Morphology-based Optimization Of Neurospora Crassa Fermentation For Cellulase Production

Neurospora crassa,a model species of filamentous fungi,secretes a range of enzymes for lignocellulosic biomass degradation,having high application value.However,the optimization of liquid fermentation conditions in stirred fermenters has not been reported.In addition,the expression and secretion of cellulase by N.crassa need to be further improved through genetic engineering approaches.The main problems in the production of cellulase by N.Crassa fermentation in stirred fermenter are the volatilization of liquid,the formation of mycelium,the consideration of the wall,etc,affecting the production of cellulase by fermentation.In this paper,wild type N.crassa was used as the initial strain to explore the liquid fermentation conditions in a 2L four-stir fermentation tank.The culture conditions were optimized to ensure the normal fermentation process.Furthermore,by selecting short mycelium mutants,the viscosity of fermentation broth can be effectively reduced and the dissolved oxygen and mass transfer efficiency can be improved.Solid additives were added to the fermentation broth of the short mycelium mutant,and the fungus was mainly in spherical shape in the later stage of the fermentation process,which could further improve the efficiency of fermentation enzyme production.This study and exploration laid a foundation for the systematic optimization of enzyme production conditions of N.Crassa fermentation,which is of great significance for the research on cellulase production and exogenous recombinant protein production by N.Crassa fermentation.The results of this study are as follows:(1)Culture in stirred fermenter.In order to reduce the liquid volatilization in the fermentation process,the sterile distilled water inlet channel was added to the intake device,and the saturated water vapor in the intake can effectively reduce the fermentation liquid volatilization.The results showed that the anti-volatilization effect of this device was better than the traditional methods such as flow addition and batch addition of sterile water.(2)Optimization of fermentation culture time and inoculation method.It was found that the maximum enzyme production was reached at 168 h in the stirred fermentation tank.Further studies showed that the inoculation method of the spore solution in sucrose medium before inoculation in the fermentation tank could shorten the adaptation period of N.Crassa in the fermentation tank and effectively improve the enzyme production level of N.Crassa in the fermentation tank.(3)Optimization of mycelium morphology based on gene mutation.The sheer force in the stirred fermenter is higher than that in the flask fermentation.By selecting short mycelium mutants,mycelium breakage can be reduced,the viscosity of fermentation broth can be reduced,and the dissolved oxygen and mass transfer efficiency can be improved.Therefore,compared with the wild type of long mycelium,the fermentation capacity of short mycelium mutant to produce cellulase was significantly improved.In addition,compared with flask fermentation,the total amount of carboxymethyl cellulase activity(CMCase)or intracellulose endonuclease activity,crystallized cellulose or filter paper enzyme activity(FPase)and secreted protein produced by short mycelium mutant A in the fermentation tank was increased by 1.2 times,1.2 times and 3.2 times respectively.The corresponding enzyme activity or protein level of short mycelium mutant B was increased by 2.3 times,1.1 times and 1.7 times respectively,all higher than that of the wild type control.(4)Optimization of mycelium morphology based on fermentation conditions.The above short mycelium mutant B was used to further optimize the mycelium morphology based on fermentation conditions.Talc,a solid additive,was added in the pre-culture process of seed liquid,so that the seeds clustered around Talc after germination,and mycelia mainly grew in small spheres after inoculation in the fermentation tank.Under this fermentation condition,the CMCase activity in flask fermentation broth was increased by 1.8 times.FPase activity increased by 4.1 times and protein secretion increased by 1.9 times.In the fermenter,CMCase activity was increased by 2.3 times,FPase increased by 1.4 times and Protein secretion increased 0.7 times.To sum up,through the optimization of mycelium morphology based on gene mutation and fermentation conditions,the concentration of reducing sugar corresponding to CMC enzyme activity was finally increased from the initial 1.4g /L to 8.4g /L,which was about 6times higher.The reducing sugar concentration corresponding to the enzyme activity of the filter paper increased from the initial 1.82 g/L to 4.9 g/L,which is 2.7 times higher.The exocrine protein concentration increased from 34.67 mg/L to 152 mg/L,which was 4.4 times higher.The short mycelium strain obtained through mutants screening showed low viscosity and other fermentation advantages at the fermentation tank level,reducing the energy input in the system and achieved the purpose of both improving the fermentation level and saving energy consumption.