Phenotypic Characteristics And Phylogenetic Analysis Of Crude Oil-degrading Bacteria

Isolation and Characteristics Of Crude Oil-Degrading BacteriaTen strains of crude oil degrading bacteria were isolated from three different soil samples by seven different mineral media. The isolates were Gram-negative, aerobic, non-spore-forming rods. All of ten isolates were able to grow on n-alkanes with medium length, diesel oil and crude oil, but not polycyclic aromatic hydrocarbons.Based on phenotypic characteristics by BioMerieux Vitek Microbiological Identification System, strain EVAS, EVA6, EVA7, EVAS, EVA9 was identified as Pseudomonas aeruginosa with 99% identity, strain EVA10, EVA11 and EVA12 expressed 99% identity to Acinetobacter calcoaceticus-baumannii complex and less than 1% identity to Sphingomonas paucimobilis, strain EVA13 expressed 95% identity to Acinetobacter calcoaceticus-baumannii complex and 4% identity to Ralstonia (Burkholderia) pickettii/thomasii; and strain EVA14 expressed 97% identity to Acinetobacter IwoffO/junii and less than 3% to Comamonas acidovorans.Phylogenetic Analysis of Crude oil-Degrading BacteriaPartial sequences of 16S rONA of ten crude oil-degrading isolates were obtained by PCR-amplification and sequencing. Based on phylogenetic characteristics of 16S rDNA, strain EVAS, EVA6, EVA7, EVAS, EVA9 was identified as Pseudomonas aeruginosa, which belonged to Bacteria, y-Proteobacteria, Pseudomonaceae/Moraxellaceae group, Pseudomonas, Pseudomonas aeruginosa group. Strain EVA10, EVA11, EVA12, EVA13, EVA14 belonged to Bacteria, y-Proteobacteria, Pseudomonaceae/Moraxellaceae group, Moraxellaceae, Acinetobacter.Based on phylogenetic tree of 16S rDNA, EVAS, EVA6. EVA7, EVAS, EVA9 clustered with Pseudomonas aeruginosa; strain EVA10, EVA11. EVA12 and EVA13 were positioned in a cluster, but clearly distributed in different subclusters. Strain EVA10 clustered with DNA group CTTU13; strain EVA11 clustered with DNA group 3; EVA12 clustered with DNA group 2; EVA13 clustered with DNA group 1-3, TU15 and some strains of DNA group 1. Strain EVA14 exhibited most close to DNA group 7 and showed great difference to other four isolates.respectively).Sequences alignment and phylogenetic analysis of 16S rDNA and ITS showed 16S rDNA and ITS of isolates belonging to Psuedomonas aeruginosa were highly conserved and ITS were more variable than 16S rDNA. Only total five variable sites were found in partial sequences of 16S rDNA (about the first 600bp from 5' end of 16S rDNA) and these variable sites appeared in a small 50bp region (region 400-450). Sequences of five isolates were identical except a single base variable in strain EVA9.The results of sequence analysis of ITS showed strain EVAS, EVA6, EVA7, EVA8, EVA9 and two other strains of Pseudomonas aeruginosa positioned in a group in 16S rDNA tree, but didn't display the differences among these strains. These differences could be clearly exhibited by ITS tree. EVAS, EVA6 and ATCC19660 showed a great similarity to each other. Strain EVA7, EVAS and PAO1 showed a great similarity to each other. Strain EVA9 showed some distance from the cluster of other six tested strains. Sequence analysis of 16S rDNA and ITS from isolates of Acinetobacter spp. (strain EVA10, EVA11, EVA12, EVA13, EVA14) revealed some discrepancy with result of phenotypic characteristics so that the determination of isolates at species level and phylogenetic relationship of Acinetobacter spp. still would depend on further study.