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Bioelectrochemical Analyses of Lactobacillus plantarum for MFC-Application

Sofia Babanova1, Krasimir Bojanov2, Yolina Hubenova2

and Mario Mitov1,3

Lactobacillus plantarum plays an important role as a natural preserve in food and canning industries. It belongs to the homofermentative microorganisms, which main metabolite is lactic acid. In this study, Lactobacillus plantarum 226-15 strain was investigated as a potential biocatalyst in mediatorless microbial fuel cell (ML-MFC). The optimal cultivation medium and conditions as well as the growth phases of the culture were examined. The cell number in suspension was determined in order to use an appropriate cell amount as anolyte in the MFC. The lactic acid quantity formed within time during cell cultivation was measured by means of neutralization titration. Polarization curves and cyclic voltammograms were taken in a two-chamber fuel cell by using carbon felt electrodes. A stable power and current density output was obtained without addition ofartificial mediator. For the first time, it is demonstrated that Lactobacillus plantarum can be used for electricity generation in a ML-MFC.

Keywords: Lactobacillus plantarum, biocatalyst, mediatorless microbial fuel cell

INTRODUCTIONLactobacillus plantarum is a widespread member of the genus

Lactobacillus that have been used for centuries for the preservation of human food. These are very flexible and versatile species commonly found in many fermented food products including sauerkraut, pickles, brinedolives, sourdough and other plant materials. The high levels of thesemicroorganisms in food also make them an ideal candidate for the development of probiotics. Researchers are studying the possibility forusing the microbes to deliver vaccines and other therapeutic compounds to people. We investigate these bacteria in a very new application, to deliver electrical energy. For that purpose we use them as a biocatalyst in mediatorless microbial fuel cell (ML-MFC).

Microbial fuel cell is a device, which convert the chemical energy in the bonds of the organic compounds directly into electrical current. The simplest used mediatorless MFC is two-chamber MFC, which can be divided into three major components: anaerobic anode chamber, cathode

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chamber and separator. It uses the bacteria as bioreactors in the anode chamber, in which through their catabolic pathways the substrate is oxidized. The electrons gained by these processes are than transferred to the electrode without addition of artificial mediators. An electrical circuit transports the electrons to the cathode, where they are consumed in the reduction reaction, most commonly by oxygen. Parallel with the electron transport, the protons pass through a separator to the cathode chamber, where with oxygen produce water [4, 5, 6, 7].

This paper describes the preliminary study of the ML-MFC performance using Lactobacillus plantarum 226-15 strain as a potential biocatalyst in the anode compartment. A set of microbiological, biochemical and electrochemical experiments was carried out in order to determine appropriate conditions for cell development and performance in MFC. Some conclusions concerning the electron transfer mechanism were also done.

MATERIALS AND METHODSMicrobiological and biochemical methodsLactobacillus plantarum 226-15 strain was cultured using 5% MRS

media (Merck) at 30°C. Under the same conditions the bacterial suspension was used as anolyte in the ML-MFC anode chamber. 3,5.103 cells were used for inoculation of 50 ml medium. The cell number was determined by serial diluting the bacteria, plating them on agar-media, counting the number of colony forming units after 30 hours and comparing them to the dilution factor. Cell growth was traced out also by spectral measuring of suspension optical density at 600 nm within time. Growth phases of the culture were established. The decreasing of reducing sugars in medium within time was determined by means of DNS method. Lactobacillus plantarum is a aerotolerant bacteria that assimilate the substrate via Embden-Meyerhof Pathway to pyruvate and then convert this metabolite to lactic acid. For 1 mol glucose the energy yield is 2 mol of ATP [2]. In the progress of fermentation the lactic acid was quantitatively measured by neutralization titration. The pH change of medium was also traced out.

Fuel cell assemblyElectrochemical experiments were performed in a two-chamber MFC

composed of anode and cathode chamber (13 ml volume each). The anode and cathode compartments were separated from each other by a proton exchange membrane (Nafion 117). Carbon felt was used for anode and cathode (surface area 4,5 cm2). Potassium ferricyanide (100 mM) in phosphate buffer (67 mM, pH=7,0) was used as cathodic electron acceptor.

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The electrodes were cleaned and activated with ethanol and acetone (1:1 volume ratio) and Nafion was pretreated by boiling in H2O2, deionized water, 0,5M H2SO4, each for 1 h [1].

Electrical measurementsPolarization curves were obtained by varying the external resistance

from 100 000 to 100 Ω and the results were monitored using multimeter (Lamar RE67).

Current was calculated according to i(µA)=E.1000/R and current density according to j(mA/m2)=i/A.1000, where E(mV) is cell measured voltage, R(Ω) - the external resistance, A(m2) is the surface area of the anode. The power density was calculated as P(mW/m2)= jE.

Cyclic voltammetry was used to characterize the redox activity of the microorganisms in the anode chamber during the fuel cell operation and to investigate the electron transfer interactions between bacteria and microbial fuel cell electrodes [3]. Cyclic voltammetry was carried out with Potentiostat-Galvanostat PJT 35-2 utilizing a three electrode arrangement, consisting of working electrode, counter electrode and Ag/AgCl reference electrode. The potential was swept from 600mV to -600mV at a scan rate of 10mV/s.

RESULTS AND DISCUSIONLactobacillus plantarum is easily cultivated under non sterile conditions,

so this is rather appropriate for MFC-application. The acid medium (pH 4)preserves from contaminations by other bacterial species. The culture reached the stationary phase at the 28th hour (Fig.1).

0 20 40 60 80 100-4

-3

-2

-1

0

1

2

3

4

log

2OD

600

Time, hours

Fig. 1 Growth of L. plantarum culture

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The glucose concentration in medium decreased three times up to 60 mM, while the lactic acid content increased during fermentation (Fig. 2).

0 20 40 60 80 1000

20

40

60

80

100

120

140

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180

200A

mM

glu

cose

im m

ed

ium

Time, hours

0 20 40 60 80 1000

1

2

3

4

5

6

7 B

g/L

lact

ic a

cid

Time, hours

Fig. 2 Determination of: A - glucose concentration (DMS method); B - lactic acid concentration (neutralization titration) changes during cultivation of L.plantarum in growth media MRS

Periodically, polarization curves were taken to estimate the dependence of the power and current within time. Typical polarization and power curve obtained with Lactobacillus plantarum are shown in fig. 3. The maximum gained power was 63,5 µW at external resistance of 1000 Ω and the maximum current was 580 µA. The maximum power density (140 mW/m2) was observed at 28th hour and for about 47 hours the electrical output was stable and constant (fig. 4). After the 75 hour it slowly diminished. These data shows that L. plantarum gives a stable power output for about 75 hours.

0 100 200 300 400 500 6000

100

200

300

400

500

0

10

20

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60

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U, m

V

I, Ax10-6

P, W

x10-6

0 20 40 60 80 100 120 140 1600

20

40

60

80

100

120

140

160

P, m

W/m

2

t, hours

Fig. 3 Polarization and power curve of Fig. 4 Dependence of power Lactobacillus plantarum in growth media MRS density (mW/m2) within time

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The cyclic voltammogram (CV) of L. plantarum, taken in the initial stage of polarization experiments (fig. 5, A), shows one reduction and one oxidation peak at a potentials of -136mV and 320 mV (vs.Ag/AgCl), respectively. At the same time, definite peaks do not appear in CV obtained with pure MRC medium as a reference (not shown). This confirms that the peaks in CV of the L. plantarum are due to a redox reaction connected with microorganisms’ metabolism. A new irreversible oxidation peak at -188 mV (vs.Ag/AgCl) appears in CV obtained with L. plantarum at the latest phase of polarization measurements (fig. 5, B). We suppose that L. plantarum transfer electrons to the solid electron acceptor either by their own exogenous special mediators or by a metabolite, which can act as a mediator. Most probably that is the lactic acid as a main metabolite of the homofermentative strain used. Further investigations aiming at identification of redox pair origin and electron transfer mechanism are in a progress.

-600 -400 -200 0 200 400 600 800-1,0

-0,8

-0,6

-0,4

-0,2

0,0

0,2

0,4

0,6

0,8 B

A

I, m

A

E (vs.Ag/AgCl), mV

Fig. 5 Cyclic voltammograms of Lactobacillus plantarum in MRS: A – at the initial phase (1 hour); B – at the final phase (214 hour) of polarization measurements

CONCLUSIONSFor the first time, Lactobacillus plantarum 226-15 strain was examined

as a biocatalyst in MFC. A stable power and current density output with maximum values of 140 mW/m2 and 1,28 A/m2, respectively, was obtainedfor about 75 hours without addition of carbohydrate source. Moreover, no artificial mediator is necessary for the electron transfer from bacteria to the anode. That makes this genus promising for application in medatorless MFCs.

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Acknowledgements: The authors would like to thank the National Science Fund of the Ministry of Education and Science of Bulgaria for the financial support through contract D002-163/2008.

REFERENCES

[1] Chae, K.J., Choi, M., Ajayi, F.F., Park, W., Chang, I.S. and Kim, I.S. (2008) Mass transport through a proton exchange membrane (Nafion) in microbial fuel cells. Energy and Fuels, 22(1), 169-176.

[2] Dimkov, R., Physiology and biochemistry of microorganisms, 1994, SU “St. Kliment Ohridski”, Bulgaria.

[3] Fricke, K., Harnisch, F. and Schröder, U. (2008), On the use of cyclic voltammetry for the study of anodic electron transfer in microbial fuel cells, Energy & Environmental Science, 1, 144-147.

[4] Logan, B.E., Hamelers, B., Rozendal, R., Schröder, U., Keller, J., Freguia, S., Aelterman, P., Verstraete, W., Rabaey, K. (2006) Microbial fuel cells: Methodology and technology. EnvironmentalScience & Technology, 40, 5181-5192.

[5] Lovley, D.R. (2006) Microbial fuel cells: Novel microbial physiologies and engineering approaches. Current Opinion in Biotechnology, 17, 327-332.

[6] Rinaldi, A., Mecheri, B., Garavaglia, V., Licoccia, S., Nardo, P., Traversa, E. (2008) Engineering materials and biology to boost performance of microbial fuel cells: a critical review. Energy & Environmental Science, 1, 417-429.

[7] Schaetzle, O., Baronian, K. (2008) Bacteria and yeasts as catalysts in microbial fuel cells: electron transfer from micro-organisms to electrodes for green energy. Energy & Environmental Science, 1, 1-24.

[8] Schröder, U. (2007) Anodic electron transfer mechanisms in microbial fuel cells and their energy efficiency. Physical Chemistry Chemical Physics, 9, 619–629.

Sofia Babanova1, Krasimir Bojanov2, Yolina Hubenova2 and Mario Mitov1,3

1Department of Chemistry, South-West University “Neofit Rilski”, 66 Ivan Mihajlov Str., 2700 Blagoevgrad, Bulgaria2Department of Biochemistry and Microbiology, “Paisii Hilendarski” University of Plovdiv, 24 Tzar Asen Str., 4000 Plovdiv, Bulgaria3Institute of Electrochemistry and Energy Systems, Bulgarian Academy of Sciences, Acad. G.Bonchev Str., bl.10, 1113 Sofia, Bulgaria