| In recent years, China has become the world’s biggest country in production, trade and consume of dye with the rapid development of dye industry. Dye wastewater always have deep color, large alkaline and high content of organic pollutants and big changing of water quality. In addition, most dyes are carcinogenic, teratogenic and mutagenic. Therefore, dye wastewater has become one of the most important pollution sources. Currently, the treatment methods of dye wastewater include physical, chemical and biological methods, the former can easily remove color but hard to remove chemical oxygen demand COD. It costs high and it is easy to cause secondary pollution.The latter is a more cost-effective and environmental method, it obtains higher COD removal efficiency and has become a hot research in dye wastewater.The laccase is a polyphenol oxidase with copper, it has broad substrates, which can oxidize various phenolic derivatives and some phenolic analogue, without which have phenolic hydroxyl groups. It is frenquently used to deal with the dye wastewater and industrial wastewater. Its biological catalysis has been widely used in the field of environment.Our subject is to select the engineered Bacillus thuringiensis ME506with surface-displayed laccase as the experimental bacteria and explore the best measure conditions of the whole cell activity and the best fermentation conditions. Here, we selected the Acid Green25as the experimental dye. We optimized the conditions and explored its mechanism of ME506. We took the polyvinyl alcohol/sodium alginate (PVA/SA) to immobilize the ME506and prepared the embedding ME506balls. The decolorization conditions were optimized and the decolorization properties of Acid Green25were investigated.Firstly, The measure conditions of the whole cell activity of ME506were optimized, the results showed that the pH2.2disodium hydrogen phosphate-citrate buffer solution was the most suitable buffer system and the suitable temperature was30℃. Cu2+could promote the whole cell activity of ME506. Afterwards, the ME506shake flask fermentation conditions were optimized. Based on the LB medium, we took the temperature, initial pH, liquid volume and inoculation volume into consideration. The results indicated that the optimal fermentation conditions were:the temperature was30℃, the initial pH was7.0, the liquid volume was30%and the inoculation volume was4%.Then we took the method of RSM to optimize the fermentation medium of ME506.First, three factors which affected fermentation were selected by single factor screening tests. The results showed that the best carbon and nitrogen source were sucrose and yeast powder respectively, the best metal ion was Mn2+.Then we optimized the initial concentration of the important factors by the orthogonal experiment. Second, MgSO4·7H2O, MnSO·H2O and the inoculation volume were chosed by Plackett-Burman design as three prime factors of the eight factors which affect the laccase activity. The path of steepest ascent experiment was adopted to approach the optimal region of the medium composition. At last, the optimal levels of these three factors were determined by the BOX-Behnken experimental design. We took SAS9.2statistical analysis software for data processing and determined the optimum fermentation medium for ME506(g/L):Sucrose10g, Yeast extract37.5g, CaCO31.0g, K2HPO4·3H2O3.75g, ZnSO4·7H2O0.25g, MgSO4·7H2O2.88g, MnSO4·H2O0.02g,3.34%of inoculation volume. The whole cell activity for ME506in the initial fermentation medium was21.55U/L, now the laccase activity achieved53.91U/L and increased1.50times.To culture the ME506under these optimal conditions, we collected ME506bacterium for decolorization of AG25. The results showed that ME506had significant decolorization effect on the AG25and the decolorization rate was63.53%.We selected PVA/SA as the embedding agent, prepared the most vintage embedding ME506balls. It showed that the decolorization rate was86.95%and increased23.42%more than ME506.Then we analyzed the adsorption isotherms and decolorization kinetics of the embedding balls. It was found to be fitted well to the Langmiur isothern models and the Freundlich isotherm models, tend to fit to the index adsorption model The decolorization behavior of the embedding balls was more in line with the pseudo-second adsorption kinetics equation.Since the pseudo-second adsorption kinetics equation contains all the adsorption process, such as external film diffusion, surface adsorption and intra-particle diffusion, so it can be more truly to reflect the mechanism of the embedding balls.The full wavelength scanning and Fourier Transform infrared spectroscopy of AG25solution before and after decolorization showed that ME506and AG25maybe functioned by ion exchange or forming hydrogen bonds or other ways. The AG25may possible be degraded. The decolorization of AG25by ME506included physical adsorption. As for the degrading effect of the surface display laccase in ME506, we need to carry out further experiments to verify. |
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