The initial reactions in the degradation of anthracene and phenanthrene are catalyzed by multicomponent enzyme dioxygenases that incorporate both atoms of molecular oxygen into the PAHs nucleus to produce dihydrodiols (Akhtar et al., 1975; Jerina et al., 1976). Enhanced degradation of PAHs was accomplished by Rhodococcus baikonurenis EN3 bacterial strain (Lee et al., 2006). Genes involved in PAH metabolism and their regulation have been described for Sphingomonas and Nocordioides (Menn et al., 1993; Sanseverino et al., 1993; Yang et al., 1994; Zylstra et al., 1994; Saito et al., 2000). The degradation of PAHs by bacteria, isolated from a beach, was examined by Rolling et al. (2004).

In the present study, soil samples were collected from the Mumbai coastal area and bacterial colonies were isolated using the enrichment culture technique (Li et al., 2000). Among the resulting isolates, the Pseudomonas strain was used for phenanthrene and anthracene degradation.

The chemicals used were of the highest purity and were obtained from Aldrich, Merck and Ranbaxy. The minimal salt medium (MSM) used for enrichment of PAHs degrading bacteria contained 1.0 gm K2HPO4, 1.0 gm KH2PO4, 1.0 gm NH4NO3, 0.02gm CaCl2, 0.05gm FeCl3 and 0.2 gm MgSO4 in 1 litre of distilled water (Lal and Khanna , 1996). The pH was adjusted to 7.0. Substrates were provided as the sole source of carbon and energy. A fifteen microliter aliquots of phenanthrene or anthracene in N, N Dimethylformamide (12mg/ml) were added to each flask for enzyme induction. MSM media containing the substrates were sterilized and incubated for 14 days at 200 rpm and 37°C. The contents of each flask were extracted with methanol. The residues were dissolved in 3 ml of methanol for analysis by HPLC.

Anthracene, phenanthrene and their metabolites were separated by HPLC using Hewlett- Packard model 1050 pump system with a Hewlett-Packard diode array model 1040. A detector at 254 nm and a 4.6, by 250 mm 5 μm C18 inertsil ODS-3 column at a flow rate of 1 ml/minute. The compounds were eluted using a linear gradient of 40 to 95% CH3OH/H2O over 40 minutes. GC-MS analyses were performed on a 5 model 4500 quadrupole mass spectrometer and a model 3400 Gas chromatography using DB-1 fused silica capillary column. N2 was used as the probe and bath gas, with an ion source temperature of 150°C. Positive or negative ion spectra were acquired in full scan mode (m/z of 100 to 400, 1.Os- cycle time) in series with a UV detector at 254 nm.Identification of phenanthrene degradation products
HPLC analysis of the neutral extract from phenanthrene incubation produced two metabolites. The EI mass spectrum of compound I, eluting at 21.4 with λmax of 201and 278nm, contained a base peak at an m/z of 212, the molecular ion (M+). Fragment ions at m/z values of 194 (M-18) +, 166 (M-18-28) + and 165 (M-18-29) + are characteristic of a dihydrodiol. The mass spectra are consistent with previously reported 9, 10 dihydroxy phenanthrene (Narro et al., 1992).

The EI mass spectrum of compound II (22.3min, λmax 202,262,270 nm) had a molecular ion at an m/z of 212 and characteristic dihydrodiol fragment ions at m/z values of 194, 166 and 165. Additional fragment ions at m/z values of 168 and 140 were present at intensities lower than in the mass spectrum of the first metabolite, indicating that compound II was a different dihydrodiol. The compound was identified as 3, 4 dihydroxyphenanthrene. The parent compound phenanthrene was eluted at 41.9 min. (Joanna et al., 2001). The HPLC profile of phenanthrene and its metabolites are shown in Figure 2(a).

Identification of anthracene degradation products
Analysis of the neutral extract from anthracene incubation produced 4 metabolites that were eluted at 22.8 min., 27.5min, 34.4 min., and 34.8min. The EI mass spectrum of compound I (retention time 22.8 min, λmax 206,25,296,308) from the neutral extract of anthracene consisted of a molecular ion at an m/z of 212 and characteristic fragment ions at m/z values of 194,166,165 resulting from losses of H2O and then either CO or HCHO. The compound was identified as 1, 2 dihydroxyanthracene by mass spectrum and comparison with data previously published (Akhtar et al., 1975; Jerina et al., 1976; Cerniglia and Yang, 1984; Sutherland et al., 1992).

Compound II was eluted at 27.5min. and had λmax values of 202,234,276,284 and 327nm. Its EI mass spectrum consisted of a molecular ion at an m/z of 196 and fragment ions at m/z values of 168 and 140 resulting from consecutive losses of CO. The compound was identified as 6, 7 benzocoumarin by comparing the results with the data previously published (Pouchert and Behnke, 1993).

Compound III was eluted at 34.4 min. with λmax of 204 and 267 nm. The EI mass spectrum of compound III had a molecular ion at m/z of 224 and strong fragment ions at m/z values of 209,181 and 159 that may be attributed to sequential losses of CH3 and CO. Metabolite III was identified as 1-methoxy -2- hydroxy anthracene by comparing result with the previous data (Joanna et al., 2001).

Compound IV (retention time 34.4 min. with λmax 203,260 and 332nm) had an EI molecular ion at an m/z of 208 and fragment ions at m/z of 180 and 152 resulting from successive losses of CO. The compound was identified as 9, 10-anthraquinone by comparing it to previously published data (Sutherland et al., 1992).

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22. Pathak H, Jain PK, Jaroli DP, Lowry ML (2008) Degradation of Phenanthrene and Anthracene by Pseudomonas Strain, Isolated From Coastal Area. Bioremediation Journal. 12:111-116