Diversity of Pyrene Degrading Microbes and Molecular Analysis of Pyrene Degrading Pathway

Abstract

In the present study, we isolated a biodiversity of 250 aerobic, mesophilic bacterial isolates from crude, diesel oil and coal-tar contaminated soil. 16S ribosomal DNA sequencing technique identified the pyrene (50 μg ml-1) utilizing bacterial isolates as members of Bacillus, Pseudomonas and Rhodococcus genera and one pyrene non-utilizing isolate belonged to Kocuria genera. Bacillus licheniformis (PK-6), isolated from coal-tar contaminated soil, showed maximum pyrene uptake (56.4 %; as assessed by HPLC technique) and biosurfactant activity (OD550nm = 1.96) in Bushnell-Haas broth medium containing 1 % (w/v) glucose in 4 days of incubation. Bacillus pumilus (PK-12) showed moderate pyrene uptake (45.6 %) and biosurfactant activity (OD550nm = 1.11). Bacterial isolates Rhodococcus rhodochrous (PK-10) showed moderate pyrene uptake (46.3 %) but higher biosurfactant activity (OD550nm = 1.45)while Pseudomonas oleovorans (PK-3) showed low pyrene uptake (25.2 %) and biosurfactant activity (OD550nm = 0.43). The four- benzene ringed compound, pyrene was essentially cometabolized along with glucose utilization in all bacterial strains. Based on six pyrene [C16H10]metabolites identified from Bacillus licheniformis (PK-6) growth extracts - Phenanthrene [C14H10], 9,10-Diphenylphenanthrene [C26H18], 9-Methoxyphenanthrene [C15H12O], 5,6,7,8- Tetrahydro-1-naphthoic acid [C11H12O2], 1,6,7-Trimethyl-naphthalene [C13H14] and Diisooctylphthalate [C24H38O4], a biochemical pathway for pyrene degradation was proposed. The seat of pyrene metabolic genes in the selected pyrene degrading isolates was presumed to be chromosomally borne as no plasmid could be detected in bacterial cells. Attempts were made to detect Rieske centers, the conserved [Fe2-S2] cluster binding region of terminal dioxygenases within the genera Bacillus, Pseudomonas and Rhodococcus, however neither Rieske centers (gene) nor ring-hydroxylated products of pyrene catabolism could be detected by gene-specific PCR or GCMS analysis, respectively. Pyrene removal by Bacillus licheniformis (PK-6) MTCC 1005 in non-sterile pyrene contaminated soil was influenced by presence of indigenous, pyrene acclimatized and pyrene metabolizing gene pool rather than bioaugmentation of Bacillus sp. (PK-6) while in autoclaved pyrene contaminated soil was influenced by bioaugmentation (introducing pyrene-degrading bacterial culture) and biostimulation (adding glucose). The pyrene removal potential of Bacillus licheniformis (PK-6) MTCC 1005 in sterile, pyrene-spiked garden soil suggests its possible use and advantage in bioremediation of sites contaminated with crude oil, coal-tar and a mixture of PAHs.