Screening Of Cellulase-producing Bacteria And Using Microbial Co-culture Strategy To Improve Degradation Of Chinese Distillers’ Grains

Chinese distiller’s grains(CDGs)are the by-products after fermentation and distillation of the mixture of grains and rice hull to obtain liquor,and it is rich in organic matter such as cellulose and protein.At present,CDGs are mainly used to produce animal feed.However,tens of millions or even hundreds of millions of tons of CDGs are produced in China every year,which brings huge challenges to the resource utilization of CDGs.Lack of efficient and cheap processing methods restricts the comprehensive utilization of CDGs and causes CDGs to be discarded at will,which further causes waste of resources and environmental pollution.It is an important task for the Chinese liquor industry to develop the treatment method of CDGs and improve economic and environmental benefits.Due to the high content of cellulose in CDGs,degradation of cellulose has become a critical step in resource utilization of CDGs.Compared with physical and chemical treatment approaches,biodegradation has the advantages of low cost and environmental friendliness.Improving enzyme activity and enhancing the adaptability of microorganisms to the environment is a difficult problem facing biodegradation.Besides,the current development and utilization of cellulaseproducing bacteria in the Chinese liquor-making process are insufficient.Based on the above actual needs and scientific problems,cellulase-producing bacteria were isolated and screened from Chinese liquor-making process(Chinese Maotai-flavor fermented grains,Chinese mild-flavor fermented grains,Chinese strongflavor Daqu starter)and soil in a bamboo forest.Moreover,the classification of bacteria,physicochemical properties and the characteristics of degrading CDGs were studied.In addition,the cellulase produced by microorganisms was characterized,and the microbial co-culture system was established to improve the degradation effect of CDGs.The main research results are as follows:(1)Using sodium carboxymethyl cellulose(CMC-Na)as the sole carbon source,39 strains of bacteria were isolated from the samples.Congo red staining and filter paper strip disintegration experiments were used for preliminary screening,and cellulase activity was determined for re-screening.The endoglucanase activities of strains A5 and B2 reached 25 U/m L and 34 U/m L,respectively.Based on morphological,physiological,biochemical,and molecular biological identification,strains A5 and B2 were identified as Bacillus licheniformis and Bacillus subtilis,respectively.The stress test further showed that Bacillus subtilis B2 was suitable for degrading CDGs,and it grew well in the environment of p H 3.0,temperature 44 ℃,alcohol volume fraction 4%.(2)Basis on the above study,Bacillus subtilis B2 strain was applied to the degradation process of CDGs.According to the single-factor test,a Box-Behnken response surface method was used to optimize the processing parameters in the treatments.The optimal process conditions were determined to be 71 g/L CDGs,the temperature of 37 ℃,p H value of 6.4,and the inoculation size of 8%,for the degradation of CDGs.In addition,the degradation rate of CDGs can reach 15.23% in this case.(3)To find thermostable cellulase-producing Bacillus,the cellulase was characterized.The results showed that the optimal reaction temperature of the cellulase produced by Bacillus licheniformis A5 was about 75 ℃,and endoglucanase was incubated at 80 ℃ for 120 min,and the residual enzyme activity was 82%.(4)By constructing a bacterial co-culture system,it was found that the cellulase activity of Bacillus subtilis A5 and Bacillus subtilis B2 was increased by 30% to 70%when co-cultured.Compared with the degradation of Bacillus subtilis B2 under optimized conditions,the degradation rate of CDGs increased by 70%,and the reducing sugar content increased to 16.6 mg/m L.In addition,the residual ethanol in CDGs did not reduce the degradation rate of CDGs.On the contrary,when 2% volume fraction of ethanol was added to the culture system,the degradation rate of CDGs increased by 15%in co-culture.Based on the above research results,we found that Chinese fermented grains and Daqu starter could be used as important biological resources for cellulase-producing bacteria.At the same time,we confirmed that Bacillus licheniformis A5 and Bacillus subtilis B2 could promote the degradation of CDGs through synergistic effect,and the two bacteria had the potential to can be used as an excellent candidate microbial consortium for lignocellulosic biomass degradation,providing new insights for the treatment of CDGs.