两株有益菌的分离、培养、鉴定及其水质调控效果评价

Isolation, Culture, and Identification of Two Strains of Probiotics and Their Effects on Water Quality Control

自河口污泥中分离到两株有益菌,通过形态分析、革兰氏染色、生理生化测定、16S rRNA序列分析和系统发育树构建,其中一株鉴定为植物乳杆菌(Lactobacillus plantarum),另一株为枯草芽孢杆菌(Bacillus subtilis)。建立了两株细菌的适宜生长条件,植物乳杆菌适宜生长pH为6.5、温度为30℃,枯草芽孢杆菌的适宜生长pH为6.0、温度为36℃。在实验室条件下,两株有益菌1∶1组合制剂对人工配制的海水中氨氮、亚硝酸盐、磷酸盐的降解率分别达到了73.2%、58.0%和52.4%;池塘养殖环境下,其对养殖水体中氨氮、亚硝酸盐、磷酸盐的降解率分别达到了86.3%、88.9%和68.3%,具有明显的水质净化效果。本研究结果可为建立两株有益菌的规模化发酵生产技术和生产应用奠定基础。

Good water quality is essential for the healthy growth of pond-cultured marine fish. Balanced microecological environment plays an important role in the maintenance of water quality and control of fish disease. To identify the mechanisms regulating the balance in microecological environment and to find specific and high-efficiency probiotics for pond culture of marine fish, here we tested two strains of bacteria isolated from sewage sludge were identified as Lactobacillus plantarum and of the estuary area of the Futuan River in Rizhao. They a Bacillus subtilis-like species according to the analysis such as morphological characteristics, biochemical properties, and 16S rRNA sequencing. The optimum growth conditions for L. plantarum and B. subtilis were pH 6.5/temperature 300C and pH 6.0/temperature 36℃ respectively. In the laboratory, the degrading effects of the mixed bacteria strains A and B （1 ： 1） on ammonia nitrogen, nitrite, and phosphate in the seawater were 73.2%, 58.0%, and 52.4% respectively. Next, we examined the degrading effects of the bacteria in two outdoor marine ponds rearing Japanese flounder. One was designated as the experimental pond added with the probiotics （mixture of strain A and strain B）, and the other one was the control. The applications of the probiotics （1：1, bacteria density 5 × 10^4-10×10^4 cell/ml） caused significant decrease in the concentrations of ammonia nitrogen, nitrite, and phosphate, and the degrading rates were 86.3%, 88.9%, and 68.3% respectively. Our results provided useful information for large-scale production of the two probiotics and for their application in aquaculture in the future.