| In plant cell walls,lignin is tightly connected with cellulose and hemicellulose with different chemical bonds to form a stable lignin-carbohydrate complex,which hinders the sufficient contact between cellulase/hemicellulase and the covalent bond structure of polysaccharides inside the cell wall and is the greatest obstacle for ruminants to utilize crude fiber raw materials.Disrupting the lignin structure and exposing the chemical bond between cellulose and hemicellulose can effectively improve the utilization rate of roughage.Previous studies in our laboratory found that buffaloes had a higher ability to degrade roughage than cattle,and were able to effectively degrade lignin,which may be related to the abundant microbial population distribution in the rumen of buffaloes.In this study,a strain of Ochrobactrum pseudintermedium numbered as CB2 isolated from the rumen of buffalo was used to study its lignin degradation ability and gene function,and the results were as follows:1,The growth characteristics of CB2 were different in aerobic and anaerobic environments,with faster aerobic growth rate and higher concentration of bacterial solution in the plateau phase;the p H of CB2 suitable growth in aerobic and anaerobic environments was 5.5 – 9.0 for aerobic and 6.0 – 7.5 for anaerobic,respectively.2,In aerobic and anaerobic environments,CB2 did not degrade cellulose in straw;in aerobic environments for 7 days,the hemicellulose and lignin degradation rates in rice straw were 25.42% and 7.65%,and the hemicellulose and lignin degradation rates in corn straw were 12.26% and 2.64%,respectively;in anaerobic environments for 7 days,the hemicellulose and lignin degradation rates in rice straw were 13.66% and 16.66%,and the hemicellulose and lignin degradation rates in corn straw were 1.82% and 8.66%,respectively.The degradation ability of CB2 to hemicellulose was significantly higher in aerobic environment than in anaerobic environment(p < 0.01),the degradation ability of CB2 to lignin was significantly higher in anaerobic environment than in aerobic environment(p<0.05);the degradation ability of CB2 to hemicellulose and lignin in rice straw was significantly higher than that in corn straw(p<0.01).3,CB2 was combined with K.pneumoniae(KL)isolated in the laboratory to degrade crop straw.The results showed that there was no significant change in cellulose content under aerobic and anaerobic environments(p > 0.05),and the degradation rate of hemicellulose in rice straw was significantly higher than that in corn straw(p < 0.01);when rice straw or corn straw was degraded,the degradation rate of hemicellulose in aerobic environment was higher than that in anaerobic environment(p < 0.01),and the degradation rate of lignin was significantly higher in anaerobic environment than that in aerobic environment(p < 0.05).4,The lignin degradation ability of CB2 under anaerobic environment was verified,and it was found that CB2 had degradation ability for kraft lignin,p-hydroxybenzoic acid,ferulic acid and p-coumaric acid,and the degradation rates were 11.48%,6.17%,5.93% and 6.79% on the 7th day,respectively.5,Aniline blue fading method was used to qualitatively detect whether CB2 secreted laccase,peroxidase and cellulase,and the results revealed that only aniline blue plates faded,indicating that CB2 could secrete peroxidase,but no secretion of cellulase and laccase was detected.6,The results of genome sequencing showed that the total length of CB2 genome sequence was 4.31 Mb,GC content was 57.90%,and two chromosomes and one plasmid were assembled,including chr1 length of 2620,480 bp,GC content of 58.02%;chr2 length of 166,4849 bp,GC content of 57.71%;and plas1 length of 20286 bp,GC content of 57.37%.Through gene function annotation and amino acid sequence alignment on NCBI,four common lignin-degrading enzyme coding genes were found,encoding multicopper oxidase(Lac),blue copper oxidase(Cue O),iron/manganese superoxide dismutase,and dye decolorization peroxidase(Dy P),and another 32 gene-coding proteins were closely related to lignin degradation,including catechol 2,3-dioxygenase,protocatechuic acid 3,4-dioxygenase,alcohol dehydrogenase,4-hydroxyphenylpyruvate dioxygenase,4-hydroxybenzoate 3-monooxygenase,salicylate hydroxylase,and3-oxoadipate Co A transferase and so on;a total of 12 aromatic compound degradation pathways were enriched by KEGG Pathway database enrichment analysis,mainly benzoate degradation,styrene degradation,and phenylalanine metabolism.In summary,straw degradation tests showed that CB2 had the ability to degrade straw lignin and hemicellulose in both aerobic and anaerobic environments,and the lignin degradation rate of rice straw was higher than that of corn straw,and the lignin degradation rate of anaerobic environment was higher than that of aerobic environment;CB2 and KL combination could reduce the degradation rate of hemicellulose by single bacteria,but did not affect the lignin degradation ability;in the pure culture system,CB2 could degrade lignin and its derived aromatic compounds in different substrates in anaerobic environment;genomics analysis revealed that CB2 genome could encode a variety of common lignin degradation enzymes such as multicopper oxidase(Lac),and the existing lignin degradation pathways mainly had multiple pathways such as benzoate degradation. |
