Abstract
In this study, a hydrocarbon-degrading mixed inoculum which is able to use used oil as sole carbon source, was selected from 15 bacterial isolates obtained from automotive service station oil-contaminated soils. Degrading microorganisms were isolated using different oils as sole carbon source and identified by the amplification and sequencing of the 16s rRNA sequences. In addition, the presence of hydrocarbon-degrading genes such as catechol 2,3 dioxygenase (nahH), alkane monooxygenase (alkB), Gram-negative (GN-RHDa) and Gram-positive PAH-Ring Hydroxylating Dioxygenase alpha (GP-RHDa) was analyzed by PCR and the molecular diversity by LSSP-PCR methods. Four (4) out of fifteen (15) isolates corresponding to Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Klebsiella pneumoniae showed significant differences regarding oil/grease removal in liquid culture after 72 hours. Subsequently, a degrading mixed inoculum composed of these isolates was constructed and its degrading potential tested in a two-liter bioreactor containing unsterile liquid oily wastes with 0.8 % (w/v) Total Petroleum Hydrocarbons (TPHs) concentration for 42 days. The use of the mixed inoculum led to a decrease of 98.4 % Biological Oxygen Demand (BOD), 97.5 % Chemical Oxygen Demand (COD) and 97.2 % TPHs after 40 days. Further scale-up of the process to five liters using 0.2 % (w/v) unsterile oily wastes produced similar results, with a reduction of 85 % BOD, 39 % COD and 87 % TPHs after 38 days. The degrading mixed microbial inoculum presented high potential for the treatment of impacted soils at automotive service stations and sites polluted with oily wastes due to its elevated growth at high hydrocarbon concentrations and its capacity to utilize oils as energy source.
