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4.3 × 1010 per L MLSS of the municipal WWTP. In contrast,
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 37, NO. 2, 2003

no amplification was observed when DNA isolated from
an industrial WWTP, where no N. oligotropha-like amoA
sequences were detected (9), was used as template (data not
shown).
Paired samples t-tests were used to determine whether
gene copies obtained in the real-time PCR assays were
equivalent to the values previously obtained using dot-blot
hybridization and competitive PCR assays for Nitrospira 16S
rDNA and N. oligotropha-like amoA (Table 2). The mean
bacterial 16S rDNA copies per liter previously obtained by
dot-blot hybridization was 2.3 ( 0.7 × 1013 (9). In this study,
the average 16S rDNA copies per liter obtained by real-time
PCR was 1 order of magnitude lower at 1.6 ( 0.7 × 1012 and
the differences in values obtained by these two methods
were statistically significant (Table 2). The mean Nitrospira
16S rDNA copies per liter previously obtained by competitive
PCR was 2.4 ( 1.3 × 1010 compared to 3.7 ( 3.2 × 1010 and
3.9 ( 6.4 × 1010 copies per liter obtained using the Nitrospira
TaqMan and Molecular Beacon assays, respectively. The
differences in the values obtained by the three assays were
not statistically significant (Table 2). The mean values
obtained using the N. oligotropha-like amoA competitive PCR
and the real-time PCR were 3.4 ( 2.3 × 108 and 1.5 ( 1.2 ×
1010 copies per liter, respectively. The differences in the mean
values obtained by the two assays were statistically significant
and may reflect the differences in the primers used for the
two assays.
Calculation of Cells/Liter from Copies/Liter and Ammonia-Oxidizing Activity in MLSS Samples. The number of
total bacteria, AOB, N. oligotropha-like AOB and Nitrospira
cells per liter MLSS were calculated from copies per liter
using several assumptions regarding gene copies per cell
(Table 3, Figure 5). First, the average 16S rDNA copies per
genome in bacterial cells were assumed to be 3.6 copies based
on the average 16S rDNA copies found in cultured bacteria
(30). Second, one cell of N. oligotropha was assumed to
contain 2 copies amoA based on the copies reported for N.
europaea (31). Third, both AOB and Nitrospira were assumed
to contain 1 copy 16S rDNA per cell based on copies 16S
rDNA found in Nitrobacter and the AOB Nitrosomonas and
Nitrosospira (32, 33).
In the 12 monthly samples, total bacteria ranged from
2.3 × 1011 to 8.4 × 1011 and Nitrospira spp. ranged from
1.7 × 1010 to 1.2 × 1011 cells per liter MLSS and were relatively
constant through the year (Figure 5). The number of
ammonia-oxidizing bacteria as determined using the AOB
16S rDNA and N. oligotropha amoA assays were not as
constant with a drop in AOB in the August, September and
October samples (Figure 5). Although the number of AOB
calculated using the 16S rDNA assay were about 2-fold the
values calculated using the amoA assays, the assays were
significantly correlated with a Pearsons coefficient of 0.901.
Ammonia-oxidizing activity per cell‚hour was calculated
from the AOB cell number and WWTP plant data using the
following formula:

activity )

+
(NH+ - NH+ - NH4OUT) × Q
4IN
4UP
AOB

NH4+IN is the average ammonia concentration (measured
in mg nitrogen/L) in the secondary influent into the WWTP
reactor. NH4+OUT is the average ammonia concentration in
the plant effluent. NH4+UP is used to account for ammonia
assimilation, because ammonia is removed by both assimilation into cells for cell growth and by autotrophic
ammonia oxidation by AOB, and equals 0.20×NH4+SI. Ammonia assimilation values of 0.1 and 0.2 were previously
used by Daims et al. (6, 34). In this WWTP an ammonia
assimilation value of 0.3 was estimated based on a Monte
Carlo analysis (35) using the steady-state ASM1 model (36).

TABLE 3. Conversion of Copies/L to Cells/L, Cells/g, and Percent of Biomass
target cellsa

copies/L

cells/Lb

cells/gc

% biomassd

bacteria 16S rDNA
N. oligotropha amoA
AOB 16S rDNA
Nitrospira 16S rDNA

1.6 ( 0.7 × 1012
1.5 ( 1.2 × 1010
1.2 ( 0.9 × 1010
3.7 ( 3.2 × 1010

4.3 ( 2.0 × 1011
7.5 ( 6.0 × 109
1.2 ( 0.9 × 1010
3.7 ( 3.2 × 1010

2.2 ( 0.97 × 1011
3.8 ( 3.0 × 109
6.1 ( 4.7 × 109
1.9 ( 1.6 ×1010

100
1.7
2.9
8.6

a Values are averaged for all 12 samples. b Cells/L ) copies/L ÷ gene copy number/cell. Assumed gene copy number/cell is 3.6 for bacterial
16S rDNA, 1 for Nitrospira 16S rDNA, and 2 for amoA gene. c Cells/g ) cells/L ÷ 1.97 g/L (mean mixed liquor volatile suspended solids (MLVSS)).
d Bacterial 16S rDNA provides normalization for comparison to the subpopulations and is set at 100%.

FIGURE 5. Total bacteria (b), N. oligotropha (1), AOB (3), Nitrospira