Hypothermia High Efficient Nitrite Aerobic-denitrifying Bacteria And Its Nitrogen Conversion Ability

Nitrite, which is known to be a natural component of nitrogen cycle, and an intermediate product in bio-nitrification and bio-denitrification processes, has also been considered as one of major pollutants of wastewater around the world. Meanwhile, nitrite can cause deterioration of water and massive death of aquatic life. Therefore, it is important to find an effective method to reduce or eliminate the nitrite nitrogen in water to ensure water security. Nitrogen removal methods from wastewater include physical, chemical and biological ways. Compared with the physical and chemical wastewater treatment method, bio-denitification is acknowledged as the most economic, effective and beneficial to convert nitrogen compounds from polluted water, which is considered as one of the most promising techniques. Up to date, most of aerobic denitrification bacterial are mesophilic and their optimal temperature for nitrogen removal range from 25 ℃ to 35 ℃. But in the cold season, the functions of biological nitrification and denitrification will be strongly inhibited. Therefore, researching and exploring a psychrotrophic nitrite denitrifying bacteria which could work efficiently at low temperature show practical significance.At 10 ℃, we isolated and purified the hypothermia nitrite denitrifying bacteria with sodium nitrite as the sole nitrogen source from the long-term flooded paddy soil by combination of enrichment culture and pH indicator purification methods. The high efficient denitrifier was screened on basis of the high removal rates of nitrite and total nitrogen, and low production of biomass nitrogen. Morphological observation, specific phospholipid fatty acid, Biolog identification, 16 S rRNA gene sequence and ITS gene sequence analysis were employed to identify strain Y-11. The results showed that twelve strains of nitrite denitrifying bacteria were isolated and purified（named Y-1 to Y-12 strain）, in which one strain naming Y-11, which has a low production of biomass nitrogen and high nitrogen removal efficiency, has been screened out to be the most efficient aerobic denitrifier. Y-11 strain can remove nitrite and total nitrogen up to 99.84% and 55.42%, and produce low biomass nitrogen of 3.32 mg/L at 20 ℃ respectively. The morphology characteristic of Y-11 was rhabditiform or beaded with dry surfaces. The specific phospholipid fatty acid of strain Y-11 shows a 0.523 similarity index with Pseudomonas-fluorescens-biotype B that was belonged to Pseudomonas tolaasii in traditional taxonomy, which was consistent with the followed 16 S rRNA and ITS gene sequences identification results of Y-11 strain. The 16 S rRNA and ITS gene sequences exhibit 98% and 99% similarity with Pseudomonas tolaasii, respectively. Strain Y-11 is corroborated as Pseudomonas tolaasii by the combination of a variety of identification methods, which offers the taxonomic assignment with higher confidence. In addition, there is no report that this kind of bacterium could conduct aerobic denitrification at home and abroad up to now, especially at low temperature, which is a further complement to the aerobic hypothermia nitrite denitrifying bacteria.Denitrification characteristics of strain Y-11, such as temperature, shaking speed, initial pH and carbon source were investigated by using shaking culture in Erlenmeyer flask, in which sodium nitrite was as sole nitrogen source to determine the optimum denitrification conditions of Y-11 strain. The experiments results indicated that the optimum conditions of Y-11 effectively removing nitrite nitrogen and total nitrogen were: initial pH 7.0, 15 ℃, shaking speed 200 r/min, inoculum size 1.5×108 CFU per 100 ml medium, sodium acetate as sole carbon source, and nitrite nitrogen 150mg/L. When sodium acetate as the sole carbon source, 15 ℃, initial pH 7.0, shaking speed 150r/min, and nitrite nitrogen 10 mg /L, the Y-11 strain can remove nitrite nitrogen and total nitrogen up to 100% and 61. 28% within 48 h.Greenhouse of CO₂ and N₂ O emission flux were measured during denitrifying process with sodium nitrite as sole nitrogen source at 15 ℃. This experiment was conducted by using sealed flask culture method. The results showed that flux of CO₂ was 24150.13 mg/m3 and it was 0.14 mg/m3 for N₂ O by strain Y-11 during denitrifying process. The large amounts of CO₂ produced by Y-11 strain indicated that the nitrogen denitrifying activity of the strain is very strong in the denitrifying medium. The small amount of N₂ O produced by Y-11 strain showed that the strain is very friendly to the gas environment because it could only produce fewer nitrogen greenhouse gases during denitrifying process. All the denitrifying characteristics of the Y-11 strain demonstrated that the strain is very securely and effectively for the environment in the actual nitrogen denitrification application in winter wastewater treatment.The abilities of strain Y-11 to remove nitrite and total nitrogen rates were analyzed preliminarily in the eutrophic landscape water by using shaking culture in the Erlenmeyer flask. Results showed that the nitrite and total nitrogen removal efficiency were up to 43.47% and 88.87% in the nature wastewater respectively, within 48 h, at 15 ℃, 150 r/min to dispose the eutrophic landscape water. Although the nitrite nitrogen removal efficiency is lower than that of total nitrogen, but achieves the purpose of the efficient nitrogen removal efficiency under the condition of complex environment.By madding high concentrations of different nitrogen wastewater, the heterotrophic nitrification and aerobic denitrification abilities of an aerobic nitrite-denitrifying bacterium, Pseudomonas tolaasii strain Y-11 were studied aerobically, which was found to display high aerobic denitrification with nitrite and total nitrogen removal capability in previous studies. The experiments results indicated that the hypothermia denitrification Y-11 strain exhibited high nitrogen removal efficiency during heterotrophic nitrification with ammonium and aerobic denitrification with nitrate or nitrite nitrogen. The ammonium, nitrate and nitrite removal efficiencies were up to 93.6%, 93.5% and 81.9% respectively when Y-11 strain was cultivated for 4 d at 15 ℃ with initial ammonium, nitrate and nitrite nitrogen concentration of 209.62, 204.61 and 204.33 mg/L and initial pH 7.2 corresponding to total nitrogen removal efficiency of 30.5%, 46.9% and 42.6% for ammonium, nitrate and nitrite respectively. The organic nitrogen few was removed and the total nitrogen removal efficiency was only 10.8%. Additionally, the removal efficiency of ammonium and nitrate ware 100% and 24.0% in the mixed ammonium and nitrate nitrogen medium, and the removal efficiency of ammonium and nitrite were 62.4% and 6.6%. Ammonium mainly removed and few nitrate（or nitrite） was removed during simultaneous nitrification and denitrification process, which suggested that high mixed concentrations of ammonium and nitrite（nitrate） nitrogen were harmful for cell growth and nitrogen removal performance. Additionally, there was no nitrite nitrogen accumulation in the all biological nitrogen removal process in addition to artificial adding nitrogen source. All of these results demonstrated than Pseudomonas tolaasii strain Y-11 has abilities to remove ammonium, nitrate and nitrite nitrogen at low temperature, which could guarantee strain Y-11 for future application in winter wastewater treatment.