Seminar Title
Multi-metal resistance mechanisms in biofilm-forming bacteria and applications of biofilm associated extracellular polymeric substances in multi-metal bioremediation
Seminar Type
Synopsis Seminar
Life Science
Speaker Name
Monika Priyadarshanee ( Rollno : 517ls2005)
Speaker Type
Life Science Seminar Room
Date & Time
28 Nov 2023 05.00 PM
Prof. Surajit Das

The thesis elucidates the multi-metal resistance potential, biofilm-forming ability, and enhanced heavy metal removal efficiency of bacteria isolated from metal contaminated sites. Soil, water, and sediment samples were collected from the Sukinda chromite mine and Paradip Port, Odisha, India. A total of 93 bacterial strains were isolated from chromium (Cr), lead (Pb), and cadmium (Cd) supplemented medium. Among them, 58 isolates showed resistance to ˃100 mg/L of all the metal ions. The biofilm screening of 58 isolates exhibited strong biofilm formation by 17 strains, moderate biofilm formation by 15 strains, weak biofilm formation by 21 strains, and no biofilm formation by 5 strains. Out of the 17 strong biofilm former, 8 bacterial strains exhibited tolerance to high concentrations of Cr, Pb, and Cd, i.e., ˃500 mg/L. The potent multi-metal resistant biofilm-forming bacterial strains were identified as Pseudomonas aeruginosa OMCS-1, Staphylococcus sp. OMCS-4, Bacillus cereus OMCS-20, ExiguobacteriumindicumOMCW-10, Staphylococcus hominis BASS-10, Bacillus cereus BASW-3, Enterobacter cloacae BASW-16 and Pseudomonas chengduensisPPSS-4. Scanning electron microscopy (SEM) unveiled closely aggregated bacterial cells embedded within the EPS matrix. Confocal laser scanning microscopy (CLSM) exhibited different biofilm components, providing a three-dimensional structure to the biofilm. The Cr, Pb, and Cd removal efficiency of bacterial strains in biofilm mode was significantly greater (P<0.0001) compared to their planktonic counterparts. The biomass of the bacteria P. aeruginosa OMCS-1 followed by. chengduensis PPSS-4 showed higher removal of Cr, Pb, and Cd at 37°C and pH 6 within 4 h of contact time. The bacterium P. aeruginosa OMCS-1 possesses multiple metal resistance genes, including chrA and chrR for Cr resistance, cadA and cadR for Cd resistance, and metallothionein, for Pb and other metal resistance. The relative expression of these genes was significantly higher (P<0.05) in biofilm mode and under different heavy metal concentrations. The adsorption behavior and interaction mechanisms of extracellular polymeric substances (EPS) of a biofilm-forming bacterium, Pseudomonas aeruginosa OMCS-1, towards Cr, Pb, and Cd were investigated. EPS-covered (EPS-C) cells exhibited significantly higher (P<0.0001 two-way ANOVA) removal of Cr (85.58±0.39%), Pb (81.98±1.02%), and Cd (73.88±1%) than the EPS-removed (EPS-R) cells, following predominant monolayer adsorption and chemisorption mechanism. Thermodynamics of binding interactions between EPS-heavy metals was spontaneous (&DeltaG < 0), primarily driven by enthalpy change (|&DeltaH| ˃ |T&DeltaS|) for both Cr(VI) and Cd(II) occurred via outer sphere complex formation, while driven by entropy change (|&DeltaH| < |T&DeltaS|) for Pb(II) through inner-sphere complex formation. The enhanced rigidity of metal treated EPS along with the accumulation of Cr, Pb, and Cd, suggested biosorption of metal ions onto EPS. The significant increase (P<0.001 one-way ANOVA) in the zeta potential of EPS after interaction with Cr, Pb, and Cd inferred the involvement of electrostatic interactions in metal binding. The unchanged crystallinity (CIXRD = 0.13) and no additional crystalline peaks in the metal treated EPS specified that complexation was the prevalent mechanism in metal ions sequestration. The hydroxyl, amide, carboxyl, and phosphate groups in EPS predominantly contributed to metal binding. Binding of metal ions altered the degree of stretching in the peptide chain, resulted in deviations in the secondary structure of EPS protein. A strong static quenching mechanism (Kq ˃2.0×1010 L mol-1 s-1) was evidenced between the tryptophan protein-like substances in EPS and Cr, Pb, and Cd, with binding constants of 3.45 M-1, 3.0 M-1, and 2.81 M-1, respectively. Cr 2p, Pb 4f, and Cd 3d peaks in Cr, Pb, and Cd loaded EPS confirmed the sequestration of metal ions by EPS. In addition, heavy metals were sequestered by EPS via the complexation with C-O, C-OH, C=O/O-C-O, and NH/NH2 group, and ion exchange by the &ndashCOOH group. Further, a multifaceted experimental design, including factorial design, Face-centered composite design (FCCD), and mixture design, was implemented to explore the competitive interaction and adsorption behavior of Cr, Pb, and Cd by the immobilized EPS-based biosorbent of Pseudomonas aeruginosa OMCS-1, in single as well as ternary metal solution. The prepared biosorbent preferentially adsorbed Cr (47.6 mg/g), Pb (46.38 mg/g), and Cd (42.02 mg/g) in the single metal system, and Pb (43.32 mg/g), Cr (40.03 mg/g) and Cd (35.9 mg/g) in the ternary metal system. The uptake behavior of all the metal ions was successfully represented by the Freundlich isotherm model (R2 ˃ 0.988), confirming the multilayer adsorption of tested heavy metal. The rate of adsorption of metal ions followed the second-order kinetics (R2 ˃ 0.997), validating chemisorption as the predominant mechanism in adsorption of tested metal ions. The declined porosity and enhanced rigidity of metal treated EPS Ca-alginate beads, along with accumulation of Cr, Pb, and Cd, suggested the adsorption of metal ions onto the immobilized biosorbent. The hydroxyl, amine, carboxyl, and phosphate functional groups of the formulated biosorbent contributed to the Cr, Pb, and Cd sequestration. Desorption study exhibited the reusability potential of immobilized EPS biosorbent after 4 cycles of adsorption-desorption reaction with significant decline (p<0.0001 one-way ANOVA) in the adsorption efficiency. Hence, the present study suggested that this multi-metal resistance biofilm-forming bacterium and its secreted polymer can be competently applied to remove heavy metals from multi-metal contaminated aqueous solution.