Study On The Denitrification Pathway And Denitrification Products Of The Cold-tolerant Heterotrophic Nitrifying And Aerobic Denitrifying Bacteria Pseudomonas Fragi EH-H

Most of the currently reported heterotrophic nitrification-aerobic denitrification microorganisms are mesophilic bacteria,which generally perform denitrification in the temperature range of 25～37℃.However,these mesophilic bacteria may encounter significant challenges and limitations in winter or in low temperature regions where biological nitrogen treatment is required,as the growth and denitrification ability of cells will be inhibited under the condition of low temperatures,especially below 10℃.Therefore,the isolation and exploration of bacteria with effectively nitrogen removal ability at low temperatures is of great practical importance for the development of novel low temperature nitrogen contaminated wastewater treatment processes.In this study,a cold-tolerant and efficient heterotrophic nitrification-aerobic denitrification bacterium Pseudomonas fragi EH-H1 was isolated and purified from soil,and the ability to convert inorganic nitrogen at low temperatures was investigated,with the aim of providing a safe and effective heterotrophic nitrification-aerobic denitrification bacterium for wastewater treatment at low temperatures:The potential cold-resistant denitrifying bacteria were isolated from soil in this investigation.Under aerobic conditions,approximately 33 denitrifying bacteria were isolated using the BTB agar plate streak method.The strain named EH-H1 with the highest nitrite and TN removal efficiencies were selected for further detection.Based on the results from the morphological and 16S r RNA analyses,the strain EH-H1 was identified as Pseudomonas fragi,which has not been reported to be able to remove inorganic compounds from wastewater,especially at low temperatures.The nitrogen removal capacity of strain EH-H1 was investigated at 15°C using inorganic nitrogen alone or mixed under laboratory conditions,and the results showed that the strain EH-H1 could remove 100%,100%and 99.95%of ammonium（54.90 mg/L）,nitrate（56.12 mg/L）and nitrite（54.15 mg/L）,respectively,and the corresponding maximum removal rates were 5.51,3.63 and 3.14 mg/L/h,respectively.In addition,strain EH-H1could also effectively carry out simultaneous nitrification and denitrification（SND）at15℃.When ammonium（52.44 mg/L）and nitrate（54.29 mg/L）were used as the mixed nitrogen sources,the removal rates of both inorganic nitrogen by strain EH-H1 reached99.58%and 96.83%respectively within 60 h.When ammonium and nitrite were used as mixed nitrogen sources,the removal rates of ammonium and nitrite nitrogen by strain EH-H1 were 98.06%and 87.90%respectively within 72 h.The above results indicated that strain EH-H1 possessed high efficiency in biological denitrification under low temperature conditions.Environmental factors play an important role in affecting the efficiency of nitrogen removal by microorganisms.In this study,the nitrogen removal conditions were investigated by setting different carbon sources,C/N,initial p H,inoculum volume and rotational speed to improve the efficiency of nitrogen loss by strain EH-H1 under low temperature.The results demonstrated that strain EH-H1 achieved the highest removal rates of nitrate with 100%、100%、99.02%、97.34%and 96.99%under the conditions of carbon source in glucose,C/N in 15,initial p H in 6.2,inoculum volume in 0.2×10⁸CFU/m L and the speed in 150 r/min,respectively.In contrast,the optimum carbon source for heterotrophic nitrification with ammonium or aerobic denitrification with nitrite by strain EH-H1 were sodium citrate,and the optimum C/N were severally 10and 25.Results from experiments on nitrogen balance after optimization of environmental conditions revealed that the nitrogen loss rates of ammonium,nitrate or nitrite by strain EH-H1 were 48.0%、48.98%and 44.10%,respectively.Furthermore,the N₂O gas produced by strain EH-H1 during the degradation of inorganic nitrogen at low temperature was examined using the sealed shake flask incubation method,and the results revealed that the N₂O produced by strain EH-H1during the degradation of ammonium,nitrate and nitrite were 0.01,0.07 and 1.64 mg/L,respectively,which.Biological nitrogen conversion is catalyzed by a series of enzymes.In this study,enzyme inhibition experiments demonstrated that the key enzymes of ammonia monooxygenase（AMO）,nitrate reductase（NR）and nitrite oxidoreductase（Ni R）for nitrification and denitrification played an important role in the degradation of inorganic nitrogen by strain EH-H1.In addition,the enzyme activity assay showed that the specific enzyme activities of AMO,NR and Ni R were 0.637,0.239 and 0.018 U/mg proteins,respectively.The whole-genome sequencing revealed that cold-tolerant genes such as csp A and cap A and nitrogen metabolism-related functional enzyme genes such as nap A、nar B、nas A、nir S and nir BD were present in strain EH-H1,further indicating that the strain was capable of denitrification under low temperature conditions.Overall,the results of this study could help to reveal the mechanism of nitrogen removal by hypothermia bacteria in the biological denitrification process,which could provide technical reference and theoretical guidance for the low-temperature wastewater treatment process.