Abstract: [Objective] The aims of the present study were to isolate and screen cellulolytic bacteria derived from spent Flammulina velutipes substrate, to assess their biological characteristics and cellulolytic enzyme activities, so as to provide technical supports for the feed utilization of spent mushroom substrate resources. [Method] Spent Flammulina velutipes substrate samples were inoculated using plate dilution coating method. The cellulolytic bacteria were then isolated and cultured on nitrate cellulose sodium medium. The cellulolytic abilities of the isolated strains were preliminarily analyzed using Congo red medium. Candidate strains were selected through 16S rDNA sequencing analysis and biosafety prediction, followed by physiological and biochemical characteristics analysis, growth curve determination at different temperatures, and dynamic analysis of cellulolytic enzyme activities. [Result] A total of 30 independent colonies were isolated after screening culture with nitrate cellulose sodium medium. All of the strains formed clear transparent circles on Congo red medium, and some strains had strong cellulolytic abilities. The 16S rDNA sequencing analysis showed that the 30 isolated strains were identified as belonging to 9 bacterial species, including Pseudomonas koreensis, Advenella mimigardefordensis, Serratia rubidaea, and so on. Taking into account both biosafety prediction and the preliminary cellulolytic abilities screening results, Pseudomonas koreensis （JK-32）, Adrenella mimigardefordensis （JK-52）, Serratia rubidaea （JK-56）, Staphylococcus equiorum （JK-57）, Staphylococcus saprophytics （JK-62） and Serratia marcescens （JK-66） were selected as candidate strains for subsequent experiments. Physiological and biochemical characteristics analysis showed that all of the 6 candidate strains were determined as positive for contact enzyme activity test, and negative for methyl red test and diacetyl test. The growth of JK-62 strain was less affected by temperature, and the growth rate and final bacterial concentration performed better among all the candidate strains. Under the low cultivation temperatures （28-34 ℃）, the growth rate and final bacterial concentration of JK-56 strain were lower than the other candidate strains, while when the cultivation temperature increased to 40 ℃, its growth rate was similar to that of the other candidate strains and the final bacterial concentration was higher. On the 1^st to 7^th days of cultivation, the endoglucanase activity of JK-56 strain was the highest among all the candidate strains. On the 2^nd, 4^th, and 5^th days of cultivation, the endoglucanase activity of JK-66 strain was significantly （P<0.05） higher than that of the CK. The exoglucanase activity of JK-66 strain was higher among the candidate strains and was significantly （P<0.05） higher than that of the CK at different detection time points. On the 2^nd to 4^th days of cultivation, the exoglucanase activity of JK-56 strain was significantly （P<0.05） higher than that of the CK. The β-glucosidase activity of JK-66 strain was higher among the candidate strains. On the 3^rd to 7^th days of cultivation, the β-glucosidase activity of JK-56 strain was next to JK-62 and JK-66 strains among the candidate strains. On the 2^nd to 5^th days of cultivation, the filter paper enzyme activity of JK-56 strain was the highest among the candidate strains. On the 5^th day of cultivation, it was significantly （P<0.05） higher than that of the CK. During this period, the filter paper enzyme activity of JK-66 strain was also at a high level. [Conclusion] Serratia rubidaea JK-56 and Serratia marcescens JK-66 strains had high overall levels of cellulolytic enzyme activities, and prominent endoglucanase and exoglucanase activities, which could be used in combination with strains exhibiting strong β-glucosidase activity for the development and utilization of spent Flammulina velutipes substrate.

Key words: cellulolytic bacterium, spent Flammulina velutipes substrate, isolation, cellulolytic enzyme activity, analysis