Screening And Nitrogen Removal Mechanism Of Heterotrophic Nitrifying Aerobic Denitrifying Bacteria And Its Treatment Effect On Slaughter Wastewater

With the rapid development of industry and agriculture in China,efficient treatment of nitrogen-rich wastewater has become one of the most important research issues.Heterotrophic nitrifying-aerobic denitrifying（HNAD）bacteria have attracted much attention of researchers in recent years because they can achieve simultaneously nitrification and denitrification under aerobic conditions.Compared with traditional biological denitrification,HNAD bacteria can denitrify wastewater in the same system,which has many advantages such as high denitrification efficiency,low treatment cost,short cycle duration,and great application prospects.In this study,an efficient heterotrophic nitrifying aerobic denitrification strain,AHP123,was screened from the activated sludge of a sewage treatment plant.The nitrogen removal characteristics of this strain were analyzed,its nitrogen removal conditions were optimized,and the genome of the strain was sequenced as well.Based on the genome information,the key genes for nitrogen removal were identified,and its nitrogen removal mechanism was revealed.In addition,the preliminary treatment of swine wastewater by this strain was also studied.The main results are as follows:（1）A total of 34 bacteria were isolated and purified from the sediment of the secondary sedimentation tank of Chengnan Sewage Treatment Plant in Wuhu City,Anhui Province.The heterotrophic nitrification-aerobic denitrification capability of the above strains was tested,and the strain numbered AHP123 was screened out for the strongest denitrification capability.After morphological observation and genome-wide sequence comparison analysis,the strain was identified as Acinetobacter oleivorans andwas named as Acinetobacter oleivorans AHP123.（2）The nitrogen removal conditions of Acinetobacter oleivorans AHP123 were optimized by selecting the best C/N ratio,carbon source type,culture temperature,shaking speed,and inoculation rate.The results showed that in the nitrification process with 0.5 g/L NH₄Cl as the sole nitrogen source,the optimum nitrogen removal conditions were C/N ratio of 20,sodium succinate as carbon sourcehe,culture temperature of 30°C,culture shaking speed of 200 rpm and inoculation rate of 2%.Under these optimum conditions,the removal rate of ammonia nitrogen reached 99.26%in 24 h.In the denitrification process with 0.5 g/L KNO₃ as the sole nitrogen source,the optimal C/N ratio is 20,the carbon source is sodium citrate,the temperature is 25°C,the speed is 120 rpm,and the inoculation rate is 2%.Under these conditions,the removal rate of nitrate nitrogen reached 92.68%in 24 h.The experimental results of simultaneous nitrification and denitrification with mixed nitrogen sources（0.2 g/L NH₄Cl,0.3 g/L KNO₃）showed that Acinetobacter oleivorans AHP123 preferentially removed ammonia nitrogen.During this process,the maximum removal rate was 6.98 mg/L/h for ammonia nitrogen and2.35 mg/L/h for nitrate nitrogen.There was no accumulation of nitrite in the process,which indicated that strain AHP123 had excellent heterotrophic nitrification aerobic denitrification ability.（3）Based on the genomic information of Acinetobacter oleivorans AHP123,twelve key genes（nor,amt,gln K,glt B,gdh A,gam,gln A,ncd2,ncd1,nir B,nas A,nar）for nitrogen removal were annotated.These genes were previously reported to be related to the nitrogen assimilation pathway and heterotrophic nitrification aerobic denitrification pathway of KEGG,so it was speculated that the strain,AHP123,had the above two nitrogen metabolism pathways.The expression levels of nitrogen removal key genes under different nitrogen source conditions were analyzed by using an RT-q PCR technology.It was found that the relative expression of key enzyme genes gam,gln A,gdh A,glt B,nir B,and nas was increased under mixed nitrogen sources.This indicates that most of the ammonia nitrogen and the nitrate nitrogen were converted into biological nitrogen through assimilation（NO₃^-→NO₂^-→NH₄⁺→biological nitrogen）.However,when ammonia nitrogen or nitrate nitrogen is used as the only nitrogen source,the gene expression of key enzymes in heterotrophic nitrification and aerobic denitrification pathway such as nar and nor was increased to some extent,which indicates that a small amount of inorganic nitrogen was converted into nitrogen-containing gas（NH₄⁺→NO₂^-→NO₃^-→NO₂^-→NO→nitrogen-containing gas）.Nitrogen balance analysis of the nitrogen removal process of this strain was conducted and could further verify the above conclusions.（4）The application of strain Acinetobacter oleivorans AHP123 in slaughterhouse wastewater treatment was preliminarily discussed.The results showed that the strain AHP123 could be quickly colonized in slaughterhouse wastewater.Under the conditions of C/N ratio of 20,sodium succinate and sodium citrate as the mixed carbon sources,temperature of 30℃,shaking speed of 200 rpm,and inoculation rate of2%,the removal rate of ammonia nitrogen（153.81 mg/L）and COD（6392.55 mg/L）in 48 h was 99.19%and 88.05%,respectively.At the same time,HNAD strain AHP123 was co-cultured with microalgae to construct a bacteria-algae symbiosis system to treat the slaughterhouse wastewater.The results showed that the best treatment of slaughterhouse wastewater was achieved when the bacteria-algae ratio was 3:1,and 5 g/L sodium succinate was added as carbon source.Under this condition,the removal of ammonia nitrogen,total phosphorus（TP）and COD were all better than those of bacteria itself.After 24 h treatment,the removal rate of 115.7 mg/L ammonia nitrogen reached 100%,the TP（32.18 mg/L）removal rate reached 95.85%,the COD removal rate reached 76.74%,and the COD concentration decreased from 5066.4 mg/L to 1178.27mg/L.