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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 Recently, electrohydrogenesis has attracted much
recent years from global scientific community. Its attention as another potential technology for
properties of high calorific value, zero carbon content achieving higher hydrogen yield in a more cost
and easy availability of substrates for its production effective manner9-11. Electrohydrogenesis is
make it an ideal replacement option for fossil fuels. It essentially a biocatalysed electrolysis procedure,
has highest gravimetric energy density than any wherein hydrogen gas is produced by organic matter
known fuel1-4 and can be used for energy conversion being degraded by bacteria in microbial electrolytic
either by electrochemical or combustion processes. cells (MECs)9. Exoelectron generating bacteria or
These features make it a promising alternate for fossil exoelectrogens are key players in either MECs or
fuels and confer it several technical, socio-economic microbial fuel cells (MFCs)12,13. These are the bacteria
and environmental benefits5,6. which utilize organic rich matter as carbon source,
Hydrogen can be produced by thermochemical oxidise them and release the electrons outside the cell.
methods such as steam reformation of natural gas, coal In a MFC, exoelectrogenic bacteria oxidize organic
gasification, and splitting water with electricity or by matter and transfer electrons to an anode which in
biological methods such as fermentation (using biomass turn travel through an external circuit to cathode
as substrate) and biophotolysis of water7,8. However, the where they combine with protons and oxygen to form
industrial methods are highly energy intensive and release water. Such exoelectron generators can effectively
carbon dioxide and other greenhouse gases and pollutants also be used for production of hydrogen gas. In
as byproducts, while biological methods suffer from the electrohydrogenesis, electrons released by
drawbacks of lower production rates and yields. These exoelectrogenic bacteria in specially designed reactors
disadvantages economically limit the usage of these (based on modifying microbial fuel cells) reduce the
methods for industrial level hydrogen production. protons (released simultaneously during metabolism)
to form hydrogen gas when a small voltage (~0.8V) is
applied through the circuit. Such modified microbial
* Correspondent author
Mobile: +91-9229557174 fuel cells are essentially called as microbial
E-mail: electrolysis cells or MEC’s. These can be effectively

used to achieve complete substrate conversion and plastic U-tube filled with KNO3 (saturated) and agar
thereby increase the total yield of hydrogen per (20 g/L). Graphite rods (surface area 14.828 sq. cm)
molecule of substrate14-16. and copper plates (surface area 15.68 sq. cm) were
In this communication, design of a microbial fuel used as electrodes. The electrodes were soaked in
cell and measurement of exoelectrogenic activity of phosphate buffer (50 mM) before placing in MFC.
bacteria isolated from different industrial waste water Copper wires (resistance 3.4 × 10-3 Ω/m) were used
and soil samples is reported. The exoelectrogens were for connecting circuits. Different cathodic electrolytes
further used in a single chambered microbial were used to study the effect of reduction potential of
electrolytic cell modified from constructed microbial catholyte on MFC performance. The cathodic
fuel cell for hydrogen production. Hydrogen electrolytes used were tap water, NaCl (10g/L) with
production was confirmed by gas chromatography. tap water, and KMnO4 (0.2 g/L) with tap water. The
Rate, yield and total volumetric hydrogen production anodic compartments were inoculated with enriched
was also determined along with energy efficiency of culture and soil samples (Fig. 1). Current and voltage
the process. measurements were recorded using a digital
multimeter (Mastech, M-830BZ, Taipei, Taiwan) after
Materials and Methods
48 h incubation.
Sampling and isolationSamples of soil and
CalculationsCurrent density of MFC was
wastewater were collected from different sources in
calculated using i (mA/m2) = I/A, where I is the
area around Raipur (21.14°N, 81.38°E), India to
current measured (mA) and A is the geometric surface
isolate electrochemically active bacteria. Soil samples
area of anode (m2). The power density of the MFC
were collected from iron-ore based industries in Urla,
was calculated using formula: P (mW/m2) = IV,
Raipur. The wastewater samples were collected from
where I is the current density and V is the voltage
industrial wastewater line and sewage tank. The
measured (mV).
samples were serially diluted and inoculated in an
enrichment media containing (gL-1) peptone: 15, yeast Construction of microbial electrolytic cell (MEC)
extract: 5, D-glucose: 5.5, NaCl: 2.5, Cysteine Single chambered MEC’s modified from MFC was
hydrochloride: 0.5, and agar: 0.75. Cysteine constructed using airtight plastic bottles of working
hydrochloride was added to reduce the redox potential volume 250 mL. Graphite rods of surface area
of the medium required for the growth of anaerobes. 14.828 sq. cm were used as electrodes and connecting
Agar was added to increase the viscosity of the wires were same as the ones used in MFC. The
medium and thus to decrease the diffusion of electrodes were connected to an external power
atmospheric oxygen into it. The autoclaved culture source which supplied a constant DC voltage of 0.8V.
tubes were flushed with nitrogen beforehand and a A gas collection unit was attached to the top of the
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 Fig.1 Schematic of setup of double chambered microbial fuel
result for catalase test. cell (MFC), where cathodic chamber (A) containing 48 h grown
Construction of microbial fuel cell (MFC)MFCs bacterial culture is connected to air tight anodic chamber
(B) through a salt bridge (C). Graphite electrodes (D) and
was constructed using two glass test tubes (E) are immersed in catholyte and anolyte respectively. A
(35 mL capacity) connected with a salt bridge multimeter (F) is connected across the cell to measure the MFC
(5 mm diameter). Salt bridge was constructed using parameters.

cell for sampling gas at regular intervals for detection To determine the rate, yield and volumetric
and analysis. Initially three MECs were operated with hydrogen production, the gas outlet from MEC’s were
different mixed cultures, (1) with mixed culture of connected to tube filled with water which was again
one isolate of water sample (WS 1) with one isolate of connected to a measuring cylinder. The schematic
soil sample (SS4), (2) with mixed culture of all diagram of the setup is shown in Fig. 4. Hydrogen gas
isolates from water sample (WS1+WS2), and (3) with produced in MEC reactor entered the falcon tube,
mixed culture of all isolates from soil sample where being insoluble in water, it displaced water into
(SS2 + SS4). Sodium thioglycolate (0.5 g/L) and the measuring cylinder. The displaced volume of
sodium acetate (1.5 g/L) was added in addition to all water in the measuring cylinder gave the volumetric
other media components as in MFC. While sodium production of H2 gas. Hydrogen yield (YH2) was
thioglycolate acts as oxygen scavenger, sodium calculated as number of moles of hydrogen gas
acetate aids in better electron transfer. Schematic set produced (nH2) per mole of glucose (nS) consumed.
up the three MEC’s which were run simultaneously is Hydrogen production rate (QH2) was calculated as
given in Fig. 2. Based on results of hydrogen volume of gas produced (m3) per unit volume of
production from the mix cultures, isolates of water reactor (m3) per day, i.e. (m3H2/m3/d). Energy
sample WS1 and WS2 were later also used recovery (η) of the whole process was calculated as
individually to run two different MECs (Fig. 3). 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. 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. 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
Fig. 3 Setup of microbial electrolytic cell with wastewater for gas chromatography can be done from ports F and G
isolates, WS1 and WS2 in monocultures. respectively.

Mumbai, India) fitted with a thermal conductivity MFC. This is evident from the results of this study as
detector (TCD) using Nitrogen (N2) as carrier gas and maximum values for all the parameters studied were
Porapak Q column. The oven temperature during GC reported when KMnO4 was used as catholyte. These
run was kept at 50 °C, while injector and detector are comparable to the results of Jadhav and
temperatures were maintained at 80 °C and 100 °C Ganghrekar23 where they also reported higher power
respectively. Pure hydrogen gas was test run through densities using KMnO4 solution as catholyte.
the column for standard. This communication deals with current generation
in a double chambered MFC using glucose as
Results and Discussion substrate. However, current generation using MFCs of
Determination of exoelectrogenic activity in different designs with different substrates such as
MFC The exoelectrogenic activity of the isolated glucose24, waste water25, and organic acids26 has been
anaerobes was confirmed by current generation in reported. The results from these reports indicate that
constructed MFC. For all the isolates current and MFCs operating on organic acids substrates such as
power density were measured along with current and acetate and butyrate are more efficient that those
voltage for each different catholyte and electrode operating on pure carbohydrates or wastewaters. This
separately (Table 1). suggests that other than pure substrates, end products
In each case maximum values of all parameters for from fermentation reactions, constituting mainly of
all isolates tested across all catholyte combinations organic acids such as acetate and butyrate can also be
were reported using graphite rods, except with WS2, used for current generation in MFC.
where maximum voltage of 506 mV was reported Hydrogen production using MECHydrogen
with copper electrode. The overall maximum power production by the exoelectrogens was carried out in
and current density of 48.85 mW/m2 and constructed single chambered MECs. Three MECs
108.57 mA/m2 respectively were obtained when WS1 were run simultaneously, first with mixed culture of
was used in MFC using graphite electrodes. Using one isolate of water sample (WS 1) with one isolate of
copper electrode the maximum power and current soil sample (SS4), second with mixed culture of both
density was 42.59 mW/m2 and 84.18 mA/m2 the isolates from water sample, and third with mixed
respectively reported in WS2. This study thus culture of both isolates from soil sample. The first and
suggests that graphite electrodes are more efficient second MEC reactors i.e. the reactor with co-culture
than copper electrodes for current generation in MFC. of one soil and one wastewater isolate and the reactor
Continuous operation of MFC leads to the with mixed culture of both wastewater isolates
development of overpotential between electrodes, showed positive hydrogen production as detected by
which reduces cell voltage (You et al22). KMnO4, gas chromatography. No hydrogen production was
being an excellent antioxidant reduces this detected in reactor with mixed culture of both soil
overpotential and thus increases the efficiency of 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
Tap water with
440 474 0.148 0.123 99.81 78.44 43.92 37.18
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
Tap water with
432 431 0.143 0.125 96.44 79.71 41.66 34.35
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
Tap water with
450 399 0.161 0.121 108.57 77.16 48.85 27.62
Tap water 172.4 120 0.075 0.027 50.57 17.21 8.72 2.06
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

individually for hydrogen production. Each isolated from industrial waste water samples. While
wastewater isolate, i.e. WS1 and WS2 were the yield of hydrogen gas reported in this
inoculated as monocultures in MEC reactor to check communication is comparable to the previously
hydrogen production and both WS1 and WS2 showed published reports, the rate of hydrogen production is
positive hydrogen production. relatively less27-30. The most probable reason for the
Reactors inoculated with WS1 and WS2 showed lower rate of hydrogen production is large MEC
hydrogen production for five and four days volume/electrode ratio. If the size of MEC would be
respectively, as shown by displacement of water from decreased similar to electrode size, substantial
centrifuge tube after which the hydrogen production increase in rate of hydrogen production can be
stopped. Figures 5 and 6 depict the rate and yield of achieved. Decreasing the distance between electrodes
hydrogen production from WS1 and WS2 is another option which can result in higher rates of
respectively. As can be seen, WS1 is efficient hydrogen production.
hydrogen producer than WS2 as rate and yield of Although the setup discussed in the current
hydrogen production for WS1 is higher than WS2. communication confirms production of hydrogen gas
Thus, by using a single chamber MEC, it was using exoelectrogenic bacteria. Alteration in design of
possible to produce hydrogen by exoelectrogens MEC setup can be done to significantly escalate the
rate and yield of hydrogen production, thereby
increasing the overall efficiency of the process.

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
Fig. 5 Comparative assessment of yield and rate of hydrogen
production of the two isolates. WS1 showed no hydrogen exoelectrogens were tested for hydrogen production
production after 120 h, while in WS2, hydrogen production in monocultures and in mixed cultures in designed
stopped after 96 h of operation. 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

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