STUDIES ON THE MICROBIAL APPROACH FOR THE BIOREMEDIATION OF AZO DYES

Abstract

Mycoremediation of textile effluents is a practical and eco-friendly approach for mineralizing synthetic dyes. The textile, food processing, chemical, leather, dyestuff, dyeing, and pharmaceutical industries discharge significant amounts of effluents including different types of dyes. One of the largest manufacturers of dyes is the textile industry, and dyes discharged from these industries directly affect the natural ecosystem by entering in to the soil and water. These industries release effluents that are carcinogenic and pose a potential threat to flora and fauna. Chapter 1 and 2 serves as an introductory gateway to our study, unveiling the promising potential isolated strains in the realm of dye degradation applications. Within this chapter, we not only provide a comprehensive background but also articulate the driving motivation and the clear objectives that underpin our research endeavors. The literature survey that maps out the historical milestones and key events in the evolution and adoption of bioremediation approaches for the treatment of dye wastewater. This chapter serves as a historical chronicle of the journey towards the discovery and implementation of these transformative methods. In Chapter 3, we provide a detailed account of the screening process conducted on fungal isolates capable of azo dye degradation. Isolate, identified as Lentinus squarusulous AF5 (MN215236.1) through ITS-based sequencing, was found to exhibit the production of both extracellular and intracellular ligninolytic enzymes for the decolorization and degradation of dyes. We also elaborate on the optimization of various physico-chemical parameters aimed at achieving the highest level of dye decolorization. Distinct fungal species are identified as dye decomposers because these can produce a variety of extracellular enzymes involved in catabolism. The strain isolated from the dye effluents was identified as Lentinus squarrosulus AF5 and had a remarkable ability for decolorization (50-96%) within 72 h using a dye mix consisting of AB10B, RB5, and RB160 (500 mg L-1). This optimization resulted in the attainment of optimal levels for key variables, including Manganese Peroxidase at 258.84 ± 0.001 IU/ml, Lignin Peroxidase at 194.98 ± 0.002 IU/ml, and Laccase at 134.33 ± 0.007 IU/ml of enzymes. In order to assess the degradation, the extractants were analysed using chromatographic and spectroscopic analysis using UV–vis, HPLC, and FTIR, 1H NMR had shown the degradation of the dyes. Chapter 4 focuses on the detailed review of biochemical and molecular factors from isolated Lentinus squarusulous AF5. Proteomics analysis was undertaken to identify differential expression of proteins with the strain incubated with dye mix (500 mg L-1). Mycelial and secretome protein profiles were analysed to find out the distinct factors that may regulate and contribute to the dye remediation. The dynamics of the protein profile was deciphered using gel-free/label-free nLC-MS/MS analysis. Distinct protein profiles in the mycelial extract of Lentinus squarrosulus AF5 were observed following exposure of the strain with dyes mix. About 90 proteins have been depicted by proteomic analysis wherein 10 proteins appear to be differentially expressed during dye treated conditions. Secretome proteome had denoted 13 proteins under similar conditions. Among the major categories of the factors, the differential patterns of expression were mainly observed for the metabolic process (30.77 %), catalytic activity (21.05%) and for the metal ion binding activity (26.32%). Besides, demarcations at chromosomal (20%) and nuclear levels (20%) and the distinct variation was observed for the cellular components. Furthermore, the in silico analysis of the enzyme's interaction with a specific compound revealed a robust binding affinity, suggesting a potential pivotal role of this interaction in the degradation of azo dyes. The molecular docking simulations were employed to elucidate the biodegradation of the dyes. The modeled structure of laccase (UniProt Id: Q5EBY5) and heme peroxidase (UniProt Id: A0A1Q3E2I9) were used for molecular docking with the three considered dye molecules including Amido Black 10B, Reactive Blue 160, and Reactive Black 5. Molecular docking analysis revealed significant binding of three azo dyes, Amido black 10B (AB10B), Reactive black 160 (RB160), and Reactive Black 5 (RB5) at the catalytic site of laccase (Q5EBY5) with significant binding energies of -7.6 kcal/mol, -7.4 kcal/mol and -6.9 kcal/mol respectively. Further interaction analysis revealed that Amido black 10B formed 3 hydrogen bonds with Gly101, Val402, and Gln441. The interactions of Reactive black 160 (RB160) had demarcated 7 hydrogen bonds with Lys71, Asp443, His401, Val402, Thr308, and Ser227. Reactive Black (RB5) upon docking with laccase had resulted in 4 hydrogen bonds with Asp443, Phe440, Thr308, and Ser227. Molecular docking analysis of A0A1Q3E2I9 heme peroxidase of Lentinula edodes resulted in high negative binding energy of -7.7, -6.4 for Amido Black 10B, Reactive Black 5. Further interaction analysis revealed that Amido black 10B formed 4 hydrogen bond with Arg980, Tyr622, Gln685, Ile03. The interactions of Reactive Black 5 had demarcated 9 hydrogen bond with Thr818, Ile803, Gln685, Asn811, Tyr622, Asp1061, Ser807. Chapter 5 depicts the proposed pathway of dye degradation and their phytotoxicity and cell toxicity analysis. LC-MS analysis have shown the putative degradation intermediates. Two agriculturally important plants Guar (Cyamopsis tetragonoloba) and wheat (Triticum aestivum) were used for evaluating the degree of toxicity of azo dyes and its degraded products. Toxicity analysis of the metabolites was performed using seed germination and plant growth on two agriculturally important plants Guar (Cyamopsis tetragonoloba) and wheat (Triticum aestivum) as well as cytotoxicity analysis using the human keratinocyte cell line (HaCaT). The dye mix appeared inhibitory for seed germination (20-40%), whereas metabolites were non-inhibitory for germination. Treatment of HaCaT cells with of dye mix and metabolites led into 45% and ~100% of cell viability of HaCaT cells respectively. Therefore, metabolites following degradation of the dye mix were observed as non-toxic. These observations suggest Lentinus squarrosulus AF5 to be a potential strain for the catabolism of azo dyes.