Bioremediation potential of biofilm forming lead resistant and naphthalene utilizing marine bacteria

Abstract

This thesis illustrates the potential of biofilm forming marine bacteria in bioremediation of naphthalene and lead. Five bacterial isolates viz. Lysinibacillus fusiformis NCW903, Pseudomonas aeruginosa N6P6, Sporosarcina luteola NR402, Pseudomonas mendocina NR802 and Pseudomonas pseudoalcaligenes NP103 were selected on the basis of naphthalene utilization and lead resistance potential. Among these five isolates, P. aeruginosa N6P6 showed highest tolerance towards Pb with a MIC of 1000 ppm followed by P. pseudoalcaligenes NP103 and P. mendocina NR802 having MIC of 750 ppm and 600 ppm. P. aeruginosa N6P6 and P. pseudoalcaligenes NP103 was able to form biofilm in presence of 1000 ppm naphthalene. The biofilm formation ability of P. aeruginosa N6P6 was increased significantly with increase in naphthalene concentration (P<0.05). The biofilm formation was found to increase significantly with increase in Pb(II) concentration upto 400 ppm in P. aeruginosa N6P6 and L. fusiformis NCW 903 (P<0.05). No significant effect of naphthalene was observed on the biochemical composition of biofilm components i.e. lipids, proteins (PN) and polysaccharides (PS) of P. aeruginosa N6P6 (P>0.05). Maximum degradation of naphthalene was observed by biofilm culture of P. aeruginosa N6P6 which degraded 99.4±0.002% naphthalene in 20 h followed by P. pseudoalcaligenes NP103 (90±1.22% naphthalene degradation). The GC-MS analysis of metabolic intermediates accumulated after 24 h of naphthalene degradation by P. aeruginosa N6P6 and P. pseudoalcaligenes NP103 and bioaugmented soil microcosm indicated the presence of catechol pathway in both the isolates. SEM analysis showed that the surface morphology of P. aeruginosa N6P6 biofilm was not significantly affected by Pb(II). However, shrinked and damaged cells were observed in the biofilm of P. pseudoalcaligenes NP103 and P. mendocina NR802 grown in presence of Pb(II). AAS analysis indicated that the Pb(II) bioaccumulation potential of biofilm culture was significantly higher than planktonic culture (P<0.05). The maximum percentage of Pb(II) was found to be accumulated inside the biofilm-EPS matrix of P. aeruginosa N6P6 (68.5%) followed by P. pseudoalcaligenes NP103 biofilm matrix which trapped 57.2% of Pb(II). The interaction of biofilm-EPS and Pb(II) was quantified using Stern-Volmer equation. The ∆G value of P. aeruginosa N6P6 (-6.1 kJ/K/mol) and P. pseudoalcaligenes NP103 (-2.78 kJ/K/mol) EPS was highly negative indicating that the EPS interacts with Pb(II) spontaneously at room temperature (298K). The maximum Pb(II) biosorption capacity (qm) of the EPS alginate biosorbent was significantly higher (416.67 mg/g) than that of alginate biomass (232.55 mg/g) and pristine alginate biosorbents (120.48 mg/g) (P<0.05; One way ANOVA). Presence of genes conferring lead resistance (bmtA) and naphthalene catabolizing ability (ndo) was detected in P. aeruginosa N6P6. Both the ndo and bmtA gene showed significant upregulation with increase in naphthalene and Pb(II) concentration respectively. The role of QS system in regulation of naphthalene catabolizing gene ndo and Pb(II) chelating gene bmtA was established by using a potent quorum sensing inhibitor tannic acid.