2.1. Isolation of hydrocarbonoclastic bacteria
100g of oil contaminated soil samples from different locations in harbor, Karaikal ONGC, Puducherry, India was aseptically collected in sterile containers and transported to the laboratory and processed immediately. 1g of each sample were serially diluted and 10− 4 dilution was spread on Bushnell Hass agar medium prepared in seawater supplemented with 1% crude oil as the only carbon source (Bushnell and Hass, 1941) and the plates were incubated at 30°C for 7 days. After incubation, morphologically different hydrocarbonoclastic bacterial colonies were streaked on Zobel Marine agar slants and used for further screening.
2.2. Screening for biosurfactant production
The six hydrocarbonoclastic isolates named as SJ1, SJ2, SJ3, SJ4, SJ5 and JS6 were further screened for biosurfactant production using different methods such as oil displacement test (Morikawa et al.,1993), drop collapse test (Youssef et al., 2004), BATH (bacterial adhesion to hydrocarbon) assay (Rosenberg et al., 1980) and emulsification index (EI24) (Cooper and Goldenberg. 1987) to select the most potent strain.
Oil displacement assay
20µl of crude oil was added to the surface of 25 ml of distilled water containing Petri plate followed by adding 10µl of cell free culture broth. If biosurfactant is present in the cell free broth; the crude oil will be displayed with the oil free clear zone is formed. The potent strain was selected based on the highest size of zone diameter.
Drop collapse test
A clean glass slide was covered with few drops of crude oil. After complete spread of the oil, a drop of cell free culture supernatant was added, the disintegration of the drop indicates positive result.
Determination of emulsification index
Emulsification index (E24) of the biosurfactant produced by the isolates was measured by mixing equal amount of cell free culture supernatant with crude oil and stirred well for 2 min then the height of the emulsification layer (EL) was measured. E24 is the ratio of the height of the EL and total height of the liquid (THL) after 24h of incubation. E24 = EL / THL×100.
BATH (Bacterial adhesion to hydrocarbons) assay
Initial optical density of cells in the mineral salt medium was determined initially at660nm using the method described by Rosenberg et al., 1980. 0.7ml of crude oil was mixed with 10 ml of suspension containing known density of cells (108 CFU/ml). After mixing for 15 min and let it stand for 30 min; the oil and water layer was allowed to separate. The aqueous phase was removed and OD was measured again. The percentage of bacteria adhered to the oil was calculated by using the formula:
Percentage (%) of bacterial adherence = [1-(OD shaken with oil / OD original)] x 100
2.3. Identification of potential biosurfactant producing SJ3 strain
Based on screening, the isolate SJ3 was selected as the most potent strain and it was identified using biochemical methods using Bergey’s Manual of Systematic Bacteriology (Bergey et al., 1974) and 16S rRNA sequencing based identification using universal Eu-bacterial primers 27F (5′AGAGTTTGATCCTGGC TCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). Sequence similarities were searched using BLAST. Phylogenetic tree was analyzed as described by Kumar et al., 2016 using MEGA software version 7.0.
2.4. Optimization and mass culture of P. stutzeri (SJ3)
Growth of the most potent biosurfactant producing P. stutzeri (SJ3) strain was optimized using one parameter at a time approach for different physicochemical parameters. As the biosurfactant production was found to be growth dependent, growth was estimated in this optimization processes. Emulsification index (E24) for the production of biosurfactant was estimated. The tested parameters were incubation periods 0–48 h(with 6 h interval), agitation (0-200 rpm), pH 4–10 (with 0.5 interval), temperatures (25, 30, 35, 37, 40 and 45°C), salinity (NaCl concentration − 0.5-3% with 0.5 interval), different carbon sources (sucrose, glucose, maltose, starch and cellulose); ideal carbon source glucose (0.5-3%) and nitrogen sources (beef extract, yeast extract, peptone, ammonium sulphate, ammonium nitrate and sodium nitrate); ideal nitrogen source yeast extract (0.5–2.5%) using crude oil as substrate (1%) were tested. Ideal conditions from the above optimization such as 36 h incubation, 150 rpm agitation, pH 7.5, 37oC, 1% salinity, 2% glucose as carbon source and 1% yeast extract as nitrogen source were used for mass culturing in a 1L conical flask containing 500 ml of medium.
2.5. Effect of different carbon sources on biosurfactant production by P. stutzeri SJ3
Apart from crude oil, seven different oil samples (1% each) such as diesel, petrol, vegetable oil, tamanu oil, peanut oil, sesame oil and Pongamia pinnata oil were tested as a sole carbon source with optimized conditions in 200ml of medium.
2.6. Recovery of biosurfactant
The cell free culture supernatant from each culture was obtained by centrifuging the culture at 12000 rpm for 20 min. followed by acid precipitation of the biosurfactant at pH 2 by adding 6N HCl and incubation at 4oC for overnight. The acidified biosurfactant precipitate was collected by centrifugation culture at 12000 rpm for 20 min. followed by neutralization using phosphate buffer (pH 7), extraction using equal volume of ethyl acetate and dried using a rotary vacuum evaporator and was tested for biosurfactant activity.
2.7. Antibacterial activity of the biosurfactant from P. stutzeri SJ3
The recovered each biosurfactant was tested for antibacterial against six different clinically important bacterial pathogens like Vibrio cholerae, Shigella boydii, Vibrio fluvialis, Shigelladysenteriae, Salmonella typhi and Salmonella paratyphi. Each strain was cultured in Muller-Hinton broth at 37oC for overnight and the cell density was adjusted to 108 CFU/ml (0.5 McFarland standard) and the activity was tested using well diffusion method on Muller-Hinton agar plates. 6mm wells were made with the sterile pipette tips. 50µl of crude cell free supernatant containing biosurfactant was loaded into the each well. Plates were incubated at 37oC for 18–24 hrs and the formation of clear zone around wells were measured and noted.
Fourier transform infrared (FT-IR) spectroscopy analysis of biosurfactant
The chemical structure and the types of functional groups (bonds) of the crude biosurfactant were determined using FT-IR analysis according to the method described by Bezza and Chirwa (2015). 1 mg of the crude biosurfactant was dried in a freeze dryer and ground with 100 mg of KBr and pressed for 30 sec. to get translucent pellets. Then it was analysed in a FTIR instrument (Thermo Niocolet, AVATAR 330 FT-IR system), with the spectrum range of 450–4000 cm− 1 at a resolution of 4 cm− 1. The data obtained from the analysis were corrected for the background spectrum.