Benz (a)pyrene Biodegradation Improvement Using the Biosurfactant Producing Bacterial Consortium

Environmental contamination by crude oil and its various processing products is becoming a common phenomenon which severely damages soil and groundwater resources. Among the constituents of oil waste, polycyclic aromatic hydrocarbons (PAHs) are of environmental concern because of their toxic, mutagenic and/or carcinogenic effects. Bioremediation involves the use of living microorganisms, bacteria or fungi, for detoxification of soil and water organic pollutants by biodegradation, biotransformation, and/or mineralization. Collaboration between different microbes under co-culture conditions such as co-metabolism or antagonism makes the system to perform better than a single microorganism. Total petroleum degradation is a result of a microbial consortium action, which is composed of different species with specific biochemical roles. On the other hand, the majority of components of petroleum products has low solubility in water and tends to bind to soil particles reducing their availability to  microorganisms for degradation. This has been well described as a major limitation to the bioremediation of hydrocarbon contamination. The surfactants can be employed to enhance hydrocarbon biodegradation by mobilization, solubilization, or emulsification. Some microorganisms synthesize a wide range of surface-active compounds, generally called biosurfactants, which increases the bioavailability of these compounds. The application of these microbial surfactants in the remediation of hydrocarbons aims to increase their bioavailability or mobilize and remove the contaminants by pseudo-solubilization and emulsification in a treatment process. This work aimed to investigate the impact of the biosurfactant producing consortium on the benzo(a)pyrene biodegradation.

Four gasoline contaminated soils were enriched in Bushnell-Hass mineral medium with Benzo(a)pyrene (200 mg/l) for three months at 30°C. After this time, to obtain Benzo(a)pyrene-degrading isolates, 0.1 ml of soil suspensions were plated on BH agar plates containing pollutant. Three colonies with different morphological distinct properties were purified on LB agar plates. The screening of the most potent surfactant strain was assayed quantitatively using measurement of surface tension by the Du Nouy ring method. For increasing the production of biosurfactant, medium conditions including pH (6, 7, 8), temperature (25, 30, 35) and carbon source (glucose, sucrose and ribose) were optimized with fractional factorial based on Taguchi. The capability of the isolates and consortium in hydrocarbon biodegradation was investigated in liquid medium of Bushnell-Hass with 150 ppm of Benzo(a)pyrene, during 14 days. Treatments included inoculation of isolates AP3 and BM1 and their consortium in presence and absence of extracted isolates biosurfactants and control (no isolate and biosurfactant). Based on the results of Benzo(a)pyrene degradation in the liquid medium, AP3 isolate, consortium and biosurfactant extracted from AP3 were selected for soil experiment. Four sets of biodegradation experiments were carried out with soil contaminated by 150 ppm of benzo(a)pyrene for 45 days, as follows: set 1: soil + AP3 isolate; set 2: soil + consortium; set 3: soil + consortium + AP3 biosurfactant and set 4: blank (soil). The residual concentrations of contaminant were extracted on days 15, 30 and 45 by dichloromethane solvent and analyzed using GC-FID.

The results revealed that strains AP3 and BM1 showed a significant potential to produce surface-active agents in the presence of Benzo(a)pyrene as substrate, reducing the surface tension to 43 and 46 mN/m, respectively. Taguchi experimental design method was applied in order to optimize the biosurfactant production by isolates. Results of experiments indicated that the optimum biosurfactant production conditions were found to be temperature of 35º C and  pH of 7, and glucose as water soluble carbon source. The produced biosurfactant reduced surface tension to 31/52 mN/m and 30/81 mN/m for BM1 and AP3, respectively. Biodegradation experiments of Benzo(a)pyrene in liquid cultures showed that the overall biodegradation efficiency of the individual isolates after 14 days was lower than consortium. Bacterial consortium enhanced degradation of contaminant to 87.3% (with addition of biosurfactant) compared to 27.6% of removal in presence of BM1 isolate. However, there was no statistically significant change in the degradation rates of contaminant in consortium with addition of AP3 and BM1 surfactant and surfactant free (87.3, 85.6 and 86.8%, respectively). The degradation of Benzo(a)pyrene was significantly enhanced in presence of AP3 biosurfactant at individual BM1 treatments (28.3 and 44.5 to 74.8%). Maximum degradation of Benzo(a)pyrene in contaminated soil was found (100%) in set 3: soil + consortium + AP3 biosurfactant. Based on GC-MS analyses, it degraded around 100% of penzo(a)pyrene, used as the sole carbon and energy source, at an initial concentration of 150 mg L-1, after 45 days of incubation, while alone consortium and isolate were able to remove 86% and 68% of hydrocarbon, respectively. Overall, these results provide evidence that consortium and AP3 biosurfactant could be potential candidates for further bioremediation.

The results revealed that the hydrocarbon removal efficiency of the consortium was higher than single species, and the final removal efficiency for the consortium could be reached in a considerably shorter time. The results suggest that biosurfactant-assisted bioremediation may be a promising practical bioremediation strategy for aged PAH-contaminated soils. It is evident from the results that the consortium alone and its producer species are both capable of promoting biodegradation to a large extent.