Microbial Diversity And Differential Protein Expression For Kenaf Bio-degumming

Bio-degumming has the advantages of low energy consumption,low pollution,low cost and high quality,which is the development direction of kenaf processing technology.However,because of the diversity of the chemical composition of kenaf and the complex mechanism of enzymatic degradation of non-cellulose,it is difficult to screen highly efficient degumming strains for kenaf.In this study,microbial diversity and function of protein through the whole degumming process were studied by metagenomic technology,cultivation technology and iTRAQ protein technology,and kenaf dominant microorganism and differences in protein were illuminated.The study provided an important basis for the screening,construction and catalytic mechanism study for kenaf bio-degumming.Based on the metagenomic technology,during the whole kenaf degumming process from 0 h to 150 h,Bacteroidetes enriched rapidly with the proportion increased from 30% to 65%;and the proportion of strain Prteobacteria decreased gradually from 55% to 20%;TM7 increased from 1% in 0 h to 11% in 150 h;and strain Firmicutes from the 0 h 2% in 40 h to 20% in 40 h,then to 2% in 150 h;actinomycete Actinobacteria reached 1%;the other strains' content was below 1%;fungi was not found in the TOP 30.To 150 h,Bacteroides sp.became the first dominant bacteria group,while Chrysebacterium sp.,Dysgonomonas sp.,Flavobacterium sp.,Acinetobacter sp.,Citrobacter sp.and Pseudomonas sp.were followed successively.From water samples,soil samples and humus,125 strains with more than 21% of the weight loss rate of.kenaf material were screened,belonging to 18 species of 13 genera.Strains K1-K5,R10 and R13 without cellulase and with non-cellulose degradation rate of more than 21%,belonged to Bacillus subtilis,Paenibacillus polymyxa,Clostridium acetobutylicum,Bacillus alcalophilus,Erwinia Chrysanthemi,Pseudomonas brassicacearum and Pectobacterium wasabiae respectively.Bacillus alcalophilus,Erwinia chrysanthemi,and Pectobacterium wasabiae were first reported for kenaf bio-degumming function.The strain R13 Pectobacterium wasabiae IBFC 2016 showed good degumming performance(Patent preservation number: CGMCC No.14601).Strains K1,K3,K4,K5 and R13 showed higher enzyme activity of pectinase,mannanase and xylanase.Strain K2 didn't produce hydrolysis circle in xylanase hydrolysis plating medium,and strain R10 didn't produce hydrolysis circle in xylanase and mannanase hydrolysis plating medium.The hydrolysates in the supernatant of kenaf bio-degumming included mannose,rhamnose,galactohydric acid,glucose,galactose and xylose,while glucuronic acid was not detected.Going further out,the galactoalonic acid continued to increase slowly,and the other monosaccharides increased first and then decreased.During the whole culture,pectinase activity,mannanase activity and xylanase activity increased continuously.For 15 h,pectinase activity,mannanase activity and xylanase activity were 130.25 IU/mL,157.58 IU/mL and 115.24 IU/mL respectively.Pectinase,mannanase and xylanase played an important role in the degumming process of kenaf bast.Based on the protein iTRAQ technology,a total of 197 proteins were identified.After comparing the 4 samples,the differential proteins were mainly located in extracellular matrix,extracellular membrane,cytoplasm and ribosomal subunit.It had many functions,including hydrolase activity,redox activity,superoxide activity,cellulose binding activity,pyruvate kinase activity,aconitase hydrolase activity,gluconate dehydrogenase activity,nucleoside phosphate kinase activity and so on.They were involved in many biological processes,including carbohydrate binding,cellulose degradation,redox balance,glycerol ether metabolism,carboxylic acid metabolism,glyoxylate cycle,tricarboxylic acid cycle,pentose phosphate pathway and biosynthesis.Proteins A0A0R0DX68,A0A0R0EAU2,I4YJF1,A0A0D6TR81,V6SPC1 series,and A0A0M9WBC6 series belonged to the esterase,glucohydrolase,alkyl glycoside hydrolase,dehydrogenase,aldehyde reductase,glucanase.As the fermentation time increasing,the relative content of esterase reached the peak in 110 h,and the others were increased continuously.