Bioelectrochemistry 87 (2012) 185191

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Bioelectrochemistry
journal homepage: www.elsevier.com/locate/bioelechem

Conversion of solar energy into electricity by using duckweed in Direct

Photosynthetic Plant Fuel Cell
Yolina Hubenova a,, Mario Mitov b
a
b

Department of Biochemistry and Microbiology, Paisii Hilendarski University of Plovdiv, 24 Tzar Asen Str., 4000 Plovdiv, Bulgaria
Department of Chemistry, South-West University Neot Rilski, 66 Ivan Mihajlov Str., 2700 Blagoevgrad, Bulgaria

a r t i c l e

i n f o

Article history:
Received 22 August 2011
Received in revised form 27 February 2012
Accepted 28 February 2012
Available online 13 March 2012
Keywords:
Direct Photosynthetic Plant Fuel Cell (DPPFC)
Lemna minuta duckweed
Electricity generation
Day/night cycle
Electron transfer mediator

a b s t r a c t
In the present study we demonstrate for the rst time the possibility for conversion of solar energy into electricity on the principles of Direct Photosynthetic Plant Fuel Cell (DPPFC) technology by using aquatic higher plants.
Lemna minuta duckweed was grown autotrophically in specially constructed fuel cells under sunlight irradiation
and laboratory lighting. Current and power density up to 1.62 0.10 A.m 2 and 380 19 mW.m 2, respectively, were achieved under sunlight conditions. The inuence of the temperature, light intensity and day/night
sequencing on the current generation was investigated. The importance of the light intensity was demonstrated
by the higher values of generated current (at permanently connected resistance) during daytime than those
through the nights, indicating the participation of light-dependent photosynthetic processes. The obtained
DPPFC outputs in the night show the contribution of light-independent reactions (respiration). The electron
transfer in the examined DPPFCs is associated with a production of endogenous mediator, secreted by the duckweed. The plants' adaptive response to the applied polarization is also connected with an enhanced metabolism
resulting in an increase of the protein and carbohydrate intracellular content. Further investigations aiming at
improvement of the DPPFC outputs and elucidation of the electron transfer mechanism are required for practical
application.
2012 Elsevier B.V. All rights reserved.

1. Introduction
Microbial fuel cells (MFCs) are intensively studied during the last
decade as a promising technology for simultaneous electricity generation and wastewater purication. A variety of bacteria as well as yeast
species have been investigated as potential biocatalysts and different
mechanisms of the electron transfer from the microorganisms to the
bioanode have been reported [111].
A promising strategy for making MFCs a feasible and sustainable
technology for electricity generation is its combination with photosynthetic processes taking into consideration the abundance of solar
energy reaching the Earth as well as the large variety of photosynthetic
living organisms on the planet highly adapted to capture this solar
energy [12].
Recently, a new concept for plant microbial fuel cell (P-MFC) has
been reported and developed [1317]. The principles of this novel
technology are based on the mutualism between plants and soil microorganisms, in particular those located within plant roots. The plants
harvest solar energy and use it to produce carbohydrates from

Corresponding author at: Department of Biochemistry and Microbiology, Paisii

Hilendarski University of Plovdiv, 24 Tzar Asen Str., 4000 Plovdiv, Bulgaria. Tel: +359
898982716.
E-mail addresses: jolinahubenova@yahoo.com (Y. Hubenova), mitovmario@mail.bg
(M. Mitov).
1567-5394/$ see front matter 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.bioelechem.2012.02.008

atmospheric CO2 via complex photosynthetic processes. A part of the

produced carbohydrates and other organic substances is excreted by
the plant roots and can be utilized by the microorganisms occupying
the rhizosphere for their own growth and development. Combining
the reducing functions of bacteria in conversion of rhizodeposits into
electrons, protons and CO2 with the fuel cell technology, continuous
green electricity could be produced without harvesting the plants. In
principle, this can be accomplished by placing the plant with its roots
in the bioanode compartment of MFC, containing electrochemically
active microorganisms. Up to now, a variety of plants including Oryza
sativa, Reed mannagras, Spartina anglica, Arundinella anomala, Dicranum
montanum, etc. were applied in P-MFCs [18,19]. Since the proof of
principles of P-MFC in 2008, the power density increased over 100
times up to 0.44 W.m 2 projected surface area [20], however, it is still
much lower than the aimed power output of 3.2 W.m 2 for these
systems [18,20]. Based on the thus far investigations, it is considered
that the interaction between the microorganisms occupying the plant
roots and the bioanode is most signicant for the P-MFC operation
[1320]. Analogous to the other MFCs, the current generation is related
to the natural electron-donating properties of bacteria, which are able
to use the anode as a nal electron acceptor.
It has to be mentioned that the electrogenicity as a driving force
not only for the ionic transport but also for the ux of important substances like sugars and amino acids [21] across the cell membranes is
essential for both prokaryotic and eukaryotic organisms. It has been

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Y. Hubenova, M. Mitov / Bioelectrochemistry 87 (2012) 185191

demonstrated that the membrane potential is generated by an ATPrequiring electrogenic proton pumps and its values correlate with
ATP levels [2225]. Plant cells exhibit a large negative membrane
potential which depends on the metabolic activity [26]. Lightdependent electrogenic pumps were established in algae and plants
[27,28]. Fischer and Lttge [29] measured the membrane potential
of L. gibba and found that it maintained more negative values in the
light than in the dark. Such transients of the membrane potential
are typical for photosynthetic cells [30], and they are related to a presence of electrogenic component [31]. Substantial depolarization of the
membrane potential with addition of different substances was also
reported [22,29]. Considering the similarity between these processes
and the ability of the anode-respiring bacteria [32] to use an electrode
instead of chemical oxidizer as a nal electron acceptor, we supposed
that at proper conditions plant cell membranes could also depolarize
on sole electrode.
In the present study, we demonstrate for the rst time the ability
of an aquatic higher plant to generate electricity in a Direct Photosynthetic Plant Fuel Cell (DPPFC). Lemna minuta, a representative of the
widespread duckweeds, has been chosen as a model plant organism.
The natural habitat of duckweeds is fresh or brackish water basins.
The duckweeds are not anchored in soil, but oat freely on the
water surface. Their unique properties such as phenomenal growth
rate, high protein content, and ability to clean wastewater, provide
opportunity for various commercial applications in elds like food
production as a source of sh and poultry feed, biofuel production
including conversion of starch in ethanol, wastewater treatment, etc.
However up until now, duckweeds have not been investigated as
plant biocatalysts in biofuel cells.
We constructed a newly designed fuel cell, in which duckweed
species can grow and develop at near natural conditions on the water
surface but in a direct contact with a at carbon-based anode. Thus constructed plant fuel cells were started at autotrophic operating conditions using CO2 from the air as a carbon source and potable water as a
growth medium.
During the experiments open circuit and polarization measurements were carried out at open-air and natural sunlight radiation, as
well as under articial light laboratory conditions. In parallel, the
temperature and the illuminance during the day/night cycle were measured. Complex microbiological, biochemical and enzymatic analyses
were also performed in order to clarify the duckweed response and
the mechanism of the electricity generation.

2. Materials and methods

2.1. Origin and growth of duckweed
L. minuta duckweed collected from a natural freshwater habitat
(pond near Banya village, Plovdiv region, Bulgaria) was used for the
plant fuel cell experiments after being thoroughly rinsed with clean
water. The plants were grown autotrophically only with an addition
of potable water and light supply. All experiments were carried out
using plants in a good physiological state.
2.2. Plant fuel cell assembly
The construction of the used plant fuel cells is schematically presented in Fig. 1a. They consist of a transparent glassy body, in which
the anode and cathode are placed. Rectangular piece of carbon felt
(SPC-7011, 30 g/m2, Weigerber GmbH & Co. KG) with dimensions
3.5 4.5 cm (geometric area of 15.75 cm2) put horizontally upon cork
pontoons served as an anode. L. minuta fronds (3 g wet biomass) were
evenly spread onto the carbon felt surface thus being in a permanent
contact with the electrode during the whole experiment (Fig. 1b, c).
The duckweed cultivation in the plant fuel cells was carried out in potable water (40 ml) at open-air conditions and only losses from evaporation and water uptake by the plants were daily compensated. The anode
was xed in such a way that the plant roots were covered by water,
while the fronds were on the water surface. Graphite granules (4.3 g
total weight) were used as an air cathode. The granules were placed
into a 100 m Christean handy water lter, which divided the biofuel
cell into two separated compartments.
Experiments under semi-natural (direct and indirect sunlight and
variable temperature) as well as laboratory conditions (variable
mixed lighting including natural illumination by capturing daylight
through the window and articial light sources at constant temperature
of 22 2 C) were carried out at minimum of two replicates. Experiments under constant temperature and illumination (800 lx) in a
Laminar box (Faster, BH-EN 2004) were also performed. The illumination was measured by a lux meter.
2.3. Electrochemical methods
Open circuit voltage (OCV) as well as anode and cathode potentials
were monitored with time while the plant fuel cells were conditioned at

Fig. 1. Plant fuel cell assembly: (a) Scheme of the constructed Direct Photosynthetic Plant Fuel Cell (DPPFC); (b) photo of the laboratory set-up; and (c) photo of anode with L.
minuta.

Y. Hubenova, M. Mitov / Bioelectrochemistry 87 (2012) 185191

open circuit. The cell voltage through a load resistance (200 ), periodically connected in the circuit for 15 min, was recorded by using a digital
multimeter. During a laboratory set of experiments, the cell voltage was
also measured when an external resistor was permanently connected
for several days. Polarization curves were produced by varying the
external resistance from 100 000 to 10 by using resistance decade
box. Each resistance value was connected for a minute and varied
through 2 units at each decade of the resistance box. The current was
calculated according to equation I/A = U.1000 / R and the current
density according to j/(mA.m 2) = I / S.1000, where U/mV is the
measured cell voltage, R/ the external resistance, and S/m2 is the
geometric area of the anode. The power density was calculated as P /
(mW.m 2) = j.U.
Cyclic voltammetry (CV) measurements of the used DPPFCelectrolyte were performed. In order to eliminate the inuence of
microorganisms, the electrolyte was ltered through a 0.2 m microporous sterile lter. Water used during duckweed cultivation in control
experiments was also analyzed by means of CV. Cyclic voltammetry
was carried out by Potentiostatgalvanostat PJT 352 (RadiometerTacussel) utilizing a three electrode arrangement, consisting of a
working electrode (Pt-wire), a counter electrode (platinized titanium
mesh) and a Ag/AgCl reference electrode. The potential was swept at
scan rates from 10 to50 mV.s 1.
2.4. Microbiological analyses
Three different selective nutrient agar media were used for the
examination of the microorganisms' proliferation in the plant fuel cell
anolyte Sabouraud agar (fungal medium), YPfru agar (yeast medium)
and meat peptone agar (bacterial medium). Aliquots (100 l) of the
anolyte were spread over the agar surface of Petri dishes using a sterile
spreader. The cultivation was carried out for 48 h at 28 C. The total
microbial number was determined by counting the colonies produced
by viable cells.
2.5. Biochemical and enzymatic analyses
Fresh duckweed (1 g mass) was mechanically disintegrated in
2 ml 50 mM phosphate buffer, pH 7, followed by centrifugation at
12000 rpm for 10 min for harvesting the chloroplasts and mitochondria
in pellet fraction, which was used for biochemical and enzymatic analyses. The results obtained with the duckweed grown in the plant fuel
cells loaded permanently by external resistance for over two weeks
were compared with those of plants grown as a control.
The protein content was estimated by using the modied Bradford
method (Merck 1.10306.0500) and BSA calibration standards. The
intracellular protein concentration was estimated in mg/ml by using
a calibration curve and recalculated as mg protein per one gram of
duckweed wet biomass.
The determination of reducing sugars (glucose equivalents) was
carried out by means of DNS method [33]. The glucose concentration
was determined in mg/ml by using a calibration curve and calculated
as mg glucose per gram wet biomass.
The iodine method of Halick and Keneaster was used for the determination of starch [34]. The small quantities of the fraction samples
(50 l) imposed modication of the method. An addition of 0.4 mg/ml
starch to the samples was necessary for compensation of the dilutions
required for the spectrophotometrical reading. This amount was subtracted from the values obtained by the calibration curve. The determined concentration is presented as mg starch per gram wet biomass.
The amylase activity was assayed by quantifying the reducing
sugars (glucose equivalents) liberated from soluble starch using the
method described by Bernfeld [35]. A commercial bacterial amylase
was used as a positive control for the enzyme assay. One unit of enzyme
activity was dened as the amount of enzyme required to release
1 mol of glucose from soluble starch per minute under the assay

187

conditions. The determined glucose amount in the pellet fraction was

subtracted from that estimated after the enzyme assay. The results are
calculated and presented as specic activity concerning the protein
content.
For the quantitative determination of inorganic phosphates, the
Molybdenum Blue method [36] was employed. The concentration is
estimated as mmol/L by using a calibration curve after subtracting
the amount of available inorganic phosphate in the buffer.
All measurements were performed in triplicates. The measurement
uncertainty was obtained by applying the uncertainty propagation
law and the prediction standard deviation was calculated according to
the Gaussian statistics [10]. The expanded uncertainty was estimated
with coverage factor k = 2 at condence level C.L.= 95%.
3. Results and discussion
3.1. Direct Photosynthetic Plant Fuel Cell performance
L. minuta duckweed was autotrophycally grown in the described
(Section 2.2) plant fuel cells under sunlight as well as under laboratory
illumination. Several minutes after the start-up of the fuel cells under
semi-natural conditions the OCV increased from 300 mV to over
600 mV. At the rst day after the start-up the OCV reached values in
excess of 700 mV and uctuated between 700 and 850 mV for most of
the experiment (see Supplementary data S1). The observed uctuation
was related to the sunshine natural cycling and the variation of the
temperature, which ranged between 18 and 36 C during the whole
experimental window of six weeks. Lower OCV values were recorded
with the plant fuel cells operating under articial illumination. The
maximum values of about 650 mV were achieved after three days and
then the OCV slowly decreased to approximately 550 mV at the end of
the experiment. In contrary to the fuel cells under semi-natural conditions, no signicant uctuation of the OCV data was observed at the
controlled laboratory conditions.
The short-term (15 min) connection of a load resistance in the
circuit resulted in a voltage drop (polarization) up to 550 mV at the
rst days after the start-up of the fuel cells under semi-natural conditions, but after the 11th day the polarization decreased to about
400 mV and varied within 50 mV during the subsequent period. The
fuel cells operated at controlled laboratory conditions showed a similar
behavior, but the lowering and stabilization of the polarization was
observed after three days of operation. The variation of the current
density, estimated from the measured terminal voltage values, with
time is plotted in Fig. 2a. As seen from the gure, the bigger polarization
at the beginning of the experiment resulted to current density values
between 0.4 and 0.6 A.m 2, while after the 11th day the achieved
values with the fuel cells operated under semi-natural conditions
reached 1.6 A.m 2. Besides the uctuation of the recorded data, relatively high current density values exceeding 1 A.m 2 were achieved
until the 45th day. One order of magnitude lower were the values
obtained with the fuel cells investigated at lab conditions (Fig. 2a
inner graph).
The observed uctuation of the current and voltage obtained with
the plant fuel cells operated at open area can be attributed to the
complex variation of the natural conditions. As seen from Fig. 2b,
the variation of the measured current density follows that of the temperature during the natural day/night sequence. The plot of the current
density versus temperature in Arrhenius coordinates (ln j vs. 1/T, see
Supplementary data S2) exhibits a linear relationship with a relatively
low correlation coefcient of 0.683, which indicates that the temperature can be considered as a signicant (as far as it inuences the rate
of photosynthesis) but not the dominating factor for the current generation processes in the system. Thus, the observed big differences
between the current and voltage obtained with L. minuta grown
under natural sunlight and at laboratory conditions can be assigned to
the greater importance of the light intensity, which was extremely

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Y. Hubenova, M. Mitov / Bioelectrochemistry 87 (2012) 185191

1,8

Day / night cycle

1,6

85

1,4

12

12

12

12

12

80

1,2

75

j / (mA.m-2)

1,0
200

0,8

j / (mA.m-2)

j / (A.m-2)

12

0,6
0,4

160
120
80
40
0

0,2

70
65
60
55

40

80

120

160

200

t / hours

50

0,0
0

10

15

20

25

30

35

40

45

45

t / days

40
0

Day / night cycle

24

1,6

28

75

1,2

26

70

24

0,8

22

0,6

20

j / (mA.m-2)

80

1,4

1,0

96

120

85

30

T / oC

j / (A.m-2)

32

72

t / hours

0 12 0 12 0 12 0 12 0 12 0 12 0 12 0 12 0 12 0 12 0 12 0

1,8

48

65
60
55
50

0,4
18
0,2
16
0

24

48

72

96 120 144 168 192 216 240 264

t / hours
Fig. 2. a) Change of current density, estimated from the measured terminal voltage of
L. minuta-DPPFC polarized by periodically connected external resistance (200 ), with
time; main graph sunlight irradiation, inner graph laboratory lighting; b) Effect of
the day/night alteration on the periodic variation of current density obtained with
L. minuta-DPPFC examined at sunlight irradiation; The top ticks present the main hours
of the day and night (light gray dotted grid line 12 o'clock at noon, black dotted grid
line 0 o'clock in the night). The variation of the temperature (right axis) during experiment is presented by blue dotted line. (The dependence of the current density on the
temperature is presented as Arrhenius plot in Supplementary data Fig. S2).

(over an order of magnitude) higher in the rst case. Putting the plant
fuel cells in darkness for 10 min resulted in the OCV decreasing by up
to 15 mV and conversely by switching on the articial illumination
the OCV values quickly recovered to previous levels. We assume that
these OCV-transients are due to light-dependent changes of the plant
cells' membrane potential. A similar effect on the electric parameters
was observed from the periodic alteration of day/night, which was
more marked in the initial period of the experiments carried out
under natural sunlight. The higher current densities measured during
daytime (Fig. 2b) specify that part of the sunlight energy captured by
plants and converted into chemical energy during the light reaction of
photosynthesis can be transferred as an electron ow to the fuel cell
anode and converted into electricity. The lower but still relatively high
generated current during night-time suggests that it is connected with
light-independent reactions (glucose oxidation during respiration).
The most cogent demonstration of the strong relationship between
the generated current and the light intensity was obtained by the
experiments, in which the plant fuel cells were permanently loaded
by using external resistors and operated at constant temperature and
daylighting in the lab (see Section 2.2). The data presented in Fig. 3a
show that the current values strictly follow the day/night cycle. The
current began to rise with the dawn reaching maxima at noon when

45
40
0

200

400

600

800

Illuminance / lx
Fig. 3. a) Change of current density, estimated from the measured terminal voltage of
L. minuta-DPPFC polarized by permanently connected external resistance (200 ),
and its dependence on the day/night cycles. The top ticks present the main hours of
the day and night (light gray dotted grid line 12 o'clock at noon, black dotted grid
line 0 o'clock in the night. The arrows indicate the addition of water to the DPPFC
anolyte; b) Dependence of the current density on the measured illuminance in the
lab, tting to equation y = y0 + A1e( x/t1), where y0 = 77.60, A1 = 32.66,
t1 = 218.20; R2 = 0.973.

the light intensity in the lab was also maximal (see Supplementary
data S3). The dependence of the current measured during the rst
half of the day on the light intensity (Fig. 3b) ts an equation very similar to those describing photosynthesis light response (PI) curves [37].
During the afternoons, however, the obtained current decreased gradually getting higher values than those measured at the mornings at equal
light intensities. The latter is considered to be connected with the natural physiological response of the duckweed to the photoperiod. As seen
from Fig. 3a, DPPFC continued to produce electricity even during nighttime, which was assigned to the light-independent processes. The
generated current densities, however, were up to 30 mA.m 2 lower
than those obtained during daytime. The observed lowest current densities at the 5th day of experiment may be explained with a diminishing
of the anolyte content in the fuel cell.
The role of the light intensity on the plant fuel cell performance can
be also seen from the polarization and power curves obtained under
semi-natural and laboratory conditions. A much higher power density
of 380 19 mW.m 2 and short circuit current density of 1.62
0.10 A.m 2 were achieved under natural sunlight (over 10 000lx),
while the corresponding results obtained under illuminance of 800lx
were approximately one order of magnitude lower Fig. 4.
The monitoring of the anode and cathode potentials strictly
showed that the fuel cell performance is determined by the phenomena
occurring on the bioanode. While the cathode potential was relatively

0,6

300

0,4

200

0,2

100

0,0
0,0

0
0,2

0,4

0,6

0,8

1,0

1,2

1,4

1,6

j / (A.m-2)
Fig. 4. Polarization and power curves obtained at the 15th day after start-up of
L. minuta-DPPFC under natural sunlight (full dots) and articial illumination (open dots).

stable with time, the anode potential steeply decreased during the rst
days reaching values between 300 and 400 mV (vs. Ag/AgCl) after
the fourth day of the experiment Fig. 5. The observed drop of the
anode potential may be assigned to a stronger interaction between
the duckweed and the anode when the plants began to grow normally
after an initial adaptive period. The results from preliminary experiments, in which it was established that the anode potential shifted to
more negative values with an increase of the fresh plant biomass spread
on the anode surface, indirectly supported such an assumption.
It has to be mentioned that the plant fuel cell outputs are dependent
on the physiological state of the duckweed. When the plants had aged
or begun to die, the OCV as well as the current and power densities decreased. An operation of the plant fuel cells in a batch-mode is therefore
likely until help plants develop normally and the requirements for their
growth and reproduction are met. The macro- as well as microscopic
observations showed that even after long term (over 6 weeks) operation of the plant fuel cells under sunlight irradiation, the anolyte was
clear and non-contaminated. Neither algal blooms nor contamination
by other aquatic microphytes were observed. An absence of fungal
and yeast proliferation on the selective agar media was noted. A very
low total microbial number of (7.2 1.1) 103 cells/ml was determined as a result of the performed cultivation on meat peptone agar
medium. The lack of fungi and yeasts as well as the veried low bacterial content in the fuel cell anolyte could be assigned, on the one hand,
to the antifungal and antibacterial properties of duckweeds [38,39]
and, on the other hand, to the disinfection properties of the sunlight
[4043].
As it has been already pointed out (Section 1), the major differences
between all other reported Plant-MFCs and our system are that

189

duckweeds do not possess typical roots xed in the soil but they oat
instead on the water surface, and the whole system is open to the air.
These specics along with the determined low total microbial number
directed our analyses towards the elucidation of the origin of observed
electrochemical activity. For this purpose, cyclic voltammetry of the
anolyte collected from DPPFCs was carried out. The recorded CVs
showed a quasi-reversible electrochemical behavior indicating the
existence of an electrochemically active component (Fig. 6a). Same CV
patterns were obtained after ltration of the electrolyte through a
microporous sterile lter, which revealed that the observed electrochemical performance is not associated with electroactive bacteria
able to shuttle electrons directly to the electrode. The linear dependence
of both cathodic and anodic peak currents on the square root of the scan
rate (Fig. 6b), typical for a diffusion-limited Nernstian reaction, supports
the presence of soluble redox couple(s) in the medium. The results from
the control experiment (CV of the water medium, in which duckweed
was normally grown) showed an absence of electrochemical activity
(Fig. 6a, inner graph), which allowed us to conclude that the appearance
of the soluble, electrochemically active compound is a response of the
living organisms in the system to the applied polarization.
To clarify whether this electrochemically active compound is
secreted by the plants or by co-existing bacteria in the medium, water
aliquots collected from the control grown duckweed vessel (without
the plants themselves) were inserted into the anodic compartment of
double-chamber MFCs [7], which were polarized by switching a load
resistance (1 k) for over a week. Such an approach is often used in
the MFC-investigations to provoke electroactive phenotype expression
of bacteria [4446]. In our case it was important to nd out whether

0,015

0,010
0,005
0,000
-0,005
I / mA

400

I / mA

0,8

P / (W.m-2)

U/V

Y. Hubenova, M. Mitov / Bioelectrochemistry 87 (2012) 185191

-0,010
-0,015

0,01
0,00
-0,01
-0,02
-0,03
-0,04
-0,05
-0,06
-1,2 -0,9 -0,6 -0,3 0,0 0,3 0,6

-0,020

E / V (vs.Ag/AgCl)

-1,2

-0,9

-0,6

-0,3

0,0

0,3

0,6

0,9

E / V (vs.Ag/AgCl)
0,08
0,06

0,04
0,3
0,02

Ip/ mA

E / V (vs. Ag/AgCl)

0,2
0,1

0,00
-0,02

0,0
-0,04

-0,1

-0,06

-0,2

-0,08

-0,3

v1/2 / (mV.s-1)1/2

-0,4
0

16

18

20

t / days
Fig. 5. Change of the anodic Ea (dots) and cathodic Ec (squares) potentials of L. minutaDPPFC with time.

Fig. 6. a) Cyclic voltammograms of the L. minuta-DPPFC anolyte before (solid line) and
after (dash line) sterile ltration compared to a control (water, in which L. minuta was
normally grown; inner graph); scan rate 10 mV.s 1; b) Dependence of anodic (dots)
and cathodic (squares) peak currents, derived from CVs, on the square root of the
scan rate; correlation coefcient 0.999.

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Y. Hubenova, M. Mitov / Bioelectrochemistry 87 (2012) 185191

Table 1
Content of proteins, carbohydrates, inorganic phosphates and amylase activity in the organelle enriched fractions of L. minuta-DPPFC against control plant.
Chloroplasts and mitochondria
enriched fraction

Protein/(mg/g
wet biomass)

Glucose/(mg/g
wet biomass)

Starch/(mg/g
wet biomass)

Inorganic
phosphates/mM

Specic amylase activity/

(Units/mg protein)

L. minuta DPPFC
L. minuta (control)

621 21
321 19

6.3 1.1
11.2 1.3

78.1 8.4
47.1 7.8

80 12
152 15

0.170 0.016
0.0028 0.0002

the polarization conditions stimulate the existing bacteria to produce

endogenous electron transfer mediators. After the polarization, CVmeasurements of the MFC-anolyte were carried out. Voltammograms
identical to that of the control, presented in Fig. 6 inner graph, were
obtained (data not shown), which proved a lack of any electrochemically active components, i.e. an absence of electroactive bacteria in the
system.
Summarizing the results from the CV studies, it may be concluded
that in the described DPPFCs the electron transfer from the living
organisms to the anode is associated with a generation of an endogenous mediator, secreted by the duckweed as a response to the applied
polarization.
3.2. Biochemical response of L. minuta to the DPPFC conditions
The response of the duckweed to the polarization conditions was
also examined by analysis of basic biochemical parameters such as
intracellular content of proteins, glucose, starch, inorganic phosphate
in fractions derived from duckweed grown in DPPFCs with permanently
connected external resistance and normally grown plants (control).
Double the amount of proteins was determined in the chloroplast and
mitochondria enriched fractions obtained from DPPFC-grown duckweed in comparison to that in the control samples (Table 1, 2nd
column). The determined glucose content in the organelle-enriched
fraction derived from duckweed grown in DPPFC was almost half that
in the pellet fraction from the control plants (Table 1, 3rd column). On
the contrary, a double increased amount of starch was determined in
the DPPFC-pellet fraction in comparison to the control (Table 1, 4th
column). The changes in protein and carbohydrate content suggest
that the polarization stimulates the synthetic processes in the plants,
which are shifted towards carbohydrate reserve generation. We assume
that this is an adaptive response of the duckweed to the enhanced
energy requirements due to the partial conversion of the produced
chemical energy into electricity. L. minuta produces reserves to keep a
positive energy balance necessary for the normal plant development
when polarization is applied. To check this hypothesis, the activity of
amylase (the enzyme, which catalyzes the hydrolysis of glycoside
bonds in the starch to produce glucose equivalents) of L. minuta organelle fraction was measured. The specic amylase activity of 0.170
0.016 Units per mg protein determined in the DPPFC-pellet fraction
against almost zero in the control (Table 1, 6th column) indicates a
need of more glucose equivalents for energy recovery of the system.
When the plant energy balance is disturbed by diverting part of the
generated electrons to the fuel cell anode, it could be compensated by
hydrolysis of accumulated starch. The hypothesis about the intensication of metabolic processes under polarization is further supported by
the decreased amount of inorganic phosphate in the DPPFC chloroplasts
and mitochondria enriched fraction (Table 1, 5th column), indicating a
higher rate of ATP production.
4. Conclusions
Conversion of solar energy into electricity can be performed by
utilization of L. minuta duckweed in plant fuel cell. The obtained electrical outputs depend on the applied illumination and temperature,
indicating the role of photosynthesis in the electricity generation.
The higher current densities correlating with the light intensities
during daytime can be associated with the light-dependent reactions

of photosynthesis, while the generated lower currents during nighttime may be assigned to the light-independent respiration. Parallel
with the plant response to the day/night cycle, the electricity generation
is also connected with a duckweed-produced endogenous electron
transfer mediator. The duckweed adapts to the DPPFC-polarization by
intensifying its metabolism towards production of more ATP, reserve
carbohydrates and proteins. The higher rate of glucose-starch interconversion in the DPPFC grown duckweed supports the hypothesis about a
plant response towards compensation of the disturbed natural energy
balance.
We hope that this study will initiate a new direction of investigations aiming at further development of plant-based fuel cells as a progressive biomimetic technology and clarifying the possible electron
transfer mechanisms in such systems.
Supplementary data to this article can be found online at doi:10.
1016/j.bioelechem.2012.02.008.
Acknowledgements
This study was supported by the National Science Fund of the Ministry of Education and Science of Bulgaria through contracts D002163/2008. We gratefully thank Prof. Dr. Ivan Kiriakov, plant physiologist at the University of Plovdiv, Bulgaria, for the duckweed species
determination, Assist. Prof. Danail Georgiev, microbiologist at the Department of Biochemistry and Microbiology of the University of Plovdiv,
for the microbiological analyses, Assist. Prof. Elitsa Chorbadjiiska and
Assist. Prof. Georgi Hristov from Department of Chemistry at SouthWest University of Blagoevgrad, Bulgaria, for CV analyses and measurement uncertainty estimation, respectively.
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Dr. rer. nat. Yolina Hubenova received M.Sc. in Biotechnology-Gene and cell engineering from St. Kliment Ohridski University of Soa (1993). She completes 3 years
specialization in Medical biology at the Medical University Plovdiv (1999). In 2005
Dr. Hubenova received doctor's degree in Neurobiochemistry at the Friedrich-Wilhelm
University, Bonn. At the present she is working at the Department of Biochemistry and
Microbiology of Paisii Hilendarski University in Plovdiv, Bulgaria. She delivers lectures
on Ecological biochemistry, Clinical biochemistry and Protein engineering at the same
university. Since 2007 her interests are directed toward bioelectrochemical investigations,
especially in the eld of biofuel cells.
Assoc. Prof. Dr. Mario Mitov graduated as a chemical engineer in Electrochemical productions and power sources at the University of Chemical Engineering and Metallurgy,
Soa, Bulgaria, in 1985. He began his professional carrier in Department of Chemistry
at South-West University, Blagoevgrad, Bulgaria in 1987. Currently, Assoc. Prof. Mitov
delivers lectures on General and Inorganic Chemistry, Physicochemistry and Electrochemistry in the same university. His research interests were initially focused on characterization of nanomaterials as potential anodes for rechargeable batteries and fuel cells.
Recently, Assoc. Prof. Mitov has redirected his research activities towards bioelectrochemical systems such as MFCs and MECs.