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40 (2006) 2416 2426

Available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/watres

Decay processes of nitrifying bacteria in biological

wastewater treatment systems
Reto Manser, Willi Gujer, Hansruedi Siegrist
Swiss Federal Institute of Aquatic Science and Technology (Eawag) and Swiss Federal Institute of Technology (ETH), CH-8600 Dubendorf,
Switzerland

ar t ic l e i n f o

A B S T R A C T

Article history:

A knowledge of the decay rates of autotrophic bacteria is important for reliably modeling

Received 15 July 2005

nitrification in activated sludge plants. The introduction of nitrite to activated sludge

Received in revised form

models also requires the separate determination of the kinetics of ammonia- and nitrite-

17 April 2006

oxidizing bacteria. Batch experiments were carried out in order to study the effects of

Accepted 18 April 2006

different oxidiationreduction potential conditions and membrane separation on the

Available online 6 June 2006

separate decay of these bacteria. It was found that decay is negligible in both cases under

Keywords:

anoxic conditions. No significant differences were detected between the membrane and

Activated sludge

conventional activated sludge. The aerobic decay of these two types of bacteria did not

Decay

diverge significantly either. However, the measured loss of autotrophic activity was only

Enzyme kinetics

partly explained by the endogenous respiration concept as incorporated in activated sludge

Membrane bioreactor

model no. 3 (ASM3). In contrast to nitrite-oxidizing bacteria, ammonia-oxidizing bacteria

Modeling

needed 12 h after substrate addition to reach their maximum growth rate measured as a

Nitrification

maximum OUR. This pattern could be successfully modeled using the ASM3 extended by
enzyme kinetics. The significance of these findings on wastewater treatment is discussed
on the basis of the extended ASM3.
& 2006 Elsevier Ltd. All rights reserved.

1.

Introduction

Autotrophic decay rates have a significant impact on the

nitrification performance and therefore on the design and
analysis of wastewater treatment plants (WWTP). Safeguarding the plants performance against wash-out or overload
depends directly on these kinetic parameters. Furthermore,
the extension of the biological treatment steps from COD
removal alone to nitrification, denitrification and biological
phosphorus removal exposes bacteria to different conditions
of oxidationreduction potential (ORP). These appear to
influence the decay rates (Lee and Oleszkiewicz, 2003; Siegrist
et al., 1999; Martinage and Paul, 2000). On the other hand,
most activated sludge models (e.g. ASM3) summarize nitrification as a single process. As a consequence, nitrite

concentrations cannot be modeled and the kinetics of AOB

and NOB are lumped into a single parameter set. However,
elevated effluent concentrations of nitrite may occur, causing
problems in the receiving water due to its toxicity. System
optimization or operation aspects may also benefit from a
detailed modeling of the nitrite dynamics in activated sludge
systems. An extension of the model to two-step nitrification
is then required, involving the separate determination of the
kinetics of ammonia-oxidizing bacteria (AOB) and nitriteoxidizing bacteria (NOB).
Decay is a general term with distinct meanings depending
on the context. From a microbiological point of view, decay
implies maintenance, endogenous respiration, degradation of
enzymes or lysis due to adverse environmental conditions
(Van Loosdrecht and Henze, 1999). In the maintenance

Corresponding author. Tel.: +41 1 823 5054; fax: +41 1 823 5389.

E-mail addresses: reto@picason.ch (R. Manser), siegrist@eawag.ch (H. Siegrist).

0043-1354/$ - see front matter & 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2006.04.019

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process, an external substrate is used to keep the current

biological activity going, but the amount of bacteria is not
altered. The endogenous respiration concept involves the
consumption of the cell-internal substrate, which leads to a
loss of activity and slightly reduced biomass. Similarly,
enzyme degradation causes a loss of activity, but this is
rapidly restored if substrate becomes available. In contrast to
the previous processes, lysis leads to death and breaking
apart of the cells and therefore to a loss of bacteria. From a
process engineering point of view, decay describes the loss of
microbial activity in general. In state-of-the-art models (such
as ASM), the activity is directly proportional to the biomass
concentration and the loss of biomass is considered as a
single process. It is important to recall that the primary
objective of activated sludge models is to model nutrient
concentrations and not the number and state of the microorganisms. In an engineering context, therefore, decay should
adequately describe the loss of biomass ( loss of activity)
and was expressed as a single process due to the lack of
experimental data. In wastewater treatment systems, loss of
activity is probably due to a combination of several of the
mechanisms mentioned above. Predation by protozoa may
also play a major role (Van Loosdrecht and Henze, 1999;
Martinage and Paul, 2000), but is hidden in the decay
processes of bacteria in current activated sludge models.
Moussa et al. (2005) presented an extended version of ASM 2d
with two-step nitrification, predation and maintenance: it
was successfully applied to the modeling of a pilot sequencing batch reactor (SBR).
Previous studies merely provide kinetic data on autotrophic
decay rates in activated sludge systems combining nitrification as one process (summarized in Martinage and Paul,
2000). Wiesmann (1994) listed the same decay rates for both
AOB and NOB at 20 1C. Due to their different temperature
dependence, NOB has a higher decay rate than AOB at
temperatures below about 20 1C and a lower one above about
20 1C (Hellinga et al., 1998; Wyffels et al., 2004). By contrast,
Morgenroth et al. (2000) found the amount of NOB reduced
more greatly than the AOB after a starvation period over
several days at 23 1C using fluorescent in-situ hybridization
(FISH). It has to be considered that the technique used for the
determination of decay rates has a significant impact on their
values (Martinage and Paul, 2000). Different methods have
been proposed to experimentally study these various mechanisms and processes that are lumped under the name of
decay. The WERF report summarizes three different experimental setups like SBR (or fill-and-draw) test, high F/M batch
test and continuous wash-out test (WERF, 2003). On top of
these methods, there is the respirometry-based method
which was used in this study. Activated sludge is placed in
a batch reactor and maximum oxygen utilization rates (OUR)
are regularly determined by adding small amounts of
substrate. Alternatively, substrate utilization rates can be
determined instead of OUR, with the advantage that the
influence of heterotrophic bacteria can be neglected. Under
aerobic conditions, autotrophic growth caused by ammonification must be taken into account. Both methods only
provide information about available autotrophic activity; no
discrimination can be made between the reduction in the
amount of bacteria (lysis or predation) and the reduction of

2417

activity (endogenous respiration). Measurement of the decrease of organic matter is insufficiently sensitive for
municipal activated sludge, because autotrophic biomass
accounts for only about 24% of the organic matter. In
contrast to previous methods, biomolecular techniques (e.g.
FISH) target active single cells. However, uncertainties in the
quantification hamper the identification of decay rates
directly from FISH measurements.
In summary, only little and inconsistent data are available
on the separate decay rates of AOB and NOB. Possible
differences between AOB and NOB may contribute to a better
understanding of nitrite dynamics. Furthermore, many
authors reported diminished decay rates under anoxic
conditions. However, the data from the literature vary
significantly and the exact reason for this decrease is not
yet known. Although the methods for the assessment of
autotrophic decay rates are mainly based on the measurement of the overall activity, the impact of heterotrophic
activity, wastewater characteristics or the treatment process
may explain the different results.
The goal of this study was to determine the decay rates of
both AOB and NOB in municipal wastewater treatment
systems. The influence of the ORP conditions and the
treatment process (conventional and membrane bioreactor,
MBR) was also examined. The technique presented by Copp
and Murphy (1995) for the determination of decay rates using
respirometry was slightly modified. The underlying processes
are discussed and consequences for modeling of activated
sludge systems are presented on the basis of the results.

2.

Materials and methods

2.1.

Activated sludge samples

Samples were taken from a conventional activated sludge

(CAS) system and a MBR pilot plant (Manser et al., 2005c),
which were operated in parallel to treat domestic wastewater
following a typical diurnal hydraulic variation. The CAS and
the MBR treated wastewater corresponding to 60 (18 m3 d
1)
and 2 (0.56 m3 d
1) population equivalents, respectively. The
oxygen concentration in the aerobic tanks was controlled
between 2.5 and 3 g m
3. Because the coarse bubble aeration
induced a cross-flow at the membrane surface, the oxygen
concentration was temporarily higher in the MBR. Both plants
were operated with pre-denitrification and the sludge retention time (SRT) was maintained at 20 days.

2.2.

Batch experiments

Activated sludge samples were placed in a batch reactor that

was either continuously aerated (decay under aerobic conditions), only stirred (decay at anoxic conditions) or a combination of both for a period of 710 days (Fig. 1). For the anoxic
experiment, nitrate (NaNO3) was manually added in order to
prevent anaerobic conditions. Autotrophic bacteria are obligate aerobic and cannot gain ATP under anoxic conditions.
The reactor was therefore aerated once a day for 5 min in
order to prevent any effects of a complete lack of ATP. An
experiment with alternating stirred (8 h) and aerated (16 h)

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Centrifugation
OUR (gO2 m-3 d-1)

Effluent MBR
Pellet
DO
T = 200.2C
T = 200.1C

pH = 7.50.1

pH = 7.50.1

NO2 NH4

700
600
500
400
300
200
100
0

8
7
6

B
A

5
4
3

0
V = 50 l

60

V=1l

120

DO (gO2 m-3)

2418

2
180

time (min)

Fig. 1 Experimental setup for batch experiments. A base oxygen utilization rate (heterotrophic rate), B max. nitratation
rate, C max. nitritation rate.

Table 1 Oligonucleotide probes and hybridization conditions applied in this study

Probe
NEUa
NmII
Nmo218
Nso1225
a

Target organisms

Reference

Most halophilic and halotolerant ammonia oxidizers in the beta-subclass of Proteobacteria

Many members of the Nitrosomonas communis lineage
Many members of the Nitrosomonas oligotropha lineage
Most known ammonia oxidizers in the beta-subclass except N. mobilis

Wagner et al. (1995)

Pommerening-Roser et al. (1996)
Gieseke et al. (2001)
Mobarry et al. (1996)

Used with an unlabeled competitor as indicated in the reference.

phases simulating a typical wastewater treatment plant with

33% anoxic volume was conducted in order to investigate a
possible effect of the feastfamine phenomenon on decay
(Lee and Oleszkiewicz, 2003). At intervals of one day, one liter
of activated sludge was withdrawn from the batch reactor and
centrifuged at 3000 rpm for 7 min. The pellet was subsequently re-suspended with effluent from the MBR (permeate)
and placed in a secondary batch reactor. The main purpose of
the centrifugation step was to provide low nitrate (ammonia
for anoxic experiment) concentrations for the measurements,
because nitrate concentrations of only 50 gN m
3 were found
to lead to a 20% reduction of the nitratation rate for the same
activated sludge (Appendix). After determining the base OUR
(Fig. 1: A), samples were successively spiked with nitrite
(NaNO2, 68 gN m
3) and ammonia (NH4Cl, 1018 gN m
3),
leading to maximum nitratation (Fig. 1: B) and nitritation
rates (Fig. 1: C). The dissolved oxygen (DO) concentration was
controlled between 3 and 4 gO2 m
3. OURs (OURi) were
calculated as the slopes of the measured DO by linear
regression (Fig. 1). The maximum OURs (OURmax) represent
mean values of at least four single OURs (Eq. (1)(3)). All
experiments were carried out at pH 7.5 and 20 1C. The pH was
controlled using HCl and NaHCO3.
P

OURHET;i
,
n
P
OURNOB;i

OURHET;m ,
n
n
P
OURAOB;i

OURmax;NOB
OURHET;m .
n
n

OURHET;m
OURmax;NOB
OURmax;AOB

(1)

(2)
(3)

2.3.

Fluorescence in situ hybridization (FISH)

Samples were taken at maximum OUR of AOB and fixed in 4%

paraformaldehyde as described by Amann et al. (1990).
Ultrasonification (35 kHz, 5 min) was applied to fixed samples
from the CAS prior to hybridization in order to break up large
flocs. In situ hybridizations of cells were performed with
fluorescently labeled rRNA-targeted oligonucleotide probes
according to Manz et al. (1992) (Table 1). Parallel hybridization
with the Nso1225 probe targeting most known AOB showed
that the applied probes covered the vast majority of the AOB
(data not shown). Oligonucleotide probes were obtained from
Microsynth (Balgach, Switzerland) and Thermo Hybaid (Interactiva Division, Ulm, Germany).
All samples hybridized with oligonucleotide probes were
embedded in Citifluor (Citifluor, Canterbury, United Kingdom)
prior to microscopic observation. Epifluorescence microscopy
was performed on an Olympus BX50 microscope equipped
with HQ-CY3 and HQ-FLUOS filters (both from Analysentechnik AG, Tubingen, Germany). The nitrifying bacteria were
quantified according to Manser et al. (2005b).

2.4.

Modeling

The activated sludge model No. 3 (ASM3, Gujer et al., 1999)

was extended with two-step nitrification to enable separate
modeling of ammonia and nitrite oxidation (Table A1).
Denitrification of nitrite and from nitrate to nitrite was not
included. The loss of activity associated with a loss of bacteria
is modeled as a single process (endogenous respiration) in the
ASM3. The endogenous respiration of heterotrophic bacteria

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4 0 (200 6) 241 6 242 6

is a lumped process accounting for the OUR due to growth on

decay products, growth on very slowly degradable COD and
probably also the respiration of protozoa (Van Loosdrecht and
Henze, 1999). In the following, heterotrophic OUR (OURHET,m)
represents the base OUR measured before the addition of
substrate (Fig. 1: B, Eq. (1)), the nitratation rates (OURmax,NOB)
after addition of nitrite (Fig. 1: B, Eq. (2)) and the nitritation
rates (OURmax,AOB) after addition of ammonia and complete
enzyme synthesis (Fig. 1: C, Eq. (3)). During the aerobic
experiments, large amounts of organic nitrogen are released,
probably due to the endogenous respiration of heterotrophic
bacteria, hydrolyzed to ammonium and immediately nitrified
to nitrate. Since this study focuses on nitrifying bacteria and
in order not to extend the model structure of the ASM3, the
heterotrophic endogenous respiration process was adapted
slightly in order to better fit the accumulation of nitrate. A
term considering the observed lag phase of the nitrate
accumulation was therefore introduced for modeling the
aerobic experiments (Table 2) and the nitrogen content of the
bacteria was assumed to be 10% referred to the COD.
All simulations and parameter estimations using a nonlinear least-squares algorithm were performed with the
AQUASIM software package (Reichert, 1994). It was assumed
that all kinetic parameters remain unchanged during the
course of the experiments. A sensitivity analysis showed that
other kinetic parameters, e.g. yield coefficients were not
sensitive on the estimation of the decay rates. Correlations
with other parameters of the extended ASM3 (e.g. fXI or iNXI)
are negligible. The default kinetic parameters provided by
Koch et al. (2000) were therefore used. Applied yield coeffi-

cients were 0.21 and 0.03 gCOD g
1

N for AOB and NOB, respectively. bHET,aer and XHET,ini were estimated from the nitrate
measurements, whereas the initial autotrophic biomass
concentrations XAOB,ini and XNOB,ini were estimated together
with the decay rates of bAOB and bNOB, respectively.

Table 2 Adaptation of ASM3 to reproduce the observed

delay of the ammonia release

Table 3 Estimated aerobic endogenous respiration rates

(with standard errors) at 20 1C using the adapted ASM3

3.1.

Decay under aerobic conditions

The results of the aerobic experiment for sludge from the CAS
are shown in Fig. 2. The activity is reduced to about 60% of the
initial value after 7 days for both AOB and NOB. Despite the
fact that nitrate accumulation and the subsequent growth of
autotrophic bacteria on the released nitrogen is well reproduced by the adapted ASM3 (Table 2), the sharp loss of
autotrophic activity during the first two days of the experiment does not seem to be consistent with the underlying
model processes. Thus, data points at t 0 were omitted for
the estimation of the parameters listed in Table 3. Experiments with sludge from the CAS and MBR yielded similar
results. The quantification of the major groups of AOB using
FISH confirmed the sharp reduction in activity at the
beginning of the experiment (Fig. 3).

3.2.

Decay under anoxic conditions

The results of the anoxic experiment for sludge from the CAS
are shown in Fig. 4. The loss of activity is negligible for both

Process rate
equation r
bH;aer K

SO
t
t0
O SO f lag t
t0

CAS
MBR

XH

XH heterotrophic bacteria. flag fit parameter. t simulation

time, t0 initial time.

200

600

150

NOB

400

100
AOB

200

50

NOB
bNOB,aerobic (d
1)

Heterotrophic
bHET,aerobic (d
1)

0.1570.02a
0.1470.01

0.1570.01a
0.1470.01

0.2870.05a
0.2370.03

Mean value from two experiments.

6000
OURNOB (gO2 m-3 d-1)

800

AOB
bAOB,aerobic (d
1)

150
SNO3

4000

100
COD

2000

50

SNO3 (gN m-3)

Aerobic endogenous respiration of XH

OURAOB, OURHET (gO2 m-3 d-1)

Results

COD (gO2 m-3)

Process

3.

heterotrophic
0

0
0

4
6
time (d)

0
0

4
6
time (d)

Fig. 2 Results of batch experiment for CAS sludge under aerobic conditions at 20 1C and pH 7.5. The lines represent the best
fit obtained by applying the adapted ASM3. Experiments with MBR sludge yielded similar trends.

ARTICLE IN PRESS
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40 (2006) 2416 2426

AOB and NOB over a period of one week. Estimated anoxic

endogenous respiration rates of autotrophic bacteria were
0.02 d
1 or lower for both CAS and MBR sludge (Table 4).
Heterotrophic endogenous respiration rates were estimated
from the measured nitrate concentrations (denitrification
was only attributed to the anoxic endogenous respiration of
heterotrophic bacteria). It was assumed that nitrite did not
accumulate during denitrification under COD limiting conditions (nitrite concentrations were not measured).

Decay under alternating anoxicaerobic conditions

The results of the alternating anoxicaerobic experiment for

sludge from the CAS are shown in Fig. 5. As in the aerobic
experiment, the ASM3 had to be adapted in order to model
the lag phase observed in the nitrate measurements (Table 2).
The estimated aerobic endogenous respiration rates of AOB
and NOB are shown in Table 5, assuming mean anoxic rates
estimated from the anoxic experiment (0.01 d
1).

N.oligotropha

N.europaea

N.communis

Sum AOB

abundance (103 m3 mmfloc-2)

20

15

Enzyme dynamics

Enzyme processes (activation, synthesis, degradation, inhibition) are comparatively fast and therefore cannot account for
the loss of autotrophic activity at the beginning of the aerobic
experiments. Enzyme activation is the fastest mechanism for
regulating a process and can be stopped or reactivated within
minutes, depending on the presence of an inhibitor. Our
measurements showed constant activation times of about
10 min for nitritation (Fig. 6A). The subsequent enzyme
synthesis took between 1 and 2 h. In contrast, the NOB
reached their maximum OUR immediately. The enzyme
saturation for every measurement was calculated as the ratio
of the OUR after the activation time and the maximum OUR
after complete enzyme synthesis. Cell growth during the
phase of enzyme synthesis was neglected in this calculation.
As shown in Fig. 6B, enzyme saturation was reduced to about
60% of its maximum level after 6 h under aerobic conditions
followed only by a marginal decline. Enzyme degradation was
more enhanced under aerobic than under anoxic conditions
(Fig. 6C). The ASM3 (with already implemented two-step
nitrification) was extended by enzyme kinetics adapted from
Wild et al. (1995) in order to test its sensitivity on the
ammonia and nitrite dynamics in wastewater treatment
systems (Table 6).
In ASM3, the growth rate of XAOB has to be multiplied by the
degree of cell saturation with nitritation enzymes. Esat is
proportional to the actual total amount of enzymes divided by
the actual biomass of XAOB. It is assumed that the anoxic

10
Table 4 Estimated anoxic endogenous respiration rates
(with standard errors) at 20 1C using the ASM3

0
0

4
time (d)

AOB

400

120

NOB
300

90

200

60
heterotrophic

100

30

0
0

6
4
time (d)

NOB
bNOB,anoxic (d
1)

Heterotrophic
bHET,anoxic (d
1)

0.01570.004
0.0170.003

o0.001
0.0270.009

0.03370.002
0.06470.002

5000

150

100
COD

4000
COD (gO2 m-3)

500

OURNOB (gO2 m-3 d-1)

OURAOB, OURHET (gO2 m-3 d-1)

Fig. 3 Quantification of ammonia-oxidizing bacteria (AOB)

in the aerobic experiment using FISH.

CAS
MBR

AOB
bAOB,anoxic (d
1)

80

3000

60

2000

40
SNO3

1000

SNO3 (gN m-3)

3.3.

3.4.

20

0
0

4
6
time (d)

Fig. 4 Results of batch experiment for CAS sludge under anoxic conditions at 20 1C and pH 7.5. The lines represent the best
fit obtained by applying the ASM3. Experiments with MBR sludge yielded similar trends.

ARTICLE IN PRESS

150

AOB
100

400
heterotrophic

50

200

COD (gO2 m-3)

NOB

600

5000

100

4000

80
COD

3000

4
6
time (d)

60

2000

40

SNO3

1000

20

SNO3 (gN m-3)

200

800

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4 0 (200 6) 241 6 242 6

OURNOB (gO2 m-3 d-1)

OURAOB, OURHET (gO2 m-3 d-1)

WAT E R R E S E A R C H

0
0

6
4
time (d)

Fig. 5 Results of batch experiment for CAS sludge under alternating anoxicaerobic conditions at 20 1C and pH 7.5. The lines
represent the best fit obtained by applying the adapted ASM3. AOB and NOB activity during anoxic periods was omitted for
better readability. Experiments with MBR sludge yielded similar trends.

Table 5 Estimated endogenous respiration rates (with standard errors) from the anoxicaerobic experiment at 20 1C using
the adapted ASM3
AOB

Heterotrophic

bAOB,aerobic (d
1)

bAOB,anoxic (d
1)

bNOB,aerobic (d
1)

bNOB,anoxic (d
1)

bHET,aerobic (d
1)

bHET,anoxic (d
1)

0.1570.01
0.1370.01

0.01a
0.01a

0.2270.01
0.1870.01

0.01a
0.01a

0.2770.02
0.2370.02

0.0670.004
0.1070.01

CAS
MBR

OUR (gO2 m-3 d-1)

1000
800
600

enzyme
synthesis

400

activation
~ 10min

200

cell
growth

0
0

30

(A)

60
90
time (min)

120

150

1.0
0.8
0.6
0.4
0.2
0.0
0

(B)

12
18
time (h)

24

Enzyme saturation AOB

Mean value derived from anoxic experiment.

Enzyme saturation AOB

NOB

1.0
0.8
0.6
0.4

aerobic
anoxic-aerobic
anoxic

0.2
0.0
0

(C)

6
4
time (d)

Fig. 6 Illustration of enzyme activation and synthesis (AOB) for a single measurement as a possible explanation of the
observed OUR pattern (A). Short-term degradation of enzymes (AOB) under aerobic conditions (B). Long-term degradation of
enzymes (AOB) during decay experiments (C). The enzyme saturation was calculated as a ratio of the OUR after the activation
time and the maximum OUR after complete enzyme synthesis. The lines represent the best fit obtained using the ASM3
extended by enzyme kinetics (Table 6). Data from experiments with conventional activated sludge are shown.

Table 6 Stoichiometric matrix and process rate equations (adapted from Wild et al., 1995).
Process
Enzyme synthesis of XAOB

Process rate (r)

Esat
1

ksynth

Aerobic enzyme decay of XAOB

1

kdecay

Growth of XAOB

mm;AOB

Esat Enzyme saturation ().

SO
SNH
SALK
KNH;AOB SNH KO;AOB SO KALK;AOB SALK
SO
KO;AOB SO

1
Esat

Esat

SO
SNH
SALK
KNH;AOB SNH KO;AOB SO KALK;AOB SALK

XAOB Esat

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decay of nitritation enzymes is negligible. The data presented

in Fig. 6B and several data sets from batch experiments
(sample shown in Fig. 6A) were used to estimate the unknown
parameters ksynth and kdecay. The values obtained at 20 1C are
as follows:

1

ksynth 30  4 d ;

1

kdecay 3:0  0:3 d .

The maximum enzyme saturation is less than 1 due to the

continuous decay of the enzymes. With the estimated
parameters, the maximum achievable enzyme saturation is
0.9 at 20 1C. The temperature dependency is assumed to be
equal to the growth of the AOB.

4.

Discussion

4.1.

Decay rates

The values of the estimated aerobic autotrophic endogenous

respiration rates correspond to the literature data for onestep nitrification (summarized in Martinage and Paul, 2000).
No differences were found between AOB and NOB. The
aerobic heterotrophic endogenous respiration rates agreed
well with the values used for modeling in previous studies
(Gujer et al., 1999; Koch et al., 2000). No shift was observed
within the AOB population during the experiment. After two
days, a significant amount of AOB had either fallen into a
dormant state (below the detection limit of FISH) or had been
grazed by protozoa.
The values of the estimated anoxic autotrophic endogenous
respiration rates contrast with previous studies (Lee and
Oleszkiewicz, 2003; Siegrist et al., 1999) that stated only a
3050% reduction of these parameters under anoxic compared to aerobic conditions. In comparison to previous
studies, our experimental setup provided measurements that
always used the same matrix (sludge washed with effluent of
the MBR) and daily aerobic periods of about 10 min. Nitrate
inhibition is a possible reason for the observed difference (see
Appendix). Additionally, starvation and OUR measurements
were carried out in the same reactor in the study of Siegrist et
al. (1999) leading to extended aerobic periods and increasing
decay, which were not considered. Finally, different wastewater characteristics and community compositions of autotrophic bacteria may also play a role here (Martinage and
Paul, 2000).
Bacteria are exposed to alternating ORP conditions in
WWTP with nutrient removal. Since nitrifying bacteria are
obligate aerobes, they are unable to store or utilize their
substrate under anoxic conditions due to the lack of oxygen.
As a result, stress and damage to their metabolisms may
occur under anoxic conditions, leading to increased substrate
requirements during the aerobic period, also known as the
feastfamine phenomenon (Chen et al., 2001). In contrast to
the completely aerobic experiment, the measured decrease of
autotrophic activity could be reasonably modeled using the
adapted ASM3 (Fig. 5). While endogenous respiration rates of
AOB and heterotrophic bacteria from the aerobic experiment
(Table 3) were confirmed, aerobic endogenous respiration
rates of NOB and anoxic endogenous respiration rates
of heterotrophic bacteria are higher. The results suggest

that NOB are more stressed than AOB under alternating

anoxicaerobic conditions.
Our experiments confirm previous studies (Lee and Oleszkiewicz, 2003; Siegrist et al., 1999; Martinage and Paul, 2000)
showing that ORP conditions (aerobic or anoxic) are crucial
for the loss of microbial activity. The extent of the reduction
of microbial activity under anoxic conditions may be influenced by the wastewater characteristics, the microbial
community composition or operational parameters. The floc
structure of the activated sludge does not seem to have an
impact on the decay, since our results exhibit no difference
between a CAS (large flocs) and a MBR (small flocs, Manser et
al., 2005a) operated at the same sludge age. The feastfamine
phenomenonstress and damage during the fasting period
and therefore enhanced substrate removal rates for repair
processes during the feasting periodresulting in lower
decay rates under alternating anoxicaerobic conditions as
previously reported (Lee and Oleszkiewicz, 2003) was not
observed in our study. However, the authors neglected
autotrophic growth on released ammonia. Therefore, their
estimated decay rate possibly does not only reflect decay but
also growth of bacteria leading to a lower observed decay rate.
We found similar decay rates for AOB and NOB, which is in
agreement with Wiesmann (1994). Different factors probably
affect the decay of bacteria. From a microbiological point of
view, it is likely that most bacteria do not dieunless they are
exposed to toxic compounds, for instancebut rather become dormant (Kaprelyants and Kell, 1996). However, predation may play a major role in activated sludge systems (Van
Loosdrecht and Henze, 1999). Studies on modeling predation
in activated sludge systems are a first step towards understanding their impact on wastewater treatment (Moussa
et al., 2005). But it remains uncertain as to how protozoa affect
nitrifiers, because the latter prefer to grow in dense aggregates
(Wagner et al., 1995). Furthermore, the transferability of the
decay parameters measured in batch experiments to continuous flow systems has not been addressed so far. Since small
amounts of substrates are always available in activated sludge
plants, the decay pattern in continuous flow systems may be
different. In this regard, in situ measurement of the decay of
radio-labeled nitrifiers in continuous flow systems, for instance, would avoid the change to batch conditions.

4.2.

Enzyme dynamics

Enzyme dynamics control the short-term change of biomass

activity. Vanrolleghem et al. (2004) already examined the
transient response of heterotrophic activated sludge activities
to sudden changes in substrate concentration. Although a
transient time of only 510 min was measured, the authors
concluded that consideration of the transience phenomenon
is important for correct data interpretation and plays a role in
plants with highly fluctuating loads.
The time needed after activation until the AOB reached
their maximum OUR was 12 h, which is in agreement with
the literature data for enzyme synthesis (Aoi et al., 2004;
Sayavedra-Soto et al., 1996). Although the AOB were able to
continuously nitrify small amounts of released ammonia,
enzyme degradation was more enhanced under aerobic
conditions (Fig. 6C). Nonetheless, the continuous release of

ARTICLE IN PRESS

ammonia due to heterotrophic decay causes the AOB to

maintain a minimal level of enzyme saturation. Our estimated half-life times of the order of hours agree with the
literature data (Sayavedra-Soto et al., 1996; Aoi et al., 2004).
The two new processes allow reasonable reproduction of the
measured values (Fig. 6). Only the long-term degradation
during the aerobic decay experiment is underestimated by
the model (Fig. 6C), but this should not play a role in the
practical operation of WWTP.

4.3.

Significance for wastewater treatment

A simulation study was carried out in order to test the

sensitivity of the reduction of anoxic autotrophic decay and
the enzyme kinetics to the nitrification process. Three
different model configurations based on the ASM3 with twostep nitrification were applied:
Model A:

ASM3 default. Anoxic autotrophic

decay 50% of aerobic decay.
Anoxic decay 0. No autotrophic decay
under anoxic conditions.
Including enzyme kinetics. Model B
extended by enzyme kinetics for AOB
(Table 6). Maximum growth rate of AOB
(mm,AOB) was increased by a factor of
1=Esat;max 0:88 (Fig. 8) in order to exclude
the effect of an overall reduced growth
rate.

Model B:
Model C:

A CAS with three completely mixed reactors was assumed.

The hydraulic retention time was chosen to be 12 h. The
temperature was set to 15 1C. A typical diurnal influent
variation of the ammonia load for municipal wastewater
was used (Fig. 7).
The sensitivity of the model predictions was investigated
on two common plant layouts (Fig. 7): fully aerobic and with a
33% anoxic volume. The DO concentration in the aerobic

Mod. A: ASM3 default

Mod. B: anoxic decay = 0
Mod. C: incl. enzyme kinetics

NH4 (gN m-3)

0.8

2000
NO2 (gN m-3)

NH4 load (gN d-1)

Influent variation
1500
1000
50

reactors was fixed at 2.5 gO2 m
3, whereas the aerobic sludge
age was set to 6 d in both layouts.
The results reveal that enzyme kinetics has an impact on
the ammonia concentrations in the effluent for typical
diurnal variation in the influent. Neglecting enzyme dynamics leads to underestimating the ammonia concentrations for fully aerobic and partly anoxic plants. Anoxic
conditions diminish the diurnal variation of the enzyme
saturation of AOB (Fig. 8, only aerobic degradation of enzymes
assumed), leading to a slightly lower ammonia peak in the
effluent (Fig. 7). Nonetheless, the enzyme saturation is always
above 75% even under fully aerobic conditions for the chosen
characteristics of the domestic influent. However, the differences are within the measurement uncertainty of ammonium
and could be compensated by, for example, a 10% increase in
the maximum growth rate of AOB. Pre-denitrification obviously decreases the nitrification performance in Model A
due to the loss of nitrifiers within the anoxic reactor, whereas
Model B (without anoxic autotrophic decay) predicts equal

0.9

0.8

0.7

fully aerobic
33% anoxic

0.6
0

0.2

0.5
time (d)

0.4
0.6
time (d)

0.8

Fig. 8 Diurnal variation of the enzyme saturation Esat of

AOB (Model C).

0.8

T = 15C

0.6

0.6

0.4

0.4

0.2

0.2

0.0

0.0

0.3

0.3

0.2

0.2

0.1

0.1
0.0

0.0

2423

4 0 (200 6) 241 6 242 6

enzyme saturation Esat (-)

WAT E R R E S E A R C H

0.2

0.4 0.6
time (d)

0.8

0.2

0.4
0.6
time (d)

Fig. 7 Simulated concentrations of ammonia and nitrite in the effluent of the third reactors.

0.8

ARTICLE IN PRESS

XSTO
KO
SNO3
O SO KNO3 SNO3

bSTO;ano K

XHET
b

KO
SNO3
O SO KNO3 SNO3
SO
STO;aer K S
STO
O O

bHET;ano K

fXI

XHET
SO
O SO

bHET;aer K

1

1

1

1

XHET

1

2:86

1
f XI
2:86

1

2:86

1
f XI
2:86

NOB

1
f XI
2:86
YNOB

1
f XI
2:86

1
f XI
2:86

iNBM
iNXI f XI

Anoxic endogenous respiration of XSTO

Aerobic endogenous respiration of XSTO

1

1
f XI

iNBM
iNXI f XI

iNBM

iNBM
iNXI f XI

1:14
YNOB
Y NOB

iNBM
iNXI f XI

3:43
YAOB
YAOB

Y

AOB

iNBM

1
Y AOB

1

SN2
SNO2
SNH
SO

Y

Anoxic endogenous respiration of XHET

Fig. A1 Relative nitratation rates (measured as maximum

OUR from three batch experiments) at different nitrate
concentrations using activated sludge from the
conventional pilot plant.

iNBM
iNXI f XI

350

Aerobic endogenous respiration of XHET

300

iNBM
iNXI f XI

NO3-N (gN

250

m-3)

Anoxic endogenous respiration of XNOB

200

1
f XI

150

Aerobic endogenous respiration of XNOB

100

50

Growth of XNOB

Anoxic endogenous respiration of XAOB

0%

1
f XI

20%

Aerobic endogenous respiration of XAOB

40%

60%

Growth of XAOB

Exp. 1
Exp. 2
Exp. 3

80%

Table A1 Extension of ASM3 with two-step nitrification

relative nitratation rate

100%

SNO3

Batch experiments using activated sludge from a conventional and a MBR pilot plant were performed in order to study
the decay of AOB and NOB. It was found that:
1. The loss of autotrophic activity was only partly explained
by the endogenous respiration concept as incorporated in
activated sludge model no. 3 (ASM3), for instance. While
the lumped model process of endogenous respiration
reasonably accounts for the overall reduction of autotrophic activity in activated sludge plants, accurate
modeling of isolated decay experiments probably requires
the introduction of processes that are mechanistically
more correct. It must also be considered that activated

1
f XI
2:86

Conclusions

Process

5.

fXI

fXI

1

1

1

fXI

SO
SNO2
NOB
O SO KNO2 SNO2
SO
NOB;aer K S
NOB
O O
KO
SNO3
NOB;ano K S
NOB
O O KNO3 SNO3

b
fXI

mNOB;m K

X
b

fXI

1

XSTO

XAOB

XNOB

XI

Process rate equation r

ammonia effluent concentrations compared to the fully

aerobic plant layout. Interestingly, the combined effect of
no anoxic decay and enzyme kinetics (Model C) equals the
effect of only a reduced anoxic decay (Model A) and leads to
almost identical concentrations of ammonia effluent for the
plant with pre-denitrification. Although Model C is more
correctly based on the experiments in mechanistic terms, the
effects of no anoxic decay and enzyme kinetics may be
reasonably lumped together into a reduced anoxic decay rate
for modeling municipal wastewater treatment with nitrogen
removal. In contrast, the effluent nitrite concentrations are
barely influenced by either anoxic decay or the enzyme
dynamics of AOB. Since nitrite production and consumption
in the denitrification process is not part of the model, the
results need to be verified with a model including denitrification over nitrate and nitrite. As a summary, it has to be
mentioned that simple state of the art models like ASM3
already reasonably allow for modeling of most municipal
WWTP where more complex models do not provide an
additional benefit. However, model extensions in the context
of this paper may lead to a better understanding of ammonia
dynamics for plants with high-fluctuating influent loads or
large anoxic volumes.

SO
SNH
AOB
O SO KNH SNH
SO
AOB;aer K S
AOB
O O
KO
SNO3
AOB;ano K S
AOB
K
S
O O NO3 NO3

40 (2006) 2416 2426

mAOB;m K

WAT E R R E S E A R C H

2424

ARTICLE IN PRESS
WAT E R R E S E A R C H

4 0 (200 6) 241 6 242 6

sludge samples were taken from continuous flow systems

and analyzed in batch systems, where other mechanisms
may occur.
2. Anoxic conditions significantly reduce the loss of autotrophic activity. The large range of this reduction from 30
to almost 100% found in different studies (Lee and
Oleszkiewicz, 2003; Siegrist et al., 1999; this study) is
partly due to the different experimental setups. It cannot
be excluded that this reduction may be influenced by
wastewater characteristics, the composition of the microbial community and operational parameters. The floc
structure of the activated sludge does not play a role, since
no differences were found between the conventional
activated (large flocs) and membrane activated (small
flocs) sludge. No additional impact of alternating anoxicaerobic conditions was detected on the autotrophic
decay rates due to the feastfamine phenomenon.
3. AOB and NOB exhibited similar endogenous respiration
rates. Since the literature data on the decay rates of twostep nitrification are scarce and inconsistent, further
research are needed to provide evidence on this issue.
4. AOB needed 12 h after substrate addition to reach their
maximum growth rate measured as a maximum OUR.
This pattern could be successfully modeled using the
ASM3 extended by enzyme kinetics. The extension of
activated sludge models by enzyme kinetics for AOB may
lead to a better understanding of ammonia dynamics in
WWTP with highly fluctuating influent loads.
Further research is needed to elucidate the processes involved
in the loss of autotrophic activity in activated sludge systems.
In particular, in situ measurement of the decay of radiolabeled nitrifiers in continuous flow systems, for example,
would avoid the change to batch conditions. The parallel
measurement of protozoa activity (e.g. Moussa et al., 2005)
would provide additional insights into the processes summarized under decay.

Appendix
This appendix gives the results from batch experiments on
the nitrate inhibition of NOB. Fig. A1 shows the measured
decrease of the maximum nitratation rate in relation to the
initial maximum nitratation rate (nitrate concentration
E5 gN m
3) as determined by respirometry. The activated
sludge was consecutively spiked with nitrate (NaNO3) and the
influence on the OUR was monitored. The nitrite concentration was always non-limiting (between 4 and 8 gN m
3) and
inhibition due to free nitrous acid should be negligible
(Wiesmann, 1994). OUR were measured between 3 and
4 gO2 m
3. All experiments were carried out at pH 7.5 and
16 1C. Three batch experiments were performed within one
month using activated sludge from the conventional pilot
plant (see Section 2.1) (Table A1).
The results show that a nitrate concentration of only
50 gN m
3 leads to a 20% reduction and about 120 gN m
3 to
as much as a 50% reduction in the nitratation rate. Analysis of
the community composition revealed that the NOB were
dominated by members belonging to the genus Nitrospira,

2425

which seems to be a typical K-strategist (Schramm et al.,

1999). This may explain the inhibition already observed at
moderate nitrate concentrations.

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