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20377

Bioelectricity Production from Different Types of Bacteria Using MFC Under Optimizing Factors and New Bacterial Strain Bioelectricity Production Isolated

from Milk Sample in Egypt

Ismaeel Y. Abdel-Gelel

1

, Mahmoud Abdel-Mongy

2

, Hanafy A. Hamza

2

, Rateb N. Abbas

2

Corresponding author, Abdelgelel, I. Y

1

1 Main Chemical Laboratories, Egypt Army, Egypt

1Department of Microbial biotechnology, Genetic Engineering and Biotechnology Research Institute University of Sadat City, Egypt.

2 Department of Microbial biotechnology, Genetic Engineering and Biotechnology Research Institute University of Sadat City, Egypt.

[email protected] [email protected] [email protected] [email protected]

ABSTRACT

Background. Microbial fuel cell is a way for clean and pure energy by exploiting the metabolism of bacteria in special conditions to produce electricity and using it in many other applications. Though the MFCs aren’t a broad used until now but many research are working on optimization of their production of energy. Our objective to investigate the electricity production using improved MFC with new Egyptian strain is collected from Cairo cow milk. Results.Streptococcus thermophilus optimized by raising the temperature38°C to give 192.2 mV than the two other bacteria were measured (E. coli is 71.8 mV and Lactobacillus acidophilus is 187.9 mV),On the other hand measurements under acidic pH elevated the bioelectricity in the case of Lactobacillus acidophilus to give 167.4 mV.while the identified bacterial sample from milk (Lactococcus lactis subs hordinea) produced highest bioelectricity score at normal conditions (418 mV). Conclusion. MFC can be optimized in structure using large volume and conductive materials.

The lactic acid bacteria have a potential to produce bioelectricity in large scale and can be optimized by different factors (Temp., pH, and Concentration of bacterial cells).

Key words: MFC, Lactococcus Lactis, Streptococcus thermophilus, Lactobacillusacidophilus

Introduction

Due to high potential of clean and renewable energy, Using microbial fuel cell is considered a new green technology for producing electricity from bacteria(Rahimnejad et al. 2011).Microbial fuel cells are producing electricity in anaerobic conditions to bacterial solution in which the single chamber fuel cell consists of anode rod that collect electrons from bacteria to be attracted to the cathode rod that works in the presence of oxygen on its plate to attract the protons from bacteria inside the cell(Sharma and Li 2010).In recent decades many researchers studied the factors that can optimize electricity production such as pH, temperature, concentration of substrate or microorganisms, type of bacteria and the cell construction(Chaudhuri and Lovley 2003).

The construction of microbial cell is depending on the three important parts anodes, the cathode, and the membrane in which the best material used for our study. The anode is a brush carbon with great surface area to collect more electrons(Liao et al. 2015). The cathode was made of stainless steel(Dumas et al. 2007)while the proton exchange membrane(PEM) is compacted to cathode layer was formed of active carbon granules (Cheng, Liu, and Logan 2006)was adhesivewith (PTFE) binderthen active carbon clothe layer and outer layer from Teflon.(Chou et al. 2010)

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Lactic acid bacteria are safe and non-pathogenic microorganisms, they differentiate with ability to live under anaerobic conditions efficiently as they are facultative anaerobic bacteria and exist in milk.(Mohania et al. 2008)

Measuring the competition ability to produce bioelectricity between lactic acid bacteria and E. coli illustrated the great difference in voltage results. Thenoptimizing bioelectricity produced by changing the factors effect on them.(Zhang et al. 2008)

The new identified bacteria that were isolated from milk from Nasr city in Egyptafter the laboratory analyzingdoing 16S rRNA to obtaining Lactococcus lactis subs hordinea bacteria and recorded in gene bank. According to the current annotation (MW857186.1,MW857181.1),That proving of the Lactococcus Lactis bacteria in which used in our experiments and give a promising results, This bacteria produced high level of voltage released from MFC.Lactococcus lactis is nonpathogenic and easily to exist in our dairy use with useful applications in which high voltage result in the first hour producing 418 mV.(Li et al. 2016)

The results of our Egyptian strain with maximum voltage are (418 mV) and proves the lactic acid bacteria are selectively substrate solution for MFC and production of the bioelectricity. The improvements that performed on MFC is optimizing the results.

Material and Methods Microorganisms used in the study

Four bacterial strains used in our MFC to measure the electricity production, three standard strains used in microbial fuel cell (Lactobacillus Acidophilus, Streptococcus Thermophilus and E. coli) in which obtained from Microbiological Resources Centre (Cairo MIRCEN) as follows:

1-E. Coli from Egypt Microbial Culture Collection designation is DSM 17257.(Wassenaar et al. 2017) 2-Lactobacillus Acidophilus from Egypt Microbial Culture Collection designation is DSM 20079.(Deraz et al. 2005)

3- Streptococcus thermophilus from Egypt Microbial Culture Collection designation is DSM 20479.(Gaus et al. 2006)

The fourth one is isolated from milk in Egypt and identified as Lactococcus lactis hordinea by 16SrRNA analysis and submitted in GenBank under access numbers(MW857186.1,MW857181.1).

Broth Media for bacterial solution

We used for E. Coli the Macconkey media to grow it from Oxoid microbiology product which is standard medium for the primary isolation of coliform bacteria and has been recommended for this purpose by public health laboratory service water committee and the world health organization.(Jung and Hoilat 2021)

For the remain three lactic acid bacteria, we used MRS broth media it obtained from bio lab for splendid isolation and has two components as fellow:

Table 1. The ingredients of MRS media.

Typical Formula Gm/liter

1.The base: 50.00

Casein peptone, 10.00

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tryptic digest

Meat extract 10.00

Yeast extract 5.00

Glucose 20.00

Tween 80 1.00

K2HPO4 2.00

Na-acetate 5.00

(NH4)2citrate 2.00

MgSO4.7H2O 0.20

MnSO4.H2O 0.05

Distilled Water 1000.00 ml 2.The

supplement

10.00 ml Adjust pH to 6.2 – 6.5

To make cultures for bacteria identification we use MRS agar media, but we used the MRS Broth media during applying on the MFC to generate electricity. To prepare 1liter from broth solution media suspend 50g in one liter of distilled water. Add 10ml of MRS supplement. Mix well and heat gently to dissolve the medium completely. Dispense into final containers and sterilize by autoclaving at 115 ˚C for 15 min. The medium is heat sensitive, no further sterilization is necessary or desirable and to ensure the homogeneity, you should shake well the supplement before use.(Islam et al. 2020)

We prepared the broth media as follow:

Macconkey broth media

We use the Macconkey media to grow Escherichia Coli from Oxoid microbiology product which is standard medium for the primary isolation of coliform bacteria and has been recommended for this purpose by public health laboratory service water committee and the world health organization. It contains 20.0 Gm/liter Peptone 10.0 Gm/liter Lactose 5.0 Gm/literBile salts 5.0 Gm/liter Sodium chloride 0.075 Gm/liter Neutral red atpH 7.4+- 0.2. then it autoclaved at 115°C for 15 min.

MRS broth media

At normal conditions measurements we pour 3ml of bacteria in the broth media at temperature 25°C and pH=7.For optimizing measurements, we applied the bioelectricity production from MFC under different conditions in which we change temperature from 25°C to 38°C, pH from 7 to 6 and concentration of bacteria from 3ml per liter to 6ml per liter.

Isolation and Identification

We isolated the fourth strain bacteria from milk cow from Nasr City in Egypt using specific agar medium MRS and doing four subcultures. To identify the sample, we proceed 16S rRNA two direction by Sigma Company for biological analysis.(Church et al. 2020)

DNA Extraction from Bacteria:

1- Added 200 ul of sample (liquid media that contain bacteria) in microcentrifugetube and add 95 ul water, 95 ul solid tissue buffer (blue) and 10 ul proteinase K.

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2- Mix thoroughly and then incubate the tube at 55ºC for 2 hours. Mix thoroughlyand centrifugation at 12,000 x g for 1 minute.

3- Transfer aqueous supernatant to a clean tube (300 ul).

4- Add 600 ul Genomic Binding Buffer. and mix thoroughly.

5- Transfer the mixture to a Zymo-Spin™ IIC-XL Column in a Collection Tube.

Centrifuge (≥ 12,000 x g) for 1 minute. Discard the collection tube with the flowthrough.

6- Add 400 µl DNA Pre-Wash Buffer to the column in a new Collection Tube andcentrifuge at (12.000 xg) for 1 minute

7- Add 700 µl g-DNA Wash Buffer and centrifuge at (12.000 xg) for 1 minute. Empty the Collection Tube.

8- Add 200 µl g-DNA Wash Buffer and centrifuge at (12.000 xg) for 1 minute.Discard the collection tube.

9- Add 03 µl elution buffer incubate for 5 minutes, and then centrifuge at (12.000xg) for 1 minute.

Then proceeding the PCR as follow:

PCR reaction set-up:

25 µ L MyTaq Red Mix 8 µ L DNA Template (change) 1 µl (20 Pico mol) Forward Primers 1 µ l (20 Pico mol) Reverse Primers 15 µ L Nuclease Free Water

B-Thermal Cycler Condition:

Table 2.Step Temperature Time Cycles.

Initial denaturation 94 °C 6 min

Denaturation Annealing Extension

94 °C 56 °C 72 °

45 s 45 s 35 cycle

1 min

Final Extension 72 °C 5 mins

The universal 16S rDNA primers are used, forward primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 1492R (5′-ACCTTGTTACGACTT-3′).(Hussain et al. 2021)

The result demonstrated as in figure (1).

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20381 Figure1.alignment results of the sample

Figure 2.ES tree for the sample identification

MFC structure

The cell designed to the best type of MFC single chamber cell in which was combat and leaked.(Wu et al. 2018) The microbial fuel cell That used in these methods was modified to be optimized the electricity production and has many advantages as follow:

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1- Made of acrylic material which is long life.

2- Has big volume to bacterial content.

3- Has two opens output and input.

4- It is made of neutral material which is not react with any chemicals or bio lives.

5- It is handmade, low cost and available material.

Collecting the parts of MFC with dimensions (9cm*9cm*12cm) from acrylic as figure (4), The front part is the cathode part with four layers from granular activated carbon, stainless steel mesh, carbon cloth and Teflon sheet as shown in figure (3). The main template of cathode using the stainless-steel mesh, the active carbon granules adhesive to the template by PTFE material. From outside part, the carbon cloth covers the cathode sheet while the outside part covered with Teflon sheet to produce complete cathode sheet which have oxygen penetration and blocking the fluid from leaking.(Cheng et al. 2006)

Figure 3.MFC construction components as: i. Stainless steel sheet. ii. Active carbon cloth. iii.

Teflon sheet. iv. Carbon brush. v. Cathode plate from MFC. vi. Full constructed MFC with nine anode rods and input open.

MFC parts are collected by strong adhesive wax that doesn’t affect chemical or biological properties of the cell or bacteria solution. The chamber has nine anode rods made from carbon brush connected consecutively to one wire. The cathode sheet with dimensions (5 cm * 8 cm) has two wires connected to give single one. There are two opens, one for entering the solution of bacteria before or during the experiment, another one for getting out the solution of bacteria from the cell.(Han et al. 2010)

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20383 Figure 4.The developed microbial fuel cell with dimensions (9cm*9cm*12cm) and it was made of

acrylic material, the cathode membrane is on front of the cell.

Production of electricity

After growing the bacteria for 24 hours in broth media in the incubator, we begin to work on it to produce electricity as follow:

1. Firstly,tie the two wires of the Avometer to the cathode and anode rods in the cell in the laminar.

2. Pour the bacterial solution (1 liter) without any additives only the bacteria and the media in our microbial fuel cell from input opening from upper side then plug off the open.

3. Turn on the Avometer and observe the read.

4. During the study of temperature effect,we proceed all the work inside the incubator at 38°C.

5. During the study of pH,we used diluted HCl with pH meter to stabilize the pH at 6.

6. While we studied the concentration effect of bacteria when we start by adding 6 ml of bacteria into broth media instead of 3 ml.

Results

Using our MFC under normal conditions (Temp. = 25 o C, pH = 7, Conc. = 3ml) by pouring the bacteria solution in it and making it completely sealed to ensure of anaerobic conditions and we notice the high change of the Avometer screen and elevation of the voltage(Mohan, Raghavulu, and Sarma 2008), the result appear in Table (2).

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Table 3. The results of bioelectricity production from different types of microorganisms (E. coli – Lactobacillus acidophilus – Streptococcus thermophilus) by MFC at normal conditions

(Temp=25°C, pH=7 and Conc.=3ml of bacteria).

Time Escherichia coli Lactobacillus acidophilus Streptococcus thermophilus

After 30 min 41.4 152 158.2

one hour 37.9 130.3 141.3

Three hours 33 108.9 122.3

Six hours 28.4 97.6 99.3

Twelve hours 26.1 89.2 90.2

one day 22 80.8 78.6

two days 16 59.1 62

three days 12.8 33.6 31.6

Four days 7.8 19.3 18.3

Five days 5.3 13 12.8

Six days 2.3 11.3 10.4

Seven days 0.2 4.9 3.9

Eight days 0 0.6 0.4

Figure 5. Chart illustrates bioelectricity production from MFC using different microorganisms for

eight days’ work at normal conditions.

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20385 Effect of changing the temperature

By elevating the temperature from 25°C to 38°Cto measure the bioelectricity production of the three bacteria which they appeared increasing in the produced voltagethat appeared in the figure (6).(Behera, Murthy, and Ghangrekar 2011)

Figure 6.Chart illustrates the temperatureeffect on bioelectricity production.

Effect of pH changing in bacterial solution

One of the important factors that effect on the bioelectricity production is the acidity parameter which it measured by increasing the acidity to notice the change in the results among the three types of bacteria that appeared in figure (7).(Behera and Ghangrekar 2009)

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Figure 7. Chart illustrates the pH change effect on bioelectricity production.

Effect of Concentration of bacteria

By changing the concentration of the bacteria that added to broth media by increase the amount of it from 3 ml of bacteria to 6 ml. this increasing that double the amount of bacteria growth in broth media which appeared in figure (8).(Chae et al. 2009)

Figure 8.Chart illustrates the concentration of microorganism’s effect on bioelectricity production.

Table 4. The results of using Lactococcus lactis subsp. Hordinea in MFC to produce bioelectricity.

Lactococcus lactis subsp.

HordniaeUnder

Temp = 25o c, PH = 7 and Conc. = 3mL

Type of Bacteria / time

Average Exp.3

Exp.2 Exp.1

Exp.No.

418 418

415 420

Start read (After 30min)

401 401

399 402

After (1h)

394 395

391 396

3h

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20387

382

382 379

385 6h

373 375

364 379

12h

357 358

352 360

24h

293 291

289 299

2d

251 250

248 255

3d

194 193

191 198

4d

115 114

112 118

5d

52 52

51 54

6d

25 25

23 28

7d

5 5

4 7

8d

In table (4) we placed the results obtained from MFC measurements for eight days from the new strain bacteria from Nasr city in Egypt that identified under access numbers in gene bank

(MW857186.1,MW857181.1).

Figure 9.Chart illustrates bioelectricity production from MFC between Streptococcus thermophilus

and Lactococcus lactis hordineaat normal conditions.

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In figure (9) There is a comparison between the bioelectricity produced from Streptococcus thermophilus (that has the highest produced volts than the two others) and the identified sample as Lactococcus lactis subs hordinea at normal conditions in which illustrate the high difference in the voltage produced to give 418 mV than 158.2 mV.(O et al. 2016)

Discussion

Single chamber microbial fuel cell is characterized with sealing of the solution and can be improved to be large in volume and optimized to generate more electricity.(Kim et al. 2009)

Our study reveals that new strain of lactic acid bacteria in improved MFC gives high voltage and conserve on the energy inside the bacteria cell for longer. Our bacteria without any motivational compounds or additives give good results should to be shown. After 30 min in the MFC will reach the read to high one until be stable then it will decrease gradually through time. This high increasing in the first as the bacteria converted completely to anaerobic conditions to achieve our equation of metabolic results to produce more electrons moved through wires in the anode chamber and attracted with the cathode plate.(Chen, Chen, and Chung 2014)In figure (5) we found that E. coli was not competitive bacteria for producing bioelectricity from MFCs asit used in combination with suitable electron mediators in MFCs, or else the production of electricity will be rather low (41.4 mV)(Zhang et al. 2006)thatcompared to (158.2 mV) from Streptococcus thermophilus, that also appear on the three factors tests. On the other hand, the lactic acid bacteria were a competitive bacterium for bioelectricity production. Streptococcus thermophilus at normal conditions gave (158.2 mV) at the starting read which is highly than Lactobacillus acidophilus (152 mV) that could return to active metabolism of Streptococcus thermophilus and the ability to live under anaerobic conditions than Lactobacillusacidophilus. The gradually decreasing of voltage through hours and days return to consuming the substrate in the bacterial solution due to metabolism that give us the actual voltage from different bacterial strain in the solution. High result appeared at raising the temperature to 38 to obtain (192.2 mV) from Streptococcus thermophilus that was affected by temperature as it increased by (34 mV), also the two other types of bacteria increased slightly due to motivating the activity of bacterial strain to produce more electrons by enhancing the metabolism, it demonstrated in figure(6).(Novik, Meerovskaya, and Savich 2017)

When we changed the pH factor, it is noticed that Lactobacillus acidophilus increased in voltage to give (167.4 mV) which indicate to the acidity of the solution is propriate to metabolism activity of the strain bacteria, which was inappropriate to Streptococcus thermophilus in which decreased clearly to give (135.7 mV) compared to at normal conditions, it was shown in figure (7).(He et al. 2008)

The concentration factor is very important as it enhance the bioelectricity production by increasing the number of electrons released from bacteria cells due to the increasing of the cells in the solution, that was shown in figure (8) when we doubled the number of bacterial cells were added to the broth media from 3 ml to 6 ml, it is increasing in the bioelectricity production from the three types of bacteriabut the increasing of volts from Lactobacillus acidophilus is

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20389

higher than Streptococcusthermophilus with (12.2 mV), that could returned to high ability of Lactobacillus acidophilus replication than the Streptococcus thermophilus.(Lu et al. 2020)

In table (3) the average results from the identified bacteria strain that collected from Nasr city in Egypt and isolated from milk sample then identified to be submitted in gene bank under accession numbers (MW857186.1,MW857181.1)to be Lactococcus lactis subs hordinea which tested under normal conditions of the three previous strains of bacteria to give the highest bioelectricity from others (418 mV) that increase due to high activity metabolism and the good conducting of electrons to transported to wiresas in L. lactis there is proton motive force- generating electron transfer chain (ETC).(Brooijmans et al. 2007)In figure (9) that appear a great difference between the highest results from the three bacterial strains of S. thermophilus (158.2 mV) at normal conditions and the identified bacterial strain L. lactis (418 mV) at the same conditions.(Kleerebezem et al. 2020)

Conclusion

From our study, we conclude that MFCs is a high potential green energy source in the future with the ability of improving and development of the MFCs to gain high performance of the microorganisms and the lactic acid bacteria is a good bacterial strain solution to produce bioelectricity under optimum conditions having the safe factor to deal with.

ACKNOWLEDGEMENT

All praise and thanks are due to ALLAH, the most merciful for assisting and directing me.

Thanks, due to all the staff of Microbial Biotechnology Department, genetic engineering and biotechnology research institute, university of Sadat city for their useful guidance.

Finally, I wish to express my deep appreciate to my parents who teaching me how to be great and all my family members especially my great wife, my brother, my sister and my two beautiful sons Eyad and Celia for supporting me and encouragement.

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