Isolation And Identification Of Aerobic Denitrifier Pseudomonas Stutzeri XX1 And Study Of Nitrogen Metabolic Pathway

Aerobic denitrification,as a novel nitrogen cycle pathway,provides a potential application possibility of biological nitrogen removal under aerobic condition.Over more than 30 years research on aerobic denitrification,it's still lack of understanding on the molecular metabolism mechanism,its pathway and affecting factors are also not clear.In this study,the research on aerobic denitrification bacteria isolation and its metabolism pathway was carried out,and the influence of several environmental factors was tested meantime.The main results are as follows:A bacteria strain named XX1 was isolated from activated sludge.The isolate showed aerobic denitrification ability and was identified as Pseudomonas stutzeri through 16S r RNA gene sequence.The whole genome of the P.stutzeri XX1 was sequenced and molecular mechanism of aerobic denitrification was analysed.The genome contains 4217 genes.According to the results of gene annotation by COG(Clusters of Orthologous Groups),NR(Non-Redundant Protein Database databases)and other databases,these genes were annotated to associate with energy production and conversion,posttranslational modification,protein turnover,chaperones,inorganic ion transportation and metabolism etc.Seven genes encoding functional enzymes related to nitrogen metabolism were identified in the genome,including:periplasmic nitrate reductase(nap AB),respiration nitrate reductase(nar GHI),assimilatory nitrare reductase(nas AB),assimilatory nitrite reductase(nir BD),dissimilatory nitrite reductase(nir S),nitric oxide reductase(nor BC),as well as nitrous oxide reductase(nos Z).By analyzing with KEGG database,three pathways were identified in the strain XX1,including complete ammonium assimilation(NH₄⁺?L-Glutamine?L-Glutamate),aerobic/anoxic denitrification(NO₃^-?NO₂^-?NO?N₂O?N₂)and DNRA(dissimilatory nitrate reduction to ammonia)(NO₃^-?NO₂^-?NH₄⁺).The nitrogen metabolism pathways of the P.stutzeri XX1 were tested using different nitrogen source(ammonium,nitrite,nitrate,ammonium+nitrite,ammonium+nitrate).Nitrogen balance calculation shown that the P.stutzeri XX1utilized ammonium completely through assimilation process.When nitrite or nitrate was used as the sole nitrogen source,30%and 45%of the consumed nitrite and nitrate volatilized from incubation system through aerobic denitrification respectively,and the left parts were converted into bacterial biomass.In contrast,the addition of ammonium promoted the efficiency of aerobic denitrification,72%and 77%of the bacterial consumed nitrite and nitrate metabolized via the aerobic denitrification pathway respectively.The effect of different environmental factors on the growth and aerobic denitrification efficiency of the P.stutzeri XX1was investigated.Results showed that the shaker speed had no effects on the aerobic denitrification efficiency,while had significant positive effects on bacterial biomass accumulation.The optimal C/N ratio for the P.stutzeri XX1 growth and nitrogen removal efficiency ranged from 10 to 15,and lower C/N ratio could only support the strain for cell growth and not for the denitrification pathway.Temperature showed significant effects on bacterial growth and denitrification efficiency.The grow and nitrite metabolism were inhibited under condition of 10?to 20?,and the highest growth and denitrification efficiency were observed under the temperature of 40?.