Study On Production Of Fungal Laccase And Its Application In Pretreatment Of Pulping And Decolorization Of Mill Effluent

Laccase is an important enzyme of lignin degradation produced by white rot fungi. It has high redox potential and takes O2 as electron receptor without the aid of H2O2. Therefore, it has more application potential than other ligninases.The influence on the production of laccase by Coriolus versicolor in different fermentation conditions and culture systems were investigated. In the liquid culture system, the optimal fermentation condition for best fungus growth and laccase producing were as follows: buffering system of pH 6.0, rotating speed 180 rpm, 100 mL culture medium per 500 ml flask. The glucose was the best carbon fountain in all the tested carbon fountains. The laccase activity was relatively high when saccharose was used as the carbon fountain in all the disaccharide. However, starch appeared minus effect on laccase production. The combination of tartaric ammonium and ammonium nitrate was found to be the best nitrogen fountain in all the tested nitrogen fountains. The effect of ammonium chloride and carbamide was number two.The effect of peptone was the worst.It was suitable to the production of laccase when the ratio of carbon and nitrogen is more than 10. The laccase activity could be improved to 1.6 IU/ml when the revulsant ABTS concentration was 0.5-1 mM.The addition of 0.5 mM guaiacol,1mM ferulic acid, 0.05 mM dimethylaniline could also improve the laccase activity obviously. The addition of 0.01 mM veraryl alcohol could also improve the laccase activity by 1.6 times.Great promotion to the reproduction of white rot fungus by adding natural extractives such as these from wood meal,maize core and potato in liquid medium was found in this research. Incubated in the liquid medium contained 10 g/L glucose as carbon source with natural extractives for ten days,the production of mycelium pellet reached more than 80 g/L.which was 5 times of that without natural extractives. After incubated in the liquid medium contained 5mg/L glucose as carbon source with natural extractives for ten days, the production of mycelium pellet could reach 69.5g/L,while the production of medium without natural extractive s was very low.When the basic medium contained 2 g/-20 g/L glucose as carbon source,the production of mycelium pellet in ten days could only reach 12.5 g/L to 14.5 g/L. Wood extractives stimulated the growth of white rot fungi instead of supply organics which fungi need.In order to reduce the environmental impact of pulping and energy consumption, improve the conventional pulping ways and take full advantage of straw.Pretreatments of straw with laccase improved the pulpability of straw and physical properties of the resultant pulp. The results indicated that the optimum conditions of pretreatment were pH value 5.0,temperature 40℃,liquor ratio l:6,pretreatment time 4 h,enzyme usage10IU/g.Compared with untreated straw,the pulp yield of straw pretreated with Laccase. With enzyme dosage of 30 IU/g and pretreatment time 4 h increased by 5.46% and the Kappa number droped by 2.6.the breaking length,tearing index increased by 25.55%,43.91%respectively. The brightness increased by 4.6 % SBD.The influence on laccase of cultivating factor was probed. For laccase production the optimum initial pH was 4.5. Addition of veratryl alcohol or elevated trace metals could both enhance the laccase activity, while Tween80 showed some improvement. The experimental results indicated that the immobilized mycelia of Coriolus versicolor in polyurethane foam had less laccase production ability than mycelia pellets. A repeated batch cultivation process was found to be a very economical way for laccase harvest. The same pellets could be used for at least 10 times and average laccase activity of each batch could reach 0.56 IU/mL. Good results were also obtained in the decolorization experiment with crude laccase and minor ABTS. Also with 0.5 IU/mL initial laccase activity, CODCr and chroma removal of paper mill wastewater reached 80% after 24h treatment when the chromaticity density was186.The molecular weight of the lignin (purified) which is from the outwater of the effluent settling chamber of a pulp mill is 31200.The molecular weight of the lignin treated for one hour by laccase increased to 46600 while the molecular weight of the lignin treated for two hours by laccase reached 58600.The results showed that the molecular weight of the lignin increased obviously after treated by laccase.In suitable condition, the laccase produced by Coriolus versicolor could polymerize the ligin and its derivatives in the wastewater of alkaline pulp mill.Then the CODCr and chroma of the wastewater would decrease.So the use of laccase could provides possibility to recycle the water in papermaking process.