Bacterial growth and hydrocarbon biodegradation analysis

The bacterial abundance was determined through the colony counting technique on nutrient agar (NA) plates. Concentration of the colony-forming units was expressed by means of logarithmic notation using the average of plate count from the triplicate flasks at the same time. Growth and degradation studies were carried out concurrently. Following the incubation times, the liquid cultures of each flask were extracted with n-hexane (1:1 media to n-hexane) as a solvent to separate the cellular material. The final residual oil extracts were transferred to tared vials and kept at 4 °C. The qualitative and quantitative analysis of residual hydrocarbons in diesel occurring in the extracts were observed with a gas chromatograph (7890A, Agilent Technologies), equipped with a DB-5MS capillary column (30 m × 0.25 mm × 0.25 µm) and a flame ionisation detector (FID). Oven temperature was initiated at 80 °C, then ramped at 20 °C/min to 280 °C and held at this temperature for 2 min. The inlet was run in split mode at 240 °C with a 50:1 split ratio. Hydrogen was used as the carrier gas with flow rate at 30 ml/min. GC peaks were characterised by injecting the standards of n-alkane (C₈–C₂₀) under the same condition. n-dodecane was used as the indicator for biodegradative losses in quantitative analysis as this n-alkane has a melting point of − 9.6 °C. This permits the incubation of cultures at any temperature above 0 °C as compared to the more common n-hexadecane. n-hexadecane has a melting point of 18.2 °C that prevent this study to compare the mineralisation at low and moderate temperature [18]. The biodegradation of hydrocarbon compounds in diesel oil was expressed as the percentage of hydrocarbons degraded with regard to the amount of the residual fractions in the appropriate abiotic control samples. The reduction of each degraded hydrocarbon was evaluated by means of biodegradation efficiency (BE) using the following expression:

where, A_s = total area of peak in sample, A_ac = total area peak in abiotic control, BE(%) = percentage of biodegradation efficiency.