decay nitrification

8/17/2019 Decay Nitrification

1/11

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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 Dü bendorf,

Switzerland

a r t i c l e i n f o

Article history:

Received 15 July 2005

Received in revised form

17 April 2006

Accepted 18 April 2006

Available online 6 June 2006

Keywords:

Activated sludge

Decay

Enzyme kinetics

Membrane bioreactor

Modeling

Nitrification

A B S T R A C T

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

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

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

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

different oxidiation–reduction potential conditions and membrane separation on the

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

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

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

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

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

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

needed 1–2 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 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). Safeguard-

ing the plant’s 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 oxidation–reduction 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 nitrifi-

cation 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 nitrite-

oxidizing 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

ARTICLE IN PRESS

0043-1354/$ - see front matter &

2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.watres.2006.04.019

Corresponding author. Tel.: +411 823 5054; fax: +41 1 823 5389.E-mail addresses: [email protected] (R. Manser), [email protected] (H. Siegrist).

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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 micro-

organisms. In an engineering context, therefore, decay should

adequately describe the loss of biomass ( ¼ 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 sequen-

cing batch reactor (SBR).

Previous studies merely provide kinetic data on autotrophic

decay rates in activated sludge systems combining nitrifica-

tion 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 me-

chanisms and processes that are lumped under the name of

‘‘decay’’. The WERF report summarizes three different experi-

mental 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 ammonifi-

cation 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

activity (endogenous respiration). Measurement of the de-

crease of organic matter is insufficiently sensitive for

municipal activated sludge, because autotrophic biomass

accounts for only about 2–4% 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 measure-

ment 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 d1)

and 2 (0.56m3 d1) population equivalents, respectively. The

oxygen concentration in the aerobic tanks was controlled

between 2.5 and 3gm3. 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 reten-

tion 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 condi-

tions), only stirred (decay at anoxic conditions) or a combina-

tion of both for a period of 7–10 days (Fig. 1). For the anoxic

experiment, nitrate (NaNO3) was manually added in order to

prevent anaerobic conditions. Autotrophic bacteria are ob-

ligate aerobic and cannot gain ATP under anoxic conditions.

The reactor was therefore aerated once a day for 5min in

order to prevent any effects of a complete lack of ATP. An

experiment with alternating stirred (8h) and aerated (16h)

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phases simulating a typical wastewater treatment plant with

33% anoxic volume was conducted in order to investigate a

possible effect of the feast–famine phenomenon on decay

(Lee and Oleszkiewicz, 2003). At intervals of one day, one liter

of activated sludge was withdrawn from the batch reactor andcentrifuged at 3000 rpm for 7 min. The pellet was subse-

quently 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 g N m3 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, 6–8 g N m3) and ammonia (NH4Cl, 10–18g N 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 g O2 m3. OURs (OURi) werecalculated 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.

OURHET;m ¼

PnOURHET;i

n , (1)

OURmax;NOB ¼

PnOURNOB;i

n OURHET;m, (2)

OURmax;AOB ¼

P

n

OURAOB;in OURmax;NOB OURHET;m. (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 samplesfrom 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 (Inter-

activa 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 equippedwith HQ-CY3 and HQ-FLUOS filters (both from Analysentech-

nik AG, Tu ¨ bingen, 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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0

100

200

300

400

500

600

700

0 60 120 180

time (min)

O

U R ( g O 2 m - 3 d

- 1 )

2

3

4

5

6

7

8

D O

( g O 2 m - 3 )

T = 20±0.2°C

V = 50 l

pH = 7.5±0.1

T = 20±0.1°C

V = 1 l

pH = 7.5±0.1

CentrifugationEffluent MBR

Pellet

DO

NO2 C

BA

NH4

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 Target organisms Reference

NEUa

Most halophilic and halotolerant ammonia oxidizers in the beta-subclass of Proteobacteria Wagner et al. (1995)NmII Many members of the Nitrosomonas communis lineage Pommerening-Ro ¨ ser et al. (1996)

Nmo218 Many members of the Nitrosomonas oligotropha lineage Gieseke et al. (2001)

Nso1225 Most known ammonia oxidizers in the beta-subclass except N. mobilis Mobarry et al. (1996)

a Used with an unlabeled competitor as indicated in the reference.

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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 non-

linear 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. f XI 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 g COD g N1 for AOB and NOB, respec-

tively. 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.

3. Results

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 repro-

duced by the adapted ASM3 (Table 2), the sharp loss of

autotrophic activity during the first two days of the experi-

ment does not seem to be consistent with the underlying

model processes. Thus, data points at t ¼ 0 were omitted for

the estimation of the parameters listed in Table 3. Experi-ments 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

ARTICLE IN PRESS

Table 2 – Adaptation of ASM3 to reproduce the observeddelay of the ammonia release

Process Process rateequation r

Aerobic endogenous respiration of XH bH;aerSO

KOþSO

tt0 f lag þðtt0Þ

XH

XH ¼ heterotrophic bacteria. f lag ¼ fit parameter. t ¼ simulation

time, t0 ¼ initial time.

0

200

400

600

800

0 2 4 6 8

time (d)

0

50

100

150

200

O U R N O B

( g O 2 m - 3 d

- 1 )

AOB

NOB

heterotrophic

0

2000

4000

6000

0 4 6 8

time (d)

C O D ( g O 2 m - 3 )

0

50

100

150

S N O 3 ( g N

m - 3 )

COD

SNO3

O U R A O B , O U R H E T ( g O 2 m - 3 d

- 1 )

2

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.

Table 3 – Estimated aerobic endogenous respiration rates(with standard errors) at 20 1C using the adapted ASM3

AOB NOB Heterotrophic

bAOB,aerobic (d1) bNOB,aerobic (d

1) bHET,aerobic (d1)

CAS 0.1570.02a 0.1570.01a 0.2870.05a

MBR 0.1470.01 0.1470.01 0.2370.03

a Mean value from two experiments.

W AT E R R E S E A R C H 40 (2006) 2416– 2426 2419


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

endogenous respiration rates of autotrophic bacteria were

0.02d1 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 condi-

tions (nitrite concentrations were not measured).

3.3. Decay under alternating anoxic–aerobic conditions

The results of the alternating anoxic–aerobic 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 d1).

3.4. Enzyme dynamics

Enzyme processes (activation, synthesis, degradation, inhibi-

tion) 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

10min 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

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0

100

200

300

400

500

0 4 8

time (d)

0

30

60

90

120

150

O U R N O B

( g O 2 m - 3 d -

1 )

0

1000

2000

3000

4000

5000

0 4 6 8

time (d)

C O D ( g O 2 m - 3 )

0

20

40

60

80

100

S N O 3 ( g N

m - 3 )

O U R A O B , O U R H E T ( g O 2 m - 3 d - 1 )

2 62

AOB

NOB

heterotrophic

SNO3

COD

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.

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

AOB NOB HeterotrophicbAOB,anoxic (d

1) bNOB,anoxic (d1) bHET,anoxic (d

1)

CAS 0.01570.004 o0.001 0.03370.002

MBR 0.0170.003 0.0270.009 0.06470.002

0

5

10

15

20

0 4 8

time (d)

N.oligotropha

N.communis

N.europaea

Sum AOB

a b u n d a n c

e ( 1 0 3 µ m 3 m m f l o c - 2 )

2 6

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

in the aerobic experiment using FISH.

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ARTICLE IN PRESS

0

200

400

600

800

0 4 6 8

time (d)

0

50

100

150

200

O U R N O B

( g O 2 m - 3 d

- 1 )

heterotrophic

AOB

NOB

O U R A O B , O

U R H E T ( g O 2 m - 3 d

- 1 )

0

1000

2000

3000

4000

5000

0 4 8

time (d)

C O

D ( g O 2 m - 3 )

0

20

40

60

80

100

S

N O 3 ( g N

m - 3 )

COD

SNO3

2 2 6

Fig. 5 – Results of batch experiment for CAS sludge under alternating anoxic–aerobic 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 anoxic–aerobic experiment at 20 1C using

the adapted ASM3

AOB NOB Heterotrophic

bAOB,aerobic (d1) bAOB,anoxic (d

1) bNOB,aerobic (d1) bNOB,anoxic (d

1) bHET,aerobic (d1) bHET,anoxic (d

1)

CAS 0.1570.01 0.01a 0.2270.01 0.01a 0.2770.02 0.0670.004

MBR 0.1370.01 0.01a 0.1870.01 0.01a 0.2370.02 0.1070.01

a Mean value derived from anoxic experiment.

0.0

0.2

0.4

0.6

0.8

1.0

0 4 8

time (d)

E n z y m e s a t u r a t i o n A O B

0.0

0.2

0.4

0.6

0.8

1.0

0 12 18 24

time (h)

E n z y m e s a t u r a t i o n A O B

0

200

400

600

800

1000

0 30 60 90 120 150

time (min)

O U R ( g O 2 m - 3 d

- 1 )

activation

~ 10min

enzyme

synthesiscell

growth

6 2 6

aerobicanoxic-aerobicanoxic

(A) (B) (C)

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 Esat Process rate (r)

Enzyme synthesis of XAOB 1 ksynthSNH

KNH;AOBþSNH

SOKO;AOBþSO

SALKKALK;AOBþSALK

ð1 EsatÞ

Aerobic enzyme decay of XAOB 1 kdecaySO

KO;AOBþSOEsat

Growth of XAOB — mm;AOBSNH

KNH;AOBþSNH

SOKO;AOBþSO

SALKKALK;AOBþSALK

XAOBEsat

Esat ¼ Enzyme saturation (–).

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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:

ksynth ¼ 30 4 d1

; kdecay ¼ 3:0 0:3 d1.

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 one-

step 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

30–50% reduction of these parameters under anoxic com-

pared 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 waste-

water characteristics and community compositions of auto-

trophic 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

feast–famine 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

anoxic–aerobic conditions.

Our experiments confirm previous studies (Lee and Olesz-

kiewicz, 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 influ-

enced 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 feast–famine

phenomenon—stress and damage during the fasting period

and therefore enhanced substrate removal rates for repair

processes during the feasting period—resulting in lower

decay rates under alternating anoxic–aerobic 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 die—unless they are

exposed to toxic compounds, for instance—but rather be-

come dormant (Kaprelyants and Kell, 1996). However, preda-

tion 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 under-

standing 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 contin-

uous 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 in-

stance, 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 5–10 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 1–2 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

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ammonia due to heterotrophic decay causes the AOB to

maintain a minimal level of enzyme saturation. Our esti-

mated 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 two-

step nitrification were applied:

Model A: ASM3 default. Anoxic autotrophic

decay ¼ 50% of aerobic decay.Model B: Anoxic decay ¼ 0. No autotrophic decay

under anoxic conditions.

Model C: 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 ¼ 0:88 (Fig. 8) in order to exclude

the effect of an overall reduced growth

rate.

A CAS with three completely mixed reactors was assumed.

The hydraulic retention time was chosen to be 12 h. The

temperature was set to 151C. 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

reactors was fixed at 2.5 g O2 m3, 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 dy-

namics leads to underestimating the ammonia concentra-

tions 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 differ-

ences 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 ob-

viously 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

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0

50

1000

1500

2000

time (d)

0 0.5 1

0.0

0.2

0.4

0.6

0.8

0.0

0.1

0.2

0.3

0 0.2 0.4 0.6 0.8 1

time (d)

0.0

0.2

0.4

0.6

0.8

0.0

0.1

0.2

0.3

0 0.2 0.4 0.6 0.8

time (d)

Mod. A: ASM3 default

Mod. B: anoxic decay = 0

Mod. C: incl. enzyme kinetics

T = 15°C

Influent variation

N H 4 l o a d ( g N

d - 1 )

N O 2 ( g N

m - 3 )

N H 4 ( g

N

m - 3 )

1

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

0.6

0.7

0.8

0.9

0 0.2 0.4 0.6 0.8

time (d)

e n z y m e s a t u r a t i o n E s a t ( - )

1

fully aerobic

33% anoxic

Fig. 8 – Diurnal variation of the enzyme saturation Esat of

AOB (Model C).

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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 auto-

trophic 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 micro-

bial 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 anox-

ic–aerobic conditions was detected on the autotrophic

decay rates due to the feast–famine phenomenon.

3. AOB and NOB exhibited similar endogenous respiration

rates. Since the literature data on the decay rates of two-

step nitrification are scarce and inconsistent, further

research are needed to provide evidence on this issue.

4. AOB needed 1–2 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 radio-

labeled 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 sum-

marized 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 g N m3) as determined by respirometry. The activated

sludge was consecutively spiked with nitrate (NaNO3) and the

influence on the OUR was monitored. The nitrite concentra-

tion was always non-limiting (between 4 and 8 g N m3) and

inhibition due to free nitrous acid should be negligible

(Wiesmann, 1994). OUR were measured between 3 and

4 g O2 m3. 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 g N m3 leads to a 20% reduction and about 120g N 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,

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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