Rhamnolipid Biosurfactants produced by Pseudomonas sp. MK5 and its efficacy on Pharmaceutical Application

 

Murugan T¹*, Murugan M¹, Albino Wins J²

¹Centre for Biological Science, Noorul Islam University, Kumaracoil - 629 180, Tamil Nadu, India

²Department of Biotechnology, Holy Cross College (Autonomous), Nagercoil - 629 004, Tamil Nadu, India

ABSTRACT:

The study was undertaken to isolate antimicrobial potent biosurfactant producing bacteria from halophilic environment. Five bacterial colonies codes as MK1-MK5 were isolated from seashore soil sample and screened for biosurfactant activity by blood haemolysis and oil spreading assay. The best biosurfactant producer of Pseudomonas sp. MK5 which produced β haemolysis and good oil displacement activity was identified by studying cultural, morphological and biochemical characteristics. The biosurfactant produced by Pseudomonas sp. MK5 was extracted and studied for their antimicrobial effect on bacterial and fungal pathogens. In this study, the biosurfactant exhibited good antibacterial activity against Gram positive than Gram negative bacteria and also showed good antifungal activity on tested fungus. The functional groups of the biosurfactant was analysed by FTIR study, the data confirms the presence of various functional groups/compounds in the biosurfactant and confirm the presence rhamnolipids. This present study substantiates the antimicrobial potential of biosurfactant produced by Pseudomonas sp. MK5. It could be gain more important in future for various pharmaceutical applications.

 

 

 

 

INTRODUCTION:

Surfactants are surface active chemical agents, which are used for many purposes in food, agricultural, industrial, cosmetic and pharmaceutical applications¹. These compounds are amphiphilic agent with both lipophilic and hydrophilic in nature². Most of surfactants are chemically synthesized, mainly from petrochemical origin³ and cause toxic problems to the environment. To rectify these problems, the alternative source for the surfactant is biological origin i.e. biosurfactant. Biosurfactants are surface active chemical metabolites produced by microorganisms such as bacteria and fungi.

They have compensation over their chemicals counterparts because they are biodegradable, have low toxicity, effective at high temperatures or pH values and have better environmental compatibility^4,5,6.

 

Biosurfactants constitute a diverse group of surface active molecules such as glycolipids, lipoproteins, fatty acids, neutral lipids, phospholipids and polymeric structures^{7 8}.

 

Biosurfactants have diverse applications includes biocontrol agent in agricultural field, health and beauty products in the cosmetic industries, etc.^9,10. It can be used as emulsifiers, de- emulsifiers, wetting and foaming agents, functional food ingredients, cosmetics and pharmaceuticals¹¹. They possess antibacterial, antifungal and antiviral properties; and they have anti-adhesive action against several pathogenic microorganisms^12,13.

 

The most prevalent biosurfactant producers, belong to the genera are Achromobacter, Acinetobacter, Aeromonas, Alkaligenes, Arthrobacter, Bacillus, Corynebacterium, Flavobacterium, Klebsiella, Micrococcus, Moraxella, Proteobacteria, Pseudomonas and Streptococcus sp¹⁴. This present study was undertaken to isolate antimicrobial potent biosurfactant producing bacteria from halophilic environment.

 

MATERIALS AND METHODS:

Soil sample:

An about 5-10 g of soil was collected from seashore at the place of Manavalakurichy, Kanyakumari District, Tamil Nadu, India. The soil sample was collected in a clean zip lock covers and immediately brought to the laboratory for the isolation of bacteria.

 

Isolation and identification of bacteria:

1 g of soil was suspended in 9 ml of saline and serial dilution was made upto 10^-6 dilution.  Each 100 µl of sample from 10^-3 to 10^-5 dilutions were taken and spread evenly over the surface of basic Nutrient agar supplemented with 5-10% of NaCl. The plates were incubated at room temperature for 48-72 h. After incubation, the morphologically diverse bacterial colonies were selected and sub-cultured in the medium for further use. The potential bacteria strain was identified by studying cultural, morphological and biochemical characteristics include Gram staining, motility, catalase, oxidase and nitrate reduction test.

 

Screening of biosurfactant activity:

Screening of biosurfactant activity was assayed by blood haemolysis and oil displacement assay.

 

For blood haemolysis test, blood agar was prepared using mineral salt medium supplemented with 5% blood. After, solidification, a loopfull of fresh bacterial culture was streaked on blood agar plates and incubated at 37°C for 24-48 h. Then, the plates were observed for the presence of haemolysis activity^{15, 16}.

 

Oil displacement assay was carried out in Petridish containing 30 ml of distilled water. 10 µl of diesel was added to the surface of water to form a thin oil layer and then, 5-10 µl of culture filtrate was gently placed on the centre of the oil layer. The oil displacement activity was observed and the diameter of the zone was measured^{13, 17}.

Production and extraction of biosurfactant:

Isolate was cultured in 50 ml of mineral salt medium (MSM) supplement with 1% of diesel in 250 ml of conical flask. The broth medium was inoculated with previously prepared 1 ml of seed culture and incubated in orbital shaker incubator at 37°C with shaking at 120 rpm for 72 h. After, suitable incubation, the cell free supernatant was obtained by centrifugation (10000 rpm for 15 min) and its pH was reduced into 2 by adding three volumes of acetone. The mixture was kept for overnight at 4°C and the precipitates was collected by centrifugation. The obtained pellet was allowed to evaporate any residual acetone and then suspended in methanol.

 

Antimicrobial activity:

Antimicrobial activity of the biosurfactant was performed by agar well diffusion method against four bacterial pathogens includes Bacillus cereus, Enterococcus faecalis, Escherichia coli and Klebsiella pneumonia, and two fungal pathogens includes Aspergillus fumigatus and Rhizopus stolonifer. 0.1ml of 18 h fresh bacterial culture was spread evenly on Nutrient agar plate using sterile cotton swab and allowed to dry for few min. Also, 0.1 ml of fungal spore suspension in distilled water was spread on Potato dextrose agar. Wells of 6 mm in diameter were punched off into medium with sterile cork borer and filled with 30 μl of biosurfactant suspension prepared in methanol (1mg/ml). All the plates were incubated at 37°C for 24 h and then the zone of inhibition was measured.

 

FTIR analysis of biosurfactant:

Fourier transform infrared spectroscopy (FTIR) analysis was carried out to elucidate the components present in the biosurfactant. The spectrum was recorded in the range of 400 to 4000 cm^-1 at a resolution of 4 cm^-1. All data were corrected for the background spectrum.

 

RESULTS:

Isolation and identification of bacteria:

A total of 5 morphologically distinct bacterial colonies were isolated from the seashore soil collected from Manavalakurichy, coded as MK1 to MK5. Among the 5 isolated colonies Pseudomonas sp. was identified by cultural, morphological and biochemical characteristics. The strain of MK5 produces light yellowish green, irregular, transparent, flat colonies; it was Gram negative, rod shaped, motile, oxidase, catalase and nitrate reduction positive (Table 1).

 

Table 1. Cultural, morphological and biochemical characteristics of potential strain

Sl. No.	Characterization	Result
1	Colony morphology	Light yellowish green, transparent
2	Gram reaction	Gram negative
3	Morphology	Rod shape
4	Arrangements	Thin Bacilli
5	Motility test	Motile
6	Oxidase test	Positive
7	Catalase test	Positive
8	Nitrate reduction	Positive

 

Screening of biosurfactant activity:

Screening of biosurfactant activity was performed by blood haemolysis and oil displacement assay. In this study, Pseudomonas sp. MK5 lyses the blood cells and formed zone of clearance in blood agar. Also the strain exhibited superior oil displacement activity (6 mm in dia.) against the layer of diesel on distilled water. 

 

Antimicrobial activity:

The biosurfactant produced by Pseudomonas sp. MK5 shows inhibitory activity against entire test organisms. B. cereus (12 mm), E. faecalis (13 mm), E. coli (10 mm), K. pneumonia (9 mm),  A. fumigatus (12 mm) and R. stolonifer (10 mm). This result showed that, highest activity among bacteria was E. faecalis (13 mm) followed by B. cereus (12 mm) and in fungi A. fumigatus (12 mm) was highly inhibited than R. stolonifer (10 mm) (Fig. 1).

 

Figure 1. Zone of inhibition of biosurfactant on microbial pathogens

 

FTIR analysis of Biosurfactant:

The FTIR analysis of the biosurfactant shows 18 peaks between 400 to 4000 cm^-1, includes 517.85, 575.71, 621.04, 666.36, 703.97, 1028.95, 1316.33, 1370.33, 1454.23, 1510.16, 1546.8, 1640.35, 1712.67, 1746.42, 2317.31, 2923.88, 3444.63 and 3739.72 cm^-1 (Fig. 2). The functional groups/compounds of respective peaks were determined by IRPAL software (Table 2).

 

Figure 2. FTIR chromatogram of biosurfactant produced by Pseudomonas sp. MK5

Table 2: Functional groups of biosurfactant produced by Pseudomonas sp. MK5

 

DISCUSSION:

Production of biosurfactants by microorganisms had been a subject of interest in recent years, becoming important biotechnology products for industrial and medical applications due to their specific modes of action, low toxicity, relatively easy preparation and widespread applicability¹¹.  In this present study, a total of five morphologically distinct bacterial colonies were obtained from the soil collected from shoreline of Manavalakurichy, Kanyakumari District, Tamil Nadu. Among these, Pseudomonas sp. was identified by studying cultural, morphological and biochemical characteristics. The strain of MK5 was confirmed as Pseudomonas sp. and then subjected to screening of biosurfactant activity, which was performed by blood haemolysis and oil displacement assay.

 

In this study, Pseudomonas sp. MK5 lyses the blood, formed β type zone of clearance in blood agar and exhibited superior oil displacement activity. Among many methods for screening of biosurfactant activity, blood agar method is often used for a preliminary screening of microorganisms for the ability to produce biosurfactants on hydrophilic media¹⁸. Displacement of oil clearly is a sign of extra cellular surfactant present in the supernatant of culture¹⁹. Pseudomonas sp. was efficient producers of biosurfactant^{20, 21}. This result was supported by Patil et al. ²², they isolated rhamnolipid biosurfactant producing P. aeruginosa strain from soil and was confirmed by studying physiological, biochemical tests and 16S rRNA sequence analysis. Also, similar result was obtained by Santhini and Parthasarathi¹¹.  

 

The antimicrobial activity of the biosurfactant produced by Pseudomonas sp. MK5 was examined by agar well diffusion method against four bacterial and two fungal pathogens. In this study, the entire bacterial pathogens such as B.cereus, E.faecalis, E.coli and K.pneumoniae, and fungal pathogens such as A. fumigatus and R.stolonifer were inhibited by the biosurfactant. Also it was observed that, Gram positive bacteria (B.cereus and E.faecalis) were more susceptible than Gram negative bacteria (E.coli and K.pneumoniae). In fungus, A.fumigatus was highly inhibited than R.stolonifer.  Das et al. ²³ found that surfactin produced by B. circulans had good antimicrobial activity against most Gram positive bacteria than Gram negative bacteria. The lipopeptide biosurfactant produced by B.licheniformis exhibited interesting antimicrobial activities against B.subtilis, B. thuringiensis, B.cereus, Staph. aureus, P.aeruginosa, E.coli, S.typhi, P.vulgaris and K.pneumoniae²⁴. The biosurfactant produced by P.aeruginosa strain F23 was found to be effective against wide range of test pathogens such as Escherichia coli, Salmonella typhimurium, Staphylococcus aureus, Proteus vulgaris and Proteus mirabilis ²². Similar results are also obtained by Karkera et al.²⁵ for biosurfactant produced by P. aeruginosa R2, which was effective against Gram positive organisms.

 

The peak at the wave numbers 1028.95, 1454.23 indicates the presence of carbohylic acid, esters and alkanes with the assignment of C-O, CH2 and CH3 stretch. Peak at 1546.8, 1640.35 indicates amides, alkenes, amine and miscellaneous compounds with the assignment NH out of plane, C=C, C=O stretch, NH2 and C=N. Also, the peak at 1712.67, 1746.42 indicates the presence of C=O stretch, these all structures confirm the presence rhamnolipids. The strong absorbance at 1638.7cm^-1 is considered to be the characteristic peak of biosurfactants by many researchers²⁶. Similar characteristic peaks were observed in FTIR spectrum for rhamnolipid produced by various Pseudomonas strains by many researchers^{25, 27, 28}. The biosurfactant produced by P.aeruginosa strain MK5 was potentially have good antimicrobial effect and it could gain more important in future for various foods, cosmetic and pharmaceutical applications.

 

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Accepted on 30.07.2017           © RJPT All right reserved