INTRODUCTION:

Today one of the major environmental pollution is caused by petrochemical industry^[1]. The process of production, refining, transport and storage are all causes of day to day leakage of petroleum products^[2]. Any exposure of hydrocarbons in the environment, caused either by daily activities or spillage is adds to a major reason of pollution^[3]. Most of the hydrocarbons like diesel, petrol, and crude oils are complex mixtures which cannot be easily degraded and they are insoluble in water ^[4]. Components of hydrocarbons have the property of neurotoxic and carcinogenic organic pollutants^[1]. These compounds can be eliminated by chemicals agents, by mechanical methods or also by biological agents.

However, the cost involved in mechanical and chemical method is high and the outcome may be poor as well^[5]. Biodegradation is one of the most capable technologies in the successful management of contaminated crude oils ^[6]. Biodegradation is a process uses naturally occurring microorganisms to breakdown toxic substances into non toxic substances^[7] .

Biosurfactants are naturally compound produced by microorganisms. They are amphipathic metabolites secreted or as element of cell structures of many prokaryotes, fungi and yeast. It has capability to reduce the interfacial tension and surface tension between two liquids as well as hydrocarbon mixtures^[8]. Bio surfactants are organic compounds which contains both hydrophobic and hydrophilic groups. Thus the biosurfactants contains both water soluble (water loving) and water insoluble (water repellent) moieties^[9]. They act in the growth medium of the microorganism producing the same and make an insoluble substrate for the microorganisms^[10].

Surfactant molecules can be categorized as synthetic (chemical) surfactants and bio-surfactant. Synthetic surfactants are produced by chemical reaction which used in the crude oil industry for cleanup of the crude oil spills. The biosurfactant is produced by biological processes instead of chemical reactions. Biosurfactants would structurally have a lipoprotein/lipopeptides /phospholipids, polysaccharide-lipid complex, glycol lipids or mycolic acids^[11]. Synthetic surfactants are non-biodegradable and are thus hazardous. Bio-surfactant molecules are more preferable than the synthetic surfactant molecules because which have several advantages like high biodegradability, selective effectiveness, ecological acceptability, active at diverse pH and temperature conditions, and low toxicity^[12;13]. Generally the biosurfactants are more eco-friendly than chemical surfactants. Biosurfactants have some physiological functions such as bioavailability of hydrophobic water insoluble substrates and increasing the surface area, bacterial pathogenesis, metal binding, quorum sensing, and bio film formation. When compared to synthetic surfactant, biosurfactant are more easily degradable^[14] .

The microbial biosurfactants are classified into two groups; first group compounds are low molecular weight surface active agents (bio-surfactant) and second group includes high molecular weight substrate (bio-emulsifier). These two major groups are further classified into six distinct classes based on their functional groups into Glycolipids, Fatty acid, Phospholipids, Lipopeptides, Lipopolysaccrides and Antibiotics^[15]. Among them, glycolipids and lipopeptides are the low molecular weight biosurfactants and the rest of them are high molecular weight biosurfactants. Some microorganisms are capable of producing different combinations of these biosurfactants^[16].

Among the different groups, glycolipids are most well studied biosurfactants. Glycolipids are carbohydrates, which contains long chain aliphatic acids or hydroxyl fatty acids. They consist of mono, di, tri, and tetrasacchrides. Rhamnolipids, Trehalolipids and sophorolipids are best known glycolipids^[17]. Some microorganisms produce complex fatty acids containing biosurfactants likecorynomucolic acid^[18]. while phospholipid biosurfactants are mainly produced by Thiobacillusthiooxidans while growing on alkane rich medium^[19]. Lipopeptides are the other major class of biosurfactant, produced by Bacillus subtilis. Lipopeptides have several advantages like enhancement of crude oil recovery, biodegradation of hydrocarbons and chlorinated pesticides^[20]. While lipopolysaccharides like Liposan, Emulsan, Mannoprotiens and polysaccharides complexes are the polymeric biosurfactants. Emulsan is produced by Acinetobacter calcoaceticus RAG-1 and liposan is produced by Candida lipolytica. The production of high amount of mannoprotien from Saccharomyces cerevisiae show high emulsifying activity against several crude oils^[21]. Finally, the antibiotic group of biosurfactants includes Decapeptide antibiotic like gramicidins have powerful surface active properties. Antibiotic TA produced from Myxococcusxanthus which has ability to inhibit peptidoglycan synthesis^[22].

Biosurfactants have a number of commercial applications such as food processing, cosmetics, pharmaceutical, agricultural use, bioremediation and aquafield. In food industry biosurfactants are act as emulsifiers, solubilizers, forming, wetting, anti-microbial agents and anti-adhesive activity^[23]. Cosmetic industrial applications of biosurfactants are anti-dandruff, anti-ageing and anti-wrinkle products, toothpaste, nail care products and deodorants^[24]. Biosurfactants have strong antifungal, antiviral and antibacterial activity. Bioremediation is a process carried out by utilizing microbes and microbial products. Now a day hydrocarbon compounds are one of the reason for environment contamination. Biosurfactant molecules have the application for treating contaminated crude oil ^[25].

MATERIALS AND METHODS:

Sample collection:

Soil samples contaminated with crude oil, petroleum and diesel were collected from different areas in Vellore (12.9165° N, 79.1325° E) Tamil Nadu, India. Three samples were collected from the localized area for the isolation of biosurfactant producing bacteria. The soil samples were collected in sterile autoclavable bags and stored in refrigerated condition.

Isolation of bacterial colony:

From the serially diluted samples, 1 mL each of 10^-3, 10^-4 and 10^-5 dilutions were inoculated by spread plate technique on R2A agar (g L^-1; 0.5 g Glucose, 0.5 g Starch, 0.3 g K₂HPO₄, 0.5 g Peptone, 0.5 g Casein, 0.3 g Sodium pyruvate, 0.05g MgSO₄ , 0.5g Yeast extract and 18g agar). These plates were incubated for 24-48 hours at 37ºC. The plates were observed for morphologically distinct colonies upon the period of incubation^[18].

Screening methods:

Blood hemolysis test:

For checking the nature of hemolysis, morphologically different isolates were inoculated on freshly prepared blood agar (5%) media. Incubation was maintained at 37ºC for 24 to 48 hours ^[9].Colonies which exhibited evident lysis were selected for further analysis.

Production of Biosurfactant:

The isolated colonies were inoculated into minimal salt medium with the following composition (g/l): KH₂PO₄ 20g, K₂HPO₄ 5.0g, (NH₄)₂PO₄ 30g, NaCl 0.1g, FeSO₄.7H₂O 0.01g, MgSO₄.7H₂O 0.2g, CaCl₂.2H₂O 0.01g, MnSO₄.7H₂O 0.2g, Glucose 0.03g, Yeast extract 0.03g. The culture was incubated at 30°C for 2 days at 120 rpm. The cultures were centrifuged at 8000 rpm at 4°C for 20 minutes. After centrifugation, cell free supernatant obtained was used for further biosurfactant studies.

Crude oil spread method:

In crude oil spreading method biosurfactant producing bacteria shows crude oil displacement activity. 1 mL of crude crude oil was placed on 30 µL of distilled water, above which 20 µL of the cell free suspension was dropped. If the tested organisms have ability to produce biosurfactant, crude oil will displace and spread in the water^[13].

Drop collapse method:

The drop collapse method was performed for detection of destabilization of liquid droplets by biosurfactant. A solid surface was coated with crude oil on which the cell free supernatant was spotted and observed after a minute. Positive result was considered for the production of biosurfactant by the isolates, when the liquid drop flat or spread over the crude oil coated surface. If the drop remains stable, it indicates the lack of biosurfactant^[14].

Blue agar plate method:

Minimal salt medium (MSM) supplemented with 0.5g m L^-1 and 0.2g mL^-1methylene blue were prepared for the detection of biosurfactant. Three different concentration of cell free supernatant 30µL, 60µL and 90µL was loaded into the well which prepared in blue ager plate. A negative control was maintained with distilled water. Incubation was observed at 37 ºC for 48- 72 hours. Anionic biosurfactants produce a bright blue halo in the vicinity of the culture^[15].

Emulsification Assay:

Emulsification activity of biosurfactant was measured by vortexing 2mL of cell free supernatant and 2mL of crude crude oil (Petrol, Diesel, and Crude oil) and kept overnight. After 24 hours the emulsion index E₂₄ was calculated based on the following formula:

Height of the emulsion layer

Emulsification index (E24)----------------------------X 100

Total height

Orcinol Assay:

The assay quantitatively measured the amount of glycolipids. To 100 µL of tested sample, 900µL orcinol (0.19%) was introduced. The tubes were heated in a water bath for 30 minutes at 80°C. The temperature of the tubes were brought down and the absorbance of the samples were measured by a spectrophotometer at 421nm. Distilled water was taken as control. Using standard curve the concentration of rhaminolipid was calculated^[16].

Hydrocarbon Overlay Agar Method:

In hydrocarbon overlay agar method (HOA) nutrient agar plates were coated with 40 µL of crude crude oil (Petrol, Diesel, and Crude oil). Pure bacterial colonies are spotted over this media. A time period of 7-10 days of incubation was carried out at room temperature. Emulsified halo around colony considered as the positive result for biosurfactant production^[17,26].

Analysis of the biosurfactant:

Fourier Transform Infra Red spectroscopy helps identify the chemical bonds forming a particular compound. It profiles the compound under screening and gives a molecular fingerprint of the compound. The FTIR spectrum of the sample was recorded by ATR-FTIR, Schimadzu Affinity 1 to determine the chemical composition of the extracted sample. 2- 3 mg of the extracted compound was of the isolated compound was blended with 200mg of potassium bromide (FTIR grade) and prepared as disc pellets.

Biochemical characterisation:

Three potential isolates were partially characterized by performing standard biochemical tests to determine the genus with reference to the Bergey’s manual of determinative bacteriology.

RESULTS AND DISCUSSION:

Upon incubation five morphologically distinct bacterial colonies were selected and designated as APB1, APB2, APB3, APB4 and APB5 for biosurfactant studies. They were studied for the presence of any biosurfactants. In primary screening, hemolysis test, three potential isolates (APB3, APB4 and APB5) exhibited β-hemolysis. The ability of hemolysis was linked to the production of biosurfactants and these three isolates were selected for further screening procedures. In emulsification assay the isolates showed an emulsification of > 40 % when tested with diesel whereas comparatively low in case of petrol. The highest emulsification index was exhibited by APB3 against crude oil reaching a percentage of 53.54±0.683 (Figure 1).

Fig 1. Emulsification assay

All the three isolates, APB3, APB4 and APB5 exhibited drop collapsing activity in less than a minute. The negative result in blue agar test indicates that, the absence of any anionic surfactant production (Table 1).

Table 1 Drop collapse and Blue agar tests

SI.No;	Sub strate	Drop collapse			Blue agar test
SI.No;	Sub strate	APB3	APB4	APB5	APB3	APB4	APB5
1	Petrol	+	+	+	-	-	-
2	Diesel	+	+	+	-	-	-
3	Crude Oil	+	+	+	-	-	-

The results obtained in the orcinol test sugeests the successful production of biosurfactants by the bacterial isolates with a high concentration of rhamnolipid detected to be 51.66±1.393 produced by APB3 when the lowest was 25.11±0.689 in comparison with the standard curve of orcinol (Fig 2).

Fig 2 Concentration of Biosurfactant produced

The isolates APB3 and APB5 showed positive results in the hydrocarbon overlay agar assay thus being able to displace the hydrocarbon above the surface of growth media. While the isolate APB3 could also emulsify the crude oil with a clear zone of around 12 mm followed by APB5 with a zone of 8 mm. All the isolates were not able to show any activity against petrol (Table 2).

The FT- IR spectra of the extracts of all the three potential isolates were similar in appearance. A broad peak was observed in the range of 3500-3100 cm owing to the presence of C-H and N-H stretching vibrations. This attributes to the presence of carbon carrying compounds with the presence of amino group. Peaks were sharper at 1406 and 1058. The spectra are as in Fig 3, 4 & 5.

Fig 3 FTIR Spectra of APB3

Fig 4 FTIR Spectra of APB4

Fig 5 FTIR Spectra of APB5

The isolates which were potential biosurfactant producers were biochemically analysed to determine their genus. The isolates APB3 and APB4 were gram negative rods and all the tests performed conclude the isolates to be of Pseudomonas sp. While the isolate APB5 was found to be a gram positive cocci, which was summarized to be in the genus Staphylococcus through various biochemical tests (Table 3).

Table 3 Biochemical test

Tests	APB3	APB4	APB5
Gram staining	Gram (-) rods	Gram (-) rods	Gram (+) cooci
Indole	-	+	-
Methyl red	-	-	+
Voges- Proskauer	-	-	+
Citrate	+	+	+
Catalase	+	+	+
Oxidase	+	+	-
Triple sugar iron agar	K/K	K/K	A/A
Gelatin liquefaction	+	+	+
Starch hydrolysis	-	-	-
Nitrate reduction	+	+	+
Urease production	-	-	+
H2S Production	-	-	-

+Positive, - negative

The production of biosurfactants by Pseudomans sp and Staphyloccous sp has been reported earlier at a larger scale. Thavasi et al.^[27] reported the production of 8.6 mg mL^-1 with peanut oil cake at 132 hours in a 3L growth chamber whereas our study reported a production of 50 µg mL^-1 at an incubation period of 72 hours.

CONCLUSION:

Our study attempted to screen and select efficient biosurfactant indigenous isolates from petroleum contaminated soil sources. The study successfully produced three potential isolates with one efficient for further investigation. Isolate APB3 which demonstrated a lead in all the assays performed and thus would be optimized for a mass scale biosurfactant production.

CONFLICT OF INTEREST:

The authors of the manuscript duly declare that there is no conflict of interest involved in the research work.

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Received on 04.04.2017 Modified on 17.04.2017

Research J. Pharm. and Tech. 2017; 10(6): 1697-1702.

DOI: 10.5958/0974-360X.2017.00300.6

SI.No;	Substrate	Hydrocarbon overlay test (zone formation in cm)			Crude oil spread method (zone formation )
SI.No;	Substrate	APB3	APB4	APB5	APB3	APB4	APB5
1	Petrol	0.63	-	0.27	-	-	0.53
2	Diesel	-	-	0.88	0.44	-	0.33