The Effect Of Cellulose Bacteria And Azotobacter Amount On In Vitro Fermentation

It has been demonstrated that the facultative anaerobic cellulolytic bacteria and nitrogen-fixing bacteria live in the rumen,but there is rarely report about its function.In early in vitro culture experiments,we found that the rumen of the internal environment and the degradation rate of feed were improved when adding facultative anaerobic cellulolytic bacteria and nitrogen fixation bacteria,which were from the rumen of cows,by ratio of 1: 4 mixed to the rumen.On this basis,we choose facultative anaerobic cellulolytic bacteria and nitrogen fixation bacteria,which were separated from the rumen of cows,as mixed bacteria.Then through in vitro fermentation method to explore the appropriate amount of mixed bacteria.And in order to reveal the possible mechanism of the mixed bacteria,we determined the amount of three kinds of cellulosic bacteria in the culture medium was by Quantitative Real-time PCR.In this experiment,we design two tests,activation of facultative anaerobic cellulolytic bacteria and nitrogen fixation bacteria and on artificial rumen fermentation test.Activation and enumeration of nitrogen-fixing bacteria and cellulose pectinolytic microorganism.The facultative anaerobic cellulose pectinolytic microorganism and nitrogen-fixing bacteria were got through the early laboratory separation,they all come from the rumen contents of cows.And they were kept in-20??Mixture of bacterial solution and 30 % glycerol 1: 1?The strain was removed by the refrigerator,thawed at room temperature,and activated with LB liquid medium.then cultured,until the bacterial count exceeded 1×10¹1 CFU/m L,the number of bacteria was determined by plate counting method.Remove part of cellulose decomposition,and put in high pressure,121 ? for 15 minutes,for diluted bacteria solution.The viable count of cellulose pectinolytic microorganism in the bacteria solution was diluted to 1.2×10¹⁰ CFU/m L,1.2×10⁹CFU/m L,1.2×10⁸ CFU/m L,1.2×10⁷ CFU/m L,1.2×10⁶ CFU/m L and 0 CFU/m L.Azotobacter bacteria also have the same dilution treatment.Then,mixing two kinds of the bacteria liquid as the ratio of 1: 4,with the same dilution concentration,to crank out mixed bacterial solution.The influence of cellulose bacteria and azotobacter additive amount on in vitro fermentation.Fermentation equipment was made of a shock water bath and 10⁰ m L syringes?called fermentation tubes?.In this test 48 fermentation tubes were chosen,and those fermentation tubes were divided into two groups,24 per group.We designed those two groups as one group and two group.The one group was finished at 12 h and the two group was finished at 48 h.Each group was divided into six treatment groups,each treated group made 4 replicates.The six processing groupper group were added to 1m L well-made mixed bacteria liquid(the addition of the six processing group were 4.0×10⁷ CFU/L,4.0×10⁸ CFU/L,4.0×10⁹ CFU/L,4.0×10¹⁰ CFU/L,4.0×10¹1 CFU/L and 0 CFU/L).To be fermented for 12 h and 48 h,the contents of NH₃-N,MCP,VFA,methane,DMD,cellulase activity?including filter paperase,CMC enzyme and ?-glucosidase?of the two group were measured.Furthermore,approximately 4 m L fermentation liquid in each fermentation tube of Group 2 was stored in liquid nitrogen and used to determine the effect of adding mixed bacteria on three cellulose bacteria?ruminococcus albus,ruminococcus flavefaciens and fibrobacter succinogenes?mount in rumen liquid.The gas production in group 2 was directly read on fermentation tubes at 2 h,4 h,8 h,12 h,24 h,36 h,48 h.According to the p H value and NH₃-N,the optimum addition amount of pectinolytic microorganism and nitrogen-fixing bacteria mixed bacteria was obtained.Selecting the control group of 48 h,optimum dosage group(4×10¹⁰ CFU/L)and the zymotic fluid of 4×10⁸ CFU/L to measure the effect of probiotics on the content of three cellulose decomposition bacteria in rumen fluid by fluorescence quantitative PCR method.The experiment result show that the p H of all test group was within the optimum p H range.And the contents of VFA and methane in the 12 h were lower than those in the control group?P<0.05?.However,the NH₃-N,MCP,DMD and cellulase activity were increased in each experimental group were higher than those in the control group?P<0.05?.Especially,in the experimental group 4.0×10¹⁰ CFU/L,the content of NH₃-N and MCP,DMD and filter paper enzyme activity were higher than those in the control group?P<0.05?.At 48 h,there was no significant difference in the p H values between the treatment groups.The contents of NH₃-N,MCP,VFA,TVFA,and methane,DMD and the activity of cellulase in the experimental group were higher than those in the control group.The content of acetic acid,propionic acid,methane,NH₃-N,MCP and DMD was significantly higher than that in the control group.The content of butyric acid was not significantly different from that of the control group,but the value of butyric acid higher than that of control group.The cellulase activity of 4.0×10¹⁰ CFU/L group was lower than that of the control group.The effect of these additions to 4.0×10¹⁰ CFU/L was most obvious.The experimental group 4×10¹⁰ CFU/L and control group were significantly different?P<0.05?.White tumor ruminococcusy in the test 4×10¹⁰ CFU/L group was significantl lower than the control group?P<0.05?,but in the experimental 4×10¹⁰ CFU/L group,it was decreased compared to the control group?P=0.0991?.Yellow ruminococcus in the experimental 4×10¹⁰CFU/L group was significantly higher than it in the control group?P<0.05?,but in the experimental 4×10⁸ CFU/L group,it was not significantly different from it in the control group?P>0.05?,but slightly higher than the control group.As for Chanhupo suansi Corynebacterium kutscheri,there were differences in all groups,the content was 4×10¹⁰ CFU/L group>4×10⁸ CFU/L group>control group.In combination,it can be concluded from in vitro test that the viable bacteria composed of strengthening and cellulose decomposing bacteria can regulate the ruminal environment,and the optimal amount is 4.0×10¹⁰ CFU/L.And the analysis of the number of three cellulase bacteria canunderstand that the viable bacteria preparation can regulate the rumen environment,may be due to the effect of the addition of live bacteria on rumen microbial population.