Medium Component Optimization And Enzyme Compostion Analysis Of A Straw-decomposing Microbial System With Low-temperature Tolerance Capacity

With the rapid development of China's new normal economy,the problem of energy shortage has become the biggest obstacle to the implementation of China's sustainable economic development strategy.Under the pressure of the energy crisis,the use of microbial fermentation technology to deal with organic waste can not only avoid the pollution of the environment can also ease the energy crisis pressure,is an effective waste resource utilization.Straw as raw material for biogas fermentation is an important way to promote the development of biogas,but the straw lignocellulose complex structure and difficult to be microbial degraded feature restricts the development of straw biogas.How to effectively realize the hydrolysis and acidification of lignocellulose has become a research direction of researchers' continuous attention.The cold and long winter in northern China is the main factor that restricts the smooth progress of biogas fermentation.In this study,a group of low temperature resistant cellulose degrading strain LTF-27 obtained by our laboratory was used as the research target.In order to obtain the optimum conditions for acid hydrolysis,the culture medium was optimized by single factor test method,Plackett-Burman?P-B?test method,steepest climbing test method,center combination test method and the response surface analysis method.And the enzyme composition of the strain was analyzed by SDS-PAGE gel electrophoresis and mass spectrometry.The main contents and results are as follows.?1?Single factor optimization of medium for low temperature resistant microbial consortium LTF-27.Under the condition of 17 ±0.5 ?,rice straw as the sole carbon source,and the ammonium sulfate was screened out as the best nitrogen source from different nitrogen sources such as soybean meal,corn flour,wheat bran,urea,ammonium nitrate,ammonium sulfate,peptone.The single-factor experiments were carried out on the inorganic ions?CaCO₃,FeSO₄,MgSO₄,ZnSO₄,CuSO₄,KH₂PO₄,K₂HPO₄?that affected the fermentation of the complex strains,and the optimum levels of the inorganic ions were 1 g·L-1,0.015 g·L-1,0.07 g·L-1,0.008 g·L-1,0.18 g·L-1,0.31 g·L-1,0.5 g·L-1 respectively.?2?The P-B test optimization of the culture medium for the low temperature resistant microbial consortium.On the basis of single factor test,P-B test was carried out on 8 factors?CaCO₃,CuSO₄,MgSO₄,ZnSO₄,CuSO₄,?NH₄?SO₄,KH₂PO₄ and K₂HPO₄?.The result showed that K₂HPO₄,CaCO₃ and MgSO₄ significantly effects the straw degradation.According to the results of the steepest climbing test of these three factors,obtained the best center combination test center for next step,which is MgSO₄ 0.055 g·L-1,CaCO₃ 1.4 g·L-1,K2HPO4 0.7 g·L-1.?3?The central composite test optimization of the culture medium for low temperature resistant strains.Applying center combination test and response surface analysis method,the best factor level was screened out as MgSO₄ 0.057 g·L-1,CaCO₃ 1.49 g·L-1,K₂HPO₄ g·L-1,the optimal medium composition as rice straw 2 g·L-1?NH₄?SO₄ 1.5 g·L-1,Na Cl 5 g·L-1,yeast extract 1 g·L-1,MgSO₄ 0.057 g·L-1,CaCO₃ 1.49 g·L-1,K₂HPO₄ 0.75 g·L-1.The predicted degradation rate of this model was 64.82%.After 5 times fermentation experimental Verification,the average degradation rate of rice straw was 64.51%,which was close to the predicted value,thus verified the correctness and accuracy of the model.Other indicators also tested in this study,the results showed that,in terms of pH,degradation of straw,microbial growth,dissolved oxygen,cellulose in the straw,hemicellulose and lignin,the straw degradation in the optimized medium was better than that of the original medium.?4?Analysis of Enzyme Composition of Low Temperature Resistant StrainsThe strain LTF-27 contains essential enzymes that degrade lignocellulose as simple sugar,that is,endoglucanase activity,exoglucanase activity,?-glucosidase,and the enzymes show a change trend of first increase and then decrease.By further analysis of the mass spectrometry,12 enzymes involved in the degradation of lignocellulose,including catalase,exoglucanase,endoglucanase,?-L-arabic aposease,?-glucosidase,?-glucuronidase and the like were identified.At the same time,a total of 57 proteins were also identified,including catalase,transferase,hydrolase,protein binding enzyme,cell signal transporter,transcriptional regulator,alcohol dehydrogenase,short chain dehydrogenase,phosphoric acid Glycerol aldehyde dehydrogenase,isocitrate dehydrogenase,nucleotide transferase,heat shock protein,ribosomal protein,kinase,chaperone protein.These proteases are directly or indirectly involved in the synthesis and catabolism of related LTF-27 degradation substances and provide a theoretical basis for further study on the mechanism of lignocellulose degradation by microbial consortiums.