Characterization of Biosurfactant From Pseudomonas Stutzeri SJ3 for Remediation of Crude Oil-Contaminated Soil


 In the present work, production of biosurfactant was studied from the bacterial strains isolated from the soil samples collected from oil contaminated sites in Karaikal ONGC, Puducherry, India. Six morphologically different hydrocarbonoclastic bacterial strains (SJ1-SJ6) isolated on oil agar plates were further screened for biosurfactant production. Based on the screening methods results of 26 mm oil displacement zone, positive results of drop collapse test, 68.14% emulsification index (E24) and 79.2% of bacterial adherence percentage, the isolate SJ3 was selected as the most potent strain and it was identified as P. stutzeri using standard biochemical and 16S rRNA gene sequencing-based methods. Optimization of the P. stutzeri strain showed 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 the ideal conditions for growth and the biosurfactant production was found to be growth dependent. The crude biosurfactant showed broad range of antibacterial activity against the bacterial pathogens tested. The P. stutzeri isolated from oil spill site showed biosurfactant with antibacterial activities.


Introduction
The revolution of modern industries and their activities has led the world affecting in many ways like soil and water pollution. Exploration, transportation, consumption, spills and disposal of petroleum hydrocarbons like crude oil, diesel and gasoline in the aquatic and terrestrial ecosystems are frequently contaminated by the petroleum hydrocarbon pollutants cause serious ecological problems (Okoh, 2006).
Even though mechanical and chemical methods reduce these issues to some extent, these practices are often expensive, time consuming and not ecofriendly (Mandri and Lin2007). Bioremediation is a method of choice to remove the hydrocarbon pollutants in the environment. Microbes produce surface active compounds as extracellular secretions or cell wall bound molecules (Plaza et al., 2006). Hydrocarbon degrading bacteria effectively degrade the hydrophobic substrates by the production of biosurfactants.
These microbial-derived biosurfactants are amphiphilic molecules produced by bacteria, fungi, yeasts and algae as extracellular component or on microbial cell surfaces. They have both hydrophobic and hydrophilic moieties that reduce the surface tension and interfacial tensions between the surfaces of the molecules and interface. The classes of the biosurfactants are mainly glycolipids, lipopeptides, lipoproteins, fatty acids, phospholipids and polymeric forms (Maneerat and Dikit, 2007) and they are mainly secreted as secondary metabolites and play vital role in the survival of the biosurfactant producer (Singh and Cameotra, 2004).
Biosurfactants are advantageous over its chemical counterpart like low toxicity, higher biodegradability and environmental compatibility and lower critical micelle concentration, easy production and synthesis from renewable sources, higher activities in various extreme conditions (Mukherjee et al., 2006). Biosurfactants are also having their applications in cosmetic, food, pharmaceutics, agriculture aspects (Makkar and Cameotra, 2002). Thus, the present study was on isolation of a potential biosurfactant producing bacterium from the oil spill site.

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.

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 ( 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 emulsi cation index
Emulsi cation 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 emulsi cation 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 (10 8 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. Identi cation of potential biosurfactant producing SJ3 strain Based on screening, the isolate SJ3 was selected as the most potent strain and it was identi ed using biochemical methods using Bergey's Manual of Systematic Bacteriology (Bergey et al., 1974) and 16S rRNA sequencing based identi cation 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.

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. Emulsi cation 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, 37 o C, 1% salinity, 2% glucose as carbon source and 1% yeast extract as nitrogen source were used for mass culturing in a 1L conical ask 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.

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 4 o C for overnight. The acidi ed 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 uvialis, Shigelladysenteriae, Salmonella typhi and Salmonella paratyphi. Each strain was cultured in Muller-Hinton broth at 37 o C for overnight and the cell density was adjusted to 10 8 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 37 o C 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.

Isolation of hydrocarbonoclastic bacteria
In the present work soil samples collected from the oil contaminated harbour sites in Karaikal ONGC, Puducherry, India was used for the isolation of hydrocarbonoclastic bacteria using oil agar plate. Six morphologically different colonies on the oil agar medium were further selected for the screening of biosurfactant production. As in the present work, several other has reported the production of

Screening for biosurfactant production
Screening for selection of potential biosurfactant producing isolate from the six hydrocarbonoclastic isolates named as SJ1, SJ2, SJ3, SJ4, SJ5 and JS6 were done using different screening methods like as oil displacement test, drop collapse test, BATH (bacterial adhesion to hydrocarbon) assay and emulsi cation index (E24). The results showed that the isolate SJ3 was the most potential one based on the following results; oil displacement showed 26mm of zone, positive for drop collapse test, 68.14% emulsi cation activity (E24) and BATH assay showed 79.2% bacterial adherence percentage and hence it was selected for further study. Emulsi cation activity is considered as one of the important criteria for the screening of biosurfactant producing strains (Rosenberg et al., 1979; Juwarkar and Khirsagar, 1991 and Carrillo et al., 1996). Molecules from microbe which exhibits emulsifying and high surface activity are classi ed as biosurfactants and these molecules are considered as potential bioremediation agents because of their surface and interfacial tensions reducing properties in both aqueous solutions and hydrocarbon mixtures (Banat et al., 2000). Thavasi et al., 2008 used BATH assay-based screening for the isolation of biosurfactant producers.

Identi cation of potential biosurfactant producing SJ3 strain
Based on the various biochemical methods and 16S rRNA gene sequencing based molecular method, the most of potential biosurfactant producing isolate SJ3 was identi ed as Pseudomonas stutzeri (Table 1 and Fig. 1)

Optimisation studies
Optimization revealed that the ideal conditions favored the maximum growth of the potential biosurfactant producing isolate P. stutzeri were 36 h incubation, 150 rpm agitation, pH 7.5, 37 o C, 1% salinity, 2% glucose as carbon source and 1% yeast extract as nitrogen source (Fig. 2). Tripathi et al., 2019 reported 96 h as the ideal incubation period for the growth of a rhamnolipid biosurfactant producing Marinobacter sp. MCTG107b strain. Bicca et al.,1999 observed the ideal conditions required for the production of a biosurfactant producing R. ruber AC 239 isolate were 37°C, 200 rpm, initial pH 7.0 and 1% Diesel (v/v). In the present study, biosurfactant production was found to be growth dependent. When different oil sources were tried for the production of biosurfactant using the SJ3 isolate crude oil was found to be best source of followed by diesel, petrol and pongamia oil, peanut oil, sesame oil and tamanu oil. Moshtaghet al. 2018 produced biosurfactant using brewery waste as the sole carbon source. Previous studies have reported the production of biosurfactant from Pseudomonas species; P. aeruginosa (Cooper et al., 1981

Recovery and FTIR analysis of biosurfactant
The recovery of biosurfactant from the mass culture medium was recovered by using acid precipitation 3.6. Antibacterial activity of the biosurfactant from P. stutzeri The crude biosurfactant was tested for the antibacterial activity; it showed a broad range of activity against the six bacterial pathogens tested. The biosurfactant obtained from crude oil as a substrate showed highest activity against the tested pathogens such as Vibrio cholerae (20 mm), Shigella boydii (18 mm), Vibrio uvialis (16 mm), Shigelladysenteriae (17 mm), Salmonella typhi (19 mm)and Salmonella paratyphi (21 mm) followed by the other sources of biosurfactants ( Fig. 3 and Table 2

Conclusion
The present study on production of biosurfactant from P. stutzeri SJ3 isolated from oil polluted sites ONGC, Karaikal, Puducherry. The growth optimization of the potent strain for biosurfactant production showed excellent productivity and antimicrobial activity against the tested bacterial pathogens. Hence, the strain can be used in bioremediation applications. Figure 1 Evolutionary analyses of Pseudomonas stutzeri and phylogenetic tree construction using neighbor-joining method Figure 2 Effect of various physicochemical parameters on growth of potent biosurfactants producing isolate P. stutzeri SJ3 Figure 3 Antibacterial activity of biosurfactants produced by P. stutzeri SJ3 against different bacterial pathogens (A-E)