Biodegradation of Polycyclic Aromatic Hydrocarbons by Soil Bacteria

Abstract

Biodegradation of organic pollutants such as polycyclic aromatic hydrocarbons using soil microorganisms where they use these compounds as carbon and energy source, is the safe, cheap and environment friendly way. In the present study bacterial samples were isolated from crude oil contaminated soil. A total of 52 isolates were initially screened, fifteen of these isolates were checked for growth on PNR medium containing up to 1200 and 800 ppm, anthracene and pyrene, respectively. Five bacterial isolates were selected on the basis of their best on PAHs growth and identified as Bacillus cereus KWS2, Bacillus cereus KWS4, Bacillus licheniformis DW3, Bacillus licheniformis Sol-10, and Pseudomonas stutzeri 10-1. Biodegradation studies of anthracene were carried out by Bacillus cereus KWS2 at different pH, temperature and incubation time. The optimum pH and temperature were 7 and 30oC respectively, for degradation and growth where 45 % of reduction in anthracene concentration was observed after seven days of incubation. Growth was maximum after 2 days of incubation (12 x 10 7 CFU/mL). Pseudomonas stutzeri 10-1 was also checked for anthracene and naphthalene degradation capability. 33.81 % reduction in naphthalene concentration was observed after one week, while 33.69 and 45.35 % reduction after two and three weeks time, respectively. In case of anthracene, 87.37 % reduction was observed after first week of incubation whereas 92.98 and 95.56 % was observed after two and three weeks, respectively. Resting cell biotransformation studies of naphthalene, phenanthrene and anthracene were carried out by Bacillus cereus KWS2 and KWS4, analyzed by GC/MS. From naphthalene biotransformation four metabolites, 2-naphthol, benzeneacetic acid, benzoic acid and benzaldehyde were detected and identified. From phenanthrene biotransformation, 9-phenanthrenol, 9,10-Dihydro,9,10-dihydroxyphenanthrene, bezeneacetic acid, 4-hydroxy-benzeneacetic acid and 2-hydroxy benzoic acid were detected and identified. Benzenecarboxylic acid, benzeneacetic acid and 4-hydroxy- benzeneacetic acid were detected and identified from anthracene biotransformation by B. cereus KWS2. The detection of both mono and dihydroxylated compounds x suggested that B. cereus strains KWS2 & KWS4 harbor both mono and dioxygenase genes. Biotransformation potential of a selected bacterial isolate, Mycobacterium PY146 for PAHs was studied. Resting and growing cell biotransformation of PAHs including phenanthrene, anthracene and pyrene by Mycobacterium PY146 one metabolite i.e, 1,2-benzenedicarboxylic acid (phthalic acid) from phenanthrene and two metabolites, 1,2-benzenedicarboxylic acid and 9,10-anthracendione from the anthracene biotransformation, where as two metabolites, 1,2-benzenedicarboxylic acid and 1- hydroxypyrene were detected and identified from pyrene biotransformation. While in growing cell biotransformation of phenanthrene, 1,2-benzenedicarboxylic acid and benzeneacetic acid, from anthracene 9,10-anthracenedione, benzenecarboxylic acid, benzeneacetic acid and 1,2-benzenedicarboxylic acid and from pyrene benzenecarboxylic acid, benzeneacetic acid and 1,2-benzenedicarboxylic acid were detected and identified. Biodegradation of PAHs including phenanthrene, anthracene and pyrene were checked in soil by Mycobacterium PY146 without and with yeast extract. After one week of incubation the detectable concentration of PAHs without yeast extract, phenanthrene (98 μg), anthracene (60 μg) and pyrene (56 μg) were decreased to 35, 18 and 27 μg, and after 2 weeks of incubation all of the detectable PAHs were 0.14, 0.45 and 0.19 μg respectively. There was no significant effect of yeast extract on degradation except phenanthrene, which decreased from 98 to 27 μg after one week. Mineralization studies of different 14 C-PAHs (naphthalene, phenanthrene, pyrene and benzo(a)pyrene) were carried out by Bacillus cereus KWS2 and Mycobacterium PY146. In case of naphthalene and phenanthrene mineralization by Bacillus cereus KWS2, no mineralization was observed. Mineralization of phenanthrene, pyrene and benzo(a)pyrene by Mycobacterium PY146 showed that 45.92, 39.89 and 4.82 % of 14 CO 2 was produced and detected in the form of Na 214 CO 3. The mineralization of 14 C pyrene and phenanthrene with the different cell densities from OD 600 0.1 to 0.5 of Mycobacterium PY146 cultures showed that the maximum mineralization of pyrene was observed in the OD 600 0.2 culture, while in the case of xi phenanthrene there is increase in mineralization between the OD 600 0.1 to 0.2 culture. There was subsequent decrease in both pyrene and phenanthrene mineralization as the cell density increases. The expression of pyrene dioxygenase gene (nidAB) from Mycobacterium PY146 was checked in E. coli by cloning the nidAB in pKK223-3 expression vector and then transformed the E. coli. The biotransformation of naphthalene, phenanthrene, anthracene and pyrene was checked using transformed E. coli cells containing nidAB. Neither any hydroxy nor any derivatized metabolite were detected of any PAH after derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide with 1 %Trimethylechlorosilane (BSTFA). Quantification of nidAB RNA transcripts in E. coli was done by RT-QPCR which revealed an ~ 2- fold increase in RNA transcripts in one sample grown in the presence of IPTG. RNA to DNA ratio of nidAB calculated by RT-QPCR was increased between OD 600 0.1 to 0.4 and then declined at and OD 600 of 0.5. Relation of phenanthrene and pyrene mineralization to RNA/DNA ratio with the OD 600 from 0.1 to 0.5 showed the positive relationship between phenanthrene mineralization and the RNA/ DNA ratio while in case of pyrene only the OD 600 of 0.3 to 0.5 corresponds to the ratio. From the present study, it is concluded that soil is the rich source of microbes having the degradative capability of pollutants like PAHs. Some microbes have the ability to degrade and mineralize the PAHs efficiently while others can only transform but can’t mineralize. The expression and the RNA transcript of pyrene dioxygenase (nidAB) copy number and RNA/DNA ratio correspond to the mineralization of PAHs.