Study On Screening The Best Combination Of Cotton Stalk And Sugar Beet Residue By In Vitro Gas Production And Nylon Bag Method

Object:To screen the best optimal combination and conditions of 10 kinds of mixed fermentation products of cotton stalk and beet residue by in vitro fermentation characteristics,rumen disappearance rate and the study of the microbial flora and number of microorganisms in the fermentation system.It provides scientific theoretical basis for the preparation,popularization and application of the mixed fermentation product of cotton stalk and beet residue.Methods:A three-factor,three-level orthogonal treatment combination were used,in order to identify the optimal fermentation conditions.Beet residue was added to cotton stalk in proportions of 10%,30%or 50%,with0.1%,0.2%or 0.3%added urea,and 0.05%,0.1%or 0.2%added bacterial liquid?Lactic acid bacteria and yeast are the main components?,with salt added 0.2%in all cases.From the 27 available combinations,9 were chosen for inclusion in the experiment,designated A–I,and a control,K,as follows:A)10%beet residue,90%cotton stalk,0.1%urea,0.05%bacterial liquid,0.2%salt;B)10%beet residue,90%cotton stalk,0.2%urea,0.1%bacterial liquid,0.2%salt;C)10%beet residue,90%cotton stalk,0.3%urea,0.2%bacterial liquid,0.2%salt;D)30%beet residue,70%cotton stalk,0.3%urea,0.05%bacterial liquid,0.2%salt;E)30%beet residue,70%cotton stalk,0.1%urea,0.1%bacterial liquid,0.2%salt;F)30%beet residue,70%cotton stalk,0.2%urea,0.2%bacterial liquid,0.2%salt;G)50%beet residue,50%cotton stalk,0.2%urea,0.05%bacterial liquid,0.2%salt;H)50%beet residue,50%cotton stalk,0.3%urea,0.1%bacterial liquid,0.2%salt;I)50%beet residue,50%cotton stalk,0.1%urea,0.2%bacterial liquid,0.2%salt and control group K?100%cotton stalk,0.00%urea,0.00%bacterial liquid,0.2%salt?.Experiment one:The purpose of this test was to study the in vitro gas production characteristics and fermentation parameters of the mixed fermentation product of cotton stalk and beet residue.A 100mL glass syringe was used as a fermentation container,and the in vitro gas production rate,gas production rate,and fermentation parameters?pH,soluble sugar,ammonia nitrogen,lactic acid,volatile fatty acid,and microbial protein?of the mixed fermentation product of cotton stalk and beet residue were measured.Experiment two:The purpose of this test was to study the microflora and the number of microorganisms in the in vitro fermentation broth of the mixed fermentation product of cotton stalk and beet residue.Illumina PE250 platform sequencing and real-time PCR technology were used to analyze the microbial flora and quantity in the fermentation broth.Experiment three:The purpose of this experiment was to study the rumen degradation rate of the mixed fermentation product of cotton stalk and beet residue.A nylon bag was used to determine the degradation rate of nutritional components of the mixed fermentation product of cotton stalk and beet residue.Results:Experiment one:The gas production in 48 hours,group I was significantly different from that group K?P<0.01?,significantly?P<0.05?higher than group A,D,and F;group G was significantly?P<0.05?higher than group K by 56.03%,there were no significant difference between the other groups?P>0.05?.Group I had the highest digestible organic matter?DOM?and metabolism energy?ME?,respectively 722.40 g·kg^-1and 9.75 MJ·kg^-1.The slow degradation parameter b in all group,there were significant difference between group I and group K?P<0.01?.The group G,H,and K were significantly different?P<0.05?.The rapid degradation parameter a and the gas production rate c were no significant difference in all group.The content of ammonia nitrogen in group H was significantly different from the other groups?P<0.05?,there was no significant difference between group K and group D,E and F?P>0.05?.Soluble sugar,group H,I and other groups were significantly different?P<0.05?,the group H was significantly higher than group B44.06%,group K 31.23%;group K and other groups were significantly different;pH,lactic acid and microbial protein were not significantly different?P>0.05?;The highest total volatile fatty acid?VFA?concentration was group H(100.31 mmol·L^-1),which was significantly higher than the other groups except?P<0.05?the group G.The concentration of acetic acid and butyric acid was also the highest,which was significantly higher than the other groups?P<0.05?except the group G and I.The concentration of propionic acid was the highest in group G,which significantly higher than the other groups except group H?P<0.05?;its acetic acid/propionic acid?A/P?is the lowest,significantly lower than the groups A,B,C and K?P<0.05?.According to the membership function analysis and comprehensive value ranking of the mixed fermentation products of cotton stalk and beet pulp,the average values of membership functions of each group were I?0.719?>H?0.692?>F?0.595?>G?0.591?>C?0.407?>E?0.400?>B?0.395?>K?0.374?>D?0.307?>A?0.243?.Experiment two:The Chao 1 index and simpson index in the fermentation products of each group were not significantly different?P>0.05?;in the shannon index of each sample,there were significant differences between group C,group I and group K?P<0.05?,and no significant difference from the other groups?P>0.05?and higher than other groups;the relative abundance above 0.1%are Bacteroides?53.91%?,Firmicutes?35.76%?,Proteobacteria?3.61%?,and Unclassified?2.10%?,Spirochaetes?1.00%?,Kiritimatiellaeota?0.92%?,Synergistetes?0.69%?,and Chloroflexi?0.52%?in phylum level.There are 18dominant bacteria with relative abundance above 1%.The top 10 dominant bacteria are Rikenellaceae RC9gut group?19.50%?,F082_norank?11.97%?,Prevotella 1?6.94%?,and Muribacaceae_norank?5.80%?,[Eubacterium]oxidoreducens group?3.58%?,Christensenellaceae R-7 group?3.48%?,Ruminococcaceae UCG-002?2.74%?,Lachnospiraceae ND3007 group?2.29%?,Prevotellaceae UCG-001?2.21%?,and Unclassified?2.10%?.There was no significant difference between Bacteroidea and Firmicutes in each sample.The proteobacteria of group B was significantly higher than the other groups except groups A and I.The Spirochaetes of groups G and H were significantly different from B,C,and D;The kiritimatiellaeota of group K and C,D,H,and group I were significant.There was no significant difference between Rikenellaceae RC9 gut group,F082_norank and Prevotella 1?P>0.05?.In Muribacaceae_norank genus,group G and F and K groups were not significantly different?P>0.05?,and were significantly different from other groups?P<0.05?.Among the[Eubacterium]oxidoreducens group,the K group was significantly different from the C group?P<0.05?,and were not significant different from other groups?P>0.05?.There was no significant difference in the total fungi and methanogens of the samples in each group?P>0.05?,the total bacteria in the A,B,D,E,I,and K groups were not significantly different?P>0.05?and the largest total bacteria in the group B;the groups G and H did not show significant differences?P>0.05?and significant differences with other groups?P<0.05?.Experiment three:The degradation rates of DM,OM,CP,NDF and ADF at different time points of thefermentation products in each group were significantly different?P<0.05?.The degradation rates of DM,OM,CP,NDF and ADF were highest in group I for 72 h.There was no significant difference in the degradation rates of DM and OM between group I and group G?P>0.05?,there was no significant difference in the degradation rate of NDF between group I and group G,H?P>0.05?,the difference in ADF degradation rate between group I and group H was not significant?P>0.05?,the other differences were significant?P<0.05?.The rapid degradation a of each group,the DM,OM and ADF of group G,H and I were significantly different from the other groups?P<0.05?,the DM and OM differences of group G,H and I were not significant?P>0.05?,the group I was significantly different from the other groups?P<0.05?in cp,and the difference of NDF between groups A,B,C and K was not significant?P>0.05?;the slow degradation b of each group,they?group I and G in OM,group I and H in CP and NDF,group I and H,G in ADF?were not significantly different?P>0.05?,while others were significantly different?P<0.05?;the potential degradation fractions of each group,the OM of group I and group G,the NDF of group H,the ADF of group G and group H were not significantly different?P>0.05?,and the other groups were significantly different?P<0.05?;The effective degradation rates in each group were the highest in group I,group I and G in DM,group I and G,H in OM and NDF were not significant;The difference of OM between group K and group A,B and C was not significant?P>0.05?,and there were significant differences among others?P<0.05?.According to the membership function analysis and comprehensive value ranking of the mixed fermentation products of cotton stalk and beet pulp,the average values of membership functions of each group were I?1.000?>H?0.911?>G?0.862?>F?0.425?>E?0.350?>D?0.310?>C?0.176?>A?0.151?>B?0.150?>K?0.004?.Conclusion:The dry matter-based cotton stalk and beet residue ratio of 50:50,the addition of 0.1%urea,0.2%compound bacteria liquid,and 0.2%salt,group I has the best rumen degradation characteristics,and its mixed fermentation product has the highest in vitro gas production.It has the largest digestive organic matter and metabolic energy,acetic acid and butyric acid concentrations are the second,and the highest comprehensive value of the fermentation combination.It is the best fermentation combination and can be used as a rough feed for ruminants.Different cotton stalk mixed fermentation products will affect the rumen microbial diversity,composition and structure to some extent.