal resistance was reduced be-
cause the electrons were being transported directly to the surface of
the anode, which acted as terminal electron acceptor. According to
Zhi et al. (2014), when EB colonize the surface of the anode and
form a thick biofilm, the rate of substrate consumption increases
and thus boosts power generation. There were three methods
by which the electrons could have been transferred to the anode:
(1) directly from the cell walls of microbes to the surface of the
anode; (2) via secondary biomolecules that shuttle electrons to the
anode; or (3) through conductive appendages called “nanowires”
(He et al. 2015). From the SEM observations, the EB attached
to the anode surface were proven to use direct transfer.
Phylogenic Analysis of EB
Three major samples of isolated bacteria were sent to the micro-
biology company Macrogen Korea and, of these, one was deter-
mined to be from the genus Pseudomonas and the other one
was identified as the species Bacillus subtilis. Researcher believes
© ASCE 04019015-5
J. Environ. Eng., 2019, 145(5): 04019015
that the presence of Pseudomonas species in MFCs can help the
systems, because the transmissions of electrons were enhanced
by their own metabolite. Pseudomonas have the ability to transfer
electrons because they secrete phenazine-based metabolites at the
surface of the anode, thus improving the generation of electricity
(Buitron and Moreno-Andrade 2014). Similarly, it has been found
that Bacillus subtilis acts as a good biocatalyst for MFC and
generates stable energy (Nimje et al. 2009). The presence of both
species helped to boost electricity generation in the ML-MFC,
as previously presented in the polarization curve (Fig. 3). The
ML-MFCs in this study were clearly using mixed bacterial cultures,
resulting in several significant advantages. As mentioned by Logan
and Regan (2006), mixed cultures have a much higher resistance to
interference with the process, higher substrate intake rates, lower
substrate specificities, and higher power generation capabilities.
To prove these hypotheses, an additional experimental was carried
out in which a ML-MFC was moistened with the dewatered sludge
using mixed culture inoculum consisting of Pseudomonas and
Bacillus subtilis bacteria. The ML-MFC was then run for 10 d,
and the results showed that it generated more electricity than a
ML-MFC that did not have inoculum containing Pseudomonas and
Bacillus subtilis bacteria added to it. The addition of EB to the
ML-MFC proved that the Pseudomonas species and Bacillus
subtilis play important roles in the performance of the ML-MFC.
Modeling EB Growth
Analysis of Modeling
To investigate the growth of EB in the ML-MFC, a mathematical
model of bacterial growth, namely the logistic equation [Eq. (3)],
which is the kinetic expression for microbial growth, was fitted
to the experimental data on EB biomass (Table 1). The logistic
model described the lag, exponential, and stationary phases well.
To determine the specific growth rate (μm ) of the EB, taking the
maximum biomass X m ¼ 31.1 mg biomass/g sample from the
experimental data and a plot of linear Eq. (3) yielded μm ¼
0.041 (h−1 ). The initial lag phase of the present study lasted from
6 to 12 h. The length of the lag phase can be affected by the several
factors including the composition of the medium (dewatered
sludge), types and ages of the EB strains, number of cells, and
J. Environ. Eng.
 Table 1. Kinetic parameters for the selected model
Model μm (1=h) M (1=h) μL (1=h)
Logistic 0.041 — —
Kono and Asai — 0.035 — —
Combined continuous logistic and fermi — — 0.049
Note: μL and μF denote specific growth for the continuous logistic and Fermi models, respectively.
physical factors such as temperature and pH (Cadenas and Sies
1998). After a lag phase, the EB entered an exponential phase
starting between 24 and 156 h. The logistic model predicted that
the maximum EB biomass would be 29.65 mg=g. Once the limit-
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ing organic compound in the dewatered sludge started to decrease,
the EB biomass stopped increasing exponentially and the stationary
phase followed. The data prediction also agreed relatively well
with the experimental data, with R2 and root mean-squared error
(RMSE) values of 0.994 and 1.082, respectively (Table 1). This
showed that the selected logistic model was sufficient to describe
both the exponential and stationary phases of EB growth in the
ML-MFC.
For the Kono and Asai model, the experimental data of the
present study showed a good fit only for the lag phase. The kinetic
model started the exponential phase after 20 h of incubation, but the
predicted value suggested by the model as the rate of increase
did not fit the experimental data well (Fig. 5). As described by
Rajendran and Thangavelu (2008), the Kono and Asai kinetic
model was modified to represent the complete cycle growth of
microorganisms, including the lag, exponential, stationary, and
death phases. However, the lack of decrement in the biomass data
for the death phase, within 10 d of incubation in the ML-MFC,
meant that the suggested data deviated slightly from the actual ex-
perimental data and resulted in the low R2 value. Likewise, the
CCLF model also usually represents the entire cycle of bacterial
growth, from lag to death phase. The model combines the continu-
ous logistic equation, for use with the earlier phase, with the Fermi
equation, for use in the later phase. In the present study, values of
R2 were very low; in fact, they were invalid to describe the growth
of EB in the ML-MFC. The data predicted by the model in
Polymath also showed that it was unreliable and did not fit well
with the experimental data (Fig. 5). Similar to the Kono and Asai
model, the CCLF model also requires sufficient data on the death
phase of the EB, before it could work satisfactorily.
35
30
Biomass, X (mg/g)
25
20
15
10
5 Acknowledgments
0
0 50 100 150 200 250
time (h)
Xexp Xlogistic Xkonoasai Xcclf
Fig. 5. Kinetic growth profiles for electrogenic bacteria using logistic,
Kono and Asai, and CCLF models.
© ASCE 04019015-6
J. Environ. Eng., 2019, 145(5): 04019015
Kinetic parameter
μF (1=h) X o (mg=g) X m (mg=g) X c (mg=g) T c (h) R2 RMSE
— 0.021 31.1 — — 0.994 0.195
— — 18.6 108 0.912 1.085
0.037 — — — 108 0.48 5.431
Model Validation
To make sure the kinetic models of EB growth were reliable, a val-
idation was required to evaluate whether the precision and accuracy
obtained were appropriate for microbial growth. An error analysis
calculation was conducted between the experimental data and pre-
dicted data using the RMSE equation, and the results are shown in
Table 1. The RMSE was calculated using the following equation
(Chai and Draxler 2014):
pX n
RMSE ¼ ðY i − Y p Þ2 ð8Þ
i¼1
where Y i and Y p are the experimental data and corresponding pre-
dicted data, respectively; and n = number of the experimental data.
The RMSE value is not standardized toward an acceptable
range, but values close to zero are considered appropriate since
RMSE values depend on the range of parameters used. In other
words, a broader range of parameters could contribute toward a
higher RMSE. As shown in Table 1, the RMSE values for all se-
lected models were low, because they were close to zero with only
the CCLF model as an exception. The lowest RMSE calculated was
from logistic model (0.195), followed by the Kono and Asai model
and CCLF with values of 1.082 and 5.431, respectively. The results
of the RMSE tallied with the values of R2 . The logistic model had a
lower RMSE value compared with the Kono and Asai and CCLF
models, proving that the former was reliable in representing EB
growth in the ML-MFC.
Conclusion
This study focused on the selection of a kinetic model to represent
the growth of EB in a ML-MFC at optimum pH, electrode distance,
moisture content, and temperature conditions. The power generated
was proportional to the growth of the EB. SEM observations proved
that the colonization of EB on the surface of the anode contributed
to the higher levels of power being generated. Phylogenetic analysis
showed the presence of bacteria from the genus Pseudomonas
and species Bacillus subtilis, which acted as biocatalysts in the
ML-MFC. In addition, three unstructured kinetic models for micro-
bial growth, namely the logistics, Kono and Asai, and CCLF mod-
els, were selected and validated. The model that fitted well with the
experimental data, and best described the behavior of EB growth in
the ML-MFC, was the logistic model.
The authors would like to thank the Universiti Sains Malaysia
for the financial support of this study via the Research University
Grant (RUI) (Account No. 1001/PJKIMIA/814267), and also a
scholarship (MyPhD) for the first author from the Ministry of
Higher Education Malaysia. The authors have declared no conflict
of interest for the manuscript.
J. Environ. Eng.
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