Research On The Proliferation Pattern Of Bacillus Subtilis BY7 In Water And Its Relationship With Water Quality Regulation

In recent years,with the rapid development of China’s national economy,China’s aquaculture industry has also been developing rapidly.The large amount of pollutants discharged from farms every day seriously damages the water environment and ecological balance,and the effect of water quality control in aquaculture waters directly determines the success or failure of aquaculture and the pollutant content of aquaculture wastewater.Bacillus subtilis,as a kind of probiotic bacteria,has the characteristics of strong vitality,metabolism and reproduction,which can degrade ammonia nitrogen,nitrite and nitrate in the waters and improve the water environment.In the present study,few studies have been reported on determining the appropriate dose of Bacillus subtilis for use in different waters,as well as studies on the correlation between its proliferation and the regulation of the water environment.Therefore,in this study,a GFP-tagged recombinant strain BY702 was constructed using Bacillus subtilis BY7,which has strong ammonia nitrogen degradation ability,and its proliferation in different waters with different nutrient levels（total nitrogen and chemical oxygen demand）was investigated to determine the appropriate dose of this strain in different waters.Based on the above,the relationship between the proliferation of this strain and water quality regulation was further analyzed to develop and promote the scientific application of Bacillus subtilis BY7 in production practice,expand the application scope of Bacillus subtilis BY7,and provide data support for the healthy development of China’s aquaculture industry.The main research contents are as follows:（1）Selection of promoters.From the promoters suitable for expression in Bacillus subtilis,three strong promoters（P43,Pamy Q and PHpa II）were selected to investigate their effects on the expression of the green fluorescent protein（GFP）gene in Bacillus subtilis.It was shown that the three promoters could achieve the expression of GFP gene in Bacillus subtilis,and all recombinant strains could emit green fluorescence.The P43 promoter mediated the best GFP gene expression,with the recombinant strain BY702 reaching a fluorescence value of 130,00RFU and high genetic stability after 16 hour incubation,which provides technical support for accurately distinguishing recombinant strain BY702 from other strains in the waters.（2）The determination of suitable application dose of Bacillus subtilis BY7.The proliferation of the recombinant strain BY702 was studied by adding different concentrations of the recombinant strain to the aquaculture water at different nutrient levels（TN and COD）.It was shown that when the aquaculture water was at high nutrient level（TN:36.70 mg/L and COD:185.60 mg/L）,the final concentration of the strain was maintained above 10~7 CFU/m L.When the aquaculture water was at medium nutrient level（TN:18.35 mg/L and COD:92.80mg/L）,the final concentration of the strain was maintained above 10~6 CFU/m L.When the culture water was at low nutrient level（TN:9.17 mg/L and COD:46.40 mg/L）,the final concentration of the strain was maintained around 10~4 CFU/m L.When the culture water was at very low nutrient level（TN:4.59 mg/L and COD:23.20 mg/L）,the final concentration of the strain was maintained around 10~3 CFU/m L.The results showed that the carrying capacity of the strain was different in different nutrient levels of the aquaculture water,and Bacillus subtilis BY7 should be applied reasonably according to the nutrient level of the waters in aquaculture production.（3）The study on the relationship between Bacillus subtilis BY7 proliferation and water quality regulation.Firstly,the initial TN and COD contents in the aquaculture water were measured,and the appropriate additive amount（10~3 CFU/m L）of the recombinant strain BY702was obtained according to the results of the above study,to study the relationship between the proliferation of strain BY702 and water quality control in the aquaculture water.The results show that the recombinant strain BY702 will first rise and then fall within 1～4 days.From the4th day,the number of recombinant strains gradually increased over time,reaching the highest number（3.4×10~4 CFU/m L）at 16th day.The ammonia nitrogen content of the aquaculture water in the test group will gradually decrease to 0.3 mg/L from the 8th day,which is significantly lower than that of the control group at 2.1 mg/L（P<0.05）,and the trend of ammonia nitrogen degradation rate was consistent with the trend of strain proliferation.The nitrite content of the aquaculture water in the test group will gradually decrease to 0.3 mg/L from the 4th day,which is significantly lower than that of the control group at 9.4 mg/L（P<0.05）,and the trend of nitrite degradation rate was consistent with the trend of strain proliferation.The overall trend of TN content in the aquaculture water of the test group was increasing（TN:17 mg/L）,but still lower than the control group TN:28 mg/L,and the trend of total nitrogen degradation rate was consistent with the trend of strain proliferation.The p H changes in the test and control groups had the same trend,both fluctuating above and below p H=8.3.The trend of strain BY702 proliferation and water quality index（ammonia nitrogen,nitrite and total nitrogen）degradation rate had a positive correlation.In this study,a GFP-tagged recombinant strain of Bacillus subtilis BY702 was constructed,with a preliminary method to evaluate the appropriate dose of Bacillus subtilis BY7 in waters with different nutrient levels,and it was discovered that the proliferation of Bacillus subtilis BY7 could directly promote the degradation of ammonia nitrogen,nitrite and total nitrogen in aquaculture waters.This study provides important theoretical and technical support to optimize the application of Bacillus subtilis in aquaculture waters.