2.1. Bacterial growth
Growth of both isolates was assessed in liquid Bushnell-Haas (BH) medium supplemented with 1% (v/v) n-hexadecane. The experiments were carried out in 100 ml Erlenmeyer flasks containing 50 ml BH medium inoculated with exponentially growing cells at a final concentration of OD600 equal to 0.035. The culture flasks were then incubated at 25 ± 0.5°C in a rotary shaker at 100 rpm under darkness. Growth was assessed as increase in cell density and total protein content (Lowry et al. 1951). Growth in BH medium without n-hexadecane served as control.
2.2. Detection of catabolic genes by PCR
Genomic DNA was isolated from BH-crude oil grown cultures using Genomic DNA extraction Kit (Bioline). The conserved fragments of catabolic alkane hydroxylase gene was amplified using the primer pairs and at annealing temperature listed in Supplementary Table 1. A 25 µl PCR reaction mixture contained 50 ng template DNA, 2.5 µl 10X reaction buffer, 1.25 µl of 25 mmol/L MgCl2, 0.5 µl of 10 mmol/L dNTPs, 0.5 µl of 10 pmol/µl each primer and 1 U Taq DNA polymerase. Final volume was adjusted by adding sterile double distilled water. PCR conditions were as follows: initial denaturation at 94°C for 4 min, followed by 30 cycles of denaturation at 94°C for 30 s, primer annealing for 30 s, elongation at 72°C for 30 s, and a final extension step at 72°C for 10 min using an ABI 2720 thermal cycler. The PCR amplicons were then separated on 1.2% agarose gel in 1x TAE buffer and visualized after staining with ethidium bromide (5 µg/ml) and photographed using a Bio-Rad Gel Doc™ XR + imaging system.
2.3. Cell surface hydrophobicity test
Cell hydrophobicity was measured according to Rosenberg (Rosenberg et al. 1980). In brief, cells were washed twice in Phosphate Urea Magnesium (PUM) buffer pH 7.1 (22.2 g K2HPO4, 7.26 g KH2PO4, 1.8 g urea, 0.2 g MgSO4.7H2O in final volume of 1000 ml sterile distilled water) and suspended in the same to an OD550 of 0.5. The cell suspension (1.0 ml) was then mixed and vortexed with heptane (0.2 ml) for 2 min followed by equilibration for 1 hour. The absorbance of the bottom aqueous phase was then measured at 550 nm. Hydrophobicity expressed as percentage adherence to heptane was calculated as follows:
% Hydrophobicity = (1- A/A0)*100 where A = OD550 of aqueous phase
A0 = OD550 of initial cell suspension
2.4. Emulsification index
The emulsification activity was determined as described (Berg et al. 1990). To culture supernatant (0.5 ml) same volume of TM buffer (20 mM Tris[tris(hydroxymethyl)-aminomethane]/HCl buffer, pH7.0 and 10 mM MgSO4) and 0.1 ml of kerosene was added. The tube was vortexed for 1 min, held stationery for 3 min, and then visually examined for turbidity of a stable emulsion. Emulsification index was calculated using the formula
E24 = (Height of emulsion formed/ Total height of solution)*100
2.5. Isolation of biosurfactant
Supernatant of bacterial culture grown for 24 and 48 hours in 0.5% (v/v) n-hexadecane amended nutrient broth were collected by centrifugation at 10,000 rpm for 15 min at 4°C. The pH of cell free supernatant was then adjusted to 2.0 using concentrated HCl and kept at 4°C overnight to allow formation of precipitate. The precipitate was collected by centrifugation at 17,000 rpm for 15 min at 4°C, dissolved in water and lyophilized (Heto LyoLab 3000 Lyophilizer, Germany). The recovery of lyophilized biosurfactant sample was expressed as g biosurfactant.g− 1 dry cell wt.
2.6. Characterization of biosurfactant
The partially purified biosurfactant was characterized based on simple colorimetric assays.
2.6.1. Carbohydrate: The carbohydrate content was measured using Phenol-sulphuric acid method with glucose as standard. In brief, 5 mg of the biosurfactant sample was dissolved in 1 ml of water. One hundred µl of this suspension was mixed with 200 µl of phenol reagent (5% v/v water) followed by addition of 1 ml concentrated H2SO4 and incubation at room temperature for 10 min. The mixture was vortexed vigorously and allowed to stand for 30 min. Finally absorbance was read at 490 nm against a reagent blank.
2.6.2. Protein: The extracellular protein in partially purified biosurfactant was measured at 750 nm using bovine serum albumin as standard according to Lowry (Lowry et al. 1951).
2.7. Crude oil degradation in liquid BH medium
Degradation of crude oil by individual bacterial isolate was carried out in biometric flasks containing 100 ml BH-medium (Himedia, India) supplemented with 1% Assam crude oil (v/v) as the sole source of carbon and energy. Bacterial cells grown in BH-medium supplemented with glucose (200 mg L− 1) were harvested, washed at 10000 rpm for 10 min in BH-medium and then re-suspended in the same. The cell suspension was inoculated at a final OD600 equal to 0.035. Non-inoculated flasks were used as abiotic controls. The culture flasks were incubated as described in Sect. 2.1. Crude oil degradation activity of bacteria was assessed in terms of CO2 evolution (Zibilski 1994). For estimation of total/residual petroleum hydrocarbons (TPH), each sample was extracted in an equal volume of dichloromethane (DCM) at regular interval and separated from aqueous phase. The DCM phase was then dehydrated with anhydrous sodium sulphate and remaining TPH was extracted using a Soxhlet apparatus. Weight of the residual TPH after rota-evaporation was determined gravimetrically.
2.8. Microcosm design and set up
Laboratory microcosm comprising 10 g sterilized sand sediment amended with 125 mg Assam crude oil and supplemented with 125 µg N.g− 1 sediment & 62.5 µg P.g− 1 sediment for P. aeruginosa AKS1, and 375 µg N.g− 1 sediment & 37.5 µg P.g− 1 sediment for Bacillus sp. AKS2 were set up. Sodium nitrate and potassium dihydrogen phosphate were used as sources of inorganic N and P respectively. Microcosms were inoculated with exponentially growing bacterial cells and incubated at a temperature of 25 ± 0.5°C under darkness. Microbial TPH degradation activity in the sediment was assessed as a function of CO2 evolution as described in Sect. 2.7 (Zibilski 1994), and in terms of residual TPH as described by Márquez-Rocha (Márquez-Rocha et al. 2001). In short, 10 g of microcosm sand sediment was mixed with equal amount (w/w) of anhydrous sodium sulphate and TPH was extracted in DCM using a Soxhlet apparatus. Non-inoculated or microcosms without crude oil were also incubated to account for any abiotic CO2 production.
Biodegradability of crude oil in both liquid culture and sand microcosms were fitted into first order kinetics as described in Eq. (1) below.
C t = C0 e− kt (1) Where, Co is the initial TPH content
C t is the residual TPH content at time t
k is the biodegradation rate constant (day− 1)
Biodegradation half-life times (t1/2) was calculated by Eq. (2)
t 1/2 = ln2/k (2)