| As the further study of rumen and the rumen microbe,the effect on in vitro rumen fermentation by inoculanting a single strain and mixed strain was gradually research emphasis,and the discovery and interaction of facultative anaerobe in the rumen wsa more research focus.Therefore,under the precondition of separation facultative anaerobe,this experiment will study the effect on in vitro rumen fermentation by inoculanting mixtures with azotobacter and celluloytic bacteria by microorganism training experiment and in vitro simulating rumen fermentation experiments,the aim was studyed the differences between the mixed bacteria.The experiment was dicided into following two parts:1Resurgence and culture of azotobacter and celluloytic bacteriaThis experiment choosed the facultative anaerobic azotobacter and cellulolytic bacteria witch were separated by the students had graduated,and they were kept-20 ?(Bacteria liquid and glycerol = 1:1);and they respectively came from soil(T),fodder(S)and rumen(L).This experiment respectively choosed 2 strains of cellulose decomposing bacteria from T,S,L,and the principle was that different source of strains had adjacent cellulase activity,but 2 strains came from the same source had different cellulase activity;The item number of T,S,Lused the number when separated,that were respectively T1 and T4,S1 and S4,L4 and L6.In addition,the facultative anaerobic azotobacter came from fodder,and the item number was S A3.The properties of selected strains were as follows:There are three of strains had high enzymatic activity and witch were separately S4(the fungus:KX588167,activity:2.824U·mL-1),T1(the fungus:KX426078,activity:2.576U·mL-1),L6(no fungus,activity:3.199U·mL-1)and there are three of strains had low enzymatic activity and witch were separately S1(the fungus:KX588165,activity:0.345 U·mL-1),T4(the fungus:KX588169,activity:0.351 U·mL-1),L4(no fungus,activity:0.503 U·mL-1)along with 1 strain of facultative anaerobic azotobacter SA3 witch came from fodder(the fungus:KU531598).Unfreezing the strains that were saved in-20 ? at the room temperature,and inoculanting in LB liquid medium to resurrection by Thermo Forma orbital shaker at 39 ?,and then inoculanting in LB solid medium to cultivate.The order of magnitude of living bacteria that was saved in the freezing temperatures(-20 ?)and after cultivated was in the normal range for 1010?1014 cfu·mL-1.After,mixing the 7 of strains into plastc ring according to the proportion of 1:4(Azotobacter:cellulose decomposition microbes),the item number of plastc ring used the number when separated(T1,T4,S1,S4,L4,L6).2 In vitro fermentationThis study included 6 of experimental groups and 1 of control group.1 mL of plastc ring in each experimental group,and 1mL of SA3(the fungus:KU531598)had been inactivated in control group.The fermentation test was processed on culture apparatus,the body of the device was composed of constant temperature water bath and syringe of 100 mL.The experiment was desigened by using one-factor and each processing including 7 times repeated(7 of syringes),there were 0.200g of fermentation substrate(refined:crude=45:55)and 30mL of mixed culture solution and corresponding mixed bacteria liquid in each syringe,the total bacteria amount was 1.2×109 cfu.Gas production was record separately at 2,4,8,12,24 h and every fermentation tubes were incubated for 24 h to measured of fermentation parameter.The results of the experiment indieated that there were no differences in pH value except as the treatment S1 and S4 that all came from fodder,in addition that the S1 was the lowest with the pH value reduce to 2.07%compared with control treatment(P<0.0001);However,the concentrations of ammoniacal nitrogen(NH3-N)for L4,S1,L6 was higher significantly than control(P<0.05),but NH3-N concentrations in T4,S4 was not different than no activity treatment;Diffience in acetic acid,propionic acid and butyric acid between the control treatment and the L4,T4,S1,L6,T1 and S4 was significant(P<0.05),and among inoculants,the concentrations of acetic acid,propionic acid and butyric acid of L4 was highest compared with control and respectively L4 67.62%,54.51%,56.85%more than control.Among inoculants,the T4,S1,S4 degraded more fermentation substrate than did control and the difference was significantly(P<0.0001)at 24h fermentation,and the degradability of DM was the highest for S1;After fermentation,FPA activity,?-glucosidase activity were similar between control and other with expection of L4,T4,S1 witch was higher than control while S1 was more CMC enzymatic activity than control(P<0.05)when there were no differences in CMC enzymatic activity between other treatment and control.There were significant increase between every treatment and control(P<0.05)in gas production.The fermentation had nothing to do with active bacteria number according to the result of RT-PCR(P>0.05).In conclution,the cluture of azotobacter from fodder and different celluloytic bacteria had signally effect on in vitro fermentation,and the low activity mixed bacteria from fodder had a good effect on fermentation. |
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