Isolation And Identification Of Major Cellulolytic Bacteria In Rumen Of Sheep And Effects Of Nitrogen Sources On Their Cellulolytic Activities

Major cellulolytic bacteria were isolated from the rumen of the sheep in Inner Mongolia. The isolates were systemically identified. The characteristics of cell growth and cellulose degradation of the cellulolytic bacteria were studied. The effects of nitrogen sources on cellulolytic activity of the rumen cellulolytic bacteria were discussed and the possible mechanisms for the effects of nitrogen sources on improving cellulolytic activity were suggested.23 strains of cellulolytic bacteria were isolated from the rumen of the sheep in Inner Mongolia and identified by morphological and physiological characteristics in the first place. 12 strains of 23 strains of cellulolytic bacteria were selected for (G+C) mol% identification. The 16S rDNA gene sequences of 11 strains of the 12 strains of cellulolytic bacteria were sequenced. Based on the 16S rDNA gene sequences, phylogenetic trees were constructed. The result of the identification showed that there were 4 strains of Butyrivibrio fibrisolvens, 1 strain of Rumincoccus albus, 2 strains of R. flavefaciens, 2 strains of Fibrobacter succinogenes, 1 strain of Clostridium polysaccharolyticum and 1 strain of Enterococcus faecalis among the 11 strains of cellulolytic bacteria. One strain of representative bacterium was selected for further research from every type of strain of Rumincoccus albus, Rumincoccus flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisolvens respectively.The following results were concluded by the studies on the characteristics for cell growth and cellulose degradation of the 4 strains. The rumen coccus grows faster than rivibrio and bacillus. The trend in cellulose degradative rate is consistent with the trend in cellulose activity for all the 4 strains. Fibrobacter succinogenes has the highest cellulose degradative rate and highest cellulase activity among the 4 strains. The exo-β-1,4-glucanases is the rate-limiting enzyme of 3 kinds of cellulase out of the 4 strains of cellulolytic bacteria. The cellulase either exists in cells or is attached to cell membrane. The ratio of cellulose to cellobiose affects the degradative rate for cellulose markedly. When the ratio of cellulose to cellobiose is 7:3 or so, the degradative rate for cellulose reaches the maximum for all the 4 strains.All the 4 strains of cellulolytic bacteria and mixed rumen microbes can grow in the chemically defined medium with ammonia-nitrogen or peptide or amino acid as the sole nitrogen source. Compared with ammonia-nitrogen, peptide and amino acid promote the degradative rate for cellulose of the 4 strains selected significantly. The influence of nitrogen source on the degradative rate for cellulose displays species differences with mixed rumen microbes, Rumincoccus albus, Rumincoccus flavefaciens, Fibrobacter succinogenes and Butyrivibrio fibrisolvens. Phe increases the degradative rate for cellulose of both the mixed rumen microbes and the 4 strains of cellulolytic bacteria, but the effects of Phe on the growth of bacteria are different with strains. Acidic hydrolysates of casein promote the degradation of cellulose of the 4 strains of cellulolytic bacteria, and enzymic hydrolysates of casein don't. Both acidic hydrolysates of casein and enzymic hydrolysates of casein have the same effect on mixed rumen microbes. Acidic hydrolysates of soybean protein, acidic hydrolysates of casein and enzymic hydrolysates of casein enhance the degradative rate for cellulose of mixed rumen microbes. Acidic hydrolysates of casein and acidic hydrolystes of soybean protein improve the degradative rate for cellulose of Butyrivibrio fibrisolvens WH-1. Met, Leu and acidic hydrolysates of soybean protein increase the degradative rate for cellulose of Rumincoccus albus VI3, Rumincoccus flavefaciens CCQ and Fibrobacter succinogenes LBG-1 respectively. The comparison between BCP concentration and cellulase activity in both the solid and liquid phases indicates that the bacteria and cellulase exist mainly by adhesion to cellulose particles. The degradative rate for cellulose is proportional to the extent of adhension of bacteria and cellulose to cellulose particles. Due to the result that there are differences between the effect of nitrogen sources on mixed rumen microbes and that on the major rumen cellulolytic bacteria, it is necessary to investigate the influence of different kinds of nitrogen sources on the major rumen cellulolytic bacteria. The research contributes to a better understanding of the mechanism of nitrogen sources working on cellulolytic activity in the rumen of ruminants.