Sample collection
Ten marine water samples were collected from hydrocarbon-contaminated sites within the Ennore Harbor, located along the Coromandel Coast in Chennai, Tamil Nadu, India (13.252851°N, 80.326744°E). Ennore Harbor is a major industrial maritime zone that experiences chronic petroleum pollution due to discharge from nearby petrochemical complexes, power plants, and shipping activities. Samples were collected aseptically from visibly oil-contaminated surface water in sterile polyethylene bags. Each bag was appropriately labelled with the site coordinates and collection date. All samples were transported immediately under cold conditions (4 °C) to the laboratory and processed within 24 h for microbiological analysis.

Enumeration of Hydrocarbon Degrading Bacteria (HDB)
The abundance of HDB in marine water samples was determined using a selective enrichment approach. Samples were serially diluted in sterile NaCl (0.85% w/v). From each dilution, 0.1 mL was spread onto sterile Bushnell Haas Agar (BHA) plates, which were pre-supplemented with 100 µL of sterile crude oil to serve as the exclusive carbon supplement. Crude oil was sourced from Chennai Harbor, filtered through a 0.45 µm membrane to ensure sterility, and stored in sterile bottles until use. The inoculated plates were aerobically incubated for 7 days at 25 °C. Following incubation, bacterial colonies that grew on the hydrocarbon-supplemented medium were counted, and the HDB population was expressed as CFU/mL^-1.⁵

Screening of biosurfactant production by HDB
All HDB were qualitatively screened for biosurfactant production using multiple phenotypic assays. These methods detect surface-active compounds through distinct physical and biochemical interactions.

Blue agar plate assay
The presence of anionic biosurfactants was measured using the blue agar plate assay described by Tahzibi et al.⁶ The screening medium consisted of basal agar supplemented with CTAB and methylene blue, adjusted to pH 7.0 and autoclaved. After solidification, 6.5 mm diameter wells were aseptically bored into the solidified agar. Culture supernatant (100 µL) from overnight-grown bacterial isolates were aliquoted into each well. The plates were incubated for 48 h at 37 °C. The development of blue halos surrounding the wells indicated the presence of anionic biosurfactants that interacted with CTAB and methylene blue to form a visible complex.

Hemolysis on Blood Agar (BA)
The biosurfactant was further evaluated based on the ability of the isolates to lyse erythrocytes using a blood agar assay as previously described.⁷ Freshly prepared BA plates with defibrinated sheep blood (5% v/v) were used as the screening medium. Each bacterial isolate was spot-inoculated onto the surface of plates under sterile conditions. The plates were incubated for 24-48 h at 37 °C. Following incubation, the appearance of hemolytic zones surrounding the bacterial colonies was examined. The observation of a clear or partially clear halo indicated hemolytic activity, suggesting biosurfactant production due to the disruption of red blood cell membranes.

Drop collapse assay
This assay was used to assess the surface-active properties of the biosurfactants present in the culture supernatants as previously described by Rani et al.⁸ A sterile 96-well polystyrene microplate lid was used as the testing surface. Each well was pre-coated with 2 µL of crude oil and allowed to equilibrate at room temperature (approximately 22 °C) for 24 h to ensure uniform spreading. Subsequently, 5 µL of clarified supernatant from each isolate was gently dispensed into the focus of the oil-coated wells. The wells were visually examined after 1-2 min. A flattened or collapsed drop indicated a reduced surface tension and was recorded as a positive result for the biosurfactant. In contrast, the retention of a rounded drop indicated a negative result.

Oil spreading assay
The experiment was performed to assess the biosurfactant activity of the bacterial isolates, following the process described by Maneerat et al.⁹ 20 mL of distilled H₂O was pipetted out into the center of a sterile petri dish, and 20 µL of crude oil was gently layered on the water surface to form a uniform oil film. Subsequently, 10 µL of the culture supernatant was carefully added to the center of the oil layer. The presence of the biosurfactant led to a reduction in the surface tension, causing the oil to move aside and resulting in the formation of a clear zone. The diameter of the zone (in mm) served as evidence of biosurfactant activity. A control consisting of culture medium without biosurfactants was included for comparative evaluation.

Emulsification Index Assay (E24)
The emulsifying ability of the biosurfactants was determined using the emulsification index technique described by Maneerat et al.⁹ In this assay, 4 mL of the supernatant was mixed with 3 mL of crude oil in a sterile test tube. The contents were vortexed vigorously for 2 min to promote thorough mixing and emulsion formation. The test tubes were kept undisturbed at ambient temperature for 24 hr. The height of the resulting emulsion layer was measured and the percentage of the total liquid column height was calculated. A higher E24 value indicates greater emulsifying capacity of the biosurfactant. A control sample in which distilled water replaced the supernatant was included to determine the baseline emulsification.

Biosurfactant production, extraction, and quantification
Bacterial isolates, Is4, Is15 and Is18, which tested positive in all preliminary biosurfactant screening assays, were selected for production studies. Each isolate was inoculated into 100 mL of sterile Bushnell Haas Broth (BHB) supplemented with 1 g of sterilized crude oil as the sole carbon supplement. Cultures were incubated at a temperature of 30 °C for 7 days under shaking at 120 rpm to facilitate hydrocarbon degradation and biosurfactant secretion. Following incubation, the culture broth was centrifuged to remove the bacterial cells, and the resulting supernatant was used for gravimetric oil degradation analysis and biosurfactant extraction. Residual crude oil was extracted using diethyl ether, and the amount of undegraded oil was quantified by weighing after solvent evaporation. Degraded oil was calculated as follows.¹⁰

Crude Oil Degradation (g) = Initial Oil Weight – Residual Oil Weight

The same supernatant was used to recover the biosurfactant via acid precipitation. The pH was adjusted to 2.0 using 11.5 N HCl and incubated overnight at 4 °C. The extracted biosurfactant was obtained from the precipitate using a 2:1 (v/v) CHCL₃-CH₃OH mixture. The organic phase was separated and evaporated in a fume hood, and the dried biosurfactant was weighed. The weight of the recovered biosurfactant was recorded in grams. These values were used to assess the biosurfactant yield of each isolate under standard production conditions.¹¹

Potential isolate identification
Identification of the potential isolate, Is15, at the species level was performed using 16S rRNA gene sequencing. Genomic DNA was extracted from Is15, and its quality was confirmed by the presence of a distinct high-molecular-weight band on a 1.0% agarose gel. Amplification of the 16S rRNA gene was performed with 16S rRNA-F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 16S rRNA-R (5′-TACGGTTACCTTGTTAC GACTT-3′), primers resulting in a single, discrete amplicon of approximately 1500 bp. The PCR product was purified to remove any residual contaminants and subjected to bidirectional sequencing on an ABI 3730xl Genetic Analyzer with BDT v3.1 Cycle Sequencing Kit. Forward and reverse sequences were assembled to generate a consensus sequence using aligner software.¹² The resulting 16S rRNA gene sequence was queried against the NCBI GenBank ‘nr’(non-redundant protein) database using BLAST, and the top ten hits based on maximum identity were aligned using ClustalW. To determine the evolutionary relationship, a phylogenetic tree along with a distance matrix was constructed using the neighbor-joining approach in MEGA 11 version.¹³

Optimization of biosurfactant production
To enhance biosurfactant yield and hydrocarbon degradation, the most promising bacterial isolate, Bacillus cereus Is15 was optimized under varying nutritional and environmental conditions. BHB supplemented with crude oil (1 g/100 mL) served as the basal production medium for all experiments. Each condition was tested in triplicate under gentle shaking for 7 days at 120 rpm at 30 °C. The combination of pH, temperature, and carbon and nitrogen sources that yields the maximum biosurfactant concentration and oil degradation efficiency is considered optimal for large-scale biosurfactant production.¹⁴

Effect of pH
To assess the effect of pH on biosurfactant production, the pH of the medium was adjusted to 6.0, 7.0, 8.0, 9.0, and 10.0 prior to inoculation. Cultures were incubated under standard conditions, and both the biosurfactant yield and crude oil degradation were determined at the end of the incubation.

Effect of temperature
Temperature optimization was carried out by incubating inoculated flasks at 17 °C, 27 °C, 37 °C, and 47 °C. After seven days, the samples were analyzed for residual oil and biosurfactant concentrations to identify the optimal temperature for metabolite production.

Effect of carbon sources
The influence of various carbon sources, such as glucose, fructose, lactose, sucrose, and dextrose, was evaluated by supplementing the production medium with these individually along with crude oil. After 7 days of incubation, oil degradation and biosurfactant yield were assessed to identify the most efficient carbon substrate.

Effect of Nitrogen sources
To optimize the nitrogen source, different organic and inorganic nitrogen supplements including casein, peptone, beef extract, yeast extract, and urea were added to the production medium at equivalent nitrogen concentrations. Following incubation, the residual crude oil and biosurfactant yield were measured gravimetrically to evaluate the influence of each nitrogen source.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of Biosurfactant
The chemical composition of the biosurfactant produced by Bacillus cereus Is15 was determined using GC-MS. The biosurfactant extract was subjected to GC-MS analysis on an Agilent GC system 7890B coupled with an MSD 5977A detector using a HP-5MS capillary column with an ID of 30 x 0.25 mm and 0.25 µm film thickness. The gas chromatography oven was programmed to begin at 60 °C with an initial hold of 5 min, then increase at a rate of 8 °C/min until it reached 280 °C, where it was held for 37 min. Helium was used as the carrier gas and flowed at a constant rate of 1.0 mL/min. The injector temperature was set to 300 °C, and 1 µL of the sample was introduced in splitless mode. For mass spectrometry detection, the interface and ion source temperatures were maintained at 310 °C and 230 °C, respectively. Spectra were acquired in electron ionization (EI) mode at 70 eV. Component identification was performed by matching the acquired spectra with entries in the NIST 20 mass spectral database.¹⁵

Biosurfactant-mediated oil removal from marine water
The effectiveness of the biosurfactant extracted from Bacillus cereus Is15 in removing crude oil was evaluated under controlled laboratory conditions using artificially contaminated seawater. The clean marine sand was first sieved and evenly distributed into three separate plastic containers (100 g per container), labelled C1, C2, and C3. Crude oil (10 g) was added to 100 mL of sterile marine water in each container to simulate oil spill conditions, and the mixture was allowed to equilibrate for 10-12 days at RT. After the contamination period, 500 mg of purified biosurfactant was added to container C2, and 10 mL of 10% SDS solution was added to container C3. Container C1 served as an untreated control. All the containers were gently agitated to ensure uniform mixing and left undisturbed for 24 h.

To quantify the residual oil content, the total petroleum hydrocarbons (TPH) were measured using the n-hexane extraction method. A 100 g sample of the water-sand mixture was combined with 200 mL of distilled hydrogen peroxide (H₂O₂), acidified with hydrochloric acid (HCl), and then extracted using n-hexane, 100 mL. The organic layer holding the oil was separated through a separating funnel and dried in a hot air oven at 103 °C. The extracted oil was weighed using the gravimetric method, and the TPH value was calculated using the following equation:

TPH (g/100 g) = W₂ – W₁

where:
W₁ = weight of empty dish
W₂ = weight of dish after oil extraction and drying, respectively.
The efficiency of oil removal was compared across the treatments to evaluate the practical applicability of the biosurfactant in marine oil spill mitigation.

Statistical analysis
All experiments were conducted in triplicate, and data were expressed as mean ± standard deviation (SD). Statistical comparisons among groups (biosurfactant, SDS, and control) were performed using one-way analysis of variance (ANOVA), followed by Tukey’s post-hoc test. Statistical significance was set at p < 0.05. Statistical analyses were performed using the IBM SPSS software version 22.