Study On Isolation And The Denitrification Performance Of An Aerobic Denitrifier,Enterobacter Cloacae Strain HNR

Aerobic denitrification was defined as that some denitrifying bacteria complete the transformation from nitrate(NO3--N) to gaseous nitrogen under aerobic condition. The discovery of aerobic denitrification technology makes it possible to complete the traditional nitrification and denitrification simultaneously in the aerobic condition. This paper took an aerobic denitrifying bacteria as the research object, and studyed the physiological and biochemical characteristics, screened nitrogen removal optimum culture conditions and nitrogen concentration load on the removal effect of nitrogen for strain HNR. It aimed to provid the theoretical and experimental basis for the practical application of aerobic denitrifying bacteria. The main results were as follows:The aim strain HNR with the aerobic denitrification function was Selected from the secondary setting tank of traditional nitrogen wastewater treatment plant, using the plate separation and liquid culture method. This strain is short and thick bacillus, the whole colony is milky white, translucent, round, smooth surface moist, colony diameter 2-5 mm. Based on the cell morphology, physiological biochemical tests and 16 S rRNA gene sequence analysis, it is concluded that the strain HNR belongs to Enterobacter cloacae.The best culture conditions were optimized with the response surface methodology. The results showed that the optimum pH range was from 7.3 to 8.3 for strain HNR aerobic denitrification, the best C/N ratio range was from 10 to 13, the optimum temperature range was from 25 to 30℃, and the optimum shaking speed range was from 120 to 140 rmp. Through Design Expert 8.05 software optimization selection function derived that strain HNR optimal aerobic denitrification conditions that C/N of 11.3, pH of 7.7, shaking speed of 137 rpm and temperature of 27.8℃. In these conditions, the TN removal rate can reach the best by strain HNR. The experiments results showed that the prediction was reliable.Logistic equation model can be used to fit the growth curve of strain HNR, and its growth was consistented with the general microbial growth rules. In the best culture conditions and glucose was the carbon source, the nitrogen concentration was set to 100, 200 and 300 mg/L, the results of different nitrogen concentration stress tests showed that the TN removal ratios were 90.9%, 81.5%, 73.6% by strain HNR, and the TOC removal ratio were 81.5%, 77.7%, 65.2%. In the whole denitrification process, pH decreased at first and then upward trend and the intermediate products had no significant accumulation.Enzyme activity assay and gene amplification results showed that the enzyme activity of Nar was 0.0688 U/mg protein. And the Nar gene fragment that experiments obtained was 880 bp in length, which can encoding contain 290 amino acid residues. The protein was basic, hydrophilicity and without the transmembrane region. The enzyme activities of Nir 0.0054 U/mg protein. And the Nar gene fragment that experiments obtained was 1331 bp in length, which can encoding contain 364 amino acid residues. The protein that was basic, hydrophilicity and without the transmembrane region. The high enzyme activities of Nar and Nir were further explained the phenomenon that the rapid degradation of NO3--N and NO2--N was not significant accumulation.