Metabolic Characteristics Of Heterotrophic Nitrifying-aerobic Denitrifying Strain Diaphorobacter Sp.PD-7

With the development of the industrialization,the problem of excessive ammonia in water has seriously affected the water environment and human health.Compared with the traditional denitrification process,the new nitrogen removal technology has always been the research direction of domestic and foreign researchers.Many studies have shown that some heterotrophic bacteria can nitrify and some bacteria can denitrify under aerobic conditions,and many aerobicdenitrifyingbacteriaareheterotrophicnitrifyingbacteria?i.e.heterotrophic nitrifying-aerobic denitrifying bacteria?.Based on the above understanding,we are actively studying and developing the simultaneous nitrification and denitrification biological nitrogen removal technology.The nitrification and denitrification can occur in the same reactor and remove ammonia nitrogen and organics simultaneously,thus solving the contradiction between the treatment efficiency and economic benefits of traditional nitrogen removal process.In this paper,the heterotrophic nitrifiying-aerobic denitrifying bacterium Diaphorobacter sp.PD-7 isolated by laboratory was studied.Single factor experiments were conducted in a 3 L fermentor.The effects of dissolved oxygen,carbon-nitrogen ratio,temperature,pH,and inoculation volume on the denitrification characteristics of the strain were studied,and the optimal culture conditions were obtained.The kinetic analysis of the enzymatic reaction of four catalytic enzymes was performed to determine the rate-limiting enzyme and rate-limiting reaction.The rate-limiting enzyme gene was over-expressed through genetic engineering methods to examine the effects of pathway enhancement on the rate of nitrogen removal.A metabolic flux analysis model was constructed to calculate the energy production levels of oxidative phosphorylation and aerobic denitrification pathways under different dissolved oxygen and reveal the metabolic characteristics of PD-7 strain.The main conclusions were as follows:?1?The effects of rotating speed,carbon-nitrogen ratio,temperature,pH and inoculation volume on the heterotrophic nitrification-aerobic denitrification by PD-7 strain were studied.The results showed that the growth and nitrogen removal performance of the strains was the best under the condition of temperature 30?,pH 7.0,C/N ratio 8,rotating speed 300 r/min and the inoculation volume 7%.?2?The enzymatic reaction rate of ammonium monooxygenase?AMO?,hydroxylamine oxidase?HAO?,nitrite reductase?NAR?,and nitrite reductase?NIR?were determined.The kinetics of the enzymatic reaction was fitted using Michaelis-Menten equation.The analysis results showed that there was a positive correlation between the rates of AMO,HAO,NAR and NIR and the correspondingsubstrateconcentrations.Accordingtothemaximum consumption rate of substrate v_max,NIR may be the rate-limiting enzyme in the process of denitrification of the strain.The reaction from nitrite to nitric oxide was the rate-limiting reaction.?3?A denitrification pathway-enhancing bacterium was constructed by over-expressing the nirS gene.The experiment results showed that the time required for the removal of 50 mg/L ammonia was reduced from 25 h to 22 h,and the nitrogen removal performance of the original strain was enhanced by amplification of the gene for the rate-limiting enzyme,and the denitrification efficiency was improved.?4?The metabolic flux network model of PD-7 strain was constructed.The intracellular metabolic flux equations and corresponding metabolic reaction equations for each metabolite in the cell were established.The energy production levels of oxidative phosphorylation and aerobic denitrification pathway under different dissolved oxygen were investigated.And the energy production level after overexpression of the gene for the rate-limiting enzyme was studied.The results showed that the proportion of oxidative phosphorylation capacity to total production capacity increased with increasing dissolved oxygen,and when dissolved oxygen was low,the proportion of denitrification production capacity to total production capacity was higher.After the amplification of the nitrite reductase gene?nirS?,the metabolic activity of the denitrification pathway increased and the energy production increased by20.7%.However,compared with the oxidative phosphorylation capacity,its production level was still very low.