Research On Laccase Production With Fungus And The Degradation Of Dyes And Lignin By Laccase-Mediator System

Laccase?EC 1.10.3.2?is a copper-containing polyphenol oxidase with a wide substrate specificity,capable of catalyzing the oxidation of various phenolic compounds and some refractory organics such as non-phenol aromatic compounds with the assistance of small molecule mediators.At present,laccase has been extensively applied in the fields of delignification,pulp bleaching,decolorization and degradation of dyes and bioremediation.In this study,the properties of laccase production by Cladosporium sp.KR14under liquid fermentation were studied,including the optimization of laccase production conditions and the investigation on the effect of single-walled carbon nanotubes?SWCNT?on the production of laccase.Then,the ability of the laccase-mediator system to decolorize and degrade refractory dyes?alizarin red,congo red and fuchsin acid?was examined with and without mediators?HBT,VA,and ACE?.Finally,the performances of laccase-ABTS systems,composed of Cladosporium sp.KR14 laccase and commercialized T.versicolor laccase,respectively,to degrade refractory substrate?alkali lignin?were compared in the presence of graphene.Subsequently,the enhancing mechanism of graphene was proposed for the commercial T.versicolor laccase-ABTS system with obvious promotion.?1?The optimal conditions for laccase production by Cladosporium sp.KR14 under liquid fermentation were determined as follows:the ratio of C/N was 20,the fermentation temperature was 30°C,the initial pH was 5.0,the shaking speed was 150r/min,the inoculum volume was 10%and the liquid volume was 80 mL/250 mL.Laccase production of Cladosporium sp.KR14 strain was promoted by the SWCNT with a concentration of 12.5 mg/L,and the enzyme productivity of the strain reached the highest level?525 U/L?on the 14^th day under the above optimal conditions.?2?The optimal temperature for the activity of Cladosporium sp.KR14 laccase was 40°C and the optimal pH was 5.0.The Cladosporium sp.KR14 laccase had low degradation efficiencies for alizarin red?43%?,congo red?25%?and fuchsin acid?15%?without mediators.For laccase-mediator systems,the most effective mediator for degradation of alizarin red?anthraquinone dye?was VA,and the decolorization degradation rate of alizarin red was up to 82.79%.Under the same conditions,the most effective mediators for congo red?azo dye?and fuchsin acid?triphenylmethane dye?were VA and HBT,respectively.?3?Suitable mediators can promote the decolorization of refractory dyes by Cladosporium sp.KR14 laccase.Different mediators exhibited varied promoting effects on the decolorization and degradation of the same dye,and the same mediator also performed differently on different dyes.?4?Graphene had a slight promoting effect on the degradation of alkali lignin by Cladosporium sp.KR14 laccase-ABTS system.On the contrary,degradation efficiency of T.versicolor laccase-ABTS system was greatly promoted by graphene.As a result,the degradation rate of alkali lignin was increased by 28%within 10 hours and the oxygen consumption rate was accelerated by 4 times.FTIR and XPS results indicated that the degradation of alkali lignin by T.versicolor laccase-ABTS system was enhanced with the addition of graphene.Contents of chemical groups of hydroxyl,methyl,methylene,conjugated carbonyl and phenolic hydroxyl were decreased,while the amount of non-etherified phenolic hydroxyl group was increased,resulting in a 13.61%increase in the oxygen-carbon ratio of alkali lignin.?5?Physical characterization was conducted to investigate the graphene involved in the T.versicolor laccase-ABTS degradation system,and results showed no obvious changes in graphene before and after the reaction.Cyclic voltammogram and ABTS intermediates test results suggested that the graphene,as an electron conductor,strengthened catalytic degradation of alkali lignin by T.versicolor laccase-ABTS system,increased the oxidation rate of ABTS²⁺to non-phenolic structural units in alkali lignin,and improved the reusability of ABTS mediator.