| Aquaculture is the fastest growing food production system in the world.In the past few decades,with the continuous increase of demand,the model of aquaculture industry has gradually shifted from traditional to intensive and large-scale development.The food residue and excreta generated by large-scale feeding gradually increase the nitrogen compounds in the water.However,excessive ammonia nitrogen content in aquaculture water can lead to a decrease in the immune and reproductive capacity of aquatic products,slow growth,and even death in severe cases,resulting in huge economic losses for the aquaculture industry.There are usually three methods for removing ammonia nitrogen:physical method,chemical method,and biological method.Among them,biological method is widely used due to its advantages of environmental friendliness,low cost,and simple operation process.Bacillus subtilis is a common beneficial bacterium that can not only degrade pollutants such as ammonia nitrogen and nitrite in water,but also have certain benefits for aquatic organisms,such as enhancing immunity and promoting growth and reproduction.This study takes strain G1000 as the starting strain and adopts physical and chemical mutagenesis methods to improve the ammonia nitrogen degradation ability as the main indicator.Dominant mutagenic strains are screened out,and the fermentation medium used for this strain is optimized to enhance the application ability of Bacillus subtilis G1000 mutagenic strain in production.The main research focuses on the following aspects:(1)Preliminary determination of the degradation ability of the original strain G1000 and single factor optimization of the cultivation conditions for ammonia nitrogen degradation determination showed that the optimal initial concentration for degradation was 20 mg/L,the optimal temperature was 37℃,the optimal p H was 8,the optimal rotational speed was180 r/min,and the optimal C/N was 10;(2)Firstly,UV mutagenesis was chosen to induce strain G1000 for breeding.Under the condition of UV irradiation at 30 cm for 120 seconds,one highly efficient mutant strain YB-41 was screened,with a degradation rate of 70.93%,which was 11.75%higher than the original strain G1000.And genetic stability testing was conducted on strain YB-41.After 5 consecutive generations,the degradation effect of strain YB-41 on ammonia nitrogen remained at around 70%,indicating that the degradation ability of strain YB-41has good genetic stability;(3)Secondly,the strain G1000 was chemically mutated and bred using diethyl sulfate(DES).Under the conditions of mutation treatment in a shaking table with DES concentration of 2.5%,37℃,and 180 r/min for 20 minutes,a highly efficient mutant strain C16 was screened,with a degradation rate of 81.83%,which was 18.99%higher than the original strain G1000.Genetic stability testing was conducted on strain C16.After 5consecutive generations,the degradation rate of ammonia nitrogen by strain C16 remained around 81%,and the degradation ability of strain C16 showed good genetic stability;(4)Finally,the fermentation medium of G1000 was optimized using response surface methodology,with biomass as the indicator.Through P-B experiments,steepest slope experiments,and response surface experiments,it was found that glucose,soybean meal powder,and manganese sulfate were the most important influencing factors.The optimal medium formula was:25 g of soybean meal powder,5 g of glucose,0.176 m L of manganese sulfate(3.08%),1 g of starch,1 g of potassium dihydrogen phosphate,0.5 g of potassium dihydrogen phosphate,and 0.2 g of magnesium sulfate,0.05 g iron trichloride,0.05 g calcium carbonate,1 g yeast extract,p H 7.2,1000 m L pure water,with a bacterial count of 3.17×10~9CFU/m L,corresponding to the software’s estimated bacterial count of3.01×10~9CFU/m L is similar;(5)Introduce the mutant strains YB-41,C16,and the original strain G1000 into the optimized fermentation medium,measure the bacterial count every 6 hours,and 2×10~3CFU/m L was added to the ammonia nitrogen degradation test medium,and the degradation rate was measured after 48 hours of cultivation.The results showed that the biomass of the mutant strain YB-41 reached its maximum at 3.51×10~9CFU/m L after 12 hours of fermentation,the highest degradation rate was observed after the cells were inserted into the degradation assay medium,reaching 72.68%;The biomass of mutant strain C16reached its maximum at 3.06×10~9CFU/m L after 12 hours of fermentation,but the degradation rate of cells can reach the highest at 79.18%after fermentation for 18 hours;Although the original strain G1000 also reached its maximum biomass at 12 hours of fermentation,it was only 2.88×10~9CFU/m L,significantly lower than the mutant strain,and at this time,although the cell degradation rate was the highest,it was only 65.71%,significantly lower than the mutant strain.In summary,this study selected highly efficient mutant strains through different mutagenic methods and optimized the fermentation medium used to expand the bacterial count in actual production,providing a theoretical basis and germplasm resources for reducing costs and expanding aquaculture production. |
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