Preliminary Study On Thermophilic Actinomycetes In Asparagus Old Stem Compost

The asparagus old stem as an agricultural waste is different from corn stalk, wheat straw, rice straw, et al. Asparagus old stem compost could be used to cultivate edible mushrooms, therefore, composting is an effective way to utilize this agricultural waste. In order to establish and improve the fermentation process of asparagus old stem compost, the microbial diversity of the compost during the fermentation should be researched. Studies have shown that the thermophilic actinomycetes were the main regnant flora in high-temperature phase of the compost. By dilution-plate, the culturable thermophilic actinomycetes in asparagus old stem compost samples at different fermentation atages were studied.By plate count, the change in values of the culturable thermophilic actinomycetes in asparagus old stem compost was investigated. Through the observation of hydrolyzed circle in CMC plate and growth situation on the filter paper,9strains of efficiently cellulolytic microorganisms were selected from132strains. After shaking flask fermentation, strain EG16showed higher cellulose activity, the CMCase activity and FPase activity were8.89U/mL and4.11U/mL respectively.By colony morphology and microscopic morphology observation, the isolated thermophilic actinomycetes through preliminary identification were Streptomyces.42strains of these Streptomyces were selected as target strains by classification, and16S rDNA sequences of them were amplified and sequenced. According to16S rDNA sequence analysis,14strains (1strain of A sample,3strains of B sample,5strains of C sample,4strains of D sample,1strain of E sample) showed the highest similarity to S. thermovulgaris,9strains (2strains of A sample,1strain of C sample,1strain of D sample,5strains of F sample) to S. albogriseolus,3strains to S. pseudogriseolus,1strain to S. espinosus,15strains could not be found matched gene sequences of Streptomyces spp. in the GenBank. The results indicated S. thermovulgaris and S. albogriseolus were the main regnant culturable thermophilic actinomycetes in the asparagus old stem compost, and existed throughout the fermentation process.Through single factor test, this paper researched the influence of the concertration of carbon, nitrogen sources, pH, temperature, inoculum age, bottling size, metal ions and NaCl on the production of cellulolytic enzymes of strain EG16. By orthogonal experiment, choosed the optimal combination of the concertration of carbon and nitrogen sources, pH and inoculum size. The results showed that the optimal culture medium and condition were follow:(1)the medium of CMCase (4.5%of asparagus old stem,0.3%of yeast extract,0.6%of (NH4)2SO4,0.1%of KH2PO4,0.03%of MgSO4·7H2O,0.01%of CaCl2,0.002g/L of ZnCl2),70mL of bottling size, shaking flask fermentation at50℃,150r/min;(2) the medium of FPase (4.5%of asparagus old stem,0.3%of yeast extract,0.6%of (NH4)2SO4,0.1%of KH2PO4,0.03%of MgSO4·7H2O,0.01%of CaCl2,0.15%of NaCl,0.002g/L of ZnCl2,0.002g/L of CoCl2),70mL of bottling size, shaking flask fermentation at50℃,150r/min.Under the optimal fermentation condition, CMCase activity and FPase activity of strain EG16reached the highest level of14.7U/mL and6.1U/mL at day5, which increased by65.37%and47.06%respectively, and the optimal fermentation time was shortened by2days.