| The cellulose degrading bacteria in the rumen of ruminant played an important role in the degradation of plant crude fiber,the cellulose degrading bacteria in rumen was dominated by anaerobic bacteria,however,there were also facultative anaerobic bacteria.Facultative anaerobic cellulose degrading bacteria were not the main cellulose bacteria in rumen,they also played a certain role in rumen,because they could tolerate a certain concentration of oxygen.Our team had proved that the adding of the mixture of cellulose degrading bacteria and nitrogen fixing bacteria which isolated from the rumen of dairy cows could promote rumen fermentation.However,the nitrogen fixing bacteria may also enter the rumen with feed intake,therefore,in this study,facultative anaerobic nitrogen fixing bacteria were isolated from soil and facultative anaerobic cellulose degrading bacteria were isolated from rumen,soil and feed,researching the effect of the mixture of two bacteria on in vitro fermentation.The strains which we used in the experiment were separated by our laboratory.T2 was nitrogen fixing bacteria which isolated from soil,nitrogenase activity was 97.0523 nmol ·mg-1 ·h-1.Facultative anaerobic cellulose degrading bacteria isolated from rumen,feed and soil were recorded as L,S and T respectively.The high cellulase activity bacteria(about 3.0 U·mL-1)were L4,S1 and T4,and the low cellulase activity bacteria(0.35 U·mL-1)were L6,S4 and T1.Then,mixed 6 cellulose degrading bacteria and nitrogen fixing bacteria with 4:1,recorded as L4,S1,L6,S4,T1 and T4.In this study,the method of in vitro rumen fermentation were used.Ruminal fluid were collected from Holstein cows which fitted with rumen fistula 2 hours before morning feeding,the rumen fluid was filtered by four layers of gauze and mixed with the artificial culture medium to form the mixed culture liquid.This experiment consisted of 7 treatment groups(contained 6 experimental groups and a control group),each group were 7 parallel samples,one time point is 24 h.The experimental group were added 6 mixed bacteria and the control group was added with the inactivated bacteria liquid.Before in vitro fermentation,0.200 g fermentation substrate and 30mL mixed medium were taken into each fermentation tube,respectively added 1mL mixed bacteria,then incubated in shaking bath at 39 ? for 24 h,measured the gas production of 2 h,4 h,8 h,12 h,24 h.At the end of the trial,all samples were collected to determine pH,dry matter degradation(DMD),ammonia-N(NH3-N),microbial protein(MCP),cellulase activity,volatile fatty acids(VFA)and fluorescent quantitation PCR(FQ-PCR).The experiment results showed that the filter paper enzyme(FPA)activity of group S1 was significantly higher than the control group(P<0.05);?-glucosidase activity of group T4 and S1 were both significantly higher than the control group(P<0.05),and group S1 was the highest;CMCase activity had no significant difference in all groups(P>0.05);the DMD values of L4,S1,T4,T1,S4 were significantly higher than control group(P<0.05),and DMD values of group S1 was the highest in these group,which increased 98.68%compared with the control group;as to pH value of the ferment liquid,the group L4 was significantly lower than the control group(P<0.05);in vitro fermentation of 2 h-24 h,the gas production in each experimental group were improved,the experimental group S1 was the highest,which was significantly higher compared with the control group significant deviation(P<0.05);the acetic acid concentration of L6 was significantly higher than that in the control group(P<0.05),however there was no difference among groups in the concentration of propionate and butyrate(P>0.05);the NH3-N concentration of experimental group L4,L6,S1,S4 were significantly higher than that in the control group(P<0.05),and the concentration of NH3-N in group S1 was the highest;the yield of MCP in the experimental group S1 was significantly higher than that of the control group(P<0.05);analysed from different enzyme activity that low enzyme activity group was better than the high enzyme activity group,and the difference was significant in the filter paper enzyme activity,?-glucosidase activity,pH value and gas production(P<0.05),other indicators were no significant difference(P>0.05).In conclusion,the mixture of soil nitrogen fixing bacteria and different sources cellulose degrading bacteria promoted the rumen fermentation.From the aspects of cellulase activity,the fermentation effect of low cellulase activity groups were better than high cellulase activity groups,and the group S1 was the best.As to three different sources of cellulose degrading bacteria,strains from the feed resulted in a more better effect than feed and rumen. |
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