SENIOR PROJECT REPORT

Study of Effects of biofilm on electrode surface using

potentiostat method

Group Members

1. Mr.Pornpawit Sanmongkol ID: 5622770691

2. Ms.Apichanat Aroonkamon ID: 5922800214

Advisor

Assoc. Prof. Dr. Rachnarin Nitisoravut

SCHOOL OF BIO-CHEMICAL ENGINEERING AND TECHNOLOGY

SIRINDHORN INTERNATIONAL INSTITUTE OF TECHNOLOGY

THAMMASAT UNIVERSITY
 Chapter 1
Introduction

In this decade, the consumption of energy has increased drastically. The main source
that is commonly being used is fossil fuels. Fossil fuels such as natural gas, oil, coal, are non-
renewable energy sources that might vanish if the consumption demand doesn’t lower.
Energy that is obtained from fossil fuels, produces by-products such as carbon dioxide,
carbon monoxide and other greenhouse gases. This environmental issue is widely considered
in many scientific fields to reduces the impact to the society and globally

Microbial Fuel Cell (MFC) is discovered to answer in the wide range of

environmental issues. It is a bio electrochemical reactor in which electroactive bacteria
convert the energy stored in various chemical substrates, into electricity [1] It is bio-
electrochemical device run by reparation mechanism by microbes converting organic
substance directly into electricity. It does this via redox reaction.

This application can be applied to many fields of environmental issues such as waste
water treatment and nutrient recovery [2,3] Chen et al. who reported an electrodialysis
system, used the MFC for alkali production as well as being upgraded of biogas. [4]

MFCs are commonly composed of an anode and a cathode chamber that are separated
by a proton exchange membrane. These elements determine the overall performance of the
MFC and susceptible changes from condition during operation. These changes include
increasing the biofilm thickness that are formed on the electrode surface. [5, 6] .The biofilm
activity on cathode is implied for increasing the oxygen reduction rates, when phototrophic or
aerobic activated sludge were used as the bio catalyst.[7,8] On the other hand, unwanted
biofilm growth on the surface of MFC component such as, membrane or the cathode, can
deteriorate the performance of MFC due to biofouling after long term operation. [9,10]

In this project, which is focused on the biofilm that is formed on the electrode’s
surface that can affect to the performance of MFC, which means that the amount of power
can be affected.

There are several goals of using MFCs ,which is nowadays , two common topic that
many scientific fields concern ,which is waste water treatment ,and another is power
generation, even it is very small amount that MFC can be able to generate it, but since the
topic ,is interested in biofouling ,so in this project are concerned about power that can be
generated based on the different mesh size of electrode to tell the performance of the most
proper mesh size of electrode that can give the best result.
 The scope of this research is that the performance of electrodes is formed on the
surface of it, when there has A biofilm. This is also influenced by the different mesh sizes of
THE electrodes. Beyond that a new technology was invented lately. It is potentiostat, an
electronic hardware. The function is to control a three electrodes cell, which is working
electrode, reference electrode, and auxiliary electrode and is run in most electroanalytical
experiments. [11,12]

Potentiostat is an equipment that can measure the performance of the electrode. The
reason to use potentiostat is that it is more practical to do in the university lab. Since the MFC
operation might be too complex for analysis. Since in this research is focused on a small part
of equipment, which is the performance of electrode during operation due to the biofouling
affect. A potentiostat should most probably give more evident and precise results.
Objectives
► To clarify the effect carbon fiber electrode modified carbon fiber electrode that affect to
the performance of MFCs.

► To study the effect of materials that are used as electrode to power output.

► To portray microbial shaping in different electrode type.

► To provide the robust data about various factors and analyze boosting and declining
factors for potentiostat efficacy.

► To lay foundation for interpretation of characteristics of electrode material in coming day

within biosystem principles.
 Chapter2
Literature Review
Introduction
Microbial respiration is based on electron transfer from electron donors to electron
acceptors, a series of reactions facilitated by a cascade of energetic substances (Stems et al.
2006; Gralnick and Newman 2007; Bird et al. 2011; Kraft et al. 2011). The donors and
acceptors of electrons are typically dissolved substances; however, some microorganisms can
use solid electron donors and/or solid electron acceptors, such as minerals and metals, in
respiration (Nealson and Finkel 2011). The exact mechanisms of electron transfer between
microorganisms and solid substances have 2 differences point of views. (1)Electrons are
transferred by conduction through extracellular materials or elongated appendages called
nanowires or pili (Reguera et al. 2005; El-Naggar et al. 2010; Malvankar et al. 2011) , and (2)
The electrons are transferred using redox mediators (Marsili et al. 2008a; Coursolle et al.
2010; Peng et al. 2010; Velasquez-Orta et al. 2010).

Biofilms with microorganisms capable of electron transfer to and from solid electron
acceptors have been used in microbial fuel cells (MFCs) to harvest energy from various
environmental processes (Sharma and Kundu 2010). The biofilms grown on the electrodes of
MFCs are called electrochemically active biofilms (EABs), which means all microorganisms
are electroactive in the respiration process (Marsili et al. 2010).

MFCs and the newer biologically catalyzed electrochemical cells have come to be
known as bioelectrochemical systems (BESs) (Rabaey et al. 2007; Arechederra and Minteer
2008; Ivanov et al. 2010; Manohar et al. 2010; Rosenbaum and Schroder 2010). BESs can
provide new insights into the fundamental mechanisms of electron transfer between
microorganisms and solid substances. Moreover, many scientific paper also use BESs as tools
of discovery in studying the process of electron transfer in EABs.

Lastly, we hope that this research can help identify and evaluate the strengths and the
limitations of BESs for both generating energy and studying the mechanisms of electron
transfer between microorganisms in biofilms and solid substances.

Potentiostat
A potentiostat is the electronic hardware required to control a cell and run
most electroanalytical experiments.[1]. The system functions by maintaining the potential of
the working electrode at a constant level with respect to the reference electrode by adjusting
the current at an auxiliary electrode.
Use of Potentiostat
This equipment is fundamental to modern electrochemical studies using three
electrode systems for investigations of reaction mechanisms related to redox chemistry and
other chemical phenomena. The dimensions of the resulting data depend on the experiment.
In voltammetry, electric current in amps is plotted against electric potential in voltage. In
a bulk electrolysis total coulombs passed (total electric charge) is plotted against time in
seconds even though the experiment measures electric current (amperes) over time. This is
done to show that the experiment is approaching an expected number of coulombs.
Most early potentiostats could function independently, providing data output through
a physical data trace. Modern potentiostats are designed to interface with a personal
computer and operate through a dedicated software package. The automated software allows
the user rapidly to shift between experiments and experimental conditions. The computer
allows data to be stored and analyzed more effectively, rapidly, and accurately than historic
methods.

A potentiostat is a control and measuring device. It comprises an electric circuit which

controls the potential across the cell by sensing changes in its resistance, varying
accordingly the current supplied to the system: a higher resistance will result in a decreased
current, while a lower resistance will result in an increased current, in order to keep the
voltage constant as described by Ohm's law.

Hickling built the first three electrode potentiostat, which is reference electrode,
working electrode, and auxiliary electrode. Hickling's device used a third electrode,
the reference electrode to control the cell potential automatically. A potentiostat measures the
potential difference between the working and the reference electrode, applies the current
through the counter electrode and measures the current.

Significant features of Potentiostat

In electrochemical experiments the electrodes are the pieces of equipment that comes
in immediate contact with the analyte. For this reason, the electrodes are very important for
determining the experimental result.
Simplified Schematic

Figure 1. Simple schematic of a

potentiostat. [
https://www.gamry.com/application-
notes/instrumentation/potentiostat-
fundamentals×1 on an amplifier
indicates that the amplifier is a unity-
gain differential amplifier. The output
voltage of this circuit is the difference
between its two inputs.

The Electrometer

The electrometer circuit measures the voltage difference between the reference and
working electrodes. Its output has two major functions: it is the feedback signal in the
potentiostat circuit, and it is the signal that is measured whenever the cell voltage is needed.

An ideal electrometer has zero input current and an infinite input impedance. Current
flow through the reference electrode can change its potential. In practice, all modern
electrometers have input currents close enough to zero that this effect can usually be ignored.

The I/E Converter

The current-to-voltage (I/E) converter in the simplified schematic measures the cell
current. It forces the cell current to flow through a current-measurement resistor, Rm. The
voltage drop across Rm is a measure of the cell current.

The Control Amplifier

The control amplifier is a servo amplifier. It compares the measured cell voltage with
the desired voltage and drives current into the cell to force the voltages to be the same.

Note that the measured voltage is input into the negative input of the control
amplifier. A positive perturbation in the measured voltage creates a negative control amplifier
output. This negative output counteracts the initial perturbation. This control scheme is
known as negative feedback.

Under normal conditions, the cell voltage is controlled to be identical to the signal
source voltage.
 Current measurement resistor (Rm) is regarded as 2 impedances z1 and z2. Z1 includes
Rm in series with interfacial impedance of Auxiliary electrode and the solution resistance
between auxiliary and reference electrode. Whereas, Z2 represents the interfacial impedance
of working electrode in series with solution resistance between working and reference
electrodes.

The Signal

The signal circuit is a computer-controlled voltage source. It is generally the output of

a Digital-to-Analog (D/A) converter that converts computer-generated numbers into
voltages.

Proper choice of number sequences allows the computer to generate constant

voltages, voltage ramps, and even sine waves at the signal circuit’s output.

When a D/A converter is used to generate a waveform such as a sine wave or a ramp,
the waveform is a digital approximation of the equivalent analog waveform, with small
voltage steps. The size of these steps is controlled by the resolution of the D/A converter and
the rate it at which it is being updated with new numbers.

Fig2.Potentiostat[https://www.gamry.com/applicationnotes/instrumentation/potentios
tat-fundamentals/]

Factor that can affect to Potentiostat

In electrochemical experiment, the electrodes are the pieces of equipment that contact
with the analyte so electrodes are very important for determine experiment result. The size of
electrodes affects the magnitude of currents passed. Which can affect signal to noise ratio.
Electrodes are not only limiting factor for electrochemical experiment, the potentiostat also
has a limited range of operation.

There is some systematic error that can caused to potentiostat such as Electric
potential range-Potential is based on solvent, electrons can also give an affect, Also, accuracy
in potential, limit deviations between the actual and reported, range of scan rate which is
about how slow or fast a potential can be scanned. Moreover, electric current range and
number of working channels also give effect to potentiostat.

Voltammetry
Voltammetry is a category of electroanalytical method, used in analytical chemisty
and various industrial process. The information of analyte is obtained by measuring the
current as the potential is varied [1,2]

Voltammetry experiment

Investigate the half-cell reactivity of an analyte. It is the study of current as a function

of applied potential (E), I=f(E) is the represent of voltammograms. Potential (E) is varied
arbitrarily and current value is measured depends on E that varied. Also, the voltammograms
curve depends on speed of potential variation or whether solution s stirred. Most experiment
control the potential (volts) of an electrode in contact with analyte while measuring the result
current

To conduct experiment, one requires at least two electrodes, the first electrode is
working electrode, which makes contact with the analyte, must apply the desired potential in
a controlled way and facilitate the transfer of charge to and from analyte. The second
electrode, that must have known the potential, which be able to gauge the potential of the
working electrode, also it must balance the charge added/removed by working electrode.
Anyway, it is very difficult for an electrode to maintain a constant potential while passing
current through working electrode. To solve this problem the roles of supporting electron and
providing a reference potential are divided to two separate electrodes which is reference
electrode and auxiliary electrode.

Reference electrode

Fig3. Ag/AgCl reference electrode

The reference electrode is a half cell with known reduction with known reduction
potential. The role is to act as reference in measuring and controlling the working electrode’s
potential. Moreover, no current allows to pass via reference electrode. It has stable electric
potential, which stability of the potential reached by a redox system with constant
concentration of each participants of redox reaction. Most common aqueous reference
electrodes are standard hydrogen electrode (SHE) has E= 0.00 V., saturated calomel
electrode (SCE) has E=+0.241V., and silver/silver chloride electrode has E=+.197 V.

Auxiliary electrode (Counter electrode)

The auxiliary electrode passes all the current needed to balance the current
observed at the working electrode. To achieve this current, it swings to extreme potentials at
the edges of the solvent window, where it oxidizes/ reduces the solvent or supporting
electrolyte. Finally, the three-electrode system includes working electrode, reference
electrode and auxiliary electrode.

The auxiliary electrode can be anything as long as it does not react with analyte
solution. Moreover, it is commonly to conduct well since the current need to be applied via
this electrode. The auxiliary electrode is different from reference electrode, it establishes the
electrical potential to balance with working electrode, moreover, potential can be adjusted to
balance the reaction occurring at working electrode so it allows the potential of working
electrode to be measured against known reference electrode without care about stability of
reference by pass current over it.

Working electrode

Electrode in system which the reaction of interest is occurring. It works together

with auxiliary and reference electrode in three electrode systems. It consists of inert material
for instance gold, silver, Platinum, inert carbon film electrode. Special type of working

Solvent window or electrochemical window (EW)

Solvent window is the voltage range between which the substance is either
oxidized or reduced. It is one of important characteristic to be identified for solvent and
electrolytes used in electrochemical applications. It is also commonly used to indicate the
potential range which calculate from the standard potential difference of anode and cathode.
This range is important to determine efficiency of electrode, out of this range, the electrical
energy is deteriorated.

In practice, it is important to have a working electrode with known surface

characteristic. As a result, it requires to clean and polish working electrode regularly, which
is related to this research, about determination of the performance of electrode with
differences mesh sizes due to the biofilm formation on electrode surface.

In most voltammetry experiment, supporting electrolyte is used to minimize

solution resistance. It is possible to run an experiment, supporting electrolyte is sued to
minimize solution resistance. It is possible to run all experiment without supporting
electrolyte, since the greatly the resistance, the badly the accuracy of the results.

Supporting electrolyte
Supporting electrolyte is an electrolyte containing chemical species that are not
electroactive within the range of potential used. Sometimes, referred to an inert electrolyte. It
is widely used in electrochemical measurements when control electrode potential is required.
It aims to eliminate the transport of electroactive specie. Moreover, it also maintains constant
ionic strength and pH.

Types of voltammetry

1. Linear sweep voltammetry

It is the voltammetry method, which current at a working electrode is measured while
the potential between the working electrode and reference electrode is linearly in time.
Experiment method

The set up utilizes a potentiator and three electrodes set up to deliver a potential to a solution
and monitor its change in current. The potential energy is delivered through working
electrode. After plot the graph between potential versus time, the graph is in linearly trend
and the slope that get from the graph is scan rate. In this set up, the working electrode, which
oxidation and reduction reaction occur the reaction occur at the surface of working electrode
A+ e- -> A- which give the standard potential (Es) zero. And the potential that has got from
monitor is as approach as standard means the current on the surface increases. As the
molecule on the surface of working electrode are oxidized or reduced, they move away from
surface and new molecule come into contact with the surface of working electrode. This flow
in and out of electron causes the current.

Current can tell the rate; which electrons are exchanged through the electrode-electrolyte
surface. When rate more than rate of oxidizing/reducing from electrolyte to electrode surface
the current reaches a plateau.

Character of linear sweep voltammetry

1. Identify unknown species from half-cell potential.

2. Determine concentration of solution from the height of current
 3. The plot between current versus potential graph can be increased by increases can
rate.
4. Higher potential means more oxidation/reduction at the surface of working electrode.

Fig4: Linear potential sweep

Linear sweep compared to cyclic voltammetry

For reversible reactions cyclic voltammetry can be used to find information about the
forward reaction and the reverse reaction. Like linear sweep voltammetry, cyclic
voltammetry applies a linear potential over time and at a certain potential the potentiostat will
reverse the potential applied and sweep back to the beginning point. Cyclic voltammetry
provides information about the oxidation and reduction reactions.

While cyclic voltammetry is applicable to most cases where linear sweep

voltammetry is used, there are some instances where linear sweep voltammetry is more
useful. In cases where the reaction is irreversible cyclic voltammetry will not give any
additional data that linear sweep voltammetry would give us. In one example, linear
voltammetry was used to examine direct methane production via a biocathode. Since the
production of methane from CO2 is an irreversible reaction, cyclic voltammetry did not
present any distinct advantage over linear sweep voltammetry. This group found that the
biocathode produced higher current densities

2. Staircase voltammetry
From linear sweep, where the current at working electrode is measured, while
potential between working and reference electrode is swept linearly in time, also, at the peak
of current signal represents oxidation and reduction reaction.
 For staircase voltammetry, potential sweep in a staircase pattern over time. Also, the
current is measured at the end of each potential change.

Fig5: Staircase potential sweep set against a linear potential sweep

Fig6: Comparison of the current

response of a platinum disc electrode in
1 M Sulphur acid given by linear sweep
voltammetry and staircase voltammetry
methods.

3. Square wave voltammetry

Square wave voltammetry is one of the form of linear potential sweep voltammetry.
The working electrode is the dropping mercury electrode (DME). Moreover, the surface area
of mercury drop electrode is varied.

In a square wave voltammetry experiment, the current at a working electrode is

measured while the potential between the working electrode and a reference electrode is
swept linearly in time. The potential waveform can be viewed as a superposition of a regular
square wave onto an underlying staircase. SWV can be considered a modification of staircase
voltammetry. The current is sampled at two times - once at the end of the forward potential
pulse and again at the end of the reverse potential pulse, which in both cases immediately
before the potential direction is reversed. As a result of having current sampling at two
different instances per square wave cycle, two current waveforms are collected. When viewed
in isolation, the forward and reverse current waveforms mimic the appearance of a cyclic
voltammogram (which corresponds to the anodic or cathodic halves.

The diffusion layer is not renewed between potential cycles. Thus, it is not
possible/accurate to view each cycle in isolation; the conditions present for each cycle is a
complex diffusion layer which has evolved through all prior potential cycles. The conditions
for a particular cycle are also a function of electrode kinetics, along with other
electrochemical considerations.

4. Cyclic voltammetry
The cyclic voltammetry has the character that the working electrode potential is ramp linearly
versus time. After the set potential is reached, working electrode ramp in opposite direction
return to the initial potential. The ramp potential cycle can repeat. The plot between current at
working and applied potential also at the working electrode will give cyclic voltammetry. The
cyclic voltammetry is used to study electrochemical of analyte in solution

Fig7. typical cyclic voltammogram where and

show the peak cathodic and anodic current
respectively for a reversible reaction. The y-
axis shows the negative current.

Fig8. Cyclic voltammetry waveform

Method of cyclic voltammetry

The rate of voltage change over time as known as scan rate (V/s). Potential is
measured between working and reference electrode, whereas, current is measured between
working and auxiliary electrode.

From the fig.8 from t0 -> t1 that reducing potential is applied so the cathodic current
increase over this time so it means there are reducible analytes in system. Also, from t1 -> t2
that reduction potential is reached then it travels in the opposite direction than the previous
stage, then the cathodic current is decreased that implies the concentration of reducible
analyte is also decreased.

In the redox reaction, the reduced analyte will start to be deoxidized rising graph of
current again, which is from the fig.8 at t2 -> t3. The more reversible the redox, the more loop
can repeatable.

Cyclic voltammetry data can provide information about redox potentials and
electrochemical reaction rates. For instance, if the electron transfer at the working electrode
surface is fast and the current is limited by the diffusion of analyte species to the electrode
surface, then the peak current will be proportional to the square root of the scan rate. This
relationship is described by the Cottrell equation.

Diffusion layer
Diffusion layer defined as the region in the vicinity of an electrode where the
concentrations are different from their value in the bulk solution. The diffusion layer thus
depends on the diffusion coefficient (D) of the analyte and for voltammetry measurements on
the scan rate (V/s). At slow scan rates, the diffusion layer is large. Relevant to cyclic
voltammetry, the diffusion layer has negligible volume compared the volume of the bulk
solution.

Diffusion coefficient
Diffusion coefficient is a proportionality constant between the molar flux due to
molecular diffusion and the gradient in the concentration of the species. The higher the
diffusivity (of one substance with respect to another), the faster they diffuse into each other.

Species pair Temperatur D Reference

(solute-solvent) e (°𝐂) (𝐂𝐂𝐂 /s)
Acetone (dis) – Water (l) 25 1.16x10−5 [3]
Air (dis) – Water (l) 25 2.00x10−5 [3]
 Ammonia (dis) – Water (l) 25 1.64x10−5 [3]
Argon (dis) – Water (l) 25 2.00x10−5 [3]
Benzene(dis) – Water (l) 25 1.02x10−5 [3]
Bromine (dis) – Water (l) 25 1.18x10−5 [3]
Carbon monoxide (dis) – Water 25 2.03x10−5 [3]
(l)
Carbon dioxide (dis) – Water (l) 25 1.92x10−5 [3]
Chlorine (dis) – Water (l) 25 1.25x10−5 [3]
Ethane (dis) – Water (l) 25 1.20x10−5 [3]
Ethanol (dis) – Water (l) 25 0.84x10−5 [3]
Ethylene (dis) – Water (l) 25 1.87x10−5 [3]
Helium (dis) – Water (l) 25 6.28x10−5 [3]
Hydrogen (dis) – Water (l) 25 4.50x10−5 [3]
Hydrogen sulfide (dis) – Water 25 1.41x10−5 [3]
(l)
Methane (dis) – Water (l) 25 1.49x10−5 [3]
Methanol (dis) – Water (l) 25 0.84x10−5 [3]
Nitrogen (dis) – Water (l) 25 1.88x10−5 [3]
Nitric oxide (dis) – Water (l) 25 2.60x10−5 [3]
Oxygen (dis) – Water (l) 25 2.10x10−5 [3]
Propane (dis) – Water (l) 25 0.97x10−5 [3]
Water (l) – Acetone (l) 25 4.56x10−5 [3]
Water (l) – Ethyl alcohol (l) 25 1.24x10−5 [3]
Water (l) – Ethyl acetate (l) 25 3.20x10−5 [3]
Table 1: Diffusion coefficient

Ref Cussler, E.L. (1997). Diffusion: Mass Transfer in Fluid Systems (2nd ed.). New York:
Cambridge Press. ISBN 0-521-45078-0

Character of cyclic voltammetry

The characteristic is depending on the analyte being used. The analyte has to be redox
within the potential window to be scanned. From fig. ipc represents the peak of cathodic
current, ipa represents the peak anodic current which is belongs to the y-axis these properties
describe about the reversible reaction properties. More over on x-axis which is represents the
potential, in this axis the difference between two peaks potentials (∆Ep) is the differences
between Epc and Epa . These differences result from the effects of analyte diffusion rates. The
waveform of cyclic voltammograms is affected by the rate of electron transfer, that an
electron relocates from an atom/molecule to another place .Typically for reversible reaction
the ratio between ipa and ipa is one, When a reversible peak is observed, thermodynamic
information in the form of a half cell potential E01/2 can be determined.

Experimental set up

It is conducted on a solution in a cell fitted with electrodes. The solution consists of

the solvent which contains electrolyte and analyte. For the set-up of the cell, cyclic
voltammetry experiment has a cell fitted with three electrodes which is reference, working,
and auxiliary electrode as known as three electrodes set up. Electrolyte usually added to the
sample solution to make a sufficient conductivity. The solvent, electrolyte, material
composition of working electrode will determine the potential range that can be performed.

The electrode is immobilized and place in unstirred solution, which is this still
solution method gives cyclic voltammetry characteristics diffusion controlled peaks, also
allow a portion of the analyte to remain after redox and oxidation, that may display the
further redox activity. By the way, stirring the solution is important in order to supply the
electrode surface with fresh analyte for each new batch of experiment.

When reduced or oxidized analyte, it is precipitate onto the electrode surface, which
can inhibit the display of redox activity on electrode surface then it gives the affect to CV
measurement. As a result, during operation it is necessary to clean electrode before start a
new batch of analysis.

Common materials for the working electrode include glassy carbon, platinum,
and gold. A regular working electrode has a radius of 1 mm. Having a controlled surface area
with a well-defined shape is necessary for being able to interpret cyclic voltammetry results.

The counter electrode, also known as the auxiliary or second electrode, can be any
material that conducts current easily and will not react with the bulk solution. To maintain the
observed current the counter electrode will often oxidize or reduce the solvent or bulk
electrolyte.

For electrolyte that being used, since the electrolyte ensures good electro conductivity
and minimizes iR drop such that the recorded potentials correspond to actual potentials.

Application

CV experiment become important electroanalytical technique. It often used to study redox

processes as well as determine stability of reaction products, electron transfer kinetics,
reversible reaction. Also, CV used for determine diffusion coefficient of an analyte, reduction
potential of analyte. Moreover, to determine concentration of unknown solution by make
calibration curve plot between current and concentration

5. Anodic stripping voltammetry

Anodic stripping voltammetry used for quantitative determine specific ionic species.
The analyte is electroplated, which is the process that use electric current to reduce dissolved
metal cation so this form a thin metal coating on electrode, the analyte is electroplated on the
working electrode during a deposition step then it oxidized from the electrode during
stripping step then measure current. The graph trend is rise up can be either linear, staircase
or square wave. Mostly the working electrode that being used is Mercury film electrode or
sometimes it can also be Ag, Au, Pt.

Fig9. A: Cleaning step, B:

Electroplating step, C: Equilibration
step, D: Stripping step

Steps of anodic stripping voltammetry

First stir the solution at the first two steps, which is cleaning and deposition of
analyte back to electrode step by lower the potential to reduce analyte back and deposit on
electrode. Then stop stirring to allow the deposited material get mixed with electrode well.
Lastly, raising the working electrode to higher potential and keep stirring analyte to let
oxidizing process occurs and repeated so the electrons transfer occurs and generate the
current.

6. Cathodic stripping voltammetry

This is a voltammetry method for quantitative determination of specific ionic
species. It is similar to anodic stripping voltammetry, except that for the plating step, the
potential is held at an oxidizing potential, and the oxidized species are stripped from the
electrode by sweeping the potential positively. This technique is used for ionic species that
form insoluble salts and will deposit on or near the working electrode during deposition. The
stripping step can be either linear, staircase, square wave, or pulse.
Electroanalytical methods
Electroanalytical methods are the technique use to study analyte by measuring the
potential and current in the electrochemical cell containing the analyte. Three main methods
are potentiometry, which is the difference in electrode potential is measure while coulometry,
that the cell’s current is measured over time and then voltammetry which is the cell’s current
is measured while actively altering the cell’s potential.

Potentiometry
Measures the potential of a solution between two electrodes which is reference
electrode, that has a constant potential and working electrode, which the potential changes
with composition of the sample so the difference of potential between the two electrodes tell
the composition of the sample. The working electrode made selectivity sensitive to the ion of
interest and the time that electrode need to make equilibrium with solution affect accuracy of
measurement

Factor that affect to the performance

1. Electrode material
The biofilm electrode material affects the measured current and open circuit
potential (OCP) of electrodes with EABs grown on them. Traditionally, cheaper graphite,
carbon paper, carbon granule, carbon brush, or carbon felt electrodes are used in MFC
practical applications (Wei et al. 2011; Zhou et al. 2011b). glassy carbon electrodes are often
used to observe electrochemical activity. One advantage of glassy carbon is that the
background currents are practically zero in the potential ranges in which EABs are studied;
another is that it is nonporous. Additionally, there is significant literature on electrochemistry
utilizing glassy carbon electrodes, potentially opening up a vast amount of literature to EAB
studies. The use of glassy carbon electrodes would provide more universal current values
when studying fundamental electron transfer of EABs. When glassy carbon electrodes are not
compatible with an experiment, common substitutes include Carbon black as an alternative
cathode material for electrical energy recovery and transfer in microbial fuel cell. The result
from this work (Logan, B.E. & Regan, J.M. et al.2006) confirm the chemical activity of
carbon black and prove its potential as a talented alternative by it low-cost, easy regeneration
and flexibility for the solid-state cathode in microbial fuel cell. Carbon black is potential-
favorable to derive electrons from biodegradable organics by microbial extraction. The
coincidence between the capacity of carbon black cathode and its abundance of C=O indicate
carbon black’s role as electron sink derived redox active C=O.

Reactors and electrode configurations used to study electrochemically active biofilms

 The positions of the biofilm electrode, counter electrode, and reference electrode
(RE) in a BES have direct effects on the measured current. (Kissinger and Heineman
1996; Logan et al. 2006; Manohar and Mansfeld 2009) the simplest system to configure to
study electron transfer in EABs could be an MFC (Fig.10). It is generally used to quantify
power production in practical research (Logan et al. 2006; Clauwaert et al. 2008). The
parameters of an MFC could be optimized to produce more power. However, an MFC cannot
obtain information about the EABs on the individual electrodes since only cell potential can
be measured. Without the individual electrode potentials, it is very difficult to determine the
fundamental reason for an increase in power in an MFC. The end result would be the
inability to study electron transfer in EABs. The MFC reactor configuration can be enhanced
by inserting a reference electrode to measure individual electrode potentials and characterize
overpotentials and potential losses (Figure 1B) (Clauwaert et al. 2008; Rismani-Yazdi et al.
2008).

The potentiostat is generally required to measure the current while fixing the biofilm
electrode potential (Figure 1C or Figure 1D). This system is often called a three-electrode
bioreactor and is used frequently to study biofilm voltammetry (Fricke et al. 2008; Marsili et
al. 2008a; Bouhenni et al. 2010; Strycharz et al. 2011). Reactor configurations with (Figure
1C) and without (Figure 1D) an ion-selective membrane have distinct advantages. For
membrane-less reactor configurations, the membrane potential loss is eliminated (Logan et al.
2006, 2008; Rozendal et al. 2008; Li et al. 2010). The disadvantage is that the supporting
electrode reaction products are free to diffuse to the working electrode. This could potentially
generate uncontrolled experimental parameters. In membrane-less MECs, the diffusion of
hydrogen from the counter electrode to the working electrode can be utilized by EABs to
produce a current higher than that expected with the supplied electron donor (Lee et al.
2009a; Parameswaran et al. 2011).

Fig10. EABs can be studied using four different

configurations: (A) an MFC with an anode and a
cathode; (B) an MFC with an anode, a cathode
and a reference electrode (RE) used to monitor
individual electrode potentials (against the RE);
(C) an MFC with an anode and a cathode and an
RE connected to a potentiostat; and (D) an
electrochemical cell with a working electrode
(WE) covered by biofilm and a counter electrode
(CE) and RE immersed in the same solution. This
is called a three-electrode bioreactor.

Current-limiting electrode
 The current-limiting electrode is the electrode which cannot pass a higher current
than the other electrode either because of its small size or because of limiting electrode
reaction kinetics. If an electrode with an EAB limits the current of the BES, this practically
means that the performance of the BES is limited by the EAB. The knowledge of which
electrode limits the current is critical when BESs are studied. In the case of MFCs (Figure
10A), it is important to confirm that the EAB under investigation is limiting the current. The
simplest way to determine which electrode in an MFC setup is the current-limiting electrode
is to monitor the individual electrode potentials using a RE (Figure 10B). The potentiostatic
mode controls the biofilm electrode potential such that perturbations of the biofilm electrode
potential cause a measurable change in the EAB under investigation. Thus, the current is
controlled by the EAB.

Potential window

The effect of the polarization potential has been studied for various BESs and EABs
(Aelterman et al. 2008; Cheng et al. 2008; Lee et al. 2009b; Torres et al. 2009; Srikanth et al.
2010). There is no consensus on the exact magnitude of potential to apply; however, there is a
clear understanding that applying a polarization potential more positive than the OCP of the
biofilm electrode is sufficient to drive electrons from the EAB to the biofilm electrode.

The polarization potential could also be used to select for different types of EABs,
with different abilities for extracellular electron transfer (Torres et al. 2009). However, for use
in MFCs and other BESs.

Electrode acclimatization
Electrode acclimatization refers to the processes that require time for EABs to
populate an electrode surface (Kim et al. 2005; Finkelstein et al. 2006; Liu et al.
2008; Rodrigo et al. 2009; Wang et al. 2009). The purpose of acclimatization is to increase
electrode performance by enhancing biofilm attachment and/or allowing the biofilm
electrode to reach a steady-state OCP prior to use in a BES (Larrosa-Guerrero et al.
2010; Cheng et al. 2011; Kassongo and Togo 2011; Renslow et al. 2011a). Four
acclimatization methods are common in the literature, (1) Closed circuit: the biofilm electrode
and the supporting electrode are short-circuited (2) open circuit: the biofilm electrode is left
disconnected; (3) controlled cell potential: a constant potential is applied between the biofilm
electrode and the counter electrode; and (4) controlled electrode potential: a constant
polarization potential is placed between the biofilm electrode and the reference electrode.
 Closed circuit acclimatization allows the biofilm electrode to reach a steady-state cell
potential, focusing on enhanced steady-state electron transfer. (Aelterman et al. 2008; Jadhav
and Ghangrekar 2009; Zhang et al. 2011). Open circuit acclimatization allows the biofilm
electrode potential to develop a steady-state OCP utilizing natural redox processes in the
environment. Controlled cell potential and controlled electrode potential acclimatization
requires powered external equipment to control cell potential.

Growing electrochemically active biofilms

Growing EABs can be achieved using two distinct acclimation methods. The first
method is to grow EABs on an electrode in the presence of a soluble electron acceptor. This
method is in line with open circuit acclimatization since no polarization potential is required
for EAB growth. Once the EAB has reached a desired state (thickness, surface coverage,
metabolic activity, OCP), the soluble electron acceptor can be removed and the EAB can be
switched to respire on the biofilm electrode where a current is measured. The second method
is to grow the EAB on an electrode which acts as the sole electron acceptor. This method is in
line with closed circuit, controlled cell potential, and controlled electrode potential
acclimatization where current produced reflects biofilm growth. Once the EAB generates a
desired state (thickness, surface coverage, metabolic activity, current), the EAB can be used
for further investigations.

Electrochemical techniques for studying extracellular electron transfer of

electrochemically active biofilms
When the correct reactor configuration has been chosen and the EAB has been
successfully grown or acclimatized on an electrode, the next step is to study the electron
transfer properties of the biofilm electrode using electrochemical techniques. (Oldham and
Myland 1994; Kissinger and Heineman 1996; Bard and Faulkner 2001).

1. Long-term electrode polarization of electrochemically active biofilms

In a long-term electrode polarization experiment, a selected polarization
potential is applied to an electrode with an EAB grown on it and the current is measured.
In this way the total charge transferred in a batch system or the steady-state current
produced by the EABs can be measured. A long-term electrode polarization experiment
identifies sustainable current generation that can be systematically related to controlled
parameters such as polarization potential. The technique appears similar to controlled
electrode potential acclimatization, in which EABs are grown on polarized electrodes. Yi
et al. (2009) allowed G. sulfurreducens DL1 to acclimatize on an electrode for 5 months
and selected for a new strain, G. sulfurreducens KN400. They then compared the
 sustainable current productions of the two strains using long-term electrode polarization
experiments. The key value of performing electrode polarization experiments is to
observe the electrochemical response of an EAB to a change in a controlled experimental
parameter.

Fig.11 Current generation by S. oneidensis MR-1 biofilm on a graphite

electrode under anaerobic conditions in the reactor configuration shown in Figure
10C. The current increased steadily over a period of 9 days. The polarization potential
was 0 mVAg/AgCl.

When a significant current is generated by an EAB such as that shown in Figure 2,

the EAB is thought to exchange electrons with the biofilm electrode. To confirm that the
current generated is related to the metabolism of the EAB, long-term electrode polarization
experiments can be performed. In batch experiments, the total consumption of the electron
donor can be correlated to the total charge transferred. If the current trends towards zero
when the electron donor concentration goes to zero, then the oxidation of the electron donor
is the source of electrons and the coulombic efficiency can be calculated (Logan et al. 2006).

Replacing the bulk solution with fresh growth medium during electrode
polarization experiments has been done to probe soluble extracellular electron transfer
mechanisms. For example, Marsili et al. (2008a) replaced the spent solution in a Shewanella
oneidensis MR-1 biofilm reactor to show that soluble redox mediators were responsible for
the steady-state current generated. Bond and Lovely (2003) replaced the bulk solution of G.
sulfurreducens biofilms with acetate-free growth medium to show acetate dependence.

Other experiments can be done to isolate controlled parameters in EAB experiments

that affect extracellular electron transfer mechanisms. As well as The pH can be adjusted to
correlate proton transfer with current generation. Both Torres et al. (2008) and Patil et al.
(2011) adjusted bulk pH and measured the current generation of their EABs.
 1. Cyclic voltammetry
When a steady-state current is identified as being the result of EABs, cyclic
voltammetry (CV) can be used to identify the biofilm electrode potential at which active
redox couples related to the EAB are oxidized or reduced. CV is an electrochemical
technique that applies a linear polarization potential scan from an initial polarization potential
to a final polarization potential and measures the current. Because redox couples can only be
reduced or oxidized at certain electrode potentials, CV can determine the biofilm electrode
potential range in which extracellular electron transfer can occur in EABs (Fricke et al.
2008; Marsili et al. 2010). CV can be used to determine whether EABs have the capability
for electron transfer, whether freely diffusing species or surface-adsorbed species contribute
to electron transfer, and whether EABs engage in catalytic activity towards specific substrates
(Fricke et al. 2008; Richter et al. 2009; Katuri et al. 2010; Carmona-Martinez et al.
2011; Strycharz et al. 2011). However, CV do not reflect the ability of EABs to produce
long-term, sustainable current, which should be reserved for long-term electrode polarization
experiments.

Often CV is coupled to long-term electrode polarization experiments in which CV

can explain how the active redox couples are affected by systematic changes in controlled
parameters such as bulk pH (Patil et al. 2011).

EABs growing on electrode surfaces can be described as a ‘well-controlled’ condition

in which CV can be applied to study reaction mechanisms. Beyond the reproducibility of the
biofilm electrode surface, simply characterizing biofilm structure itself has been difficult
(Yang et al. 2000, 2001; Renslow et al. 2011b). The result of biofilm heterogeneity is the
local variation of not only diffusion coefficients, but local flow velocities as well (Beyenal et
al. 1998; Beyenal and Lewandowski 2001, 2002; Renslow et al. 2010). The unknown mass
transfer conditions suggest that not all cells in the EAB contribute equally to current
production.

Fig12. (A) In situ CV of S. oneidensis MR-1 (black trace). The biofilm was physically
removed from the biofilm electrode and a second CV was performed (red dashed
trace). A glassy carbon electrode (10 mm × 10 mm) was used. The scan rate was 10
mV s71. (B) In situ CV of G. sulfurreducens (black trace). The biofilm was physically
 removed from the biofilm electrode and a second CV was performed (red dashed
trace). A glassy carbon electrode (25 mm × 25 mm) was used. The scan rate was 10
mV s71 for the first CV and 1 mV s71 for the second CV.
Advanced techniques in CV can provide further information about the
electrochemical responses of EABs under various conditions. These include the scan rate
dependence of peak currents, altering the reversal potential of the CV scan, and altering the
potential window of the CV scan (Bard and Faulkner 2001; Snider et al. 2011; Strycharz-
Glaven et al. 2011).

Fig.13: Example scan rate dependence of the

peak potentials of the CV in Figure 12A. Red
arrows show the peak potentials used for scan
rate analysis. The inset shows that the peak
potential is a linear function of scan rate.

One aspect of CV in EAB research is varying the initial potential and the potential
window to study anodic and cathodic peak coupling.

Figure 14(A). Choosing the initial potential of the CV can yield information on anodic and
cathodic peak coupling. (B) Choosing different specific potential windows can show different
CV behaviors that may be interpreted in different ways.

Figure 14(B). shows that scanning in the cathodic direction initially can mask the cathodic
peak response. Only after the scan in the anodic direction is completed is the cathodic peak
response observed. By manipulating the initial potential of the CV, anodic peaks can be
coupled to cathodic peaks. Figure 14B shows further that different voltammograms can be
obtained depending on the potential window used for the CV.
Limitation of electrochemical technique
Electrochemical investigations in complex systems such as EABs require more
physical and chemical evidence to determine if an observed electrochemical response was
caused by a change in an experimental parameter. Electrochemical techniques such as CV
accurately describe the nature of the electron transfer event in the presence of EABs
(reversibility, mass transfer limitations, properties of redox couples, and reaction steps).
however, they do not give any evidence on how EABs participate in the electron transfer or
what aspect of EABs promotes electron transfer. More importantly, the presence or absence
of electrochemical activity (current peaks observed in CV) does not necessarily imply that it
is or is not the source of long-term, sustainable current in EABs. An example of this is the
ability of certain microorganisms to utilize soluble exogenous electron shuttles in their
surroundings for extracellular electron transfer. For example, Milliken and May
(2007) showed that Desulfitobacterium hafniense strain DCB2 could utilize exogenous
quinone-like mediators to produce sustainable current in MFCs.

Coupled techniques
The coupling of electrode polarization, CV with methods that directly measure
physical or chemical parameters varying inside EABs in situ addresses issues with the
interpretation of electrochemical data. The goal of this coupling is to correlate the properties
that vary within the EAB under various electrode-respiring conditions. future EAB research
and advanced techniques should focus on the variation that occurs within the biofilm and not
just in the bulk solution. It can be done by: (1) directly measuring the kinetics of redox
mediators inside biofilms; (2) resolving local concentrations of chemical species inside the
biofilm; (3) resolving the physical location of electrochemically active species in situ; and (4)
correlating limiting current with local biofilm parameters. There are several available tools
and techniques that can be used, such as microelectrodes, Spectro electrochemical methods,
and microscopy.

1. Microelectrode
Microelectrodes have been used to study various biofilm properties in biofilms
and water-sediment interfaces since the early 1990s (Cronenberg and Vandenheuvel
1991; Glud et al. 1992; Vanhoudt et al. 1992; Zhang and Bishop 1994). Microelectrodes have
been used to measure concentrations of oxygen, hydrogen, hydrogen sulfide, and carbon
dioxide, as well as pH, redox potential, and local flow velocities (Lee and Debeer 1995; Yang
and Lewandowski 1995; Xia et al. 1998; Yu and Bishop 1998; Beyenal et al. 2004). Because
microelectrodes are minimally invasive and have dimensions that can be as small as 1-5 mm,
they are well suited to studying changes in both the EAB and the bulk solution above the
EAB during electrode respiration. A distinct advantage of microelectrodes is that they can
correlate local biofilm properties with the bulk biofilm properties, electron transfer rates and
electron transfer mechanisms. On the other hand, the data should be critically interpreted
when a highly heterogeneous EAB is investigated because the results can vary between
locations (Nguyen et al. 2012a).

Fig15. The redox potential inside a S.

oneidensis MR-1 biofilm grown on a
graphite electrode. Reprinted (adapted)
with permission from Babauta et al.
(2011). Copyright (2011) American
Chemical Society.

Microelectrodes can be used with voltammetry techniques in two modes. The first, in
which depth profiles are taken during constant polarization, which is shown in Figure 15, At
each step, redox potential was measured, producing a redox potential depth profile inside the
biofilm. In the presence of soluble redox mediators, redox potential is expected to increase
towards the biofilm electrode.

Microscopy

The in-situ imaging of biofilms using fluorescent microscopes is a standard technique

in biofilm research (Lewandowski and Beyenal 2007). The development of current with the
biofilm has been observed for thick biofilms (McLean et al. 2010). It provides the ability to
monitor biofilm parameters such as surface coverage, biovolume, and biofilm roughness with
current. The commercial availability has allowed the use of in situ confocal scanning laser
microscopy (CLSM) for EABs., Nevin et al. (2009) used CLSM to monitor the difference in
biofilm structure between G. sulfurreducens biofilms grown on an electrode. McLean et al.
(2008) used CLSM to locate individual strains of S. oneidensis MR-1 in a mixed culture
biofilm. Additionally, microscopic techniques allow the determination of single-cell electron
transfer rates (McLean et al. 2010). They were able to measure pH profiles that developed
within the biofilm during electrode respiration, providing evidence that pH changes
appreciably inside EABs. The use of microscopy would critically allow investigators to
correlate biofilm structure with its function related to electron transfer.
 Fig16. in situ confocal scanning laser microscopy (CLSM)

Scaling up the current density generated by electrochemically active biofilm

One of the limitations of MFCs, and of BESs generally, is that the current density
does not scale up linearly with the active surface area of the biofilm electrode to meet the
demand.
Extracellular electron transfer mechanism electrochemically active biofilm

The role of extracellular polymeric substances (EPS) in electron transfer. Cao et al.
(2011) found redoxactive proteins in the EPS of Shewa nella sp. HRCR-1, which were
commonly found on the cell surface. EPS is known to facilitate oxidation/reduction reactions
to minerals (Sand and Gehrke 2006; Gralnick and Newman 2007).

(Junil et al.2011) show the cyclic voltammograms that were caused by the bacterial
adhesion on the platinum electrode in a 10 mM potassium ferricyanide solution (scan rate 250
mV/s, potentials between 0.1 and 0.5 V vs. the Ag/AgCl reference electrode, pH 9.2).

Fig17. The cyclic voltammograms that

were caused by the bacterial adhesion on
the platinum electrode in a 10-mM
potassium ferricyanide solution (scan rate
250 mV/s, potentials between 0.1 and 0.5
V vs. the Ag/AgCl reference electrode, pH
9.2).
 Fig 18. image of the biofilms captured using CLSM on the platinum electrode s that
were caused by the biofilm formation for 24 h (in the condition of nutrients supply) on
the platinum electrodes in a 0.1 mM phosphate buffer solution (scan rate 250 mV/s,
potentials between 0.6 and 1.2 V for the platinum electrodes vs. the Ag/AgCl
reference electrode, pH 7.1).
 Chapter3
Methodology

Preparation Phase
1. Reactor design configuration
For our project, we focus on an electrode in the anaerobic chamber of microbial fuel
cell (MFC) system only.
1.1.Materials needed for the system per one condition
1.1.1. Triple head flask 500 mL 1 piece
1.1.2. Electrical alligator 1 piece
1.1.3. Stopper rubber 3 pieces
1.1.4. Carbon fiber 1 piece
1.1.5. Sludge 100 grams
1.1.6. Substrate 400 grams

Figure: Draft design of the system

1.2.System setting up steps
1.2.1. According to the previous figure (figure), we set up the system which set
the carbon fiber in the middle neck of the flask.
1.2.2. Put 100 mL of sludge and 400 mL of substrate into the flask and let them
settle down
1.2.3. Close 2 sides neck with stopper rubber and in the middle, we pierce the
stopper rubber and put the alligator inside in order to hang the carbon fiber
1.2.4. Before closing the system, we have to purge nitrogen gas in order to let
oxygen in the system out, then closed.

Figure: Setting up system

 2. Carbon fiber preparation
For our project, we vary 3 mesh sizes of carbon fiber (as a working electrode) and
also vary the 4-different preparation condition of the carbon fiber in order to observe the
efficiency of the electrode.

Mesh Condition Dope with Coated with Coated with

size no. Nitric Acid Carbon black Teflon spray
no. powder
1
2 √
3 √ √
1 4 √ √
5 √
6 √
1
2 √
2 3 √ √
4 √ √
5 √
6 √
1
2 √
3 3 √ √
4 √ √
5 √
6 √
Table: Carbon fiber preparation

Steps of Carbon fiber preparation

 Figure: Prepared carbon fiber
Preparation Condition Steps of preparation
condition no.
1 Pure carbon fiber Cut carbon fiber into 2.5 cm. width x 5.5 cm. length
for every mesh size and hold the all 4 edges with
cotton tape 0.5 cm width. So, the actual area for each
carbon fiber is 2x5 cm2

Figure: Carbon fiber size preparation

2 Carbon fiber doped with 1. Prepare carbon fiber same as the first condition
nitric acid 2. Prepare 30% nitric acid 300mL with 700 mL
DI water

Figure: Preparing nitric acid

3. Put the carbon fiber into test tube one by one
and fill the test tube with prepared nitric acid,
then close the tube with its lid

Figure: Carbon fiber with nitric acid in the test tube

4. Put all of the them into an oven operate with
50℃ 1 hour
 Figure: Carbon fiber with nitric acid in the oven
5. After that, wash with water about 3-5 times in
order to take nitric out

Figure: Washing the carbon fiber

6. Put them into the oven again with 100℃ and 1
hours to dry them
7. Drain the waste nitric acid in a heavy metal
tank

Figure: Heavy metal tank

3 Carbon fiber doped with 1. Firstly, do them same steps as the previous
nitric acid and coated with condition (condition2)
carbon black powder 2. Coat the dry carbon fiber with carbon black
powder on the side that have a cotton tape with
the same weight for every piece
 Figure: Carbon fiber coated with carbon black powder
3. After that, put them into the oven with 200℃
and 2 hours

4 Carbon fiber doped with 1. Firstly, do the same steps as the 2𝑛𝑛 condition
nitric acid and coated with 2. Coat the carbon fiber with Teflon spray by
Teflon spray spraying it far from each other about 30 cm.
and only 1 time on the side that have cotton
tape

Figure: Spraying Teflon spray on the carbon fiber

3. Dry them by the oven with 100℃ and 1 hour

5 Carbon fiber coated with 1. Do the same steps as the 1𝑛𝑛 condition
carbon black powder 2. Coat them with carbon black powder on the
side that have a cotton tape with the same
weight for every piece

Figure: Prepared carbon fiber for the 5th and

th
6 condition
3. Put them into an oven with 200℃ and 2 hours

6 Carbon fiber coated with 1. Do the same steps as the 1𝑛𝑛 condition
Teflon spray
 2. Coat the carbon fiber with Teflon spray by
spraying it far from each other about 30 cm.
and only 1 time on the side that have cotton
tape
3. Dry them by the oven with 100℃ and 1 hour

Table: Preparation steps of carbon fiber

Weight of the carbon fibers in different condition

Figure: Weight the carbon fibers

Condition no.1 Carbon fiber

Mesh size no. Weight (g) Average weight (g)

1.92
1.81
1.88
1.60
1 1.73 1.83
1.87
1.87
1.93
1.83
1.80
1.96
1.76
2.06
1.98
 2.09
1.98
2 1.91
2.04
2.19
1.96
2.05
2.02
1.99
2.04
2.10
2.98
2.84
3.14
2.96
3 2.88 3.05
3.05
3.04
3.08
3.33
2.96
3.16
3.17
Table: Weight of carbon fiber with preparation condition no.1

Condition no.2 Carbon fiber doped with Nitric Acid

Mesh size no. Weight (g) Average weight (g)

1 0.76 0.80
0.85
2 0.82 0.88
0.94
3 1.31 1.29
1.28
Table: Weight of carbon fiber with preparation condition no.2

Condition no.3 Carbon fiber doped with Nitric Acid, then coated with Carbon
black powder

Mesh size no. Weight (g) Average weight (g)

0.72
0.80
1 0.79
0.88
0.74
 0.86
0.75
1.06
1.16
2 1.12
1.15
1.22
1.04
1.08
1.57
1.44
3 1.53
1.55
1.50
1.61
1.52
Table: Weight of carbon fiber with preparation condition no.3

Condition no.4 Carbon fiber doped with Nitric acid, then coated with Teflon
spray

Mesh size no. Weight (g) Average weight (g)

1 0.64 0.75
0.77
2 0.86 089
0.91
3 1.26 1.29
1.32
Table: Weight of carbon fiber with preparation condition no.4

Condition no.5 Carbon fiber coated with Carbon black powder

Mesh size no. Weight (g) Average weight (g)

1 0.77 0.81
0.85
2 0.97 0.96
0.94
3 1.35 1.32
1.28
Table: Weight of carbon fiber with preparation condition no.5

Condition no.6 Carbon fiber coated with Teflon spray

Mesh size no. Weight (g) Average weight (g)

 1 0.73 0.74
0.75
2 0.86 0.88
0.89
3 1.30 1.34
1.37
Table: Weight of carbon fiber with preparation condition no.6

3.Substrate Preparation
Materials preparation
1. Glucose 200g
2. NaHCO3 3.13 g
3. NH4 Cl 0.15 g
4. Methylene blue 100 mL
5. Buffer 150 mL
6. Diluted trace element
Trace element preparation
1. Drop 1 mL of stock trace element into 10 mL cylinder, and then fill with DI water
until 10 mL
2. Take out 1 mL of first diluted trace element and drop into 100 mL cylinder, and then
fill with DI water until 100 mL
Substrate preparation
1. Put all 6 materials that were prepared into 1 L beaker
2. Put 100 mL diluted trace element into the beaker also
3. Fill the beaker with DI water until 1 L
4. Stir the solution until them turn into homogeneous phase (all dissolved, no
precipitation)

Electrode material and treatment

Carbon fiber electrode

The chemically inert carbon fiber (CF) has outstanding mechanical and electrical
properties and provides an excellent base electrode for electrophysiological, electrochemical
and biosensor applications. Activated carbon fiber. (Leitner et al., 2006; Shiraisgi et al., 2002;
Kim et al., 2004) have been widely used as electrode materials due to their high specific
surface area and electric conductivity. The carbon fiber electrode has several unique
characteristics to give it an advantage over other techniques. Carbon fiber electrodes have the
ability to monitor in a sub second time frame and record in real time. Because they are so
small. Furthermore, the fast response time and the excellent biocompatibility of these
electrodes make them a powerful tool for real-time monitoring of biological events with high
resolution.
Nitric acid oxidation of activated carbon fabric in combination with calcination at
optimum temperature was conducted to explore the influence of surface carbon–oxygen
complexes on the performance of electrochemical capacitors fabricated with the carbon fabric
(Nian and Teng, 2002). The specific capacitance of the carbon was found to increase upon
oxidation. The micropore resistance for ion migration was low for these carbons. A
capacitance increases of more than 40% has been achieved, without increasing IR drop, by
nitric acid oxidation followed by 300 C calcination for 1 hr. that was shown to remove the
majority of the CO2-desorbing complexes while retaining the CO-desorbing complexes.
The presence of oxygen surface complexes on carbon electrodes of an EC has been
shown to affect the performance of the capacitor.13-16 In acidic solutions, increasing the
population of oxygen surface complexes generally results in an increase of the double layer
capacitance (K. Kinoshita, Carbon: Electrochemical and Physicochemical Properties, p. 302,
John Wiley & Sons, New York ~1988). The capacitance increase can be partially ascribed to
the specific adsorption of ions and solvent molecules, which is caused by the enhanced
affinity toward protons due to the electronic density change in the neighborhood of surface
complexes. Some types of oxygen functional groups may provide redox activity to enhance
the pseudo capacitance.

Carbon Calcinations Surface Pore volume Pore size distribution

Sample Temp (℃) area (𝐂𝐂𝐂 𝐂−𝐂 ) Micro (% ) Meso (%)
(𝐂𝐂 𝐂−𝐂 )
CFN150 150 925 0.44 100 0
CFN300 300 1030 0.49 100 0
CFN450 450 1080 0.52 100 0
CFN600 600 1140 0.55 100 0
CFN750 750 1060 0.51 98 2
CFT* 900 1170 0.56 100 0
Note: * No nitric acid oxidation executed prior to thermal treatment
Teflon

Figure: Fabrication and characterization of CFP electrode. (A) Schematic showing the
fabrication of a CFP-based electrode. (B) The cyclic voltammograms of ferricyanide
on the CFP (curve a) and the CFPP (curve b) electrodes were determined in a reaction
chamber containing PBS, pH 6.5 within a potential between 0.6 V and 0.8 V vs.
Ag/AgCl. The scan rate was 120 mV s1.

The remaining area (0.3 cm · 0.5 cm) was then covered with Teflon glue to form an insulating
layer. CFP (0.3 cm · 1.4 cm) was then partially bound on the supporting strip (0.7 cm in
length) at the side covered with Teflon film, allowing a 0.2 cm wide area of CFP to directly
contact with copper foil. The remaining part of the CFP (0.7 cm in length) overhung from the
supporting strip and acted as a working electrode. The surface of CFP outside the working
area (0.3 cm · 0.7 cm) was insulated with the Teflon film to prevent unwanted redox reactions
((Liu et al., 2008; Mathur et al., 2006; Saha et al., 2009, 2007).

Method analysis
Modeling electrochemically active biofilms
Predicting the current generation from growing EABs through both mediated and
conductive electron transfer mechanisms was a way to confirm the experimental results. Two
models were developed that accounted for the growth kinetics of biofilms and mass transport
inside biofilms during current generation (Marcus et al. 2007; Picioreanu et al. 2007). Each
model was able to fit experimental data from both mediated electron transfer, modeled using
Butler-Volmer kinetics (Equation 1), and conductive electron transfer, modeled using the
Nernst-Monod equation (Equation 2), were possible.
 1. Butler-Volmer equation is given by Equation 1:

n is the number of electrons transferred; I is the current density (A m2)

k0 is the standard heterogeneous rate constant (m s−1)

Xred is the concentration of the reduced form of the redox couple at the biofilm electrode
surface (mM)
Xox is the concentration of the oxidized form of the redox couple at the biofilm electrode
surface (mM)
α is the charge transfer coefficient

f is the grouped term of nF/RT

Faraday constant, F (s A mol-1)

Temperature, T (K)

The universal gas constant, R (J/K.mol)

E is the biofilm electrode potential (V);

𝑛0 𝑛 is the standard reduction potential of the redox couple (V)

The Butler-Volmer equation is useful for modeling mediated electron transfer

mechanisms because it couples the concentration of the electron mediator in both the
oxidized and the reduced form at the biofilm electrode surface and the biofilm electrode
potential to the production of current. This equation is often coupled to the diffusion of
electron mediators through biofilm to provide a method of calculating of current.

The critical parameter in this equation

1. k0, the standard heterogeneous rate constant (m s−1). It has been shown to control the
dependence of current in MFCs (Renslow et al. 2011a). This parameter is a function
of both the biofilm electrode material properties and the redox couples present in the
system.
2. The charge transfer coefficient (α), It is often assumed to be 0.5, which signifies a
symmetric energy barrier for the forward and reverse redox reactions (Verhagen and
Hagen 1992).
 3. the standard reduction potential is critically important for EAB modeling because it
can dictate the amount of energy that is available for a microorganism and also which
terminal electron acceptors are available for that microorganism to utilize

2. The Nernst-Monod equation is used to model conductive electron transfer:

Imax is the limiting current density (A m−2)

EKA is the potential at which the current is half the limiting current (V)

S is the concentration of the electron-donating substrate (mM)

KS is the Monod half saturation constant for that substrate (mM).

The Nernst-Monod equation applicable when the electron acceptor is assumed to be a

solid electron acceptor, accessible via instantaneous conduction, in this equation, EKA is the
critical parameter because it defines where the inflection point on an ideal slow-scan CV will
occur.

3. Randles – Sevcik
The surface coverage can be calculated from the Randles–Sevcik equation. The
Randles–Sevcik equation expresses that the current of the cyclic voltammogram is
proportional to the square root of the scan rate.
3
i p  (2.69 x10 )n Aact D 0.5C0v 0.5
5 2
(3)

Ip: peak current (A)

n: number of electron

Aact: active surface area (cm2)

D: diffusion coefficient (cm2 /s)

C0: bulk concentration (mol/cm3)

n: scan rate (mV/s)

 From Eq. (2), the active surface area (Aact) and the diffusion coefficient (D) can be
calculated using the slope of curves representing the relationship between the peak current
(ip) and the square root of various scan rates (n1/2).
To obtain the active surface area, diffusion coefficient must be calculated
antecedently. Since diffusion coefficient is mainly dependent on temperature and pressure,
the influence of Aact affecting on diffusion coefficient is thought to be minimal at the
condition of fixed temperature and pressure. Therefore, the diffusion coefficient of non-
colonized electrode was calculated in advance and it is considered that this value would be
almost identical with that of colonized electrode.
Possible outcome
► Better understanding of the role of different characteristics of materials that being used in
MFCs.
► Effect of electrode’s materials on the power output will be clarified
► Microbial shaping in different type of modification electrode

► Feasibility of installation of MFC with carbon-based fiber will be evaluated

► Lay out the foundation for modeling and understanding the performance of the
modification of electrode that affect to MFC.
Summarization of Study Factor

Study Factors

Bioprocess Favorable operating condition

● Support medium ● Design configuration

● Operating parameter

Ex. pH, conductivity, dissolved oxygen,

temperature,

Output: Power

Data
Processing

Optimization of system

Identification of boosting and declining factors for MFCs performance

Materials required

Material Purpose

1. Triple head flask System

2. Stopper rubber

3. Electrical alligator

4. Carbon fiber

5. Cotton tape

6. Sludge

7. Glucose

8. NaH𝐂𝐂𝐂

9. N𝐂𝐂 Cl

10. Methylene blue

11. Trace elements

12. DI water

13. Buffer

14. Regulator for Nitrogen tank

15. Nitrogen gas

16. Flask standee

17.

18.
Cost Estimation

Parameter Amount Price (Baht) Total Price

(Baht)

1. Triple neck flask 3 750 2,250

2. Stopper rubber, size 09 6

3. Stopper rubber, size 12 3

4. Regulator for Nitrogen tank 1 1,123 1,123

5. Nitrogen gas

6. Carbon fiber, mesh size 01 1

7. Carbon fiber, mesh size 02 1

8. Carbon fiber, mesh size 03 1

9. Cotton tape 3

10. Electrical alligator 3

11. Pt-electrode 1 17,800 17,800

12. Ag-AgCl electrode 1 12,300 12,300

13.

14.

15.

16.