Study On The Denitrification Mechanism Of Pseudomonas Taiwanensis EN-F2 And The Effect Of Hydroxylamine On Its Denitrification Product

Hydroxylamine and nitrite,as the intermediate metabolites of heterotrophic nitrification-aerobic denitrification（HN-AD）,were toxic to most nitrogen-transforming microorganisms,which made wastewater treatment more difficult.Most of the currently reported HN-AD microorganisms can not simultaneously grow in the presence of hydroxylamine and nitrite,nor do they have the abilities to remove hydroxylamine and nitrite.Moreover,there is a lack of studies on the effects of hydroxylamine on the nitrogen removal products of HN-AD microorganisms,leading to the multiple limitations of HN-AD microorganisms in the actual wastewater treatment.Therefore,it is urgent to isolate HN-AD nitrogen conversion microorganisms that can efficiently remove various inorganic nitrogen sources,especially hydroxylamine and nitrite in wastewater,and clarify the effects of hydroxylamine on the nitrogen removal process of the strain EN-F2,so as to provide a theoretical basis for further improving the efficiency of wastewater treatment.（1）In this manuscript,twenty-four strains were isolated from vegetable fields and named as EN-A1～EN-J1 by using hydroxylamine and nitrite as mixed nitrogen sources.Subsequently,a bacterium strain EN-F2 which was capable of excellent hydroxylamine,nitrite and total nitrogen removal abilities was obtained at 25℃.The colony morphologies of strain EN-F2 were yellow,circular,middle bulge,wet with smooth surfaces;The cells were rod-shaped under the scanning electron microscope;The strain EN-F2 was identified as Pseudomonas taiwanensis by 16S r RNA sequences and phylogenetic tree.（2）The removal efficiencies of hydroxylamine and nitrite from wastewater by strain EN-F2 can be affected by environmental factors.To improve the removal abilities of hydroxylamine and nitrite,the nitrogen transformation conditions of the strain EN-F2 were optimized.The results demonstrated that the optimal conditions for the removal of hydroxylamine and nitrite were as follows:25℃,sodium butyrate,150 rpm,0.5×10⁸ CFU/m L,C/N=20,p H=7.40.Under the optimal conditions,the strain EN-F2 could completely convert hydroxylamine and nitrite within 30 h.（3）To study the HN-AD ability of strain EN-F2,ammonium,hydroxylamine,nitrate and nitrite were selected as sole nitrogen source.Strain EN-F2 could convert100%,100%,89.21%and 86.99%of ammonium,hydroxylamine,nitrate and nitrite,corresponding to the peak conversion rates of 8.72,2.12,4.55 and 5.80 mg/L/h,respectively.When hydroxylamine and ammonium were mixed,the strain EN-F2could also completely and simultaneously remove them within 12 h.Nitrite and nitrate were mixed as nitrogen sources,the removal efficiencies were 85.44%and57.88%,respectively,and nitrite was preferentially removed,indicating that the strain EN-F2 had outstanding HN-AD ability and the heterotrophic nitrification（HN）was stronger than aerobic denitrification（AD）ability.（4）The specific activities of ammonia monooxygenase（AMO）,hydroxylamine oxidoreductase（HAO）,nitrate reductase（NR）and nitrite reductase（NIR）,which were essential for HN-AD of strain EN-F2,were detected and analyzed at levels of0.54,0.15,0.14 and 0.01 U/mg protein,respectively.It was confirmed that several enzymes existed in strain EN-F2 and could catalyze the HN-AD process.The HN-AD specific enzyme inhibitors of DDC,OCT,PYD and Na₂WO₄further confirmed that the removal of inorganic nitrogen depended on the action of HN-AD enzymes.The nitrogen balance results showed that the strain EN-F2 could convert 90.45%,25.73%,43.63%and 63.84%of ammonium,hydroxylamine,nitrate and nitrite into gaseous nitrogen,in which the N₂O productions of ammonium,nitrate and nitrite were only0.37,0.87 and 0.93 mg/L,respectively,indicating that most inorganic nitrogen was converted to N₂.Combination with the intermediate metabolites of HN-AD,Key enzyme,specific enzyme inhibitors,nitrogen balance and gas detection of the strain EN-F2,the nitrification pathways of the strain EN-F2 may be inferred as NH₄⁺-N→NO₂^--N→NO₃^--N→NO₂^--N→N₂O/N₂ and NH₄⁺-N→NO₂^--N→N₂O/N₂and NH₄⁺-N→biomass nitrogen;NH₂OH→NO₂^--N→NO₃^--N→NO₂^--N→N₂O/N₂and NH₂OH→NO₂^--N→N₂O/N₂ and NH₂OH→NH₄⁺-N→biomass nitrogen.The denitrification pathways were predicted to be NO₃^--N→NO₂^--N→N₂O/N₂ and NO₃^--N→NO₂^--N→NH₄⁺-N→biomass nitrogen;NO₂^--N→N₂O/N₂ and NO₂^--N→NH₄⁺-N→biomass nitrogen.（5）The simultaneous nitrification and denitrification（SND）ability of strain EN-F2 was studied.Results indicated that the strain EN-F2 was capable of simultaneous removing ammonium and nitrate/nitrite,hydroxylamine and nitrate/nitrite mixed nitrogen sources.Even though high concentrations of nitrate and nitrite existed in wastewater,the strain EN-F2 could still remove ammonium and hydroxylamine preferentially and completely.（6）The optimal carbon sources for the removal of hydroxylamine and ammonium/nitrate were sodium succinate and glucose,respectively.Under the optimal carbon source condition,the supplemation of hydroxylamine at different concentrations had no remarkable effect on ammonium oxidation efficiency and the types of intermediate metabolites,but promoted the formation of N₂O;Furthermore,the addition of hydroxylamine could promote the removal of nitrate and nitrite,the cells growth and the emission of N₂O,and had no effect on the types of nitrogen metabolizing intermediates.Therefore,the addition of hydroxylamine was beneficial to the growth of strain EN-F2 and the conversion of other inorganic nitrogen.In conclusion,the strain EN-F2 had excellent HN-AD and SND abilities.Under optimal conditions,the strain EN-F2 could completely remove hydroxylamine and nitrite within 30 h.The addition of hydroxylamine promoted the cell growth and the removal of ammonium,nitrate and nitrite nitrogen,which was conducive to improve the efficiency of wastewater treatment.