Journal of Biosensors &

Bioelectronics

Quek et al., J Biosens Bioelectron 2014, 5:2

http://dx.doi.org/10.4172/2155-6210.1000152

Research Article

Open Access

Bio-Electrochemical Sensor for Fast Analysis of Assimilable Organic Carbon in

Seawater
Soon Bee Quek, Liang Cheng* and Ralf Cord-Ruwisch
School of Engineering and Information Technology, Murdoch University, Australia
*Corresponding

author: Liang Cheng, School of Engineering and Information Technology, Murdoch University, Australia, Tel: +618 9360 2804; E-mail:

[email protected]
Rec date: May 09, 2014, Acc date: May 30, 2014, Pub date: June 05, 2014
Copyright: 2014 Cheng L, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract
A Microbial Fuel Cell (MFC) based biosensor for the determination of Assimilable Organic Carbon (AOC) in
seawater was developed by establishing an anodophilic marine biofilm on the surface of an electrode poised at +250
mV (vs Ag/AgCl) rather than the traditionally used potentials of about -300 mV. A linear correlation (R2>0.99)
between electrochemical signals (peak current) and acetate concentration ranging 10 to 55 M was achieved.Using
the positive anodic potential enabled the rapid establishment of the electrochemically active anodophilic biomass
within a period of less than 8 days, a higher sensitivity (0.017 mA/M acetate added) and a lower detection limit (2.5
M acetate, 0.16 mg O2/L of Biological Oxygen Demand (BOD)) compared to the negative anodic potential. Further,
it was shown that this bio-electrochemical AOC sensor could tolerate the presence of low concentrations of
dissolved oxygen. The established potentiostat controlled MFC biosensor could be used for the purpose of online
water quality monitoring for seawater desalination plants prone to biofouling of RO membranes.

Keywords: Biosensor; Microbial fuel cell; Seawater desalination;

Biofouling; Potentiostat; Assimilable Organic Carbon

Introduction
In the past few decades, clean water supplies have become a lot
more critical due to the excessive usage and increasing contamination
of natural water sources. In recent years, Reverse Osmosis (RO) has
become an important technology to provide clean water from
seawater. It is reported that membrane-based seawater desalination
accounts for about 44% of the installed capacity of water desalination
in the world [1].
Biofouling is referred to as the deposition and growth of bacterial
biofilms on membranes, which is a universal phenomenon occurring
in membrane water purification process. Biofouling has been reported
as a serious problem for seawater RO systems [2], which introduces an
additional hydraulic resistance, increases the feed channel pressure
drop, and enhances concentration polarization. This causes significant
deterioration in the performance and efficiency of the RO membranes.
The presence of organic pollutants in seawater is one of the main
causes of RO membrane biofouling. The attached bacterial cells
produce exopolymers and multiply by the uptake of soluble organics
from the feed water. Therefore, monitoring the nutrient content, such
as Assimilable Organic Carbon (AOC) one of the main food sources
for the bacteria, in the feed water is important to predict the biofouling
potential of particular seawater and enable possible measures to
minimize biofouling.
Several methods have been reported to quantify AOC in wastewater
or seawater. The techniques based on the Biochemical Oxygen
Demand (BOD) detection are widely used, such as traditional BOD5
test requiring a 5 day off-line laboratory incubation making it not
suitable for a fast and online monitoring system [3]. Recently, other
types of BOD biosensors based on measuring the change in Dissolved

J Biosens Bioelectron
ISSN:2155-6210 JBSBE, an open access journal

Oxygen (DO) by a suspended or immobilised microbial biomass were

reported [4,5]. Mediator modified BOD tests also gained interest
because of the much faster analysis time [6-11]. The test bacteria
would reduce the mediator, which can be detected
photospectrometically or electrochemically. However, those methods
are not designed for online measurement, as regular sample harvesting
is required.
In a MFC, microorganisms growing on the surface of anode
electrode generate an electrical current by transferring electrons from
the oxidation of organic matter present in the solution through an
electrical circuit to a cathode electrode [12-14]. No transducer is
needed to convert the measured signal to an electrical signal because
the measured signal is already an electrical current. The current study
aims at developing a potentiostat-controlled MFC-based AOC
biosensor and optimizing it for the detection of low AOC in seawater.
The potentiostat control of the anode is expected to provide greater
accuracy than the use of traditional MFC.

Material and Methods

Marine Microbial Fuel Cell Biosensor
Bacterial Inoculum and Growth Medium
The bacterial inoculum was extracted from ocean sediment,
collected from Coogee Beach, Coogee, South Fremantle, Western
Australia. The sediment was mixed with seawater with a weight ratio
of 1 to 5 followed by continuously stirring for 24 hours. After settling
for 2 hours the supernatant with OD600nm value of about 0.2 was
collected and used as inoculum for the establishment of the marine
anodophilic biofilm. Seawater was used as anolyte (working electrode
compartment) and catholyte (counter electrode compartment). In RO
plants, suspended solids that are present in the feed-water will be
removed by ultra-filtration. Therefore, this study utilised real seawater

Volume 5 Issue 2 1000152

Citation:

Quek S B, Cheng L, Ralf Cord-Ruwisch (2014) Bio-Electrochemical Sensor for Fast Analysis of Assimilable Organic Carbon in
Seawater. J Biosens Bioelectron 5: 152. doi:10.4172/2155-6210.1000152

Page 2 of 4
with no suspended solids (OD600nm<0.01) to demonstrate the
applicability of this method in industry. Yeast extract solution was
periodically added (ca. every 2-5 days) to the anolyte (50 mgL-1 final
concentration) as bacterial growth supplement.

were used as the reference set point in the LabBIEWTM feedback

control program.

Microbial Fuel Cell Sensor Set up

The current production, which is with mA level and proportional to

the rate of acetate oxidation by the anodophilic bacteria, can be
retrieved by the potentiostat. Cumulative charges were calculated by
integrating the electrons transferred by the biofilm as current
throughout the detection period [15]. The signals (current peak/
cumulative charges) obtained from the acetate addition were
calculated by subtracting the background values. Steady state was
defined as no changes in current ( 0.1 mA). The recovery time was
defined as the time required for the current returns to the original level
after the depletion of acetate.

A two-chamber MFC (made of transparent Perspex) was used in

the present study. The compartments of the fuel cell (anode and
cathode) having equal dimension of (9 cmx6 cmx1 cm) were
physically separated by a cation selectively exchange membrane
(CMI-6000, Membrane International INC.) with a size of 59.4cm2.
Both chambers were filled with conductive graphite granules (EI Carb
1000, Graphites Sales, Inc., Chagrin Falls, OH, USA) of about 2-6 mm
in diameter. As current collectors, two graphite rods (3 mm diameter
and 10 cm length) were inserted into the anodic and cathodic
chambers, which acted as the working and counter electrodes,
respectively. A potentiostat was connected to the electrodes and was
used to maintain the anodic potential. The potentials of the electrodes
were measured and controlled against a saturated Ag/AgCl reference
electrode (BASi, MF-2079) placed inside the anodic chamber.

Microbial Fuel Cell Sensor Operation

Start-up Procedure
The MFC was operated in a fed-batch mode with both catholyte
and anolyte continuously re-circulating over the cathodic and anodic
compartments, respectively. The anodic chamber (as described in
Section 2.1.2) was inoculated with 100 mL of seawater containing 50
mL of the inoculum (prepared according to the procedure described
above), 50 mgL-1 yeast extract and 10 mM acetate. A 10 mL bottle was
connected in the anolyte-circulating loop for pre-mixing of acetate
addition and the anolyte prior to introducing to the anodic
compartment. The cathodic compartment was filled with 50 mL
seawater as the catholyte (Section 2.1.1).
After the anodophilic biofilm had been successfully established
(indicated by a steady current production), the anodic compartment
was flushed with fresh seawater to remove all suspended biomass.

Acetate Detection Procedure

Analytic Methods

Results and Discussion

The Development of Marine-MFC Biosensor and the Effect
of Anodic Potentials
Ideally, a rapid start-up of MFC-biosensor is desired for practical
applications. In the current study, the biosensor was ready to be used
after operating the inoculated anodic compartment at a fixed potential
of +250 mV (vs Ag/AgCl) for about 7 days. This start-up time was
about 2 to 4 times faster compared to that needed when using negative
anodic potential conditions (data not shown). This finding is in line
with previous studies, which demonstrated that a higher anodic
potential increased the growth rate of anodophilic bacteria, resulting
in faster microbial colonisation and quicker start-up of MFCs [16-19].
The enhanced bacterial growth rate at positive anodic potential is
probably due to the greater available energy [16,17,20]. Moreover, it
has also been suggested that at positive anodic potentials, the positive
charged electrode surfacecan enhance the adhesion of bacteria with
negative charged cell membranes (e.g. Geobacter) [21]. In the current
study, the MFC operated at +250 mV anodic potential resulted in an
about 2-fold higher current peak and cumulative charges compared to
results obtained at -250 mV anodic potential (Figure 1). This finding is
consistent with previous published works, which has demonstrated
that anodophilic bacteria produce higher currents at higher anodic
potentials due to the faster substrate oxidation rate [22-25].

For calibration purposes specified concentrations of sodium acetate,

which represents readily assimilable organic carbon, were added into
the anolyte-circulating loop via a septum-sealed injection port. The
anode was controlled at different potentials ranging from -250 mV to
+600 mV (vs Ag/AgCl) using the potentiostat.

Control and Monitoring

The anolyte and catholyte were maintained at room temperature.
The anodic compartment was kept under anaerobic conditions unless
stated elsewhere. The anodic potential, current and pH were
monitored continuously using LabVIEWTM 7.1 software interfaced
with a National InstrumentTM Data Acquisition Card (DAQ).
Control and monitoring of the biosensor was automated and
online. In the experiments of testing the response of the biosensor to
acetate additions, automated acetate dosing was implemented using a
computer feedback-controlled peristaltic dosing pump. The online
interpretations of a steady anodic potential and/or baseline current

J Biosens Bioelectron
ISSN:2155-6210 JBSBE, an open access journal

Figure 1: Effect of anodic potential (-250 and +250 mV (vs Ag/

AgCl)) on the responses of the MFC-biosensor to the acetate
addition (50 M).

Volume 5 Issue 2 1000152

Citation:

Quek S B, Cheng L, Ralf Cord-Ruwisch (2014) Bio-Electrochemical Sensor for Fast Analysis of Assimilable Organic Carbon in
Seawater. J Biosens Bioelectron 5: 152. doi:10.4172/2155-6210.1000152

Page 3 of 4

Standard Curves and Detection Limits

Sensitivity to Dissolved Oxygen

The reliability of the established MFC biosensor and the correlation

between the signal production and acetate concentration were tested at
+250 and -250 mV anodic potentials. The peak current values
(maximum current minus background current) were plotted against
the acetate concentrations (Figure 2) revealing a linear relationship
with high R2 values (> 0.99) for both tested anodic potentials.
However, the sensitivity (mA/M acetate added) and detection limits
were improved by 4 and 2 times respectively at +250mV anodic
potential (Figures 2 and 3). The lowest detection limit was based on a
signal-to-noise ratio of 3 [26]. The use of an even higher anodic
potential up to +400 mV did not improve detection limit (2.5 M
acetate, 0.16 mg O2/L BOD equivalent) (Figure 3).

In typical MFC, the presence of oxygen completely suppresses the

metabolic activity of electrochemically active anodophilic bacteria and
hence signal formation [27]. This would possibly be a critical problem
for the application of the biosensor in the real desalination plant as the
MFC-biosensor should also be able to operate in the presence of
Dissolved Oxygen (DO). In order to overcome this outcompeting
effect of oxygen over the anode as electron acceptor by the bacteria,
the anode potential was increased to a level that is higher than the
redox potential of oxygen (about +80 mV (vs Ag/AgCl) considering
the effect of overpotential) [28]. Then, the biosensor was tested for its
response (current generation) to the addition of low concentration of
acetate (20 M) in the presence of DO (0 to 3 mg/L) (Figure 4).

Figure 2: Correlation between acetate concentrations and current

peak at different anodic potentials, -250 and +250 mV (vs AgAgCl).

Figure 3: The lowest acetate concentration that can be detected by

the established MFC biosensor at different anodic potentials.
From fundamental considerations, the linear correlation between
current peak value and acetate concentration only holds true if the
acetate oxidation reaction is of first order with respect to the acetate
concentration. However, this is known to be not the case for microbial
or enzyme based reactions when the substrate concentration is
increased and saturation behavior sets in. In those higher
concentrations, dilution of seawater prior to detection might be
necessary.

J Biosens Bioelectron
ISSN:2155-6210 JBSBE, an open access journal

Figure 4: Current production from the addition of 20 M acetate at

different fixed anodic potentials (-250 to +600 mV (vs Ag/AgCl)) in
absence and presence of various dissolved oxygen (0, 1.5, 2, 3
mg/L).
In the presence of low DO concentrations (< 2 mg/L), the anodic
biofilm produced current production at high anodic potentials from
+150 to +600 mV (vs Ag/AgCl) but not at anodic potentials lower than
+100 mV (vs Ag/AgCl). This suggests that the anodophilic biofilm that
had been enriched during the 7 days of operation at +250 mV was able
to transfer electrons to the positive electrode in the presence of low
dissolve oxygen (< 2 mg/L). However, the reduced current peak (3fold lower) associated with the oxygen consumption (data not shown)
suggested that a portion of the acetate was used for the aerobic
respiration at positive anodic potentials. The observation that at low
oxygen concentrations and high potentials the anode is used
simultaneously with oxygen suggests that the flow of electrons to
either oxygen or anode is of competitive nature. Using potentials
higher than +250 mV did not further eliminate the interfering effect of
oxygen. This result suggests that the anodic potential is an important
factor in collecting electrons from microbial organics oxidation as it
can influence the attractiveness of the anode compared to oxygen.
The more positive redox potential of a terminal electron acceptor (i.e.
higher anodic potential) the higher energy gains for a microorganism.
As the signal production from AOC was suppressed at DO
concentrations higher than 2 mg/L, the elimination of DO from
seawater is still necessary for the practical applications of the
biosensor. The combination of an electrochemical online oxygen
removal with the sensor described here is currently in progress in our
laboratory.

Volume 5 Issue 2 1000152

Citation:

Quek S B, Cheng L, Ralf Cord-Ruwisch (2014) Bio-Electrochemical Sensor for Fast Analysis of Assimilable Organic Carbon in
Seawater. J Biosens Bioelectron 5: 152. doi:10.4172/2155-6210.1000152

Page 4 of 4

Conclusions
The sensitive and accurate online monitoring of low levels of
organic pollutant (i.e. AOC) in seawater is essential to predict the
biofouling potential of the feedwater to RO desalination plants. The
use of positive anodic potentials for development of the anodophilic
biofilm and operation is of advantage compared to the traditionally
negative anodic potentials used as it allows a faster development of the
sensor biofilm and improved signal quality.
In the current study acetate was only used as a preliminary substrate
to establish a proof of concept and optimize the sensor performance
under the well-controlled conditions. In the further, we plan to
improve this sensor by testing complex organics and develop a
disposable-type anode for the real application.

Acknowledgement
The authors acknowledge the financial support of the National
Centre of Excellence in Desalination Australia, which is funded by the
Australian Government through the National Urban Water and
Desalination Plan. The authors would also like to thank Murdoch
University, AquaMen, Valoriza Agua (Spain) and Nanyang Technical
University (Singapore) for their advice and financial support.

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