Resistant Rhodococcus for Biodegradation of Diesel Fuel at High Concentration and Low Temperature

Abstract

The resistance of 16 Rhodococcus strains to diesel fuel was studied. The minimal inhibitory concentrations of diesel fuel against Rhodococcus were 4.0-64.0 vol. % and 0.5-16.0 vol. % after 7 days of incubation in Luria-Bertani broth and a mineral "Rhodococcus-surfactant" medium, respectively. The three most resistant strains (R. ruber IEGM 231, IEGM 442 and Rhodococcus sp. IEGM 1276) capable of overcoming the toxicity of diesel fuel at a high (8.0 vol. %) concentration and at a low (4 °C) temperature were selected. Respiration activities, growth kinetics, and changes in the diesel fuel composition during the biodegradation process were elucidated using gas chromatography with mass spectrometry, respirometry, and Bradford analysis. Growth conditions were optimised for the improved biodegradation of diesel fuel by Rhodococcus cells using multifactor analysis. They included the simultaneous addition of 1.3 g·L^-1 of granular sugar and 0.25 g·L^-1 of yeast extract. The twofold stimulation of the biodegradation of individual hydrocarbons in diesel fuel (n-pentadecane, n-hexadecane and n-heptadecane) was demonstrated when glycolipid Rhodococcus-biosurfactants were added at a concentration of 1.4 g·L^-1. A total removal of 71-91% of diesel fuel was achieved in this work.

Keywords: Rhodococcus; biodegradation; biosurfactants; diesel fuel; growth kinetics; heavy contamination; low temperatures; no catabolite repression; resistance; respirometry.

Figure 1

Calibration curve between concentration of bovine serum albumin and A_{595 nm} for Bradford analysis.

Figure 2

Dynamics of total removal of diesel fuel at a concentration of 8.0 vol. % and temperature of 28 °C in the presence of R. ruber IEGM 442 cells.

Figure 3

GC-MS chromatograms of original (a) and residual diesel fuel after 8 days of biodegradation using R. ruber IEGM 442 cells at 28 °C (b).

Figure 4

Biodegradation of individual hydrocarbons in diesel fuel at its concentration of 8.0 vol. %, at 28 °C in 8 days by R. ruber IEGM 442 cells without (a) and in the presence (b) of Rhodococcus-biosurfactants added at a concentration of 1.4 g·L⁻¹.

Figure 5

Respiratory activities of Rhodococcus strains during the biodegradation of different concentrations of diesel fuel at 28 °C. The parameters measured were as follows: (a) consumption of O₂, μL; (b) production of CO₂, μL; (c) rate of O₂ consumption, μL·min⁻¹; and (d) rate of CO₂ production, μL·min⁻¹.

Figure 6

Growth kinetics of Rhodococcus sp. IEGM 1276 cells in the presence of 2.0 and 3.0 vol. % diesel fuel at 4 °C.

Figure 7

Pareto diagrams showing the effects of sugar, diesel fuel, and yeast extract concentrations on the number of R. erythropolis IEGM 587 cells (a) and the biodegradation of diesel fuel (b).