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suggesting that both assays were useful for AOB quantification. The reason for the drop in AOB is unclear as the percent
nitrification remained constant and there was no apparent
link to the basin temperature. The AOB cell numbers
calculated by the 16S rDNA assay were approximately 2-fold
higher than the N. oligotropha cell numbers using the amoA
assay. Although AOB have not been cultured from this plant,
these results suggest that N. oligotropha-like AOB comprise
at least 50% of the AOB population in this WWTP.
The percent of the AOB population determined using
either the AOB 16S rDNA assay (2.9%) or the N. oligotropha
amoA assay (1.7%) were 3-4-fold lower than reported for an
350

9

ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 2, 2003

activated sludge sample obtained from the second stage of
a 2-stage WWTP measured by fluorescence in situ hybridization (FISH) (8.4%) (6). Additionally, the AOB percent was
approximately 2-3-fold lower than reported for a sludge
sample from an industrial plant connected to a rendering
factory (7%) (7). Differences in the percent of ammoniaoxidixing bacteria between these studies may reflect the
differences in the operation and design of the WWTPs, e.g.
single-sludge nitrification versus two-stage rectors or sludge
with a high nitrogen load. Alternatively, the differences may
reflect differences in the methods, FISH versus real-time PCR.
Interestingly, the percent Nitrospira (8.6%) calculated in this
study is consistent with values obtained by FISH in the sludge
from an industrial plant connected to a rendering factory at
9% to 12% Nitrospira population (7, 40).
The ammonia-oxidizing activity per cell per hour was
calculated based on the estimated number of ammoniaoxidizing cells in the basin and the estimated amount of
ammonia oxidized per hour. The estimated ammoniaoxidizing rate of 7.7 fmol/hr/cell based on AOB 16S rDNA,
or 12.4 fmol/hr/cell based on the N. oligotropha amoA, were
in the range of values reported using FISH (2.3 ( 0.4 fmol/
hr/cell) (6), a cPCR assay (16 to 43 fmol/hr/cell) (34) and for
pure cultures (4 to 23 fmol/hr/cell) (42). Given that the
estimated ammonia-oxidizing rates are in the expected range,
it is likely that the real-time PCR assays used in this study
detects one of, if not, the major organisms mediating
ammonia oxidation in the WWTP under study.
The results of this study indicate that real-time PCR
technology is a valuable tool for quantification of uncultivable
or difficult to culture microbes in environmental samples,
offering high throughput, analytical sensitivity, and precision.
The bacterial 16S rDNA assay and the Nitrospira 16S rDNA
assays described in the study and the previously published
AOB 16S rDNA may have broad utility to other wastewater
treatment plants and environmental samples. The N. oligotropha amoA assay will be more useful in wastewater systems
where the N. oligotropha is a known member of the AOB
population. The amoA assay may also prove useful in
developing mRNA based reverse transcriptase real-time PCR
assays to measure physiological responses of N. oligotropha
to changes in environmental conditions.

Acknowledgments
This work was funded by a Water Environment Research
Foundation research grant (WERF project #98-CTS-2) and
by the University of Tennessee, Waste Management Research
and Education Institute. H.D. is a recipient of a postdoctoral
fellowship from CONICET. We thank Arthur Meyers of
Eastman Chemical Company (Kingsport, TN) for technical
advice and Neil Quigley at the Molecular Biology Resource
Facility (University of Tennessee, Knoxville, TN) for DNA
sequencing. We thank Knoxville Utilities Board (KUB,
Knoxville, TN) for providing samples.

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