Fermentation Of White Rot Fungus And The Application Of Its Laccase In The Treatment Of Wastewater From Chlorine Bleaching Process

CEH bleaching process is a main traditional bleaching technology used in our county. The wastewater discharged to the environment not only contains high duty of COD and BOD, but also carcinogens materials, such as chlorphynols etc. The white-rot fungus can not only degrade lignin and its derivatives with the released enzyme, but change inorganic chloride into chlorine and CO2 and destroy color group and structure. Laccase is a ligninolytic enzyme that uses O2 as the electron receptor and has high redox potential. Therefore its application in paper industry in more promising as compared to ligninolytic enzyme.The effect of inoculating time and differene culture methods on the production of laccase by Trametes hirsuta BYBF were investigated. The optimal condition for incubation of the fugus was as follows: inoculation time 5 days, inoculum concentration 10%, shaking speed 180 rmp/min, liquid volume 100 mL/mL, pH of the midUm 6.5 in the shaking culture system. Glucose was the best carbon source. AmmonUm nitrate and tartaric ammonUm combined by 1:1 (w/w) was found to be the best nitrogen source. Cu2+ was the best accelerant in all tested metal ions.The Laccase production was promoted obviously when 0.1 mmo1/L Cu2+ added to the midUm. Tween 80 was the best to increase the Laccase production in all tested surfactant and the optimal concentration was 5mL/L. In all used inducing agents, 0.5mM of ABTS had obvious effect on Laccase production and the enzyme activity reached about 2 times of the blank. The addition of 0.5mM ferulic acid, 0.1 mM guaiacol and 0.05mM dimethylaniline could significantly improve the activity of laccase produced.The 2-chlorophenol (2-CP) was treated by laccase as model of chlorinated lignin to find the optimal polymerization conditions. The reaction products were analyzed by GPC, FT-IR and 13C-NMR. The polymerization of 2-CP by Laccase not only was concerned with types of organic solvents, organic solvents concentration and reacting time but affected by temperature, pH and Laccase concentration. Under a suitable condition, acetone was superior to dioxane and the optimal concentration was 50%(v:v).With the extension of processing time and increasing of enzyme dosage, the removal ratio of 2-CP was increased. The optimal condition was as follows: processing time 24h, temperature 40℃, pH6. When the initial concentration was 500mg/L, removal of 2-CP achieved 88% under the optimal polymerization condition. The final product had a high molecular(31926) after polymerization catalyzed by laccase. The analysis of 13C NMR showed that 2-chlorophenol formed high molecule polymer at first and then a part of the aromatic ring was opened to form aliphatic structures.The bleaching effluent was treated by laccase from Trametes hirsuta BYBF. It was found that laccase could remove lignin and reduce CODCr and chroma of effluent effectively under oppropriate condition. The processing time, enzymatic dosage, reaction temperature, pH value etc had great effect on the result of treatment of the bleaching effluents treated by laccase produced from Trametes hirsuta BYBF. With the extension of treatment time, the removal rates of CODcr, chroma and lignin increased gradually. But with the increase of enzyme dosage, the removal rate increased at first and then decreased because of the CODCr and colour of laccase itself. The optimal treatment condition was as follows: 24h, 16 U/mL of enzyme dosage, 30℃and pH 6.64. The removal rate of lignin, CODCr and chroma was 83.36%, 83.78% and 87% respectively under the optimal treatment condition.The polymerization with laccase of 2-CP and the structure of the products were analyzed by GPC and FT-IR. It was found that the molecular weight of lignin which was from bleaching effluent was enhanced from 15,000 to 41,000 through laccase treatement. The increase of molecular weight of lignin indicated that the lignin were polymerized by the catalysis of laccase. The laccase of Trametes hirsuta BYBF could catalyze the polymerization of lignin and its derivation in bleaching effluent to be high molecular weight polymer under suitable condition and was advantage to the subsequent flocculation which reduced the lignin and its derivation of bleaching effluent and let CODCr and chroma down. Furthermore, it has the possibility to recycle the bleaching effluent after this treatement.