Laccase Production By Recombinant Trichoderma Reesei Under Solid State Fermentation And Its Application In Treatment Of Organic Pollutants

Laccase plays an essential role in environmental pollution abatement.Aimed at solving the problems related to the high cost of laccase production,straw accumulation and increasing levels of organic pollutants,in this study,a strain of recombinant Trichoderma reesei was used to consume rice straw during solid-state fermentation to achieve efficient straw degradation and high activity laccase production simultaneously.The laccase-mediated catalytic reactions of several typical organic pollutants were studied.For the 2,4,5-trichlorophenol-contaminated soil samples,the enzymes obtained via solid fermentation were directly used for soil remediation.Furthermore,the experiment of remediation coupled with straw degradation was carried out.The conditions of laccase production by recombinant T.reesei were studied.The recombinant T.reesei had wide temperature(24-32 C),humidity(60-75%)and pH(4.6-6.6)adaptation.The suitable conditions were:30?,substrate moisture content 70%(w/w),natural pH.With the addition of 30%bran and 0.025%CuSO4,the growth of recombinant T.reesei was promoted,and the cellulase,xylanase,laccase were secreted.Cellulase activity increased rapidly,reaching 98.05 IU/g dry koji on the 6th day of the fermentation,and the degradation rate of straw cellulose reached 64.44%.Xylanase activity peaked at 5407.7 IU/g dry koji on the 10th day of the fermentation,and the hemicellulose degradation rate was 54.17%.The laccase activity increased slowly.On the 12th day,the laccase activity peaked at 10.12 IU/g dry koji,and the lignin degradation rate was 31.16%.At this time,the xylanase and cellulase activities reached 4267 IU/g dry koji and 116.96 IU/dry koji,the degradation rates of cellulose and hemicellulose were 78.02%and 55.1 7%,respectively,and the total weight loss rate of straw was 55.23%.The degradation of lignin by recombinant T.reesei has great significance,since its degradation intermediates can be used as natural mediators by laccase,which is conducive to the treatment of pollutants.The solid-state fermentation simulation scale-up experiment was carried out in a shallow dish(35 cm×25 cm×10 cm),and 6 g enzymes obtained via solid fermentation was added to 300 g matrix.The laccase activity peaked at 8.77 IU/g dry koji after culturing at room temperature and natural pH for 10 days.And the cellulase activity reached 125.68 IU/g dry koji on the 12th day,and the weight loss rate of straw reached 50.46%.Acid green 25 is a typical anthraquinone dye,causing severe environmental pollution.The acid green 25 was decolorized by laccase at the conditions of laccase dosage 0.05 IU/mL,pH 4.0,60? for 30 min,and the decolorization rate was up to 95.5%.No external mediators were required for this reaction.Nonylphenol is an endocrine disruptor that poses a huge hazard to people and environment.The laccase-syringaldehyde system was used to degrade nonylphenol.The suitable conditions were as follows:laccase dosage 0.2 IU/mL,syringaldehyde dosage 0.2 mM,pH 4.6,60?,and the degradation rate reached 95.6%after reacting for 140 min.Syringaldehyde is a natural mediator produced during the degradation of lignin and is a good alternative to the synthetic mediator ABTS.The results have good practical application prospects.2,4,5-trichlorophenol is a typical chlorophenolic pesticide with strong toxicity and difficulty in natural degradation.The degradation test of 2,4,5-trichlorophenol was carried out by laccase-syringaldehyde system,and the degradation rate of 2,4,5-trichlorophenol reached 90.11%in 180 min.The remediation of 2,4,5-trichlorophenol-contaminated soil using koji was carried out at the conditions of 10%(w/w)koji with 10%(w/w)water addition every 2 days.The degradation rate reached 68.11%at 12th day without adding mediators,and the residual laccase activity kept above 28.3%.The coupling test of straw degradation and soil remediation was carried out in a shallow pan(20 cm in diameter and 10 cm in height):100 g of 2,4,5-TCP contaminated(89.7 mg/kg)soil was weighed,and the straw substrate(30 g)was laid on the upper layer of contaminated soil.Koji of T.reesei was inoculated into straw and then cultured in natural environment.After 12 days,the laccase activity was 5.06 IU/g dry koji,and the cellulase activity was 75.63 IU/g dry koji.The resulting straw degradation rate was 47.26%with the degradation rate of 2,4,5-trichlorophenol up to 52.11%.The research results in this paper lay a solid foundation for further research on rapid degradation of straw and soil pollution abatement.