Corn Stalk Pretreatment And Fermentation By Neurospora Crassa

One of the bottlenecks of cattle and sheep breeding in China's agricultural areas and southern China is the lack of high quality roughage.The fermentation of corn stalk can effectively improve its nutritional value and palatability,and the fermentation of feedstuffs from corn stalk is one of an effective way to improve the utilization ratio of corn stalk and solve the problems of shortage of animal feedstuff at the same time.However,corn stalk cannot bio-transformed directly and efficiently because of the complex chemical composition and structural resistance of corn stalk.Pretreatment,detoxification and fermentation were taken as a route to overcome the problems of indigestibility and low nutritional value of corn stalk in this research.Firstly,the corn stalks were treated with calcium hydroxide and the most economical and appropriate treatment temperature?80??and concentration of calcium hydroxide?5g/L?were selected with the degradation of lignin and hemicellulose as the index.The phenolic compounds in the hydrolysate were extracted by dichloromethane,and the five phenolic inhibitors with higher content in the hydrolysate were analyzed qualitatively and quantitatively by high performance liquid chromatography?HPLC?.Then the laccase was used to degrade the toxic compounds in the alkaline pretreatment hydrolysates.Because laccase has a good degradation effect on ferulic acid,the optimum laccase content was screened with the degradation rate of ferulic acid as the index.In order to degrade ferulic acid to the greatest extent in a short period of time,the single factor experiment was used to select three factors:pH,concentration of Cu²⁺and concentration of Mn²⁺.The response surface methodology was used to analyzed the optimal detoxification condition with the degradation rate of ferulic acid as the index.The optimum conditions of the three factors were determined as:p H 3.91,concentration of Cu²⁺29.3mg/L,and concentration of Mn²⁺30.8mg/L.Under the optimum conditions,the degradation rate of ferulic acid reached 85.11%within 1 h.The results showed that the detoxification effect of laccase was greatly enhanced in a short time after optimization.But at the same time,one unknown phenolic compound was detected in the hydrolysates during the detoxification of laccase.Secondly,the alkaline hydrolysates were detoxified by Phanerochaete chrysosporium for 24 hours.It was found that the degradation of vanillin?77.19%?and p-hydroxybenzaldehyde?63.82?)was better,but the degradation rate of ferulic acid was relatively weak?15.34%?.Finally,vanillin,p-hydroxybenzaldehyde,ferulic acid 2-methoxy-4-vinylphenol and salicylic acid were all detoxified by laccase and P.chrysosporium.The degradation rates of vanillin,p-hydroxybenzaldehyde,ferulic acid 2-methoxy-4-vinyl phenol and salicylic acid were 89.66%,90.99%.98.00%,88.46%,58.13%,respectively.And the unknown compound was also degraded in this process.The results showed that the detoxification of phenols by laccase combined with P.chrysosporium was effective.The results inferred that P.chrysosporium might generate some substance which could cooperate with laccase to decompose the phenolic compounds during its spore germination and growth period?but no lignin peroxidase or manganese peroxidase?.Neurospora crassa is a simple and rapid growing fungus,which can be safely used in production.There are not only complete lignocellulose-degrading enzymes,but also abundant proteins,vitamin B12 and many enzymes in the N.crassa.Compared with the growth of N.crassa on detoxified and undetoxified corn stalks,it was found that the growth status of N.crassa on corn stalks after detoxification was obviously improved and the content of true protein was also increased.After detoxification,the inhibition of phenolic compounds on the growth of fungi was obviously reduced.The detection of lignocellulose content before and after pretreatment showed that the degradation rate of hemicellulose reached 39.00%and the degradation rate of lignin reached 21.17%.The content of true protein increased2.73 times after fermentation.This study provides a reference for the degradation of phenols in corn stalk after alkaline pretreatment and has potential application value in large-scale production.