Multi-metal Resistance Mechanisms in Biofilm Forming Bacteria and Applications of Biofilm Associated Extracellular Polymeric Substances in Multi-metal Bioremediation

Abstract

The thesis elucidates the multi-metal resistance, 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, and 58 isolates were found to show resistance to ˃100 mg/L of all the metal ions. The biofilm screening of 58 isolates exhibited strong biofilm formation in 17 strains, moderate biofilm formation in 15 strains, weak biofilm formation in 21 strains, and the remaining isolates showed no biofilm formation. Out of the 17 strong biofilm formers, 8 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, Exiguobacterium indicum OMCW-10, Staphylococcus hominis BASS-10, Bacillus cereus BASW-3, Enterobacter cloacae BASW-16 and Pseudomonas chengduensis PPSS-4. These strains showed viable growth in the presence of Cr, Pb, and Cd and efficiently formed moderate to strong biofilm in different concentrations of multi-metal ions. In addition, these strains exhibited tolerance to various other heavy metals, including Ni, Zn, Cu, Mn, and Hg. 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; two-way ANOVA) compared to their planktonic counterparts. The biofilm culture of P. aeruginosa OMCS-1 exhibited greater removal of heavy metals, followed by P. chengduensis PPSS-4. The biomass of P. aeruginosa OMCS-1 showed higher removal of Cr, Pb, and Cd compared to P. chengduensis PPSS-4 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 (mt) for Pb and other metal resistance. The relative expression of these genes was significantly higher (p<0.05; one-way ANOVA) in biofilm mode and under different heavy metal concentrations. The adsorption behavior and interaction mechanisms of extracellular polymeric substances (EPS) of P. 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 and followed predominant monolayer adsorption and chemisorption mechanism. Thermodynamics of binding interactions between EPS-heavy metals were spontaneous (ΔG < 0). EPS-Cr(VI) and EPS-Pb(II) binding were exothermic (ΔH < 0), while EPS-Cd(II) binding was endothermic (ΔH ˃ 0) process. The enhanced rigidity of metal treated EPS along with the accumulation of Cr, Pb, and Cd, suggested the 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 sequestration. The hydroxyl, amide, carboxyl, and phosphate groups in EPS predominantly contributed to metal binding. The binding of metal ions altered the degree of stretching in the peptide chain, resulting in deviations in the secondary structure of EPS protein. A strong static quenching mechanism (Kq ˃ 2.0×1010 L M-1 s-1) was evidenced between the tryptophan protein-like substances in EPS and Cr, Pb, and Cd, with binding constants of 3.38 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, EPS sequestered heavy metals via the complexation with C-O, C-OH, C=O/O-C-O, and NH/NH2 groups and ion exchange by the –COOH 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 P. 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 the adsorption of tested metal ions. The declined porosity and enhanced rigidity of metal treated EPS Ca-alginate beads, along with the 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. The desorption study exhibited the reusability potential of immobilized EPS biosorbent after four cycles of adsorption-desorption reaction with significant decline (p<0.0001; one-way ANOVA) in the adsorption efficiency. However, the biosorbent efficiently adsorbed 61.52±0.13% of Cr, 70.27±0.12% of Pb, and 42.64±0.04% of Cd in the single metal system after the 4th adsorption cycle with regeneration efficiency of 72.2±0.45%, 78.65±0.6%, and 66.96±0.02%, respectively. Similarly, in the ternary metal system, the adsorption-desorption efficiency retained by the biosorbent was 35.41±0.2% and 51.44±0.98% for Cr, 51.58±0.15% and 63.98±0.24% for Pb, and 30.68±0.13% and 60.39±0.46% for Cd, respectively. Hence, the present study suggests that multi-metal resistant biofilm-forming bacterium P. aeruginosa OMCS-1 and secreted polymer (EPS) can be competently applied to remove heavy metals from multi-metal contaminated wastewater.