Screening And Application Of Proteinase Producing Bacillus Adapted To Different Aquaculture Temperature And Salinity

The main source of nitrogen-containing organic pollutants in mariculture water and waste water is the protein added in mariculture feed.Bacillus with high protease activity can be used in mariculture system to solve the problem of soluble and insoluble protein pollution in water.However,the strains of commercially available Bacillus preparations in Hainan are mainly from foreign countries or other regions in China,survivability and water purification effects of that Bacillus preparation are poor in the unique aquaculture environment of Hainan province.The main reason is that the protease activity and survival ability of Bacillus are related to the natural conditions of the aquaculture environment,especially the temperature.However,most of the exogenous Bacillus strains are not suitable for the special tropical climate conditions in Hainan province and cannot have effective water purification effects in applications.Therefore,according to different mariculture conditions in Hainan province,in this research we first screened indigenous Bacillus strains with high adaptability ability and high protease activity under different water temperature conditions,and then we have obtained higher protease activity strains through mutagenesis.Finally,the survival time and water purification effect of the selected strains were tested under different aquaculture conditions(temperature and salinity).The research results of this paper lay the foundation for the development of water quality improver,and provide potential strains for the development of Bacillus related microbial preparations,which is conducive to the accurate,efficient and safe application of Bacillus microbial preparations in Hainan province.In this study,18 Bacillus strains with high protease activity were selected from the“Microorganisms Collection of South China Sea” constructed previously in our laboratory.Based on the common aquaculture water temperature in different seasons in Hainan(20°C,24°C and 30°C),among the selected strains,Bacillus strains adapted to different temperatures were screened.4,6,and 18 strains were screened at 20°C,24°C,and 30°C respectively.The strain Bacillus licheniformis X1 has strong proliferation ability at different temperatures.Subsequently,we used X1 as the initial strain to further screen strains with higher protease activity through UV mutagenesis.After UV mutagenesis,the protease activity of the mutant strain UV115 is 31.14 U/m L at 30°C,and the enzyme activity increase by 49%;The protease activity of mutant strain UV110 is 28.8 U/m L at24°C,and the enzyme activity increase by 42%;The protease activity of the mutant strain UV116 is 33.43 U/m L at 20°C,and the enzymatic activity increase by 40%.Finally,two mutant Bacillus strains(20°C: UV116,UV123;24°C: UV109,UV110;30°C: UV112,UV115)with significant increase in protease activity at each temperature are selected for genetic stability and biological safety experiment.The results show that the protease-producing genetic stability of these 6 mutant strains are relatively stable and safe to the environment and aquaculture animals.According to the growth characteristics of 6 strains of mutant Bacillus under different temperature and salinity conditions,all the strains were applied under different combination of aquaculture conditions(temperature and salinity)to test their water purification effect by compared with commercially available Bacillus preparations.The results show that the strain UV116 has a better water purification effect under the conditions of 20°C and 10‰ salinity,and the survive period is 4 days;Strain UV123 has a better water purification effect at 20°C and 30‰ salinity,with a survive period of 5 days;Strain UV109 has a better water purification effect under the conditions of 24°C,10‰salinity and 20‰ salinity,and the survive period is 7d and 3d respectively;The strain UV112 has a better water purification effect at 30°C and 10‰ salinity,and the survive period is 2d.Under the conditions of 20°C 20‰ salinity,24°C 30‰ salinity,30°C 20‰salinity,and 30°C 30‰ salinity,the water purification effect of the corresponding strains is not significantly improved compared with the control group,so it is necessary to further screen strains with excellent performance.