Introduction

There is a world-wide concern about the liberation of hydrocarbon in the environment of both from industrial activity and accidental spills of oils. Most of the hydrocarbons are insoluble in water. Presence of these hydrocarbons in the environment is of considerable public health and ecological concern due to their persistence and toxicity. Different physical and chemical technologies are employed to clean the contaminated site such as excavation, thermal evaporation, soil flushing and soil vapour extraction¹. Surfactants are surface active substances which reduces the surface tension and increase spreading and wetting properties. Surfactants includes synthetic surfactants and biosurfactants². Majority of the synthetic surfactant production is from petrochemicals. These synthetic surfactants increase the level of environmental pollutions as they are hardly degraded by microorganisms and toxic in nature. Biosurfactants are surface active agents that are amphiphilic in nature which has hydrophilic region that consists of carbohydrate, phosphate, amino acid, alcohol or carboxylic acid and hydrophobic region that contain long fatty acid chain or hydroxyl fatty acids which reduce the surface and interfacial tensions between two immiscible liquids to form emulsions³. Biosurfactants can breakdown the hydrocarbon into other less toxic compounds and eventually to water and carbon dioxide. They have many advantages over synthetic surfactants such as lower toxicity, specificity, biodegradability and they can function in wide and extreme conditions and their bio compatibility⁴. Biosurfactants are synthesized by different microorganisms such as bacteria, yeast and fungi by utilizing hydrocarbons as carbon source.

Chemical composition and microbiological origin can be used to categorise different types of biosurfactants. High molecular weight biosurfactants, which show higher surface and interfacial tensions, and low molecular weight molecules, which show lower surface and interfacial tensions, are divided into two classes. Glycolipids like rhamnolipids, sophorolipids, lipopeptides, and phospholipids like gramicidens and subtilysin are examples of low-molecular-weight surfactants, whereas polymeric and particulate surfactants like emulisan, liposan, vesicles, and fimbriae are examples of high-molecular-weight surfactants.

Biosurfactants has many applications in cosmetics, food processing, pharmaceuticals, nanotechnology, textile industries and agriculture⁵. It has important role in pharmaceutical industry because antiviral, anti-adhesive, anti-microbial, spermicidal, hemolytic, anti-inflammatory and immune modulatory properties⁶. They have vital role in agriculture because it can be used for destroying plant pathogen and for increasing the availability of nutrients to the plants⁷. They have many applications in food industry such as solubilizing vegetable oils, improving the antimicrobial and antibiofilm properties and ensures emulsion of food during transport and storage⁸. In nanotechnology biosurfactants act as reducing agents, functionalization, emulsifier and provide self – assembly structures in encapsulation.

The aim of this research is to isolate and screen the native Bacillus sp that produces biosurfactants.  The biosurfactant was further characterized using, UV – Vis, TLC, FTIR and GC – MS analysis and compared with standard surfactin. 

Materials and Method

Collection of Soil Samples

Soil samples were collected from oil spilled surfaces of different automobile workshops and petrol pumps of Kerala and Tamil Nadu, India. Sample sites were dug around 5cm to collect the contaminated soil. Three samples were collected from Kalamassery, Aluva, Palakkad of Kerala, India. Another three samples were obtained from Eachnari,Coimbatore, Madukkarai of Tamil Nadu, India. The samples were collected in sterilized containers, well packed and were taken to the laboratory for analysis.

Isolation of Bacteria from Contaminated soil

The microorganisms of soil were isolated by serial dilution method and plated on nutrient agar and incubated at 37⁰C for 24 hrs.⁹.One gram of soil sample was suspended in 100mL. of distilled water and vortexed well. Each sample was serially diluted from 10^-1 to 10^-4. After serial dilution, pour plate technique was carried out to isolate bacterial cells colony.  The prominent colonies with different morphological characteristics were sub-cultured on nutrient broth (1.3g in 100mL distilled water) and incubated at 37⁰C for 24 hrs. and stored at 4⁰ C for further studies.

Morphological and Biochemical analysis of the isolates

The isolated bacterial cells were perceived with Gram staining under a compound microscope. (Unicon compound microscope). Bacterial cell’s shape such as bacilliwas observed and selected.  Biochemical analysis of the bacterial isolates such as Gram staining, Glucose Fermentation, Colonial Characters, Voges Proskauer, Starch Hydrolysis, Methyl Red Test, Catalase Activity, Lipid Hydrolysis, Indole Production, Casein Hydrolysis, Gelatin Hydrolysis, Spores were carried out. All biochemical assays were performed according to Bergey’s Manual of Systematic Bacteriology¹⁰.

Confirmation of Bacteria Using Specific Medium

Screening of the Bacillus sp was done on Starch agar (25g in 100mL), Casein agar (6.3g in 100mL) and Bacillus agar (4.1g in 100mL) medium. Bacterial strains were inoculated to differential medium by streaking method and incubated at 37⁰C for 24 hrs. Bacterial colonies which produced clear zone in starch agar and casein agar were inoculated in Hi Chrome Bacillus agar medium and incubated at 37⁰C for 24 hrs. for further confirmation of bacteria. After incubation, based upon the morphology and pigment production, Bacillus sp was confirmed and used for further studies¹¹.

Screening of Bacteria for Biosurfactant Production

The bacterial isolates were inoculated into 100 mL of Minimal Salt Medium (MSM) [ D-glucose 3g, NaNo₃ 0.4g, K₂HPo₄ 0.34g, KH₂PO₄ 0.06g, Yeast Extract 0.12g, (Himedia, Mumbai, India) Water 100mL] and supplemented with diesel (1%) w/v) as the carbon source and incubated at 37⁰C for 7days. After 7 days incubation in an orbital shaker, the extraction of Biosurfactant was done by centrifugation. Minimal Salt Medium (MSM) and Zobell medium can be used for the production of biosurfactant by Bacillus sp has proved in the studies of Saritha et al¹⁰.   The culture media were centrifuged at 8000 rpm for 10 min (Remi R-8C) and supernatant was collected for screening methods. This cell free culture broth was used for screening tests such as Hemolytic activity, Oil spreading assay, Emulsification ability assay, Bacterial Adherence to Hydrocarbon Activity, Drop collapse assay¹².

Hemolytic activity

Hemolytic activity is the preliminary screening test to recognize the biosurfactant producing bacteria. Hemolytic activity was performed using the procedure by Ewida& Mohamed¹³. Human blood (5%) was added to nutrient agar medium for preparing blood agar plates. Isolated bacteria were streaked in blood agar plates and incubated for 24 hrs. at 37⁰C. Then the bacterial colonies were visually analyzed for the presence of zone of inhibition. The presence of zones around the colonies indicated the presence of biosurfactants with hemolytic activity.

Oil spreading assay

Oil spreading assay was accomplished as narrated15. by Phulpoto et al¹⁴. with slight modifications. The hypothesis of this method is that biosurfactants can change the angle of reciprocal action of oil and water. 50 µL oil was added to the surface of 30mL of distilled water in a Petri plate (70x15mm) to form a thin oil layer. 5 µL of the culture supernatant are gently pipetted on the center of the oil coated thin film layer and after 5minutes, observations were recorded. If the biosurfactant activity is present, a clear zone will be produced by displacing oil. The diameter of zone corresponds the biosurfactant activity. The biosurfactant will produce a surface pressure to displace the oil. It is called as oil displacement activity. Distilled water without cell free supernatant was maintained as negative control, in which no clear zone was detected. 

Emulsification ability assay

The Emulsification ability was assayed by calculating the emulsion index as described by Saritha et al.¹², 500 µL oil was added to 1mL cell free supernatant and vortexed at high speed for 3minutes. Then kept undisturbed at room temperature for 24hrs.  After 24hrs the height of the stable emulsion layer and height of the liquid were determined. The emulsion index E was estimated as the percentage of the height of the emulsion layer after 24hours with the total height of liquid. Distilled water was maintained as negative control.

Bacterial Adherence to Hydrocarbon Activity

Bacterial adherence to hydrocarbons was assessed by measuring the hydrophobicity of cells as described by Ambrin et al¹⁵. The cell culture was centrifuged for 10000 rpm for 10min. (Remi R-8C). The cell pellets were collected and supernatant discarded. The pellets were washed twice with Phosphate buffer to eliminate the interfering solutes. Then cell pellets were resuspended in Phosphate buffer solution (pH 7.0). Optical Density of this suspension was measured at 600nm in a Spectro photometer. 50µL of oil was added to 1mL of cell suspension and vortexed for 3min. After vortexing, crude oil and aqueous phase were allowed to separate for 2hours. OD of the aqueous phase was measured with a UVSpectro photometer at 600nm. Hydrophobicity was calculated by following formula.

 % Bacillus adherence= [1- (OD of aqueous phase ÷ OD of the initial suspension)] x 100

Drop collapse assay

Drop collapse assay was carried out with a modified procedure of Rani et al¹⁶. Drop collapse assay explains about the destabilization of a liquid drop by biosurfactant containing cell free supernatant on the oil surface. Sterile Petri plates were coated with oil and stabilized for 1hr to make sure a consistent oil coating. 1 µL cell free supernatants were put onto the center of the petri plate. The reactions were observed visually with the help of a magnifying glass after 15min. If the drop of the supernatant was collapsed, the result was marked as positive. If the drop persisted beaded, the result was marked as negative. A negative control was kept with distilled water without biosurfactant.

Identification of Bacteria Using Molecular Techniques

After screening techniques, the potential biosurfactant producing cultures were selected for molecular techniques. Bacterial genomic DNA was isolated using Phenol Chloroform method¹⁷ and used as template to amplifying the 16S rRNA gene by Polymerase Chain Reaction (PCR). The specific primer 16SF (5’– AGAGTTTGATCMTGGCTCA G -3’) and 16SR (5’-ACGGYTACCTTGTTACGACTT-3’) was used to amplify the 16S rRNA. The PCR was run with Applied Biosystems Thermal Cycler by following thermal cycle; initial denaturation for 5 min at 94^o C, denaturation for 30 sec at 94^o C, annealing for 30 sec at 55 ^o C. and extension for 1.30 min at 72 ^o C and the final extension for another 5 min. at 72 ^o C. The cycles were repeated for 35 times. Amplified products were segregated in 1.5 % of agarose gel with 0.5 µg/ mL Ethidium bromide and was imaged using Biotech Submarine Gel System.

Amplified 16S rRNA fragments were purified and the products were sent to Chromus Biotech, Bangalore for sequencing analyses. The obtained16S rRNA gene sequences were uploaded in Basic Local Alignment Search Tool (BLAST) to determine the identity of the isolates based on gene sequence similarity. Phylogenetic tree and molecular evolutionary analyses were done using MEGA10 software. The obtained 16S rRNA gene sequences were submitted to NCBI Gene Bank for accession number.

Extraction and purification of biosurfactant

The extraction of biosurfactant was done using the acid precipitation procedure by Phulpoto¹⁴. The biosurfactant extraction was carried out by centrifugation (10,000rpm for 15minutes) of MSM broth culture. Cell free supernatant was precipitated by 6N HCl with pH 2.0. This was stored in refrigerator at 4⁰C for overnight.   Precipitated cell free supernatant was centrifuged at 10,000rpm for 15minutes (Remi R-8C) and obtained pellet was maintained at pH 7.0 by adding deionized water. The crude biosurfactant was retrieved from pelleted precipitates by adding methanol and chloroform (1:2 ratio) and vortexed for 5minutes. The mixture was kept undisturbed for 30minutes and collected the lower layer containing biosurfactants and evaporated to dry at 37⁰C for 1hour. This was used for further characterization of biosurfactants.

Biomass Estimation

The biomass was estimated by dry mass method as explained by Araujo et al³. Weight of empty Eppendorf tube was taken initially. 1mL crude broth culture was added to the same Eppendorf tube. These tubes were centrifuged at 10,000 rpm for 15 minutes (Remi R-8C). Supernatant was discarded and pellet was washed with distilled water and evaporated to dry by heating at 50⁰C. Then measured the weight of the Eppendorf tube with the pellet. Difference of empty tube’s weight and Eppendorf tube with pellet were taken for estimation of the biomass.  Cell biomass was estimated by the following formula

Cell biomass= [(mass of the tube with pellet – mass of empty tube) ÷2]x1000

Analytical Method

Thin Layer Chromatography

The preparatory characterization of the extracted biosurfactant was done with thin layer chromatography after calibrating the Retention factor value. The extracted biosurfactant (10µL) was loaded on TLC plate using capillary tubes and the biosurfactant was separated with the mobile phase of chloroform: Methanol: Water (60:15:2 – v/v/v). After three quarter movement of the sample, the plate was dried in room temperature and sprayed 1% ninhydrin for the clear visualization of biosurfactants. Commercial Surfactin from Merck, Mumbai was used as a positive control in TLC biosurfactant characterization. Retention factor (Rf) values of each sample was calculated by the following formula.

UV-Visible spectroscopy analysis

UV spectrophotometer is a widely used analytical technique to estimate the quantity of chemicals in the solution. The chemical’s reflective properties are based on the wavelength of radiated light. The extracted biosurfactants was further characterized by UV-Visible analysis (Labtronics LT2204 Spectrophotometer) in the range of 200-800nm with the scanning interval of 100mm¹⁸.

Fourier Transform Infrared Spectroscopy (FTIR)

Biosurfactants were analyzed using FTIR spectrum for the detection of various types of functional groups. It can be utilized to recognize the components of the unknown composition mixture. The FTIR spectrum was analyzed using dried sample with potassium bromide pellets by Shimadzu FTIR -8200 (Japan). About 1mg of dried biosurfactant was ground with 100mg of potassium bromide and pressed to produce transparent pellets.  Biosurfactant samples were placed directly under the infrared beam which were analyzed by FTIR for detailed structural analysis with a resolution of 4cm ^-1 in the spectral region of 4000 – 550 cm ^-1. The comparison studies were carried out with the standard Surfactin (Merck Mumbai).

Gas Chromatography – Mass Spectrometry Method

Gas chromatography – mass spectrometry (GC – MS) is an analytical method which combines the properties of gas chromatography and mass spectrometry to recognize the various substances with in a sample. 5% HCl methanol was assorted with 10mg of sample biosurfactant. 1mL of sterile water added to put out the reaction.  Methanol was added to retrieve the sample. 1µL of biosurfactant sample was placed into a gas chromatograph machine (Agilent). The carrier gas, Helium was utilized at the constant flow rate of 1.5mL min ^-1 and the GC injector’s functioning temperature was at 260 ⁰C. The ion source temperature was synchronized at 280 ⁰C. 90.67 minutes were the total GC running time which was scheduled by GC – MS analyst. The identifications of peaks in chromatographs by comparing with references of mass spectra and NIST08 mass spectral data base¹⁹.

Antimicrobial assay

The antimicrobial activity of the biosurfactant was measured using well diffusion method²⁰. The antibacterial activity was done against Gram positive and Gram-negative bacteria such as Paeruginosa, E coli, K pneumoniae, S aureus and antifungal activity against Aspergillus niger, Aspergillus terreus, Aspergillus flavus. Muller Hinton Agar medium (3.8g in 100mL) was prepared and 70µL of bacterial culture P aeruginosa, E coli, K pneumoniae, S aureus were spread. 5mm diameter agar well was prepared with the help of a sterilized stainless cork-borer. 10µL of extract was added to each well. Ampicillin disc (10mg) was kept as a positive control. DMSO (Dimethyl sulfoxide) was set as negative control and plates were incubated at 37⁰C for 24hrs. Diameter of zoneof inhibition indicates the antibacterial activity.

Malt extract agar medium (5g in 100mL) was prepared according to the manufacturer’s instructions (Hi media) and poured in petri plates. After solidification, 10mm diameter wells were made using sterilized stainless cork-borer. 80µL of the fungal spores were added to well and incubated at 30⁰C for 2-5 days.  5µL Fluconazole (antifungal drug) was kept as standard and DMSO was set asnegative control. Antifungal activity was evaluated by measuring the zones of inhibition in mm²¹.

Results and Discussion

Isolation and confirmation of bacteria from oil contaminated soil.

Six bacterial strains were isolated by serial dilution plate procedure from oil contaminated soil of different places of Kerala and Tamil Nadu, India. The result showed highest bacterial colony count (10.6 x 10⁵ CFU/mL) from Kalamassery, Kerala, India. and least bacterial colony count from Madukkarai (1 x 10³ CFU /mL.) Tamil Nadu, India, as it was the soil of a new petrol pump. In various previous studies such as Deblina et al²², Ambrin et al¹⁵, nutrient agar was used for the isolation of bacteria. In this study also we used nutrient agar for the isolation of bacteria from contaminated soil. Many researchers have isolated bacteria from soil. Ndibe et al²³ has isolated bacteria which produces biosurfactants from the river. Various bacteria such as Bacillus sp and Arthrobacter sp. were secluded from clayey soil reported by Prashanthi et al²⁴. Identical results were noted in the writings of Naif et al²⁵. The sampling site location was depicted (Fig.1). The individual viable bacterial cells in the soil sample multiplied to form an easily visible colony. The number of colonies counted and reported in Colony Forming Units (CFUs). The results of Colony Forming Units were represented in bar graph to visualize the number of colonies of different sampling sites easily (Fig.2).

Figure 1: Map of Sampling Site Click here to view figure

Biochemical analysis

The bacterial strains were identified as Bacillus sp using biochemical reactions and specific medium like Starch agar, Casein agar and Bacillus agar with the reference of Bergey’s manual. For further confirmation 16S rRNA study was carried out.  The colonial morphological characteristics of isolates were noticed as circular large slightly raised off white smooth colonies. According to cellular morphology, the isolates were Gram positive endospore forming rod shaped cells. Previous literatures such as Ndibe et al²³, Anuraj et al¹² revealed that Bacillus sp has these morphological and cellular characteristics. The same features of Bacillus sp. were observed in the finding of Prashanthi et al²⁴.  Ndlovu et al²⁶ utilized Bacillus amyloliquifaciens for biosurfactant production. The biochemical results were outlined (Table1).  These identical characteristics and recognition clues were discussed as Bacillus sp in the paper of Santos, E. C. L et al ²⁷.

Table 1: Biochemical analysis of Microorganisms

Screening of Bacteria for biosurfactant production

All the bacterial strains were screened for identifying the bacteria which is more capable to produce biosurfactants.  The strains which showed superior results were selected for further studies. The Bacillus sp showed extremely positive results for various screening tests as mentioned by Deblina et al.²² Saravanan & Vijayakuamar²⁸ reported that more screening procedures should incorporate in screening techniques to recognize the capable biosurfactant producers.  John Paul et al⁹detailed that Bacillus sp. isolated from oil contaminated soil are proficient to produce biosurfactant.

Hemolytic activity

The results of hemolytic activity of three samples were positive (Fig.3). They showed a transparent zone formation around the colonies. The sample B1 and B2 exhibited the topmost hemolytic activity as 2cm. zone. Two samples B4 & B5 were negative, as they didn’t show any zone formation. The positive activity of hemolysis by various Bacillus strains were described by Ambrin et al¹⁵. The hemolytic study of Bacillus by Rajesh et al²⁹. revealed the identical results. NDIBE et al²³. suggested that biosurfactant producing organisms can be screened by hemolytic activity. The present result of hemolytic activity was authenticated with the result of M. Garg et al³⁰. The organisms which produce biosurfactants will lyse the erythrocytes and produce a transparent zone on all sides of colony. Ewida& Mohammed¹³.  reported that the transparent zone of hemolysis is equivalent to the biosurfactant concentration. Based on the literature of Dhouha et al³¹.2012, Lipopeptide and Rhamnolipid biosurfactants can be screened by hemolytic assay.

Figure 3: Hemolytic activity. Click here to view figure

Oil spreading assay

Oil spreading assay is more precise and less time-consuming method to disclose the biosurfactants. There is a straight relationship between the diameter of zone and the presence of biosurfactants¹⁰. The existence of biosurfactants persuade clear zone formation by oil displacement. The supernatants of all bacterial strains were added to the plates containing oil. All the strains except sample 5 were positive as they expelled the oil to form a clear zone (Fig.4). Based upon the article of Chikodili et al¹⁹,the oil spreading positive results indicate that the isolates can produce biosurfactants efficiently. Parthipan et al³².  detailed that the efficiency of oil spreading assay is used to evaluate the production of biosurfactant. The greatest oil displacing activity indicated by sample B1. The sample B5 specified a negative activity result. Similar results were illustrated by Thavasi et al³³. Saritha et al¹⁰ detailed that biosurfactant produced by Bacillus sp are potential to produce oil displacement clear zone.

Figure 4: Oil Spreading Assay. Click here to view figure

Emulsification ability assay

It is an indirect technique employed to screen the biosurfactant production. If the cell free supernatant consists of biosurfactants, it will form emulsion with the hydrocarbons which carry in the test solution. When a liquid phase is diffused in a continuous liquid phase in the form of droplets, there will be a formation of amalgam. Emulsification ability assayed with cell free supernatant and two organic solvents such as petrol and coconut oil. The emulsification index E is measured as the correlation of height of the emulsion with the liquid’s total height. Bacterial strain (B2) showed more emulsification index in petrol (Fig.5). The outcome acquired from this procedure are thoroughly supported by the description given by M. Garg et al³⁰. The present result of emulsification assay was verified with the literature of Magalhaeset al³⁴. The potential of biosurfactants can be determined by emulsification ability. Shoeb et al³⁵ narrated emulsifying ability can decide   the yield of bio-emulsifier. Saritha et al¹⁰ justified that biosurfactant has high emulsification ability

Figure 5: Emulsification ability Assay. Click here to view figure

Bacterial adherence to hydrocarbons activity

The positive cell hydrophobicity described as the sign of biosurfactant production. Cell adherence with hydrocarbon such as petrol is observed as an indirect method to screen biosurfactant producing bacteria. Biosurfactant producing cells can affix themselves with hydrocarbons. The ability of bacterial cells to adhere to hydrocarbons were tested using BATH assay. The results were noted down (Table 2). The sample B1 showed highest cell adherence. BATH assay revealed positive results which are close to the studies detailed by Arif et al³⁶. Strains of Bacillus sp has cell adherence to crude oil which is corroborated with the findings of Ambrin et al¹⁵. Thavasi et al³³ validated that the positive results of cell hydrophobicity are the sign of biosurfactant production. Anuraj et al¹² authenticated that Bacillus sp has the highest cell adherence compared to other bacteria such as E coli and Staphylococcus.

Table 2: BATH assay

Drop collapse assay

Drop collapse assay is a liable test and it is not a complicate test to perform. A little amount of cell free broth is needed for this test. The bacterial strains which are potential to generate extracellular biosurfactants indicate the positive result. The results acquired by drops collapse assay was accurate and authentic when compared to the result recorded by Ndibe et al²³. The drop collapse assay is based on the disruption of liquid drop by biosurfactant containing cell free bacterial extract. The positive collapse test assures the surface activity against oil. If the supernatant contains surfactants, the drops of liquid will collapse as interfacial tension between liquid and hydrophobic surface was decreased. All the bacterial strains except sample 5 showed positive result (Table 3). Collapsed drop approved the presence of biosurfactant in the sample as suggested by Rani et al¹⁶. Biosurfactant produced by Pseudomonas also showed positive result for drop collapse assay correlate the findings of Sumathi et al¹. Arif et al³⁶ has done research in biosurfactant production by Gordonia sp which work also disclosed that biosurfactant can collapse the drops as interfacial tension decreases.  Bacillus drop can collapse within 58 seconds which is explained by Anuraj et al¹²., that is verifying current study. The triumphant collapse test guarantees the extracellular production of biosurfactant and destabilization cum capability as mentioned by Ewida& Mohammed¹³.

Table 3: Drop collapse assay

“+ + + “- Excellent, “+ + “- Good, “+ “   – Fair, “ – “-  Absent

Biomass estimation

The dry mass estimation normally gives a much more reliable result than the wet weight. The cell biomass was estimated by taking the difference of empty tube’s weight and the Eppendorf tube with pellet. Day 4 showed more cell biomass for all 3strains (Fig. 6). The biomass was determined using dry mass method which was adopted from Araujo et al³. The growth of bacterial cells can be tracked by computing the cell biomass³⁷. Dry weight of biosurfactant almost close to the result which was narrated by Anuraj et al¹².

Figure 6: Biomass Estimation. Click here to view figure

Identification of bacteria using molecular techniques

After screening and preliminary identification, DNA of selected bacterial strains were extracted (Fig.7) and 16S rRNA was amplified using Polymerase Chain Reaction (PCR). 1.5 % of Agarose gel with 0.5 µg /mL. Ethidium Bromide was used to verify the PCR products which showed the fragment of 760 base pairs (Fig.8). The amplified 16S rRNA of B2 was purified and performed 16S rRNA gene sequence. Based on BLAST analysis, B2 isolate showed maximum identity 99%. The phylogenetic tree was constructed using MEGA 10 software (Maximum likelihood tree) with 0.1 distance. The Phylogenetic tree for bacterial isolate is shown (Fig.9) Santos E.C.L. et al²⁷. has outlined that biosurfactant producing bacteria as Bacillus sp by molecular characterization and phylogenetic tree as in the present study. The molecular analysis done by Nurul et al³⁸. showed that 16S rRNA gene sequence and phylogenetic studies of genus Bacillus, which is more or less similar to the current research work. In hydrocarbon contaminated soil, the presence of Bacillus sp and their molecular studies were discussed in the literature of Aabed et al³⁹. which corresponds current study. In previous research, molecular study of bacteria from soil by John et al⁹ revealed that bacteria as Bacillus sp. The gene sequences were submitted to National Center for Biotechnology Information (NCBI) for accession number. Accession numbers were granted from NCBI as M N 243657 – Bacillus sp.

Figure 7: DNA of Bacillus spLane 1: DNA ladder ( 2kb) Lane 2: Bacillus sp (760 bp) Click here to view figure

Figure 8: PCR Product. Click here to view figure

Figure 9: Phylogenetic Analysis of Bacillus sp. Click here to view figure

Analytical methods

Thin Layer Chromatography

The crude biosurfactant was scrutinized on the TLC plate that suggested the presence of biosurfactant with lipopeptide nature. This could hypothesis that the extract has different composition. The Rf value of derived biosurfactant was measured as 0.68 in TLC chromatogram (Fig.10). The similar Rf value of the lipopeptide biosurfactants were reported by Joy et al⁴⁰. This preliminary profile revealed that the extracted biosurfactant has the similarities with the standard surfactin (0.71Rf value). Ramyabharati et al⁴¹ also reported similar results with biosurfactants produced by Bacillus sp while contrasting with standard surfactin. Rf value was calculated by the ratio of distance travelled by solute and distance travelled by solvent. Lipopeptide biosurfactant can be explored using ninhydrin as red spots which was outlined by Ndibe et al²³. The derived biosurfactant from Bacillus sp is closely associated to lipopeptide type according to Rf value data base. The similar findings were observed by the report of MBS Donio et al⁴².Sumathi et al¹reported that biosurfactant such as rhamnolipids produced by Pseudomonas shows yellow spots in TLC. Okore etal⁴³. authenticated that lipopeptide biosurfactant produced by Bacillus sp will develop red spot while spraying Ninhydrin reagent in TLC plate. Biosurfactant with Rf value 0.68 was recorded in previous literature of Deepansh et al⁴⁴.Surfactin and Iturin by Bacillus subtilis were corroborated on TLC by Phulpoto et al¹⁴ with the Rf value of 0.3and 0.75 respectively.

Figure 10: TLC Analysis of Biosurfactant with surfactin. Click here to view figure

UV-Visible spectroscopy analysis

The production of biosurfactants were confirmed by UV-Visible spectrophotometer. UV-Vis Spectroscopy is a quantitative technique used to measure how much a chemical substance absorbs light. The existence of certain type functional groups can be checked by UV- Vis spectroscopy (Fig.11). The extracted biosurfactant showed maximum peak for Bacillus sp at 415nm.The UV absorptions at 340nm revealed the presence of azo chromophore with n- π* transition. The peak of absorption at 375nm indicates the existence of arene group such as anthracene with n – π* transition. The results revealed that the biosurfactant is sizeable with complex molecule which consist of many functional groups¹⁸ Dharman et al⁴⁵ published that biosurfactant from Bacillus safensis displayed 530nm of maximum absorption.

Figure 11: UV analysis of biosurfactant Click here to view figure

Fourier Transform Infrared Spectroscopy (FTIR)

The extracted biosurfactant of Bacillus sp had extreme peak at 3340 cm ^-1 which correlates -OH groups. The existence of this peak denotes that sample carries alcohol group in agreement with M Garg et al³⁰.Peaks representing C=C alkene group was received at wave number 1635 cm^-1. This peak indicates the sample comprises peptides. Another vibration at 1211 cm^-1 approved the presence of glycosidic bond C-O-C ether groups¹⁸. The absorption frequency at 686 cm^-1 and 601 cm^-1 specifies the presence of C-Cl group³⁷. The C-Br stretching vibration at 563.21cm^-1 designated halogen compound. The presence of carbon iodine bond was revealed by the peak at 401 cm^{-1 42}(Table 4).  The peaks produced by biosurfactants were compared to the corresponding functional groups. The characteristic vibrations of stretching peaks indicated the presence of lipopeptide biosurfactant⁴⁵. The identical functional groups were disclosed in previous works done by M Garg et al³⁰.  The characteristics of biosurfactants was assessed by contrasting the IR spectra of sample to commercial surfactin⁴⁶. (Fig.12)

Table 4: Functional groups identified by (FTIR)

Gas Chromatography – Mass Spectrometry

The GCMS characterization further exposed that biosurfactant produced by Bacillus sp was lipopeptide in nature. After performing GC -MS of biosurfactant, the great peaks were monitored at various retention time of 12.75, 10.22, 4.98, 3.87 respectively. The evaluation of biosurfactant fraction exhibited the existence of a prime peak at 12.75 retention time. Fatty acid nature compounds such as methyl ester, carboxylic acid were the vital compounds in the sample which was similar to the result of Parthipan et al³². The compounds such as methyl ester, silane, 3-Quinoline carboxylic acid, methyl- dodecyl ester which were present in the sample favour tenso-active property for the biosurfactant. Methyl ester compounds are long chain fatty acids derived from vegetable oil or animal fats which has magnificent biosurfactant properties are easily bio degradable in nature. Dimethyl silane diol is used as a surface modification regent such as hydrophobization of oxidized silicon and oxidized metal surfaces. Silane is used to increase resistance to abrasions as well as increased adhesion thermal stability and cross linking in paints, inks and coatings. Dharman et al⁴⁵ disclosed the occurrence of fatty acids such as 3- hydroxy decanoic acid in rhamnolipid biosurfactant. GCMS analysis of biosurfactant from soil bacteria which degrade oil was carried out by Santhakumar et al⁴⁷.Parthipan et al³² was disclosed that Bacillus subtilis can produce lipopeptide biosurfactant. Bacillus sp produce lipopeptide nature biosurfactant was detailed in the study of Deshmukh et al⁴⁸. Retrieval of these constituents such as 2 trimethyl silyl ethanol, 2 methoxy -5-phenol of biosurfactant produced by Bacillus sp Lv13 was narrated by Yinghi et al⁴⁹. Halophilic Bacillus sp produced biosurfactant consist of glycolipids which was detailed by MBS Donio et al⁴².Santhakumar et al⁴⁷ summarized that biosurfactant extracted from bacteria hydrocarbon contaminated soil corresponds to fatty acids and fatty acid esters. The major functional compounds recognized by GCMS analysis were inscribed (Table5).

Table 5: Major functional compounds recognized by GCMS analysis.

Antimicrobial activity

The antimicrobial activity of biosurfactants against various groups of pathogenic microorganisms has attained more consciousness in the latest years. Antimicrobial assay of biosurfactants was carried out using well diffusion method against bacteria such as P aeruginosa, E coli, K pneumoniae, S aureus and fungi Aspergillus niger, Aspergillus terreus, Aspergillus flavus. The extracted biosurfactant showed antimicrobial activity in contrast to food borne pathogens. In our recent study, biosurfactants from Bacillus sp exhibited inhibitory results against fungal and bacterial human pathogens. The elevated antibacterial activity was noticed in K pneumoniae when compared with commercial surfactin. The greatest antifungal inhibition zone observed against Aspergillus terreus..The zone of inhibition against clinical pathogens were specified (Fig.13 &14) The biosurfactant from Bacillus sp exhibited good inhibitory zone against Gram negative and Gram-positive bacteria (Fig.15 &16). These results are in accordance to pioneer reports by Sharma et al⁴⁶.  Prasanthi et al²⁴ reported that soil bacteria have antibacterial activity opposed to pathogenic bacteria. Lipopeptide biosurfactants from Bacillus sp has antibacterial, antifungal and antiviral activity which was revealed by the study of MBS Donio et al⁴².M Garg et al³⁰ noted that refined biosurfactant from Candida parapsilosis displayed antimicrobial activity. The study by Araujo et al³ signified that biosurfactant of which comes under Rhamnolipids has antifungal activity.

Figure 13: Antifungal Activity of Biosurfactant with commercial surfactin against human pathogens. Click here to view figure

Figure 14: Antibacterial Activity of Biosurfactant with commercial surfactin against human pathogens. Click here to view figure

Figure 15: Antibacterial Activity. Click here to view figure

Figure 16: Antifungal Activity Click here to view figure

Conclusion

In the present study, six bacterial strains from hydrocarbon contaminated soil of Kerala and Tamil Nadu, India were screened for potential biosurfactant producer. The capable isolate was identified by 16S rRNA analysis as Bacillus sp(MN243657). The nature of biosurfactant was characterized by TLC, UV, FTIR and GC-MS analysis and concluded as Lipopeptide nature. The extracted biosurfactant has promising antimicrobial activity. This bacterial strain can be used for bioremediation of contaminated sites. This can be utilized in pharmaceutical industry as it has antimicrobial activity. Further research on vital applications of this biosurfactant are recommended.

Aknowledgement

The authors of this paper wish to acknowledge Centre for Bioscience and Nanoscience Research (CBNR), 461/ 3, Amman Nagar, Near Chandran Steels, LT BY Pass Road, Eachanari, Post, Coimbatore, Tamil Nadu 641021 for the assistance and well-equipped facilities for the completion of entire study.

Conflict of Interest

The authors declared that there is no conflict of interest in this research paper.

References 

1. R.Sumathi & N.Yogananth. Isolation and identification of Biosurfactant producing Pseudomonas aeruginosa from marine sediment samples and its Antimicrobial Properties. Int. J. Adv. Res. Biol. Sci.2016; 3(12): 200-212.
2. Mariela Bustamante., Nelson Duran & Cristina Diez. Biosurfactants are useful tools for the bioremediation of contaminated soil. J. Plant Nutr. Soil Sci. 2012; 12(4):667-687.
3. Jaciara S. de Araujo., Juliene da C. Rocha., Marcos Antonio Oliveira Filho., Vitor Troccoli Ribeiro., Luan T. C. deP. Vasconcelos., Nathalia K. de Araujo., Eduardo L. de B. Neto &Everaldo Santos. Production of Rhamnolipids by Pseudomonasaeruginosa AP029-GLVIIA and Application on Bioremediation and as a Fungicide.Biosci. Biotechnol. Res. Asia.2020; 17(03):467-477.
   CrossRef
4. P. Singh., Y. Patil & V. Rale. Biosurfactant production: emerging trends and promising strategies. Appl. Microbiol. 2019; 126: 2-13.
   CrossRef
5. Thiago R. Bjerk ., Patricia Severino., Sona Jain., Conrado Marques., Amélia M. Silva., Tatiana Pashirova& Eliana B. Souto. Biosurfactants: Properties and Applications in Drug Delivery, Biotechnology and Ecotoxicology.Bioengineering 2021; 8(8):115.
   CrossRef
6. Constantina Eleni Drakontis&SamiulAmin. Biosurfactants: Formulations, properties, and applications. COCIS. 2020; 48:77-90.
   CrossRef
7. Fakruddin Md. Biosurfactant: production and application. J. pet. environ. biotechnol.2012; 3(4):12
8. S. Vijayakumar & V. Saravanan. Biosurfactants types sources and applications. Res. J. Microbiol. 2015; 10:181-192.
   CrossRef
9. John Paul Matthew D., Guzman Alba., Jeun Marianne T., Torres & Mark Louie S. Isolation, screening & characterization of biosurfactant-producing Bacillus sp. from soil and their potential biofilm inhibitory activities against Pseudomonas aeruginosa. J. Microbiol. Biotechnol. Food Sci.2020; 10 (2):245-248.
   CrossRef
10. Saritha Pendyala& Madhuri Kulkarni. Screening, Isolation and Molecular Identification of Biosurfactant Producing Bacteria from Rhizosphere Soil Samples. Nat. Volatiles & Essent. Oils. 2021; 8(6): 702-711.
11. Dr. R Ananthanarayan&Dr. CK Jayaram Paniker. Textbook of Microbiology.2020.
12. AnurajNayarisseri., Poonam Singh & Sanjeev Kumar Singh. Screening, isolation and characterization of biosurfactant producing Bacillus subtilis strain ANSKLAB03. Bioinformation. 2018; 14(6): 304-314.
    CrossRef
13. Ayman Youssef Ibrahim Ewida&Walaa Salah El-Din Mohamed. Isolation and characterization of biosurfactant producing bacteria from oil-contaminated water. Biosci. Biotechnol. Res. Asia. 2019;16(4):833-841.
    CrossRef
14. Irfan Ali Phulpoto., Zhisheng Yu., Bowen Hu., Yanfen Wang., Fabrice Ndayisenga., Jinmei Li., Hongxia Liang & Muneer Ahmed Qazi. Production and characterization of surfactin-like biosurfactant produced by novel strain Bacillus nealsonii S2MT and it’s potential for oil contaminated soil remediation. Microb Cell Fact. 2020; 19:145.
    CrossRef
15. Ambrin Sarwar., Gunter Brader., Erika Corretto., GajendatAleti., Muhammad Abaidullah., AngelamSessitsch& Fauzia Yusuf Hafeez. Qualitative analysis of biosurfactants from Bacillus spexhibiting antifungal activity. PLoS One. 2018; 13(6):1-15.
    CrossRef
16. Mamta Rani., Joel T. Weadge&SuhaJabaji. Isolation and Characterization of Biosurfactant-Producing Bacteriafrom Oil Well Batteries with Antimicrobial Activities Against Food-Borne and Plant Pathogens. Front. Microbiol. 2020; 11:1-17.
    CrossRef
17. Mitchell Henry Wright., Joseph Adelskov and Anthony Carlson Greene. Bacterial DNA Extraction Using IndividualEnzymes and Phenol/Chloroform Separation. J MicrobiolBiol Educ.2017; 18(2): 18-48.
    CrossRef
18. Ekramul Haque., Mohd Aamir Bin Riyaz & Sriram Shankar Saqib Hassan. Physiochemical and structuralcharacterization of biosurfactant produced by halophilic pseudomonas aeruginosa ENO14 isolated from seawater. Int. J. Pharm. Investig. 2020; 10(4):437-444.
    CrossRef
19. Chikodili G. Anaukwu., Chioma M. Ogbukagu& Ikechukwu A. Ekwealor. Optimized Biosurfactant Production byPseudomonas aeruginosa Strain CGA1 Using Agro-Industrial Waste as Sole Carbon Source. J. adv. microbiol.2020; 10:543-562.
    CrossRef
20. JesteenaJohney., S. Radhai Sri & R. Ragunathan. Extraction of Chitin and Chitosan from Wild Type PleurotusSpp and its Potential Application – Innovative Approach. J Pure ApplMicrobiol. 2018;12(3):1631-1640.
    CrossRef
21. JesteenaJohney., Kannan Eagappan& R.R Ragunathan. Microbial extraction of chitin and chitosan from Pleurotusspp, its characterization and antimicrobial activity. Int. J. Curr. Pharm. 2017; 9(1):88-93.
    CrossRef
22. Deblina D., Namrata M, Taishee P., Veena S &Bhaskara Rao K.V. Evaluation of Biosurfactant producing activity of Bacillus thuringiensis-VITNDUVNB04 isolated from oil contaminated soil by various methods. Research J.Pharm. and Tech. 2016; 9(12):2186-2190.
    CrossRef
23. Ndibe., Eugene & Usman. Screening of biosurfactant-producing bacteria isolated from river Rido, Kaduna, Nigeria. Appl. Sci. Environ. Manage. 2018; 22(11): 1855–1861.
    CrossRef
24. R. Prashanthi., Shreevatsa G K. Krupalani S & Manoj L. Isolation, characterization and molecular identification of soil bacteria showing antibacterial activity against human pathogenic bacteria. J Genet EngBiotechnol. 2021; 19:1-14.
    CrossRef
25. Naif Abdullah Al-Dhabi., Galal Ali Esmail &MariadhasValanArasu. Enhanced Production of Biosurfactant from Bacillus subtilis Strain Al-Dhabi-130 under Solid-State Fermentation Using Date Molasses from Saudi Arabia for Bioremediation of Crude-Oil-Contaminated Soils. J. Environ. Res. PublicHealth. 2020; 17(22):1-20.
    CrossRef
26. Thando Ndlovu., Marina Rautenbach., Johann Arnold Vosloo., Sehaam Khan &Wesaal Khan. Characterisation andantimicrobial activity of biosurfactant extracts produced by Bacillus amyloliquefaciens and Pseudomonas aeruginosa isolated from a wastewater treatment plant. AMB Express. 2017; 7: 1-19.
    CrossRef
27. Elane Cristina Lourenço Dos Santos., Daniele Alves Dos Rela Miranda., Amanda Lys Dos Santos Silva & Ana Maria Queijeiro Lopez. Biosurfactant production by Bacillus strains isolated from sugar cane mill wastewaters. Braz. Arch. Biol. Technol. 2019;62: 1-12.
    CrossRef
28. V. Saravanan & S. Vijayakumar. Isolation and screening of biosurfactant producing microorganisms from oil contaminated soil. J. Acad. Indus. Res. 2012; 1(5) :264-268.
29. M. Rajesh., M. Samundeeswari& B. Archana. Isolation of biosurfactant producing bacteria from garbage soil. Appl. Environ. Microbiol. 2017; 5:74-78.
    CrossRef
30. Mayank Garg., Priyanka & Mary Chatterjee. Isolation, characterization and antibacterial effect of biosurfactant fromCandida parapsilosis. Biotechnol Rep. 2018; 18:1-6.
    CrossRef
31. DhouhaGhribi., LobnaAbdelkefi-Mesrati., Ines Mnif., RadhouanKammoun., ImenAyadi., ImenSaadaoui., SamehMaktouf&SemiaChaabouni-Ellouze. Investigation of antimicrobial activity and statistical optimization of Bacillussubtilis SPB1 biosurfactant production in solid-state fermentation. J.BiomedBiotechnol. 2012; 10:1-12
    CrossRef
32. PunniyakottiParthipan., Elumalai Preetham., Laura Machuca., Pattanathu K S M Rahman., KadarkaraiMurugan&Aruliah Rajasekar. Biosurfactant and Degradative Enzymes Mediated Crude Oil Degradation by Bacterium Bacillussubtilis A1. Bio-corrosive microbial communities in petroleum facilities. Front. Microbiol. 2017; 8:1-12.
    CrossRef
33. RengathavasiThavasi., Shilpy Sharma &Singaram Jayalakshmi. Evaluation of screening methods for the isolation of biosurfactant producing marine bacteria. J. pet. environ. biotechnol. 2017; 2:1-6
34. EmiliannyRafaely Batista Magalhães., FrancinaldoLeite Silva., Magna Angélica dos Santos Bezerra Sousa &EveraldoSilvino Dos Santos. Use of Different Agroindustrial Waste and Produced Water for BiosurfactantProduction. Biosci Biotech Res Asia. 2018;15(1):17-26.
    CrossRef
35. ErumShoeb., Nuzhat Ahmed., Jameela Akhter., UzmaBadar., Khaizran Siddiqui., Faiza A. Ansari., Maheen Waqar., Samina Imtiaz., Nimrah Akhtar., Quratul Ain Shaikh., RoaunqBaig., Saba Butt., Saema Khan., Sidra Khan., Shahid Hussain., Bashir Ahmed& Maqsood A. Ansari. Screening and characterization of biosurfactant-producing bacteria isolated from the Arabian Sea coast of Karachi. Turk J Biol.2015; 39:210-216
    CrossRef
36. ArifNissarZargar., Sarthak Mishra., Manoj Kumar &Preeti Srivastava. Isolation and chemical characterization of the biosurfactant produced by Gordonia sp. IITR100. PLoS One. 2022;17(4):1-27.
    CrossRef
37. Aparna A., G. Srinikethan& Smitha Hedge. Isolation, Screening and Production of Biosurfactant by Bacillus clausii 5B. Res. J. Biotechnol.2012; 3(2): 49-56.
38. Nurul Hanisah Md Badrul Hisham., Mohamad Faizal Ibrahim., Norhayati Ranti &Suraint Abd-Aziz. Production of biosurfactant produced from used cooking oil by Bacillus sp. HIP3 for heavy metals removal. Molecules. 2019; 24(14): 1-16.
    CrossRef
39. KawtherAabed., Abeer Almutairi., Alaa Al-Shwuair., Amal Al-Otaibi., Arwa Alhazzani., Areej Al-Shbi., Hind Al-Moegelth., Lama Al-Assaf &Sultanah Al-Omri. Diversity and distribution of thermophiles and thermotolerant bacteria in the soil samples obtained from different regions in Saudi Arabia. Biosci Biotech Res Asia. 2021;18(1):163-172.
    CrossRef
40. Sam Joy., Pattanathu K.S., M.Rahman& Shashi Sharma. Biosurfactant production and concomitant hydrocarbon degradation potentials of bacteria isolated from extreme and hydrocarbon contaminated environments. Chem. Eng. J.2017; 317:232-241.
    CrossRef
41. Ramyabharathi S., Meena KS., Rajendran L., Karthikeyan G., Jonathan E &Raguchander T. Biocontrol of wilt-nematode complex infecting gerbera by Bacillus subtilis under protected cultivation. Egypt J Biol Pest Co. 2018; 28:186-190.
    CrossRef
42. M B S Donio., S F A Ronica., V Thanga Viji., S Velmurugan., J Adlin Jenifer., M Michaelbabu& T Citarasu. Isolation and characterization of halophilic Bacillus sp. BS3 able to produce pharmacologically important biosurfactants. Asian Pac J Trop Med. 2013;6(11):876-883.
    CrossRef
43. OkoreChioma., MbanefoOgechukwa., Onyekwere Bright., Oneywenjo Simon & Abba-Father Chinyere. Isolationand characterization of biosurfactants producing bacteria from oil polluted soil. Asian Pac J Trop Med. 2013;6(11):876-883.
44. Deepansh Sharma., Baljeet Singh Saharan., Nikhil Chauhan., Anshul Bansal & Suresh Procha. Production and structural characterization of Lactobacillushelveticus derived biosurfactant. Sci. World J. 2014; 14:548-557.
    CrossRef
45. DharmanKalaimurugan., Balasubramanian Balamuralikrishnan., Rasiravathanahalli., KaveriyappanGovindarajan.,Naif Abdullah Al-Dhabi., MariadhasValanArasu., ChithravelVadivalagan., Srinivasan Venkatesan., HesamKamyab., ShreeshivadasanChelliapan&ChartchaiKhanongnuch. Production and Characterization of a Novel Biosurfactant Molecule from Bacillus safensis YKS2 and Assessment of Its Efficiencies in Wastewater Treatment by a Directed Metagenomic Approach. Sustainability.2022; 14:1-20.
    CrossRef
46. Deepak Sharma., Mohammad Javed Ansari., Sonam Gupta Ahmad Al Ghamdi., ParulPruthi&Vikas Pruthi. Structural Characterization and Antimicrobial Activity of a Biosurfactant Obtained from Bacillus pumilus DSVP18 Grown on Potato Peels. Jundishapur J Microbiol. 2015; 8(9):1-8.
    CrossRef
47. Santhakumar., Muthukamalam., SivalingamSivagangavathi., DharmapalDhrishya&Sadras Sudha Rani. Characterization of dioxygenases and biosurfactants produced by crude oil degrading soil bacteria. Braz J Microbiol. 2017; 48(4):637-647.
    CrossRef
48. Deshmukh C., Jagtap C. B., Titus S. & Kumar P. Isolation and characterization of fatty acid esters and phosphatidylethanolamine surfactants from a consortium of marine bacteria. Indian J. Geomarine sci. 2012; 41: 398–404.
49. YinghaiLyu., Tingting Zhang., Baojuan Dou., Guijiang Li., Chengxin Maa &Yangyang Lia. A lipopeptidebiosurfactant from Bacillus sp. Lv13 and their combined effects on biodesulfurization of dibenzothiophene. R. Soc. Chem. 2018; 8: 38787–38791.
    CrossRef