Identification And Nitrogen Removal Characteristics Of Heterotrophic Nitrification And Aerobic Denitrification Bacterium Pseudomonas Sp. ADN-42

Ammonia is an important pollutant in wastewater. Biological methods, with the advantages of cost-effectiveness and no pollution, are considered to be one of the most effective ways to remove nitrogen from wastewater. Traditional biological nitrogen removal theory suggests that the nitrification reaction is conducted by autotrophic nitrification bacteria under aerobic conditions, while the denitrification reaction was performed by heterotrophic denitrification bacteria under anaerobic conditions. Therefore, nitrification and denitrification processes must be accomplished in separated reactors. However, the newly discovered heterotrophic nitrification and aerobic denitrification bacteria can perform simultaneous nitrification and denitrification in the same reactor, thus providing a new approach for nitrogen removal.Herein, a novel heterotrophic nitrifying-aerobic denitrifying bacterial strain was isolated from Hymeniacidon perleve in the seawater of Dalian. Strain identification, optimization of nitrogen removal conditions, determination of nitrogen removal pathways and the balance analysis of N elements were carried out. The key gene responsible for nitrogen removal was also analyzed to clarify the mechanism.The strain, named ADN-42, was bacillus and gram negative based on the morphology, scanning electron microscope and Gram staining analysis. The average length of the strain was1.37μm and the diameter was0.34μm. According to16S rRNA gene sequence analysis, strain ADN-42showed99%similarity with Pseudomonas sp., thus it was identified as Pseudomonas sp.The optimum heterotrophic nitrifying-aerobic denitrifying conditions were found to be as follows:temperature of30℃, C/N ratio of12, rotating speed of150r/min, initial ammonium concentration of300mg/L, salinity of40g NaCl/L and inoculum volume of5%. Under the above conditions, the ammonium removal rate was6.3mg/(L-h). Nearly75.4%of ammonium was removed in84h, while the accumulation of nitrite was8.3mg/L and nitrate was lower than detect limit. Strain ADN-42grew well during the process and reached logarithmic growth phase in48h (OD600=1.97). In the meantime, the CODcr-concentration decreased from4203.3mg/L to1240.5mg/L. Strain ADN-42was also capable of simultaneous nitrification and denitrification in the presence of pure oxygen. N2and CO2were the main products, whereas no greenhouse gas N2O was produced. The effect of dissolved oxygen (DO) on denitrification process was investigated. It was found that low level of DO (about3mg/L) was more preferable, and ammonia was completely removed. The balance analysis of N elements indicated that37.5%of the ammonia was converted into the intracellular nitrogen, and N2accounted for30.5%of denitrification products. A nitrogen balance was found between the initial and the final phases of the process.The N2O reductase gene (nosZ) was cloned from strain ADN-42, leading to a453bp product. Sequence analysis indicated that the nosZ gene of strain ADN-42had92%similarity with a previously reported nosL gene. The results showed that Pseudomonas sp. ADN-42could be an efficient bacterial strain for simultaneous nitrification and denitrification under high saline conditions.