Study On Nitrogen Removal Characteristics And Its Nitrogen Metabolism Mechanism Of Pseudomonas Putida Y-9

As the nitrogen pollution in the water environment becomes more and more serious,biological denitrification is characterized by high efficiency,low cost and no secondary pollution.In recent years,this technology has attracted more and more attention of scholars.So far,the biological denitrification pathways reported include anaerobic ammonia oxidation?ANAMMOX?involving anammox bacteria,autotrophic microorganisms involved in the nitrification process and heterotrophic microorganisms involved in the anaerobic denitrification process and heterotrophic nitrification-aerobic denitrifiers involved in the coupled heterotrophic nitrification aerobic denitrification process.Due to the ability to convert ammonium to gaseous products under aerobic conditions,more and more heterotrophic nitrifying microorganisms are being isolated.Although most of these heterotrophic nitrifying bacteria have the ability of aerobic denitrification,they involve different genus and still have their own characteristics in terms of heterotrophic denitrification performance and nitrogen removal pathways.Therefore,it is necessary to screen new heterotrophic denitrifying bacteria from different environment and conduct in-depth studies on their denitrification characteristics to enrich the denitrifying microbial species and biological denitrification theories.In this study,the hypothermia nitrite denitrifying bacteria Y-9 was separated from the long-water flooded winter paddy field in Guizhou.Strain Y-9 is a gram-negative bacterium.The colonies of the strain Y-9 are convexly rounded and have a smooth,moist surface with neat,opaque and pale blue edges.Strain was identified as Pseudomonas putida by specific phospholipid fatty acid detection and 16S rRNA gene analysis and was named Pseudomonas putida Y-9.On this basis,the denitrification characteristics,heterotrophic nitrification performance,aerobic denitrification performance and nitrogen removal mechanism were studied.The main findings are as follows:?1?When sodium nitrite was used as the sole nitrogen source,the effects of physicochemical properties on the growth and nitrogen removal capacity of strain Y-9 were examined.The results showed that the optimal denitrification conditions were temperature of 15?,shakingspeedof100 rpm,sodium citrate as the carbon source and pH of 7.0.In addition,strain Y-9 is tolerant to dissolved oxygen and can utilize multiple carbon sources for cell growth and denitrification.Under optimal conditions,The removal rates of nitrite nitrogen and total nitrogen within 48 hours were as high as 100%and 77.13%.?2?Under 15?and aerobic conditions,strain Y-9 could grow well and metabolically denitrify with ammonium salt,nitrate and nitrite as sole nitrogen sources.When 208.94 mg/L ammonium nitrogen was used as the nitrogen source,the removal efficiencies of ammonium nitrogen and total nitrogen?TN in suspension?by strain Y-9 were 98.3%and 29.63%,respectively,and the nitrification products?NO₃^-,NO₂^-?were not detected.Nitrate and nitrite nitrogen removal efficiencies of strain Y-9 were 74.7%and 85.6%,respectively,when strain Y-9 was cultivated at the initial 205.93 mg/L nitrate nitrogen and 205.32 mg/L nitrite,the corresponding TN removal efficiencies were 18.3%and 54.5%,respectively.In addition,when NH₄⁺-N and NO₃^--N or NO₂^--N were used as a mixed nitrogen source,strain Y-9 preferentially utilized NH₄⁺-N for metabolism,and the presence of nitrate had no effect on the ammonium removal rate of strain Y-9,and yet the presence of nitrite would inhibit the cell growth and decrease the ammonium removal efficiencies.?3?Allylthiourea?ATU?as a typical nitrification inhibitor can inhibit the activity of ammonia monooxygenase?AMO?and also inhibit the activity of copper nitrite reductase?NirK?.The experiment found that the addition of ATU could not inhibit the ammonium removal of strain Y-9,but it could inhibit nitrite removal,indicating that the ammonia oxidation process of strain Y-9 is not via the coupling of heterotrophic nitrification with nitrite denitrification,and the nitrite reductase?NIR?of strain Y-9 is copper nitrite reductase?NirK?.In addition,the results of isotope experiments indicate that strain Y-9 can oxidize NH₄⁺-N to N₂O under aerobic conditions,but no N₂is released during this process,whereas N₂ was known as the end product of aerobic denitrification.Therefore,The pathway of ammonium removal was significantly different between P.putida Y-9with heterotrophic nitrification-aerobic denitrification bacteria.?4?Hydroxylamine oxidase?HAO?,nitrate reductase?NAR?and nitrite reductase?NIR?are the key enzymes in the process of heterotrophic nitrification and aerobic denitrification.The determination of these enzyme activities contributes to further evidence of nitrogen metabolism in strain Y-9.HAO activity was not detected in the crude enzyme solution extracted by ultrasonic fragmentation,but the activity of NAR and NIR was detected,and and the enzyme activity was 1.8and 5.6m U/mg protein respectively,respectively,it is further proved that the aerobic ammonium oxidation of strain Y-9 is not via nitrification but a novel metabolic pathway.