Screening Of Denitrifying Phosphorus Accumulating Organisms And Their Action Pattern In Wastewater Treatment

Nitrogen and phosphorus pollution has become a serious problem,which is generally attributed to the excessive discharge of agricultural wastewater,improper treatment of industrial and domestic sewage,and the metabolism of aquatic organisms.In recent years,with the continuous promotion of industrialization,various industries have produced a large amount of wastewater,among which the indiscriminate discharge of some wastewater with high concentration of nitrogen and phosphorus has caused a series of problems such as water eutrophication,aggravating the current situation of water environment.Therefore,how to complete wastewater denitrification and phosphorus removal in a green and efficient way to improve the existing traditional wastewater treatment process,has become one of the key factors to solve the water pollution problem.This study aimed to screen out the high-efficiency denitrifying phosphorus-polymerizing bacteria from an activated sludge in the secondary sedimentation tank of AAO process,optimize the denitrifying and phosphorus removal conditions,explore its process pathway,and complete the denitrification and phosphorus removal of wastewater in combination with the sequencing batch reactor.The main results are as follows:（1）Twenty-one bacterial species were isolated using R2A medium,mainly belonging to Actinobacteniota（61.9%）,Proteobacteria（33.3%）and Firmicutes（4.8%）.Among them,there were 19 strains possess the removal abilities for nitrogen and phosphorus,which were affiliated to the genus Tsukamurella,Pantoea,Aeromonas,Staphylococcus,Acinetobacter,Pseudoxanthomonas,Gordonia,and so on.The strains Gordonia sp.D4,Tsukamurella sp.D14,and Pantoea sp.D6 had the removal efficiency of higher than 80%for medium containing high concentrations of nitrate nitrogen（40 mg/L）,ammonia nitrogen（80 mg/L）and phosphorus（10 mg/L）.Strain D4 was selected as the subsequent research object.（2）The optimal conditions for the denitrifying and phosphorus removal of D4 were investigated by single-factor and response surface experiments.The results showed the optimal conditions for nitrogen and phosphorus removal were sodium acetate（3.32 g/L）as carbon source,5%inoculum,initial phosphorus content of 18.61 mg/L,31.3℃,and p H 7.9.Under these conditions,the removed amounts of total phosphorus,nitrate nitrogen and ammonia nitrogen by strain D4 were 14.18 mg/L,39.67 mg/L and 69.71 mg/L,respectively;and the corresponding removal rates were 84.50%,97.67%and 96.22%,respectively.（3）The transformation of different nitrogen sources by strain D4 and nitrogen balance experiments demonstrated that D4 has the effects of simultaneous nitrification denitrification and phosphorus aggregation.In HNM medium,the removal of NH₄⁺-N by D4 reached more than 134 mg/L,of which 19.58%of nitrogen escaped in the gaseous form.In ADM-1medium,the removal of NO₃^--N by D4 amounted to more than 154 mg/L,of which 21.30%was converted to its gaseous form,and in ADM-2 medium,the removal of NO₂^--N by D4amounted to more than 86 mg/L,of which 27.86%was converted to its gaseous form.From the detected intermediate metabolites and nitrogen balance results,it was speculated that the possible denitrification pathways of D4 were synchronous nitrification denitrification and allochthonous reduction to ammonium.Among them,the simultaneous nitrification denitrification pathway is,NH₄⁺-N→（NH₂OH）→NO₂^--N→NO₃^--N→NO₂^--N→（NO→N₂O→N₂）;the allochthonous reduction to ammonium pathway is,NO₃^--N→NO₂^--N→NH₄⁺-N→biological nitrogen.（4）The bacterial genome of strain D4 was sequenced and analyzed biometrically.The results showed that the total genome sequence length of strain D4 was 5348752 bp,the GC content was 67.75%,the number of CDS was 4924,the number of t RNA was 47,and the number of r RNA was 5.The whole genome sequence of D4 was annotated using six databases,and among them,ppk1,ppk2,nar K,nar I,nar J,nar H,nar G,nir B,nir D and other genes related to the function of nitrogen removal and phosphate removal of the strain were annotated in both COG functional analysis and KEGG metabolic pathway analysis.（5）The results showed that compared with the blank group,the experimental group improved the removal of total phosphorus,total nitrogen and COD by 3.33,13.60 and 36.93mg/L,respectively,at the feed water levels of 20,200 and 2000 mg/L,and the removal efficiency was increased by 18.91%,7.35%and 2.16%.The results of macrogenomic analysis showed that strain D4 could colonise in the reactor well and implemented nitrogen and phosphorus removal by altering the microflora of the activated sludge,which increased theremovalefficiencyofunclassified＿f＿＿Saprospiraceae,Thauera,unclassified＿f＿＿Cyclobacteriaceae,Pararhodobacter and unclassified＿o＿＿Rhodospirillales,and reduced the species abundance of unclassified＿p＿＿Chloroflexi and unclassified＿f＿＿Caldilineaceae.