Kinetics, diffusional limitation and microscale distribution

of chemistry and organisms in a CANON reactor

Michael Nielsen a,*, Annette Bollmann a,1, Olav Sliekers b, Mike Jetten b,c,Markus Schmid b, Marc Strous c, Ingo Schmidt c, Lars Hauer Larsen d,

Lars Peter Nielsen a, Niels Peter Revsbech a

a Department of Microbiology, University of Aarhus, Ny Munkegade, Bldg. 540, 8000 Aarhus C, Denmarkb Department of Biotechnology, TU Delft, Julianalaan 67, nl2628 BC Delft, The Netherlands

c Department of Microbiology, University of Nijmegen, Toernooiveld 1, NL 6525 ED Nijmegen, The Netherlandsd Unisense A/S, Science Park, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark

Received 29 June 2004; received in revised form 31 August 2004; accepted 1 September 2004

First published online 21 September 2004

Abstract

In the Completely Autotrophic Nitrogen removal Over Nitrite (CANON) process, aerobic and anaerobic ammonia oxidizing

bacteria cooperate to remove ammonia in one oxygen-limited reactor. Kinetic studies, microsensor analysis, and fluorescence in situ

hybridization on CANON biomass showed a partial differentiation of processes and organisms within and among aggregates. Under

normal oxygen-limited conditions (�5 lM O2), aerobic ammonia oxidation (nitrification) was restricted to an outer shell (<100 lm)

while anaerobic ammonia oxidation (anammox) was found in the central anoxic parts. Larger type aggregates (>500 lm) accounted

for 68% of the anammox potential whereas 65% of the nitrification potential was found in the smaller aggregates (<500 lm). Anal-

ysis with O2 and NO�2 microsensors showed that the thickness of the activity zones varied as a function of bulk O2 and NO�

2 con-

centrations and flow rate.

� 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Keywords: CANON; Anammox; Nitrification; Microscale distribution; Kinetics; Diffusional limitation

1. Introduction

Nitrogen removal is one of the most important and

costly elements in wastewater treatment and is normally

carried out by sequential nitrification–denitrification

processes. Ammonium ðNHþ4 Þ is oxidized to nitrate

ðNO�3 Þ followed by a reduction of NO�

3 to free nitrogen

(N2). In the mid-1990s, an alternative process of nitro-

gen removal was described, the so-called anammox

process [1,2]. Under completely anoxic conditions

NHþ4 is oxidized with nitrite ðNO�

2 Þ as electron acceptor

to N2 and small amounts of NO�3 [3]. The anammox

process was first discovered in a wastewater treatment

plant in Delft, The Netherlands, and today anammox

activity has been reported from several other treatment

plants [4–8]. Recently the process has also been shown

to occur in nature in marine sediments and anoxic water

columns [9–11]. Anammox is carried out by autotrophic

bacteria belonging to the order Planctomycetales, like

Candidatus Brocadia anammoxidans and Candidatus

Kuenenia stuttgartiensis [4,12].

Since the very first description the focus has been on

optimization of the anammox process for wastewater

0168-6496/$22.00 � 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

doi:10.1016/j.femsec.2004.09.003

* Corresponding author. Tel.: +45 89423329.

E-mail address: mnielsen@biology.au.dk (M. Nielsen).1 Present address: Department of Biology, Northeastern Univer-

sity, 360 Huntington Avenue, Boston, MA 02115, USA.

www.fems-microbiology.org

FEMS Microbiology Ecology 51 (2005) 247–256

treatment. Compared to alternative treatment methods

the anammox process offers several advantages and is

especially suited for the treatment of high ammonia

wastewater [13,14]. However, to work optimally the

anammox process has to be supplied with an approxi-

mately 1:1 mixture of NHþ4 and NO�

2 . One approach

is partial nitrification of NHþ4 to NO�

2 in an aerated

reactor operated as a chemostat by the so-called SHA-

RON process [15,16]. In this reactor a combination of

short residence time and high temperature prevents the

growth of NO�2 oxidizing bacteria, and the effluent con-

taining NHþ4 and NO�

2 is subsequently fed to an anoxic

anammox reactor [13,17]. Recently, a new approach has

been described, the CANON reactor, where aerobic

ammonia oxidation and anammox are occurring within

the physical constrains of the same reactor [18–20]. This

reactor is operated at oxygen-limited conditions allow-

ing both aerobic and anaerobic processes at the same

time. Further oxidation of NO�2 to NO�

3 is prevented

by reactor operation at high NHþ4 concentration (5

mM) and oxygen-limited conditions. A nitrogen re-

moval efficiency of up to 85% has been reported [18].

However, in terms of wastewater treatment, the actual

nitrogen removal rate is crucial and dependent on O2

mass transfer efficiency from the gas to the liquid phase.

Using a gas-lift reactor with more efficient mass transfer

than a sequencing batch reactor (SBR) it is possible to

increase nitrogen removal rate from 0.07 to 1.5 kg N/

m3/day [19].

For future optimization of the CANON process, it is

important to obtain detailed information about kinetics,

diffusional limitation, and microscale distribution of

chemistry and organisms. We have obtained such infor-

mation by analysis of the reactor processes with micro-

and macroscale sensors for O2 and NO�2 . Microbial

aggregates were furthermore analyzed with fluorescence

in situ hybridization (FISH) so that the microdistribu-

tion of organisms could be compared with the microdis-

tribution of chemistry and processes.

2. Materials and methods

2.1. Mineral salt medium

For culturing and experiments a mineral salt medium

supplemented with different amounts of NHþ4 and NO�

2

has been used: 1.25 g/l KHCO3, 0.025 g/l KH2PO4, 0.3

g/l CaCl2 · 2H2O, 0.2 g/l MgSO4 · 7H2O, 6.25 mg/l

FeSO4, 6.25 mg/l EDTA and 1.25 ml/l trace elements

according to Sliekers et al. [18].

2.2. CANON reactor

The CANON reactor has been described in detail by

Sliekers et al. [18]. Briefly, it is a SBR with a working

volume of between 1 and 2 L, operated at 12 h cycles

including a 11.5 h filling period, a 0.25 h settling period,

and a 0.25 h draining period. Biomass in the reactor pri-

marily consisted of aggregates up to a size of �1.5–2 mm

in diameter and some biofilm on reactor walls. The reac-

tor was kept at a temperature of 30 �C. During the filling

period the system was stirred to keep the aggregates in

suspension. The reactor was fed with mineral salt medi-

um containing 9:4 mM NHþ4 , and oxygen was supplied

from a 91.8% Ar/8.2% O2 mixture with a bubbling rate

of 30.5 ml/min. During normal operation typical con-

centrations of O2;NHþ4 and NO�

2 in the reactor were

about 5 lM, 5 mM, and 45 lM, respectively.

2.3. NO�2 and O2 microsensors

NO�2 concentrations were measured with a new NO�

2

biosensor consisting of an immobilized culture of a

NO�2 -reducing=N2O-producing bacterial strain (Steno-

trophomonas nitritireducens) coupled to a Clark type

N2O-microsensor [21]. The sensor was constructed in

micro- and macroscale versions. At 30 �C the macro-

scale sensors had 90% response time of about 1–2 min,

a detection limit of about 1 lM NO�2 , and a linear range

up to 0:5–1:5 mM NO�2 . Microscale sensors were con-

structed with a tip diameter of about 30–35 lm and were

operated with a positive tip potential to enhance sensi-

tivity [22,23]. Operated at +0.4 V tip potential the detec-

tion limit was about 1 lM NO�2 and the range for linear

response was 0–0:25 mM NO�2 . The 90% response time

was 30–50 s. Two point in situ calibrations were used for

both types of biosensors. During our investigations of

the CANON reactor we had unexpected problems with

the long-term stability of the NO�2 biosensors as they be-

came sensitive also to NO�3 due to rapid bacterial con-

tamination. This problem that limited both the

duration of continuous measurements and the number

of replicates has subsequently been solved by addition

of tungstate to the biosensor interior (M. Nielsen,

unpublished).

Clark-type O2-microsensors equipped with guard

cathode [24] were used for microscale analysis of O2.

The sensors were constructed with tip diameters of

about 10 lm and had 90% response time <1 s. Bulk

O2 concentrations in the CANON reactor were meas-

ured with STOX (Switch able Trace amount OXygen)

sensors (Unisense, Aarhus, Denmark) with 90% re-

sponse time of about 5 s. The STOX sensor is a modified

Clark type O2 sensor with two front cathodes designed

for measurements of very low O2 concentrations down

to 0.01–0.02 lM. An electrically mediated activation/de-

activation of the cathode closest to the tip gives a very

precise in situ zero calibration and explains the very high

sensitivity of the sensor. The O2 sensors were two point

calibrated in air saturated reactor water and anoxic

alkaline ascorbic acid solution.

248 M. Nielsen et al. / FEMS Microbiology Ecology 51 (2005) 247–256

2.4. CANON reactor studies

On-line monitoring of NO�2 and O2 concentrations in

the CANON reactor was performed with macroscale

NO�2 biosensors and STOX sensors connected to a data

logger (ADC-16, Pico Technology). A sampling fre-

quency of 30 data points per hour was used. Experi-

ments with the CANON reactor were performed

during two experimental periods: 2001 and 2002 cam-

paigns. Identical reactors were used but with some var-

iation in biomass composition possibly due to a

difference in reactor operational lifetime. The first set

of measurements were performed on a reactor character-

ized by relatively large aggregates and with significant

wall-growth of biofilm while the second reactor had

been in operation for a shorter time and had relatively

smaller aggregates and less biofilm growth.

Oxygen perturbation experiments were performed on

the CANON reactor when operated as a SBR-reactor

(2001 campaign) and when operated as a 1.6 L batch

system (2002 campaign). A partial pressure of O2 in

the inflowing gas ranging between 5% and 90% was

used. To maintain a high ammonia concentration dur-

ing batch operation where the flow feed into the reactor

was stopped, NHþ4 was supplied manually at regular

intervals. At different oxygen supply rates complete 12

h cycles for O2 and NO�2 were recorded during SBR

operation, while steady state values for O2 and NO�2

were obtained during batch operation.

2.5. Mini-reactor studies

A mini reactor was used to determine the kinetic

characteristics of aerobic and anaerobic ammonia oxi-

dation of the CANON biomass. The mini reactor was

constructed as a closed system and consisted of a stop-

pered conical flask with a liquid phase of 110 ml and a

gas phase of �20 ml. Stirring of the system was accom-

plished with a magnetic stirring bar (4 cm long, 100 rpm)

and temperature was kept at 30 �C (±1 �C). The gas

composition of the headspace was controlled through

a valve. The concentrations of NO�2 and O2 in the bulk

liquid were monitored by insertion of a macroscale NO�2

biosensor and a microscale O2 sensor into the reactor.

Liquid with aggregates (110 ml) was retrieved from the

operating CANON reactor (2002 campaign) and sepa-

rated by sieving through a screen into aggregates <500

lm and >500 lm. Each of the two size fractions were

subsequently re-suspended into 110 ml of fresh mineral

salts medium.

Aerobic and anaerobic ammonia oxidizing activities

were determined from O2 and NO�2 consumption rates

during oxic and anoxic incubations, assuming that nitri-

fication and anammox were the only O2 and NO�2 con-

suming processes in the reactor. Aerobic and anaerobic

ammonia oxidizing bacteria have been shown to consti-

tute about 85% of the CANON biomass [18]. Further-

more, general lack of organic electron donors in the

reactor limits alternative O2 and NO�2 consuming proc-

esses. Oxygen was supplied to the reactor by bubbling

the liquid phase with pure O2, and the headspace was

subsequently flushed with N2. A correction factor to

compensate measured O2 consumption rates for O2 loss

at the gas/water interface was determined in a separate

experiment where there was no biomass in the mini reac-

tor. Nitrite was added to the anoxic reactor after bub-

bling the liquid phase with N2.

2.6. Microscale analysis

Analysis of the spatial distribution of metabolic proc-

esses in CANON aggregates was performed with

O2 and NO�2 microscale sensors in a flow chamber

(Fig. 1). The flow chamber was flushed with mineral

medium supplemented with 5 mM NHþ4 and either

30 or 100 lM NO�2 . The flow inside the chamber was

controlled with a peristaltic pump and the effluent from

the flow chamber was re-circulated to a 10 L medium

reservoir. The temperature was kept at 30 �C (±1 �C)

and the O2 concentration in the system was adjusted

with a mass flow controller (Model 5878, Brooks Instru-

ment B.V) mixing air with N2.

Larger aggregates (1–1.3 mm) were taken directly

from the CANON-reactor (2002 campaign) and placed

on a metal grid where they were exposed to a downward

flow. Two different flow settings were applied: �0.2 and

1 cm s�1. The flow field was not totally homogeneous, so

we can only indicate the approximate flow at the loca-

tion of the aggregate. With the sensor penetrating verti-

cally from above, profiles were made on the flow side of

Fig. 1. Schematic drawing of the flow chamber set-up used for

microscale analysis of NO�2 and O2 distribution in aggregates from the

CANON reactor. Arrows indicate direction of the water flow.

M. Nielsen et al. / FEMS Microbiology Ecology 51 (2005) 247–256 249

the aggregate. A pre-incubation period of 1 h was used

to create (semi-) steady state conditions. Concentration

profiles were recorded by introducing the sensors into

the aggregates at 50 lm steps using a micromanipulator.

The sensor signal was continuously recorded on a strip-

chart recorder. A dissection microscope was used to

determine the position of the aggregate/water interface.

Rates of O2 and NO�2 uptake by the granules were

calculated from the concentration gradients in the diffu-

sive boundary layer using Fick�s first law of diffusion,

J = �D(dC/dx), where J is the flux, D is the diffusion

coefficient for O2 (2.73 · 10�5 cm2 s�1, [25]) or NO�2

(2.02 · 10�5 cm2 s�1, [26]) in water at a temperature of

30 �C, and dC/dx is the concentration gradient.

2.7. Fluorescence in situ hybridization

Aggregates from the CANON reactor were fixed in

paraformaldehyde solution and cryosectioned as de-

scribed previously [27]. Fluorescence in situ hybridiza-

tions were performed according to Schmid et al. [4].

The probes used in this study were S-*-Neu-0653-a-A-

18 (NEU) for halophilic and halotolerant Nitrosomonas

spp. (N. europaea, N. eutropha, N. mobilis, N. halophila),

S-P-Betao-1225-a-A-20 for most betaproteobacterial

aerobic ammonia oxidizers and S-*-Amx-0820-a-A-22

for anammox bacteria (Candidatus ‘‘Kuenenia’’, Can-

didatus ‘‘Brocadia’’). Information about all probes can

be found in probeBase.net [28]. After hybridization

slides were washed briefly with ddH2O, air-dried and

embedded in Vectashield. For image acquisitions a Zeiss

Axioplan microscope (Zeiss, Jena, Germany) was used

together with the standard software packages delivered

with the instruments.

3. Results

3.1. CANON reactor studies

Recorded cycles of NO�2 and O2 during 12 h cycles in

the CANON reactor (Fig. 2) showed a consistent pat-

tern that was present at all oxygen supply rates exam-

ined. The O2 concentration in the reactor remained at

a stable level during the complete cycle whereas the

NO�2 concentration showed an initial increase followed

by a leveling off and a subsequent slow decrease.

Increasing the oxygen supply rate led to higher levels

of both O2 and NO�2 in the reactor. Compared to nor-

mal operational setting the highest supply rate tested re-

sulted in a 2.5-fold increase in bulk O2 concentration

and an almost 3-fold increase in NO�2 concentration

(Table 1). However, a saturation of either aerobic or

anaerobic ammonia oxidation was not observed, as such

saturation would have caused rising O2 or NO�2 concen-

trations during the cycle. Steady state concentrations for

O2 and NO�2 obtained during batch operation of the

reactor (Table 1) showed the same effect of enhanced

oxygenation. At steady state the NO�2 concentration

was about 7–10 times higher than the O2 concentration.

3.2. Mini-reactor studies

The O2 depletion rate was most rapid for the flasks

containing small aggregates (<500 lm) with initial rates

of 15–16 lmol L�1 min�1, while the rate for the large

0 3 6 9 12

0 3 6 9 12

0

50

100

150

ON

2–)

Mµ(

Time (h)

(a)

(b)

0

10

20

30

O2

)M

µ(

0

50

100

150

ON

2–)

Mµ(

0

10

20

30

O2

)M

µ(

Fig. 2. Concentrations of O2 (- - - -) and NO�2 (––) in the CANON

reactor during SBR operation with 12 h cycles at two different oxygen

supply rates: (a) 30.5 ml/min 16.4% O2/83.6% Ar; (b) 30.5 ml/min

32.8% O2/67.2% Ar.

Table 1

Concentrations of O2 and NO�2 in CANON reactors at different

oxygen supply rates

Campaign O2 (lM) NO�2 ðlMÞ

2002 0.59 7.8

2002 3.28 20.7

2002 5.46 37.8

2001a 5.66 45.3

2001 7.30 50.1

2001 14.17 125.2

Presented data are steady state values during batch operation (2002)

and reactor concentrations during SBR operation (2001) at time 6.9 h

(volume �1.6 L).a SBR operation at normal O2 supply rate.

250 M. Nielsen et al. / FEMS Microbiology Ecology 51 (2005) 247–256

aggregates (>500 lm) was about 8 lmol L�1 min�1 (Fig.

3(a)+(c), Table 2). It was difficult to estimate an appar-

ent Km value for the fraction consisting of the larger

aggregates due to the rather heterogeneous rate of de-

crease, but it was close (about 12 lM) to the apparent

Km value of 14 lM that could be estimated for the small

aggregates. The O2 concentrations observed in the CA-

NON reactor were always below or near these apparent

Km values (Table 1) and calculated from the slopes in

Fig. 3(c) the turnover time for oxygen in the reactor

was found to be about 1 min. The initial NO�2 depletion

rate under anoxic conditions was higher for the large

aggregates (5.1 lmol L�1 min�1) than for the small

aggregates (2.4 lmol L�1 min�1), and the apparent Km

value was also higher (23 lM) for the large aggregates

than for the small aggregates (14 lM) (Fig. 3(b)+(d),

Table 2). The larger difference in apparent Km for

NO�2 uptake indicates a distribution where annamox

bacteria are found within the anoxic central parts of

the aggregate, and where nitrite thus has a long diffusion

path before it reaches all active zones in large aggre-

gates. The largest (65%) aerobic ammonia oxidizing

capacity (O2 uptake) was located in the aggregates

<500 lm, whereas the largest (68%) anaerobic ammonia

oxidizing capacity ðNO�2 uptakeÞ was found for the

aggregates >500 lm (Table 2). This result shows a size

dependent skewed distribution of processes among

aggregate. Calculations of weight specific activity rates

yield an almost three times higher O2 uptake rate for

the smaller aggregates while NO�2 uptake rates where

about 40% higher for the larger sized aggregates as com-

pared to the small aggregates.

3.3. Microscale analysis

The microprofiles were performed under as realistic

conditions in terms of water flow, ambient chemistry,

and temperature as we could obtain. The applied flow

system made it possible to perform microscale analysis

at very low O2 concentrations (2–12 lM) comparable

to in situ reactor levels.

O2 and NO�2 profiles were determined in large (1.0–

1.3 mm diameter) aggregates with variation in external

O2 and NO�2 concentrations (Fig. 4). The profiles ob-

tained at O2 concentrations of 2 and 9 lM (Fig. 4(a)

0 10 20 30 400

40

80

120

ON

2–)

Mµ(

Time (min)

0 2 4 6 80

20

40

60

Time (min)

O2

)M

µ(

0 15 30 45 60

O2

Mµ(

nim

–1)

O2 (µM)

0

5

10

15

20

0 25 50 75 100 1250

2

4

6

8

ON

2–i

mM

µ(n–

1 )

NO2

– (µM)

(a) (b)

(d)(c)

Fig. 3. Kinetic experiments performed in mini reactors with CANON biomass separated into two size fractions: <500 lm (h) and >500 lm (s).

Decrease in O2 (a) and NO�2 (b) after addition of either substrate and calculated O2 (c) and NO�

2 (d) uptake rates as a function of substrate

concentrations.

Table 2

Kinetic characteristics of biomass from the CANON reactor with the

aggregates separated into size fractions <500 and >500 lm

<500 lm >500 lm Total

Vmax–O2 (lMmin�1) 15 8 23

V max–NO�2 (lMmin�1) 2.4 5.1 7.5

Apparent Km–O2 (lM) 14 12

Apparent Km–NO�2 (lM) 14 23

Aerobic ammonia oxidation (%) 65 35 100

Anaerobic ammonia oxidation (%) 32 68 100

Dry weight (g L�1) 0.255 0.385

Activity (lMO2 g�1 min�1) 58.8 20.8

Activity ðlMNO�2 g�1 min�1Þ 9.4 13.2

M. Nielsen et al. / FEMS Microbiology Ecology 51 (2005) 247–256 251

and (b)) represent ‘‘extremes’’ as compared to a concen-

tration of about 5 lM O2 during normal reactor opera-

tion. In both cases there was a spatial separation of

processes with the O2 reduction zone found in the upper

<100 lm and the NO�2 reduction zone starting at the

oxic/anoxic interface and penetrating to a depth of

250–300 lm. Elevation of the external NO�2 concentra-

tion from 30 to 100 lM (Fig. 4(c)) led to a very signifi-

cant increase in NO�2 penetration depth to about 550

lm. We did several replicate measurements at this high

NO�2 concentration, and some profiles showed complete

NO�2 penetration of the aggregates. There were, how-

ever, pronounced difficulties with these measurements,

as the CANON aggregates turned out to be extremely

tough to penetrate to greater depth with the relatively

thick (about 30–35 lm tip diameter) and relatively con-

ical NO�2 biosensors, and the insertion thus caused a

variable compression of the aggregate. The profiles

(Fig. 4(d)) measured at a lower flow rate (about 0.2

cm s�1 as compared to 1 cm s�1) deviate from the other

profiles by a 100 lm thicker (�250 lm) diffusive bound-

ary layer.

O2 and NO�2 uptake rates (Table 3) were strongly

influenced by bulk water concentrations and boundary

layer conditions. Significantly higher uptake rates were

found at elevated O2 concentrations with a 5-fold in-

crease in O2 uptake rate at 9 lM as compared to 2

lM. At similar high O2 concentration the NO�2 uptake

rate at 100 lM NO�2 was 180% of the uptake rate at

30 lM NO�2 . Increasing the boundary layer thickness

(a) (b)

(d)(c)

0 5 10 15

600

400

200

0

-200

-400

0 25 50 75 100

NO2 (µM)

O2 (µM)

0 5 10 15

600

400

200

0

-200

-400

0 10 20 30

NO2

(µM)

O2 (µM)

0 5 10 15

600

400

200

0

-200

-400

0 10 20 30

O2 (µM)

NO2 (µM)

0 5 10 15

600

400

200

0

-200

-400

NO2

– –

––

(µM)

O2 (µM)

0 10 20 30

Fig. 4. Profiles of O2 (h) and NO�2 ðsÞ in large type aggregates from the CANON reactor shown at 4 different experimental conditions: (a) low O2;

(b) high O2; (c) high NO�2 ; (d) low flow. The aggregate surface is represented as depth = 0.

Table 3

Rates of O2 and NO�2 uptake by large type CANON aggregates at different experimental conditions

Experimental condition Bulk O2 (lM) Bulk NO�2 (lM) O2 uptake (lmol cm�2 s�1) NO�

2 uptake (lmol cm�2 s�1)

Low O2 (Fig. 4(a)) 2 30 2.35 · 10�6 2.16 · 10�5

High O2 (Fig. 4(b)) 9 30 1.28 · 10�5 2.33 · 10�5

High NO�2 (Fig. 4(c)) 12 100 1.64 · 10�5 4.13 · 10�5

Low flow (Fig. 4(d)) 10 30 1.07 · 10�5 1.41 · 10�5

Uptake rates were calculated from concentration gradients in the diffusive boundary layer.

252 M. Nielsen et al. / FEMS Microbiology Ecology 51 (2005) 247–256

from about 150 lm to about 250 lm caused 20–40%

reduction in O2 and NO�2 uptake rates.

At the high O2 concentrations of 9 lM (Fig. 4(b)) the

O2 flux was about 1.28 · 10�5lmol cm�2 s�1 (Table 3).

This flux corresponds to a NO�2 production of

0.85 · 10�5lmol cm�2 s�1, assuming that all O2 was

used for aerobic ammonia oxidation. The NO�2 con-

sumption rate by anaerobic ammonia oxidation calcu-

lated from the NO�2 profile (Fig. 4(b)) was 2.33 · 10�5

lmol cm2 s1. Nitrification in the oxic region of the

aggregate thus supplied about 36% of the NO�2 used

for the anammox process in the deeper anoxic layers.

At the low O2 of 2 lM (Fig. 4(a)) the rate of O2 uptake

decreased to 2.3 · 10�6lmol cm�2 s�1 and nitrification

within the aggregate could thus only supply about

11% of the NO�2 uptake of 2.16 · 10�5

lmol cm�2 s�1.

It should be stressed that these experiments were done

in a flow-cell with fixed concentrations of

O2 and NO�2 . In the CANON reactor, the concentration

of NO�2 is strongly affected by the O2 concentration as

shown in Table 1. The conditions applied for the data

shown in Fig. 4(b) are, however, not far from actual

reactor conditions, and the data thus show that large

aggregates such as the one analyzed consume more

NO�2 than they produce.

3.4. Fluorescence in situ hybridization

Fluorescence in situ hybridisation indicated a distinct

distribution of aerobic ammonia oxidizing bacteria and

anaerobic ammonium oxidizing bacteria within the

aggregate (Fig. 5).While aerobic ammonia oxidizers were

located at the surface layer of the granule, anaerobic

ammonia oxidizers occupied most of the interior parts.

4. Discussion

4.1. Distribution of nitrification and anammox within

aggregates

The combination of reactor (Fig. 3), microsensor

(Fig. 4) and, FISH (Fig. 5) data show that aerobic

ammonia oxidation by N. europaea affiliated bacteria

was limited to an oxic <0.1 mm thin surface layer of

the CANON reactor aggregates, and that anaerobic

ammonia oxidation by anammox bacteria occurred in

the deeper anoxic layers. The CANON reactor granules

thus functioned according to the ‘‘magic bead concept’’

[29], where one aggregate can mediate two types of reac-

tions based on differences in chemical conditions at the

periphery and in the aggregate center. This pronounced

stratification of organisms and reactions is different

from the spatial distribution of organisms in a rotating

biological contractor operated at oxygen-limited condi-

tions analyzed by Pynaert et al. [7] where both aerobic

and anaerobic ammonia oxidizing bacteria were found

throughout the biofilm. The exact thickness of the nitri-

fying layer in the CANON reactor was governed by the

O2 concentration in the bulk liquid and by the degree of

turbulence (Fig. 4). O2 concentrations up to 11 lM,

which is about double the concentration measured in

the reactor under normal operational conditions, did

not saturate the O2 uptake of the nitrifying bacteria so

that oxygen only penetrated to about 0.1 mm depth

(Fig. 4). There thus seemed to be a large over-capacity

by the aerobic ammonia oxidizing bacteria under nor-

mal operational conditions that was also verified by

the O2 perturbation experiments with the CANON reac-

tor (Fig. 2). The microscale analysis performed at

100 lM NO�2 caused very deep NO�

2 penetration in

the aggregate (Fig. 4(c)) approaching full penetration,

and the maximum possible anaerobic ammonia oxidiz-

ing activity was apparently approached at this concen-

tration. This conclusion is also supported by the

experiment with small and large aggregates in mini reac-

tors (Fig. 3), where even the large aggregates exhibited

saturation at 100 lM NO�2 . The NO�

2 uptake kinetics

experiments conducted during the 2002 campaign (Fig.

3, Table 2) actually indicate that the high NO�2 concen-

trations of >100 lM measured in the CANON reactor

at 14 lM O2 during the 2001 campaign (Fig. 2(b))

should have caused reactor failure due to an inability

of the anammox biomass to reduce all the nitrite pro-

duced by aerobic ammonia oxidation. This absence of

reactor failure was probably due to an average larger

aggregate size and extensive wall growth of anammox

biomass in the intact reactor during 2001, as both situa-

tions would result in higher apparent Km values for NO�2

than the ones shown in Table 2.

Concentration profiles such as those shown in Fig. 4

may seem more defined than they actually are and the

Fig. 5. Fluorescence in situ hybridization of an aggregate from the

CANON reactor. Aerobic ammonia oxidizers appear purple, because

of a simultaneous hybridization of Neu (labeled with Cy3, red) and

NSO1225 (labeled with Cy5, blue). Green colour indicates anaerobic

ammonia oxidizing bacteria hybridized with probe Amx820.

M. Nielsen et al. / FEMS Microbiology Ecology 51 (2005) 247–256 253

aggregate surface is less well defined. Microscopy of

the aggregates showed a somewhat fluffy surface layer

where a visual definition of ‘‘surface’’ within ±50 lm

was difficult. It is consequently not possible to tell

whether the active zone is 50 or 100 lm thick. What

can be seen from the profiles is, however, that the dif-

fusive boundary layer is extremely important for the

mass transport of chemical species to the aggregate,

and that the chemical conditions in the active layers

of the aggregate are very different from the bulk condi-

tions. We, unfortunately, do not have comparable

O2 and NO�2 microsensor data from the normal opera-

tional conditions of 5–6 lM O2 and 45 lM NO�2 , but

the shown data for 9 lM O2 and 30 lM NO�2 (Fig.

4(b)) are sufficiently close to illustrate the point. At

these bulk concentrations, O2 within the nitrifying zone

varied between 0% and 50% of the bulk concentration,

and NO�2 in the zone with anammox activity varied be-

tween 0% and 25% of the bulk concentration. Micro-

profiles as shown in Fig. 4(b) are measured in an

immobilized aggregate exposed to a high flow rate of

about 1 cm s�1, and the free-floating aggregates in

the reactor are presumably exposed to less shear as

exemplified with the flow at about 0.2 cm s�1 (Fig.

4(d)). At low flow almost all the diffusion limitation

of O2 supply occurred in the diffusive boundary layer,

and even at the applied bulk O2 concentration of 12

lM, the O2 concentration experienced by the bacteria

in the outermost layers of the aggregate was only about

2–3 lM. It should be realized that the microgradients

within aggregates in a reactor are dynamic due to ran-

dom turbulences, collisions with other aggregates, etc.

However, the extremes in flow rate applied in Fig.

4(b) and (d) probably give a realistic representation

of the range in chemical gradients experienced within

the reactor. There were no O2 gradients in the bulk liq-

uid as shown by the constant signal from the only 50

lm thick O2 microsensor (Fig. 2).

The low O2 and NO�2 concentrations experienced by

the bacteria within the aggregates favor bacteria with

low Km values. Even for the diffusion-limited intact

aggregates, the experiments with mini reactors resulted

in apparent Km values of 12 lM for O2, and 14 lM

for NO�2 for the small aggregates (Table 2). For

non-diffusion-limited cells, the real Km values must

then be far lower. In another study with CANON

reactor biomass (O. Sliekers et al., unpublished re-

sults), a Km value as low as 2.3 lM O2 was found,

which is in line with our data. Other authors have also

found relatively low Km values for N. europaea [30,31].

The apparent Km value of 14 lM NO�2 for the small

aggregates, which were less diffusion-limited than the

large aggregates (apparent Km of 23 lM), indicate a

real Km < 10 lM NO�2 for anammox, which is compa-

rable with the Km of <7 lM reported by Strous et al.

[32].

4.2. Distribution of nitrification and anammox among

aggregates

Even at the highest oxygen concentration there was

no visible peak of nitrite within the nitrifying layers of

the analyzed large aggregates (Fig. 4). The concentra-

tion gradients thus indicated a NO�2 flux to the aggre-

gate from the bulk liquid, and the rate of aerobic

ammonia oxidation within these aggregates was conse-

quently lower than the rate of anaerobic ammonia oxi-

dation. Fluxes of O2 and NO�2 into the aggregate as

calculated from the data (Fig. 4, Table 3) show an al-

most 10-fold lower O2 as compared to NO�2 uptake at

2 lM O2. Even at an elevated O2 concentration of 9

lM, the NO�2 uptake was almost double that of the

O2 uptake. The theoretical ratio is an uptake of 1.5 O2

for each NO�2 produced and the imbalance must be

due to our selection of large aggregates only for micro-

sensor studies. The biological reactions in the small

aggregates were, however, investigated by the mini-reac-

tor studies (Table 2), where reactors were operated with

biomass <500 and >500 lm, respectively. This experi-

ment showed that the small aggregates, that only consti-

tuted 40% of the biomass, were responsible for roughly

two thirds of the overall aerobic ammonia oxidation

capacity (Vmax), while the opposite was the case for

anaerobic ammonia oxidation. A skewed distribution

of processes with respect to aggregate size was expected

because of the extreme O2 sensitivity of the anammox

bacteria [3]. Even the low bulk O2 concentration (about

5 lM) during normal reactor operation prevents anam-

mox activity in newly formed small aggregates, which

become purely aerobic ammonia oxidizing compart-

ments and thus only sources for nitrite. At a certain size

anoxic zones develop and the aggregate will support

anaerobic ammonia oxidation and growth of anammox

bacteria. As the aggregate gradually increases in size the

lower surface to volume ratio yields a progressively

higher anoxic fraction and the aggregate will eventually

transform to a net sink for nitrite. However, depending

on prevailing conditions the inner parts of the aggregate

will eventually become nitrite-limited as evidenced by

the microscale analysis (Fig. 4), and this diffusion limita-

tion probably prevents the formation of very large

aggregates. The distinct organization in the aggregates

of these immotile and slow growing bacteria with dou-

bling times of >7 h and >10 days for aerobic and anaer-

obic ammonia oxidizing bacteria, respectively [14,33],

also suggests that the aggregates must be stable over

very long time.

Partial differentiation of organisms and processes

among different sized aggregates may be of relevance

in the context of full-scale reactor control and stability.

Adjusting the efficiency of the sludge retention can be

used to control the relative ratios of aerobic and anaer-

obic ammonium oxidizing bacteria within the reactor.

254 M. Nielsen et al. / FEMS Microbiology Ecology 51 (2005) 247–256

Low biomass retention efficiency will select for larger

aggregates and promote anammox activity whereas a

high nitrification potential can be obtained by efficient

settling so that only very small aggregates leave with

the reactor effluent. By always keeping the anammox

potential higher than the nitrification potential the

occurrence of fatal nitrite build-up and following irre-

versible nitrite inhibition [32] of the anammox biomass

can be reduced.

4.3. Feed-back control of CANON reactors

This study has confirmed that significantly elevated

process rates in the sequencing batch CANON reactor

can be obtained by elevation of the oxygen supply.

Very high rates of up to 1.5 kg N/m3/day have thus

been obtained in a CANON gas lift reactor [19], where

the mass transfer between gas and liquid is more effi-

cient. Recent studies [20] showed that the CANON

biomass is very resilient against disturbances in waste-

water composition and by proper control the CANON

process may be a good alternative to existing nitrifica-

tion–denitrification systems for treatment of liquid

waste rich in ammonia. However, it may be critical

to have on-line information about the chemical condi-

tions within the reactor. A recent simulation study of a

CANON type biofilm system performed by Hao et al.

[34] showed that oxygen regulation according to varia-

tions in ammonia load was essential for optimal reac-

tor performance. The continuous threat of reactor

failure due to oxygen overloading and subsequent ni-

trite poisoning of the anammox biomass probably

necessitate information about NO�2 status. It may also

be relevant to have on-line information about nitrate

(e.g., [21]) as a warning system about increasing NO�3

concentration due to growth of NO�2 oxidizing bacte-

ria. This study also showed that size distribution and

amount of aggregates greatly affects steady state con-

centrations of O2 and NO�2 , and monitoring of both

parameters may be used to manage sludge retention

as discussed above.

Acknowledgements

We thank Preben Soerensen for technical assistance.

This study was performed as part of the Icon project un-

der the Fifth Framework Programme of the European

Commission (EVK1-CT-2000-00054).

References

[1] Mulder, A., Van de Graaf, A.A., Robertson, L.A. and Kuenen,

J.G. (1995) Anaerobic ammonium oxidation discovered in a

denitrifying fluidized-bed reactor. FEMS Microbiol. Ecol. 16,

177–183.

[2] Van de Graff, A.A., Mulder, A., Debruijn, P., Jetten, M.S.M.,

Robertson, L.A. and Kuenen, J.G. (1995) Anaerobic oxidation of

ammonium is a biologically mediated process. Appl. Environ.

Microbiol. 61, 1246–1251.

[3] Jetten, M.S.M., Strous, M., van de Pas-Schoonen, K.T., Schalk,

J., van Dongen, U.G.J.M., van de Graaf, A.A., Logemann, S.,

Muyzer, G., van Loosdrecht, M.C.M. and Kuenen, J.G. (1998)

The anaerobic oxidation of ammonium. FEMS Microbiol. Rev.

22, 421–437.

[4] Schmid, M., Wachtmann, U.T., Klein, M., Strous, M., Jure-

tschko, S., Jetten, M.S.M., Metzger, J.W., Schleifer, K.H. and

Wagner, M. (2000) Molecular evidence for genus level diversity of

bacteria capable of catalyzing anaerobic ammonium oxidation.

System. Appl. Microbiol. 23, 93–106.

[5] Egli, K., Ranger, U., Alvarez, P.J.J., Siegrist, H., van der Meer,

J.R. and Zehnder, A.J.B. (2001) Enrichment and characterization

of an anammox bacterium from a rotating biological contactor

treating ammonium-rich leachate. Arch. Microbiol. 175, 198–207.

[6] Helmer, C., Tromm, C., Hippen, A., Rosenwinkel, K.H.,

Seyfried, C.F. and Kunst, S. (2001) Single stage biological

nitrogen removal by nitritation and anaerobic ammonium oxida-

tion in biofilm systems. Water Sci. Technol. 43, 311–320.

[7] Pynaert, K., Smets, B.F., Wyffels, S., Beheydt, D., Siciliano,

S.D. and Verstraete, W. (2003) Characterization of an auto-

trophic nitrogen-removing biofilm from a highly loaded lab-scale

rotating biological contactor. Appl. Environ. Microbiol. 69,

3626–3635.

[8] Schmid, M., Walsh, K., Webb, R., Rijpstra, W.I.C., van de Pas-

Schoonen, K., Verbruggen, M.J., Hill, T., Moffett, B., Fuerst, J.,

Schouten, S., Damste, J.S.S., Harris, J., Shaw, P., Jetten, M.S.M.

and Strous, M. (2003) Candidatus ‘‘Scalindua brodae’’, sp nov.,

Candidatus ‘‘Scalindua wagneri’’ sp nov two new species of

anaerobic ammonium oxidizing bacteria. System. Appl. Micro-

biol. 26, 529–538.

[9] Dalsgaard, T., Canfield, D.E., Petersen, J., Tharmdrup, B. and

Acuna-Gonzalez, J. (2003) N-2 production by the anammox

reaction in the anoxic water column of Golfo Dulce, Costa Rica.

Nature 422, 606–608.

[10] Thamdrup, B. and Dalsgaard, T. (2002) Production of N-2

through anaerobic ammonium oxidation coupled to nitrate

reduction in marine sediments. Appl. Environ. Microbiol. 68,

1312–1318.

[11] Kuypers, M.M.M., Sliekers, A.O., Lavik, G., Schmid, M.,

Jorgensen, B.B., Kuenen, J.G., Damste, J.S.S., Strous, M. and

Jetten, M.S.M. (2003) Anaerobic ammonium oxidation by

anammox bacteria in the Black Sea. Nature 422, 608–611.

[12] Strous, M., Fuerst, J.A., Kramer, E.H.M., Logemann, S.,

Muyzer, G., van de Pas-Schoonen, K.T., Webb, R., Kuenen,

J.G. and Jetten, M.S.M. (1999) Missing lithotroph identified as

new planctomycete. Nature 400, 446–449.

[13] Jetten, M.S.M., Horn, S.J. and vanLoosdrecht, M.C.M. (1997)

Towards a more sustainable municipal wastewater treatment

system. Water Sci. Technol. 35, 171–180.

[14] Jetten, M.S.M., Wagner, M., Fuerst, J., van Loosdrecht, M.,

Kuenen, J. and Strous, M. (2001) Microbiology and application

of the anaerobic ammonium oxidation (‘‘anammox’’) process.

Curr. Opin. Biotechnol. 12, 283–288.

[15] Hellinga, C., Schellen, A.A.J.C., Mulder, J.W., van Loosdrecht,

M.C.M. and Heijnen, J.J. (1998) The SHARON process: an

innovative method for nitrogen removal from ammonium-rich

wastewater. Water Sci. Technol. 37, 135–142.

[16] Logemann, S., Schantl, J., Bijvank, S., van Loosdrecht, M.,

Kuenen, J.G. and Jetten, M.S.M. (1998) Molecular microbial

diversity in a nitrifying reacter system without sludge retention.

FEMS Microbiol. Ecol. 27, 239–249.

[17] Van Dongen, U., Jetten, M.S.M. and van Loosdrecht, M.C.M.

(2001) The SHARON ((R))–Anammox ((R)) process for treat-

M. Nielsen et al. / FEMS Microbiology Ecology 51 (2005) 247–256 255

ment of ammonium rich water. Water Sci. Technol. 44, 153–

160.

[18] Sliekers, A.O., Derworth, N., Gomez, J.L.C., Strous, M., Kue-

nen, J.G. and Jetten, M.S.M. (2002) Copletely autotrophic

nitrogen removal over nitrite in one single reactor. Water Res.

36, 2475–2482.

[19] Sliekers, A.O., Third, K.A., Abma, W., Kuenen, J.G. and Jetten,

M.S.M. (2003) CANON and Anammox in a gas-lift reactor.

FEMS Microbiol. Lett. 218, 339–344.

[20] Third, K.A., Sliekers, A.O., Kuenen, J.G. and Jetten, M.S.M.

(2001) The CANON system (competlely autotrophic nitrogen-

removal over nitrite) under ammonium limitation: interaction and

competition between three groups of bacteria. System. Appl.

Microbiol. 24, 588–596.

[21] Nielsen, M., Revsbech, N.P., Larsen, L.H. and Lynggard-jensen,

A. (2002) On-line determination of nitrite in wastewater treatment

by use of a biosensor. Water Sci. Technol. 45, 69–76.

[22] Larsen, L.H., Kjaer, T. and Revsbech, N.P. (1997) A microscale

NO3-biosensor for environmental applications. Anal. Chem. 69,

3527–3531.

[23] Kjaer, T., Larsen, L.H. and Revsbech, N.P. (1999) Sensitivity

control of ion-selective biosensors by electrophoretically mediated

analyte transport. Anal. Chim. Acta 391, 57–63.

[24] Revsbech, N.P. (1989) An oxygen microsensor with a guard

cathode. Limnol. Oceanogr. 34, 474–478.

[25] Broecker, W.S. and Peng, T.H. (1974) Gas-exchange rates

between air and sea. Tellus 26, 21–35.

[26] Li, Y.H. and Gregory, S. (1974) Diffusion of ions in sea-water and

in deep-sea sediments. Geochim. Cosmochim. Acta 38, 703–714.

[27] Lee, N., Nielsen, P.H., Andreasen, K.H., Jureschko, S., Nielsen,

J.L., Schleifer, K.H. and Wagner, M. (1999) Combination of

fluorescent in situ hybridization and microautoradiography – a

new tool for structure–function analysis in microbial ecology.

Appl. Environ. Microbiol. 65, 1289–1297.

[28] Loy, A., Horn, M. and Wagner, M. (2003) ProbeBase – an online

resource for rRNA-targeted oligonucleotide probes. Nucl. Acid

Res. 31, 514–516.

[29] dos Santos, V.A.M.P., Bruijnse, M., Tramper, J. and Wijffels,

R.H. (1996) The magic-bead concept: an integrated approach to

nitrogen removal with co-immobilized micro-organisms. Appl.

Microbiol. Biotechnol. 45, 447–453.

[30] Laanbroek, H.J. and Gerards, S. (1993) Competition for limiting

amounts of oxygen between Nitrosomonas-europaea and Nitrob-

acter-winogradskyi grown in mixed continuous cultures. Arch.

Microbiol. 159, 453–459.

[31] Laanbroek, H.J., Bodelier, P.L.E. and Gerards, S. (1994) Oxygen-

consumption kinetics of Nitrosomonas-europaea and Nitrob-

acter-hamburgensis grown in mixed continues cultures at different

oxygen concentrations. Arch. Microbiol. 161, 156–162.

[32] Strous, M., Kuenen, J.G. and Jetten, M.S.M. (1999) Key

physiology of anaerobic ammonium oxidation. Appl. Environ.

Microbiol. 65, 3248–3250.

[33] Prosser, J.I. (1989) Autotrophic nitrification in bacteria. Adv.

Microb. Physiol. 30, 125–181.

[34] Hao, X., Heijnen, J.J. and Van Loosdrecht, M.C.M. (2002)

Model-based evaluation of temperature and inflow variations on

a partial nitrification-ANAMMOX biofilm process. Water Res.

36, 4839–4849.

256 M. Nielsen et al. / FEMS Microbiology Ecology 51 (2005) 247–256