Study On The Production Of Feeding-protein In Rice Straw Using Neurospora Crassa By Solid State Fermentation

In China,the quantities of rice straw?RS?and other agricultural waste are huge and the utilization rate of RS is low,by using the technology of solid-state fermentation?SSF?RS can be microbiologically converted into animal feed with high nutrition to achieve the sustainable use of resources,thus also providing a new way for the development of feed industry.Among a variety of filamentous fungi involved in lignocellulose degrading,Neurospora crassa has the ability to degrade and utilize lignocellulose for the production of ethanol,which also has been reportedly used forgenetic and molecular studies,however,few literatures were reported in the field of the optimization of fermentation conditions using N.Crassa to maximize feeding-protein yield.It has been reported that N.Crassa has the ability to synthesize all three cellulase enzymes,various hemicellulases and some ligninolytic enzymes.N.crassa grows more rapidly and has strong ability of protein expression and secretion.Moreover,its safety has been verified,so it can be used for SSF.In this paper,RS was chosed as raw material,and N.Crassa 14-8 was used as the key strain in SSF together with other fungi.The true protein?TP?content of fermentation products was improved by optimizing the fermentation conditions.Firstly,the effect of different fermentation conditions on TP yield by N.Crassa 14-8 was studied.Factors,including temperature,initial pH,incubation period,inoculum size,substrate/water ratio,soluble starch addition,nitrogen sources and urea addition were optimized through the methods of single factor test and orthogonal test successively.Finally,after mensurating and analysising TP yield,it was found that when the medium was provided for 0.2% urea and 1% soluble starch in SSF,the initial pH was adjusted to 6 and the substrate/water ratio ratio was 1:2.N.crassa 14-8?10%?was added into the fermentation medium and cultivated at 32°C for 120 h,the higher TP production?7.35%?was obtained.The mixed fermentation system of N.crassa 14-8 and Candida utilis was used to degradation RS,the effects of these three factors,including inoculation ratios of the mixed strains,inoculation time of C.utilis and incubation period,on TP yield were investigated,the optimum conditions in SSF were determined.In order to further improve TP content,the response surface methodology?RSM?was adopted to optimize the different cations and Tween 80.The results of Plackett-Burman Design?PBD?indicated that Mn2+,Zn2+ and Cu2+ were the significant variables,three kinds of cations can affect the enrichment of TP significantly.Based on the results of PBD,the optimum values for the three cations determined by the BBD and steepest ascent experiments were as follows: Mn2+ 0.06 g/L,Zn2+ 0.15 g/L,Cu2+ 0.2 g/L.Under this optimum condation the TP content increased from 3.98% to 9.84%,with 147.24% of its increase rate.The results showed that the addition of cations could promote the enzyme activity,improve the degradation rate of RS and increase the accumulation of microbial protein.Four strains including N.crassa 14-8,C.utilis,Aspergillus oryzae and Phanerochaete chrysosporium with different combinations were used for SSF.The results showed that the highest TP content could be obtained by using N.crassa 14-8,C.utilis and P.chrysosporium as fermentation strains.After studying the effects of different inducers on ligninase production of P.chrysosporium,5 mM Mn2+ and 50 mM veratryl alcohol were used as inducers of lignin peroxidase?LiP?to improve the efficiency of enzymatic hydrolysis.Meanwhile,the conditions in the process of SSF were optimized,when N.crassa 14-8 was inoculated 1 day later than P.chrysosporium,the total inoculum size was 10%,and the optimum ratio of N.crassa 14-8 to P.chrysosporium was 1:2,the maximum TP yield was obtained.The effects of different concentrations of Ca?OH?₂ and detoxification process on the degradation of RS and enrichment of protein were discussed.The results showed that the pretreatment with 5 g/L Ca?OH?₂ could effectively decompose lignin and increase the degradation rate of RS.After alkali pretreatment RS was washed with water repeatedly,2% laccase was added into the fermentation medium.The lignin content decreased significantly,and TP content of fermentation product increased.It shows that the method of washing and adding laccase before SSF can remove parts of inhibitors.This study proposed an eco-friendly and economical way to convert RS into protein-enriched livestock feed using SSF by N.crassa 14-8,which also showed a potential application in large-scale feedstuff protein conversion.