Molecular and Physiological Studies on Bacterial Degradation of Polynuclear Aromatic Hydrocarbons
Abstract:
McDoel Switchyard, an old industrial site in Bloomington, Indiana, US inundated with extensive levels of organic pollutants, was screened for the presence of Polynuclear aromatic hydrocarbons (PAHs) degrading bacteria. The incidence of the PAHs and other organic pollutants was evaluated using the US EPA methods 8270 and 3546. The technique of continual enrichment of selected PAHs- naphthalene, chrysene, pyrene, fluoranthene and anthracene yielded eleven unique bacterial isolates tentatively named OC-1, OC-2, OC-3, OC-4, FB-1, FB-2, FB-3, CB-1, CB-2; PB-1 and PB-2. Following the isolation of pure bacterial strains, each of the bacterial strains was screened against four different PAHs. Their degradative abilities on the PAHs were determined using a GC- FID HP 5890 series II gas chromatograph connected to an HP 3396 Series II Integrator. Epifluorescent microscopy was used to measure the cell numbers via a PAH-dependent growth study. For the molecular characterization of the bacterial strains, genomic DNA was extracted. The polymerase chain reaction was carried out using 8FM as forward primer, and as reverse primers 926R and 1387R to amplify the 16S rDNA. The amplified fragments of 16S rDNA were sequenced with an ABI 3730 sequence machine. The results were analyzed using the following bioinformatic tools: Stuffit Expander 2009, Chromas Lite 2.0, BioEdit,7.0.9, Codon Aligner and BLAST algorithm. The bacterial strains evolutionary relatedness were performed on the basis of 16S rDNA gene analysis by comparison of the obtained sequences data with known sequences in the GenBank. From the environmental audit carried out at the site of study, two- to three-ring PAHs which were seven in number were recorded. From the values obtained, the 4-ring PAHs Surface Benzo (a) anthracene had the highest incidence of about 63,000 μg/kg with minimum and maximum values between 13.35-63000 μg/kg while the lowest recorded PAH was Sub surface dibenz(a,h) anthracene with values between 1-1249 μg/kg. Following the screening on the different PAHs, all the strains showed an ability to utilize a broad spectrum of the different PAHs as carbon and energy sources. They have also shown an ability to utilize the PAHs under anaerobic conditions. The biodegradation and PAH-dependent studies showed rapid exponential increase in cell numbers in some PAHs with about 99% disappearance of some of the PAHs at different volume biodegradation rates. The 16S rDNA analysis classified the organisms (OC-1, OC-2, OC-3 and OC-4) as species of uncultured bacterium OC-1, Bacterium OC-2 with about 99% homology to the type strain of Pseudomonas putida, strain OC-3 as Pseudomonas sp. strain OC3 and OC-4 as xxvi Pseudomonas putida strain OC4. Furthermore, strains of FB-1 FB-2 and FB-3 were identified as Lysinibacillus sp. FB1, Bacterium FB2 with 99% homology to the type strain of Paenibacillus sp. and Lysinibacillus fusiformis strain FB3 respectively. Strains CB-1 and CB-2 were identified as Bacterium CB1 with 100% homology to the type strain of an uncultured bacterium and Stenotrophomonas maltophila strain CB2 respectively. Strains PB-1 and PB-2 were identified as Pseudomonas plecoglossicida strain PB1 and Pseudomonas sp. strain PB2. The obtained 16S rDNA gene sequences have been deposited at the GenBank with the accession numbers issuedORDER COMPLETE MATERIAL (CHAPTER 1-5)
