Study on the Antibacterial Activity and Identification of cellulolytic bacteria from Cow Urine

 

Bharathirajan M¹, L. Jeyanthi Rebecca^1*, M. Jothi Muniyandi²

¹Department of Industrial Biotechnology, Bharath Institute of Higher Education and Research, Selaiyur, Chennai-600 073

²Department of Biotechnology, Sourashtra College, Madurai

 

ABSTRACT:

Cow urine has been used as an antimicrobial agent from time immemorial. The present study was carried out to explore the presence of cellulase producing bacteria from cow urine thus making it a cheaper substrate for the large-scale production of enzymes. Cellulase producing Pseudomonas aeroginosa was isolated and characterized using biochemical and molecular characterization. There were significant levels of cellulase in the urine sample. The presence of the cellulase enzyme might be a reason for the antifungal activity of cow urine. The antibacterial activity of the urine sample showed more inhibition with respect to Staphylococcus aureus when compared to Klebsiellapneumonia and Escherichia coli.

 

 

 

INTRODUCTION:

Cowpathy, is a method of treating diseases by using cow urine and products from cattle. Cow Urine (Gomutra) is used as an effective medicine¹. Recent researches prove that cow urine therapy is capable of curing several diseases including certain types of cancer. Ancient literature in Ayurveda states that cow urine is one of the best natural remedies to cure many bacterial and fungal diseases especially skin diseases. Cow urine is said to contain Nitrogen, Sulphur, Phosphate, Sodium, Manganese, Carbolic Acid, Iron, Silicon, Chlorine, Magnesium, Calcium, Salts, Vitamins A, B, C, D & E, Minerals, Lactose, enzymes, creatinine, hormones, urea and Gold acids².

 

Large amounts of agricultural, industrial and municipal cellulosic/organic wastes is accumulated or used inefficiently due to the high cost of their utilization processes or recycling process³.

 

 

Cellulolytic strains are extremely resistant to environmental stresses and are thermophilic, psychrophilic alkaliphilic or acidophilic as well as  halophilic⁴. Bacteria and fungi are an attractive potential agent for the exploitation of cellulases and hemicellulases and lipases from organic waste using solid state fermentation due to their rapid growth rate, enzyme complexity and extreme habitat variability⁴.The antimicrobial activity of cow urine was tested onpathogens like Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia². Bacteria capable of producing cellulases are widely distributed as carbon and energy sources.  Cellulolytic bacteria are also found to be associated with the aquatic weeds of Kerala^{5, 6}.National Environmental Engineering Research institute (NEERI), Nagpur, India had reported that the slurry obtained from aquatic weeds like water hyacinth could be used as a source of cellulose for microbial growth and enzyme production⁷.Cellulolytic strains that are extremely resistant to environmental stresses include strains that are thermophilic, psychrophilic alkaliphilic or acidophilic as well as strains that are halophilic⁴.  Bacteria also present an attractive potential for the exploitation of cellulases and hemicellulases due to their rapid growth rate, enzyme complexity and extreme habitat variability⁴.

The present study was carried out to isolate and identify cellulase producing bacteria from the cow urine sample. The antibacterial activity of cow urine was analyzed against three bacterial pathogens.

 

MATERIALS AND METHODS:

Biochemical and morphological characterization:

Collection of the Sample:

Cow Urine sample was collected using pre-sterilized Sterile Container from the cow shed located in Nallochanpattiwhich is located in Madurai.

 

Isolation of cellulolytic Bacteria:

The cellulase producing bacterial strain was isolated using serial dilution and pour platetechnique. Primary screening was performed by growing the isolates on minimal agar medium containing: K₂HPO₄7g; K₂HPO₄ 2g; (NH₄)₂So₄ 0.5g, glucose, 1g; sodium citrate, 0.5g; MgSO₄, 0.1g; Agar agar, 20g and distilled water 1000 ml at pH 7, supplemented with 1% car boxy methyl cellulose (CMC)at 37 C for 48 hours.

 

Identification of the Isolates:

The bacterial isolates obtained after the primary screening were maintained in pure culture on CMC supplemented minimal agar slants. All the agar slants were refrigerated at 4 C until used. Study of colony morphology of the isolated cultures was carried out using Gram’s staining.

 

Biochemical Characterization:

Methyl Red Test:

Cultures were inoculated in two sets of tubes containing MR-VP Broth and incubated for 2 days at 30 C.One set of tube approximately 5-10 drops of Methyl red solution has been added and allowed to run slowly downside the tube to form a layer on the surface of the culture.

 

Voges-Proskauer Test:

In another set of tube 0.6ml of naphthol solution and 0.2ml of KOH solution has been added, mixed and allowed to stand for some time.

 

Catalase test:

Loop full of culture grown on nutrient agar plate in 1% H₂O₂ on the glass slide.  Then the slide was observed for the production of air bubbles with effervescence.

 

Citrate Ulilization Test:

The Bromothymol blue indicator in simmons citrate agar, changes from green to Prussian blue color indicated a positive reaction in all samples.

 

Starch Hydrolysis Test:

The organisms which utilize the starch are grown in the media. This can be confirmed by adding iodine to it. Blue color is formed all over the plate except at the place of growth of bacteria. This is due to the utilization of starch.

Cellulase Production:

CMC supplemented modified mineral salts solution medium contain: CMC, 5g; CaCl₂, 0.005; FeCl₃, 0.00005g; ZnSo₄ 0.0005g; MnCl, 0.000126g; NH₄Cl 1g; MgSO₄, 0.82g; K₂HP0₄, 0.82g; K₂HPO₄, 1.25g; KH₂PO₄ 3g yeast extract, 5g; and distilled water 1000 ml at pH 7; the culture tubes containing sterile broth was separately inoculated with the incubated at 37̊C for 48 hours in a shaker incubator (Orbitek) at150rpm. The broth was centrifuged at 14000xg for 30 min at 4̊C. the clear supernatant was collected separately and the cellulase enzymes assay was carried out using DNS method.

 

Cellulase Assay:

Cellulase activity was assayed by using 1% CMC as the enzyme substrate. The reaction mixture contained 1ml of the substrate solution of 0.25g of CMC with 24ml of distilled water in a beaker and covered with aluminum foil and boiled to dissolve on ot plate at 100̊C for 10 min and made up the volume to 25 ml with distilled water. To this solution 0.5 ml of the culture supernatant crude enzyme and 1ml of sodium acetate buffer was added. The mixture was incubated at 37̊C in water both with shaking for 30 min. Released reducing sugar was measured using 3,5- DNS and glucose as standard. The color was developed by boiling in water both for 5 min and read using spectrophotometer at 540nm. One unit of enzyme activity was defined as the amount of enzyme required to liberate of glucose per mg of enzymeprotein per min under the standard assay conditions. One unit of specific activity was defined as the amount of enzyme required to liberate mol of glucose per mg of enzyme protein per min under the standard assay conditions.

 

Molecular characterization:

Isolation of genomic DNA:

The DNA was isolated from the pure culture of bacterial isolate using the standard phenol-chloroform method. The isolated DNA was elctrophoresed and was used for 16s rRNA sequencing.

 

PCR Amplification of 16s rRNA Gene:

Test:

(Total volume 50µl), Genomic DNA- 3µl, 8F Primer- 2.5µl, 1490 R- 2.5µl, Genei master mix - 25µl, Deionized water - 17µl

 

Control:

(Total volume 50µl), Genomic DNA- 3µl, Forward Primer - 2.5µl, Reverse Primer - 2.5µl, Deionized water- 17 µl

 

Sequencing:

The amplified PCR products were purified and it was sequenced.

Database searching and deposition of Gene in Gen Bank:

Nucleotide database was searched with the sequences obtained with NCBI (National Centre for Biotechnology Information) BLAST (Blastn) tool http://www.ncbi.nlm.nih.gov/BLAST.

 

Antimicrobial activity:

The antimicrobial activity of cow urine and its distillates was tested by agar well diffusion method for the following strains of microbes like Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae. Size of the well was 10 mm and 0.5 ml of urine and its distillate was introduced. Ofloxacin 10 µg mol^-1 was used as the standard for the study. The petri dishes were then incubated at 37^◦C for 24 hr and the zone of inhibition was measured.

 

RESULTS:

The sample was collected from the Nallochanpatti, Madurai (Fig-1).Cellulase producing organism was isolated in minimal salt agar medium supplemented with carboxy methyl cellulose from cow urine sample (Fig-2).

 

 

Fig- 1 Sample collection

 

 

Fig- 2 Single colony plate

 

The morphological characterization of the organism was observed using gram staining method (Fig-3). It showed positive in Indole test, Catalase test, simmon citrate test (Fig-4).  All the other tests namely, starch hydrolysis, Methyl red and Voges proskauer test gives negative results (Table-1).

 

 

Fig- 3 Gram stain

 

Fig- 4 Biochemical Test

 

Table 1: Biochemical Characterization

Methyl red	Negative
Voges Proskaeur	Negative
Simmons Citrate	Positive
Indole	Positive
Starch	Negative
Catalase	Positive

 

Cellulase enzyme was estimated by DNS method (Table-2). The genomic DNA of cow urine isolates was resolved in 1% agarose gel with marker (Fig-5).The 16s rRNA segment was amplified and it was found to have a size of 1.5kb when compared with the marker(Fig-6).

 

 

 

 

Table 2: Estimation of cellulase activity using DNS method

S. No	Vol of Std maltose solution (ml)	Concentration (mg)	Vol of dist. Water (ml)	Vol of DNS reagent (ml)	Incubation (min)	OD at 540 nm
Blank	0	0	2	2 ml	15 min in boiling water bath	0
S1	0.4	0.8	1.6			0.2
S2	0.8	1.6	1.2			0.6
S3	1.2	2.4	0.8			0.14
S4	1.6	3.2	0.4			0.21
S5	2.0	4.0	0			0.26
T1	0.5	-	1.5			0.29

 

 

 

Fig-5 Isolated bacterial genomic DNA

 

 

Fig-6 Amplified PCR product

 

 

The sequenced DNA was analyzed using bioinformatics tools as Pseudomonas aeruginosa. Antimicrobial activity was tested against four major pathogens such as Klebsiella pneumoniae, Staphylococcus aureus, and Escherichia coli. All of them were found to be sensitive to cow urine (Table-3; Fig-7).

 

     

Klebsiella pneumonia

 

 

 

E. coli

 

 

 

Staphylococcus aureus

 

Fig-7 Antimicrobial activity of cow urine

 

 

 

 

Table-3 Antimicrobial activity of cow urine

Volume of urine	Escherichia coli	Klebsiella pneumoniae	Staphylococcus aureus
60 µl (Ofloxacin control)	8mm	7mm	8mm
20  µl	3mm	2mm	4mm
40 µl	5mm	5mm	6mm
60 µl	7mm	7mm	6mm

 

 

DISCUSSION:

The cow urine is used in the rural villages of India. Cow urine is an effective antiseptic for wounds, skin diseases, etc.  In rural houses, people dilute cow urine with water and use it as a natural disinfectant¹.  It was revealed that cellulose degrading bacteria was rod shaped. The molecular characterization was done using 16s rRNA sequencing method. The sample was sequenced and BLAST analysis was done and the organism was identified as Pseudomonas aeruginosa. The antifungal activity of cow urine is studied extensively^8-10. The presence of cellulolytic enzymes might be a reason why the cow urine has the antifungal activity. The cattle feed also has an imprint on the microflora of cow urine. The Antimicrobial activity against some pathogenic organism was done using cow urine sample. It has been studied using other pathogens also^11-15. Cow urine is claimed to possess antidandruff effect⁹ apart from having wound healing properties^16-17. It has been found to exhibit anti-helmintic properties also¹⁸. This study also shows that cow urine can be used as a cheap source of industrially important enzyme.  The cellulase enzyme is used in food, laundry, detergent industries, etc. 

 

CONFLICT OF INTEREST:

The authors certify that they have NO affliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

 

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Accepted on 20.06.2018          © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(9): 3993-3997.