Study On Breeding Of Heterotrophic Nitrification-aerobic Denitrification Bacteria And Its Nitrogen Removal Characteristics And Pathway

Industrial and agricultural wastewater and domestic sewage contain a large amount of nitrogen-containing substances,which has caused enormous pressure on China’s sewage treatment system.Once the nitrogen-containing substances in waste water cannot be treated in a timely manner and discharged up to standard,the nitrogen element entering natural water bodies and drinking water will endanger human and animal health,hinder the development of industry and agriculture,reduce environmental quality,and lead to ecological disasters.The increasing number of nitrogen containing substances in wastewater puts forward higher requirements for the ability of biological nitrogen removal methods.Researchers have been exploring new and efficient nitrogen removal bacteria to enable them to meet both the basis of biological science and practical engineering needs in terms of nutritional and respiratory patterns,and to study their nitrogen removal characteristics and mechanisms on this basis.Researchers have proposed the concept of heterotrophic nitrification aerobic denitrification（HN-AD）.Research has shown that HN-AD microorganisms can achieve simultaneous removal of NH₄⁺-N,NO₃^--N,and NO₂^--N under aerobic conditions,without nitrite residue.Subsequently,more than 20 functional genera of HN-AD bacteria were isolated from nature.These microorganisms have received increasing attention due to their high tolerance to NH₄⁺-N and efficient nitrogen removal ability.Based on the existing HN-AD theory,this study designed experiments to select and breed HN-AD strains.After obtaining a strain with efficient growth and nitrogen removal capacity,the optimization of nitrogen removal performance and analysis of nitrogen removal mechanism of the strain were conducted,providing a reference for the development of HN-AD theory.The main results and conclusions of this study are as follows:（1）A strain of Acinetobacter johnsonii,named Acinetobacter johnsonii sp.N26,with HN-AD performance,was screened from sheep manure compost through the selection of HN-AD strains.The colony shape of this strain is circular,with a smooth and glossy surface.The colony is low convex,with neat edges,and its color is opaque milky white.The strain is rod-shaped,without spores or capsule,and is a Gram negative bacterium.（2）The growth and nitrogen removal characteristics of strain N26 were studied.The results showed that strain N26 had a fast growth rate and high nitrogen removal efficiency in NH₄⁺-N,NO₃^--N,NO₂^--N,and their combined nitrogen sources with initial concentrations of50 mg/L.In 36 hours,the removal rates of NH₄⁺-N,NO₃^--N,and NO₂^--N were all above99.0%,and the removal rates of combined nitrogen sources were also nearly 100%.The maximum removal rate of NH₄⁺-N was 5.330 mg/（L·h）;The maximum removal rate of NO₃^--N was 3.147 mg/（L·h）.The maximum removal rate of NO₂^--N was 3.159 mg/（L·h）.（3）The survival and nitrogen removal ability of strain N26 under different initial concentrations of NH₄⁺-N were studied.The results showed that strain N26 had a high removal rate under low NH₄⁺-N concentrations（less than 300 mg/L）,and could grow and remove nitrogen under high NH₄⁺-N concentrations（800 mg/L）.However,high concentrations of NH₄⁺-N would inhibit the growth rate and nitrogen removal efficiency of N26.（4）Through growth and nitrogen removal curves and nitrogen balance analysis,when the three nitrogen sources exist separately and simultaneously,the nitrogen removal priority of strain N26 is NO₂^--N>NH₄⁺-N>NO₃^--N.（5）In the study on the optimization of nitrogen removal performance of strain N26,it was found that the optimal conditions for the growth and nitrogen removal of strain N26 were as follows:the nitrogen source was ammonium chloride,the carbon source was sodium succinate,the temperature was 30℃,the inoculation amount was 5%,the p H was 8.0～9.0,the C/N was 15,and the rotational speed was 120 rpm.（6）The results of functional gene identification showed that the strain Acinetobacter johnsonii sp.N26 had functional genes such as amo A,nap A,nor B,nos Z,and nir S.（7）The results of nitrogen balance analysis and functional gene identification indicate that nitrogen removal by this strain is not only a HN-AD process,but also a short-range nitrification denitrification process.NH₄⁺-N,NO₃^--N,and NO₂^--N in water can be simultaneously removed in the same reactor under aerobic conditions,and the metabolic pathway of nitrogen removal is as follows.NH₄⁺→NH₂OH→NO₂^-→NO→N₂O→N₂NO₃^-→NO₂^-→NO→N₂O→N₂Therefore,the HN-AD strain Acinetobacter johnsonii sp.N26 has excellent nitrogen removal characteristics and has application potential in the treatment of nitrogen pollution in water bodies.This result can provide a theoretical basis for the application of HN-AD microorganisms in biological nitrogen removal engineering.