Environ
.
Sci
.
Technol
.
2003
,
37
,
343-351
Real-Time
PCR
Quantification
of
Nitrifying
Bacteria
in
a
Municipal
Wastewater
Treatment
Plant
G
E
R
D
A
H
A
R
M
S
,
†
,
§
A
L
I
C
E
C
.
L
A
Y
T
O
N
,
†
,
§
H
E
B
E
M
.
D
I
O
N
I
S
I
,
†
,
§
I
G
R
I
D
R
.
G
R
E
G
O
R
Y
,
†
,
§
V
I
C
T
O
R
I
A
M
.
G
A
R
R
E
T
T
,
†
,
§
S
H
A
W
N
A
.
H
A
W
K
I
N
S
,
‡
,
§
K
E
V
I
N
G
.
R
O
B
I
N
S
O
N
,
‡
,
§
A
N
D
G
A
R
Y
S
.
S
A
Y
L
E
R
*
,
†
,
§
Department
of
Microbiology
,
Department
of
Civil
and
Environmental
Engineering
,
and
Center
for
Environmental
Biotechnology
,
The
University
of
Tennessee
,
Knoxville
,
Tennessee
,
37996
Real-time
PCR
assays
using
TaqMan
or
Molecular
Beacon
probes
were
developed
and
optimized
for
the
quantification
of
total
bacteria
,
the
nitrite-oxidizing
bacteria
Nitrospira
,
and
Nitrosomonas
oligotropha-like
ammonia
oxidizing
bacteria
(
AOB
)
in
mixed
liquor
suspended
solids
(
MLSS
)
from
a
municipal
wastewater
treatment
plant
(
WWTP
)
using
a
single-sludge
nitrification
process
.
The
targets
for
the
real-time
PCR
assays
were
the
16S
rRNA
genes
(
16S
rDNA
)
for
bacteria
and
Nitrospira
spp
.
and
the
amoA
gene
for
N.
oligotropha
.
A
previously
reported
assay
for
AOB
16S
rDNA
was
also
tested
for
its
application
to
activated
sludge
.
The
Nitrospira
16S
rDNA
,
AOB
16S
rDNA
,
and
N.
oligotropha-like
amoA
assays
were
loglinear
over
6
orders
of
magnitude
and
the
bacterial
16S
rDNA
real-time
PCR
assay
was
log-linear
over
4
orders
of
magnitude
with
DNA
standards
.
When
these
real-time
PCR
assays
were
applied
to
DNA
extracted
from
MLSS
,
dilution
of
the
DNA
extracts
was
necessary
to
prevent
PCR
inhibition
.
The
optimal
DNA
dilution
range
was
broad
for
the
bacterial
16S
rDNA
(
1000-fold
)
and
Nitrospira
16S
rDNA
assays
(
2500-fold
)
but
narrow
for
the
AOB
16S
rDNA
assay
(
10-fold
)
and
N.
oligotrophalike
amoA
real-time
PCR
assay
(
5-fold
)
.
In
twelve
MLSS
samples
collected
over
one
year
,
mean
cell
per
L
values
were
4.3
(
2.0
×
1011
for
bacteria
,
3.7
(
3.2
×
1010
for
Nitrospira
,
1.2
(
0.9
×
1010
for
all
AOB
,
and
7.5
(
6.0
×
109
for
N.
oligotropha-like
AOB
.
The
percent
of
the
nitrifying
population
was
1.7
%
N.
oligotropha-like
AOB
based
on
the
N.
oligotropha
amoA
assay
,
2.9
%
total
AOB
based
on
the
AOB
16S
rDNA
assay
,
and
8.6
%
nitriteoxidizing
bacteria
based
on
the
Nitrospira
16S
rDNA
assay
.
Ammonia-oxidizing
bacteria
in
the
wastewater
treatment
plant
were
estimated
to
oxidize
7.7
(
6.8
fmol
/
hr
/
cell
based
on
the
AOB
16S
rDNA
assay
and
12.4
(
7.3
fmol
/
hr
/
cell
based
on
the
N.
oligotropha
amoA
assay
.
*
Corresponding
author
phone
:
(
865
)
974-8080
;
fax
:
(
865
)
974-8086
;
e-mail
:
sayler
@
utk.edu
.
†
Department
of
Microbiology
.
‡
Department
of
Civil
and
Environmental
Engineering
.
§
Center
for
Environmental
Biotechnology
.
10.1021
/
es0257164
CCC
:
$
25.00
Published
on
Web
12
/
04
/
2002
©
2003
American
Chemical
Society
1
.
Introduction
Chemolithotrophic
nitrification
is
a
two-step
process
involving
two
groups
of
bacteria
:
ammonia-oxidizing
bacteria
(
AOB
)
oxidize
NH3
to
NO2-
and
nitrite-oxidizing
bacteria
(
NOB
)
oxidize
NO2-
to
NO3-
(
1
)
.
Nitrification
is
an
important
process
in
biogeochemical
nitrogen
cycling
and
in
controlling
effluent
toxicity
in
wastewater
treatment
.
The
physiological
activity
and
abundance
of
these
organisms
in
wastewater
processing
is
critical
in
the
design
and
operation
of
waste
treatment
systems
,
particularly
since
these
organisms
display
low
growth
rate
and
high
sensitivity
to
environmental
disturbances
and
inhibitors
(
2
)
.
An
important
aspect
of
activity
relates
to
reactor
design
.
Single
sludge
wastewater
treatment
designs
accomplish
nitrification
concurrent
with
removal
of
carbonaceous
oxygen
demand
in
one
reactor
;
activated
sludge
in
these
reactors
contains
both
heterotrophs
and
nitrifiers
that
necessarily
compete
for
resources
such
as
oxygen
(
3
)
.
Single
sludge
reactors
are
a
dominant
design
in
the
United
States
(
4
)
.
Alternatively
,
nitrification
can
be
accomplished
using
a
series
of
reactors
,
the
first
dedicated
to
carbonaceous
oxygen
demand
removal
and
the
second
to
nitrification
.
To
date
,
molecular
quantification
of
nitrifying
populations
in
activated
sludge
from
full-scale
wastewater
treatment
plants
(
WWTPs
)
has
been
performed
on
samples
obtained
from
facilities
with
high
nitrogen
loads
(
5
)
or
twostage
reators
(
6
,
7
)
.
Given
that
single-sludge
nitrification
processes
may
contain
fewer
nitrifiers
and
are
more
susceptible
to
plant
upsets
,
attempts
at
quantification
of
nitrifiers
in
these
plants
is
warranted
.
In
recent
studies
,
a
competitive
Polymerase
Chain
Reaction
(
cPCR
)
procedure
was
developed
to
quantify
nitrifying
bacteria
by
PCR
amplification
of
ammonia
monooxygenase
(
amoA
)
and
Nitrospira
spp
.
16S
rRNA
genes
(
16S
rDNA
)
,
respectively
(
8
,
9
)
.
The
cPCR
procedure
is
a
well
established
quantitative
method
that
has
been
used
to
enumerate
both
culturable
and
nonculturable
organisms
,
including
nitrifying
bacteria
(
10-13
)
.
This
method
relies
on
the
measurement
of
PCR
products
at
the
endpoint
,
after
gel
electrophoresis
,
and
it
has
a
log-linear
detection
range
of
only
2
to
3
orders
of
magnitude
(
14
,
15
)
.
The
cPCR
technique
is
robust
due
to
the
presence
of
a
stringent
internal
control
.
However
,
cPCR
is
difficult
to
use
in
routine
process
monitoring
of
populations
in
wastewater
treatment
processes
because
it
is
labor-
and
cost-intensive
with
low
throughput
.
In
the
current
study
,
real-time
PCR
was
investigated
for
applications
in
monitoring
nitrifying
populations
in
wastewater
treatment
because
it
combines
high
throughput
with
high
analytical
sensitivity
and
precision
,
offering
a
dynamic
detection
range
of
6
orders
of
magnitude
or
more
(
15
,
16
)
.
Although
real-time
PCR
has
been
widely
applied
in
medical
research
,
its
application
to
environmental
research
has
been
slower
(
17-21
)
.
In
real-time
PCR
,
amplicons
are
detected
by
measurement
of
a
fluorescence
signal
without
post-PCR
sample
processing
such
as
gel
electrophoresis
(
Figure
1
)
.
Several
different
fluorescent
probes
can
be
used
in
real-time
PCR
including
TaqMan
(
22
)
or
Molecular
Beacon
(
23
,
24
)
.
A
TaqMan
probe
is
a
linear
oligonucleotide
complementary
to
a
target
nucleic
acid
sequence
,
with
a
fluorophore
attached
to
the
5
′
end
and
a
quencher
to
the
3
′
end
(
22
)
(
Figure
1
)
.
The
TaqMan
probe
is
cleaved
by
the
5
′
exonuclease
activity
of
Taq
DNA
polymerase
as
the
primer
is
extended
,
resulting
in
the
separation
of
the
reporter
dye
from
quencher
dye
and
an
increase
in
fluorescence
signal
emitted
by
the
reporter
.
In
each
cycle
,
additional
reporter
dye
molecules
are
cleaved
,
resulting
in
an
increase
in
fluorescence
intensity
proportional
to
the
amount
of
amplicon
produced
(
22
)
.
In
contrast
,
a
VOL.
37
,
NO
.
2
,
2003
/
ENVIRONMENTAL
SCIENCE
&
TECHNOLOGY
9
343
FIGURE
1
.
Schematic
representation
of
the
quantification
of
specific
targets
from
mixed-liquor
suspended
solids
(
MLSS
)
samples
.
After
DNA
extraction
,
the
DNA
template
is
used
in
real-time
PCR
assays
.
(
A
)
TaqManprobe
assay
.
A
TaqMan
probe
is
a
linear
oligonucleotide
probe
complementary
to
the
target
sequence
,
with
a
fluorescent
dye
(
green
circle
,
F
)
attached
to
the
5
′
end
and
a
quencher
(
blue
triangle
,
Q
)
to
the
3
′
end
.
The
proximity
of
these
two
dyes
quenches
the
signal
.
Steps
:
(
1
)
Denaturation
of
the
DNA
at
95
°
C
separates
the
template
.
(
2
)
Annealing
of
primers
(
usually
18-22
nucleotides
)
and
probe
to
target
DNA
at
temperatures
of
50
°
C
to
60
°
C.
(
3
)
Extension
and
synthesis
of
the
DNA
strand
results
in
the
5
′
exonuclease
activity
by
Taq
DNA
polymerase
(
red
oval
,
P
)
separating
the
fluorescent
dye
from
the
quencher
resulting
in
an
increase
in
fluorescence
.
Steps
2
and
3
are
often
performed
simultaneously
.
The
spectrofluorimetric
thermal
cycler
measures
the
relative
fluorescence
at
the
end
of
step
3
(
eye
)
.
Steps
1-3
are
repeated
45
to
50
times
.
In
each
cycle
,
additional
reporter
dye
molecules
are
cleaved
,
resulting
in
an
increase
in
fluorescence
intensity
proportional
to
the
amount
of
amplicon
produced
(
22
,
43
)
.
(
B
)
Molecular
beacon
assay
.
A
Molecular
beacon
probe
has
a
stem
and
loop
structure
.
The
loop
section
of
the
probe
is
complementary
to
the
target
and
the
stem
results
from
the
annealing
of
artificially
designed
arm
sequences
(
23
,
24
,
43
)
.
A
fluorescent
dye
(
green
circle
,
F
)
and
a
quencher
(
blue
triangle
,
Q
)
are
attached
to
the
arm
sequences
.
Step
:
(
1
)
Denaturation
at
95
°
C
results
in
the
separation
of
the
template
DNA
and
separation
of
the
stem
on
the
molecular
beacon
probe
,
separating
the
quencher
from
the
dye
allowing
fluorescence
.
(
2
)
Annealing
at
60
°
C
allows
the
probe
to
hybridize
to
the
template
resulting
in
fluorescence
.
Alternatively
,
if
the
probe
does
not
hybridize
with
the
template
the
stem-and-loop
structure
is
reformed
and
fluorescence
is
quenched
.
The
relative
fluorescence
is
measured
at
the
beginning
of
this
step
(
eye
)
and
is
proportional
to
the
number
of
target
sequences
.
(
3
)
Extension
and
synthesis
of
DNA
at
72
°
C
results
in
dissociation
of
the
probe
from
the
target
and
fluorescence
is
quenched
.
Steps
1-3
are
repeated
45
to
50
times
.
The
final
output
for
both
assays
shows
relative
fluorescence
as
a
function
of
cycle
number
.
Amplification
curves
for
template
concentrations
between
107
and
10
copies
per
PCR
reaction
are
shown
(
left
to
right
)
.
The
threshold
is
calculated
as
10
times
the
standard
deviation
of
the
background
fluorescence
(
dashed
line
)
.
The
point
where
the
fluorescence
signal
crosses
the
threshold
is
the
threshold
cycle
(
CT
)
(
dotted
line
)
and
is
lower
when
more
copies
of
the
template
are
present
at
the
beginning
of
the
reaction
.
344
9
ENVIRONMENTAL
SCIENCE
&
TECHNOLOGY
/
VOL
.
37
,
NO
.
2
,
2003
TABLE
1
.
Primers
and
Probes
Used
in
This
Study
assay
target
primer
/
probea
sequence
(
5
′
-3
′
)
b
TM
(
°
C
)
c
reference
1055f
1392r
16STaq1115
amoNo550D2f
amoNo754r
amoNoTaq729
5
′
-ATGGCTGTCGTCAGCT-3
′
5
′
-ACGGGCGGTGTGTAC-3
′
5
′
-
(
6-FAM
)
-CAACGAGCGCAACCC-
(
TAMRA
)
-3
′
5
′
-TCAGTAGCYGACTACACMGG-3
′
5
′
-CTTTAACATAGTAGAAAGCGG-3
′
5
′
-
(
6-FAM
)
-CCAAAGTACCACCATACGCAG
(
TAMRA
)
-3
′
5
′
-GGAGRAAAGCAGGGGATCG-3
′
5
′
-GGAGGAAAGTAGGGGATCG-3
′
5
′
-CGTCCTCTCAGACCARCTACTG-3
′
5
′
-
(
6-FAM
)
-CAACTAGCTAATCAGRCATCRGCCGCTC-
(
TAMRA
)
-3
′
5
′
-CCTGCTTTCAGTTGCTACCG-3
′
5
′
-GTTTGCAGCGCTTTGTACCG-3
′
5
′
-
(
6-FAM
)
-AGCACTCTGAAAGGACTGCCCAGG
(
TAMRA
)
-3
′
5
′
-
(
6-FAM
)
-GCTGCACC
|
AGCACTCTGAAAGGACTGCCCAGG
|
GGTGCAGC-
(
DABCYL
)
-3
′
57.7
58.9
62.9
55.4
55.0
64.1
(
25
)
(
26
)
this
study
this
study
this
study
this
study
63.5
62.3
62.8
71.0
(
20
)
(
20
)
(
20
)
(
20
)
64.7
67.8
73.9
(
9
)
(
9
)
this
study
bacterial
16S
rDNA
bacterial
16S
rDNA
N.
oligotrophalike
amoA
N.
oligotropha
amoA
gene
AOB
ammonia-oxidizing
bacterial
16S
rDNA
CTO
189fA
/
B
CTO
189fC
RT1r
TMP1
Nitrospira
16S
rDNA
Nitrospira
spp
.
16S
rDNA
NSR1113f
NSR1264r
NSR1143Taq
NSR1143Beac
this
study
a
Primer
/
probe
abbreviations
:
f
)
forward
primer
,
r
)
reverse
primer
,
Taq
)
TaqMan
probe
,
Beac
)
beacon
probe.
b
5
′
-Fluorophore-probequencher-3
′
in
case
of
TaqMan
probe
;
5
′
-fluorophore-arm
|
probe
|
arm-quencher-3
′
in
case
of
Molecular
Beacon
.
6-FAM
)
6-carboxyfluorescein
;
TAMRA
)
carboxytetramethylrhodamine
;
DABCYL
)
4-
(
4-dimethylaminophenyl
)
azo
)
benzoic
acid.
c
Melting
temperatures
were
calculated
using
the
oligo
calculator
from
Sigma
Genosys
(
http
:
/
/
www.genosys.com
/
cgi-win
/
oligo
_
calconly.exe
;
Sigma
Genosys
,
The
Woodlands
,
TX
)
.
Molecular
Beacon
probe
has
a
stem
and
loop
structure
,
with
the
loop
section
of
the
probe
complementary
to
the
target
and
the
stem
formed
by
the
annealing
of
an
artificially
designed
arm
sequence
(
24
)
.
A
fluorescent
dye
and
a
quencher
are
attached
to
both
ends
of
the
molecule
(
Figure
1
)
.
When
free
in
solution
the
Molecular
Beacon
adopts
a
hairpin
structure
,
which
results
in
fluorophore
quenching
.
In
the
presence
of
a
complementary
target
,
the
hairpin
structure
unfolds
and
the
separation
of
the
fluorophore
and
the
quencher
leads
to
emission
of
fluorescence
.
Using
either
a
TaqMan
or
a
Molecular
Beacon
probe
the
threshold
cycle
of
a
sample
is
inversely
proportional
to
the
logarithm
of
the
amount
of
template
DNA
initially
added
to
the
PCR
reaction
for
both
types
of
probe
(
22-24
)
.
Although
the
design
,
chemistry
and
portion
of
the
PCR
cycle
in
which
fluorescence
is
detected
differ
for
these
two
probes
,
similar
results
should
be
obtained
in
the
enumeration
of
molecules
.
The
primary
objectives
of
the
current
study
were
to
develop
methods
and
to
quantify
ammonia-oxidizing
bacteria
and
nitrite-oxidizing
bacteria
(
Nitrospira
)
in
a
single-stage
type
municipal
WWTP
.
Real-time
PCR
assays
for
Nitrospira
16S
rDNA
and
Nitrosomonas
oligotropha
amoA
genes
were
developed
from
existing
cPCR
assays
for
monitoring
nitrifying
bacteria
,
and
a
real-time
PCR
assay
developed
to
enumerate
AOB
16S
rDNA
in
soil
(
20
)
was
applied
to
mixed-liquor
suspended
solids
(
MLSS
)
.
A
bacterial
16S
rDNA
TaqMan
assay
was
also
developed
to
monitor
total
biomass
in
the
MLSS
samples.
Secondary
objectives
of
this
study
were
to
compare
real-time
PCR
assays
using
different
probe
designs
(
molecular
beacon
versus
TaqMan
)
,
compare
real-time
PCR
assays
directed
toward
ribosomal
RNA
genes
versus
catabolic
genes
(
amoA
)
,
and
validate
real-time
PCR
assays
by
comparison
with
previous
data
in
copies
per
liter
obtained
by
dot-blot
hybridization
and
competitive
PCR
.
The
results
of
this
study
indicate
that
real-time
PCR
can
be
implemented
as
a
tool
to
facilitate
molecular
monitoring
and
quantification
of
critical
sub-populations
,
such
as
nitrifying
bacteria
,
in
wastewater
treatment
processes
.
(
NH4
+
-N
)
removal
designed
with
average
and
peak
flow
capacities
of
40
million
gallons
per
day
(
MGD
)
and
70
MGD
,
respectively
.
In
the
year
2000
,
this
single
stage
reactor
system
was
operated
at
an
average
solids
retention
time
of
12
days
.
The
reactor
system
consists
of
six
identical
basins
arranged
in
parallel
,
each
basin
being
183
feet
long
,
32
feet
wide
,
and
unusually
deep
at
33
feet
.
The
basins
are
in
turn
sub-divided
into
five
compartments
in
series
along
the
flow
path
.
Each
compartment
is
aerated
with
ceramic
fine
bubble
diffusers
that
provide
for
complete
mixing
.
The
basins
are
fed
via
a
common
influent
channel
.
Gravity
overflow
from
the
basins
first
combines
and
then
splits
to
flow
to
a
series
of
circular
clarifiers.
MLSS
samples
were
collected
from
the
upwell
at
the
center
of
one
of
the
clarifiers
prior
to
the
effluent
flow
entering
the
clarifier
quiescent
zone
.
Reactor
specific
flow
and
influent
ammonia
data
along
with
plant
effluent
data
was
obtained
.
The
mean
influent
BOD5
was
302
(
(
47
)
mg
/
L
,
and
mean
mixed-liquor
volatile
suspended
solids
(
MLVSS
)
was
1971
(
178
mg
/
L
over
the
one-year
period
.
The
mean
monthly
influent
temperature
for
the
year
was
17
°
C
,
with
a
low
of
9
°
C
and
a
high
of
25
°
C.
Genomic
DNA
was
extracted
in
triplicate
from
2
mL
of
MLSS
samples
using
a
FastDNA
kit
(
BIO
101
,
Vista
,
CA
)
with
minor
modifications
as
described
by
Dionisi
et
al
.
(
9
)
.
2
.
Methodology
Real-Time
PCR
Assays
.
Real-time
PCR
assays
were
developed
for
the
quantification
of
bacterial
16S
rDNA
,
Nitrospira
spp
.
16S
rDNA
,
and
N.
oligotropha-like
amoA
.
Three
TaqMan
probes
,
16sTaq1115
,
amoNoTaq729
,
and
NSR1143Taq
,
were
designed
(
Table
1
)
using
the
guidelines
provided
by
Applied
Biosystems
(
http
:
/
/
home.appliedbiosystems.com
;
Applied
Biosystems
,
Foster
City
,
CA
)
.
The
primers
and
probes
were
synthesized
by
Sigma
Genosys
(
Sigma
Genosys
,
The
Woodlands
,
TX
)
.
A
Molecular
Beacon
probe
,
NSR1143Beac
,
was
designed
using
the
guidelines
provided
at
http
:
/
/
www.molecular-beacons.org
/
protocol.html
and
synthesized
by
Stratagene
(
Stratagene
,
La
Jolla
,
CA
)
.
The
optimal
artificial
arm
sequences
were
determined
by
using
the
Zuker
DNA
folding
program
(
mfold
)
(
http
:
/
/
bioinfo.math.rpi.edu
/
∼
mfold
/
dna
/
form1
/
cgi
)
.
Samples
.
MLSS
samples
were
collected
monthly
for
one
year
from
a
local
municipal
WWTP
treating
mainly
municipal
wastewater.
The
WWTP
employs
single
stage
reactors
for
carbon
(
biological
oxygen
demand
(
BOD5
)
)
and
nitrogen
The
real-time
PCR
assay
for
AOB
used
two
forward
primers
CTO
189A
/
B
and
CTO189C
,
one
reverse
primer
RT1r
and
the
TaqMan
probe
TMP1
(
Table
1
)
as
described
by
Hermansson
and
Lindgren
(
20
)
.
VOL.
37
,
NO
.
2
,
2003
/
ENVIRONMENTAL
SCIENCE
&
TECHNOLOGY
9
345
Real-Time
PCR
for
Quantification
of
Bacterial
16S
rDNA
.
Bacterial
16S
rDNA
was
amplified
using
primers
1055f
(
25
)
and
1392r
(
26
)
(
Table
1
)
.
The
TaqMan
probe
16STaq1115
was
modified
from
the
1114f
primer
(
26
)
.
The
PCR
mix
with
a
total
volume
of
25
µL
contained
Platinum
Quantitative
PCR
SuperMix-UDG
(
Life
Technologies
,
Inc
.
,
Gaithersburg
,
MD
)
with
5
mM
MgCl2
,
15
pmol
primers
1055f
and
1392r
,
6.25
pmol
TaqMan
probe
16STaq1115
,
3.2
to
7.0
ng
of
sample
DNA
or
dilutions
of
plasmid
pCR2.1
vector
(
Invitrogen
,
Carlsbad
,
CA
)
carrying
a
16S
rRNA
gene
for
Nitrospira
(
GenBank
accession
number
AF420301
)
(
9
)
as
standard
(
from
4.5
×
103
to
4.5
×
108
copies
of
the
16S
rDNA
gene
)
.
The
PCR
program
was
3
min
at
50
°
C
,
10
min
at
95
°
C
,
45
cycles
at
95
°
C
for
30
s
,
50
°
C
for
60
s
,
and
72
°
C
for
20
s
.
Real-Time
PCR
for
Quantification
of
Nitrospira
16S
rDNA.
The
Nitrospira
16S
rDNA
primers
NSR1113f
and
NSR1264r
(
Table
1
)
were
previously
designed
and
tested
using
genomic
DNA
extracted
from
municipal
and
industrial
MLSS
as
templates
(
9
)
.
The
TaqMan
probe
NSR1143Taq
(
Table
1
)
was
derived
from
a
conserved
sequence
region
between
the
primers
NSR1113f
and
NSR1264r
.
The
probe
portion
of
the
Molecular
Beacon
NSR1143Beac
(
Table
1
)
is
identical
to
that
one
used
in
the
TaqMan
probe
.
The
probe
region
of
NSR1143Beac
has
a
TM
of
68.0
°
C
,
and
the
stem
region
has
a
TM
of
70.2
°
C
(
with
5.0
mM
MgCl2
)
.
Real-time
PCR
assays
using
NSR1143Taq
were
performed
in
a
total
volume
of
25
µL
with
5
mM
MgCl2
,
Platinum
SuperMix
,
15
pmol
of
primers
NSR1113f
and
NSR1264r
,
6.25
pmol
TaqMan
probe
NSR1143Taq
,
and
3.2
to
7.0
ng
of
sample
DNA
or
30
to
3
×
107
copies
of
the
standard
(
151
bp
fragment
of
Nitrospira
16S
rDNA
from
AF420301
)
(
9
)
.
PCR
amplification
consisted
of
2
min
at
50
°
C
,
10
min
at
95
°
C
,
55
cycles
at
95
°
C
for
30
s
,
63
°
C
for
60
s
.
The
Molecular
Beacon
assay
contained
Platinum
SuperMix
with
5
mM
MgCl2
,
25
pmol
of
primers
NSR1113f
and
NSR1264r
,
8.5
pmol
Molecular
Beacon
probe
NSR1143Beac
,
3.2
to
7.0
ng
of
sample
DNA
or
30
to
3
×
106
copies
of
standard
DNA
in
25
µL
.
PCR
amplification
consisted
of
5
min
at
95
°
C
,
55
cycles
at
95
°
C
for
30
s
,
60
°
C
for
60
s
,
and
72
°
C
for
10
s
.
Real-Time
PCR
for
Quantification
of
N.
oligotrophalike
amoA
Gene
.
The
primers
amoNo550D2f
and
amoNo754r
(
Table
1
)
were
designed
to
target
the
amoA
gene
of
ammoniaoxidizing
bacteria
found
in
the
full-scale
municipal
WWTP
(
9
)
based
on
alignment
of
amoA
gene
sequences
using
the
CLUSTAL
W
program
(
27
)
.
Alignments
consisted
of
amoA
sequences
from
clonal
libraries
obtained
from
the
WWTP
(
9
)
,
four
bench-scale
municipal
wastewater
treatment
systems
(
8
)
,
and
amoA
sequences
available
in
GenBank
(
28
)
.
The
forward
primer
amoNo550D2f
contained
two
degenerate
bases
in
order
to
amplify
all
amoA
clones
from
the
libraries
,
as
well
as
N.
urea
(
AJ388585
)
and
N.
oligotropha
(
AF272406
)
amoA
genes
.
The
TaqMan
probe
amoNoTaq729
was
derived
from
a
conserved
sequence
region
within
the
primer
pair
amoNo550D2f
and
amoNo754r
(
Table
1
)
.
The
25
µL
PCR
mix
contained
TaqMan
Universal
PCR
Master
Mix
(
PE
Applied
Biosystems
,
Foster
City
,
CA
)
with
7.5
pmol
primers
amoNo550D2f
and
amoNo754r
,
6.25
pmol
TaqMan
probe
amoNoTaq729
,
0.3
to
0.7
ng
of
sample
DNA
.
Standards
consisted
of
the
plasmid
pCR2.1
carrying
the
M-20
amoA
gene
(
GenBank
accession
number
AF420299
)
(
9
)
adjusted
to
10
to
1.0
×
107
copies
per
PCR
.
PCR
amplification
consisted
of
3
min
at
50
°
C
,
10
min
at
95
°
C
,
55
cycles
at
95
°
C
for
30
s
,
56
°
C
for
60
s
.
Real-Time
PCR
for
Quantification
of
AOB
16S
rRNA
Gene
.
Real-time
PCR
assays
were
performed
as
described
by
Hermansson
and
Lindgren
(
20
)
in
a
total
volume
of
25
µL
with
Universal
PCR
Master
Mix
(
PE
Applied
Biosystems
)
,
7.5
pmol
of
a
2
:
1
ratio
of
primers
CTO
189fA
/
B
and
CTO
189fC
,
7.5
pmol
of
the
reverse
primer
RT1r
,
3.125
pmol
TaqMan
probe
TMP1
,
and
0.3
to
0.7
ng
of
sample
DNA
or
60
to
6
×
346
9
ENVIRONMENTAL
SCIENCE
&
TECHNOLOGY
/
VOL
.
37
,
NO
.
2
,
2003
107
copies
of
the
standard
(
Nitrosomonas
europea
16S
rDNA
cloned
into
pCR2.1
)
.
PCR
amplification
consisted
of
2
min
at
50
°
C
,
10
min
at
95
°
C
,
40
cycles
at
95
°
C
for
30
s
,
60
°
C
for
60
s
.
Acquisition
and
Data
Analysis
.
Real-time
PCR
assays
for
bacterial
16S
rDNA
and
Nitrospira
16S
rDNA
were
run
on
a
Bio-Rad
iCycler
with
the
iCycler
iQ
fluorescence
detector
and
iCycler
software
version
2.3
(
Bio-Rad
,
Hercules
,
CA
)
.
Plate
well
factors
were
determined
prior
to
each
PCR
run
to
normalize
background
fluorescence
intensities
from
each
single
well.
amoA
and
AOB
16S
rDNA
real-time
PCR
assays
were
run
using
a
DNA
Engine
Opticon
Continuous
fluorescence
Detection
System
(
MJ
Research
,
Waltham
,
MA
)
.
The
threshold
was
determined
by
the
computer
software
as
10
times
the
standard
deviation
of
the
background
fluorescence
averaged
over
at
least
5
cycles
at
the
start
of
the
run
.
The
threshold
cycle
(
CT
)
of
each
PCR
reaction
was
automatically
determined
by
detecting
the
cycle
at
which
the
fluorescence
exceeded
the
calculated
threshold
.
For
the
Molecular
Beacon
probe
,
the
data
window
was
adjusted
to
capture
roughly
10
%
of
all
data
,
chosen
among
the
data
points
in
the
beginning
area
of
the
annealing
step
.
During
each
PCR
run
,
the
CT
values
obtained
from
the
DNA
standards
were
used
for
the
construction
of
standard
curves
.
Data
for
bacterial
16S
rDNA
,
Nitrospira
16S
rDNA
,
and
N.
oligotropha-like
amoA
was
previously
collected
for
the
same
set
of
samples
used
in
this
study
using
dot-blots
and
hybridization
with
a
P32
labeled
universal
16S
rDNA
probe
and
competitive
PCR
assays
for
the
Nitrospira
16S
rDNA
and
amoA
genes
(
9
)
.
These
data
were
compared
to
the
data
obtained
by
real-time
PCR
for
each
gene
using
paired
samples
t-tests
.
Paired
samples
t-tests
compute
the
differences
between
the
values
of
the
two
variables
for
each
case
and
tests
whether
the
average
differs
from
zero
(
SPSS
version
11.01
,
SPSS
Inc
.
,
Chicago
,
IL
)
.
The
null
hypothesis
was
that
there
were
no
significant
differences
between
the
copies
/
L
on
each
sample
date
obtained
by
the
real-time
PCR
assay
and
the
analogous
competitive
PCR
assay
or
dot-blot
hybridization.
Application
of
Real-Time
PCR
Assays
to
MLSS
Samples
.
All
real-time
PCR
assays
were
performed
using
three
replicates
per
sample
,
and
all
PCR
runs
included
control
reactions
without
template
.
The
effect
of
sample
concentration
on
PCR
performance
was
determined
using
dilutions
of
sample
DNA
(
initial
concentration
of
50
ng
/
µL
)
containing
5
pg
to
50
ng
in
sterile
water
followed
by
real-time
PCR
analysis
for
16S
rDNA
,
Nitrospira
16S
rDNA
,
AOB
16S
rDNA
,
and
N.
oligotropha-like
amoA
as
described
above
.
Gene
copies
were
initially
calculated
by
comparison
of
threshold
cycles
obtained
in
each
PCR
run
from
known
standard
DNA
concentrations
.
To
reduce
variability
between
PCR
runs
,
data
were
recalculated
using
a
second
standard
curve
generated
from
11
standard
curves
for
bacterial
16S
rDNA
(
r2
)
0.94
)
,
22
standard
curves
for
Nitrospira
16S
rDNA
(
r2
)
0.84
)
,
3
standard
curves
for
AOB
16S
rDNA
(
r2
)
0.98
)
,
and
5
standard
curves
for
N.
oligotropha
amoA
(
r2
)
0.90
)
.
In
the
case
of
the
Nitrospira
16S
rDNA
and
bacterial
16S
rDNA
assays
,
one
universal
standard
curve
was
applied
for
calculations
,
because
both
assays
were
shown
to
function
alike
with
standard
plasmid
AF420301
as
template
(
Figure
2
)
.
3
.
Results
Development
and
Optimization
of
Real-Time
PCR
Assays
.
The
real-time
PCR
assays
were
validated
using
known
concentrations
of
standard
DNA
.
The
linear
range
of
detection
for
the
real-time
PCR
assay
for
bacterial
16S
rDNA
was
4
orders
of
magnitude
,
from
4.5
×
104
to
4.5
×
108
copies
per
PCR
and
the
detection
limit
for
this
assay
was
4.5
×
103
target
DNA
copies
.
The
linear
range
of
detection
for
the
real-time
PCR
assays
for
Nitrospira
16S
rDNA
,
N.
oligotropha
amoA
,
FIGURE
2
.
Comparison
of
bacterial
16S
rDNA
and
Nitrospira
16S
rDNA
real-time
PCR
TaqMan
assays
.
The
plasmid
containing
Nitrospira
16S
rDNA
was
used
as
the
target
for
both
assays
.
The
dashed
line
represents
the
95
%
confidence
interval
.
Error
bars
indicate
the
standard
deviation
of
3
PCRs
and
are
smaller
than
the
symbols
.
and
AOB
16S
rDNA
were
at
least
6
orders
of
magnitude
,
from
30
to
3.0
×
107
,
10
to
1.0
×
107
,
and
60
to
6
×
107
copies
per
PCR
,
respectively
.
The
regression
coefficient
(
r2
)
values
for
standard
curves
for
all
real-time
PCR
assays
in
each
run
were
always
above
0.90
.
In
addition
,
copies
per
PCR
calculated
for
the
standard
plasmid
AF420301
by
the
bacterial
16S
rDNA
and
Nitrospira
16S
rDNA
assays
were
highly
correlated
and
demonstrated
a
linear
relationship
(
r2
)
0.97
)
with
a
slope
of
1.14
,
indicating
that
the
assays
for
bacterial
16S
rDNA
and
Nitrospira
16S
rDNA
were
functioning
alike
(
Figure
2
)
.
Application
of
Real-Time
TaqMan
PCR
Assays
to
WWTP
Samples.
In
environmental
samples
,
PCR
techniques
can
be
biased
by
the
presence
of
inhibitory
compounds
that
copurify
with
the
DNA
or
low
target
concentrations
in
a
high
background
of
heterologous
DNA
(
15
,
29
)
.
Inhibition
effects
by
the
environmental
samples
on
the
real-time
PCR
assays
were
tested
using
serial
dilutions
of
genomic
DNA
extracted
from
MLSS
.
PCR
amplification
was
completely
inhibited
in
the
undiluted
sample
at
50
ng
per
PCR
assay
for
all
four
real-time
PCR
assays
and
also
in
diluted
samples
containing
10
ng
per
PCR
assay
for
the
bacterial
16S
rDNA
and
the
N.
oligotropha-like
amoA
assays
.
CT
values
were
proportional
to
DNA
at
concentrations
ranging
from
5
pg
to
5
ng
for
the
bacterial
16S
rDNA
(
r2
)
0.98
,
slope
)
-3.87
)
,
at
concentrations
ranging
from
5
pg
to
5
ng
for
the
Nitrospira
16S
rDNA
(
r2
)
0.98
,
slope
)
-3.45
)
,
at
concentrations
from
250
pg
to
2.5
ng
for
the
AOB
16S
rDNA
(
r2
)
0.98
,
slope
)
-3.15
)
,
and
at
concentrations
ranging
from
200
pg
to
1
ng
for
the
N.
oligotropha-like
amoA
assay
(
r2
)
0.97
,
slope
)
-
3.01
)
(
Figure
3A
)
.
In
the
no-template
control
reactions
no
CT
values
were
obtained
for
the
Nitrospira
16S
rDNA
,
AOB
16S
rDNA
,
and
N.
oligotropha-like
amoA
assays
.
In
the
no
template
control
reactions
using
the
16S
rDNA
assay
a
CT
value
of
29.7
(
0.43
was
obtained
.
Conversion
of
the
CT
value
to
copies
per
PCR
based
on
the
standard
curve
indicated
that
there
were
3.8
(
0.9
×
103
copies
bacterial
16S
rDNA
in
the
control
reaction
without
sample
DNA
.
This
background
value
may
result
from
bacterial
DNA
contamination
of
the
Taq
enzyme
or
other
reagents
in
the
PCR
mix
.
The
MLSS
sample
containing
greater
than
5
pg
DNA
per
PCR
assay
was
20-fold
higher
than
the
detection
limit
(
Figure
3B
)
so
the
effect
of
background
contamination
on
the
calculated
value
would
be
less
than
5
%
in
the
most
dilute
samples
.
Conversion
of
the
CT
values
to
copies
per
PCR
for
the
other
3
assays
indicated
that
the
detection
limit
for
quantifying
these
targets
in
MLSS
samples
were
180
copies
for
Nitrospira
16S
rDNA
,
1.2
×
103
for
N.
oligotropha-like
amoA
,
and
2.6
×
103
AOB
16S
rDNA
assays
(
Figure
3
B
)
.
FIGURE
3
.
(
A
)
Threshold
cycle
(
CT
)
measurements
in
diluted
DNA
from
a
MLSS
sample
and
(
B
)
calculated
copies
of
target
genes
in
diluted
DNA
from
a
MLSS
sample
.
TaqMan
real-time
PCR
assays
shown
are
Bacterial
16S
rDNA
(
b
)
,
N.
oligotropha-like
amoA
gene
(
1
)
,
AOB
16S
rDNA
(
3
)
,
and
Nitrospira
16S
rDNA
(
9
)
.
Error
bars
indicate
the
standard
deviation
of
3
PCRs
.
Comparison
of
TaqMan
and
Molecular
Beacon
Probes
in
the
Nitrospira
16S
rDNA
Real-Time
PCR
Assay
.
Fluorescence
obtained
with
NSR1143Taq
was
twice
as
high
as
the
fluorescence
obtained
with
NSR1143Beac
(
Figure
4a
)
,
indicating
a
higher
signal-to-noise
ratio
using
the
TaqMan
probe
.
The
r2
values
of
standard
curves
obtained
using
NSR1143Beac
and
NSR1143Taq
were
similar
,
ranging
from
0.97
to
0.99
and
0.92
to
0.99
,
respectively
.
When
MLSS
samples
were
analyzed
using
both
probes
,
the
copies
obtained
with
the
Molecular
Beacon
probe
were
up
to
12.5
times
lower
than
those
obtained
with
the
TaqMan
probe
,
except
for
the
last
3
samples
in
which
the
values
were
almost
equivalent
(
Figure
4b
)
.
Nitrospira
16S
rDNA
copies
per
liter
in
the
municipal
WWTP
ranged
from
1.7
×
1010
to
1.2
×
1011
using
the
TaqMan
probe
and
from
2
×
109
to
2.1
×
1011
using
the
Molecular
Beacon
probe
.
In
a
paired
t-test
the
differences
in
results
obtained
by
the
Molecular
Beacon
probe
and
the
TaqMan
probe
assays
were
not
significant
(
t
)
-0.125
,
p
)
0.903
)
(
Table
2
)
.
Validation
of
N.
oligotropha
amoA
,
Nitrospira
16S
rDNA
,
and
Bacterial
16S
rDNA
Real-Time
PCR
Assays
.
To
verify
the
specificity
of
the
amoA
primers
,
PCR
product
obtained
with
genomic
DNA
extracted
from
MLSS
of
the
municipal
WWTP
as
template
was
cloned
and
sequenced
as
described
previously
(
9
)
.
Amplification
of
genomic
DNA
from
suspended
solids
using
the
primers
amoNo550D2f
and
amoNo754r
produced
a
product
with
the
expected
size
of
approximately
205
bp
(
data
not
shown
)
.
In
a
clonal
library
,
VOL
.
37
,
NO
.
2
,
2003
/
ENVIRONMENTAL
SCIENCE
&
TECHNOLOGY
9
347
FIGURE
4
.
(
A
)
Measurements
of
Nitrospira
16S
rDNA
copies
during
real-time
PCR
using
TaqMan
probe
NSR1143Taq
(
9
)
or
molecular
beacon
probe
NSR1143Beac
(
b
)
in
DNA
extracted
from
MLSS
.
The
fluorescence
intensity
was
measured
during
the
annealing
step
of
each
temperature
cycle
.
No-template
controls
are
shown
as
(
0
)
and
(
O
)
for
the
TaqMan
and
molecular
beacon
probes
,
respectively
.
Error
bars
indicate
the
standard
deviation
of
3
reactions
.
The
dashed
line
represents
the
threshold
value
(
42
RFU
)
calculated
for
the
molecular
beacon
assay
.
The
threshold
value
for
the
TaqMan
assay
was
20
RFU
.
(
B
)
Nitrospira
16S
rDNA
copies
per
liter
as
determined
by
real-time
PCR
using
TaqMan
probe
NSR1143Taq
(
9
)
or
Molecular
Beacon
probe
NSR1143Beac
(
b
)
in
DNA
extracted
from
MLSS
.
TABLE
2
.
Comparison
of
Real-Time
PCR
Assays
with
Other
Molecular
Methods
Using
Paired
Samples
t-Test
compared
groups
microbial
16S
dot-blota
vs
bacterial
16S
TMb
amoAc
TM
vs
amoA
cPCRd
Ntspaf
TM
vs
Ntspa
cPCR
Ntspa
MBg
vs
Ntspa
cPCR
mean
paired
differences
t
df
significance
(
2-tailed
)
2.2
×
1013
10.79h
11
0.000
1.5
×
1010
4.324h
11
1.4
×
1010
1.456
11
1.5
×
1010
0.813
11
0.001
0.173
0.433
a
dot-blot
)
dot-blot
hybridization.
b
TM
)
TaqMan
based
real-time
PCR
assay
.
c
amoA
)
N.
oligotropha-like
amoA
.
d
cPCR
)
competitive
quantitative
PCR
.
e
AOB
)
ammonia-oxidizing
bacteria
16S
rDNA.
f
Ntspa
)
Nitrospira
spp
.
16S
rDNA.
g
MB
)
Molecular
Beacon
based
real-time
PCR
assay.
h
Significant
at
the
prescribed
R
.
23
randomly
selected
clones
were
identified
as
amoA
sequences
.
These
sequences
were
89-94
%
similar
to
clone
M20
and
92-94
%
similar
to
clone
M379
,
which
were
amoA
clone
sequences
previously
isolated
from
this
WWTP
(
9
)
.
N.
oligotropha-like
amoA
gene
copies
ranged
from
2.6
×
109
to
4.3
×
1010
per
L
MLSS
of
the
municipal
WWTP
.
In
contrast
,
348
9
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
(
9
)
cells
per
L
MLSS
in
a
municipal
WWTP
determined
using
the
bacterial
16S
rDNA
,
N.
oligotropha
amoA
,
AOB
16S
rDNA
and
Nitrospira
real-time
PCR
Taqman
assays
.
Error
bars
indicate
the
standard
deviation
of
3
reactions
.
Quantitative
detection
limits
for
each
assay
were
as
follows
:
bacterial
16S
rDNA
,
1.0
×
1010
,
N.
oligotropha-like
amoA
,
1.5
×
109
,
AOB
16S
rDNA
2.6
×
109
and
Nitrospira
16S
rDNA
,
9
×
107
cells
per
L
.
An
intermediate
value
of
0.2
was
used
for
activity
calculations
.
Q
is
the
average
influent
flowrate
to
the
WWTP
reactor
(
L
/
hr
)
.
AOB
is
the
total
AOB
cell
number
in
the
basin
as
determined
by
real-time
PCR
.
Mean
monthly
ammonia-oxidizing
activity
based
on
the
N.
oligotropha
amoA
cell
calculations
ranged
from
3.5
to
56.2
fmol
/
hr
/
cell
.
The
highest
activity
value
,
which
was
greater
than
twice
the
standard
deviation
of
the
data
set
,
was
excluded
resulting
in
a
mean
activity
of
12.4
(
7.3
fmol
/
hr
/
cell
.
The
mean
ammonia-oxidizing
activity
based
on
the
AOB
16S
rDNA
cell
values
was
7.7
(
6.8
fmol
/
hr
/
cell
.
4
.
Discussion
Real-time
PCR
using
a
fluorescent
internal
probe
combines
high
throughput
with
high
analytical
sensitivity
for
the
detection
of
specific
genes
present
in
low
concentrations
in
complex
and
variable
DNA
mixtures
,
such
as
those
extracted
from
environmental
samples
.
The
log-linear
detection
range
using
standards
in
the
real-time
PCR
assays
was
very
broad
,
with
up
to
6
orders
of
magnitude
and
detection
limits
of
10
to
60
copies
for
the
Nitrospira
16S
rDNA
,
16S
AOB
rDNA
,
and
N.
oligotropha
amoA
assays
.
These
values
were
comparable
to
other
real-time
PCR
assays
(
15-20
)
.
The
bacterial
16S
rDNA
assay
showed
a
log-linear
detection
range
across
4
orders
of
magnitude.
The
higher
detection
limit
(
4.5
×
103
copies
per
PCR
)
in
the
bacterial
assay
was
comparable
to
the
detection
limit
for
other
bacterial
real-time
PCR
assays
(
21
,
37
)
.
These
high
detection
limits
in
bacteria
real-time
PCR
assays
are
generally
attributed
to
DNA
contamination
of
the
Taq
DNA
polymerase
enzyme
or
other
sources
such
as
water
and
plasticware
(
37
)
.
The
high
detection
limit
in
the
bacterial
real-time
PCR
assay
did
not
affect
the
use
of
this
assay
in
DNA
extracted
from
MLSS
because
these
samples
contained
approximately
108
bacterial
16S
rDNA
copies
per
µg
of
DNA
.
The
bacterial
16S
rDNA
real-time
PCR
assay
was
designed
to
be
a
broad-based
assay
for
the
estimation
of
total
bacterial
biomass
in
the
MLSS
.
This
assay
may
also
serve
as
an
internal
standard
,
since
bacterial
16S
rDNA
in
the
MLSS
remained
relatively
constant
.
Although
the
PCR
primers
and
probes
used
in
this
study
have
zero
base
pair
mismatches
with
more
than
9000
bacterial
16S
rDNA
sequences
found
in
GenBank
,
the
number
of
bacterial
cells
calculated
needs
to
be
considered
an
estimate
because
some
bacterial
strains
/
and
or
species
may
not
hybridize
to
the
primers
or
probes
and
thus
may
not
be
detected
.
The
cell
/
L
values
obtained
using
the
real-time
PCR
assay
were
10-fold
less
than
those
obtained
by
dot-blots
using
a
single
probe
.
This
may
result
from
the
reduction
of
background
due
to
the
combined
specificity
levels
of
the
primers
and
probe
used
for
the
16S
rDNA
assay
.
In
addition
the
probe
1392r
used
for
the
dot-blot
hybridizations
(
9
)
is
a
universal
probe
that
hybridizes
to
eukaryotic
DNA
found
in
the
MLSS
and
measures
total
microbial
population
including
both
eukaryotic
and
bacterial
cells
.
The
bacterial
real-time
PCR
assay
used
in
this
study
has
not
been
compared
to
other
bacterial
16S
rDNA
real-time
PCR
assays
(
21
,
37
,
38
)
.
Application
of
PCR
assays
to
environmental
samples
is
complicated
by
several
factors
such
as
low
concentration
of
targets
and
the
possible
presence
of
PCR
inhibitors
.
The
inhibitory
effects
of
complex
genomic
DNA
at
concentrations
of
10
to
50
ng
/
ul
and
other
PCR
inhibitors
can
be
minimized
by
dilution
of
the
DNA
extracts
.
However
,
over-dilution
may
result
in
low
total
DNA
and
target
concentrations
resulting
in
high
variability
and
potential
over-
or
under-estimation
of
the
target
.
In
this
study
,
the
acceptable
DNA
per
PCR
range
varied
between
the
assays
with
the
bacterial
16S
rDNA
and
Nitrospira
16S
rDNA
assays
producing
similar
results
over
a
broad
DNA
concentration
range
(
1000
to
2500-fold
)
.
In
contrast
,
the
N.
oligotropha-like
amoA
and
AOB
16S
rDNA
assay
produced
consistent
results
over
a
narrow
DNA
concentration
range
(
5
to
10-fold
)
.
These
results
suggest
that
in
DNA
extracts
from
environmental
samples
real-time
PCR
assays
may
need
to
be
performed
using
several
dilutions
.
Other
factors
that
may
affect
real-time
PCR
assays
are
a
loss
in
fluorescence
signal
due
to
a
large
excess
of
complex
DNA
or
an
overestimation
of
target
DNA
due
to
limited
probe
specificity
(
15
)
.
The
design
of
specific
PCR
assays
for
Nitrospira
16S
rDNA
was
straightforward
because
regions
of
the
Nitrospira
16S
rDNA
are
well
conserved
between
Nitrospira
species
and
distinct
from
nearest
phylogenetic
groups
(
39
,
40
)
.
The
primers
used
for
the
Nitrospira
real-time
PCR
assays
in
this
study
were
previously
demonstrated
to
be
specific
for
Nitrospira
sp.
in
the
cPCR
assay
(
9
)
.
The
Molecular
Beacon
and
TaqMan
probes
were
designed
using
the
same
target
sequence
used
in
the
cPCR
assay
.
The
differences
in
values
obtained
previously
in
individual
MLSS
samples
using
cPCR
and
more
recently
using
the
Molecular
Beacon
and
TaqMan
real-time
PCR
assays
were
statistically
insignificant
.
This
suggests
that
quantitative
PCR
methods
are
robust
and
that
real-time
PCR
assays
can
be
adapted
from
cPCR
assays
.
Slight
differences
in
numbers
obtained
with
cPCR
and
real-time
VOL
.
37
,
NO
.
2
,
2003
/
ENVIRONMENTAL
SCIENCE
&
TECHNOLOGY
9
349
PCR
using
the
TaqMan
probes
have
also
been
reported
for
the
quantification
of
Mycobacterium
tuberculosis
DNA
in
sputum
(
41
)
.
In
contrast
to
the
Nitrospira
assay
,
designing
a
real-time
PCR
assay
to
detect
AOB
is
more
complicated
for
two
reasons
.
First
,
wastewater
treatment
plants
may
contain
multiple
species
of
AOB
(
28
)
.
Second
,
most
AOB
are
phylogenetically
closely
related
to
other
activated
sludge
bacteria
within
the
beta-subdivision
class
of
Proteobacteria
.
Both
the
16S
rDNA
and
amoA
gene
provide
well-studied
genetic
markers
for
the
characterization
of
AOB
(
28
)
.
DNA
probes
and
primers
targeting
AOB
16S
rDNA
have
been
used
to
detect
essentially
all
known
beta-subdivision
AOB
species
(
2
,
5-7
,
11
,
12
,
28
)
.
The
16S
rDNA
assay
described
by
Hermansson
and
Lindgren
(
20
)
is
broad
based
and
can
quantify
a
range
of
AOB
species
including
N.
oligotropha
,
the
presumptive
AOB
species
at
this
WWTP
.
One
potential
problem
with
probes
and
primers
designed
to
target
AOB
16S
rDNA
is
that
they
may
hybridize
with
closely
related
non-AOB
species
resulting
in
false
positives
.
Indeed
,
BLAST
analysis
of
the
primers
and
probe
used
in
the
AOB
16S
rDNA
assay
indicated
that
one
of
the
degenerate
permutations
of
each
of
the
RT1r
and
TMP1
sequences
had
zero
mismatches
to
Ralstonia
eutropha
,
a
non-AOB
species
.
However
,
in
realtime
PCR
assays
,
3
oligonucleotides
(
2
primers
and
1
probe
)
must
bind
for
efficient
amplification
and
detection
,
so
the
effect
of
nonspecific
hybridization
by
one
primer
or
probe
is
reduced
.
The
amoA
gene
can
be
used
as
an
alternative
phylogenetic
marker
or
target
molecule
for
the
detection
of
AOB
(
28
)
.
The
amoA
gene
is
found
only
in
AOB
and
thus
serves
as
a
more
specific
marker
than
the
16S
rDNA
.
However
,
the
amoA
gene
has
higher
sequence
variability
between
AOB
than
the
16S
rDNA
,
thus
making
it
more
difficult
to
design
a
single
assay
to
detect
all
ammonia
oxidizing
bacteria
.
In
this
study
,
the
real-time
PCR
assay
was
designed
to
detect
N.
oligotrophalike
bacteria
because
it
was
the
only
AOB
previously
found
within
the
municipal
WWTP
used
in
this
study
(
9
)
.
The
primers
amoNo550D2f
and
amoNo754r
were
redesigned
from
the
previously
published
cPCR
primers
based
on
additional
sequence
information
obtained
from
amoA
clonal
libraries
made
to
MLSS
obtained
from
a
bioreactor
system
(
8
)
.
The
specificity
of
the
primer
pair
was
confirmed
by
additional
sequence
analysis
of
clonal
libraries
obtained
after
PCR
amplification
and
cloning
of
the
product
.
When
compared
to
the
cPCR
assay
,
the
real-time
PCR
with
the
redesigned
primers
detected
about
50-fold
more
amoA
copies
in
the
municipal
MLSS
samples
.
The
specificity
of
the
N.
oligotropha-like
amoA
real-time
PCR
assay
was
confirmed
by
the
lack
of
amplification
found
in
industrial
MLSS
samples
which
lack
N.
oligotropha
but
contain
N.
nitrosa
AOB
(
9
)
.
Because
the
AOB
16S
rDNA
assay
has
the
potential
to
produce
false
positives
and
the
N.
oligotropha
amoA
assay
has
the
potential
to
produce
false
negatives
,
the
use
of
the
two
assays
in
these
samples
provides
complimentary
data
for
the
detection
of
AOB
.
The
16S
rDNA
assay
and
the
amoA
assay
were
highly
correlated
and
both
assays
indicated
a
drop
in
AOB
during
August
,
September
,
and
October
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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