Study On Metabolic Mechanism Of Pseudomonas Putida Strain NP5 Possessing Simultaneous Nitrogen And Phosphorus Removal Ability

With the development of social economy,the discharge of sewage containing nitrogen,phosphorus and other nutrients has increased significantly.Excessive nutrients have led to eutrophication of water bodies and the destruction of water ecosystems.How to effectively control nitrogen and phosphorus in water has become an urgent problem to be solved in environmental protection and sustainable social development.Conventional nitrogen and phosphorus removal processes separated the denitrification and phosphorus removal processes,which cuased the disadvantages of complex process,high energy consumption and harsh reaction conditions.In recent years,researchers have found that some heterotrophic bacteria can not only achieve simultaneous nitrification and denitrification,but also remove phosphorus during denitrification.The discovery of such heterotrophic bacteria with simultaneous nitrogen and phosphorus removal ability provides a new idea for biological nitrogen and phosphorus removal.A heterotrophic nitrifying bacteria?HNB?capable of simultaneous nitrification,denitrification and phosphorus accumulation was screened from a SBR reactor for long-term treatment of high ammonia-nitrogen organic wastewater in the laboratory.Based on this strain,the characteristics of heterotrophic nitrification,aerobic denitrification and phosphorus accumulation were investigated,and the mechanism of nitrogen and phosphorus removal was explored as well.The main results are as follows:?1?A HNB was screened from the SBR reactor and named as NP5.After physiological and biochemical and 16s rRNA identification,the strain was identified as Pseudomonas putida.The optimum reaction conditions for nitrogen and phosphorus removal were determined by environmental factor test:succinate as carbon source,C/N10,P/N 0.2,temperature 30?,shaking speed 160 r/min,pH 7.0 and salinity 0%.?2?The strain NP5 can tolerate high ammonia concentration.When the initial NH₄⁺-N concentration was 50?200 mg/L,NH₄⁺-N was completely removed within 48h.When the initial NH₄⁺-N concentration was increased to 500?1000 mg/L,strain NP5could still remove about 60%of NH₄⁺-N,which implied that strain NP5 had the potential to treat high ammonia concentration wastewater.Cultivated under the initial conditions of NH₄⁺-N concentration of 100 mg/L and PO₄^3--P concentration of 20 mg/L for 48 h,the maximum specific growth rate of NP5 was 0.41 h^-1.The maximum removal rates of NH₄⁺-N and TN were 99.16% and 98.32% respectively,and the maximum degradation rates were 10.47 mg/L/h and 10.33 mg/L/h,correspondingly.?3?Strain NP5 could be used for aerobic denitrification and denitrification by using NO₂^--N and NO₃^--N as the sole nitrogen?N?source,and the removal of N was accompanied with the growth of the strain,simultaneously.When the initial concentration of N was 100 mg/L,99.44%of NO₂^--N and 99.63%of NO₃^--N were removed within 24 h,and the maximum removal rates were 8.59 mg/L/h and 7.96mg/L/h,respectively.?4?Strain NP5 could achieve phosphorus?P?removal by using NH₄⁺-N,NO₂^--N and NO₃^--N as the sole N source,and the maximum removal rate of PO₄^3--P was found in the source of NH₄⁺-N.About 91.72%of PO₄^3--P was removed,and the maximum removal rate was 1.71 mg/L/h in NH₄⁺-N within 48 h.In the processes of aerobic denitrification to remove NO₂^--N and NO₃^--N,the PO₄^3--P removal rates were 87.9% and 83.9% within 48 h,with the maximum removal rates of 1.59 mg/L/h and 1.42mg/L/h,respectively.?5?The addition of ATU in heterotrophic nitrification process did not inhibit the ammonia oxidation of NP5,but significantly inhibited its denitrification.No significant changes of N and P removal process by strain NP5 was obtained when NaClO₃ was added into the substrate.It was speculated that strain NP5 could directly convert NH₂OH or NO₂^--N to gaseous nitrogen to achieve N removal.Anaerobic/aerobic induction experiments demonstrated that the strain NP5 was capable of P release under anaerobic condition and was able to accumulate excess P under aerobic condition.No significant effect was observed of single induction on the performance N and P removal by strain NP5,and repeated exposure to the anaerobic/aerobic conditions could significantly improve the nutrient removal efficiency.?6?The successful expression of HAO,NiR,and NAR enzymes indicated that the strain had a complete denitrification pathway.The successful expression of PPX and PPX demonstrated that NP5 was capable of P release/absoption under anaerobic/aerobic condition.The successful expression of the functional enzymes related to N and P removal confirmed the simultaneous nitrification and denitrification phosphorus accumulation ability of strain NP5.