Production of biosurfactant by B. velezensis KLP2016 cells
A fresh loopful culture of B. velezensis KLP2016 was inoculated in 100 mL of Luria bertani broth and incubated at 200 rpm under shaking at 30ºC to get 1.0 OD of cells at 620 nm. Bacterial growth was monitored regularly and 1.0 OD cells obtained at 9 hrs incubation. For production of biosurfactants, 1000 mL of LB broth was prepared in which 4 % (v/v) of bacterial inoculum (1.0 O.D cells) was inoculated and the flasks were incubated for 72 h at 30ºC at 200 rpm. After incubation, the culture broth was centrifuged at 10,000 rpm for 10 min at 4ºC . Biosurfactants containing supernatant/ cell free broth was collected for further experiments.
Measurement of emulsification index, surface tension and critical micelle concentration
Biosurfactant containing culture broth was evaluated by measuring the emulsification index (E24%) using various chosen organic hydrocarbon compounds (benzene, pentane, cyclohexane, xylene, n-hexane, toluene and engine oil) as the substrate. In a test tube, 1.5 mL of each hydrocarbon was added to 1.5 mL B. velezensis cell-free broth. This combination was mixed by using a vortex for 2 min, and the content was left undisturbed for 24 h. The percentage of the emulsification index (E24%) was calculated by using the following equation .
The surface tension of cell-free broth of B. velezensis strain was determined by the drop weight method at 25°С and 35°C temperatures; and Luria bertani broth and Minimal Salt medium (MSM) . Cell-free broths of B. velezensis KLP2016 grown in Luria Bertani (LB) and Minimal Salt Medium (MSM) for 72 h incubation, were used to measure the surface tension. The uninoculated LB and MSM broth (g/l) (KH2PO4, 1.4; Na2HPO4, 2.2; (NH4) SO4, 3; MgSO4, 0.6; NaCl, 0.05; yeast extract, 1; CaCl2 0.02) was taken as negative control. Critical micelle concentration (cmc) is the concentration of biosurfactant above which micelle form and further no reduction in surface tension occurs. The surface tension (γ) and critical micelle concentration (cmc) was calculated by using the following equation ;
Where γ0 is surface tension, n0 is number of drops and ρ0 is density of uninoculated broths, while γ is surface tension, n is number of drops and ρ is density of cell-free fermentation broth.
Purification and identification of active compound extracted from culture broth of B. velezensis KLP2016
Ammonium sulfate (NH4SO4) mediated protein precipitation and dialysis
The cell-free broth was introduced with 0-20, 20-40, 40-60, 60-80 and 80-100% saturation of NH4SO4 at 4°C, further mixed and kept overnight at 4°C. Thereafter, the precipitates were deposited after centrifugation at 12,000 rpm for 15 min. The precipitates were reconstituted in 1 mL of 20 mM sodium phosphate buffer at pH 7.5 and checked for emulsification activity against engine oil. One unit of emulsifying activity was explicated as the quantity of emulsifier that yielded an absorbance (600 nm) of 0.1 in the assay mixture .
Ion exchange chromatography
The DEAE cellulose packed glass column (height 10 cm; diameter 1.5 cm) was equilibrated with 20 mM sodium phosphate buffer (pH 7.5) after activation by 0.5 M NaOH. Five mL of dialyzed biosurfactant preparation (4.0 mg protein) was loaded on the matrix in the column . Column was equilibrated with 20 mM sodium phosphate buffer (pH 7.5). Unbound proteins were eluted with low ionic strength buffer (sodium phosphate buffer; pH 7.5) at a flow rate of 1 mL/min and discarded. The bound biosurfactant molecules eluted with the stepwise gradient of 0.5 M NaCl, 1 M NaCl and 1.5 M NaCl in sodium phosphate buffer (pH 7.5; 20 mM), respectively . Emulsification activity and A280 values were evaluated against the engine oil.
Size exclusion chromatography
Sephadex G-25 packed matrix was washed off with several column volume of 20 mM sodium phosphate buffer (pH 7.5). Pooled active fraction of the DEAE was loaded on the bed surface of Sephadex G-25 column and eluted with the sodium phosphate buffer (20 mM; pH 7.5) and fractions were collected . Absorbance at 280 nm and emulsification activity was evaluated against the engine oil. Active fractions were further checked with UV-vis spectrophotometer and TLC, as detailed below.
TLC and UV-VIS spectrophotometry
The fractions obtained from size exclusion chromatography, were analysed and mixed on the basis of their OD. A solvent system of chloroform: methanol: water (39:15:3; v/v) was prepared, and 5 µl sample of mixed biosurfactant fractions was applied at the point of origin of the TLC plate . Lipid moiety of the molecule was detected by TLC plate sprayed with water and thereafter kept for drying. The Rf values of the biosurfactant spot on the TLC plate were evaluated using the following formula and results recorded accordingly.
The purified biosurfactant was also analysed for ultraviolet absorbance spectrum  at range of 190-800 nm (UV-VIS Spectrophotometer, CARY, VARIAN).
High performance liquid chromatography analyses
The presence of biosurfactant in the purified molecule was confirmed by HPLC using an HPLC pump (Waters, USA) by a reverse phase column (Lichrosorb C18-5 µm; Merck, Germany) and 2998 photodiode assay detector . The mobile phase contained acetonitrile (ACN): ammonium acetate (10 mM) in the ratio of 40: 60 (v/v) and mobile phase flow rate was adjusted at 2 mL / min. Biosurfactant sample 5 µl was injected each time and analysed at 254 nm wavelength with comparing standard biosurfactants, i.e., surfactin and iturin.
ESI‑MS of purified biosurfactant
A mass spectrometer (Q-TOF micro Waters 2795 UK) was used to find the molecular weight of the purified biosurfactant. The conditions for used MS were temperature source, 100οC; 3000 V in positive mode; capillary voltage, cone voltage, 30 V; current source, 80.0 A and capillary voltage of 7.0 V in positive mode . About 20 μl of purified biosurfactant was injected into the MS and gently ionized with CH3OH and H2O (80:20) using electrospray (ESI) with flow rate of 1.0 mL/min. ESI-MS results were compared with the authentic surfactin biosurfactant molecule to identify the molecular mass of the purified biosurfactant of B. velezensis.
Hydrocarbon degradation activity of B. velezensis KLP2016
Biodegradation of engine oil (K 15W-40) by B. velezensis KLP2016 in a biometric system
For the biodegradation of engine oil, 5% (v/v) starter inoculum of 7 h of B. velezensis KLP2016 culture was inoculated in the 250 mL capacity sterilized flasks each containing 100 mL MSM and LB broth. Hydrocarbon substrate (K 15W-40 Engine oil) was added at 1% (v/v) concentration in each of the sterilized flasks. One test tube containing fresh KOH (10 mL; 0.05 M) was placed in each of the flasks, and these flasks were incubated at 30°C under shaking (100 rpm) from 5 to 20 days. Inoculated and uninoculated broths were observed for their absorbance (A600) and CO2 content at 5-day intervals up to 20 days. The CO2 gas trapped in the KOH solution was titrated by introducing 100 µl of barium chloride (w/v; saturated) and three drops of phenolphthalein with 0.05 M HCl until the appearance of the end point as the colourless solution. The difference in millilitres of HCl used to titrate KOH containing solution of control (placebo) and B. velezensis KLP2016 inoculated media was converted into ppm of fixed carbon dioxide as per method [17, 25]. Hydrocarbon degradation of engine oil facilitated by B. velezensis KLP2016 was also confirmed by Gas chromatography-mass spectrometry (GC-MS) analysis of the engine oil treated with bacterial cells.
Hydrocarbon analysis by GC-MS of K 15W-40 engine oil treated with B. velezensis KLP2016
In order to analyse the hydrocarbon products of engine oil broken down by B. velezensis KLP2016, the culture broth (5, 10, 15 and 20 days) was centrifuged at 10,000 rpm, at 4°C for 10 min. From the supernatant, the upper layer was collected, filtered with syringe filter (0.22 µm) and the filtrate was analysed using GC-MS to evaluate engine oil products. The GC-MS analyses were performed using an MS5973 spectrometer with a ULBON HR-1 column (25 mm x 50 mm), with thickness of 0.25 micron, 1 mL/min flow rate of the sample injected (5 µL) with the carrier gas helium, ion source temperature 230οC at 18.5 psi pressure and 20% split ratio . Results were observed and recorded accordingly.
Statistical analysis: All methods are statistically analysed.