water research 43 (2009) 4599–4609

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Distributions and activities of ammonia oxidizing

bacteria and polyphosphate accumulating organisms
in a pumped-flow biofilm reactor

Guangxue Wua,b, Michael Nielsena,*, Ketil Sorensena, Xinmin Zhana, Michael Rodgersa,b
a
Department of Civil Engineering, National University of Ireland, Galway, Ireland
b
National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland

article info abstract

Article history:
Received 14 January 2009
Received in revised form
3 July 2009
Accepted 7 July 2009
Published online 3 August 2009
Keywords:
Pumped-flow biofilm reactor
Microbial distributions
Ammonia oxidizing bacteria
Polyphosphate accumulating
organisms
Fluorescence in situ hybridization
Micro-sensors
The spatial distributions and activities of ammonia oxidizing bacteria (AOB) and poly-
phosphate accumulating organisms (PAOs) were investigated for a novel laboratory-scale
sequencing batch pumped-flow biofilm reactor (PFBR) system that was operated for carbon,
nitrogen and phosphorus removal. The PFBR comprised of two 16.5 l tanks (Reactors 1 and 2),
each with a biofilm module of 2 m2 surface area. To facilitate the growth of AOB and
PAOs in the reactor biofilms, the influent wastewater was held in Reactor 1 under stagnant
un-aerated conditions for 6 h after feeding, and was then pumped over and back between
Reactors 1 and 2 for 12 h, creating aerobic conditions in the two reactors during this period;
as a consequence, the biofilm in Reactor 2 was in an aerobic environment for almost all the
18.2 h operating cycle. A combination of micro-sensor measurements, molecular tech-
niques, batch experiments and reactor studies were carried out to analyse the performance
of the PFBR system. After 100 days operation at a filtered chemical oxygen demand (CODf)
loading rate of 3.46 g/m2 per day, the removal efficiencies were 95% CODf, 87% TNf and 74%
TPf. While the PFBR microbial community structure and function were found to be highly
diversified with substantial AOB and PAO populations, about 70% of the phosphorus
release potential and almost 100% of the nitrification potential were located in Reactors
1 and 2, respectively. Co-enrichment of AOB and PAOs was realized in the Reactor 2 biofilm,
where molecular analyses revealed unexpected microbial distributions at micro-scale, with
population peaks of AOB in a 100–250 mm deep sub-surface zone and of PAOs in the
0–150 mm surface zone. The micro-distribution of AOB coincided with the position of the
nitrification peak identified during micro-sensor analyses. The study demonstrates that
enrichment of PAOs can be realized in a constant or near constant aerobic biofilm envi-
ronment. Furthermore, the findings suggest that when successful co-enrichment of AOB
and PAOs occur in biofilm environments, such as in the PFBR system, they do so at different
zone depths in the biofilm.
ª 2009 Elsevier Ltd. All rights reserved.

* Corresponding author. Tel.: þ353 91 493 107; fax: þ353 91 494 507.
E-mail address: michael.nielsen@nuigalway.ie (M. Nielsen).
0043-1354/$ – see front matter ª 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2009.07.007
4600 water research 43 (2009) 4599–4609

phosphorus (P) removal has been investigated in detail

1. Introduction (Rodgers et al., 2006; Zhan et al., 2006; O’Reilly et al., 2008;
Rodgers et al., 2008). In the present study, distributions and
In order to limit eutrophication of receiving water bodies, activities of AOB and PAOs were examined for a PFBR system
removal of nitrogen and phosphorus is a priority in modern that was operated for C, N and P removal. Detailed insight into
wastewater treatment plants. Nitrogen and phosphorus are population structures and system function was obtained
routinely removed through activity of different groups of through application of high-resolution micro-sensor and
micro-organisms. Biological nitrogen removal is a multi-step molecular techniques in combination with reactor phase
process, where ammonium (NHþ 4 ) is first oxidized to nitrite studies and batch experiments. The results revealed the
(NO 
2 ) or nitrate (NO3 ) during nitrification, and then subse- presence of distinct distributional patterns of AOB and PAO
quently reduced to dinitrogen gas through denitrification or populations inside the PFBR system, both at micro-scale and
ANaerobic AMMonium OXidation (ANAMMOX, Ahn, 2006). macro-scale levels.
Oxidation of NHþ 4 , the initial and rate-limiting step of nitrifi-
cation and nitrogen removal, is performed by aerobic
ammonia oxidizing bacteria (AOB). AOB are chemo-
lithoautotrophs that gain energy by oxidation of NHþ 
4 to NO2 .
2. Materials and methods
Biological phosphorus removal is achieved through harvest-
ing of polyphosphate accumulating organisms (PAOs), 2.1. Pumped-flow biofilm reactor (PFBR)
a process known as enhanced biological phosphorus removal
(EBPR) (Mino et al., 1998). PAOs are heterotrophic organisms The laboratory PFBR system (Fig. 1a) comprised two 16.5 l
characterized by their ability to store large amounts of both reactor tanks (Reactors 1 and 2), each containing a PVC biofilm
polyphosphate and organic carbon sources like poly- module (R1 and R2, respectively) 200  200 mm in plan and
hydroxyalkanoate (PHA) intracellularly (Kuba et al., 1993). In 180 mm high with a surface area of approximately 2 m2
order to selectively enrich a system with PAOs, the system is (Rodgers et al., 2006). The biofilm module consisted of irreg-
usually alternated between anaerobic ‘‘feast’’ conditions with ular hexagonal-shaped vertical columns (with longest and
available dissolved biodegradable organic carbon and aerobic shortest plan dimensions of 15 and 10 mm, respectively) and
‘‘famine’’ conditions with little dissolved biodegradable its base was supported on a metal grid 70 mm above the tank
organic carbon. invert. An operational cycle lasted 18.2 h and included four
Co-enrichment of AOB and PAOs in a single system is phases: Seven-min fill into Reactor 1, 6-h stagnant in Reactor
complicated by the different physiologies of autotrophic AOB
and heterotrophic PAOs. For example, an elongated anaerobic
period, which is traditionally used to promote the growth of
PAOs, could be detrimental to the oxygen-requiring AOB. Also, a
in a combined nitrogen and phosphorus removal system,
insufficient organic carbon for PAOs may remain for effective
phosphorus reduction after the denitrification of NO 2 and
NO 3 , has taken place (Seviour et al., 2003). A number of labo-
ratory-scale systems, in particular biofilm systems with fixed
or suspended carriers, have been developed to overcome such
potential problems (e.g. Gieseke et al., 2001; Terada et al.,
2006). In biofilm systems, a long biomass retention time R1 R2
promotes enrichment of slow-growing organisms such as
AOB and NOB. In addition, the presence of chemical micro-
gradients inside biofilms favours co-establishment of aerobic/
anoxic/anaerobic conditions, which can benefit simultaneous
nitrogen and phosphorus removal. However, the factors that
regulate the distributions of AOB and PAOs within biofilm
systems remain poorly understood. A spatial separation of
AOB and PAOs was suggested in a moving-bed sequencing
batch biofilm reactor (Pastorelli et al., 1999). In contrast,
detailed micro-scale studies have indicated a coexistence of
AOB and PAOs in the upper layers of both biofilms and aerobic
b
granules (Gieseke et al., 2002; de Kreuk et al., 2005). 6
0 Stagnant Pumping 18
A two-tank pumped-flow biofilm reactor (PFBR) system Hours
was developed for nutrient removal at the Civil Engineering R1 Water covered Water covered/Air exposed
Department, National University of Ireland, Galway (Rodgers R2 Air exposed Water covered/Air exposed
et al., 2004). The system is flexible and can be used efficiently
to readily create anoxic, anaerobic and oxic conditions by the Fig. 1 – Schematic diagram of the PFBR system (a), and
PLC control of standard pump technology. The application of overview of the conditions in Reactors 1 and 2 during the
this biofilm technology for carbon (C), nitrogen (N) and two cycle phases (b).
 water research 43 (2009) 4599–4609
1, 12-h continuously pumping over and back between Reac-
tors 1 and 2, and 5-min draw from Reactor 1. At the end of the
draw phase, the total residual bulk fluid volume in the system
was approximately 5.8 l, with about 1.4 l in Reactor 1 and 4.4 l
in Reactor 2 (including pipe volumes). The PFBR treated bulk
fluid volume was controlled by an upper water level switch in
Reactor 1, and this treated volume decreased with time from
about 13.3 to 8.0 l due to the thickness of the Reactor 1 biofilm.
After filling Reactor 1 with an average 10.4 l volume of new
synthetic wastewater, the biofilm module (R1) in Reactor
1 was fully submerged, and the module (R2) in Reactor 2 was
completely exposed to the air (Fig. 1b). During the pumping
phase, a total average volume of 16.2 l wastewater was
circulated between the two reactor compartments, with
a total cycling time of about 60 s. Biofilm air exposure times of
the upper and lower boundaries of the module sections,
during this cycling time, were about 45 and 15 s, respectively.
At the end of the pumping phase, most of the treated water
resided in Reactor 1, and this reactor was decanted without
settling so that the detached biofilm suspended in the effluent
water was removed.
The system was seeded with activated sludge from Tuam
Municipal Wastewater Treatment Plant, Galway, Ireland, and
was operated for 140 days in a temperature-controlled room at
11  C. The composition of the synthetic wastewater was:
1000 mg/l CH3COONa, 120 mg/l yeast extract, 480 mg/l dried
milk, 120 mg/l urea, 240 mg/l NH4Cl, 200 mg/l Na2HPO4 $ 12H2O,
200 mg/l KHCO3, 520 mg/l NaHCO3, 200 mg/l MgSO4 $ 7H2O,
8 mg/l FeCl3 $ 6H2O, 12 mg/l CaCl2 $ 6H2O and 8 mg/l
MnSO4 $ H2O. The influent chemical oxygen demand (CODf),
total nitrogen (TNf), NHþ4 and total phosphorus (TPf) concentra-
tions were 1108  61, 118  11, 74.0  23.6 and 19.9  2.5 mg/l,
respectively.
The phase performance of the PFBR system was investi-
gated 4 times after the start-up of the reactor, on Days 89, 95,
110 and 135, in order to describe the bulk fluid dynamics of NO3,
3
NO þ
2 , NH4 , orthophosphate (PO4 –P) and CODf during the
reaction cycle. During the phase studies, water samples for
chemical analyses were taken from Reactor 1 at 0.5–1 h inter-
vals. The oxygen dynamics were monitored during the initial
30 min period of the pumping phase, using a fabricated Clark-
type O2 mini-sensor with a response time (t90) of about 5 s.
2.2. Batch experiments
For batch experiments (two replications), biofilm samples were
removed on Day 132 from inside columns of the upper and lower
half sections of R1 and R2 (upper and lower sections refer to 0–90
and 90–180 mm from the top of the biofilm modules, respec-
tively). The samples were collected at the end of the pumping
phase and had a biofilm area of 45 cm2. The samples were sus-
pended in a total volume of either 400 (nitrification experiments)
or 200 ml (phosphorus release experiments) of liquid medium
with the same chemical composition as the synthetic waste-
water, but excluding organic carbon and nitrogen sources.
Nitrification rates were evaluated under aerobic conditions
(air bubbling) following the addition of 16 mg NHþ 4 –N/l to the
liquid medium. Aeration was started 0.5 h before the addition
of NHþ 4 . Phosphorus release rates were determined under
anaerobic conditions (N2 purging of headspace) after adding
4601
1000 mg acetate/l to the liquid medium. The nitrification and
phosphorus release experiments were performed twice and
lasted 240 and 120 min, respectively. Water samples of 2 ml
were taken at 10- and 30-min intervals for chemical analyses.
At the end of the experiment, mixed liquor suspended solids
(MLSS), mixed liquor volatile suspended solids (MLVSS) were
analyzed. Nitrification and phosphorus release rates were
described from increases in total oxidized nitrogen (NOx ) and
PO3
4 , respectively.
2.3. Micro-sensor studies
Biofilms for micro-sensor profiling were grown on detachable
PVC strips (20  300 mm), which were placed inside the
vertical columns of the biofilm modules from the start-up of
the system. For sample preparation, small sections of biofilm
(2–3 cm2) were transferred from the PVC strips to glass slides
primed with a thin fluid agar film (w100 mm) for fixation.
Micro-sensor measurements were performed in a flow
chamber with the water level above the horizontal biofilm
samples adjusted to approximately 5 mm. Aerated liquid
medium (total 1.5 l) was re-circulated by a peristaltic pump
from a 1 l reservoir that was placed in a refrigerated water
bath to keep the temperature in the flow chamber at 11  C. The
basic medium composition was identical to the synthetic
wastewater fed to the PFBR system, excluding nitrogen and
organic components. For the different experiments, selected
amounts of NHþ 
4 , NO3 and COD were added to the liquid
medium from stock solutions. For all test conditions, an
acclimation period of a minimum of 2 h was applied before
sensor profiling.
Clark-type O2 micro-sensors (tip diameter ¼ 10–15 mm) and
NO x micro-biosensors (tip diameter ¼ 25–30 mm) were con-
structed in the laboratory, for analyses of O2 and NO x
concentration profiles, respectively (Revsbech, 1989; Larsen
et al., 1997). The experimental set-up, which allowed for
automatic micro-profiling and data collection, consisted of
a motorized micro-manipulator, a two-channel picoammeter,
an A/D converter, profiling software (Profix 3.09, Unisense A/S),
and a laptop computer. A dissection microscope (PZMIV,
World Precision Instruments) was used for micro-sensor
positioning at the biofilm surface and for the determination of
biofilm thickness.
Oxygen micro-profiles were measured for biofilm samples
from the upper and lower sections of R1 and R2 after 50 and
80 days, and again for samples from R2 after 120 days. The bio-
film O2 dynamics were initially characterized in media without
any organic carbon and NHþ 4 , and subsequently in the presence
of 25 mg NHþ 
4 –N/l. After about 135 days of operation, O2 and NOx
profiles were determined for biofilm samples from the upper
and lower sections of R2, at experimental conditions with either:
(i) 5 mg NHþ þ
4 –N/l and 12.5 mg COD/l; or (ii) 25 mg NH4 –N/l and
50 mg COD/l in the liquid medium. In both experimental
conditions, the water phase was supplemented with 0.5 mg
NO 
3 –N/l for in situ NOx biosensor calibration. During all exper-
iments the bulk O2 concentration was kept close to saturation
level (10.9 mg/l at 11  C). To control the water flow condition
above the biofilm sample, a gentle air stream was blown across
the water surface and adjusted to obtain a diffusive boundary
layer thickness of about 150–200 mm.
4602 water research 43 (2009) 4599–4609

2.4. Fluorescence in situ hybridization (FISH)
About 1 cm2 of biofilm pieces were sampled on Day 80 from the
upper and lower sections of R1 and R2 and again on Day 130 from
the lower section of R2. The samples were fixed overnight at 4  C
in PBS buffer (1.09 g/l Na2HPO4, 0.32 g/l NaH2PO4, 9.0 g/l NaCl, pH
7.2) containing 4% paraformaldehyde. Fixed samples were
washed twice in PBS buffer and finally stored at 20  C in a 1:1
ethanol/PBS buffer solution. The biofilm was prepared for
sectioning by overnight immersion in OCT tissue freezing
medium. It was frozen and sliced into 20 mm thick transects on
a cryomicrotone (Leica Microsystems). Transects were trans-
ferred to gelatinized (0.1% gelatine þ 0.01% CrKSO4) glass
microscope slides and immersed in distilled water for 10 min to
remove remaining OCT medium. Finally, samples were dehy-
drated by immersion in 50, 70, and 100% ethanol solutions (5 min
each). CY3- or fluoresceine-labelled oligonucleotide probes were
used to specifically stain AOB or PAOs as well as general Bacteria.
The probes employed were NSO1225 (50 -CGCCATTGTAT-
TACGTGTGA-30 ) specific for AOB belonging to Beta-proteobacteria
(Norton et al., 1996), PAO651 (50 -CCCTCTGCCAAACTCCAG-30 )
specific for Rhodocyclus-related PAOs (Crocetti et al., 2000), and
EUB338 (50 -GCTGCCTCCCGTAGGAGT-30 ) for most Bacteria
(Amann et al., 1990). Hybridization and washing were performed
as described previously (hybridization with 35% formamide at
46  C, washing in 80 mM NaCl at 48  C). Microscopy was per-
formed on a Nikon epifluorescence microscope equipped with
suitable filters and a digital imaging system. The biomass frac-
tions of AOB and PAOs to general Bacteria in biofilm samples
were evaluated by quantifying the area fraction of cells hybrid-
izing with the respective probes on at least 20 randomly
distributed microscope images of each sample taken at 1000
magnification. Depth distribution profiles of AOB and PAOs were
studied by quantifying the distribution of fluorescence on
micrographs taken at 100 magnifications. Standard UVI geltec
software (Uvitec) was used for this quantification.
2.5. Analytical methods
MLSS, MLVSS and COD were determined according to stan-
dard methods (APHA/AWWA/WPCF, 1995). NHþ 
4 , NO2 , NO3

3
and PO4 were tested using a Konelab analyzer (Thermo
Clinical Labsystems, Vantaa, Finland). TN and TP were
analyzed with Hach TN and TP equipment (Hach, USA).
Filtration was conducted using Whatman GF/C filter papers
(1.2 mm pore size).
removed in the effluent was 45 g, while the removed total
CODf was 1987 g giving a net biomass production of 0.13 g
biomass/g CODf removed.
Chemical parameter dynamics in Reactor 1 during the
phase studies provided detailed information on reactor
processes and overall performance of the PFBR system. Phase
study results on Day 130 are shown in Fig. 2. Throughout the
6 h-long stagnant phase, there was a gradual decrease in CODf
concentration (about 40 mg/l per h) and a simultaneous
phosphorus release from the biofilm to the water, averaging
4 mg PO34 –P/l per h during the first 3 h. With a stagnant water
volume of approximately 9.8 l in Reactor 1 on Day 130 and
a biofilm surface area of 2 m2, this phosphorus release rate
was 19.6 mg/m2 per h (Table 1).
During the first few cycles of the pumping phase the
O2 concentration in the bulk water rose sharply due to efficient
aeration, reaching 55% saturation within 5 min and 70% after
3
30 min. There was an instantaneous drop in NHþ 4 , PO4 and
CODf concentrations due to dilution with the 4.4 l previously
treated wastewater in Reactor 2. The relative dilution effect
was less for PO3 þ
4 (85%) compared to NH4 and CODf (65%),
indicating that phosphorus was released from the biofilm in
Reactor 2 during the stagnant phase. During the first hour of the
pumping phase, the CODf concentration decreased rapidly
from about 400 to 100 mg/l, but subsequently slowed. Phos-
phorus removal decreased gradually from an initial rate of
4.5 mg PO34 –P/l per h to close to zero. At the end of the aerobic
phase, the phosphorus released during the stagnant phase and
the phosphorus added in the feed were almost entirely taken
up by the biofilm biomass. The increase in the NO 3 concen-
tration within the first hour of the pumping phase showed an
early onset of nitrification. The initial NHþ 4 removal rate was
about 10 mg NHþ 4 –N/l per h, and gradually decreased until NHþ
4
was nearly entirely depleted after 9–10 h of pumping. The
maximum NO 
3 production rate was about 3 mg NO3 –N/l per h,
corresponding to a nitrification rate in 14.2 l wastewater of
2
21.3 mg NO 3 –N/m per h assuming that nitrification was
restricted to Reactor 2 (Table 1). This value is the net production
rate and thus represents a minimum value. During the
pumping phase, the NHþ 4 concentration decreased by more
than 40 mg NHþ 4 –N/l whereas the NO3 concentration increased
70 1000
Anaerobic Aerobic
60
N and P concentrations (mg/l)

800
CODf concentration (mg/l)

50 NH4-N
NO2-N
3. Results NO3-N 600
40
PO4- P
30 CODf
3.1. Reactor performance 400

20
After 100 days operation, at a CODf loading rate of 3.46 g/m2
200
per day, removal efficiencies of 95% CODf, 87% TNf and 74% TPf 10
were obtained for the laboratory PFBR unit. Near the end of the
study period (Day 130) TNf and TPf removal efficiencies of 0 0
0 4 8 12 16 20
about 89 and 92%, respectively, were observed. As there was
Time (hour)
no settlement phase in the PFBR system, excess sludge was
removed in the effluent. On Day 130, the total biofilm biomass Fig. 2 – Water phase dynamics in Reactor 1 of the PFBR
was 222.1 g. During this 130-day period, the total biomass system during a full reactor cycle on Day 130.
 water research 43 (2009) 4599–4609 4603

Table 1 – Area-specific rates of nitrification and phosphorus release measured during this study.
Reactor Section Nitrification Phosphate release
2 a 2 b 2 c
mg N/m per h mg N/m per h mg N/m per h mg P/m per ha
2
mg P/m2 per hb

Reactor 1 Upper 10.2  14.4 n.d. <1 240.7  15.5 23.7  10.7
Lower 2.5  3.6 <1 243.8  27.4

Reactor 2 Upper 93.9  13.4 18.9  5.2d 16.6  2.2 (19) 68.9  5.8 n.d.
Lower 129.7  14.7 14.0  6.5 (34) 151.7  32.5

n.d.: not determined.

a Rates measured during batch experiments after 132 days.
b Rates measured during phase studies of the PFBR system after 95, 110 and 130 days.
c Rates calculated from oxygen micro-profiles after 50, 80 and 120 days – values in brackets from NO
x profiles after 135 days.
d Rate calculated assuming that nitrification was restricted to Reactor 2.

by about 12 mg N/l, which showed that more than 70% of the
NHþ4 was either assimilated or removed through simultaneous
nitrification–denitrification.
3.2. Batch experiments
The batch experiments on Day 132 showed large variations in
both nitrification and phosphorus release rates for biofilm
samples taken from different reactor sections (Table 1). The
area-specific nitrification rate was highest in the lower section
of the R2 bio-module (129.7  14.7 mg N/m2 per h) and less in
the upper section (93.9  13.4 mg N/m2 per h). The nitrification
rates were an order of magnitude smaller for the R1 samples,
with higher activity in the upper section (10.2  14.4 mg N/m2
per h) in comparison to the lower section (2.5  3.6 mg N/m2
per h) where activity was close to the detection limit. Area-
specific phosphorus release rates were high in Reactor 1, with
240.7  15.5 mg P/m2 per h in the upper section of R1 and
243.8  27.4 mg P/m2 per h in the lower section. Biomass from
the lower section of R2 exhibited two-fold higher P-release
rates (151.7  32.5 mg/m2 per h) compared to biomass from
the upper section of this module (68.9  5.8 mg/m2 per h).
3.3. Micro-sensor analyses
Biofilms on the PVC strips developed faster in Reactor 1 than
in Reactor 2, e.g., average biofilm thicknesses of 1600–1900 mm
(Reactor 1) and 1000–1200 mm (Reactor 2) at Day 80. The faster
growing biofilms in Reactor 1 were less stable than those in
Reactor 2; complete biofilm detachment from the PVC strips in
Reactor 1 occurred between Days 80 and 130. Biofilm detach-
ment from the PVC strips was not observed in Reactor 2.
The general trends in biofilm oxygen dynamics were
consistent for all 3 sampling days (Days 50, 80 and 120) and are
exemplified by results from the second experimental round on
Day 80 (Fig. 3). A large variation in oxygen micro-distribution
was found between biofilm samples from R1 and R2 at test
conditions with and without NHþ 4 in the overlying water
phase, reflecting substantial differences in microbial pop-
ulations. In the absence of organic carbon and nitrogen
substrates, the average oxygen utilization rate (OUR) was
markedly higher in Reactor 1 biofilm samples, with average
rates ranging between 180 and 182 mg O2/m2 per h compared
to 64–111 mg O2/m2 per h in Reactor 2 (Table 2). In the
presence of NHþ 4 , the average OUR in Reactor 2 biofilms
increased by 43–96 mg O2/m2 per h, corresponding to nitrifi-
cation rates (from ammonia to nitrate) of between 9 and 21 mg
N/m2 per h (Tables 1 and 2). The change in OUR in Reactor
1 biofilms was always below 5 mg O2/m2 per h, corresponding
to nitrification rates below 1 mg N/m2 per h.
Combined analyses of O2 and NO x micro-scale distributions
in biofilm samples from Reactor 2 showed the presence of
distinct nitrification zones within oxic surface layers (Fig. 4).
Similar dynamics were found for samples from upper and lower

0 5 10 0 5 10 0 5 10 0 5 10
-400 R1-Up R1-Low R2-Up R2-Low -400

0 0
Depth (µm)
Depth (µm)

400 400

800 800

1200 1200
0 5 10 0 5 10 0 5 10 0 5 10
O2 (mg/l)

Fig. 3 – Oxygen micro-profiles measured in biofilm samples from the PFBR after 80 days of operation, at water phase
conditions without NHD4 (triangles, filled) and with 25 mg/l NH4 –N (squares, open). Sampling locations: Reactor 1 upper
D

(R1 – Up) and lower (R1 – Low); Reactor 2 upper (R2 – Up) and lower (R2 – Low).
4604 water research 43 (2009) 4599–4609
Table 2 – Area-specific oxygen utilization rates of biofilm samples 50, 80 and 120 days after reactor start-up.
Sample Conditions
R1 – upper Background
25 mg/l NHþ
4 –N
R1 – lower Background
25 mg/l NHþ
4 –N
R2 – upper Background
25 mg/l NHþ
4 –N
R2 – lower Background
25 mg/l NHþ
4 –N
Rates were calculated from the measured oxygen micro-gradients in the diffusive boundary layer.
biofilm module sections, but the results indicated somewhat
higher nitrification activity in biofilms from the lower section.
Incubated with 5 mg NHþ 4 –N/l and 12.5 mg COD/l in the water
phase, the average NO x production rate, calculated from fluxes
across the biofilm-water interface, were about 19 and 34 mg N/m2
per h for upper and lower samples, respectively (Table 1).
In both samples nitrification activity was greatly affected by
nutrient availability. When water phase concentrations were
increased to 25 mg NHþ 4 –N/l and 50 mg COD/l, oxygen
penetration depths decreased from about 250–300 mm to about
150–175 mm, which coincided with displacements of NO x peak
depth position from about 200 to 300 mm to about 100 mm and
reductions in average maxima sub-surface concentrations
from 2.2–2.9 to 0.6–0.9 mg NO x –N/l.
3.4. Enumeration of AOB and PAOs
The biomass fractions of AOB and PAOs to total Bacteria at the
four locations on Day 80 are summarized in Fig. 5. AOB were
most abundant in Reactor 2. In R2, biofilm samples between
1.5 (upper section) and 3.0% (lower section) of the cells that
hybridized with EUB338 were also stained with probe
NSO1225. In R1 biofilm samples, less than 0.4% of the EUB338-
positive cells were also stained with NSO1225, and there was
no significant difference in AOB fractions between upper and
lower sections. PAOs were more numerous than AOB
throughout the two reactors. In Reactor 1, 29% (upper section)
and 38% (lower section) of the EUB338-positive cells were
stained by probe PAO651, while the corresponding PAO frac-
tions in Reactor 2 were 22% (upper section) and 22% (lower
section).
The depth distributions of AOB and PAOs were examined
in biofilm samples from the lower section of R2 on Day 130
(Fig. 6). AOB were most abundant in a layer about 100–200 mm
below the surface, whereas PAOs appeared to be most abun-
dant near the biofilm/water interface. The distribution of each
group in the 450 mm biofilm surface layer was further quan-
tified by integrating fluorescence signals over 30 mm depth
sections on micrographs obtained at 100 magnification
(Fig. 7). These results indicate that AOB peaked in abundance
between 150 and 250 mm below the biofilm surface, while
PAOs were most abundant from the surface of the biofilm
down to 150–200 mm depth.
Oxygen utilization
rate (mg O2/m2 per h)
Day 50 Day 80 Day 120
180  12 182  16 –
182  15 182  12 –
140  36 140  34 –
139  10 145  6 –
80  5 111  12 76  14
159  35 176  12 160  7
64  6 99  9 90  6
107  23 150  10 188  22
It should be noted that although the applied FISH probes
EUB338 and PAO651 are good indicators of the targeted groups
they did not completely cover the Bacteria and Accumulibacter
populations, respectively.
4. Discussion
The present study focuses on the organisation of microbial
populations in wastewater treatment biofilms. For efficient
biological nitrogen and phosphorus removal, simultaneous
enrichment of AOB and PAOs is required, but this is compli-
cated by the marked physiological differences, i.e. metabolic
requirements, growth rates and substrate kinetics, of the two
functional groups. Successful co-enrichment in biofilm
reactor systems can be achieved at a macro-scale level in
separate reactor compartments or at a micro-scale level
within the same biofilm compartment.
A characteristic of the studied PFBR system is the estab-
lishment of highly variable chemical conditions in time and
space during operation, which will support growth of different
types of microbial communities, and hence favour the
simultaneous enrichment of AOB and PAO populations. In the
preliminary stagnant phase, the high carbon availability and
prolonged anaerobic conditions in Reactor 1 are in contrast
with the low nutrient levels and aerobic conditions in Reactor
2, thus creating highly selective conditions for PAOs and AOB
in the two reactor compartments. During the subsequent
pumping phase, both reactors are experiencing the same
alternating mode of water coverage and air exposure (thin
water film condition), transient dynamics that are similar, but
slower, to the conditions in rotating biological contactors
(RBC; Patwardhan, 2003). Variable growth conditions are also
found inside the reactor compartments along the vertical axis
of each biofilm module. Due to longer air exposure periods,
upper sections are thus supplied with more oxygen but will at
the same time experience reduced availability of nutrients
such as NHþ 4 and organic substrates.
To describe the spatial distribution of AOB and PAO pop-
ulations in the PFBR system a combination of several experi-
mental approaches was used to evaluate biofilm activity. In this
respect it should be stressed that the conditions in batch and
 water research 43 (2009) 4599–4609 4605

O2(mg/l)
0 5 10 0 5 10
-400 -400

0 0

Depth (µm)

Depth (µm)
400 400

800 800

1200 a b 1200
0 1 2 3 0 1 2 3
NOx- -N (mg/l)

O2 (mg/l)
0 5 10 0 5 10
-400 -400

0 0

Depth (µm)
Depth (µm)

400 400

800 800

1200
c d 1200
0 1 2 3 0 1 2 3
NOx- -N (mg/l)

Fig. 4 – Micro-profiles of O2 (squares, open) and NOL

x (triangles, filled) in biofilm samples from upper (a D b) and lower (c D d)
sections of R2, measured after about 135 days at water phase conditions of 5 mg/l NHD 4 –N D 12.5 mg/l CODf (a D c) and
25 mg/l NHD 4 –N D 50 mg/l CODf (b D d).

micro-sensor experiments were not completely identical to in
situ reactor conditions. Batch and micro-sensor studies of
nitrification rates were performed with the biofilm material
permanently water-covered, and measurements were per-
formed in the absence of organic substrates. Batch studies
furthermore involved rate analysis of suspended biofilm mate-
rial in contrast to an attached film, and at highly turbulent
conditions, whereby a higher fraction of the biomass was
exposed to substrates and therefore contributing to the rate
measurement. For these reasons, variation in activity rates was
expected, and in particular, the measured activities during
batch studies represent potential rates that were markedly
higher than for the attached biofilm. However, the overall trends
in the measured activities and hence reactor distributions of
AOB and PAO populations were highly consistent.
4.1. Distribution and activity dynamics of AOB
The growth conditions in Reactor 1 did not favour any
significant enrichment of AOB populations, so the high rates
of nitrification in the PFBR system were linked almost
exclusively to the activity of the substantial AOB population
numbers in Reactor 2.
According to reactor data, the conditions were favourable
for nitrification throughout most of the 12 h pumping phase,
with high activity inside Reactor 2, but clearly the prolonged
anaerobic/anoxic conditions had a detrimental effect on the
growth of AOB in Reactor 1. In environments with large
heterotrophic populations like in the Reactor 1 biofilms, the
competition for oxygen is strong in comparison with more
autotrophic biofilm environments. During the turbulent
pumping phase, phosphate uptake lasted for about 9 h, which
suggests extended periods with oxygen limiting conditions for
AOB as a result of high heterotrophic activity (Gieseke et al.,
2001). It is therefore likely that active nitrification in Reactor
1 was restricted for a prolonged period to shallow oxic surface
layers; in fast developing biofilms these are areas that are
unsuitable for slow-growing organisms like AOB (Nogueira
et al., 2002; Meng and Ganczarczyk, 2004). Even during the
later stages of the turbulent phase, when competition for
oxygen was lessened, NHþ 4 limiting conditions were devel-
oping in Reactor 1 due to the unrestricted activity of AOB
populations in Reactor 2 throughout the pumping phase.
4606 water research 43 (2009) 4599–4609
6 60
5 50
4 40
AOB (%)
PAO (%)
30
3
2 20
1 10
0 0
Upper Lower Upper Lower
R1 R2
Fig. 5 – Biomass fractions of AOB (white bars) and PAOs
(grey bars) to general Bacteria determined in biofilm
samples from the upper and lower sections of R1 and R2 on
Day 80.
Consequently, the period where neither of oxygen or NHþ 4 under alternating water-covered and air-exposed conditions
limitation prevailed in Reactor 1 was short, and this was
apparently critical for the growth of AOB.
Although AOB communities in biofilms are predominantly
associated with oxic surface layers due to their inherent
requirement for oxygen, population structures within the oxic
zone can vary considerably, either showing a decline in
numbers from the biofilm surface, the presence of a sub-
surface maximum, or a more or less homogeneous distribu-
tion (Okabe et al., 1999; Okabe and Watanabe, 2000; Gieseke
et al., 2001). FISH data revealed a characteristic sub-surface
peak in AOB populations within biofilms from the lower
section of R2 that correlates well with the described active
nitrification zone from the micro-sensor analyses (low COD
condition). Evidence suggests that the occurrence of AOB
population maximum in sub-surface regions, where oxygen
and NHþ 4 availability for nitrification is suboptimal compared
to in surface layer, is linked primarily to the availability of
organics (Okabe and Watanabe, 2000; Nogueira et al., 2002).
Increasing organic levels stimulate the growth of heterotro-
phic populations, resulting in faster biofilm development and
covering growth over slow-growing organisms like AOB that
are unable to compete for space. Consequently, a layering will
develop with heterotrophs (e.g. PAOs) predominantly at the
surface and AOB in deeper regions of the oxic zone, as
observed in this study.
The presence of maximum AOB populations in the 150–
200 mm sub-surface layers may explain the observed dramatic
change in micro-scale nitrification dynamics upon COD
amendment, with greatly reduced activities at 50 compared to
12.5 mg/l. Even though the increased COD availability only
displaced oxygen penetration depth by approximately 100 mm,
from about 250–300 to 150–200 mm, the micro-sensor
measurements confirmed that critical oxygen limitation was
developing in otherwise active nitrifying biofilm layers. This
result demonstrates how even minor transitions in micro-
chemical regimes, in response to a change in substrate
availability, may impose a marked shift in biofilm function.
Inconsistent with this finding, the cycle reactor data gave no
clear indication of such a negative correlation between water
COD level and nitrification activity, even at concentrations in
excess of 100 mg CODf/l. These conflicting results may reflect
that permanently water-covered conditions were adopted in
the micro-sensor analysis in contrast to the reactor dynamics
that involved alternating water-covered and air-exposed
conditions on the biofilm. Because transient dynamics, as
already discussed, simultaneously increase oxygen supply
while reducing organic availability, nitrification can proceed
at comparable higher organic levels, thus increasing the water
COD threshold limit for activity of nitrifiers. This conclusion is
further supported by evidence from NO x micro-sensor
measurements in the PFBR biofilm at 200 mm depth, and in the
presence of 75 mg CODf/l (unpublished data). When this bio-
film was transiently exposed to the air to simulate reactor
dynamics, the sensor signal revealed high nitrification
activity, but when the conditions were changed to permanent
coverage with oxygen saturated water, nitrification activity
completely ceased. This emphasises that in further studies or
modelling of PFBR and RBC biofilm systems for wastewater
treatment, the improved nitrification activity in the biofilm
should be considered.
4.2. Distribution and activity dynamics of PAOs
Both cell counts and batch experiments indicated that
although PAOs were most abundant in Reactor 1, they also
constituted a significant part of the biomass and microbial
activity in Reactor 2.
With its alternating levels of oxygen and organic material,
Reactor 1 constitutes a classical example of PAO enrichment
(e.g. Li et al., 2003; Gieseke et al., 2002). During the aerobic
pumping phase PAOs assimilate and store available phos-
phate as intracellular polyphosphate. This polyphosphate is
subsequently used as an energy reserve during the stagnant
phase, allowing the PAOs to assimilate organic material while
other aerobic organisms are limited by the absence of oxygen.
The assimilated organic material is stored as PHA and used for
growth when water phase COD is low during the aerobic
turbulent pumped phase. Thus, the ability to alternately store
energy (phosphate) and organic material (PHA) give the PAOs
a selective advantage over other aerobic heterotrophs in the
PFBR. The phase study showed that phosphorus release
slowed down during the stagnant phase in Reactor 1, in spite
of a high proportion of PAOs being detected within the R1
biofilms. A possible explanation for this scenario is that a large
fraction of COD was oxidized to CO2 by ordinary heterotrophs
rather than used for storage as PHA and glycogen, which is
supported by the very low net yield coefficient of 0.13 g
biomass/g CODf for the system. In this respect, data on PHA
and glycogen levels in the reactor would have helped explain
the reasons for the reduced organic carbon uptake/phos-
phorus release rates observed during the later stages of the
stagnant phase.
Reactor 2, on the other hand, constitutes a more unusual
environment for PAO enrichment. While the PAO population in
Reactor 1 assimilates organic material and releases phosphate
to the water during the stagnant phase, PAOs in Reactor 2 are
subjected to a prolonged aerobic carbon starvation period. As
pumping starts, organic material becomes available, but only
 water research 43 (2009) 4599–4609 4607

Fig. 6 – Micrographs showing distributions of bacteria (a, c), PAOs (b), and AOB (d) in two transects of biofilm sampled at the
lower section of R2 on Day 130. The samples were stained with fluoresceine-labeled probe EUB338 (a, c) and CY3-labeled
probes PAO651 (b) or NSO1225 (d). Scale bar indicates 0.1 mm.

for a relatively brief period (1 h or less), followed by another
long aerobic starvation period. In Reactor 2, the PAOs were
particularly abundant close to the biofilm/water interface
above the AOB peak where the availability of organic carbon is
high in comparison to the deeper biofilm regions. Hence,
enrichment of PAO occurred in either constant or nearly
constant aerobic micro-environments. Because the oxygen
status in the 0–100 mm deep biofilm layers is uncertain at the
higher COD conditions during the initial pumping phase, it is
possible that the PAOs briefly experience anaerobic/anoxic
conditions as pulses of organic material enter the biofilm
during water coverage. Activities of other types of hetero-
trophs, which likely accounted for a large proportion of the
surface biofilm population, could have facilitated the devel-
opment of the required anaerobic micro-niches. In that case,
the ability to anaerobically assimilate and store carbon
substrates may represent a selective advantage compared to
other aerobic heterotrophs as is the case in Reactor 1. In any
event, this study indicates that the ability to rapidly store PHA
while carbon is available and subsequently use it for growth
during nutrient limiting conditions may provide a selective
advantage, which allows PAOs to compete with other hetero-
trophs in predominantly aerobic, low COD environments like
Reactor 2. Enrichment of PAOs was previously reported in
constantly aerated reactors by separating the availability of
carbon and phosphorus, but in this system the redox condi-
tions within flocs and aggregates were not characterized (Ahn
et al., 2007). Enrichment of PAO within constantly aerobic
micro-niches in wastewater environments therefore remains
to be demonstrated.
4.3. Suggestions for optimizing the PFBR system for
nutrient removal
The performed study showed that N and P removal efficien-
cies of 87 and 74%, respectively, could be achieved for the PFBR
when the system was operated with a simple cycle regime,
consisting of 6 h stagnant and 12 h pumping phases. As
4608 water research 43 (2009) 4599–4609
Fig. 7 – Depth distributions of PAOs (a) and AOB (b)
communities in biofilm sampled at the lower section of R2
on Day 130. Numbers for each depth zone of 30 mm are
given as a percentage of the total PAO or AOB population in
the top 450 mm of the biofilm.
already discussed, this operation facilitated the simultaneous
enrichment of substantial populations of AOB and PAOs that
were partially separated between the two reactors.
The information on reactor dynamics and microbial
distribution patterns suggests that one simple way to improve
system performance may be through adjustment of the cycle
phase lengths. By extension of the un-aerated stagnant phase,
phosphorus removal in Reactor 1 may be further enhanced. If
a larger fraction of the organic carbon is used up during the
anaerobic phase in Reactor 1, a lower enrichment level of
heterotrophs in Reactor 2 is to be expected. Consequently, the
longer stagnant phase may enhance nitrification in Reactor
2 as the micro-distribution of the nitrifying community will
likely shift from the observed sub-surface biofilm layers to
surface layers, thus reducing the degree of substrate diffu-
sional limitation. As a result, this may induce a further sepa-
ration of heterotrophic populations and AOB populations into
Reactors 1 and 2, respectively, whereby nitrification and
phosphorus removal may be completely separated. However
this change may adversely affect denitrification performance
of the system and hence overall N removal. When operated
with a 6 h stagnant phase, high rates of denitrification
occurred during the early part of the pumping phase because
soluble organic carbon was still available in the bulk water at
this time. According to reactor dynamics, most N and P
concentrations had stabilized during the later stages of the
pumping phase, indicating no or little activity. This suggests
that improved system performance could be achieved by
reducing the duration of the pumping phase to about 10 h.
5. Conclusions
(1) In the studied PFBR system, simultaneous nitrification and
biological phosphorus removal were achieved through
employment of a simple operational cycle. The specialized
water flow dynamics inside the reactor system created
diverse biofilm growth niches that governed co-enrich-
ment of substantial AOB and PAO communities.
(2) After 100 days operation at a filtered chemical oxygen
demand (CODf) loading rate of 3.46 g/m2 per day, the
removal efficiencies of the PFBR system were 95% CODf,
87% TNf and 74% TPf.
(3) The overall function of the PFBR system was found to be
highly diversified due to preferential selection for AOB and
PAOs in separate compartments. Nitrification was almost
exclusively restricted to Reactor 2 that was almost
continuously aerobic, while most of the phosphorus
removal potential was found in the aerobic/anaerobic
Reactor 1.
(4) Unusual micro-distributions of AOB and PAOs were
encountered inside the PFBR biofilm from Reactor 2 where
co-enrichment of populations was realized. Peak pop-
ulations of AOB were found in the 150–200 mm sub-surface
layers – areas of the biofilm with suboptimal availability of
substrates for nitrification. The PAO community showed
a distribution with maximum populations at 0–150 mm
depth, indicating that enrichment took place in a highly
atypical environment for PAOs, characterized by constant
or nearly constant aerobic conditions.
Acknowledgements
This research was supported by the EU Marie Curie Transfer of
Knowledge (TOK) project under the Sixth Framework Research
Programme and the Irish Higher Education Authority through
the National Centre for Biomedical Engineering Sciences
(NCBES), NUI Galway. The authors are grateful to Edmond
O’Reilly for his expertise and technical help.
references
Ahn, Y.-H., 2006. Sustainable nitrogen elimination
biotechnologies. Process Biochem 41, 1709–1721.
Ahn, J., Schroeder, S., Beer, M., McIlroy, S., Bayly, R.C., May, J.W.,
Vasiliadis, G., Deviour, J., 2007. Ecology of the microbial
community removing phosphate from wastewater under
continuously aerobic conditions in a sequencing batch
reactor. Appl. Environ. Microb. 73, 2257–2270.
Amann, R.I., Krumholz, L., Stahl, D.A., 1990. Fluorescent-
oligonucleotide probing of whole cells for determinative,
phylogenetic, and environmental studies in microbiology.
J. Bacteriol. 172, 762–770.
APHA/AWWA/WPCF, 1995. Standard Methods for the
Examination of Water and Wastewater, 19th ed. Washington
DC, USA.
Crocetti, G.R., Hugenholtz, P., Bond, P.L., Schuler, A., Keller, J.,
Jenkins, D., Blackall, L.L., 2000. Identification of
polyphosphate-accumulating organisms and design of 16S
rRNA-directed probes for their detection and quantitation.
Appl. Environ. Microb. 66, 1175–1182.
de Kreuk, M.K., Pronk, M., van Loosdrecht, M.C.M., 2005.
Formation of aerobic granules and conversion processes in an
aerobic granular sludge reactor at moderate and low
temperatures. Water Res. 39, 4476–4484.
 water research 43 (2009) 4599–4609
Gieseke, A., Purkhold, U., Wagner, M., Amann, R., Schramm, A.,
2001. Community structure and activity dynamics of nitrifying
bacteria in a phosphate-removing biofilm. Appl. Environ.
Microb. 67, 1351–1362.
Gieseke, A., Arnz, P., Amann, R., Schramm, A., 2002.
Simultaneous P and N removal in a sequencing batch biofilm
reactor: insights from reactor – and microscale investigations.
Water Res. 36, 501–509.
Kuba, T., Smolders, G.J.F., van Loosdrecht, M.C.M., Heijnen, J.J.,
1993. Biological phosphorus removal from wastewater by
anaerobic-anoxic sequencing batch reactor. Water Sci.
Technol. 27, 241–252.
Larsen, L.H., Kjær, T., Revsbech, N.P., 1997. A microscale NO 3 Revsbech, N.P., 1989. An oxygen microsensor with a guard
biosensor for environmental applications. Anal. Chem. 69,
3527–3531.
Li, J., Xing, X.-H., Wang, B.-Z., 2003. Characteristics of phosphorus
removal from wastewater by biofilm sequencing batch reactor
(SBR). Biochem. Eng. J. 16, 279–285.
Meng, Q.E., Ganczarczyk, J., 2004. Full scale comparison of
heterotrophic and nitrifying RBC biofilms. Environ. Technol.
25, 165–171.
Mino, T., van Loosdrecht, M.C.M., Heinen, J.J., 1998. Microbiology
and biochemistry of the enhanced biological phosphate
removal process. Water Res. 32, 3192–3207.
Nogueira, R., Melo, L.F., Purkhold, U., Wuertz, S., Wagner, M.,
2002. Nitrifying and heterotrophic population dynamics in
biofilm reactors: effects of hydraulic retention time and the
presence of organic carbon. Water Res. 36, 469–481.
Norton, J.M., Low, J.M., Klotz, M.G., 1996. The gene encoding
ammonia monooxygenase subunit A exists in three nearly
identical copies in Nitrosospira sp. NpAV. FEMS Microbiol. Lett.
139, 181–188.
Okabe, S., Watanabe, Y., 2000. Structure and function of nitrifying
biofilms as determined by in situ hybridization and the use of
microelectrodes. Water Sci. Technol. 42, 21–32.
4609
Okabe, S., Satoh, H., Watanabe, Y., 1999. In situ analysis of
nitrifying biofilms as determined by in situ hybridization and
the use of microelectrodes. Appl. Environ. Microb. 65,
3182–3191.
O’Reilly, E., Rodgers, M., Zhan, X.-M., 2008. Pumped flow biofilm
reactors (PFBR) for treating municipal wastewater. Water Sci.
Technol. 57, 1857–1865.
Pastorelli, G., Canziani, R., Pedrazzi, L., Rozzi, A., 1999.
Phosphorus and nitrogen removal in moving-bed sequencing
batch biofilm reactors. Water Sci. Technol. 40, 169–176.
Patwardhan, A.W., 2003. Rotating biological contactors: a review.
Ind. Eng. Chem. Res. 42, 2035–2051.
cathode. Limnol. Oceanogr. 34, 474–478.
Rodgers, M., Mulqueen, J., Zhan, X., O’Reilly, E., 2004. A method
and apparatus for biologically processing fluid. Patent
Application No. S2004/0462.
Rodgers, M., Zhan, X., O’Reilly, E., 2006. Small-scale domestic
wastewater treatment using an alternating pumped
sequencing batch biofilm reactor system. Bioproc. Biosyst.
Eng. 28, 323–330.
Rodgers, M., Wu, G., Zhan, X., 2008. Nitrogen and phosphorus
removal from domestic strength synthetic wastewater using
an alternating pumped flow sequencing batch biofilm reactor.
J. Environ. Qual. 37, 977–982.
Seviour, R.J., Mino, T., Onuki, M., 2003. The microbiology of
biological phosphorus removal in activated sludge systems.
FEMS Microbiol. Rev. 27, 99–127.
Terada, A., Yamamoto, T., Tsuneda, S., Hirata, A., 2006.
Sequencing batch membrane biofilm reactor for simultaneous
nitrogen and phosphorus removal: novel application of
membrane-aerated biofilm. Biotechnol. Bioeng. 94, 730–739.
Zhan, X., Rodgers, M., O’Reilly, E., 2006. Biofilm growth and
characteristics in an alternating pumped sequencing batch
biofilm reactor (PFBR). Water Res. 40, 817–825.