Hydrocarbonoclastic bacteria isolated from petroleum contaminated sites in Tunisia: isolation, identification and characterization of the biotechnological potential

Petroleum hydrocarbons are important energy resources used by industry and in our daily life, whose production contributes highly to environmental pollution. To control such risk, bioremediation constitutes an environmentally friendly alternative technology that has been established and applied. It constitutes the primary mechanism for the elimination of hydrocarbons from contaminated sites by natural existing populations of microorganisms. In this work, a collection of 125 strains, adapted to grow on minimal medium supplemented with crude oil, was obtained from contaminated sediments and seawater from a refinery harbor of the Bizerte coast in the North of Tunisia. The diversity of the bacterial collection was analyzed by amplification of the internal transcribed spacers between the 16S and the 23S rRNA genes (ITS-PCR) and by 16S rRNA sequencing. A total of 36 distinct ITS haplotypes were detected on agarose matrix. Partial 16S rRNA gene sequencing performed on 50 isolates showed high level of identity with known sequences. Strains were affiliated to Ochrabactrum, Sphingobium, Acinetobacter, Gordonia, Microbacterium, Brevundimonas, Novosphingobium, Stenotrophomonas, Luteibacter, Rhodococcus, Agrobacterium, Achromobacter, Bacilllus, Kocuria and Pseudomonas genera. Acinetobacter and Stenotrophomons were found to be the most abundant species characterized by a marked microdiversity as shown through ITS typing. Culture-independent approach (DGGE) showed high diversity in the microbial community in all the studied samples with a clear correlation with the hydrocarbon pollution rate. Sequencing of the DGGE bands revealed a high proportion of Proteobacteria represented by the Alpha and Gamma subclasses. The predominant bacterial detected by both dependent and independent approaches were the Proteobacteria. The biotechnological potential of the isolates revealed a significant production of biosurfactants with important emulsification activities useful in bioremediation. The highest emulsification activity was detected in Pseudomonas geniculata with 52.77% of emulsification. Our overall results suggest that the obtained bacterial isolates may constitute potential candidates for bioremediation and can be useful for biotechnological applications.