Indian Journal of Experimental Biology

Vol. 51, October 2013, pp. 860-865

Design of a microbial fuel cell and its transition to microbial electrolytic cell for
hydrogen production by electrohydrogenesis
Pratima Gupta *, Piyush Parkhey, Komal Joshi & Anjali Mahilkar
Department of Biotechnology, National Institute of Technology, Raipur, Chhattisgarh 492 010, India
Received 8 November 2012; revised 10 July 2013

Anaerobic bacteria were isolated from industrial wastewater and soil samples and tested for exoelectrogenic activity by
current production in double chambered microbial fuel cell (MFC), which was further transitioned into a single chambered
microbial electrolytic cell to test hydrogen production by electrohydrogenesis. Of all the cultures, the isolate from industrial
water sample showed the maximum values for current = 0.161 mA, current density = 108.57 mA/m2 and power
density = 48.85 mW/m2 with graphite electrode. Maximum voltage across the cell, however, was reported by the isolate
from sewage water sample (506 mv) with copper as electrode. Tap water with KMnO4 was the best cathodic electrolyte
as the highest values for all the measured MFC parameters were reported with it. Once the exoelectrogenic activity of the
isolates was confirmed by current production, these were tested for hydrogen production in a single chambered microbial
electrolytic cell (MEC) modified from the MFC. Hydrogen production was reported positive from co-culture of isolates
of both the water samples and co-culture of one soil and one water sample. The maximum rate and yield of hydrogen
production was 0.18 m3H2/m3/d and 3.2 mol H2/mol glucose respectively with total hydrogen production of 42.4 mL and
energy recovery of 57.4%. Cumulative hydrogen production for a five day cycle of MEC operation was 0.16 m3H2/m3/d.

Keywords: Exoelectrogenic activity, Hydrogen gas, Microbial Electrolytic Cell, Microbial Fuel Cell.

Hydrogen, as a fuel has attracted much attention in
recent years from global scientific community. Its
properties of high calorific value, zero carbon content
and easy availability of substrates for its production
make it an ideal replacement option for fossil fuels. It
has highest gravimetric energy density than any
known fuel1-4 and can be used for energy conversion
either by electrochemical or combustion processes.
These features make it a promising alternate for fossil
fuels and confer it several technical, socio-economic
and environmental benefits5,6.
Hydrogen can be produced by thermochemical
methods such as steam reformation of natural gas, coal
gasification, and splitting water with electricity or by
biological methods such as fermentation (using biomass
as substrate) and biophotolysis of water7,8. However, the
industrial methods are highly energy intensive and release
carbon dioxide and other greenhouse gases and pollutants
as byproducts, while biological methods suffer from the
drawbacks of lower production rates and yields. These
disadvantages economically limit the usage of these
methods for industrial level hydrogen production.
___________
* Correspondent author
Mobile: +91-9229557174
E-mail: prati_biotech@yahoo.co.in
Recently, electrohydrogenesis has attracted much
attention as another potential technology for
achieving higher hydrogen yield in a more cost
effective manner9-11. Electrohydrogenesis is
essentially a biocatalysed electrolysis procedure,
wherein hydrogen gas is produced by organic matter
being degraded by bacteria in microbial electrolytic
cells (MECs)9. Exoelectron generating bacteria or
exoelectrogens are key players in either MECs or
microbial fuel cells (MFCs)12,13. These are the bacteria
which utilize organic rich matter as carbon source,
oxidise them and release the electrons outside the cell.
In a MFC, exoelectrogenic bacteria oxidize organic
matter and transfer electrons to an anode which in
turn travel through an external circuit to cathode
where they combine with protons and oxygen to form
water. Such exoelectron generators can effectively
also be used for production of hydrogen gas. In
electrohydrogenesis, electrons released by
exoelectrogenic bacteria in specially designed reactors
(based on modifying microbial fuel cells) reduce the
protons (released simultaneously during metabolism)
to form hydrogen gas when a small voltage (~0.8V) is
applied through the circuit. Such modified microbial
fuel cells are essentially called as microbial
electrolysis cells or MEC’s. These can be effectively
 GUPTA et al: MICROBIAL FUEL CELL & HYDROGEN PRODUCTION
used to achieve complete substrate conversion and
thereby increase the total yield of hydrogen per
molecule of substrate14-16.
In this communication, design of a microbial fuel
cell and measurement of exoelectrogenic activity of
bacteria isolated from different industrial waste water
and soil samples is reported. The exoelectrogens were
further used in a single chambered microbial
electrolytic cell modified from constructed microbial
fuel cell for hydrogen production. Hydrogen
production was confirmed by gas chromatography.
Rate, yield and total volumetric hydrogen production
was also determined along with energy efficiency of
the process.
Materials and Methods
Sampling and isolationSamples of soil and
wastewater were collected from different sources in
area around Raipur (21.14°N, 81.38°E), India to
isolate electrochemically active bacteria. Soil samples
were collected from iron-ore based industries in Urla,
Raipur. The wastewater samples were collected from
industrial wastewater line and sewage tank. The
samples were serially diluted and inoculated in an
enrichment media containing (gL-1) peptone: 15, yeast
extract: 5, D-glucose: 5.5, NaCl: 2.5, Cysteine
hydrochloride: 0.5, and agar: 0.75. Cysteine
hydrochloride was added to reduce the redox potential
of the medium required for the growth of anaerobes.
Agar was added to increase the viscosity of the
medium and thus to decrease the diffusion of
atmospheric oxygen into it. The autoclaved culture
tubes were flushed with nitrogen beforehand and a
2 cm layer of autoclaved oil was applied on top of
medium and sealed to ensure anaerobic conditions.
Isolation of amaerobic bacteriaA total of four
anaerobic bacteria, two from soil and two from
wastewater were isolated from the collected samples.
Each bacterial isolate was given a separate code as
Soil Sample from Mahamaya Ispat industry - SS2,
Soil Sample from Abhishek Industry - SS4,
Wastewater sample from Amleshwar industrial region
– WS1 and Wastewater sample from Urla industrial
region – WS2. The isolates SS2 and WS2 were found
to be Gram positive, whereas isolates SS4 and WS1
were Gram negative. All the isolates showed negative
result for catalase test.
Construction of microbial fuel cell (MFC)MFCs
was constructed using two glass test tubes
(35 mL capacity) connected with a salt bridge
(5 mm diameter). Salt bridge was constructed using
861
plastic U-tube filled with KNO3 (saturated) and agar
(20 g/L). Graphite rods (surface area 14.828 sq. cm)
and copper plates (surface area 15.68 sq. cm) were
used as electrodes. The electrodes were soaked in
phosphate buffer (50 mM) before placing in MFC.
Copper wires (resistance 3.4 × 10-3 Ω/m) were used
for connecting circuits. Different cathodic electrolytes
were used to study the effect of reduction potential of
catholyte on MFC performance. The cathodic
electrolytes used were tap water, NaCl (10g/L) with
tap water, and KMnO4 (0.2 g/L) with tap water. The
anodic compartments were inoculated with enriched
culture and soil samples (Fig. 1). Current and voltage
measurements were recorded using a digital
multimeter (Mastech, M-830BZ, Taipei, Taiwan) after
48 h incubation.
CalculationsCurrent density of MFC was
calculated using i (mA/m2) = I/A, where I is the
current measured (mA) and A is the geometric surface
area of anode (m2). The power density of the MFC
was calculated using formula: P (mW/m2) = IV,
where I is the current density and V is the voltage
measured (mV).
Construction of microbial electrolytic cell (MEC)
Single chambered MEC’s modified from MFC was
constructed using airtight plastic bottles of working
volume 250 mL. Graphite rods of surface area
14.828 sq. cm were used as electrodes and connecting
wires were same as the ones used in MFC. The
electrodes were connected to an external power
source which supplied a constant DC voltage of 0.8V.
A gas collection unit was attached to the top of the
Fig.1 Schematic of setup of double chambered microbial fuel
cell (MFC), where cathodic chamber (A) containing 48 h grown
bacterial culture is connected to air tight anodic chamber
(B) through a salt bridge (C). Graphite electrodes (D) and
(E) are immersed in catholyte and anolyte respectively. A
multimeter (F) is connected across the cell to measure the MFC
parameters.
862 INDIAN J EXP BIOL, OCTOBER 2013
cell for sampling gas at regular intervals for detection
and analysis. Initially three MECs were operated with
different mixed cultures, (1) with mixed culture of
one isolate of water sample (WS 1) with one isolate of
soil sample (SS4), (2) with mixed culture of all
isolates from water sample (WS1+WS2), and (3) with
mixed culture of all isolates from soil sample
(SS2 + SS4). Sodium thioglycolate (0.5 g/L) and
sodium acetate (1.5 g/L) was added in addition to all
other media components as in MFC. While sodium
thioglycolate acts as oxygen scavenger, sodium
acetate aids in better electron transfer. Schematic set
up the three MEC’s which were run simultaneously is
given in Fig. 2. Based on results of hydrogen
production from the mix cultures, isolates of water
sample WS1 and WS2 were later also used
individually to run two different MECs (Fig. 3).
Fig. 2 Schematic setup of three microbial electrolytic cells,
MEC (A) mixed culture of WS1 and SS4, MEC (B) mixed culture
of WS1 and WS2, and MEC (C) mixed culture of SS2 and SS4.
The MECs are connected in parallel to a 0.8 V supply and
hydrogen produced in the three reactors was collected in D1, D2
and D3 respectively.
Fig. 3 Setup of microbial electrolytic cell with wastewater
isolates, WS1 and WS2 in monocultures.
To determine the rate, yield and volumetric
hydrogen production, the gas outlet from MEC’s were
connected to tube filled with water which was again
connected to a measuring cylinder. The schematic
diagram of the setup is shown in Fig. 4. Hydrogen gas
produced in MEC reactor entered the falcon tube,
where being insoluble in water, it displaced water into
the measuring cylinder. The displaced volume of
water in the measuring cylinder gave the volumetric
production of H2 gas. Hydrogen yield (YH2) was
calculated as number of moles of hydrogen gas
produced (nH2) per mole of glucose (nS) consumed.
Hydrogen production rate (QH2) was calculated as
volume of gas produced (m3) per unit volume of
reactor (m3) per day, i.e. (m3H2/m3/d). Energy
recovery (η) of the whole process was calculated as
ratio of energy produced in the form of hydrogen
(WH2) to the energy input in form of glucose as
substrate (WS), where WH2 = nH2∆H H2, and Ws =
nS∆HS where nH2 and nS are number of moles of
hydrogen gas produced and number of moles of
glucose consumed respectively, and ∆HH2 and ∆HS are
heat of combustion of hydrogen gas (285.83 kJ/mol)
and substrate (2805 kj/mol, for glucose) respectively.
Gas chromatographyThe gas produced was
analysed by Gas chromatography17-21 (Thomas
Scientific, Ceres 800 Plus, Chemito instruments,
Fig. 4 Schematic set up microbial electrolytic cell (MEC) for
determination of rate and yield of hydrogen gas production. Single
chambered MEC (A) with graphite electrodes is connected to
external power supply of 0.8 V. Gas produced in MEC gets
collected into an air tight chamber (B) filled with water via saline
tube (C). Hydrogen being insoluble in water pushes it through
another saline tube (D) into a measuring cylinder (E). Water
collected in measuring cylinder is equal to hydrogen gas collected
on top of chamber B. Fresh media inoculation and gas sampling
for gas chromatography can be done from ports F and G
respectively.
 GUPTA et al: MICROBIAL FUEL CELL & HYDROGEN PRODUCTION 863

Mumbai, India) fitted with a thermal conductivity
detector (TCD) using Nitrogen (N2) as carrier gas and
Porapak Q column. The oven temperature during GC
run was kept at 50 °C, while injector and detector
temperatures were maintained at 80 °C and 100 °C
respectively. Pure hydrogen gas was test run through
the column for standard.
Results and Discussion
Determination of exoelectrogenic activity in
MFC The exoelectrogenic activity of the isolated
anaerobes was confirmed by current generation in
constructed MFC. For all the isolates current and
power density were measured along with current and
voltage for each different catholyte and electrode
separately (Table 1).
In each case maximum values of all parameters for
all isolates tested across all catholyte combinations
were reported using graphite rods, except with WS2,
where maximum voltage of 506 mV was reported
with copper electrode. The overall maximum power
and current density of 48.85 mW/m2 and
108.57 mA/m2 respectively were obtained when WS1
was used in MFC using graphite electrodes. Using
copper electrode the maximum power and current
density was 42.59 mW/m2 and 84.18 mA/m2
respectively reported in WS2. This study thus
suggests that graphite electrodes are more efficient
than copper electrodes for current generation in MFC.
Continuous operation of MFC leads to the
development of overpotential between electrodes,
which reduces cell voltage (You et al22). KMnO4,
being an excellent antioxidant reduces this
overpotential and thus increases the efficiency of
MFC. This is evident from the results of this study as
maximum values for all the parameters studied were
reported when KMnO4 was used as catholyte. These
are comparable to the results of Jadhav and
Ganghrekar23 where they also reported higher power
densities using KMnO4 solution as catholyte.
This communication deals with current generation
in a double chambered MFC using glucose as
substrate. However, current generation using MFCs of
different designs with different substrates such as
glucose24, waste water25, and organic acids26 has been
reported. The results from these reports indicate that
MFCs operating on organic acids substrates such as
acetate and butyrate are more efficient that those
operating on pure carbohydrates or wastewaters. This
suggests that other than pure substrates, end products
from fermentation reactions, constituting mainly of
organic acids such as acetate and butyrate can also be
used for current generation in MFC.
Hydrogen production using MECHydrogen
production by the exoelectrogens was carried out in
constructed single chambered MECs. Three MECs
were run simultaneously, first with mixed culture of
one isolate of water sample (WS 1) with one isolate of
soil sample (SS4), second with mixed culture of both
the isolates from water sample, and third with mixed
culture of both isolates from soil sample. The first and
second MEC reactors i.e. the reactor with co-culture
of one soil and one wastewater isolate and the reactor
with mixed culture of both wastewater isolates
showed positive hydrogen production as detected by
gas chromatography. No hydrogen production was
detected in reactor with mixed culture of both soil
isolates and therefore they were not tested

Table 1 Characterisation of microbial fuel cell for current generation operated with different bacterial isolates.

Voltage (mV) Current (mA) Current density (mA/m2) Power density (mW/m2)
Isolate Cathodic electrolyte
Graphite Copper Graphite Copper Graphite Copper Graphite Copper
Tap water 418 284 0.132 0.048 89.02 30.61 37.21 8.69
Tap water with NaCl 116 312 0.057 0.090 38.44 57.39 4.46 17.91
SS2
Tap water with
440 474 0.148 0.123 99.81 78.44 43.92 37.18
KMnO4
Tap water 376 215 0.120 0.056 80.92 35.71 30.43 3.68
Tap water with NaCl 22 53 0.009 0.018 6.06 11.47 0.133 0.61
SS4
Tap water with
432 431 0.143 0.125 96.44 79.71 41.66 34.35
KMnO4
Tap water 248 224 0.096 0.048 64.74 30.61 16.05 6.86
Tap water with NaCl 88.5 51 0.038 0.015 25.63 9.56 2.27 0.49
WS1
Tap water with
450 399 0.161 0.121 108.57 77.16 48.85 27.62
KMnO4
Tap water 172.4 120 0.075 0.027 50.57 17.21 8.72 2.06
WS2
Tap water with NaCl 112.4 233 0.055 0.100 37.09 63.77 4.17 14.86
Tap water with 392 506 0.146 0.132 98.46 84.18 38.59 42.59
864 INDIAN J EXP BIOL, OCTOBER 2013
individually for hydrogen production. Each
wastewater isolate, i.e. WS1 and WS2 were
inoculated as monocultures in MEC reactor to check
hydrogen production and both WS1 and WS2 showed
positive hydrogen production.
Reactors inoculated with WS1 and WS2 showed
hydrogen production for five and four days
respectively, as shown by displacement of water from
centrifuge tube after which the hydrogen production
stopped. Figures 5 and 6 depict the rate and yield of
hydrogen production from WS1 and WS2
respectively. As can be seen, WS1 is efficient
hydrogen producer than WS2 as rate and yield of
hydrogen production for WS1 is higher than WS2.
Thus, by using a single chamber MEC, it was
possible to produce hydrogen by exoelectrogens
Fig. 5 Comparative assessment of yield and rate of hydrogen
production of the two isolates. WS1 showed no hydrogen
production after 120 h, while in WS2, hydrogen production
stopped after 96 h of operation.
Fig. 6 Volumetric hydrogen production and energy efficiency
of the isolates WS1 and WS2 for hydrogen production over a 5
day cycle. Vol WS1 and Vol WS2 show the volumetric hydrogen
production while E EF WS1 and E EF WS2 show the energy
efficiency for WS1 and WS2 respectively.
isolated from industrial waste water samples. While
the yield of hydrogen gas reported in this
communication is comparable to the previously
published reports, the rate of hydrogen production is
relatively less27-30. The most probable reason for the
lower rate of hydrogen production is large MEC
volume/electrode ratio. If the size of MEC would be
decreased similar to electrode size, substantial
increase in rate of hydrogen production can be
achieved. Decreasing the distance between electrodes
is another option which can result in higher rates of
hydrogen production.
Although the setup discussed in the current
communication confirms production of hydrogen gas
using exoelectrogenic bacteria. Alteration in design of
MEC setup can be done to significantly escalate the
rate and yield of hydrogen production, thereby
increasing the overall efficiency of the process.
Conclusion
Exoelectrogens were successfully isolated from the
soil and waste water samples. Their exoelectrogenic
activity was characterised using the designed double
chambered microbial fuel cell. The MFC inoculated
with the exoelectrogens was characterised for
parameters such as voltage and current production,
along with current and power density using copper
and graphite electrodes and different catholytes. The
exoelectrogens were tested for hydrogen production
in monocultures and in mixed cultures in designed
single chambered microbial electrolytic cell. The
isolates from the wastewater samples showed positive
hydrogen production while the soil isolates showed no
hydrogen evolution. The total volumetric hydrogen
production, along with rate and yield of hydrogen
production and energy efficiency of the whole process
was also evaluated for both wastewater isolates. The
MFC and MEC were designed at basic lab scale and
can further be optimized for enhanced current
production and subsequent hydrogen production
respectively.
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