Study On Laccase Production From Trametes Versicolor By Solid-state Fermentation With Tea Residue As Substrate And Its Application

China is a large tea producer.It results in a large amount of tea residue waste every year.The current utilization of tea residue was poor and it brought low economic benefits.In this study,the solid-state fermentation?SSF?technology with microbes was applied in the production of high value-added product—laccase using tea residue as substrate.It provided a new idea for the comprehensive utilization of tea residue.For this purpose,laccase was produced from Trametes versicolor with tea residue by SSF and the fermentation conditions were optimized to increase laccase production.The promotion effect of tea polyphenols on laccase production was investigated.The decolorization of diazo dyes was also studied by using solid laccase on tea residue.The main conclusions are as follows:1.The medium composition and culture condition were optimized by one-factor-at-a-time design and response surface method for the SSF with tea residue.The following was the optimal fermentation condition:glucose 13.84 g/L,?NH₄?₂SO₄ 4.90g/L,KH₂PO₄ 1.99 g/L,MgSO₄·7H₂O 1 g/L,CaCl₂·2H₂O 0.20 g/L,CuSO₄·5H₂O 1.92g/L,water content 83.57%?w/w?,pH 5,inoculum 4.40%?w/w?,incubation temperature26°C.The laccase activity reached 25.68 U/g dry substrate after 7-days culture under the optimized condition,which increased by 2.74 times compared to that before optimization.The scale-up culture was conducted in shallow tray by SSF with tea residue and the laccase production of 32.15 U/g dry substrate was achieved.These results indicated that the laccase production for SSF with tea residue was greatly improved by the optimization of fermentation condition.The optimal strategy can be used for the laccase production in large scale.2.The compositions and their content of tea polyphenols in tea residue were analyzed.Based on it,the promotion effect of tea polyphenols in tea residue was investigated in the laccase production by SSF.The laccase production by SSF reached26.53 U/g dry substrate and 18.64 U/g dry substrate after 7-days culture in tea residue and tea residue without polyphenols,respectively.The laccase production in tea residue without polyphenols was only 70%of that in tea residue.The laccase activity of 26.22U/g dry substrate in SSF was obtained after the crude extract of tea polyphenols was added to tea residue without polyphenols,which recovered to the level of the laccase production in tea residue.It preliminarily demonstrated that tea polyphenols can enhance laccase production.The mixture of different polyphenol standards exhibited the induction effect in the SSF of tea residue without polyphenols,which was similar to the case with the addition of the crude extract of tea polyphenols.Among them,catechin gallate was the most effective inducer of laccase production with the best induction ability per dosage unit.When catechin gallate of 4.16 mg/L was added to tea residue without polyphenols,the laccase production by SSF was enhanced to 53.05 U/g dry substrate and the laccase gene expression was increased by 3.20 times compared to the control.Therefore,catechin gallate in tea polyphenols is a good laccase inducer with high efficiency.3.Solid laccase on tea residue was prepared from the laccase produced from T.versicolor by SSF with tea residue.The solid laccase was used for the decolorization of disazo dyes.Decolorization mechanism was also evaluated using Direct Fast Yellow RS as the representative example.The optimal dosage of solid laccase was 160 U/L with respect to the dye concentration of 100 mg/L.After 2 h incubation,the decolorization efficiency of Direct Fast Yellow RS reached 81.1%.The dye decolorization by solid laccase on tea residue was contributed from three parts:degradation by crude free laccase,adsorption by tea residue and degradation by immobilized laccase,where the decolorization efficiencies of Direct Fast Yellow RS were 16.40%,43.20%and 19.70%,respectively.The solid laccase on tea residue exhibited good reusability for the dye decolorization.The decolorization efficiency kept more than 50%after 4 recycles.The toxicity test showed that the germination rate of mung bean seeds in dye solution increased from 36.67%to 85%after decolorization.It indicated that the toxicity of dyes solution decreased significantly after decolorization.