4. MATERIALS AND METHODS
4.1. MFC DESIGN
A prototype of the MFC was first designed using AutoCAD. This basic design was
then constructed as a working model in the laboratory
VACCUM
G
INSULATED
MULTIMETER
SALT BRIDGE
DELIVERY TUBE
ARRANGMENT
FOR AERATION
RUBBE
R DISC
SAMPLE (II)
ELECTRODE
WASTE WATER
SUSPENSION
ELECTRODE
SAMPLE (I)
SUSPENSION
DURAN BOTTLE
DURAN BOTTLE
(ANAEROBIC
CHAMBER)
(AEROBIC
CHAMBER)
MAGNETIC
Fig 8. AutoCAD design of Prototype MFC
4.2. MFC CONSTRUCTION AND OPERATION
The basic design of the microbial fuel cell consists of two distinguished
compartments: Aerobic and Anaerobic chambers. 1l glass reagent bottle (Duran) sealed with
the punched rubber cork was used to function as the anaerobic chamber and another 1l glass
reagent bottle (Borosil) served as the aerobic chamber. A Vacuum pump was connected
through an L-glass rod in the punched cork to the glass bottle and serves to rid the chamber of
all oxygen present and produced. Also the anaerobic chamber which is to carry the substrate
of growth is place on a magnetic stirrer to maintain agitation in the sample to assure
homogeneity and proper mixing of reactor feed as also to prevent settling of the medium in
the bottle. The aerobic chamber is left uncovered to allow hollow glass tube with terminal
membranes. This operates as the salt-bridge once supplied with the necessary entities in
optimum concentrations (2-5% of agar heated with 1M KCl) A platinum electrode in the
aerobic chamber is used as the cathode and a carbon rod fitted thorough the punched cork
functions as the anode. The multimeter is connected to check for any deflection and measure
the electricity generated.
The sample is introduced into the anaerobic chamber under sterile conditions. The
magnetic stirrer is used to facilitate optimum agitation in the substrate. Extreme care is
necessary while setting the speed for the magnetic stirrer to avoid upsetting the electrode
setup in the chamber. This setup is left undisturbed but with continuous monitoring for over
24 hours; till the exponential after the setting up of the cell a slight deflection is noticed and
attempts were made to measure the potential across the circuit.
Fig 9. Built working model based on the AutoCAD design
4.3. MFC Components
Microbial Fuel Cell majorly constitutes Electrodes, Anodic and Cathodic Chamber
and Salt Bridge. The Anodic chamber is an anaerobic chamber, which holds the substrate and
the biocatalyst-Microorganisms. The cathodic chamber was maintained aerobically with
catholyte in it. The salt bridge that forms a bridge between cathodic and anodic chamber
facilitates the transfer of ions (protons).
4.3.1. Electrodes
4.3.1.1 Carbon/Platinum electrodes
Two chamber MFC with carbon serving as anode and platinum serving as cathode
was used. Wastewater served as the substrate. The reactor was run for 21 days and readings
were noted at regular intervals.
Fig 10. Carbon Electrodes
4.3.1.2. Carbon/Carbon Electrode
The similar set up as mentioned above was used excepting the change in the cathode
electrode from platinum to carbon.
4.4. Surface Area of Electrode
A Two Chamber MFC set up was adopted with two different surface area of
electrode. The Surface area of the electrodes that was used was 40 cm 2 and 85 cm2. The
reactors were run for 21 days and readings were noted at regular intervals.
4.5. MFC Construction
4.5.1. Two Chamber MFC
1L Schott Duran glass reagent bottle served as the anodic chamber. To function as
medium for the other half of the redox reaction, 1L of distilled water was taken in another
Schott Duran glass reagent bottle (1L). The terminal electron acceptor, carbon rod (anode)
was placed in the anodic chamber containing the substrate and which was connected to the
external circuit which leads to the platinum cathode.
The two chambers are linked physically by a salt bridge which serves to transport the
protons produced during microbial metabolism to the H+ sink, Pt electrode. The salt bridge
was filled with 5M NaCl; in 10% agar while practicing extreme caution to avoid formation
of air bubbles. The cork which seals the anodic chamber is reinforced using paraffin wax to
contain any cracks, fissures or gaps hence making it impermeable to atmospheric oxygen.
An external circuit was established by connecting a multimeter which measures the
voltage and current across the cell. The set up (Fig.11) was left undisturbed for 21 days and
the results were recorded periodically.
Fig 11: Two Chamber MFC with multimeter.
4.5.2. Two Chamber Stacked MFC
Three setups of the above mentioned model were stacked and connected in series.
An external circuit was established by connecting a multimeter which measures the voltage
and current across the cell. The set up (Fig.12) was left undisturbed for 21 days and the
results were recorded periodically.
Fig.12: Stacked Two Chamber MFC with multimeter.
4.5.3. Two chamber H Shaped MFC
500 ml capacity plastic containers were used as anode and cathode. The Salt Bridge
was placed as a bridge between both the chambers as shown (Fig. 13). The salt bridge was
caste with Polyvinyl Chloride (PVC) of 2cm internal diameter and 12 cm length. The salt
bridge was made of 10% agar and 5 M NaCl. The rest of the operating conditions remained
the same as mentioned above.
Fig.13: Two Chamber H shaped MFC with multimeter.
4.5.4. Air Cathode MFC
A plastic container of capacity 2 liters served as the anodic chamber. The anodic
compartment contains the substrate and the carbon electrode (anode ~85cm 2). A similar
carbon electrode which was used as anode served as cathode. The salt bridge serves as an
electrolyte in transfer of protons. The cathode was placed over the salt bridge. Salt bridge
employed here was made with 5M NaCl and 10% Agar. The salt bridge was cast in a PVC
pipe (12 cm X 2cm). The cathode surface is exposed to atmospheric air. Proper precautions
were taken to ensure complete sealing of anodic chamber by means of applying epoxy and
wax to ensure anaerobic conditions. The external circuit was completed by connecting a
resistor (270 Ω) between the two leads of the electrodes. The complete setup is shown (Fig.
14)
Fig.14: Air Cathode MFC
4.5.5. Salt Bridge-Immersed-Air Cathode MFC
Salt Bridge-Immersed-Air Cathode MFC (SBIAC-MFC) consists of a plastic
container of capacity 2 liters served as the anodic chamber. The anodic compartment
contained the substrate and the carbon electrode (anode ~85cm2). A similar carbon electrode
which was used as anode served as cathode. The salt bridge served as an electrolyte in
transfer of protons. The cathode was immersed in the salt bridge when it was in molten stage
to ensure complete surface contact. The 50% cathode surface was exposed to atmospheric air.
Salt bridge employed here was made with 5M NaCl and 10% Agar. The salt bridge was cast
in a PVC pipe (12 cm X 2cm) Proper precautions were taken to ensure complete sealing of
anodic chamber by means of applying epoxy and wax to ensure anaerobic conditions. The
external circuit was completed by connecting a resistor (270 Ω) between the two leads of the
electrodes. The setup is depicted in figure 15 and the schematic representation of the design is
given in figure 16.
Fig 15: SBIAC MFC
Fig 16: Schematic representation of design and working principle of SBIAC-MFC
4.6. MFC Operation
Substrate was added in the anodic chamber and was completely sealed to maintain
anaerobic condition. The reactor was sparged with CO 2 before sealing completely to ensure
complete removal of oxygen. A batch configuration was employed and readings were taken
for a period of 21 days. The rise and decline in readings was noted for this period and
readings were taken every day. A standard reactor (SBIAC-MFC) was always employed for
comparison of the modified parameter with that of the unaltered. A graph was generated
(Current Vs Time) to visually represent the comparison at the end of the experiment.
4.6.1 Optimization of Salt Bridge
4.6.1.1 Various strong salts for Salt Bridge preparation
Two well known strong salts Sodium Chloride (NaCl) and Potassium Chloride (KCl)
were tested for efficacy to transport H+ ions in the salt bridge. Two reactors (SBIAC-MFC)
with wastewater as substrate were setup with respective strong salt used for salt bridge
fabrication. The reactors (Fig.17) were run for 21 days and readings were noted at regular
intervals.
4.6.1.2. Molar Concentration of Salt
Salt bridges were prepared with various Molar concentrations 1, 3, 5, 7, 9M NaCl and
with constant agar concentration of 10%. The five reactors (SBIAC-MFC) with wastewater
as substrate were setup with various molar salt concentrations in salt bridge. The reactors
(Fig.18) were run for 21 days and readings were noted at regular intervals.
4.6.1.3. Concentration of Agar
Salt bridges were prepared with various agar concentrations 2, 5, 10, 15, and 20%.
5M NaCl was used in all these salt bridges with varied agar concentrations. The five reactors
(SBIAC-MFC) with wastewater as substrate were setup with each of these variations. The
reactors (Fig.19) were run for 21 days and readings were noted at regular intervals.
Fig.17: NaCl and KCl SBIAC MFC
Fig.18: SBIAC MFC with 1, 3, 5, 7, 9M Salt Bridge
Fig 19: SBIAC MFC with 2, 5, 10, 15, 20% Salt Bridge Agar Concentrations
4.7. Salt Bridge Modifications
4.7.1. Length of Salt Bridge
A PVC pipe of length 24 cm and 12 cm was used for preparing the salt bridge with
5M NaCl and 10% Agar. Care was taken to ensure that the salt bridge did not touch the
bottom of the reactor. The reactors (Fig.20) were run for 21 days and readings were noted at
regular intervals.
4.7.2. Supportive End-Cap Method
The PVC pipe used in salt bridge was fitted with an end-cap to retain the salt bridge
cast in it. The end cap was perforated to ensure the contact of the salt bridge with the
substrate. Salt bridge (12cm) devoid of an end-cap was employed as standard for comparison.
The reactors (Fig.20) were run for 21 days and readings were noted at regular intervals.
4.7.3. Perforated Salt Bridge
A perforated PVC pipe was employed as Salt Bridge. The salt bridge was cast in a
perforated PVC pipe. Wastewater was employed as the substrate and a normal salt bridge
(12cm) was employed as standard for comparison. The reactors (Fig. 20) were run for 21
days and readings were noted at regular intervals.
Fig 20: Standard SBIAC MFC, Perforated SBIAC MFC, End Cap SBIAC MFC, Long
SBIAC MFC
4.8. MFC with varied number of salt-bridges and electrodes
1. Two salt bridge MFC with 1 and 3 electrodes
2. Three salt bridge MFC with 1 and 3 electrodes
3. Four salt bridge MFC with 1 and 3 electrodes
4. Five salt bridge MFC with 1 and 3 electrodes
5. Eight salt bridge MFC with 1 and 3 electrodes
The procedure for construction involves the same basic set up as two chamber salt bridge
MFC (Fig. 21).
4.9. Acclimatized Anode
The anode of a MFC setup previously run was used as an anode for this experiment.
The effect of the bio-film present in the anode was studied in the MFC. The bio-film coated
anode was employed in the MFC and wastewater served as the substrate. Then unaltered
MFC served as a standard for comparison and the two reactors (SBIAC-MFC) were run for a
period of 21 days.
Fig. 21. (clockwise) 3,4,5 and 8 salt bridge MFC
4.10. Substrates
4.10.1. Substrate Collection
Waste water which served as the substrate of the MFC was collected from SRM
University- Sewage Treatment Plant (100000 cubic meter capacity). The source of the plant
includes wastes from laboratories, toiletries etc. The inlet of the lamellar filter served as the
substrate. This phase was selected so as to avoid the coarse and large particles found in raw
waste water. The natural consortium present in the waste water was majorly used in all
studies (unless mentioned).
4.10.2. From various Stages of Wastewater Treatment
The substrate was collected from various stages of wastewater treatment plant. The
samples were collected from the inlet of Lamella Separator, effluent of Carbon Filter and
effluent of Sand Filter. The substrate samples were collected in sealed containers. These
substrates were used in three different SBIAC-MFC setups. The reactors were run for 21 days
and readings were noted at regular intervals.
4.11. Synthetic Media
Three synthetic medium were used as substrate in anodic chamber.
4.11.1 Medium A (Logan et al., 2005)
-
NaCl
NaHCO3
MgCl2.7H2O
CaCl2
KH2PO4
K2HPO4
Na2HPO4 . 7H2O
FeSO4 .7H2O
MnSO4 .H2O
NH4Cl
KC1
CoCl2 . 6H2O
ZnCl2
CuSO4 .5H2O
H3BO3
Na2MoO4
NiCl2 .6H2O
EDTA
8800 mg/L
3000 mg/L
330 mg/L
275 mg/L
14 mg/L
21 mg/L
56 mg/L
10 mg/L
5 mg/L
3.1 mg/L
2 mg/L
1 mg/L
1 mg/L
0.1 mg/L
0.1 mg/L
0.25 mg/L
0.24 mg/L
1 mg/L
The solution pH was 7. Cysteine (L-Cysteine HC1- Sigma Chemicals) was added to a final
concentration of 385 mg/L.
4.11.2. Medium B (Venkata Mohan et al., 2007)
-
Glucose
NH4Cl
H2PO4
K2HPO4
MgCl2
CoCl2
ZnCl2
CuCl2
CaCl2
MnCl2
3 g/L
0.5 g/L
0.20 g/L
0.20 g/L
0.25 g/L
20 mg/L
10 mg/L
10 mg/L
4 mg/L
10 mg/L
4.11.3. Medium C (Bond and Lovley, 2003)
-
KCl
NaH2PO4
NaCl
NaHCO3
Glucose
0.1g/L
0.6 g/L
2.9 g/L
2 g/L
30g/L
Three reactors with above mentioned synthetic wastewaters were setup. The three reactors
(Fig.22) were run for 21 days and readings were noted at regular intervals.
Fig 22: SBIAC MFC with various synthetic media (Logan et al; Lovley et al; Venkata
Mohan et al)
4.12. Paddy Straw
Naturally available paddy straw (Agricultural waste) was collected and dried. The
dried paddy straw was soaked in water and 2% glucose was added. The paddy straw infusion
was left undisturbed for three days for microbial proliferation. The reactors (Fig.23) (SBIACMFC) were run for 21 days and readings were noted at regular intervals.
Fig 23: Standard SBIAC MFC and SBIAC MFC with Paddy Straw as substrate
4.13. Tannery effluent
About three liters of untreated tannery effluent and four sludge samples were
collected from “Vaasan Tanneries Pvt. Ltd.” Naagalkeni, Chromepet, Chennai. The effluent
was devoid of any large solid chunks owing to the primary solid separation at the plant.
However the sludge primarily consisted of the solids filtered off the effluent. The samples
could be stored no longer than 24 hours and hence all processing was carried out using
freshly collected samples.
4.13.1. Microbial analysis of tannery effluent
The sludge and effluent samples were evaluated for microbial activity. Serial
dilution of samples was done by consecutive transfer of 1ml of solution till a concentration
of 10 -7 was achieved. (Table 1)
Serial dilution is done in an aseptic environment to ensure sterility. Three
concentrations 10-5, 10-6, 10-7 of each of the four samples S1, S2, S3, and S4 were
inoculated in Petri dishes with autoclaved LB agar media. Spread plate technique was
carried out using an L rod to ensure uniform distribution of innoculant over the media
surface. Critical care is taken to prevent tearing of the agar. Also aseptic conditions in the
laminar air flow prevent contamination of the plates.
Table 1. Serial dilution of tannery effluent for microbial analysis
SAMPLE
DILUTION
S1
10 -1
10 -2
10 -3
10 -4
10 -5
10 -6
10 -7
S2
10 -1
10 -2
10 -3
10 -4
10 -5
10 -6
10 -7
S3
10 -1
10 -2
10 -3
10 -4
10 -5
10 -6
10 -7
S4
10 -1
10 -2
10 -3
10 -4
10 -5
10 -6
10 -7
Effluent samples diluted to various concentrations with autoclaved distilled water
The inoculated plates are incubated overnight at 37 oC and the plated are checked for
growth after 18-24 hours.
4.14. Analysis of pH Variation in MFC
The pH of the wastewater adjusted to 6, 7 and 8 were maintained in three separate
reactors. The initial pH of wastewater was found to be 6.5. The reactors (Fig.24) (SBIACMFC) were run for a period of 21 days and readings were noted at regular intervals.
4.15. Analysis of Ionic Strength Variation in MFC
Ionic strength of the wastewater was varied by means of adding NaCl salt in
wastewater. The Ionic Strength of waste water was adjusted to 200 mM, 400 mM, 600 mM,
800 mM and 1000 mM. These substrates were used in SBIAC-MFC reactors. The wastewater
(unaltered) served as a standard for comparison of results. The reactors (Fig.25) were run for
21 days and readings were noted at regular intervals.
Fig 24: SBIAC MFC operated at pH of 6, 7 & 8
Fig 25: SBIAC MFC with 200, 400, 600, 800, 1000mM Ionic Strength Substrate
4.16. Physical Parameters
4.16.1 Removal of gas from head space
The gas headspace was removed using a vacuum pump connected to the SBIAC-MFC
under the pressure of 60 mbar. The reactor (Fig.26) was run for 21 days and readings were
noted at regular intervals.
Fig. 26: Vacuum based SBIAC MFC
4.16.2. Mixing
Mixing in MFC was studied by employing a magnetic stirrer in SBIAC-MFC.
Wastewater was used as the substrate and an unaltered MFC served as the standard. The
reactor (Fig.27) was run for 21 days and readings were noted at regular intervals.
Fig 27: Magnetic Stirrer based SBIAC MFC
4.16.3. Effect of Heat Treatment
The wastewater collected from treatment plant was heat treated at a temperature 90 oC
to kill the non-spore forming aerobes. The heat treated wastewater was cooled down to room
temperature and then used as in MFC (SBIAC-MFC). The unaltered wastewater was used as
a substrate in control MFC (SBIAC-MFC). The reactors were run for 21 days and readings
were noted at regular intervals.
4.16.4. Effect of Aerobic inhibitors
The wastewater collected from treatment plant and 2 % of Cysteine HCl and Sodium
azide which served as the oxygen scavenger were added in the setup. The unaltered
wastewater was used as a substrate in control SBIAC-MFC. The reactors were run for 21
days and readings were noted at regular intervals.
4.17. Optimized Reactor
Optimized Salt Bridge-Immersed-Air Cathode MFC (SBIAC-MFC) consists of a
plastic container of capacity 2 liters served as the anodic chamber. The anodic compartment
contains the substrate and the carbon electrode (anode ~85cm 2). A similar carbon electrode
which was used as anode served as cathode. The cathode was immersed in the salt bridge
when it was in molten stage to ensure complete surface contact. Approximately 50% of
cathode surface is exposed to atmospheric air. Salt bridge employed here was made with 1M
NaCl and 10% Agar. The salt bridge was cast in a PVC pipe (12 cm X 2cm). The external
circuit was completed by connecting a resistor (270Ω) between the two leads of the
electrodes. The pH of the wastewater was adjusted to 8. The ionic strength of the solution
was adjusted to 2000 mM. 2% of Cysteine HCl was added to remove Dissolved Oxygen and
to avoid aerobes and then sparged with CO2. The gas headspace was removed using a
vacuum pump connected to the MFC under the pressure of 60 mbar. Mixing was facilitated
using a magnetic stirrer. The reactor (Fig.28) was run for 21 days and readings were noted at
regular intervals. The results were compared with the standard SBIAC-MFC which was run
simultaneously.
Fig.28: Optimized SBIAC MFC
4.18. Stacked Microbial Fuel Cell
Connecting several microbial fuel cell units in series or parallel can increase voltage
and current; the effect on the electricity generation was yet unknown. The use of series or
parallel stacked MFCs are essential to increase its voltage and current. Connecting several
fuel cells in series adds the voltages while one common circuit flows through all fuel cell. In
case several power sources are connected in parallel, the voltages averages and the current
are added.
Any desired current or voltage can be obtained by combining the appropriate no. of
series and parallel connected fuel cells. However, the electricity production in a MFC is a
microbial process and is compliant to external conditions .Hence,the external circuit should
have an effect on the microbial electricity production .Furthermore ,the MFC units in a series
or parallel connection might not work independently and might be influenced by electricity
production of other MFCs. In this research, stacked MFC was used to investigate the
influence of the electrical circuit (series or parallel) on the power, voltage and current output
of the overall stacked MFC and to monitor the evolution between the microbial community
and electrochemical characteristics of the individual MFC.
4.19. Stacked Air Cathode MFC.
Stacking air cathode MFCs using either wires or a conductive plate successfully
increased the voltage of two cell stacks .However, voltage reversal occurs when one cell does
not generate sufficient voltage relative to other cells. It was shown that fuel starvation in an
active cell or a lack of power generation in the absence of bacterial activity in a cell produces
voltage reversal. A better understanding of the effects of voltage reversal on the bacterial
biofilm and the factors that lead to voltage reversal will help to enhance the utility of using
MFCs in stacks. Further studies should examine methods to control voltage reversal and to
temporarily isolate cells in the stack until stable power generation can be restore
4.20. Microbial Consortium
Naturally available microorganisms present in the waste water catalyze the oxidation
of fuels in MFC at room temperature at which microbial life is possible. Addition of aerobic
inhibitors like cysteine HCl, casein inhibits aerobic organisms in the anodic chamber,
increasing the efficiency from 50-60%.In the absence of the inhibitor, there were chances for
aerobic organisms to thrive in the medium and hence the current generation was less. It is
clear that the application of the biofilm from an existing MF anode to a new electrode is an
effective method of enriching electrochemically active bacteria “Electricigens”.
4.21. Sediment Battery SMFC
4.21.1. Substrate collection-sediment and water
Sediment (mud) from bottom (at the depth of 3 feet) of Potheri Lake and water from
the same location was collected.
4.21.2. SMFC CONSTRUCTION
SMFC majority constitutes of electrodes, anodic and cathodic chambers in same
container. The anodic chamber consists of sediment and anodes are buried inside it and
cathodes are suspended in water column above anode in same container. The transfer of ions
takes place externally through circuit and internally through potential difference created by
oxygen gradient.
One plastic container of height 27 cm was used. 5/7 of the container was filled with
sediment. Then terminal electron acceptor graphite electrodes were buried and then again 5/7
of container mud was placed and covered. Rest was filled with water and cathode was
suspended partially. The setup was left for half hour to settle the sediment. An external circuit
was established by connecting a multimeter which measures the voltage and current across
the cell. The set up left undisturbed for 21 days and results were recorded periodically.
Fig. 29. Sediment Microbial Fuel Cell
4.21.3. SMFC OPERATION
Anaerobic anodic chamber consisting of anode buried in sediment was prepared and
cathode was suspended in water above. The container was sealed on the top and made
airtight. Geobacters present in sediment breaks down the organic substrate present .This leads
to release of electron which are captured by anode in anaerobic chamber and protons which
gets transferred to cathode due to potential difference created by oxygen gradient. This makes
the circuit complete and electricity is generated.
4.21.4. Stacked sediment microbial fuel cells
Connecting several sediment microbial fuel cells units in series or parallel can
increase the voltage or current. The use of series or parallel stacked SMFCs are essential to
increase its voltage and current. Several fuel cells in series add the voltage while one
common circuits flows through all fuel cells. In case several power sources are connected in
parallel the voltage averages and current are added.
By using this, desired current or voltages can be obtained by combining the
appropriate number of series and parallel connected fuel cells. However the electricity
production in SMFC is a microbial process and is compliant to external conditions. Hence,
the external circuit should have an effect on the microbial electricity production. Furthermost,
the SMFC units in a series or parallel connection might not work independently and might be
influenced by electricity production of SMFC. In this research stacked SMFC was used to
investigate the influence of the electrical circuit (series or parallel) on the power, voltage and
current output of the overall stacked SMFC and to monitor the evolution between the
microbial community and electrochemical characteristics of the individual SMFC.
4.21.5. Microbial consortium
Naturally available microorganism present in the mud catalyzes the oxidation of fuel
in SMFC at room temperature at which microbial life is possible. It has been observed that
the sediment has enormous number of Geobacters which are capable of generating electricity
and are called electrigens.
4.22. Measurement of Output:
The output of the MFC is expressed by means of current (mA). For this purpose
multimeter was used and calibrated each time before use. The multimeter was also used to
measure Open Circuit Voltage (OCV). Ohm’s Law was use to calculate Power (P) and hence
the power density was also calculated. The formula used is given below:
V=IR
P=VI
PD= VI/A
4.23. Notation
x
V- Volts (V)
x
I – Current (mA)
x
P- Power (mW)
x
PD- Power Density (mW/m2)
x
Surface Area of electrode (m2)
Resistance of 270Ω was employed in all experiments and hence calculations were
based on it. Readings from the multimeter were noted only after a steady and constant value
was obtained, which took 3-4 hours. The multimeter was connected in series with MFC when
measuring current and was connected in parallel while measuring OCV.