Study On Isolation And Identification Of Aerobic Denitrifiers

At present, eutrophication has become a global environmental problem. Attention for treatment of nitrogen pollution is growing at home and abroad, and biological denitrification technology is popular because of the rapid and safe. As one of biologicaldenitrification, aerobic denitrification technology has become a hot topic because of its unique advantages in certain areas comparing with the traditional anoxic denitrification technology.Through long term of sludge domestication in aerobic nitrate system, the expression of strain nitrate reductase was induced and aerobic denitrifiers were accumulated effectively. In this study, intial isolation of aerobic denitrifiers was finished from aerobic denitrification sludge by BTB media. 20 BTB-positive bacteria were isolated from two different reactor A and B in laboratory, except one Gram-positive cocci, the remaining 19 bacteria were Gram-negative and rod. The result of determination of aerobic denitrifying activity showed that the nitrate removal rate of 12 bacteria were more than 50%, and TIN removal rate of 9 bacteria were more than 50%, and it not only confirmed that the sequencing batch reactor achieved a true sense of aerobic denitrification from the perspective of biolog, but also provided the basis for the follow-up study of aerobic denitrifiers.The results of physiological and biochemical tests of the 6 bacteria that their nitrate and TIN removal rates were more than 50% showed that their characteristics were basically the same: oxidase positive, contact enzyme positive, ethanol oxidation positive, indole negative, glucose ferment negative, not hydrolyzed starch, and not deliquesce gelatin. But there were difference between the use of different carbon sources for growth: The 4 bacteria (WXZ-4, 7, 13, 19) grew best by using of sodium citrate and WXZ-9 and WXZ-15 grew best by use of sodium succinate.By PCR amplification and homology analysis of 16S rDNA sequence, the taxonomic status of the various strains wer determined that the 15 strains belonged to Pseudomonas,Delftia,Bacillus,Herbaspirillum and Comamonas, and the degree of homology with the standard strains were revealed.Use two extract DNA methods: boiling water method and chemical-biological method. By comparing, although boiling water method was simpler relatively, but chemical-biological method had higher success rate. Using boiling water method for the initial extraction, and then using the chemical-biological method was feasible, it could simplifier a number of steps.Refering to literature we found that Pseudomonas, Bacillus and Comamonas were three speces, which aerobic denitrifier had found in the genus, but so far has not been reported that aerobic denitrifier had found in speces of Herbaspirillum and Delftia. This study first disvoer that Delftia has efficient aerobic denitrifying activity, and it should be the focus of the aerobic denitrification study in our lab.