Identification of a Strain Degrading Ammonia Nitrogen, Optimization of Ammonia Nitrogen Degradation Conditions, and Gene Expression of Key Degrading Enzyme Nitrite Reductase

Abstract

In recent years, nitrogen pollutants have become one of the main causes of water pollution and eutrophication; thus, it is very important to increase the research on nitrogen removal in wastewater. In this study, a bacterium with outstanding ammonia nitrogen degradation capability was isolated from piggery wastewater and identified as Bacillus tequilensis (designated as A2). The ammonia nitrogen degradation rate of A2 reached the highest level (95%) when the incubation temperature was 42 °C, the initial pH was 7, the seed volume was 5%, the rotation speed was 160 r·min−1, the C/N was 10:1, and the carbon source was sodium citrate. A new nitrite reductase gene was successfully expressed in E. coli BL21 (DE3), and the result showed that the enzyme gene contained 2418 bp and 805 encoding amino acids, the recombinant enzyme was purified through an Ni2+ affinity chromatography column, it had a molecular weight of about 94 kDa, it displayed the maximum enzyme activity at 40 °C and pH 6.0, it exhibited good stability in the range of 25 °C to 35 °C, and it showed a pH of 6.0 to 7.0. A 1 mM concentration of Fe3+ promoted the enzyme activity, followed by a 1 mM concentration of Fe2+ and Mg2+. The kinetic parameters of Km, Kcat, and the Vmax of NiR-A2 were calculated to be 1.37 μmol·mL−1, 4.9 × 102 s−1, and 23.75 μmol·mg−1·min−1, respectively. This strain shows good prospects for wastewater treatment, especially in the treatment of high concentration ammonia nitrogen and nitrite degradation, because of its tolerance to and high degradation rate of high concentrations of ammonia nitrogen and high nitrite.