Screening Of Alkaline Cellulase High Producing Bacteria

Alkaline cellulases(CMCase), refers to the cellulases those exhibited higher activity in alkaline environment than in the neutral and acid environments. For their preference on alkaline environment, alkaline cellulases were broadly used in the industrial fields such as paper making, textile, laundry, environmental pollution remediation, etc.In order to get a alkaline cellulose high-yielding strain, we initially isolated about 100 cellulase-produce strains from waster of a paper mill. The community compositions and the pHylogeny of cellulolytic bacteria in the papermaking wastewater oxidation pond were studied. Through Congo-red transparent circle method and cellulose activity assay, an alkaline cellulose high-yield strain Bacillus pumilus SS-1-4 was isolated. Optimum conditions were examined for the alkaline Cellulase producing catalyzing. We further using nitrosoguanidine to improving SS-1-4 cellulose producing capacity, and a mutants No. 103,with the enzyme activity was 1.18 times of the starting strain was get. Through single factor combined with response surface experiments, the best fermentation conditions and fermentation medium was optimized. The results were listed as follows :1) More than 100 strains were isolated from waster belongs to a paper making mil through the Congo-red plate method. A system research on relationship between the sample sources and the cellulase production community composition was studied.2) By submerged fermentation in flask, a strain SS- 1-4 with relative higher alkaline-cellulose-producing capacity, was isolated. And then was identified as Bacillus pumilus basing on 16 s rRNA sequence information.3) In order to improve the yield of alkaline cellulose, we screened a mutants strain hence force referred to as No.103 from the mutant library produced by treating Bacillus pumilus with NTG. The best mutagenesis condition is 0.8 mg/ml nitrosoguanidine processing for 30 min. Compared with the original strain Bacillus pumilus SS- 1-4, the alkaline cellulase activity of mutant No.103 has a significant increase.4) We optimized fermentation conditions of No.103 by single-factorial combined with response surface experiments. The optimal compositions of fermentation medium were starch 30.0g,peptone 30.0g,NaCl 8.0g,CMC-Na 0.5 g,KH2PO4 2.0 g,(NH4)2SO42.5g, CaCl2 0.5 g, MgSO4 2.0g, with 1000 ml volumn.5) The pilot-scale fermentation was carried in 10 L fermenter. And the parameters were fluid medium 3 L, rotation speed was 180 r/ min, temperature was 28℃, pH 8.0. The agitation speed and aeration rate should be adjusted according to the dissolved oxygen,after 24 h fermentation, the yield of alkaline cellulose activity is 13.46U/ml.