| In the rumen of ruminant,it was very efficient to use forage as the main energy source,it mainly because there are a lot of cellulose decomposing bacteria in the rumen.Most of the rumen cellulose bacteria are strictly anaerobic bacteria,but there are also facultative anaerobic bacteria.Our laboratory finds that it can regulate the rumen internal environment and improve the degradation rate of feed in vitro by mixed facultative anaerobic cellulolytic bacteria and nitrogen fixing bacteria.However,it was still a doubt that facultative anaerobic bacteria are inherent in the rumen,or enter from the outside world.Because the rumen was an open environment for feed,some facultative anaerobic bacteria and nitrogen fixing bacteria may enter the rumen followed with feed.We speculate that facultative anaerobic bacteria may play an important role in the fermentation of aerobic environment after a certain period time of take food.In this study,a facultative anaerobic nitrogen fixing bacteria isolated from rumen fluid and a mixture of facultative anaerobic cellulose from three sources of rumen fluid,soil and feed were selected.Experiment 1: Resurrection and enumeration of bacteriaThe selected strains were isolated from feed,rumen and soil in the early stage of our laboratory,including facultative anaerobic bacteria and nitrogen fixing bacteria.The mixture of bacteria liquid and 30% glycerin was kept in the refrigerator at-20 ?by 1 : 1 ratio.A higher cellulase activity and a lower cellulase activity were selected for each source,and the strains of different sources had similar cellulase activity.The selected strains were isolated from soil T1(2.576 U m L-1)and T4(0.351 U m L-1),from the feed separation of S1(2.824 U m L-1)and S4(0.345 U m L-1),and separated from the rumen L6(3.199 U m L-1)and L4(0.593 U m L-1).In addition,a facultative anaerobic nitrogen fixing bacterium L5(17.637 U m L-1)was isolated from rumen fluid.The selected strains were removed from the refrigerator and thawed,and then resurgence and cultured.Experiment 2 : Effect of mixed bacteria on in vitro fermentation,,Mixed experiment 1 cultured out 6 cellulose decomposing bacteria and nitrogen fixing bacteria(ratio of 4:1)to form 6 kinds of mixed bacteria,marked separately L4,S1,T4,L6,S4 and T1(Because they are all mixed with the same nitrogen fixing bacteria,they are all expressed with cellulose decomposing bacteria for the sake of labeling).In this study,the study method was in vitro fermentation.In vitro fermentation experiments were performed on the in vitro culture system.The in vitro culture system was composed of HZS-H type constant temperature water bath shaking table and 100 m L syringe.The study was divided into 6 experimental groups,1 control groups,each group with 7 replicates(ie,7 syringes).Each syringe contains 0.2000 g(accurate to 0.0001 g)fermentation substrate(concentrate : roughage=45 : 55)and with mixed bacterial liquid of 1m L(the amount of added bacteria was 1.2×109 CFU/L),then add culture medium to 30 m L,and the mixture mix well into the in vitro fermentation culture device to experiment.When fermentation to 2,4,8,12,24 hours,record the syringe reading(ie,gas production).Culture for 24 hours,fermentation was over,all the cultures were removed from injector and pH was determined by pH.The culture was centrifuged at 3500 r/min for 15 min,and the supernatant was added into different centrifuge tubes and stored at-80 ?,which was ready for testing three cellulose bacteria.NH3-N,MCP,volatile fatty acid(VFA)and filter paper enzyme activity,CMC enzyme activity,and the changes of beta glucosidase and flora to detect.The sediment after centrifugation was used to measure the dry matter degradation rate(DMD).The experimental result indicated that the mixed bacteria group compared with the control,there was a significant increase in gas production for experiment group(P<0.05).T1 had the highest gas production rate and the lowest T4 gas production rate,and the low enzyme activity test group and the high enzyme activity gas production were slightly higher.Compared with the control group,the DMD of fermentation 24 h in each experiment group increased significantly(P<0.05).There was no significant difference between the high enzyme activity test group and the low enzyme activity test group(P>0.05).The DMD in the T1 group was the highest,while the DMD in the T4 group was the lowest,and the DMD in the L6,L4 and S4 groups were very close.Test group and control group were significantly increased in pH(P<0.05),wherein L4 highest pH of the test group,the test groups T1 lowest pH,low enzyme activity group pH higher than high enzyme activity group.Add mixed bacteria have a significant effect on NH3-N concentrations(P<0.05),L4,L6,T4 group have high NH3-N concentrations,and low enzyme activity group produced NH3-N concentration was higher than that of high enzyme activity group.Compared to the experimental group and the control group on the concentration of MCP has improved significantly(P<0.05),L6 and T4's MCP concentration group higher than the control group,and high enzyme activity group have higher concentration of MCP than low enzyme activity group.Compared with control,the VFA concentration in the experimental group was significantly increased after adding the mixed bacteria(P<0.05),T1's VFA lowest concentration and lower than the control group.The concentrations of VFA in L4,T4 and L6 were higher.Low enzyme activity group have higher concentration of MCP than high enzyme activity group.The cellulase activity of the experimental group was higher than that of the control group.The filter paper enzyme activity of L4 experimental group was the highest,the filter paper enzyme activity of S1 was the lowest,and the activity of CMC enzyme in the L4 group was the highest,and the activity of beta glucosidase was similar.After adding the mixed bacteria,the number of Ruminobacter albus in the rumen was reduced,however,the increase in the number of Ruminococcus flavefaciensand and Fibrobacter succinogenes was significantly increased.The addition mixed bacteria experiment group all promoted the in vitro fermentation;Low enzyme activity facultative anaerobic cellulolytic bacteria isolated(L4)from rumen fluid mixed with facultative facultative anaerobic nitrogen fixing bacteria(L5)isolated from rumen works best;The fermentation activity of facultative anaerobic celluose with low cellulase activity was better than that of High Cellulase Activity facultative anaerobic bacteria. |
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