Screening And Nitrogen Removal Characteristics Of Heterotrophic Nitrification-Aerobic Denitrification Bacteria

Nitrogen is a widespread basic element in nature and an important part of biogeochemical cycles.In recent years,due to the rapid development of national industrialization,a large number of gaseous nitrogen is fixed to nitrogen compounds,which makes the nitrogen content in the water exceed the standard,resulting in water eutrophication and water environment pollution.Nitrogen is the main pollutant in water,and there are usually three methods for nitrogen removal:physical,chemical and biological.Biological nitrogen removal is considered to be an economical and effective method to solve nitrogen pollution,but the traditional biological nitrogen removal needs two processes:nitrification and denitrification,strictly controlling oxygen,and the process is more complex.Therefore,screening heterotrophic nitrification-aerobic denitrification（HN-AD）strains to achieve low-cost denitrification has become a research hotspot.The aim of this study is to provide theoretical basis and germplasm resources for bioremediation of nitrogen pollution.In this study,two heterotrophic nitrification-aerobic denitrification strains were obtained.After strain identification,the nitrogen removal characteristics of the two strains were analyzed,including the optimization of influencing factors,the exploration of nitrogen removal mechanism,and the whole genome analysis,and finally applied in actual wastewater.The main findings were as follows:（1）Screening of heterotrophic nitrification-aerobic denitrification strains.After the initial screening,504 strains of bacteria were obtained.A total of 32 strains with strong nitrification ability were obtained through secondary screening,and the denitrification function of these 32strains were determined qualitatively and quantitatively.The results showed that the ammonia nitrogen conversion rate of strain T2-1 was 97.33%,nitrate nitrogen conversion rate was61.12%and nitrite nitrogen conversion rate was 54.34%.In strain F1-6,the conversion of ammonia nitrogen,nitrate nitrogen and nitrite nitrogen were 95.48%,61.31%and 79.43%,respectively.Strains T2-1 and F1-6 were highly heterotrophic nitrification-aerobic denitrifying bacteria.（2）Identification of highly efficient HN-AD strains.Through colony observation,gram staining and other morphological characteristics,physiological and biochemical characteristics analysis,and 16S r RNA gene phylogenetic analysis,strain T2-1 was identified as Acinetobacter johnsonii and strain F1-6 was identified as Acinetobacter junii.（3）Optimization of influencing factors for efficient HN-AD strains.Single-factor optimization experiments were conducted to analyze the factors affecting the denitrification performance of strains T2-1 and F1-6（carbon source,C/N ratio,p H,temperature,and rotation speed）,and the optimal denitrification conditions for nitrification and denitrification processes were obtained.Based on the results of the single-factor experiments,the response surface analysis of p H,temperature and rotation speed of the two strains showed that the optimal conditions for strain T2-1 were p H 6.7,culture temperature of 29.4℃and rotation speed of184 r/min,resulting in an actual NH₄⁺-N conversion rate of 98.57%.The optimum conditions for strain F1-6 were as follows:p H 6.8,culture temperature of 31.19℃,and rotation speed of130 r/min.resulting in an actual NH₄⁺-N conversion rate of 98.83%.（4）Analysis of denitrification characteristics of efficient HN-AD strains.The denitrification characteristics of the two strains were analyzed at the optimal denitrification conditions with a1%inoculation rate.With ammonia as the only nitrogen source,the conversion efficiency of strain T2-1 and F1-6 reached the highest at 36 h,which were 98.02%and 98.13%,respectively.When using nitrate as the only nitrogen source,the conversion efficiency of strain T2-1 and F1-6 reached the highest at 42 h and 18 h,respectively,which were 95.84%and 92.37%.When using nitrite as the only nitrogen source,the conversion efficiency of strain T2-1 and F1-6 reached the highest at 36 h and 30 h,respectively,which were 54.84%and 82.27%.（5）Whole genome analysis of efficient HN-AD strains.To understand the genomic components and gene functions of strains in a more in-depth and detailed way,strains T2-1and F1-6 were sent to Guangdong Meig Gene Technology Co.,Ltd.for whole genome sequencing.（6）Mechanism of denitrification by efficient HN-AD strains.Through the genome analysis of strains T2-1 and F1-6,such as KEGG,GO,COG,etc.,it was found that genes encoding enzymes related to nitrogen metabolism:nitrate reductase gene:nas A;Nitrite reductase gene:nir B.At the same time,functional gene extraction was performed,and the band of the hydroxylamine oxidase gene hao was amplified,further verified the HN-AD function of the strain.（7）Practical application of high-efficiency HN-AD bacteria strains in wastewater.Strains T2-1 and F1-6 were applied to actual domestic wastewater.The results showed that strain T2-1 achieved conversion efficiencies of 94.19%and 73.02%for ammonia nitrogen and nitrate nitrogen,respectively,in urban domestic wastewater after 48 hours.Strain F1-6 achieved conversion efficiencies of 93.25%and 85.84%for ammonia nitrogen and nitrate nitrogen,respectively,after 48 hours.These results indicate that strains T2-1 and F1-6 have certain potential applications in wastewater treatment.Through the optimization of influencing factors,analysis of nitrogen removal characteristics,whole genome analysis and exploration of potential nitrogen removal mechanism of the two dominant strains,the theoretical basis for the study of HN-AD bacteria is enriched,which is of great significance in solving the process of nitrogen pollution of water resources.