Characterization, phylogenetic distribution and evolutionary trajectories of diverse hydrocarbon degrading microorganisms isolated from refinery sludge

Abstract

Phylogenic association between bacteria living under harsh conditions can provide important information on adaptive mechanism, survival strategy and their potential application. Indigenous microorganisms isolated from toxic refinery oily sludge with ability to degrade a diverse range of hydrocarbons were identified and characterized. The strains including Pseudomonas aeruginosa RS1, Microbacterium sp. RS2, Bacillus sp. RS3, Acinetobacter baumannii RS4 and Stenotrophomonas sp. RS5 could utilize n-alkanes, cycloalkanes, polynuclear aromatic hydrocarbons (PAHs) with 2-4 rings and also substituted PAHs as sole substrate. The phylogenetic position of Bacillus sp. RS3 and Pseudomonas sp. RS1 was tested by applying the maximum likelihood (ML) method to the aligned 16S rRNA nucleotide sequences of PAH and aliphatic hydrocarbon degrading strains belonging to the corresponding genus. The base substitution matrix created with each set of organisms capable of degrading aromatic and aliphatic hydrocarbons showed significant transitional event with high values of transition: transversion ratio (R) under all conditions. The guanine-cytosine (GC) content of the hydrocarbon degrading test strains was also found to be highest for the clade which harbored them. The test strains consistently occupied a distinct terminal end within the phylogenetic tree constructed by ML analysis. This study reveals that the refinery sludge imposed environmental stress on the bacterial strains which possibly caused significant genetic alteration and phenotypic adaptation. Due to the divergent evolution of the Pseudomonas and Bacillus strains in the sludge, they appeared distinctly different from other hydrocarbon degrading strains of the same genus.

Keywords: Aliphatic and aromatic hydrocarbons; Bacteria, phylogeny; MEGA-6; Maximum likelihood method; Refinery oily sludge.

Fig. 1

Phylogenetic tree for a RS1, b RS2, c RS3, d RS4 and e RS5

Fig. 1

Phylogenetic tree for a RS1, b RS2, c RS3, d RS4 and e RS5

Fig. 1

Phylogenetic tree for a RS1, b RS2, c RS3, d RS4 and e RS5

Fig. 2

Growth profiles of strains isolated from refinery sludge on n-alkanes and cycloalkanes, a n-hexadecane, b n-octadecane, c n-nonadecane, d docosane, e cyclohexane and f decalin. Here, continuous line with diamond Pseudomonas aeruginosa, continuous line with square Acinetobacter baumannii, dashed-dotted line with triangle Bacillus sp., dashed lines with asterisk Microbacterium sp., and continuous line with cross Stenotrophomonas sp.

Fig. 3

Growth profiles of strains isolated from refinery sludge on 2, 3 and 4 ring PAHs, a naphthalene, b 1-methyl naphthalene, c phenanthrene, d anthracene, e fluoranthene, and f pyrene. Here, continuous line with diamond Pseudomonas aeruginosa, continuous line with square Acinetobacter baumannii, dashed-dotted line with triangle Bacillus sp., dashed lines with asterisk Microbacterium sp., and continuous line with cross Stenotrophomonas sp.

Fig. 4

a Phylogenetic tree constructed with 16S rRNA sequence of 52 strains of Bacillus sp. based on a maximum likelihood analysis. The pyrene degrading type strains are marked with superscript “T” and reference strain as “R”. b Phylogenetic tree constructed with 16S rRNA sequence of 30 strains of Bacillus sp. based on a maximum likelihood analysis. The n-hexadecane degrading type strains are marked with superscript “T” and reference strain as “R”

Fig. 4

a Phylogenetic tree constructed with 16S rRNA sequence of 52 strains of Bacillus sp. based on a maximum likelihood analysis. The pyrene degrading type strains are marked with superscript “T” and reference strain as “R”. b Phylogenetic tree constructed with 16S rRNA sequence of 30 strains of Bacillus sp. based on a maximum likelihood analysis. The n-hexadecane degrading type strains are marked with superscript “T” and reference strain as “R”

Fig. 5

a Phylogenetic tree constructed with 16S rRNA sequence of 36 strains of Pseudomonas sp. based on a maximum likelihood analysis. The pyrene degrading type strains are marked with superscript “T” and reference strain as “R”. b Phylogenetic tree constructed with 16S rRNA sequence of 37 strains of Pseudomonas sp. based on a maximum likelihood analysis. The n-hexadecane degrading type strains are marked with superscript “T” and reference strain as “R”

Fig. 5

a Phylogenetic tree constructed with 16S rRNA sequence of 36 strains of Pseudomonas sp. based on a maximum likelihood analysis. The pyrene degrading type strains are marked with superscript “T” and reference strain as “R”. b Phylogenetic tree constructed with 16S rRNA sequence of 37 strains of Pseudomonas sp. based on a maximum likelihood analysis. The n-hexadecane degrading type strains are marked with superscript “T” and reference strain as “R”

Fig. 6

Percentage of transition and transversion base substitution measured by maximum likelihood estimate of substitution matrix pattern using the Tamura–Nei (1993) model constructed with 16S rRNA nucleotide sequence of polynuclear aromatic and aliphatic hydrocarbon degrading strains. Here a PAH degrading Bacillus sp., b Aliphatic hydrocarbon degrading Bacillus sp., c HMW-PAH degrading Pseudomonas sp., d Aliphatic hydrocarbon degrading Pseudomonas sp. A discrete Gamma distribution was used to model evolutionary rate differences among sites [five categories, +G]. For estimating ML values, a tree topology was automatically computed. All positions containing gaps and missing data were eliminated. All analyses were conducted in MEGA6. Here, stripes represents % transition and dashes represents % transversion

Fig. 7

GC Content of housekeeping gene (16S rRNA) constructed based on a maximum likelihood analysis for a PAH degrading Bacillus sp., b aliphatic hydrocarbon/oil degrading Bacillus sp., c HMW-PAH degrading Pseudomonas sp. and d aliphatic hydrocarbon/oil degrading Pseudomonas sp. GC content ratio of individual clades with respect to the reference strain for e PAH degrading Bacillus sp., f HMW-PAH degrading Pseudomonas sp. and g aliphatic hydrocarbon degrading Pseudomonas sp.