Studies On The Pretreatment And The Neutral Protease Production Of Flammulina Velutipes Spent Mushroom Compost

Biomass is one of the most abundant renewable resources on the earth.The development and utilization of biomass resources has been a research hotspot.In recent years,with the booming development of edible fungus industry,a large amount of spent mushroom compost?SMC?,as a kind of biomass,was produced.In addition,the improper disposal of SMC will also cause some environmental problems.Therefore,a widespread concern has been aroused.Protease is one of the three major industrial enzymes.Finding a cheap substrate for protease production is of great significance.Due to the nutrients in SMC,it is possible to be converted into protease.However,researches on the recycle of SMC nowadays less focus on its conversion into a protease.Firstly,this study applied an efficient pretreatment method to improve the digestibility of SMC.Secondly,Bacillus subtilis protease fermentation process was investigated.Finally,Clostridium thermocellum cellosome was used as cellulase to reduce the amount of celluase in protease fermentation process.A large amount of water extract was found in the SMC.When the solid to liquid was1:10 in water extraction,1.38 g/L protein,0.31 g/L xylose,and 0.25 g/L arabinose was detected in water extract liquid.Na Cl O₂ was applied to pretreat water-extracted SMC.The optimal pretreatment conditions?2:25,4 h,80°C,8%?w/v?Na Cl O₂?obtained by orthogonal design.The water extraction process before pretreatment would effect the component content in the raw material of SMC.Additionally,the yield of reducing sugar and the enzymolysis efficiency of the water extracted-pretreated SMC increased by 19.3%and 10.2%,respectively,compared with the pretreated SMC without water extraction.The pretreated SMC was used for protease fermentation.Moreover,the effect of fermentation process on protease fermentation efficiency and product yield was investigated.The hydrolysate obtained at a high substrate loading was converted into protease by separate hydrolysis fermentation?SHF?.When 30 mg/m L of enzymatic hydrolysis sugar solution and 1g/L of urea were used as carbon and nitrogen sources,the protease activity in the fermentation broth was 4250 U/m L.The water extract liquid was successfully recycled in the simultaneous saccharification and co-fermentation?SSc F?process for protease production of pretreated SMC.Compared with SHF,the protease productivity was significantly improved.In addition,the comparable protease was obtained at a lower enzyme loading.The protease fermentation based on Clostridium thermocellum was investigated.The addition of Tween 20 and cellulase?CTec2?could synergistically promote the saccharification of Clostridium thermocellum.The concentration of glucose and xylose was increased in the fermentation broth.Subsequently,the saccharification liquid was used for protease production.The yield of reducing sugar was 149.3 U/mg of saccharification liquid,which was comparable to the yield of hydrolysate obtained from the enzymolysis using cellulase only.Moreover,the addition of Tween 20 could promote the protease production by Bacillus subtilis.The cellulosome was applied for protease production in SSc F.Ultimately,the fermentation residue was used for feed application to realize the sustainable development of agricultural wastes.