Inhibition of Electricity Generation by Micrococcus Luteus 1-I in a

Biofuel Cell by Respiratory Poisons

I. A. Topchiy

, D. Yu. Amsheev

, D. I. Stom

1,2,3 c

A. B. Kupchinsky

and M. Yu. Tolstoy

Irkutsk State University, Irkutsk, Russia

Baikal Museum of the SB RAS, Listvyanka, Russia

Irkutsk National Research Technical University, Irkutsk, Russia

Keywords: Micrococcus luteus 1-I, biofuel cells, electricity inhibition, respiratory poisons.

Abstract: The article studied the effect of three respiratory poisons (sodium fluoride, sodium azide, 2,4-dinitrophenol)

at concentrations of 5 and 20 mmol/L on the electrical characteristics of BFC operating on model wastewater

with the bioagent Micrococcus luteus 1-I. It has been demonstrated that the inhibitors used in all tested

concentrations had a negative effect on the power generation of BFC. It was shown that the degree of influence

of respiratory poisons on voltage and current increased in the series sodium fluoride > sodium azide > 2,4-

dinitrophenol.

1 INTRODUCTION

Biofuel cells (BFCs) are unique bioelectrochemical

installations that convert the energy of chemical

bonds in organic and inorganic molecules into

electrical energy. Microorganisms are biocatalysts for

this process (Idris et al., 2016). BFCs can use various

wastes as a substrate, for example, sewage. This

makes BFC technology extremely useful in terms of

environmental conservation (Munoz-Cupa et al.,

2021).

Today, very little is known about the mechanisms

of electron transfer from microbial cells to the

electrode and about the enzymes involved in these

processes. It was expected that the study of the effect

of enzymatic poisons would provide material for a

possible interpretation and understanding of the

sequence of events unfolding in the processes of

electrogenesis in BFC. For these purposes, the use of

poisons that can block individual elements of the

respiratory chain, inhibit glycolysis, uncouple the

processes of oxidative phosphorylation, lead to

irreversible disturbances in ion exchange, and also

http://orcid.org/0000-0002-9091-4062

http://orcid.org/0000-0003-1909-1240

http://orcid.org/0000-0001-9496-2961

http://orcid.org/0000-0001-8884-8636

http://orcid.org/0000-0002-1573-060X

induce oxidative stress is especially promising. The

list of substances with the above properties primarily

includes sodium fluoride, sodium azide, and 2,4-

dinitrophenol (Parthasaradh and Kumari, 2018,

Pradiptama et al., 2019, Johnston and Strobel, 2019,

Zhang et al., 2022, Johnston and Strobel, 2020,

Suresh et al., 2020).

The purpose of the study: to study the inhibitory

effect of sodium fluoride, sodium azide and 2,4-

dinitrophenol on the power generation of BFC with

Micrococcus luteus 1-I.

2 MATERIALS AND METHODS

2.1 BFC and Electrodes

The effect of inhibitory substances on the generation

of electricity was carried out in BFCs designed at the

Research Institute of Biology of ISU (Kuznetsov et

al., 2021). This is a two-chamber structure made of

Plexiglas (Plexiglas XT 20070, 3 mm). The MF-4SK

proton-exchange membrane (JSC Plastpolimer,

322

Topchiy, I., Amsheev, D., Stom, D., Kupchinsky, A. and Tolstoy, M.

Inhibition of Electricity Generation by Micrococcus Luteus 1-I in a Biofuel Cell by Respiratory Poisons.

DOI: 10.5220/0011571200003524

In Proceedings of the 1st International Conference on Methods, Models, Technologies for Sustainable Development (MMTGE 2022) - Agroclimatic Projects and Carbon Neutrality, pages

322-326

ISBN: 978-989-758-608-8

 2023 by SCITEPRESS – Science and Technology Publications, Lda. Under CC license (CC BY-NC-ND 4.0)

Russian Federation) separates the chambers with a

volume of 0.35 liters. The electrodes were URAL T-

22P A carbon cloth (OAO Svetlogorskkhimvolokno,

Republic of Belarus). The electrode dimensions were

16 × 4 cm (Fig. 1).

Figure 1: BFC design: 1 – anode chamber; 2 – cathode

chamber; 3 – carbon cloth electrodes; 4 – rubber cap for

anolyte sampling; 5 - proton exchange membrane.

2.2 Strains and Culture Conditions

Sterile model wastewater (MW) served as the anolyte

(Stom et al., 2021). Tap water was used for catholyte

(water intake in the Angara River upstream of the

HPP dam). Micrococcus luteus 1-I was chosen as a

bioagent. It was isolated from the activated sludge of

the Angarsk petrochemical plant (Russia). The strain

was deposited in the All-Russian Collection of

Microorganisms (VKM) under the number VKM Ac-

2637D. Bacteria were cultivated on fish peptone agar

(FPA) (Stom et al., 1992).

In all experiments, 5 ml of washings from a one-

day culture in saline (0.85% NaCl) were used. The

washings were carefully suspended before

inoculation. Sodium fluoride (NaF), sodium azide

(NaN3) and 2,4-dinitrophenol (2,4-DNP) at

concentrations of 5 and 20 mmol/L were chosen as

biochemical inhibitors.

2.3 Generation of Electricity in BFC

The current in the MFC was recorded with a digital

multimeter "DT-266". The voltage was measured

using an automatic data recording system based on

the microprocessor board "Arduino Mega 2560".

BFC were thermostated at 30°C for 3 days.

2.4 Statistical Processing of Results

All experiments were performed in at least 5

independent experiments with 3 parallel

measurements each. Statistical processing of

experimental data was carried out using the Microsoft

Excel software package. The results are the mean

values for the sample. and their standard errors.

Conclusions are made with the probability of an

error-free forecast P ≥ 0.95

3 RESULTS AND DISCUSSION

The used inhibitors at all concentrations had a

negative effect on the power generation of BFCs with

the bioagent M. luteus 1-I. However, the degree of

this influence was different for each compound. So,

NaF at a concentration of 20 mmol/L on day 2

reduced the voltage from 341 mV to 75 mV (about 5

times) and the short circuit current from 0.371 to

0.051 mA (more than 7 times), respectively. (Fig. 2

and Fig. 3).

Sodium azide had a stronger effect. Compared to

fluoride, at the same NaN3 concentration on day 2,

the voltage suppression was 3 times greater, and the

current strength was 40 times greater (Fig. 4 and Fig.

5). 2,4-DNP at all concentrations completely

suppressed the work of BFC (Fig. 6 and Fig. 7).

When discussing the data obtained in connection

with the results of other authors, the works of J. K.

Jan et al. They studied the effect of poisons on the

operation of BFCs. It has been shown that the

introduction of sodium azide at a concentration of 0.4

mmol/L into the biocathode leads to a decrease in the

generated current from 3 mA to 2.8 mA. The authors

suggested that the inhibitory effect is achieved due to

disruption of extracellular electron transport (Jang et

al., 2013). Based on this, one could expect an increase

in the inhibitory effect on power generation in BFC at

higher concentrations of sodium azide, which was

confirmed in our experiments.

Influence of respiratory poisons on the power

generation of BFC with Chlorella pyrenoidosa.

studied Yi S. Hu et al. In the presence of the ATP

synthase inhibitor resveratrol and the protonophore

2,4-DNP at a concentration of 1000 ppm (which is

approximately equal to 5 mmol/L in our study), the

current strength was not only not inhibited, but even

increased. However, this change in the operation of

the BFC was insignificant (Xu et al., 2015).

Inhibition of Electricity Generation by Micrococcus Luteus 1-I in a Biofuel Cell by Respiratory Poisons

323

Figure 2: Dynamics of voltage generated in BFC by M. luteus 1-I strain in the presence of sodium fluoride (t = 30° C, electrode

– carbon cloth, substrate – MW).

Figure 3: Dynamics of current generated in BFC by M. luteus 1-I strain in the presence of sodium fluoride (t = 30 °C, electrode

– carbon cloth, substrate – MW).

Figure 4: Dynamics of voltage generated in BFC by M. luteus 1-I strain in the presence of sodium azide (t = 30 °C, electrode

– carbon cloth, substrate – MW).

y = 166,5x + 37,5

R² = 0,9139

y = 71x + 1,6667

R² = 0,9983

y = 39,5x - 3,1667

R² = 0,9987

Voltage, mV

Time, day

Control NaF 5 mM NaF 20 mM

y = 0,1855x - 0,0352

R² = 0,9027

y = 0,043x - 0,022

R² = 0,9264

y = 0,0285x + 0,0015

R² = 0,828

Current, mA

Time, day

Control NaF 5 mM NaF 20 mM

y = 11,75x - 6,75

R² = 0,766

y = -19,5x + 51,667

R² = 0,7308

y = 80,5x + 130,39

R² = 0,9684

Voltage, mV

Time, day

NaN3 5 mM NaN3 20 mM Control

MMTGE 2022 - I International Conference "Methods, models, technologies for sustainable development: agroclimatic projects and carbon

neutrality", Kadyrov Chechen State University Chechen Republic, Grozny, st. Sher

324

Figure 5: Dynamics of current generated in BFC by M. luteus 1-I strain in the presence of sodium azide (t = 30 °C, electrode

– carbon cloth, substrate – MW).

Figure 6: Dynamics of voltage generated in BFC by M. luteus 1-I strain in the presence of 2,4-DNP (t = 30 °C, electrode –

carbon cloth, substrate – MW).

Figure 7: Dynamics of current generated in BFC by M. luteus 1-I strain in the presence of 2,4-DNP (t = 30 °C, electrode –

carbon cloth, substrate – MW).

y = 0,018x - 0,0173

R² = 0,8322

y = -0,0538x + 0,1061

R² = 0,8808

y = 0,1192x + 0,1489

R² = 0,9311

Сurrent, mA

Time, day

NaN3 5 mM NaN3 20 mM Control

y = -19,5x - 21,833

R² = 0,1112

y = 10,5x - 22,833

R² = 0,0522

y = 132,67x + 116

R² = 0,9615

Voltage, mV

Time, day

2,4-DNP 5 mM 2,4-DNP 20 mM Control

y = 0,0135x - 0,0558

R² = 0,0518

y = -0,0025x - 0,0242

R² = 0,002

y = 0,0188x + 0,0931

R² = 0,8093

Current, mA

Time, day

2,4-DNP 5 mM 2,4-DNP 20 mM Control

Inhibition of Electricity Generation by Micrococcus Luteus 1-I in a Biofuel Cell by Respiratory Poisons

325

4 CONCLUSIONS

In this work, we studied the effect of three respiratory

poisons and electron transfer inhibitors (NaF, NaN3,

2,4-DNP) on the power generation of BFC with M.

luteus 1-I as an anode bioagent. It was found that all

compounds in all tested concentrations (5 and 20

mmol/L) inhibit voltage and current. Moreover, the

degree of inhibition of these indicators increases in

the series sodium fluoride > sodium azide > 2,4-

dinitrophenol.

ACKNOWLEDGEMENTS

The authors are grateful to E. Yu. Konovalova for

providing the M. luteus 1-I strain. The study was

carried out with the financial support of the Russian

Foundation for Basic Research within the framework

of the scientific project 19-58-44003 mong_t.

REFERENCES

Idris, S. A., Esat, F. N., Rahim, A. A., Rizzqi, W. A.,

Ruzlee, W., Razali, M. Z., 2016. Electricity generation

from the mud by using microbial fuel cell. MATEC Web

Conferences. 69. 02001.

Munoz-Cupa, C., Hu, Yu., Xu, C., Bassi, A., 2021. An

overview of microbial fuel cell usage in wastewater

treatment, resource recovery and energy production,

Science of The Total Environment. 754. 142429.

Parthasaradh, S. P., Kumari, J. P., 2018. Fluoride effect on

minimum inhibitory concentration and growth

dynamics of Lactobacillus acidophilus and

Lactobacillus salivarius. World Journal of

Pharmaceutical Research. 7 (17). pp. 1024–1036.

Pradiptama, Y., Purwanta, M., Notopuro, H., 2019.

Antibacterial Effects of Fluoride in Streptococcus

mutans Growth. Vitro, Biomolecular and health science

journal. 02 (01). pp. 1-3.

Johnston, N. R., Strobel, S. A., 2019. Nitrate and Phosphate

Transporters Rescue Fluoride Toxicity in Yeast.

Chemical Research in Toxicology. 32 (11). pp. 2305-

2319.

Zhang, Z., Yu, Y., Xi, H., Zhou, Y., 2022. Inhibitory effect

of individual and mixtures of nitrophenols on anaerobic

toxicity assay of anaerobic systems: Metabolism and

evaluation modeling. Journal of Environmental

Management. 304. 114237.

Johnston, N. R., Strobel, S. A., 2020. Principles of fluoride

toxicity and the cellular response: a review. Archives of

Toxicology. 94. pp. 1051–1069.

Suresh, A., Pan, C., Ng, W. J., 2020. Sodium azide

inhibition of microbial activities and impact on sludge

floc destabilization. Chemosphere. 244, 125452.

Kuznetsov, A. V., Khorina, N. N., Konovalova, E. Yu.,

Amsheev, D. Yu., Ponamoreva, O. N., Stom, D. I.,

2021. Bioelectrochemical processes of oxidation of

dicarboxylic amino acids by strain Micrococcus luteus

1-I in a biofuel cell. IOP Conference Series: Earth and

Environmental Science. 808. 012038.

Jang, J. K., Kan, J., Bretschger, O., Gorby, Y. A., Hsu, L.,

Hong, K. B., Nealson, K. H., 2013. Electricity

Generation by Microbial Fuel Cell Using

Microorganisms as Catalyst in Cathode. J. Microbiol.

Biotechnol. 23 (12). pp. 1765-1773.

Xu, C., Poon, K., Choi, M. M. F, Wang, R., 2015. Using

live algae at the anode of a microbial fuel cell to

generate electricity. Environmental Science and

Pollution Research. 22 (20). pp. 15621–15635.

Stom, D. I., Zhdanova, G. O., Kalashnikova, O. B., Bulaev,

A. G., Kashevskii, A. V., Kupchinsky, A. B.,

Vardanyan, N. S., Ponamoreva, O. N., Alferov, S. V.,

Saksonov, M. N., Chesnokova, A. N., Tolstoy, M. Yu.,

2021. Acidophilic Microorganisms Leptospirillum sp.,

Acidithiobacillus sp., Ferroplasma sp. As a Cathodic

Bioagents in a MFC. Geomicrobiology Journal. 38(4).

pp. 340–346.

Stom, D. I., Geel, T. A., Balayan, A. E., Shachova, G. I.,

Kuznetsov, A. M., Medvedeva, S. E., 1992.

Bioluminescent method in studying the complex effect

of sewage components. Archives of Environmental

Contamination and Toxicology. 22. pp. 203–208.

MMTGE 2022 - I International Conference "Methods, models, technologies for sustainable development: agroclimatic projects and carbon

neutrality", Kadyrov Chechen State University Chechen Republic, Grozny, st. Sher

326