Isolation and identification of a Bacillus subtilis strain for water purification

Abstract:

[Background] With the development of the aquaculture industry and the improvement of intensive aquaculture, the aquaculture environment deteriorates seriously and the aquatic animal diseases take place frequently. Aquatic probiotics are widely used in aquaculture for their environmental friendliness and safety. [Objective] Bacillus subtilis was isolated from the bottom mud of Litopenaeus vannamei breeding pond. The in vitro biological characteristics and water purification effect were then explored to expand the germplasm resources of microecological preparations. [Methods] Firstly, the strains were isolated by the dilution spread plate method and its identification and classification were determined successively by morphological observation, physiological and biochemical assays, phenotypic test by microbial identification system, and 16S rRNA gene sequence analysis. Secondly, biological characteristics in vitro were studied by a single factor control method. Lastly, the contents of nitrite (NO2?-N), ammonia nitrogen (NH3-N), and chemical oxygen demand (COD) of the aquaculture water were separately measured by N-(1-naphthyl)-ethylenediamine spectrophotometry method, Nessler?s reagent method, and alkaline potassium permanganate method. [Results] The isolated strains were identified as Bacillus subtilis WH1 by the phenotypic identification and 16S rRNA gene sequence analysis. During a series of in vitro biological characteristics test studies, WH1, compared with the model bacteria, had better performances in resistance of high temperature, artificial gastric juice, and bile salts, as well as the inhibitory ability of Staphylococcus aureus and Vibrio parahaemolyticus. Especially, it was sensitive to 8 kinds of antibiotics that must be checked for Bacillus used as a feed additive according to the European Food Safety Agency rules. This also met the in vitro evaluation standard of aquatic probiotics used as feed additives. Additionally, the water purification studies showed that the strain WH1 could significantly reduce the contents of NO2?-N, NH3-N, and COD in water (P<0.05). [Conclusion] The screened Bacillus subtilis WH1 can be used as a water quality regulator and has the advantage of in vitro biological characteristics used as a feed additive, which can effectively expand the germplasm resources of microecological preparations.