INTRODUCTION:

Urbanization and the expansion of anthropogenic activities are undermining the environment's natural biodiversity. The introduction of contaminants into the environment leads to pollution that causes chaos, unpredictability, and distress to physical and chemical processes, including living creatures. According to Ogilvie and Hirsh, a variety of human activities such as deforestation and mining have damaged the natural ecosystem by adversely affecting flora and fauna¹.

In addition to the chemical industry, pathology and research labs also discharge harmful chemicals into the environment. When such hazardous chemicals like agarose gels that contain EtBr, acrylamide, sodium azide, sodium hypochlorite, and dimethyl sulfoxide (DMSO) are disposed inadequately, it leads to air, water and soil pollution. More importantly, these compounds have been confirmed to be carcinogenic, neurotoxic, and reproductively toxic in animal species². As a result, there is an urgent need to reduce environmental contamination that is brought on by regional, national, and international activities³. There are several carcinogens in the environment which are associated with DNA damage including single- and double-stranded breaks, DNA cross links, oxidative stress, and DNA- DNA or DNA- protein breaks⁴.

There are numerous strategies for mitigating environmental contaminants that have been studied and reported, such as biodegradation. The biologically mediated reduction in the complexity of chemical compounds is known as biodegradation, a process by which living microbial organisms break down organic molecules into smaller ones^5-6. In most cases, however, the word biodegradation is used to refer to nearly any biologically mediated alteration in a substrate⁷. Understanding the biodegradation process necessitates knowledge of the microorganisms that make it possible. The compounds that are to be biodegraded are transformed by microbial organisms through metabolic or enzymatic mechanisms⁸. The biodegradation process involves several microorganisms, including fungi, bacteria, and yeasts. The role of algae and protozoa in the biodegradation process is poorly understood⁹. However, microbial remediation procedures have improved over time, resulting in better, more eco-friendly, safer, and biodegradable measures for the complete dispersal of these harmful xenobiotic chemicals. Bioremediation is the process of detoxifying harmful xenobiotic compounds using living creatures such as plants (phytoremediation), microbes (microbial remediation), and their enzymes. Harmful xenobiotics include synthetic organochlorides such as plastics and insecticides), naturally occurring organic molecules, polyaromatic hydrocarbons (PAHs), crude oil, and coal fraction¹⁰. The most successful and useful method for xenobiotic chemical breakdown is microbial-assisted degradation. Different bacteria such as Rhodococcus¹¹, Alcaligenes¹², Methanospirillum¹¹, Bacillus¹³, Micrococcus¹³, Pseudomonas¹¹and Flavobacterium¹¹have shown the biodegradation potential of xenobiotic compounds which were isolated from contaminated soil or water. Some of the keratinolytic fungus have also been used to remove or reduce livestocks feather waste; which plays important role in maintain the environmental balance like Mucor and Aspergillus Flavus¹⁴. Several pesticides and insecticides are also being reduced like pesticide cypermethrin (cypersul) through the action of enzyme produced by Actinomycetes¹⁵. Likewise insecticide Chlorpyrifos, which is popular organophosphorus used in agriculture produce neurotoxic chemicals which is being neutralize by the strain Pseudomonas sp. B5-2^16-17. Heavy metals like copper and lead were deteriorated by Staphylococcus aureus^18-19. Chromium is another heavy metal which is deteriorated by microbes²⁰. Pseudomonas fluorescens was able to degrade Eriochrome Blue Black, a commercial diazo dye used in tannery and textile industry at high concentration²¹. Rhodotorola sp. NS01 shows the potential of degrading Benzo[a]pyrene (BaP) which is a polycyclic aromatic hydrocarbon²².

Ethidium bromide (EtBr):

EtBr is a red color powder and water-soluble fluorescent dye. It absorbs light in the UV range and shows absorbance maxima at 210 and 285nm and gives bright orange fluorescence in the visible range with a wavelength of 605nm²³. It is an intercalating agent commonly used as a fluorescent tag (nucleic acid stain) in molecular biology experiments²⁴. EtBr shows orange fluorescence when exposed to UV which intensifies about 20-folds after binding to DNA²⁵. Since, the 1950s, it has been widely used in veterinary medicine to treat trypanosomiasis in cattle under the name homidium²⁶. The dye intercalates between adjacent base pairs in the double-stranded DNA molecule²⁷. Depending on the organism and exposure conditions, EtBr is regarded as a serious biohazard because of its mutagenicity, carcinogenicity, and teratogenicity²⁸.

Chemical characteristics and mechanism of EtBr:

EtBr is a widely used fluorescent dye in biotechnology research. The excitation peak of EtBr is observed at 301nm while its emission peak is observed at 605nm. It is an intercalating agent which intercalates into the minor grooves of the DNA strand; thus, it is widely used as a nucleic acid fluorescent tag. After the intercalation of EtBr in DNA, intense fluorescence is observed due to the presence of a hydrophobic environment between the base pairs. The EtBr cation in the hydrophobic environment releases the fluorescent quencher, which causes the water molecules to be removed, enabling the EtBr to fluoresce²⁹. EtBr was also well known for its activity against Trypanosomes, as it was used as first line of defence against the infection in cattle³⁰.

Biodegradation:

Biodegradation is the degradation of the chemicals into the environmentally acceptable products such as water, carbon dioxide, and biomass by the action of naturally available microorganisms under normal environmental conditions. The current disposal practices like the use of activated charcoal, dumping EtBr in the ground, and the use of bleach like sodium hypochlorite have various harmful effects on the environment as well as on human health³¹. The management and disposal of the hazardous substance can be made simple and affordable by discovering a unique bacterium that can successfully breakdown the EtBr. In the current study, we aim to identify the biodegradation potential of different microbial isolates for the bioremediation of EtBr.

MATERIALS AND METHODS:

In current study, different bacterial isolates were obtained from sewage water, tap water and soil and they were successfully evaluated for their biodegradation potential against EtBr. The samples were inoculated and revived on the nutrient agar and nutrient broth. Further, their biochemical tests were done by using different media and chemical reagents to identify the bacterial isolates.

Chemicals:

Agar (UM888), Nutrient agar (NA) (M001), Nutrient Broth (NB) (M002), Luria Bertani broth (LB) (M1245), LB agar (M1151), MacConkey Agar (M008), Eosin Methylene Blue (m70186), Buffered Glucose Broth (MR-VP Medium) (M070S), Kovacs’ Indole Reagent (R008), Gordon-McLeod Reagent (Oxidase Reagent) R026, Barritt’s Reagent A (VP test) (R029), Barritt’s Reagent B (VP test) (R030), Methyl Red solution (MR) (08714), Gram’s staining kit (M65092), Sulfide Indole Motility (SIM) (M85438), Simmon’s Citrate Agar (M85463), Ethidium Bromide (EtBr) (E7637), Glycerol (AS101) were procured from Hi Media (Mumbai, India). All the other chemicals used in the current investigation were of analytical grade.

Methods:

In the current study, morphological, biochemical, and cytological approaches were used to identify the potency of the bacterial isolates for studying the biodegradation potential at 30mg/ml and 60mg/ml concentrations of EtBr.

Sample collection:

All the samples were aseptically collected in duplicates from different sources like tap water, sewage water, and soil. The tap water was collected from two distinct laboratories of our department and labeled as sample 1 and 2. Similarly, sewage water samples were collected from the sewage lines outside the hospitals and were labeled as sample 3 and 4.

Screening and identification of bacteria:

All the collected samples were serially diluted up to ten dilutions and 100µl of each dilution was grown on the nutrient agar (NA) to obtain different types of bacterial strains. All the bacterial colonies that were morphologically different, were picked aseptically and inoculated separately in the LB broth to obtain the pure isolate. All the isolates were incubated for 24 hours at 37°C.

Morphological detection of various bacterial isolates:

All the obtained colonies were observed and evaluated by their colony forming characteristics like morphology on the plate, width of the colony, color of the colony, etc. Each pure isolate was streaked on the NA, EMB, and, MacConkey agar, followed by incubation at 37°C for 24 hrs.

Maintenance of pure culture of various isolates:

For the preservation of the pure isolates, glycerol stocks were prepared. The broth culture containing the pure isolate and autoclaved 80% glycerol was mixed in 1:1 proportion (2 ml) each. A total of 4 ml of stock culture was prepared and that was stored at -20°C for future use. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive.

Biochemical characterization of the bacterial isolates:

The reaction of catalase, oxidase, indole, methyl red and Voges-Proskauer (MR-VP), and Simmon’s citrate agar were performed by the method of Kumar et al³². The utilization of sulfide, indole and the motility of the test organisms was observed by the SIM media³³. The biochemical identification of the bacterial isolates was carried out as follows:

Oxidase test:

The Kumar et al³². methodology was used to carry out the oxidase test. For the oxidase test, each pure isolate was inoculated in the LB broth and incubated at 37˚C for 24 hours. After the incubation period, Gordon and Mcleod’s reagent (2-3 drops) was added to the sample followed by gentle mixing. The reaction is observed within 5-10 sec at room temperature.

Methyl Red-Voges Proskauer (MRVP) test:

MRVP test was performed by the method of Kumar et al.³². The isolates were inoculated aseptically in the glucose phosphate broth (MRVP broth) followed by incubation for 48 hours at 37˚C. After the incubation period, broth tubes were vortexed gently and divided into two portions for performing the MR and VP tests separately. For the MR test, 2-3 drops of methyl red (pH indicator) were added to the half culture tubes. Other half culture tubes were used for the VP test by adding 6 drops of Barritt’s reagent A and then 3 drops of Barritt’s reagent B. The test tubes were allowed to incubate at room temp for 30 minutes to observe the color change.

Citrate utilization test:

The Citrate utilization test was performed by the method of Kumar et al.³². The citrate utilization test was conducted by preparing a test tube slant of Simmon’s citrate media. The streaking of a pure isolate was done in a zig-zag pattern on the slant. The inoculated media was incubated at 37˚ C for 24 hours and color change was observed.

SIM (Sulfide, Indole, Motility) test:

According to Thai ³⁴, SIM (Sulfide, Indole, Motility) test was performed. Pure colonies from an 18-24-hour old culture were inoculated in the SIM medium by stabbing in the center of the medium to a depth of half an inch. The inoculated medium was incubated aerobically at 37°C for 18-24 hours and observed for motility of the test organism and H₂S production. To test the indole production ability, a few drops of Kovac’s reagent were added to the same medium followed by incubation for a few minutes. The medium was observed for the development of a pink-to-red color.

Gram’s staining:

This method was performed using Bartholomew and Mitter³⁵. A smear of each isolate was prepared on the clean slide which was air-dried and fixed. Crystal violet was poured onto the slides for 1 minute and then it was rinsed with water.After that iodine was flooded onto the slides for 1 minute and then it was rinsed with water.Later, the slides were washed with a decolorizer for 10-20 seconds and were rinsed with water. Safranin was added for a minute which was rinsed afterward. The slides were allowed to airdry. Further, all the slides were studied under fluorescence microscope.

Evaluation of the biodegradation potential of EtBr:

Biodegradation of EtBr by the bacterial isolates at different concentrations was analyzed on the NA plate. The zone of degradation was selected as the benchmark for assessing the efficacy of various isolates on NA plate supplemented with 30 mg/ml and 60 mg/ml of EtBr. Different zones of degradation were observed in different isolated species.

a) Degradation of EtBr at 30 mg/ml for sewage and tap water sample:

According to the preferred measurement indicated on the Hi-media bottle, the NA was measured. To eliminate any additional contamination, the media, and the petri plates to be used were autoclaved and irradiated to UV light. EtBr powder measuring 30mg/ml concentration was weighed and dissolved in the autoclaved lukewarm NA media. The media containing the EtBr was poured into the petri-plates and were allowed to cool down. Each isolate was taken with the aid of a sterilized loop and placed in the center of the solidified agar plate to observe the pattern of degradation. The NA-EtBr supplemented media plates with bacterial inoculates were incubated for the different time duration of 24, 48, and 120hours at 37°C. All the plates were monitored daily during the incubation period to ensure adequate evaluation of bacteria that degrade EtBr.

b) Degradation of EtBr at 60mg/ml for sewage and tap water sample:

The preparation of the nutrient media was done in a similar way as stated above. EtBr powder measuring 60 mg/ml concentration was weighed and dissolved in the autoclaved lukewarm NA media. The media containing the EtBr was poured into the petri-plates and were allowed to cool down. Each isolate was taken with the aid of a sterilized loop and placed in the center of the solidified agar plate to observe the pattern of degradation. The NA-EtBr supplemented media plates with bacterial inoculates were incubated for the different time durations of 24, 48, 120, and 192hours at 37°C. During the incubation period, all the plates were observed every day for the evaluation of EtBr degrading bacteria.

c) Degradation of EtBr at 30 mg/ml for soil sample:

1gm of soil sample was weighed and was serially diluted till 10^-10 concentration in normal saline water. The normal saline solution was prepared at a concentration of 0.9%. To eliminate any additional contamination, the media, and the petri plates to be used were autoclaved and irradiated with UV light. EtBr powder measuring 30 mg/ml concentration was weighed and dissolved in the autoclaved lukewarm NA media. 100ml of each dilution of the soil sample was taken and spread over the EtBr -NA plate. The plates having isolates with NA-EtBr supplemented media were incubated for 24, 48, 120, and 192 hours. During the incubation period, all the plates were observed every day for the evaluation of EtBr degrading bacteria.

RESULTS:

In the current investigation, the potential of the various bacterial isolates to degrade the EtBr was studied and screened. Morphological and biochemical parameters were examined to further characterize these isolates.

Screening and identification of bacteria:

From sample 1, several small, circular-shaped pink colonies were observed on the MacConkey agar medium that confirms the presence of Enterobacteriaceae bacteria (Fig.1A). On the Eosin-Methylene Agar plate (EMB agar), metallic-green and black colored colonies were observed (Fig.1B); while on Nutrient Agar (NA) medium, it developed dense mucoid colonies were observed (Fig.1C).

From sample 2,same small circular colonies which were distinctively placed from each other were observed on the MacConkey agar medium which reconfirmed the presence of Enterobacteriaceae bacteria (Fig.1D). On EMB agar medium, S2 showed different blackish colonies (Fig.1E); while on NA agar medium, the colonies were elliptical in shape and less in number as compared to sample 1 (Fig.1F).

From sample 3,distinct morphology with punctiform colonies were observed on the MacConkey agar medium (Fig.1G). On EMB agar medium, S3 exhibited colonies with a smooth texture and irregular shape (Fig.1H). On NA agar medium, streaked culture exhibited large, circular, and smooth colonies (Fig.1I).

From sample 4, punctiform colonies were observed on the MacConkey agar medium which were distantly placed from each other (Fig.1J). On the EMB agar medium, colonies with undulated edges and green metallic sheen were observed (Fig.1K). On NA agar medium, S4 exhibited circular, large, and smooth colonies (Fig.1L).

Figure 1: (A-L)Screening of different bacterial isolates from tap water and sewage water.

(A-F) Screening of different isolates from Tap water sample 1 and 2 on MacConkey, Eosin methylene Blue (EMB) and Nutrient agar (NA)- A: Sample 1 streaked on MacConkey agar plate for morphological screening, B: Sample 1 streaked on EMB agar plate for morphological screening, C: Sample 1 streaked on NA plate for morphological screening, D: Sample 2 streaked on MacConkey agar plate for morphological screening, E: Sample 2 on EMB agar plate for morphological screening, F: Sample 2 on NA plate for morphological screening, (G-L): Screening of different isolates from sewage water sample 3 and 4 on MacConkey, EMB and NA- G:Sample 3 on MacConkey agar plate for morphological screening, H: Sample 3 on EMB agar plate for morphological screening, I: Sample 3 on NA plate for morphological screening, J: Sample 4 on MacConkey agar plate for morphological screening, K: Sample 4 on EMB agar plate for morphological screening, L: Sample 4 on NA plate for morphological screening.

Biochemical analysis:

All the 13 isolates were revived from the glycerol stocks. The revived culture stocks were streaked on NA media for biochemical analysis (Fig.2).

Figure 2: Different pure isolates obtained from tap water, sewage water and soil samples.

All the 13 isolates were streaked on NA plate to visualize different morphological characters.

Citrate utilization test: Biochemical analysis showed that no color shift was observed in the test tube slant streaked with IS12 (Fig.3 A1) whereas isolates IS1, IS2, IS3, IS4, IS5, IS6, IS7, IS8, IS9, IS10, IS11, and IS13 demonstrated the citrate fermentation ability by changing the color of the citrate agar from green to blue (Fig.3A2). The above results indicate that except IS12, all other isolates have citrate permease enzyme which converted citrate to pyruvate.

Sulfide, Indole, Motility (SIM) test: The result of the SIM test indicates that isolates IS1, IS2, IS3, IS4, IS5, IS6, IS7, IS8, IS9, IS10, IS11, and IS13 were motile. The motility was observed by diffused hazy growths that cover the entire medium, making it slightly opaque (Fig.3B2). However, the growth of IS12 was restricted to the stab-line with distinct margins, demonstrating IS12's non-motility. The surrounding medium in the test tube was transparent in comparison to other isolates. (Fig.3 B4), Fig.3 B1 and B3 are the negative control tubes.

All the isolates were negative for the H₂S test as blackening of the medium was absent indicating that microbes could not reduce thiosulfate and release sulfide gas (Fig.3 C2). Following the addition of Kovac’s reagent, no color change was observed in the tubes as none of the isolates produced a red band at the top of the medium (Fig.3 D2) Fig.3 C1 and D1 are the negative control tubes.

Oxidase test: The test determines the presence of oxidase enzymes, which is performed by using the Gordon-Mcleod reagent (oxidase reagent). The oxidase test is performed to detect the synthesis of an intracellular oxidase enzyme by bacteria. All the 13 isolates were analyzed for the presence of the enzyme cytochrome oxidase. The oxidase test indicated that all the isolates were not able to oxidize the Gordon-MacLeod reagent and hence there was no color shift observed from black to blue. None of the isolates developed deep purple-blue/blue color indicative of oxidase production. The findings suggest that the bacteria may be anaerobic, aerobic, or facultative (Fig. 3 E2). Fig.3 E1 is the negative control.

MRVP test: The Methyl Red (MR) test is used to identify the mixed acid fermenting bacteria that yield a stable acid end-product whereas the Voges-Proskauer (VP) test is used to identify that the bacteria are capable of fermenting 2,3-butanediol following mixed-acid fermentation. All the isolates changed the color of broth from yellow to red upon the addition of methyl red. Thus, all the isolates were MR-positive as they can utilize glucose and convert it into pyruvic acid (Fig.3 F2). All the isolates were negative for the VP test as no change in the color of the medium was observed (Fig.3 G2). Fig.3 F1 and G1 are the negative control tubes.

Gram’s staining: Isolates IS1, IS3, IS4, IS6, IS7, IS8, IS9, IS10, IS11, IS12, and IS13 were found to be negative for gram staining as pink color colonies were observed under a fluorescent microscope (Fig. 3H). Isolate IS2 and IS5 were positive for gram staining as purple color colonies were observed under the microscope (Fig.3I). The summary of biochemical analysis is depicted in Table no. 1.

Table 1: Results of different biochemical tests shown by all the bacterial isolates.

Isolate number	Citrate Test	Oxidase Test	SulfideTest	Motility Test	Indole Test	Methyl red Test	Voges Proskauer Test	Gram staining
IS 1	+	-	-	Motile	-	+	-	-
IS 2	+	-	-	Motile	-	+	-	+
IS 3	+	-	-	Motile	-	+	-	-
IS 4	+	-	-	Motile	-	+	-	-
IS 5	+	-	-	Motile	-	+	-	+
IS 6	+	-	-	Motile	-	+	-	-
IS 7	+	-	-	Motile	-	+	-	-
IS 8	+	-	-	Motile	-	+	-	-
IS 9	+	-	-	Motile	-	+	-	-
IS 10	+	-	-	Motile	-	+	-	-
IS 11	+	-	-	Motile	-	+	-	-
IS 12	-	-	-	Non- Motile	-	+	-	-
IS 13	+	-	-	Motile	-	+	-	-

Figure 3: (A-I) Different biochemical tests and Gram’s staining done for 13 isolates.

(A1 and A2): Citrate utilization test-A1 and A2 represents Citrate negative (IS-12) and positive (IS-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13) isolates, respectively; (B1 – B4): Motility test for isolates- B1 and B2 show control and experimental tubes. The result indicated that isolates 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 13 were motile. B3 and B4 shows control and experimental tubes. The result indicated that isolate 12 was non motile; (C1 and C2): Sulfide test- C1 and C2 show control and experimental tubes. The result indicated that all the isolates were negative for sulfide test; (D1 and D2): Indole test- D1 and D2 show control and experimental tubes. The result indicated that all the isolates were negative for Indole test; (E1 and E2): Oxidase test- E1 and E2 show control and experimental tubes. The result indicated that all the isolates were negative for Oxidase test; (F1 and F2): Methyl red test- F1 and F2 show control and experimental tubes. The result indicated that all the isolates were positive for MR test; (G1 and G2): Voges- Proskauer test- G1 and G2 show control and experimental tubes. The result indicated that all the isolates were negative for VP test; (H and I): Gram’s staining of all bacterial isolates- H: Gram negative isolates (1, 3, 4, 6, 7, 8, 9, 10, 11, 12, 13), I: Gram positive isolates (2 and 5).

Evaluation of biodegradation potential of different isolates against EtBr:

a) EtBr degradation at a concentration of 30mg/ml:

The isolates underwent a 6-day incubation period on an NA medium. None of the isolates were able to break down EtBr till 2 days of incubation at 37°C on an NA medium (Fig 4). After 3 days, the isolates showed a modest deterioration of EtBr. Under a UV transilluminator, the colonies were bright orange in colour. On day 6, the isolates showed a clear zone of degradation depicted as blue colour around the bacterial colony The findings suggest that the isolates have the potential to accumulate as well as degrade EtBr, thus, utilising it as a fuel source (Fig 4).

Figure 4: Degradation of 30 mg/ml concentration of EtBr after 48 hrs (2 days) by all the isolates; degradation of 30 mg/ml concentration of EtBr after 144 hrs (6 days) by all the isolates.

b) EtBr degradation at a concentration of 60mg/ml:

The isolates were incubated on an NA medium for 8 days. None of the isolates were able to degrade EtBr post 96 hours of incubation at 37°C on the NA medium (Fig 5).From day 5 onward, the isolates showed a minor degradation of EtBr. The colonies were seen as a bright orange color under the UV transilluminator. On day 8, the isolates showed a clear zone of degradation depicted as blue color around the bacterial colony. The results indicate that the isolates have the capacity of accumulating or using EtBr as a source of energy (Fig 5).

Figure 5: Degradation of 60mg/ml concentration of EtBr in 120hrs (5 DAYS) by all the isolates; degradation of 60mg/ml concentration of EtBr in 192hrs (8 DAYS) by all the isolates.

DISCUSSION:

The objective of the current research was to assess the ability of the bacterial isolates collected from tap water, sewage water, and soil samples to degrade EtBr. Citrate test of various isolates showed that IS 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 13 were found positive as they were able to use citrate as a sole source of energy as these bacteria metabolize the citrate through the enzyme citrate lyase, which breaks down citrate to oxaloacetate and acetate. The isolate IS 12 showed negative results which showed that the bacteria were not able to use citrate as a sole source of energy. All the isolates gave an oxidase negative test The results of the MRVP test demonstrate that all the isolates were MR positive which shows that bacteria produce acid as a stable end-product while all the isolates were found to be VP negative, which indicates that they are not capable of fermenting 2, 3-butanediol after mixed-acid fermentation. Except for IS 12, which was non-motile, all the isolates tested in the SIM medium test were sulphide negative, indole negative, and motility positive. The gram-staining procedure showed that IS 2 and 5 are Gram-positive while all other isolates are Gram-negative. The EtBr could not be broken down by any soil-derived bacterial isolate used in the current study. Bacterial isolates from the tap water (IS1, IS2, IS3, IS4, IS5, IS6) and sewage water (IS7, IS8, IS9, IS10, IS11, IS12, IS13) have shown degrading potential against EtBr at concentration 30µg/ml after 2 and 5 days. Bacterial isolates from tap water (IS1, IS2, IS3, IS4, IS5, IS6) and sewage water (IS7, IS8, IS9, IS10, IS11, IS12, IS13) have shown degrading potential against EtBr at concentration 60µg/ml after 5 days and 8 days. EtBr deterioration activities of bacterial isolates revealed that all the isolates from the water samples were able to degrade the EtBr despite having a wide degradation range. When examined with a UV transilluminator, isolates taken from the soil sample did not exhibit any EtBr biodegradation.

LIST OF ABBREVIATIONS:

EMB: Eosin methylene blue agar, EtBr-NA: Ethidium bromide nutrient agar, EtBr: Ethidium Bromide, IS: Isolate, LBA: Luria Bertani Agar, LBB: Luria Bertani broth, MR: Methyl red, NA: Nutrient agar, NB: Nutrient broth, SIM: Sulfide indole Motility, VP: Voges-proskauer.

AVAILABILITY OF DATA AND MATERIALS:

All supporting data are presented in the main paper

COMPETING INTERESTS:

The authors declare that they have no competing interests.

AUTHOR’S CONTRIBUTION:

Shaleeni (S) and Vandana Jhalora (VJ): S has conducted the wet lab experiments with the help of VJ. S wrote the manuscript. VJ analyzed the results and helped in reviewing the manuscript.

Shubhita Mathur (SM): SM Evaluated the manuscript.

Renu Bist (RB): RB was the supervisor to conduct all experiments mentioned in the manuscript. She planned and helped in execution of experiments, evaluated the manuscript, and communicated too.

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Received on 14.07.2023 Modified on 28.09.2023

Research J. Pharm. and Tech 2024; 17(6):2541-2548.

DOI: 10.52711/0974-360X.2024.00397