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Applied and Environmental Microbiology, January 2002, p. 245-253, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.245-253.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Quantification of Nitrosomonas oligotropha-Like Ammonia-Oxidizing Bacteria and Nitrospira spp. from Full-Scale Wastewater Treatment Plants by Competitive PCR

Hebe M. Dionisi,1,2 Alice C. Layton,1,2 Gerda Harms,1,2 Igrid R. Gregory,1,2 Kevin G. Robinson,2,3 and Gary S. Sayler1,2*

Department of Microbiology,1 Center for Environmental Biotechnology,2 Department of Civil and Environmental Engineering, The University of Tennessee, Knoxville, Tennessee 379963

Received 15 June 2001/ Accepted 10 October 2001

Utilizing the principle of competitive PCR, we developed two assays to enumerate Nitrosomonas oligotropha-like ammonia-oxidizing bacteria and nitrite-oxidizing bacteria belonging to the genus Nitrospira. The specificities of two primer sets, which were designed for two target regions, the amoA gene and Nitrospira 16S ribosomal DNA (rDNA), were verified by DNA sequencing. Both assays were optimized and applied to full-scale, activated sludge wastewater treatment plant (WWTP) samples. If it was assumed that there was an average of 3.6 copies of 16S rDNA per cell in the total population and two copies of the amoA gene per ammonia-oxidizing bacterial cell, the ammonia oxidizers examined represented 0.0033% ± 0.0022% of the total bacterial population in a municipal WWTP. N. oligotropha-like ammonia-oxidizing bacteria were not detected in an industrial WWTP. If it was assumed that there was one copy of the 16S rDNA gene per nitrite-oxidizing bacterial cell, Nitrospira spp. represented 0.39% ± 0.28% of the biosludge population in the municipal WWTP and 0.37% ± 0.23% of the population in the industrial WWTP. The number of Nitrospira sp. cells in the municipal WWTP was more than 62 times greater than the number of N. oligotropha-like cells, based on a competitive PCR analysis. The results of this study extended our knowledge of the comparative compositions of nitrifying bacterial populations in wastewater treatment systems. Importantly, they also demonstrated that we were able to quantify these populations, which ultimately will be required for accurate prediction of process performance and stability for cost-effective design and operation of WWTPs.

* Corresponding author. Mailing address: Department of Microbiology, The University of Tennessee, 676 Dabney Hall, Knoxville, TN 37996-1605. Phone: (865) 974-8080. Fax: (865) 974-8086. E-mail: sayler{at}utk.edu.

Applied and Environmental Microbiology, January 2002, p. 245-253, Vol. 68, No. 1
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.1.245-253.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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