| The rough feeding resistance of buffalo may be related to the ability of rumen flora to degrade lignocellulose.Various external factors such as feeding time,season and other periodic factors and the influence of environmental microorganisms on rumen flora cannot be ignored.In this study,based on three crossbred buffaloes fitted with rumen fistulas,rumen samples(0h before feeding and 2h after feeding)and various environmental samples(soil,feed,stool,and litter)were collected in four different seasons to explore the changes in rumen bacteria structure,environmental relevance,and the abundance of lignocellulose-degrading microorganisms,environmental relevance,and degradation ability of lignocellulose-degrading microorganisms in different seasons under housing conditions by sequencing analysis of the bacterial 16 S r RNA gene V3 – V4 region of various samples,laying the foundation for the in-depth study of crude tolerance mechanism in buffaloes,and the main results are as follows:(1)Seasonal fluctuation of rumen bacteria in buffaloes: The abundance of rumen bacteria fluctuated between different seasons,and the difference was extremely significant(p < 0.001).At the phylum level,the relative abundance of Fibrobacteres in spring and winter was extremely significantly higher than that in summer and autumn(p < 0.01),the abundance of Proteobacteria in spring was extremely significantly higher than that in other seasons(p < 0.001),and other phyla also had seasonal dynamic changes in relative abundance,at the genus level,the relative abundance of Rikenellaceae＿RC9＿gut＿group in the spring was significantly lower than that in summer and autumn(p < 0.05),the relative abundance of Ruminocaceae＿UCG-011 in the summer was significantly higher than that in spring and autumn(p < 0.05),the abundance of Fibrobacter in spring and winter was extremely significantly higher than that in summer and autumn(p < 0.01),and other dominant genera also showed large abundance differences in different seasons;there was no significant difference in rumen bacterial diversity and richness at different times during the feeding cycle(p > 0.05).(2)Environmental relevance of rumen flora: The number of OTU shared by rumen samples with soil,feed,stool,and litter was 4617,3835,3577,and 3547,respectively,accounting for 39.69%,32.97%,30.75%,and 30.49% of the total OTU of the rumen,respectively,with more microbial species coincidence and a high degree of similarity in bacterial diversity;feces were the most similar to the rumen in terms of flora structure and evolutionary relationship,followed by feed.(3)Functional analysis of the buffalo rumen flora: The functional prediction of rumen bacteria in buffaloes showed that the rumen microbial function was concentrated on amino acid transport and metabolism,carbohydrate transport and metabolism and other related genes,and the rumen was rich in microorganisms with protein transport,metabolism and cellulose and lignin degradation related functions.(4)Correlation between the lignin-degrading strains in the rumen of buffalo and the environment: Based on the results of the above functional prediction,a fungus strain of Lichtheimia ramosa(F9)and five bacteria strain of Escherichia coli(B20),Bacillus circulans(B36),Bacillus licheniformis(B44,B46)and Bacillus pumilus(B58)with lignin-degrading function was isolated from buffalo rumen by using lignin-only carbon source medium,choose B58 with strong enzyme production ability and good growth as the test bacteria for subsequent studies;these 5 strains of bacteria can be clustered into 4 OTUs of environmental sample sequencing data.and these microorganisms colonized in the rumen may come from an external environment.(5)Biological characteristics and probiotic potential of B58: B58 can grow rapidly at p H 6.5-9.0,5.44% and 9.29% of the degradation of sodium lignosulfonate and rice straw cellulose for 7 days,fermentation in the production medium for 5 days,acid production reduces the p H of the medium to 5.6,the maximum enzyme activity of lignin manganese peroxidase is 1.47 U/m L,and the enzyme activity of cellulose degrading enzyme is 3.69 U/m L;10 common virulence genes are detected in its genome,of the 25 common antibiotics,only 6 antibiotics such as cefotaxime are resistant,12 antibiotics such as ganciclovir are sensitive,the adhesion of porcine small intestinal epithelial cells IPEC-J2 is 4.32%,and the environment with p H 2.0 and bile salt concentration of 0.30% can be tolerated.In summary: there was a significant difference in the abundance of rumen bacteria between different seasons,no significant difference in rumen bacteria at different sampling times during the feeding cycle,and there was a species diversity association between the rumen and the environment such as feces and feed.Prediction of bacterial function revealed that the rumen was rich in degrading lignocellulosic microorganisms,and all six strains isolated from the rumen verified this function,and there was a genetic association between these strains and the external environment.Bacillus pumilus B58,which has strong enzyme production ability,was selected for further study and found that this strain was able to degrade lignin and cellulose,had high manganese peroxidase and cellulase production ability,had high safety and stress resistance,and had the potential to be developed as a probiotic. |
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