The Characteristics And Mechanism Of Dye Decolorization By Pseudomonas Sp.DY1 And Micrococcus Sp.BD15

Azo dyes and triphenylmethane dyes are extensively used in textile, food and paper industries, but considered to be toxic and recalcitrant. The pollution caused by the dyes is serious nowadays. The conventional physicochemical treatments are not only expensive but also may cause secondary pollution; as an eco-friendly method, the biological treatments are received more and more attentions. Some bacteria maybe have a great potential in application because these strains could grow quickly and may decolorize the dye efficiently. Studying the characteristics and mechanism of dye decolorization by these strains could be helpful for the bioremediation of the dye.In this research, Pseudomonas sp. strain DY1 and Micrococcus sp. strain BD15 were isolated from the soil with rotten woods and sludge in aeration tank; the characteristics of the decolorization was studied systematically and the optimal conditions of the decolorization was obtain by the statistical method; the mechanism of the decolorization was investigated by different analysis; and finally, the practical application potential of these two strains was confirmed by the immobilization and toxicology tests. The results of the reseach can be devided into the following parts:(1) The isolation and identification of dye-degrading bacteria:Pseudomonas sp. strain DY1 and Micrococcus sp. strain BD15 were isolated from the polluted soil and identified by the 16S rDNA and biochemical tests. The strain DY1 could decolorize both Acid Black 172 and Malachite Green efficiently, whereas strain BD15 could only decolorize Malachite Green efficiently.(2) The characteristics and optimization of Acid Black 172 decolorization by the strain DY1:It was observed that most of the tested nitrogen source enhanced decolorization, while carbon source inhibited or had no influence on decolorization. Efficient decolorization was still observed when high concentrations of Fe2+, Fe3+ and Ca2+ existed. Based on the above results, a Plackett-Burman design was used to select the operational parameters for decolorization of Acid Black 172. Four significant parameters, including pH, temperature, concentrations of Fe3+ and NaH2PO4, were optimized. A quadratic model obtained from the response surface design was constructed on experimental data. The optimal condition for decolorization was found to be pH 6.23,30℃,8.0 mM of Fe3+, and 10.0 g/L of NaH2PO4. The confirmatory experiments (86.0% decolorization percentage within the confidence interval) subsequently verified the accuracy of the experimental model. Moreover, the decolorization under the optimal condition fitted the logarithmic model well (R2= 0.964).(3) Biosorption mechanisms of the metal-complex dye Acid Black 172 by live and heat-treated biomass of strain DY1:Kinetic data for Acid Black 172 adsorption by strain DY1 fit a pseudo-second-order model. Increased initial dye concentration could significantly enhance the amount of dye adsorbed by heat-treated biomass in which the maximum amount of dye adsorbed was as high as 2.98 mmol/g biomass, whereas it had no significant influence on dye sorption by live biomass. As treated temperature increased, the biomass showed gradual increase of dye sorption ability. Experiments using potentiometric titration and FTIR indicated that amine groups (NH2) played a prominent role in biosorption of Acid Black 172. SEM, AFM and TEM analysis indicated that heat treatment of the biomass increased the permeability of the cell walls and denatured the intracellular proteins. The results of biosorption experiments by different cell components confirmed that intracellular proteins contributed to the increased biosorption of Acid Black 172 by heat-treated biomass.(4) The characteristics, degradation products and enzyme analysis of Malachite Green by strain DY1:The results showed that this strain demonstrated high decolorizing capability (90.3-97.2%) at high concentrations of MG (100-1000 mg/L) under shaking condition within 24 h. In static conditions, lower but still effective decolorization (78.9-84.3%) was achieved. The optimal pH and temperature for the decolorization was pH 6.6 and 28-30℃, respectively. Mg2+ and Mn2+(1 mM) were observed to significantly enhance the decolorization. The intermediates of the MG degradation under aerobic condition identified by UV-Visible, GC-MS and LC-MS analysis included malachite green carbinol, (dimethyl amino-phenyl)-phenyl-methanone, (methyl amino-phenyl)-phenyl-methanone, (amino phenyl)-phenyl methanone, di-benzyl methane and N, N-dimethylaniline. The enzyme analysis indicated that Mn-peroxidase, NADH-DCIP and MG reductase were involved in the biodegradation of MG.(5) The characteristics and optimization of Malachite Green by strain BD15: Optical microscope and UV-visible analysis were carried out to determine whether the decolorization was due to biosorption or biodegradation. Plackett-Burman design was employed to investigate the effect of various parameters on decolorization, and response surface method was then used to explore the optimal decolorization conditions. The results indicated that the decolorization by the strain was mainly due to biodegradation. Concentration of MG, urea and yeast extract, and inoculums size had significantly positive effect on decolorization, while concentration of CuCl2 and MgCl2, and temperature had significantly negative effect. The optimal conditions for decolorization were 1.0 g/L urea,0.9 g/L yeast extract,100 mg/L MG,0.1 g/L inoculums (dry weight), and at 25.2℃. Under the optimal conditions,96.9% MG was removed by the strain within 1 h, which was highly efficient microbial decolorization according to our knowledge. Moreover, the kinetic data for decolorization fit a second-order model well.(6) The degradation products of Malachite Green by strain BD15 and enzymes analysis:The results of UV-Visible, FTIR, GC-MS and LC-MS analysis indicated that the intermediates of the MG degradation by strain BD15 included:(dimethyl amino-phenyl)-phenyl-methanone, Michler's ketone, (methyl amino-phenyl)-phenyl-methanone, (amino phenyl)-phenyl methanone, 4-methylaminobenzoic acid,4-hydroxyl-N, N-dimethylaniline, N, N-dimethylaniline, hydroxyl-(dimethyl amino-phenyl)-phenyl-methanone and 4- hydroxyl-aniline. Based on the above results, the possible degradation pathway of MG was proposed. The enzymes analysis indicated that laccase and NADH-DCIP reductase were involved in the degradation of MG.(7) The cell immobilization and toxicology tests:The results indicated that the immobilized cells of strain DY1 and BD15 could be used repeatedly, and the decolorization efficiency was effective. The toxicity of the dye solution after decolorization was significantly decreased compared to before decolorization.