Decolorization Of Azo Dye Congo Red By White Rot Fungus Trametes Pubescens

Recently, synthetic dyes extensively used in many industries can be found in wastewaters. Such effluents lead to a reduction in sunlight penetration, which in turn decrease photosynthetic activity, dissolved oxygen concentration, and water quality, and have acute toxic effects in aquatic flora and fauna, causing severe environmental problems worldwide. Cleaning up of the environment by removal of hazardous contaminants from textile effluents is a crucial and challenging problem needing numerous approaches to reach long-lasting suitable solutions. Accordingly, main objectives of the present study were:(i) to screen the white rot fungal strain with higher dye decolorization capability,(ii) to study the biodegradation and adsorption during decolorization process and (iii) to purify the extracellular laccase from the target strain by a three-step method.The experimental results displayed that, among the42white rot fungal strains, Trametes pubescens Cui7571was the target strain with higher decolorization capacity for azo dye Congo Red. During the decolorization process, the strain’s biomass could be continuously utilized for three times with declining decolorization rates. The biodegraded metabolites of azo dye Congo Red were identified as naphthalene amine, biphenyl amine, biphenyl and naphthalene diazonium by fourier transform-infrared spectroscopy (FT-IR) and gas chromatography-mass spectroscopy (GC-MS). Phytotoxicity studies revealed that the dye biodegradation by T. pubescens resulted in its detoxification.During the liquid cultivation of T. pubescens, increases in the contents of malondialdehyde (MDA)(P<0.01), hydrogen peroxide (H2O2)(P<0.05) and ascorbic acid (AA)(P<0.05) were observed. The higher activities of superoxide dismutase (SOD)(P<0.05) and total antioxidative capacity (T-AOC)(P <0.01) still could be detected during this period. However, higher restraining ability to hydroxyl free radical (RAHFR)(P<0.05) and catalase activity (CAT)(P<0.01) had negative influence on laccase activity. It was concluded that a relationship between laccase synthesis and antioxidative capability exists in fungal cells, which can be regulated by reactive oxygen.Extracellular laccase (Tplac) from T. pubescens was purified to homogeneity by a three-step method, which resulted in a high specific activity of18.543U/mg,16.016-fold greater than that of crude enzyme at the same level. Tplac is a monomeric protein that has a molecular mass of68.0kDa. The enzyme demonstrated high activity toward1.0mmol/L2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS) at an optimum pH of5.0and temperature of50℃, and under these conditions, the catalytic efficiency(kcat/Km) is8.34s-1μM-1. Tplac is highly stable and resistant under alkaline conditions, with pH ranging from7.0to10.0. Interestingly, above88%of initial enzyme activity was maintained in the presence of metal ions at25.0mmol/L, leading to an increase in substrate affinity, which indicated that the laccase is highly metal-tolerant. These unusual properties demonstrated that the new fungal laccase Tplac has potentials for the specific industrial or environmental applications.In order to improve the stability and recovery, chitosan as carrier and glutaraldehyde as cross-linking agent were employed for immobilization of T. pubescens purified laccase Tplac. During the immobilization process, optimal concentration of cross-linking agent glutaraldehyde, cross-linking time, enzyme solution and immobilized time were0.8%(v/v),3h,2.0mL (about43.672U/mL) and4h respectively. Additionally, pH adaptability and thermal stability of immobilized Tplac were much higher than those of free laccase. These findings promoted the development of immobilized laccase Tplac for environmental and other fields.Meanwhile, a novel design of dye decolorization was conducted with the sorbents obtained from T. pubescens biomass in submerged cultivation. The results demonstrated that the sorbents prepared by heat treatment (121℃,20min) exhibited preferable decolorization capacity for azo dye Congo Red. Moreover, the decolorization rate-dependent influencing factors, i.e. salinity, Tween80, temperature, pH and dye concentration, were optimized by Box-Behnken full factorial design. When they were1.1%(w/v),3.5%(v/v),41℃,6.2and114.3mg/L respectively, the decolorization rate was up to99.23%after a7-day incubation period. Adsorptive decolorization was caused by the electrostatic forces between dye molecules and sorbents obtained from T. pubescens biomass, as evidenced by FT-IR, chemical modifications and scanning electron microscope (SEM) data.The T. pubescens biomass immobilized by sodium alginate or macroporous resin D201enhanced the sorption capacity for azo dye Congo Red approximately4-fold of the free biomass. Dye uptake was favored by acidic conditions at pH2.0with increasing initial dye concentration up to100mg/L using less biomass at room temperature and agitation speed. Adsorption of dyes onto the biomass was weakly dependent on ionic strength. Additionally, the adsorption process followed the pseudo-second-order kinetic and Freundlich isotherm models. During this process, the morphological changes on the biomass surface occurred, the amine functional groups present on the cell surface were mainly responsible for this process, and reduction in crystallinity of the biomass was observed, as confirmed by SEM, FT-IR and XRD respectively. Desorption experiments were performed to regenerate the sorbent, making the process more economic and environment friendly.Compared to the general treatment with fresh biomass, no dramatic variations were observed from the dye removal efficiencies by T. pubescens biomass without addition of nutrition. Thus, the results indicated that the treatment by fungal biomass without addition of nutrition performed well at lowering the capital and running cost, and enhancing convenient availability for dye decolorization. During the decolorization process, the biomass presented good persistence in repetitive decolorization operations, as well as high potentials towards the biodegradation of dyes which could be attributed to the presence of the biotransformation enzymes. After a7-day incubation period, a higher dye removal efficiency of80.52%was obtained with the optimal parameters initial pH at2.0, temperature at30℃, dye concentration at80mg/L and salinity at2.5%(w/v) respectively. In addition, the biodegraded metabolites were identified as naphthalene amine, biphenyl amine and naphthalene diazonium by GC-MS. Phytotoxicity studies displayed the less toxic nature of the biodegraded metabolites with respect to plant seeds.In this study, white rot fungus T. pubescens was observed to decolorize azo dye Congo Red more efficiently. During the decolorization process, the two simultaneous steps, adsorption between the biomass and the macromolecular dyes and enzymatic biodegradation, were elucidated systematically. The results may facilitate a better application of pollution-free treatment of various dyes and support the utilization of fungi in many industrial fields.