| This experiment was intended to isolate the fiber degrading bacteria from the rumen contents（including two parts of rumen fluid（L） and residue（N））, soil（T） and diets（S）.The bacterial strains were identified by conventional and molecular biological methods, and the cellulase activity of the strains was determined. The aim was to compare the properties of the cellulose decomposing bacteria from the rumen contents and the cellulase activity, which would provide experimental data for the understanding and utilization of the rumen facultative anaerobic bacteria.Test 1 Isolation, purification and screening of decomposing cellulose bacteriaCarboxymethyl cellulose was used as isolation medium with culture process of anaerobic and aerobic to get bacteria with facultative character. Decomposing cellulose ability for the bacteria was deterimined by congo red plate method Methods of morphological characteristics,physiological and biochemical characteristics and 16 Sr DNA sequence analysis were adopted, for classifying and identifying the bacteria.Results showed that 29 strains of decomposing cellulose bacteria（DCM） were isolated from the rumen contents, soil and diets.It was found that 6 of the 29 strains had a strong ability to decompose cellulose, which the transparent circle diameter was more than 10 mm for. According to the sequence of 16 Sr DNA, 25 were Bacillus, including 12 strains of Bacillus tequilensis（ 2 strains rumen fluid, 4 strains from rumen residue, 5 strains from, and 1 strains from soil）. Seven strains of Bacillus methylotrophicus（2 strains from rumen fluid, 2 strains from rumen residue, and 3 strians form diet and soil, respectivly）, 5 strains of the lake of Bacillus amyloliquefaciens（3 form residue,and 1 strain from soil and diet, respectively）, and 1 strain of Bacillus licheniformis from rumen fluid). However, only 2 were Acinetobacter（from rumen fluid and soil）.Test 2 Determination of cellulase activity of decomposing cellulose bacteria.Five m L enzyme producing medium was loaded into a 50 m L triangle bottle, and then 1 Ml standard seed fluid（with purified bacteria）was added. The bottles were culture 48 h at 39℃ and speed of 220r/min. After that, activity of cellulase was gotten based onamount of reducing sugar production determined using 3,5- two nitro salicylic acid. The cellulase included three kinds of filter paper enzyme（FPE）, CMC and β-glucosidase.FPE activities of L5, L7, N5 from rumen residue were higher than that from soil T1 and diet S6（P<0.05）. That of N9 was higher than that of S1 in the rumen（P<0.05）, S6 had significantly higher activity, comparing with T1（P<0.05）. Moreover the L5 had biggest value（P<0.05）. For CMCactivity, L5, L7, N5 and N9 were higher than T1 and S6（P<0.05）, and however L5 enzymeactivity was higher than other strains（P<0.05）. But there was no difference between S6 strain and T1 strain of soil（P>0.05）.The There was no different β-glucosidase activities between L5 and S6（P>0.05）, and however there was obvious differenceamongother strains（P<0.05）.Based on the above two experiments, the followings were gotten:（1） 29 strains of bacteria isolated from the rumen contents,soil and diet were all facultative anaerobic bacteria, and they almost belonged to the genus bacillus.（2） the bacteria from the soil and the diets were isolated from the same species in the rumen contents, which indicated that the cellulose decomposing bacteria in the rumen contents may enter the rumen with the outside soil or the diet.（3） the rumen bacterial strain L5 filter paper enzyme activity and CMC enzyme activity was the highest, which were 6.2741U·m L-1 and 7.9809U·m L-1, respectively, but the T1 strain of the soil strain was highest for 6.1753U·m L-1... |
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