Screening, Identification and Fermentation Optimization of a Acid-protease Strain
YANG Cheng1,2, YAO Shan-Jing2, YANG Zhi-Jian1,3, ZHENG Gang1*
1 Ocean Research Center of Zhoushan, Zhejiang University, Zhoushan 316021; 2 College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027; 3 College of Life Science, Zhejiang University, Hangzhou 310012
Abstract Excessive use of fertilizers leads to serious degradation of cultivated lands which greatly affects crop yield and quality. Through the screening of acid-protease strains, combined with modern fermentation techniques, the present study attempted to develop new amino acid organic fertilizers. An acid-protease strain named Zju-c was screened from the hydrolytic liquid of fish meal using the transparent hydrolysis circle method and the Folin-Phenol method. Morphological observation and 26S rRNA molecular identification were performed, and the fermentation condition was optimized. Results of physiological and biochemical reactions showed that most sugars and a small portion of alcohols could be utilized directly, and casein, starch, gelatin, and tartrates could be hydrolyzed, and the growth rate was fast. The concentration of NaCl can be tolerated upto 13%. The BLAST of 26S rRNA sequence in GenBank indicated that the strain belonged to Pichia pastoris and the phylogenic tree of the strain, which was generated by Vector NTI Suite 7 software based on 26S rRNA sequence, showed that it was most close to Pichia galeiformis (GenBank No. JF781422.1) strain with 100% of identity. In addition, the optimum fermentation conditions were 300 g/L fish meal, 50 g/L glucose, 30 g/L soy peptone, 30 g/L NaCl and 0.9 mg/mL Mg2+ with 3% inoculation quantity, with the culture condition of 30 ℃, pH 5.0, and 180 r/min for 7 d.The contents of free amino acids and enzymic activity were the fermentation index, total amino acids content in the fermentation broth eventually increased from initial 29.4±2.1 mg/mL to 88.4±1.4 mg/mL, and the enzymic activity was optimized from 190.2±2.9 U/mL to 400.1±3.8 U/mL. Therefore, the strain has good application potential for the development of liquid bio-organic fertilizer.
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