| The resources of stalks and straws were abundant in our country, but small parts of them were used as animal roughage. A quite great part of them were abandoned and burn out. This was a great resource waste for human. On the other hand, the nutritive value of stalks and straws was questioned. Bad palatability, low N, P, Ca and digestive energy resulted in high reject rate of cow. Poor quality roughage had counteracted cow industry development.Cellulolytic microbes can improve oligosaccharide and protein content in the roughage. Adding cellulolytic bacteria is thought to be effective method to improve the nutritive value of roughage. There are amount of bacteria in the cow rumen that can decompose cellulose effectively. Of them, anaerobic cellulolytic microbes had been well studied in abroad. Facultatively anaerobic cellulolytic bacteria can grow better in aerobic and anaerobic environment, so they will be the best candidates for roughage fermentation. But few researches had been done about ruminal facultatively anaerobic cellulolytic bacteria.Our study is to screen facultatively anaerobic cellulolytic bacteria which can be explored as Microbial Feed Additives from dairy cow ruminal fluid .The study consists of two parts:Part I : Isolation and characterization of ruminal facultatively anaerobic cellulolytic bacteria in Holstein CowsRuminal fluid was streaked aseptically on sterilized CMC-Na agar plates, then the plates were incubated under anaerobic conditions. The isolates which can form big clear zones surrounding individual colony in CMC–Na agar plates when dyed by Congo red were selected for identification. Biochemical characterization and 16SrDNA sequence analysis were used for the assignment of the strains. Physiological properties and Pathogenicity were also primarily studied.63 cellulolytic strains were isolated and 29 of them behave strong abilities in degrading cellulose. Of 24 Gram-negative isolates, 18 strains related to the genera Klebsiella pneumoniae, 3 strains related to Pseudomonas aeruginosa, 2 strains related to Escherichia coli and 1 related to Klebsiella oxytoca. Of 5 Gram-positive isolates, 2 strains were belonging to Bacillus thuringiensis, 2 strains were belonging to Bacillus megaterium and 1 strain was belonging to Bacillus cereus. The strains we screened show better temperature (20-41℃) and PH (5-9) tolerance. Pathogenicity studies show 3 bacteria of the isolates can not cause death in rats.PartⅡ: The cellulase activities study of Klebsiella Pneumoniae and Bacillus thuringiensisOne strain of Gram-negative bacteria (No.2, Klebsiella Pneumoniae) and one strain of Gram-positive bacteria (No.9, Bacillus thuringiensis) were selected to study their enzyme activities.Ultrasonic wave was used to break bacteria in order to ascertain whether the bacteria produce cell-internal enzyme or cell-external enzyme. Filter paper activity, exo-β-1, 4-glucanase activity,β-1,4-glucosidase activity and endo-β-1,4-glucanase activity were measured to determine enzyme category that the bacterium could sercret. The results showed that supernatant liquid behaved highβ-1,4-glucosidase activity and endo-β-1,4-glucanase activity. At the same time, the enzyme activities of broken fermentation broth and unbroken fermentation broth did not show significant difference. The results suggested that the bacteria could produceβ-1,4-glucosidase and endo-β-1,4-glucanase, and the enzymes were secreted to supernatant liquid.The bacteria precipitations behaved high exo-β-1,4-glucanase activity ,while the supernatant liquid behaved extremely low exo-β-1,4-glucanase activity , which suggested that exo-β-1,4-glucanase can be produced but could not be transported to supernatant liquid. Maybe the enzyme had poor dissociation ability, and the enzyme adhered in cell membrane or cell wall.The results indicated that Klebsiella Pneumonia and Bacillus thuringiensis both could secret endo-β-1, 4-glucanase andβ-1, 4-glucosidase, and the two enzymes were cell -external enzymes. The two bacteria also could produce exo-β-1,4-glucanase, but the enzyme could not be transported to supernatant liquid. However, both the bacteria have better cellulase system.The results of the study suggest that the strains isolated could be candidates for Microbial Feed Additives.
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