Biodegradation of p-nitrophenol by P. putida

A strain of Pseudomonas putida was found capable of metabolizing p-nitrophenol (PNP) as a sole source of carbon, nitrogen and energy. To explore the applicability of this strain for bioremediation for controlling environmental PNP pollution, its degradation potential at 300 and 500 ppm was examined in a medium devoid of carbon and nitrogen source (minimal medium). At A600, 0.5 OD inoculum, the strain metabolized 300 and 500 ppm within 36 and 72 h, respectively. The degradation was accompanied by release of stoichiometric amount of nitrite. Effect of glucose and nitrogen on PNP degradation under similar conditions revealed that (i) glucose (0.4 g/l) at 20 and 50 ppm PNP did not accelerate the rate of PNP degradation, while glucose (0.4 g/l) at 300 ppm PNP inhibited its degradation, (ii) nitrogen supplement viz. sodium nitrate and ammonium sulphate (0.04 and 0.4 g/l) in minimal medium with PNP (300 ppm) showed no effect on PNP degradation, while glutamate alone (0.04 and 0.4 g/l) showed mere rise in biomass (from 0.5 to 1.6 OD units), and (iii) acidic pH (4.0-6.5) did not support PNP degradation, while alkaline pH (7.5-9.5) significantly enhanced the rate of PNP degradation. The complete degradation of PNP at high concentration (300 ppm) was confirmed by HPTLC analysis. In order to probe root cause of higher PNP degradation, preliminary studies on genetic analysis of P. putida were undertaken, which revealed the prevalence of a degradative plasmid of approximately 15 kb, while cured derivatives of P. putida (PNP-) did not show ability to degrade PNP. Further conjugal transfer of PNP+ phenotype from P. putida to standard strain of E. coli Nova blue (PNP-) confirmed the degradative type of plasmid.