Screening And Characterization Of High-yield Cellulolytic Bacillus Sp. And Preliminary Application Study On Solid-state Fermentation Of The Mixture Of Pretreated Corn Straw And Soybean Meal

China is rich in maize straw,which is often regarded as agricultural waste without being fully utilized,resulting in resource waste and environmental problems.In order to further strengthen the comprehensive utilization of maize straw,the Chinese government published a plan of“13th Five-Year”comprehensive utilization of maize straw.Namely,we should constantly improve the storage and transportation system of maize straw,and promote the utilization of straw fertilizer,feed,fuel,base material and raw material.Microbes play a key role in straw feed and fertilizer,and screening,constructing,analysising with a specific function of microorganisms in the comprehensive utilization of straw resource is of great significance.At present,a large number of high-yield cellulase strains have been successfully screened,and most researches have been focused on fungi.Lignocellulosic degrading fungi can produce high-yield cellulase,but long incubation time,poor temperature and pH tolerance have restricted its industrial production.Probiotics have a stronger advantage over fungi,especially Bacillus sp.Bacillus sp.is easy to be screened and expresses strong stress resistance（acid and alkali resistant,high temperature resistant）,which is potential for industrial application and production.Several Bacillus strains can produce abundant lignocellulosic enzymes and play an important role in biomass conversion.Thus,we used a variety of lignocellulosic enzymes-medium for screening cellulosic degrading strain.We isolated an endophytic strain named Bacillus velezensis 157 from Eucommia bark,which has abundant lignocellulosic enzymes and the ability to inhibit a large number of pathogenic bacteria and fungi.In addition,we analyzed partial biological characteristics of it,the optimization of its CMCase production conditions,and enzymatic properties.At the same time,we carried out the whole-genome sequencing and bioinformatics analysis of Bacillus velezensis 157,exploring the lignocellulose degrading enzyme gene.Moreover,we also conduct a comparative genomics analysis of 24 Bacillus velezensis strains to explore the important commonality and specificity involving lignocellulolytic enzyme genes in genomics of one species.Finally,we used Bacillus velezensis 157 for producing cellulase,xylanase,pectinase and amylase under single solid-state fermentation,consisting of three groups:pretreatment of maize straw,and soybean meal and mixture of pretreatment of maize straw and soybean meal（AWC）respectively.The enzyme conditions of mixed solid-state fermentation were optimized by single factor method.We used scanning electron microscopy（SEM）,Fourier transform infrared spectrum（FTIR）,GC-MS product analysis and content determination of lignocelluloses to evaluate the degradation effect of three groups under solid-state fermentation.This study can provide new innovative ideas for biomass conversion and utilization.The main results are as follows:1.Isolation and identification of high-yield cellulase Bacillus sp.and analysis of partial biological characteristics analysisIn this study,we successfully screened an endophytic strain 157 from Eucommia bark.The strain 157 was identified as Bacillus velezensis by bacterial colony morphology,traditional physiological,biochemical identification,automatic microbiological identification system and 16S rRNA molecular biology technology.Partial biological characteristics of Bacillus velezensis 157 showed that Bacillus velezensis 157 can grow under the condition of pH 2-12,and its optimal pH is 6.At the same time,this strain can also tolerate 0.4%bile salt and high temperature of 85℃;Bacillus velezensis 157 also have rich lignocellulosic enzymes:xylanase,amylase,protease,pectinase and lignin enzyme activity.The CMCase and FPAse were 4.78IU/mL and 0.73 IU/mL,respectively.Bacillus velezensis 157 is susceptible to 19antibiotics commonly used in veterinary medicine;Animal testing is safe and non-toxic.The crude extract of Bacillus velezensis 157 showed a broad inhibitory spectrum against several animal pathogenic bacteria（Staphylococcus aureus CVCC519,Escherichia.coli CVCC233,Escherichia.coli CVCC236,Escherichia.coli CVCC196,Escherichia.coli CVCC205,Escherichia.coli BNCC125988,Salmonella Typhimurium ATCC25241,Salmonella choleraesuis CVCC3383,Salmonella choleraesuis CVCC3775,Salmonella choleraesuis CVCC79102,Salmonella choleraesuis CVCC503,Salmonella choleraesuis CVCC3780,Salmonella enteritidis CVCC3378,Clostridium perfringens,Proteus hauseri）,aquatic pathogenic bacteria（Aeromonas hydrophila ATCC7966,Aeromonas veronii ATCC35624,Aeromonas veronii TH0426,Aeromonas veronii AV115,Aeromonas veronii AV75,Aeromonas caviae ATCC 15468,Streptococcus agalactiae）and fungal plant pathogens（Botrytis cinerea and Fusarium oxysporum）2.Optimization of CMCase production and analysis of enzymatic properties of Bacillus velezensis 157The optimized conditions of CMCase production by Bacillus velezensis 157 are as allows:CMC as carbon source（3 g/L）,yeast powder as nitrogen source（3 g/L）,3%inoculation,and pH=7 under the condition of 37℃48h.After the optimization,the CMCase of Bacillus velezensis 157 is up to 5.08±0.14 IU/mL.The CMCase properties analysis of Bacillus velezensis 157 found that the optimal temperature and pH of enzyme reaction is 60℃and 5,respectively.It also has good stability and tolerance of NaCl and pH,which belongs to endoglueanases.K_m=5.59 mg/mL,V_max=5.81μg/mL·min;Metal ions Na⁺,Mg²⁺,Ca²⁺can increase Bacillus velezensis157 CMCase,Co²⁺,Hg²⁺,Fe²⁺,Cu²⁺and SDS inhibited CMCase;The surfactant Tween 80,Tween 20 and Triton X-100 had no effect on Bacillus velezensis CMCase.In addition,this study successfully constructed the prokaryotic expression strain of recombinant CMCase from pET32a-157-CMCase BL21（DE3）.The enzymatic properties of the recombinant CMCase were analyzed.The optimal enzyme reaction temperature and pH of recombinant CMCase were 50℃and pH6,respectively,expressing good pH tolerance.The enzymatic properties of pET32a-157-CMCase BL21（DE3）is similar to the CMCase of wild Bacillus velezensis 157.However,the recombinant CMCase has better temperature stability,as well as substrate affinity,which provides convenience for further experimental research.3.Whole-genome sequencing and comparative genomic analysis of Bacillus velezensis 157Here,we report the complete genome sequence of the Bacillus velezensis 157,which is composed of a circular 4,013,317bp chromosome with 3789 coding genes and a G+C content of 46.41%,one circular 8439bp plasmid and a G+C content of40.32%.The genome contained a total of 8 candidate gene clusters and dedicates over15.8%of the whole genome to synthesize secondary metabolite biosynthesis,including Surfactin,Butirosin,Macrolactin,Bacillaene,Fengycin,Difficidin,Bacillibactin,Bacilysin。In addition,the genes encoding enzymes involved in degradation of cellulose,xylan,lignin,starch,mannan,galactoside and arabinan were found in the Bacillus velezensis 157 genome.The comparative genomic analysis of 24sequenced Bacillus velezensis indicated that the genes encoding lignocellulolytic enzymes among all Bacillus velezensis strains mainly acted on cellulose and hemicelluloses.The common GH family involved in cellulose degradation,mainly including GH1,GH4,GH5,GH13,GH16,GH30 and GH32.The shared GH and CE families involved in the degradation of hemicelluloses,including GH11,GH26,GH43,GH51,GH53,CE3 and CE7.But the capacity for lignin degradation is limited,including AA4,AA6,AA7,AA10.4.Enzyme production function on mixed solid-state fermentation of pretreated maize straw and soybean meal by Bacillus velezensis 157The optimum substrate of Bacillus velezensis 157 under solid-state fermentation was detected.Soybean meal appeared to be the most efficient substrate for the single fermentation of Bacillus velezensis 157.The highest yields of pectinase（19.15±2.66U g-1）,cellulase（46.69±1.19 U g-1）and amylase（2097.18±15.28 U g-1）were achieved on untreated soybean meal.In order to solve the problem of low utilization ratio and enzyme production of maize straw,we pretreated maize straw with dilute alkali and then conducted mixed fermentation of pretreated maize straw with soybean meal,and the conditions under solid-state fermentation were optimized.The results showed that the proportion of alkali maize straw and soybean meal is 1:1,and the ratio of substrate and water is 1:0.5,at 37℃for 24h fermentation,and CMCase,amylase,xylanase and pectinase of AWC group are higher than those of single fermented soybean meal group and pretreated maize straw group.The enzyme activity can reach 56.83±1.47 U/g,1949.28±14.41U/g,16.52±0.79 U/g and 14.53±0.56 U/g,respectively.We used SEM,FTIR,GC-MS product analysis and content determination of lignocelluloses to evaluate the degradation effect of the three groups under solid-state fermentation.The results of SEM showed that in AWC group a large area of lignocellulose degradation pores appeared,the number of bacterial strains increased significantly and the strains were densely packed;The results of FTIR,GC-MS product analysis and content determination of lignocelluloses showed that in AWC group the degradation of cellulose and hemicellulose emerged in a large amount.After 10d fermentation,the degradation rate of cellulose was 9.06%,and the degradation rate of hemicellulose was 43.61%;The results of GC-MS showed that the degradation of AWC group mainly acted on the amorphous region of cellulose,hemicellulose and starch,the degradation products consisted of glucose,mannitol,galactose,xylose,cellobiose,three malt sugar alcohol and glucuronic acid substances.In summary,this study screened an endophytic strain named Bacillus velezensis157157 from Eucommia bark,which has been verified to have the potential to degrade cellulose and hemicelluloses by experiment.In addition,the studies on partial biological characteristics and CMCase properties analysis and whole-genome sequencing of Bacillus velezensis 157 will lay the foundation for the subsequent industrial application.Bacillus velezensis 157 under solid-state fermentation for disposing agricultural waste has a wide application prospect and practical significance.