Screening And Identification Of Cellulase-producing Strains And Optimization Of Their Fermentation Conditions

Cellulolytic enzymes are endo-glucanases(EG), exoglucanases (CBH) and cellobiases (BG), and cellulose is completely hydrolyzed enzymatically through the synergistic action of three types of enzymes. Filter paper enzyme activity (FPase) represents the total enzyme activity of various enzyme activity. Cellulase has a very broad application prospects including energy, feed, textile, food, industrial washing, oil exploration, agriculture, medicine and so on, so the identification and screening of cellulase-producing strains and enzyme production conditions research have important ecological benefits, economic and social significance. Fungi, bacteria and actinomycetes can all produce cellulase, the components of cellulase produced by micro-organisms are different due to the different types of micro-organisms, which constitute different cellulose degradation characteristics. In this paper, on the one hand, we studyed the screening, identification and the optimum conditions for celluase-producing fungi, on the other hand, the screening and the polyphasic taxonomy of actinomyces isolated from the desert which can produce cellulase were studyed.The screening, identification and the optimum conditions for celluase-producing fungi were studied in the first part of the paper. More than 50 strains which could produce celluase were isolated by enriching culture from 9 samples of Cow dung compost, collected from the Huazhong Agricultural University, Wuhan. Finally a efficient celluase-producing strain(HS-F9) was screened through the determination of filter paper degradation capability and the liquid fermentation of celluase production.The strain HS-F9 could completely degradate the filter paper in 4 days and the activity of endo-1,4-β-glucanase (EG), exo-1,4-β- glucanase (CBH) and filter paper enzyme (FPase) was up to 3724.2 U/mL, 6287.3 U/mL and 2731.9 U/mL.The colony of the strain HS-F9 could grow rapidly on PDA plate at 30℃, the diameter of the colony was about 40-60 mm after 36 h culturing, and there was a round-shaped green zone for spore production about 30mm in diameter of the circle, with the expansion of colonies, spore production area expanded gradually. After 48 hours culturing, the colony occupied almost the entire plate. Aging colony was filamentous which has the dark green positive contrary to the colorless back. The hyphae with horizontal septum had more branch, and Spores terrier was ring arrangement. Oval conidia with rough surface directly growed in Spores sub-Terrier.The temperature for growth of the strain HS-F9 ranged from 20℃to 37℃, the optimum growth temperature was 30℃, and the pH ranged from 3.0 to 7.5, the optimum pH for growth was 5.0.The 18S rRNA gene of 1823bp was coloned from HS-F9 by PCR amplication and sequenced (Genebank No: FJ598872). By nucleic blast system, it had high Homology to all the 18S rRNA gene sequences of Trichoderma. The results of multiple sequence alignment and the construction of phylogenetic trees by using the software of Mega showed that the strain HS-F9 and Trichoderma viride gathered together and their homology had reached 99.82% through software of DNAMAN. Based on the identification of physiological and biochemical as well as the molecular identification, we determined preliminarily the strain HS-F9 as Trichoderma viride.The strain HS-F9 was optimized for enzyme production by culturing in the liquid CMC-Na medium. The optimum cultural conditions of the strain HS-F9 for enzyme production were as follows: the optimum carbon source for EG, CBH and FPA production was CMC-Na; the optimum nitrogen source was peptone for EG and CBH, but Soy flour for FPase. The optimum tempreture for EG,CBH and FPA production was 30℃,30℃,33℃and the optimum initial pH was respectively 3.0, 3.0 and 4.0, the optimum amount of inoculum for EG and FPA was 2%,while the optimum amount of inoculum for CBH was 8%, the culture time was appropriate for 5 to 6 days.Under the optimum conditions, the enzyme activity of EG, CBH and FPA was up to 5275.3 U/mL,8502.1 U/mL and 3619.1U/mL, which was 1.42,1.35 and 1.32 times respectively than un-optimized conditions.The second part of this study was about the screening and the polyphasic taxonomy of actinomyces which could produce celluase. more than 60 actinomycetes strains were isolated from the typical desert soil samples in ShanShan country XinJiang province, there were nine actinomycetes could grow in the CMC-Na (as the sole carbon source)medium which indicated qualitatively they could produce cellulase. The actinomycetes which had been isolated were classfied by the polyphasic taxonomy technique including its morphology, physiological and biochemical characteristics, chemotaxonomy and 16S rRNA gene sequence analysis and so on.The results showed that: seven actinomyces belonged to Streptomyces among nine cellulase- producing actinomycetes, only the strain XJSS-08 belonged to Moraxella while the strain XJSS-39 belonged to Nocardia. Both XJSS-08 and XJSS-39 were alkali-resistant bacteria ,which were different from the other seven kinds of Streptomyces, and they had two different types of cell wall.