| ObjectiveLignocellulose is the most inexpensive and abundant renewable natural resources worldwide, therefore, which evokes great interest in the use of cellulosic biomass as a renewable source of energy. In the process of transformation, cellulose is degenerated into monosaccharide that can then be converted to available products such as soluble oligosaccharides, glucose, alcohols, and other industrially important chemicals, and so on. Currently, an effective way to degrade and transform the cellulose is to isolate and identify the microorginasm producing the cellulase. Cellulases can be divided into three main groups of enzymes: endocellulase, exocellulase (also called cellobiohydrolase), andβ-glucosidase. The first two enzymes degrade cellulose to oligosaccharides and cellobiose, and the glucosidase hydrolyzes cellobiose to glucose monomers. Cellulases have attracted much interest because of their enormous potential to convert cellulose to liquid fuel .The screening for the strains with high cellulase activity and the studies on how to improve strains cellulase activity are very important for ecology, economy and our society. In the present study, we intended to obtain some cellulase- producing bacteria with high activity by a modified screening medium from some collected samples, and this study will provide the new theory basis for further industrialization production of cellulase.Material and Methods Material source: Soils containing rich cellulosic material and waste paper pulp were collected from Zhengzhou University and a paper mill in Zhengzhou city, respectively.Screening methods: Using CMC-Na as main carbon source, enrichment culture was carried out with the collected samples, then primary screening with Congo red identification culture medium by diluting and smearing slab methods were further performed, it was evaluated by the diameter size of the transparent zone around the colony on plates. In order to obtain pure culture strains with high cellulase activity, secondary screening was being underway with primary screening cultures by plate streaking.Identification methods: Combined with the results of morphological and physiological, biochemical characterization, the 16S rDNA were further amplified by PCR method and sequenced. The 16S rDNA of isolated strains was compared with GenBank and the phylogenetic tree was constructed with related bacteria. Based on the above results, the isolated strains were then identified.Shaking flask culture: Shaking flask fermentation of isolated strains of the optimum conditions of CMCase production were separately carried out according to the experimental results on plates. In order to investigate the process of CMCase production and activity, enzyme activity was measured during different culture interval.Result①After primary and secondary screening steps, two CMCase producing bacterial strains were isolated from the collected samples.②Based on 16S rDNA sequences analysis, strain CP4 and L1 appeared the highest nucleotide homology with Bacillus sp and Arthrobacter sp, respectively.③Preliminary study revealed the strain CP4 was successfully grown with rapid growth rate and a high relative enzymatic activity in the pH range from 5 to 11 and temperature range from 20 to 45℃. Its relative CMCase activity was 5.43 mm/d under 35℃and pH9 during 3 days plate culture. ④Strain L1 was an alkalophilic bacteria, which could produce CMCase in the pH range from 7 to 11, and its relative CMCase activity was 3.25 mm/d under pH10.⑤The result of the shaking-flask fermentation were as follows: CMCase activity of CP4 and L1 strains reached 104.34 U/ mL and 88.33 U/mL, respectively, when the strain was cultivated with1.0 g/L CMC-Na as main carbon source at 180 r/min for 48h accompanied with inoculum size 2%, initial pH value 8.0 for CP4 and 11.0 for L1, 35℃for CP4 and 30℃for L1.ConclusionCP4 and L1 strains are identified as Bacillus megaterium and Arthrobacter sp, respectively, which can both produce alkaline cellulase with high activity. |
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