Biofilm Modulation and Catabolic Gene Expression in Marine Bacteria for Biodegradation of Polycyclic Aromatic Hydrocarbon

Abstract

This thesis illustrates the biofilm formation of marine bacteria and biofilm-mediated degradation of phenanthrene. Sediment samples were collected from Paradip Port and Chilika Lake, Odisha coast, India. A total of 132 marine bacterial strains were isolated by selective enrichment with 100 mg/L phenanthrene. Biofilm formation was observed in 69 marine bacterial isolates, out of which 41 formed weak biofilms, 23 formed moderate biofilms, and 5 isolates formed strong biofilms. Among the 28 moderate to strong biofilm formers, 8 strains harbored the nahAc gene responsible for the catabolism of phenanthrene. Biochemical and molecular identification grouped the isolates into Pseudomonas and Brucella genera. Scanning electron micrographs (SEM) analysis of the bacterial biofilms revealed rod-shaped cells glued together within a slimy matrix of extracellular polymeric substances (EPS). Chemotaxis movement towards phenanthrene was observed in 4 marine bacterial isolates, Pseudomonas mendocina AS2-P3, Pseudomonas aeruginosa AS2-P13, Pseudomonas guguanensis OS-P8, and Pseudomonas aeruginosa PFL-P1. The degradation of phenanthrene was significantly higher in the biofilm mode of growth than in the planktonic counterparts (p<0.05). The highest degradation of ~74% phenanthrene (100 mg/L) was achieve within 5 d using biofilm cultures of P. aeruginosa PFL-P1 grown on glass beads. The biofilm-EPS of P. aeruginosa PFL-P1 exhibited high emulsification activity against different hydrophobic compounds. Fluorescence spectroscopy analysis revealed the presence of two protein-like fluorophores at (Ex/Em) wavelengths of 280/351 nm and 220/350 nm. The fluorescence intensity of the fluorophore gradually decreased with the increasing concentration of phenanthrene. The high binding constant (4.42 L/mol) and binding site number (1.38) indicated strong interaction between EPS and phenanthrene. Biofilm formation in P. aeruginosa PFL-P1 evaluated on glass, polystyrene, steel, ceramic and rubber substrata revealed maximum arithmetic mean biofilm height of 611 nm on the ceramic substratum. Analysis of the bare substratum indicated the highest surface roughness of 545 nm on the ceramic substratum. The increased roughness promoted biofilm growth, and the biofilm biomass increased to ~18 μm3/μm2 on ceramic substratum with a maximum biofilm thickness of ~50 μm in the aqueous state. Molecular docking studies revealed that 3OC8-HSL interacted with LasR with a binding energy of -10.19 kcal/mol, and C6-HSL bound to RhlR with a binding energy of -8.59 kcal/mol. Exogenous addition of 10 μM C6-HSL and 3OC8-HSL improved the biofilm formation ability in P. aeruginosa PFL-P1. Significant improvement in cell surface hydrophobicity, auto-aggregation ability, swimming, and swarming motility was observed in the presence of AHLs (p<0.05). The biofilm cultures of P. aeruginosa PFL-P1 grown over ceramic beads achieved 85% degradation of 100 mg/L phenanthrene within 5 d. The phenanthrene degradation ability of the bacterium increased to 97% in the presence of 10 μM 3OC8-HSL with a degradation rate constant of 0.7027/d and t1/2 of 0.986 d. Exogenously supplemented AHLs significantly improved the relative expression of nahAc, pslB, lasI, and rhlI genes in P. aeruginosa PFL-P1 (p<0.05). Further, next-generation sequencing of P. aeruginosa PFL-P1 revealed the presence of 145 genes involved in xenobiotics biodegradation and metabolism. Common gene clusters, benABCD, xylXYZ, and catAB, playing crucial roles in the degradation of toxic aromatic compounds, were present in the genome. Metabolic pathway analysis revealed P. aeruginosa PFL-P1 utilized both the naphthalene route as well as the phthalic acid route for the degradation of phenanthrene. It also encodes several catabolic genes for the degradation of naphthalene, benzoate, aminobenzoate, fluorobenzoate, toluene, xylene, styrene, atrazine, caprolactam, etc., which indicate that this biofilm-forming marine bacterial strain can adapt to heavily contaminated environments and is a potential organism to be used for bioremediation purposes.