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Performance of Microbes Consortium on Single-chamber Microbial Fuel Cell

as Electricity Generation

Conference Paper · August 2014

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 PERFORMANCE OF MICROBES CONSORTIUM ON
SINGLE-CHAMBER MICROBIAL FUEL CELL AS
ELECTRICITY GENERATION
Diana Rahayuningwulana,*, Dani Permanaa and Herlian Eriska Putraa
a
Research Center for Chemistry, Indonesian Institutes of Sciences,
LIPI Campus Cisitu, Bandung, Indonesia

Abstract
Microbial Fuel Cell (MFC) systems use microbes to convert organic compounds, as in food
wastewater treatment, and could produce direct current. Single microbe has showed their
performance as good biocatalysts on previous researches, both on synthetic media or wastewater.
This research studied 1-liter single-chamber MFC (SCMFC) using microbes as consortium on
tofu wastewater, since it has high organic pollutant value (COD as O2). Variation of consortium
concentration consists of three single microbe, Saccaromyces cereviceae, Saccaromycopsis fibuligera,
and Escherichia coli that previously acclimated with the wastewater. Result shows that tofu
wastewater, as substrate for the consortium decreases 76% COD value compared to the blank on
variation 1. This SCMFC system also produced maximum current at 0.25 mA with consorsium
SF2SC1:EC1 during 40hours.
Key words: SCMFC, Microbes, Consortium, Tofu wastewater, Current

I. INTRODUCTION
Microbial fuel cell would be an alternative of renewable energy source, where
bacteria as biocatalyst source on oxidizing organic and/or inorganic matter and
produce electricty. Electron produced by bacteria activity from its substrate would
be transferred into anode (negative pole) to cathode (positive pole) by conductor
and resistor.
In MFC, bacteria catalyse oxidation process from reducted substrate release
electrons from respiration cell to anode, which flows by external circuit loop to
cathode chamber and produce current. Every electron produced, a proton could be
transferred via electrolyte (liquid phase) to maintain current continuity. Electron
and proton react with oxygen in cathode chamber, which catalyzed by common
catalyst, as platinum, to form water.
Based on previous research [1], electrons can be transferred to the anode by
electron mediators or shuttles, direct membrane associated electron transfer, or

* Corresponding author. Phone: +62-222503051. Email: dian009@lipi.go.id

187
188 Proceeding ASEAN COSAT 2014

so-called nanowires produced by the bacteria, or undiscovered means. Bacteria

gain the energy by electron transfer from substrate reduction on low potential
(as glucose) to electron acceptor on higher potential (as oxygen).
Basic and economic design consists of two chambers MFC of H-shape, i.e.
two bottles connected completed by separator, e.g. cation exchange membrane or
simple salt bridge. Main component consists of anode, cathode, and electrolyte.
Factors that influence MFC operation system are temperature, pH, electron
acceptor, surface area, reactor dimension and operation time. Substrates also
determine the performance of MFC, for example acetate, glucose, biomass
lignocellulose, beer wastewater, starch, cellulose and chitin [2,3]. Most of MFC
operating on neutral pH to maintain growth condition of microbes on current
production [4]. Ionic strength also influences the conductivity of suspension on
MFC that impacts to internal resistance and its performance [4].
Wastewater could be functioned as electron donor, to be placed in anode
chamber, and have large potential energy, for example domestic wastewater
with–32.80 kJ/eq [5].
Based on recent research using different types of wastewater and inoculum
source [6], current density showed below 2 mA/cm2. High current density resulted
from two-chamber MFC using chocolate wastewater and graphite as electrodes, ie.
0.302 mA/cm2 [7], which COD 1,459 mg/L and using activated sludge inoculum.
Single inoculum of microbe has been studied and showed their result on
previous research. S. cereviceae on double chamber MFC using glucose yeast
extract medium with riboflavin mediator [8], S. cereviceae on salt-brigde MFC
using rice-rinsing wastewater [9], E. coli on salt bridge MFC on glucose and
brewey wastewater [10]. S. fibuligera application on decomposed amylum and
produced amylase had been studied [11], and potential as biocatalyst on MFC.
Since mixed culture gives better organic degradation in wastewater treatment
performance [12,13], this research aim to determine the influence of consortium
microbes variation to current production and organic removal on aerobic single-
chamber MFC using tofu industry wastewater. Tofu industry as one of traditional
food industry in Indonesia produced 15–20 liter wastewater/kg soybean with
BOD dan COD concentration approximately 65 gr and 130 gr/kg soybean
[Potter, C.S., M and Gani A., 1994 in [14].

II. METHOD/M ATERIAL

A. Reactor
This research used MFC reactor consists of single 1 liter-glass tube, which anode
chamber filled with 900 mL wastewater and 100 mL isolate microbes suspension
and using air cathode. Tofu industry wastewater was from process PT X, Bandung
 Innovation for Better ASEAN Community 189

Figure 1. Scheme of Single Chamber MFC

with COD concentration 7,600–10,000 mg/L (pH 4.0–5.6 and conductivity

9.6 mS).
Batch reactor operated aerobically on temperature 25+0.1°C. Electrodes
connected to multimeter SANWA and data collected every 4 hours as shown
Figure 1, with COD sample collected every 12 hours.
Carbon plate 5 mm of 4 cm x 7 cm was placed as the anode and cathode, as
their good performance and inexpensive material electrode for MFC [15].
Salt-bridge, as cation exchange, was clamped between anode and cathode
chamber. Salt bridge was prepared from 11.6 gram sodium chloride added on 5%
(b/v) commercial agar powder and diluted in 100ml aquadest, and then boiled
and stirred homogeniously. Then, agar solution was poured to the 3 cm diameter
pipe and allowed to solidify.

B. Microbes
Microbes were consortium of S. cereviceae, S. fibuligera, and E. Coli, obtained
from Biochemistry Laboratory, University of Padjadjaran, Bandung, Indonesia.
Enrichment of microbes consortium used standard methods [16]. Before operat-
ing, 100% wastewater on MFC, activation and acclimatization was conducted
for each microbes with comparison from 25, 50, until 75% of tofu wastewater
to MFC medium.
190 Proceeding ASEAN COSAT 2014

The medium and inoculum medium were used for S. cereviceae, S. fibuligera,
and E. coli cultivation was YEPD (Yeast Extract Potato Dextrose) medium.
Liquid YEPD medium placed in closed Erlenmeyer. It was sterilized at 121 °C of
temperature and 15 psi of pressure for 15 minutes with an autoclave (Hirayama
HL36 AE, Japan) [17].
Composition of YEPD (yeast extract peptone dextrose) medium for MFC
medium and inoculums medium were 0.5% (w/v) of yeast extract (Becto and
Dickinson), 0.5% (w/v) of bacteriological peptone (Becto and Dickinson),
0.3% (w/v) of ammonium sulfate (Merck), 0.3% (w/v) of potassium dihydrogen
phosphate (Merck), 2% (w/v) of glucose (Sygma-Aldrich), and 1.5% (w/v) of
agar (Becto and Dickinson) [17].
Inoculum of S. cereviceae, S. fibuligera, and E. coli was made by took a loop
of S. cereviceae, S. fibuligera, and E. coli pure culture from agar slant, and then
inoculated into sterile YEPD inoculum medium with 25 mL of volume. Then,
inoculum medium incubated for 18 hours at 30°C and shaken at 150 rpm in a
shaking incubator (Certomat B Braun). The ratio of Erlenmeyer size of to the
volume of the culture volume was maintained at 4:1 to maintain the availability
of dissolved oxygen. The entire MFCs inoculum was transferred to the medium
of MFCs. The volume of MFCs inoculum that added to MFCs medium was
10 % (v/v) of 1000 mL of MFCs medium. Concentration of microbes was 106
CFU/mL.
Since consortium concentration might influenced the electricity generation,
microbe of S. fibuligera (SF), S. cereviceae (SC), and E. coli (EC), was made on
consortium ratio between 1) SF2 : SC1 : EC1, and 2) SF1 : SC2 : EC1. Those
consortiums were tested on 100% tofu wastewater on single chamber MFC and
compared to control reactor without consortium addition.

C. Analysis
Data collection of Optical Density (OD) was set every 4 hours and 12 hours
for Chemical Oxygen Demand (COD) analysis sample. Microbes growth was
approached by detecting its optical density, where the measurement of OD (O 560
nm) using spektrofotometer LW Scientific V325XS. COD sample was analized
using closed reflux titrimetry SNI 6989.2:2009 method.
Collected data was analized for microbes performance on acclimatization and
running MFC, COD removal, current, and voltage measurement.

III. R ESULT AND DISCUSSION

A. Acclimatization
On acclimatization, microbes were adapted step by step into wastewater char-
acteristic, in order to prevent any shock loading. This 0% wastewater, increased
 Innovation for Better ASEAN Community 191

into 25%, 50%, and 75% wastewater was done until OD concentration showed
constant graph, where indicated their growth not significantly different between
each microbes, as shown in Figure 2. On 0% wastewater ratio, S. cereviceae was
the easiest adaptable microbe than two other microbes. It reached its maximum
growth in exponential phase on 20 hours, where the microbe growth, the cell
division and increasing cell numbers run very fast, and also cell biomass increased.
Although suddenly decreased into death phase.
At 75% wastewater ratio, those three microbes need only 12 hours for
exponential phase before entered the stationary phase, as the indicator of limited
nutrient and demand of substrate supply [18]. This phase also indicate that
microbes consortium ready for wastewater substrate.
S. cereviceae also reached maximum growth better than S. fibuligera and E.coli,
either on ratio 25% wastewater or 50%. On 75% ratio, E. coli and S. fibuligera
showed their performance approached to S. cereviceae.

;ĂͿ ;ďͿ

;ĐͿ ;ĚͿ
Figure 2. Growth Curves in Acclimatization Stage of (a) 0% ; (b) 25%; (c) 50%; (d) 75%
wastewater to substrate
192 Proceeding ASEAN COSAT 2014

S. cerevisiae or Baker’s yeast was able to produce bioethanol in medium of

acclimatization and medium of MFC. As we know, ethanol is toxic for micro-
organism. During ethanol fermentations, yeast cells suffer from various stresses.
Not only yeast cells suffered various stresses, but also bacteria cells like E. coli.
S. cerevisiae have ethanol tolerance and can survive in medium that ethanol
existed. However, high ethanol concentrations cause a problem, in that the
fermentative microorganisms have limited ethanol tolerance. S. cerevisiae can
survive in maximum concentration of ethanol of 18 % (v/v) [19]. Growth curve
and cells amount of S. cerevisiae are higher than E. coli and S. fibuligera. It is
possibly caused by the ability of S. cerevisiae to growth in ethanol stress but E.
coli and S. fibuligera were not.

B. MFC Operation
At the end of acclimatization stage, the consortium microbes were tested into
single-chamber MFC with 100% tofu wastewater. Organic matter in wastewater
was oxidized by microbes, the electron released and transferred to electrode (anode)
[20]. This electron was transferred (determined as current), and open circuit
voltage (OCV) was compared with control reactor without microbes applied.
Current production on both variations was shown in Figure 3. Compared to the
control reactor, there was a significant difference and variation with SF1:SC2:EC1
gave better result in current production. Control reactor produced 0.11 mA
current at the maximum performance.

Figure 3. Current Production

 Innovation for Better ASEAN Community 193

In this study, compared to control reactor, consorsium addition increased

current into 0.15 mA for SF2:SC1:EC1 and into 0.25 mA for SF1:SC2:EC1 in
16 hours. The consortium addition influenced current production, almost 127%.
Mix culture as biocatalyst sinergy treated on the substrate. Utilization consortium
culture consists of S. fibuligera and E.coli in tofu wastewater as substrate, which
resulted maximum current 0.23 mA, and it had increased from 0.19 mA maximum
current when using E.coli only [21], this study gave higher result. The advantages
of using microbes consorsium are microbes could degrade the substrate sequently,
consortium can increase the rate of subsrate degradation overall, and consortium
allow microbes find the easiest thermodynamic path or process [22].
S.cereviceae is a facultative microbe that could respirated under aerob and
anaerob condition, and could use media containing high glucose and protein, as
in tofu wastewater, as source of its metabolism.
Based on Figure 4, maximum voltage 0.432 volt and 0.505 volt were resulted
by SF2:SC1:EC1 and SF1:SC2:EC1 variation respectively, in 16 hours. Compared
to double chamber using potassium fericyanate (Rohan, 2013), maximum voltage
using E. coli reached 0.534 V in 2 days.
Consortium allows microbes to find the easiest thermodynamic path or
process [22], so that consortium could oxidize the wastewater substrate faster
than single microbes.
Other fuel cell application, i.e. hydrogen fuel cell and dan Direct Ethanol Fuel
Cell (DEFC) resulted 0.95 volt and 0.6 volt, respectively [23]. Base on that fuel
cell, MFC system could be developed by optimatization of reactor configuration,
microbes performance, and electrode material.
To calculate the electricity energy produced during 40 hours, average of
current and voltage in Joule’s law multiplied by time during MFC operation P
(mW-hour) = V(volt) x I_(mA) x time (hour). In control reactor yielded 0.60
mWh, in SF2:SC1:EC1 and SF1:SC2:EC1 are 1.14 and 2.03 mWh, respectively.

C. COD Removal
As the current generated during reactor running, the tofu wastewater as substrate
tend to decrease on its organic concentration by the time, expressed as COD. This
advantage of MFC system was applied as one of wastewater biological treatment.
According to growth curve during acclimatization stage, in first 12 hours the
consortium entered growth phase rapidly so that the degradation of substrate
was maximum. After that, rate of cell decay and divide tend to similar, indicated
stationer phase had began. The substrate was utilized by microbes for their
metabolism. Since available substrate was limited, it caused the tendency of
microbes to decay, so that the COD value became fluctuated.
194 Proceeding ASEAN COSAT 2014

Figure 4. Voltage Measurement during Reaction

Overall COD removal efficiency resulted from SF2:SC1:EC1 and

SF1:SC2:EC1 variations were 76% and 58% respectively, gave better result than
51% on control reactor during 48 hours, as shown in Figure 4.
There is still challenge to increase the current and voltage production by
single chamber MFC system, either by effective proton exchange, reactor design
and reactor configuration or stacking, considering the volume of tofu wastewater
that produced every day.

IV. CONCLUSION
Performance of microbes consortium, S. cereviceae, S. fibuligera and E. coli, on
single-chamber microbial fuel cell as electricity generation with tofu wastewater
as substrate has been studied. Acclimatization was an important stage to adapt the
microbes to wastewater in order to increase the overall removal efficiency. Overall
COD removal efficiency with SF2:SC1:EC1 variations were 76%; it gave better
result than 51% on control reactor during 48 hours. Addition of SF2:SC1:EC1
into substrate resulted maximum current 0.25 mA in 16 hours.

V. ACKNOWLEDGEMENT
We would like to thank to Djaenudin for his valuable outlook, as well as Oman
Rohman and Mahyar Ependi for wastewater sampling.
 Innovation for Better ASEAN Community 195

Figure 5. COD Removal

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