INTRODUCTION:

In the recent years, tremendous energy consumption has been observed in synchronization with the vast growth of the population. On the basis of mode of origin and reliability, energy sources can be classified into fossil fuels, nuclear sources and renewable sources¹. The electricity generation from fossil fuels and coal which are non-renewable resources are depleting at a very fast rate and can lead to global energy crisis². The nature is negatively influenced by fossil fuels owing to the emissions of carbon dioxide. The use of fossil fuels has severely jeopardized human life through its drastic repercussions, such as global warming³.

In spite of that, various countries tried to find a plausible solution for energy crisis from renewable energy resources such as solar energy, wind energy and water. As an aftermath of these efforts, an alternative for energy production has been found i.e., energy production by Microbial fuel cells (MFC). In addition MFC contains plethora advantages as compared to other energy generators, e.g. higher efficiency, and it does not release any environmental polluting gases (such as CO₂ and CO)⁴.

It is known for almost one hundred years that the microorganisms could be used to generate electricity⁵.

Microbial fuel cells (MFCs) are bio-electrochemical devices, an emerging technology which uses living microorganisms as biocatalysts for the conversion of complex organic and inorganic material which generates electric current^6,7,8,9,10. The idea of generating electric current using bacteria was observed in the year 1911 by Potter¹¹. A typical MFC consists of anode and cathode compartments, which are separated by a cationic membrane. The anodic chamber consists of microorganisms where they metabolize organic compounds such as glucose which acts as an electron donor. The electrons and protons are generated through the metabolism of organic compounds. The electrons then move to the anode surface. From the anode surface the electrons then transfer to the cathode compartment via electric circuit. The protons migrate firstly through electrolyte and then through the cationic membrane. In the cathode chamber the electrons and protons are consumed by a soluble electron acceptor¹².

In the anodic chamber of the microbial fuel cell, the microorganisms oxidize various organic substrates such as glucose^13,cystine¹⁴, actate¹⁵, sucrose¹⁶, wastewater and many other substances. The protons that are generated are transferred through the salt bridge the cathode compartment. There is some new type of developed MFC which omits the use of salt bridge. Microbial fuel cells can be classified into single chamber microbial fuel cells (SC-MFC) and double chamber microbial fuel cells (DC-MFC).

The electrons generated in the anode compartment are transferred to the electrode with the help of various mediators such as methyl viologen, thionine, and methylene blue etc. The MFC which doesn’t use any exogenous mediator are termed as mediator less MFC which utilizes electrochemically active bacteria (EAB).

The bacteria present in the anode compartment degrade the organic waste biologically hence acting as a bioremediation agent. Different electrodes can be used in MFC such as graphite, carbon paper, platinum etc. Moreover, an ideal electrode should provide good electrical conductivity, large surface area, mechanical strength and so forth. In recent decades, MFC technology has been improved remarkably. There are various factors which affect the MFC performance for instance, electrode material, equipment design and so on.

MATERIALS AND METHODS:

Organic Raw Material and Microorganism Selection:

The organic waste material used for the experimentation was vegetable waste which was collected from kitchen (vegetable peels and vegetables leftover) from a number of households. The organic waste was washed properly and shredded into smaller pieces and was treated in acidic and basic medium separately for 24 hours before adding it into the anodic chamber. Culture containing different bacteria was provided by the lab MRD Life sciences, Lucknow, India.

Screening of Cellulose Degrading Microorganism:

This test was performed to check the ability of the microorganism to degrade cellulose because vegetables are rich in cellulose content. To enhance the electricity production cellulose was degraded as the polysaccharides that are present in vegetable waste require a higher consumption of energy for degradation and involving in metabolic routes¹⁷.The bacteria were streaked on CMC media and incubated for 48 hours, after that it was stained with Congo red media and washed with 1M NaCl and the zone of hydrolysis was observed.

Biochemical Characterization of the Bacteria:

The bacterial species were determined by standard bacteriological identification methods according to Bergey’s manual. Various biochemical tests were performed by using broth culture which mainly included Gram staining, Endospore staining, Catalase test, Methyl red test, Mannitol test, and Indole test.

Strain Improvement and Media Optimization:

Physical strain improvement was performed by treating the bacteria with UV light for 2min, 4min, 6min, 8min. Optimization of the media was done to prepare a suitable growth media for the bacteria by using minimal salt media supplied with different concentration of Carbon and Nitrogen sources in separate conical flasks to measure the highest absorbance after the inoculation of bacteria. Temperature and pH were also optimized.

Effect of Temperature and pH:

After the streaking of bacteria in Nutrient agar media, the Petri plates were incubated at different temperatures viz. 4°C, 32°C (room temperature), 35°C and 40°C.

After the inoculation of bacteria in the Nutrient broth, it was set at various pH viz. 4, 5, 6, 7, 9 and 11 by adding 1N NaOH and 1N HCl was incubated for 24 hours. After 24 hours O.D. was measured at 620 nm.

Construction of MFC:

The microbial fuel cell consisted of a cathode and an anode, which were two plastic containers placed on the opposite sides with a volume of 300mL, joined together on either side of the salt bridge. A hole was drilled on the lid of plastic containers for copper wires. The copper wires were put into the drilled holes and were sealed with a duct tape to provide anaerobic condition. The copper wires are then are attached to a multimeter to measure the electricity generated.

RESULTS:

Screening of Cellulose Degrading Bacteria and Strain Improvement:

The use of Congo-Red as an indicator for cellulose degradation in an agar medium provides the basis for a rapid and sensitive screening test for cellulolytic bacteria. The zone of hydrolysis was observed.

Biochemical Characterization of the Bacteria:

After performing various staining methods and biochemical characterization prescribed by Bergey’s Manual the potential isolate was identified as Bacillus megaterium. The biochemical characteristics of the bacterium were shown in Table 1.

Table 1- Biochemical characterization of the bacteria


TESTS	RESULT
Gram Staining	Positive
Endospore Staining	Positive
Catalase Test	Positive
Methyl-Red Test	Positive
Mannitol Test	Positive
Indole Test	Negative

Strain Improvement and Media Optimization:

In strain improvement (physical mutation) the UV treatment given for 8 min showed the best results. In media optimization different concentrations of various nutrients such as carbon, nitrogen and salts (Table 2) were added in the CMC broth. The various concentrations taken were CMC (0.1 g/lt to 0.5 g/lt); Beef extract (1 g/lt to 9 g/lt); Peptone (1 g/lt to 9 g/lt); MgSO₄ (0.2 g/lt to 0.5 g/lt); NaCl (4 g/lt to 9 g/lt) and p H (4 to 11). The media was optimized for both UV treated bacteria and wild strain of bacteria.

Table 2- Optimization of media

Factors	Modified Media	Standard Media
		CMC- 0.1gm/lt
		BEEF EXTRACT-8g/lt
		PEPTONE- 8g/lt
		MgSO₄- 0.25g/lt
		NaCl – 5g/lt
CMC	MM1(BLANK)	0.1g/lt
	MM2	0.2g/lt
	MM3	0.3g/lt
	MM4	0.4g/lt
	MM5	0.5glt
BEEF EXTRACT	MM6 (BLANK)	8g/lt
	MM7	1g/lt
	MM8	2g/lt
	MM9	4g/lt
	MM10	9g/lt
PEPTONE	MM11 (BLANK)	8g/lt
	MM12	1g/lt
	MM13	2g/lt
	MM14	4g/lt
	MM15	9g/lt
MgSO₄	MM16 (BLANK)	0.2g/lt
	MM17	0.1g/lt
	MM18	0.3g/lt
	MM19	0.4g/lt
	MM20	0.5g/lt
NaCl	MM21(BLANK)	5g/lt
	MM22	4g/lt
	MM23	6g/lt
	MM24	8g/lt
	MM25	9g/lt
pH	MM26 (BLANK)	pH 7
	MM27	pH 4
	MM28	pH 5
	MM29	pH 6
	MM30	pH 7
	MM31	pH 9
	MM32	pH 11

Effect of Temperature and pH:

In the optimization of temperature, the growth of the bacterium was observed after 24 hours in which the plate which was incubated at room temperature (32°C) showed the best results. For optimization of pH O.D. was measured after 24 hours in which pH 4 showed the best results with O.D. 0.34 at 620nm (Fig 3).

Electricity Generation by MFC:

A dual chamber MFC was designed using waste material. In the cathode chamber different electrolytes such as 1% Copper sulphate, 1% potassium nitrate were added and in the anodic chamber bacteria was inoculated and incubated at room temperature. The electricity generation was measured at an interval of 24 hrs. Various MFC were prepared using different carbon sources, agar salt bridges and electrolytes (Table 3) by inoculating UV mutated strain and in (Table 4) by inoculating wild strain. The maximum power generation was found to be 229mV and 220 mA.

Table 3- MFC for UV treated (mutated bacteria)


Electrolyte	Salt Bridge	Substrate	VOLTS (V)	AMPERE (I)	POWER
ZnSO₄(1%)	MgCl₂₊Agar	CMC+B.E.+NaCl +Peptone+ MgSO₄	197mV	122 mA	24mW
NaCl(1%)	NaCl + Agar	Vegetable waste	156 mV	13 mA	02mW
KCl(1%)	NaCl + Agar	CMC+B.E.+NaCl +Peptone+ MgSO₄	174 mV	136 mA	06mW
CuSO₄(1%)	NaCl + Agar	CMC+B.E.+NaCl +Peptone+ MgSO₄	229 mV	220 mA	50mW
KNO₃(1%)	KCl + Agar	Dextrose+ B.E.+ NaCl +Peptone+ MgSO₄	146 mV	186 mA	27mW
CH₃COONa (1%)	KCl + Agar	Dextrose+ B.E.+ NaCl +Peptone+ MgSO₄	172 mV	161 mA	27mW
NaCl (1%)	MgSO₄+ Agar	Dextrose+ B.E.+ NaCl +Peptone+ MgSO₄	68 mV	59 mA	04mW
KCl (1%)	MgSO₄+ Agar	Dextrose+ B.E.+ NaCl+ Peptone+ MgSO₄	03 mV	03 mA	09mW
CuSO₄(1%)	MgSO₄+ Agar	Dextrose+ B.E.+ NaCl +Peptone+ MgSO₄	0 mV	0mA	0mW
KNO₃(1%)	MgSO₄+ Agar	Maltose+ B.E.+ NaCl +Peptone+ MgSO₄	44 mV	55 mA	02mW
CH₃COONa (1%)	MgSO₄+ Agar	Maltose+ B.E.+ NaCl +Peptone+ MgSO₄	108 mV	101 mA	10mW
NaCl (1%)	KNO₃ + Agar	Lactose+ B.E.+ NaCl +Peptone+ MgSO₄	46 mV	25 mA	01mW
KCl (1%)	KNO₃ + Agar	Lactose+ B.E.+ NaCl +Peptone+ MgSO₄	86 mV	84 mA	07mW
CuSO₄(1%)	KNO₃ + Agar	Lactose+ B.E.+ NaCl +Peptone+ MgSO₄	231 mV	133 mA	30mW
KNO₃(1%)	KNO₃ + Agar	Fructose+ B.E.+ NaCl +Peptone+ MgSO₄	150 mV	28 mA	04mW
CH₃COONa (1%)	KNO₃ + Agar	Fructose+ B.E.+ NaCl +Peptone+ MgSO₄	150 mV	83 mA	12mW
Distilled water (Electrode used-Graphite)	MgSO₄+ Agar	Maltose+ B.E.+ NaCl +Peptone+ MgSO₄	61 mV	38 mA	02mW
CuSO₄(2%)	NaCl + Agar	Dextrose+ B.E.+ NaCl +Peptone+ MgSO₄	21 mV	29 mA	06mW
CuSO₄(2%)	NaCl + Agar	Lactose+ B.E.+ NaCl +Peptone+ MgSO₄	196 mV	192 mA	37mW
CuSO₄(3%)	NaCl + Agar	Sugarcane juice	191 mV	180mA	34mW
CH₃COONa (1%)	MgSO₄+ Agar	Maltose+ B.E.+ NaCl +Peptone+ MgSO₄	124 mV	71 mA	8mW
CH₃COONa (1%)	MgSO₄+ Agar	Maltose+ B.E.+ NaCl +Peptone+ MgSO₄	176 mV	30 mA	5mW

Table 4- MFC for wild strain of bacteria

Electrolyte	SALT BRIDGE	SUBSTRATE	VOLTS (V)	AMPERE (I)	POWER
CuSO₄(1%)	NaCl + Agar	CMC+B.E.+ NaCl + Peptone+ MgSO₄	264 mV	188 mA	49mw
CuSO₄(4%)	NaCl + Agar	Sugarcane juice	173 mV	161 mA	27mw

DISCUSSION:

This study is based on designing and optimization of microbial fuel cell for the production of bioelectricity. Bacteria producing cellulose degrading enzymes were isolated and further used for experimentation. In future, MFC may set a confident baseline for waste management and can act as an alternative for energy source. MFC should replace methanogenesis in India particularly benefitting the rural areas¹⁸. The current study was carried out to generate electricity from vegetable waste by degradation of cellulose present in the vegetable waste. The potential isolate was identified as Bacillus megaterium on the basis of morphological, biochemical and staining techniques. Double chambered microbial fuel cell utilizing Bacillus megaterium was tried for its performance. Bacillus megaterium is one of the best strain or producing bioelectricity up to 440mV¹⁹. Similarly in our study, maximum current of 229mV was generated by Bacillus megaterium. Most of the previous results showed that Proteus vulgaris²⁰, Clostridium butyricum²¹, Clostridium acetobutylicum²² are known for their bioelectricity production. Similarly, our current study showed that Bacillus megaterium are also able to produce bioelectricity.

Different electrodes were used in MFC such as graphite rods, the results obtained were 00mV and 00 mA, but according to many literature the maximum power density obtained was 2400mW/m² because the graphite rods used were of great surface area²³. In our study, the cellulose degrading bacteria was identified to be Bacillus megaterium and the maximum electricity generation was observed 50mW and the electrolyte used was 1% CuSO₄, the salt bridge used was NaCl agar but according to some literature, the cellulose degrading bacteria was Enterobacter cloacae, a specially designed U- shaped MFC was used in which reactors were run for 19 cycles and the maximum electricity generation observed was 4.9mW^24,25,26.

In our current study, sugarcane juice was used as substrate or anodic chamber, it was observed that the electricity generation increased within 2 days (180 mA 191 mV) but it gradually decreased after 3-4 days and after 13-14 days the electricity generation was increased (281 mA and 315mV) but according to some literature the substrate used was molasses, and the electricity output of the MFC increased with time for 4 days, the electricity output decreased until the 7^th day, further study has not been provided. The maximum voltage obtained was (365mV) with molasses as substrate.

One of the main reasons that MFC is still not used at larger scale because after few days there is a decrease in the current output, this decrease in the electricity output can be assigned to the decrease in the organic matter.

Figure 1: Optimized media for UV treated bacteria.

Figure 2: Graphical representation for optimized media of wild strain of bacteria.

Figure 3: Graph representing growth of bacteria at different pH.

ACKNOWLEDGMENT:

The author is thankful to MRD life sciences laboratory, and ITM University Raipur, for allowing me to carry out this research work and providing me the required support.

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Received on 16.11.2021 Modified on 23.04.2023

Research J. Pharm. and Tech 2024; 17(8):4002-4006.

DOI: 10.52711/0974-360X.2024.00621