Isolation,Screening And Nitrogen Metabolism Characteristics Of Heterotrophic Nitrification-Aerobic Denitrifying Bacteria Pseudomonas Alcaligenes S303

Heterotrophic nitrification aerobic denitrification（HN-AD）bacteria are a kind of microorganisms that can use organic matter as carbon source and energy,and transform NH₄⁺into N₂ by nitrification and denitrification under aerobic conditions.They have the advantages of rapid reproduction and strong environmental adaptability,which have great application potential in the treatment of nitrogen-containing wastewater.This study isolated and screened an efficient HN-AD bacteria,and investigated its nitrogen metabolism characteristics and influencing factors,in order to enrich the strain resources of HN-AD bacteria and provide experimental basis for its practical application.The main research results of this study are as follows:43 heterotrophic bacteria were isolated from soil and activated sludge,which can grow well with NH₄⁺-N or NO₃^--N as the sole nitrogen source.Among them,the HN-AD bacteria with the best removal rate of total nitrogen（TN）and NH₄⁺-N was selected,named S303.After morphological observation and 16S r RNA gene sequencing,and it was preliminarily identified as Pseudomonas alcaligenes.This study cultured strain S303 under heterotrophic and aerobic conditions in different inorganic nitrogen sources.When strain S303 is cultured with NH₄⁺-N as the sole nitrogen source,the highest nitrogen removal rate is achieved at 16 hours,with NH₄⁺-N removal rate of 100%,total dissolved nitrogen（TDN）removal rate of 84.6%,and TN removal rate of 16.7%.When strain S303 is cultured with NO₂^--N as the sole nitrogen source,the highest nitrogen removal rate was achieved at 28 hours,with NO₂^--N removal rate of 84.3%,TDN removal rate of 57.2%,and TN removal rate of 13.3%.When strain S303 is cultured with NO₃^--N as the sole nitrogen source,the highest nitrogen removal rate is achieved at 24 hours,with NO₃^--N removal rate of 78.5%,TDN removal rate of 56.3%,and TN removal rate of 14.1%.When strain S303 is cultured with NH₄⁺-N,NO₂^--N,and NO₃^--N as the nitrogen sources,the basic removal of three nitrogen forms is basically completed within 28 hours,and the utilization order is:NH₄⁺-N,NO₂^--N,and NO₃^--N.It was determined that strain S303 could grow well under heterotrophic and aerobic conditions,with NH₄⁺-N,NO₂^--N,and NO₃^--N as nitrogen sources,and had heterotrophic nitrification and aerobic denitrification capabilities.Meanwhile,nitrogen products of the HN-AD process was detected by membrane inlet mass spectrometry,which indicated when strain S303 is cultured with NH₄⁺-N as the sole nitrogen source,the concentration of N₂ was always about 50%of the TN removal amount.By adding denitrification inhibitor to explore the change of intermediate products in nitrogen metabolism of strain S303,it was found that strain S303 can remove part of nitrogen through the denitrification pathway of NO₂^-being reduced to gaseous nitrogen by nitrite reductase.By setting different initial NH₄⁺-N concentrations,carbon to nitrogen ratios,temperatures,shaking speeds and initial p H values,the effects of environmental factors on the growth and metabolism of strain S303 under heterotrophic and aerobic conditions were explored.Strain S303 was able to grow normally within the initial NH₄⁺-N concentration range of 50-1000 mg/L.The optimal conditions for TN removal were carbon to nitrogen ratio of 8,temperature of 30℃,shaking speed of 170 rpm,and initial p H value of 8.