Optimization Of Biodegradation Of Polycyclic Aromatic Hydrocarbons (pahs) By Bacteria

Polycyclic aromatic hydrocarbons (PAHs) are a large group of organic compounds mainly consisting of benzene rings which have become the common threat to our environment as the grievous pollutants.The only cost effective and eco-friendly way to remove these pollutants from the environment is their degradation via microbes. In the present study bacterial cultures were isolated for degradation of Naphthalene (Nap) and Phenanthrene (Phe) representing low molecular weight (LMW) PAHs, Fluoranthene (Fla) representing high molecular weight (HMW) PAH and Iranian light crude oil (ILCO) representing mixture of hydrocarbons. Forty-four bacterial strains (CMG2001-CMG2044) were isolated from water and mud samples enriched on Nap. Two bacterial consortia HP and LP and one bacterial strain (CMGCZ) of LP consortium were isolated from oil contaminated soil samples by enrichment on Fla. Bacterial strains CMG2001-CMG2044 were screened for growth on Nap, Phe and Fla by 96 well microtiter plate assay. Among them eighteen bacterial strains exhibited growth on one or more tested PAHs while two of them (CMG2028 and CMG2042) were selected for further studies on PAHs degradation.CMG2028 and CMG2042 were identified by 16S rRNA gene sequencing as Kocuria flavus and K. rosea, respectively. In minimal medium 36% and 53% Nap (500mg l-1) was degraded in ten days of incubation by K. rosea CMG2042 and K. flavus CMG2028, respectively.Addition of yeast extract (YE) in medium as an additional carbon source resulted in enhanced degradation (59%) of Nap in K. rosea CMG2042 and reduced degradation (45%) in K. flavus CMG2028 within ten days.Although both the strains exhibited growth on Phe (10mg l-1) and Fla (10mg l-1) in YE added and omitted medium but only Phe (9%) was degraded by K. rosea CMG2042 as a sole carbon source.K. flavus CMG2028 and K.rosea CMG2042 exhibited growth on YE added and omitted minimal agar plates coated with ILCO and their colonies accumulated oil but did not grow in liquid medium with 0.5% ILCO. Bacterial strain CMGCZ, isolated from LP consortium, was identified as Rhodococcus erythropolis by 16S rRNA gene sequencing. R. erythropolis CMGCZ formed clear zones on Fla sprayed minimal and LB agar plates.In minimal medium degradation of Nap (500mg l-1), Phe (100mg l-1) and Fla (100mg l-1) by R.erythropolis CMGCZ in one week of incubation was 13.2%, 13.1% and 99.3%, respectively however YE addition in medium resulted in complete inhibition of Nap degradation, slightly enhanced degradation of Phe (14.8%) and a more rapid degradation of Fla (100%). R.erythropolis CMGCZ was capable of growing on 1% ILCO in liquid medium and degraded 13.2% and 11% aiphatic fraction of ILCO in YE added and omitted medium, respectively. LP and HP consortia enriched in minimal medium (HPMO/LPMO) and in YE added medium (HPMM/LPMM) were tested for degradation of Fla (100mg l-1).LPMM and HPMM consortia degraded 100% and 25.5% Fla, respectively in YE added medium in twenty days. In minimal medium 51.5% Fla was degraded by LPMO consortium but HPMO consortium failed to degrade Fla.Further subculturings of both the consortia in YE added medium resulted in improved Fla degradation by LPMM consortium but Fla degradation by HPMM consortium ceased. LPMM consortium degraded 98.6% and 95.7% Fla in YE added and omitted medium, respectively within a week of incubation.When LPMO consortium was incubated longer (35 days) in minimal medium without further transfer it degraded 97.6% Fla in a week which proved that longer incubation compensated deficiency of additional carbon source.Comparison of aromatic ring dioxygenase expressing bacteria (ARDB) of both the consortia revealed that increase in ARDB was observed only in LPMM and longer (35 days) incubated LPMO consortium.LPMM consortium was proved to be completely bacterial by adding microeukaryotic and different prokaryotic inhibitors in the growth medium. The optimized temperature and pH of the medium for LPMM consortium were found to be 30Ċ and 7.0, respectively.Degradation optimization for Fla concentrations ranging from 100mg l-1 to 1000mg l-1 with the difference of 150mg l-1 Fla resulted in 98.8%, 94.6%, 85.6%, 77.5% and 44.6% Fla degradation by LPMM consortium, respectively.Fla degradation optimization by R. erythropolis CMGCZ for same concentrations of Fla resulted in 100%, 100%, 57.2%, 19.1% and 12.6% degradation of Fla, respectively. PCR amplification of Rieske [Fe2-S2] center of PAH dioxygenase genes resulted in 100bp PCR product in HPMM and LPMM consortia, R. erythropolis CMGCZ and only one type of blue colony (CMGBL) in ARDB population of LPMM consortium.Amplified PCR product of R.erythropolis CMGCZ exhibited homologies at nucleotide and deduced amino acid level mainly with Rieske [Fe2-S2] domain protein of Mycobacterium species and pahAC gene of uncultured bacterium clones, known for degradation of PAHs.Bacterial strains and LP consortium isolated in the present study are efficient degraders of different PAHs and promising candidates for use in bioremediation operations at hydrocarbons contaminated sites