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POST PRINT of the paper which is printed in Water Research 2012 46(4) Pages 1671175.

The publishers version is available at: http://dx.doi.org/10.1016/j.watres.2011.12.003



Suspended biofilm carrier and activated
sludge removal of acidic pharmaceuticals
P. Fals*, A. Baillon-Dhumez*, H. R. Andersen**, A. Ledin*, J. la Cour Jansen*
* Water and Environmental Engineering, Department of Chemical Engineering, Lund University, P.O. Box 124,
SE-221 00 Lund, Sweden
**Department of Environmental Engineering, Technical University of Denmark, B113, DK-2800 Kgs Lyngby,
Denmark

Keywords: Pharmaceuticals; Activated sludge; Biofilm carriers

Abstract - Removal of seven active pharmaceutical substances (ibuprofen, ketoprofen,
naproxen, diclofenac, clofibric acid, mefenamic acid, and gemfibrozil) was assessed by batch
experiments, with suspended biofilm carriers and activated sludge from several full-scale
wastewater treatment plants. A distinct difference between nitrifying activated sludge and
suspended biofilm carrier removal of several pharmaceuticals was demonstrated. Biofilm
carriers from full-scale nitrifying wastewater treatment plants, demonstrated considerably
higher removal rates per unit biomass (i.e. suspended solids for the sludges and attached
solids for the carriers) of diclofenac, ketoprofen, gemfibrozil, clofibric acid and mefenamic
acid compared to the sludges. Among the target pharmaceuticals, only ibuprofen and
naproxen showed similar removal rates per unit biomass for the sludges and biofilm carriers.
In contrast to pharmaceutical removal, the nitrification capacity per unit biomass was lower
for the carriers than the sludges, which suggests that neither the nitrite nor the ammonia
oxidizing bacteria are primarily responsible for the observed differences in pharmaceutical
removal. The low ability of ammonia oxidizing bacteria to degrade or transform the target
pharmaceuticals was further demonstrated by the limited pharmaceutical removal in an
experiment with continuous nitritation and biofilm carriers from a partial nitritation/anammox
sludge liquor treatment process.

1. Introduction
Detection of pharmaceuticals in wastewater effluents and receiving waters has increased the
awareness of the potential threat that pharmaceuticals pose on the aquatic environment.
Removal efficiency of pharmaceuticals at wastewater treatment plants (WWTPs) is often
highly dependent on the biological treatment step (Carballa et al., 2004; Zorita et al., 2009)
and it has been indicated that the biological treatment design can influence the overall
removal of estrogenic activity (Kirk et al., 2002; Svenson et al., 2003) and pharmaceuticals
(Kasprzyk-Hordern et al., 2009). Several comparative studies on pharmaceutical removal in
biological systems have been presented, often focusing on activated sludge systems and
membrane bioreactors (Bernhard et al., 2006; Clara et al., 2005 a; De Wever et al., 2007;
Lesjean et al., 2005; Radjenovi et al., 2009).

The importance of solid retention time (SRT) on pharmaceutical removal in systems with
suspended bacteria cultures is widely discussed and it has been indicated that a prolonged
SRT can promote removal of some pharmaceuticals (Clara et al., 2005 b; Fals et al., in press;
Kimura et al., 2007). The biomass retention time in biofilm systems is not easily controlled,
but low loaded biofilm processes tend to favor slow-growing bacteria, which seems promising
for the pharmaceutical removal. In spite of this, todays knowledge on pharmaceutical
removal in biofilm systems is rather limited. However, parallel examinations of a full-scale
submerged biofilm reactor with Biostyr and an activated sludge process showed slightly
lower removal of estrogens in the biofilm reactor (Joss et al., 2004) and comparable removal
of several other pharmaceuticals (Gbel et al., 2007; Joss et al., 2005), despite a much longer
hydraulic retention time (HRT) in the activated sludge process. These findings suggest that
the shorter reaction time in the biofilm reactor is compensated by a higher biomass
concentration and/or a higher pharmaceutical removal capacity per unit biomass. Little is still
known about the biomass capacity to remove pharmaceuticals in biofilm systems and whether
this capacity differs from that of activated sludge.

This study aims to assess the removal rate of seven acidic pharmaceuticals (ibuprofen,
ketoprofen, naproxen, diclofenac, clofibric acid, mefenamic acid and gemfibrozil) by
suspended biofilm carriers and to compare the removal rate per unit biomass to that of
nitrifying activated sludge. Carriers and sludge from several full-scale WWTPs with
nitrification were investigated in order to see whether general trends in the pharmaceutical
removal potential exist. Further comparison was also made with carriers from a partial
nitritation/anammox sludge liquor treatment process. The ability of the carriers and sludges to
remove seven pharmaceuticals was studied in batch reactors and the results are discussed with
regards to biomass origin and nitrification.

2. Materials and Methods
2.1. Characteristics of WWTPs and sample collection
Seven full-scale WWTPs were selected for activated sludge or carrier sampling. Table 1 gives
main characteristics of the biological processes sampled and additional information on the
plants is given in supplementary information, S1.

Table 1 Characteristics of sampled processes

Klippan
WWTP
Nykping
WWTP
Saleboda
WWTP
Himmerfjrdsverket
WWTP
Kllby
WWTP
Klagshamn
WWTP
resundsverket
WWTP
Treatment

Combined
MBBR
Activatedsludge
PreDN
Twoaerated
compartments*
MBBR
PreDN
Twoaerated
compartments

MBBR
Twoaerated
compartments
MBBR
Sludgeliquor
treatment
Partial
Nitritation/anammox
Activated
sludge
PreDN
EBPR
Activated
sludge
Activated
sludge
PreDN
EBPR
HRT(h) 12 67 1112 35 18 56 14
SRT(d) 1718 7 2530
Filingratio(%)
Carriertyp
3040
BiofilmChipM
A,B
5060
K1
A
4060
K1
A
30
K1
A

ZonesVolumetricratio
(anaerobe:anox:aerobe) (0:1:1)
(0:1:2)

(0:0:1) Intermittent
aeration
(1:1:7) (0:0:1) (1:4:5)
PreDNPredenitrification
EBPREnhancedbiologicalphosphorousremoval
*Onewithcarriersandactivatedsludgeandonewithactivatedsludgeonly
A
AnoxKaldnes
B
Aeratedcompartmentwithcarriersandactivatedsludge


Carriers were collected from the two aerated compartments at Nykping WWTP, the first
aerated compartment at Saleboda WWTP, the combined process at Klippan WWTP and the
sludge liquor treatment at Himmerfjrdsverket WWTP. Activated sludge was collected from
the last aerated zone at Kllby WWTP, Klagshamn WWTP and resundsverket WWTP. At
the time of sampling, the wastewater temperature was 10-15 C in the full-scale plants, while
the temperature was 25-30 C in the sludge liquor treatment at Himmerfjrdsverket WWTP.
Carriers and sludge were transported directly to the laboratory and stored at 4-8 C. Batch
experiments were then started within 48 h after the sample collection.

2.2. Batch experiments with carriers and activated sludge from full-scale plants
Batch experiments were performed in 5 L reactors containing either activated sludge or
carriers plus wastewater. For the experiment with carriers from Klippan WWTP treated
wastewater was used, whereas wastewater and carriers were taken at the same point in
Saleboda WWTP and Nykping WWTP. Pharmaceuticals dissolved in methanol were added
to the reactors to attain an initial concentration of 100 g/L. For reactors with low initial
alkalinity, NaHCO
3
was added to reach an initial alkalinity of 3-6 mmol/L, HCO
3
-
. The
reactors were completely mixed, fully aerated (5-9 mg/L, O
2
) and kept at 183 C. Samples
for pharmaceutical analysis were withdrawn from the reactors over 24 h, 9 for the carrier
reactors and 12 for the sludge reactors. Samples for determination of COD, nitrification
(NH
4
+
-N, NO
3
-
-N, NO
2
-
-N) and biomass were collected from the reactors and oxygen levels
and pH were measured. When water samples were withdrawn from the reactors, biomass
either as carrier biofilm or as sludge was removed to maintain the initial biomass to water
ratio.

2.3. Batch experiment with carriers from a partial nitritation/anammox sludge liquor
treatment process
The batch experiment was made with carriers and wastewater from the single reactor partial
nitritation/anammox sludge liquor treatment process. The experimental setup was similar to
that described for the carriers from full-scale plants, but a few adjustments were required.
Water instead of methanol was used as pharmaceutical solvent, since methanol inhibits
anammox bacteria (Gven et al., 2005; Isaka et al., 2008). Furthermore, the reactor
temperature was raised (282 C) and the oxygen level was lowered (1.5-2 mg/L, O
2
) in order
to have normal process conditions for the biofilm and to allow simultaneous nitritation and
anammox acitivity. At the start of the experiment, NaNO
2
was added (40 mg/L, NO
2
-N) to the
reactor to confirm simultaneous removal of NO
2
-

and NH
4
+
.

2.4. Batch experiment with carriers without biomass
The control experiment was made with tap water and new carriers (K1, AnoxKaldnes)
without biomass. The reactor setup was identical to that described for the carriers from full-
scale plants, and when water samples were withdrawn from the reactor a corresponding
fraction of carriers was removed to maintain constant filling ratio.

2.5. Sample preparation and pharmaceutical analysis
Samples for pharmaceutical analysis were centrifuged and filtered, GFC-filters (Glass fiber
filter, 1.2 m, Whatman). 250 mL of the filtered samples were acidified to pH 3 with a
phosphate buffer and internal standard, mecoprop (40 g/L), was added before extraction.
The SPE-cartridges, Oasis HLB 3cc, were conditioned serially with 3 mL each of methanol,
ethyl acetate and acidified water. The extraction rates were below 2 mL/min and the
cartridges were dried completely after extraction. Samples were eluted with 1.5 mL ethyl
acetate and the extracts were evaporated under a gentle stream of N
2
at 35 C to a volume of
approximately 250 L. Remaining eluates were transferred to GC-vials and 25 L of the
derivatisation agent, N-(t-butyldimetylsilyl)-N-methyltrifluoroacetamid (MTBSTFA), was
added and allowed to react for 60 minutes at 60 C.

Quantification of the derivates of each pharmaceutical was made with a GC/MS, Agilent
5973N Mass Selective Detector. The capillary column was an Agilent HP 5-MS (30.0 m
250 m 0.25 m) with 1 L injection in splitless mode. The limit of detection for the
method on effluent wastewater samples was determined according to DIN 32645 based on
linearity and was found at 1.0 g/L for naproxen, 1.1 g/L for diclofenac, 1.2 g/L for
ketoprofen, and 0.8 g/L for ibuprofen, clofibric acid, mefenamic acid and gemfibrozil. The
method has a linear response between the detection limit and at least 100 g/L and a relative
standard deviation (n=9) of 5% for ibuprofen and gemfibrozil; 6% for clofibric acid and
mefenamic acid; 7% for naproxen and ketoprofen, and 8% for diclofenac. Further information
on the analytical method is given by Hey et al. (in press).

2.6. Nitrification capacity measurements
Nitrification capacity measurements were in general made as described by Kristensen et al.
(1992). Fully aerated (5-9 mg/L, O
2
) and completely mixed reactors with activated sludge or
carriers plus wastewater were kept at 20 C. Nutrients and alkalinity (0.672 g/L, NaHCO
3
;

0.236 g/L, (NH
4
)
2
SO
4
; 0.044 g/L, KH
2
PO
4
) were added and samples were taken every 15
minutes for 2 hours. Samples were filtered, Munktell 1002, and nitrogen species (NH
4
+
-N,
NO
3
-
-N and NO
2
-
-N) measured.

2.7. Measurements of nitrogen species, COD, oxygen levels, pH and biomass
Nitrogen and COD fractions were measured spectrophotometrically. Dr-Lange cuvettes were
used to measure NH
4
-N (LCK 303) and COD (LCK 114), while NO
2
-
-N, and NO
3
-
-N were
measured either with Technicon Autoanalyzer II or Dr Lange cuvettes (LCK 341 and LCK
339, respectively). With respect to NO
2
-
-N and NO
3
-
-N, only the sum of the two species was
measured in the reactor with carriers from Klippan. Samples for determination of nitrogen
species and COD in the batch reactors were filtered with GFC-filters (Glass fiber filter, 1.2
m, Whatman) and Munktell 1002 filters (Cellulose filter, 6-10 m, Munktell),
respectively. Oxygen and pH levels were recorded, with electrodes connected to WTW Oxi
730 and WTW pH 320, respectively.

Biomass was measured as suspended solids for the reactors with sludge and as biofilm solids
for the reactors with carriers. The suspended solids concentration was determined, according
to European Standard (EN 872), by the difference in weight of filtered samples, GFA-filters
(Glass fiber filter, 1.6 m, VWR), before and after heating at 105 C for 1 h. Biofilm
solids were determined by the difference in weight of dried carriers (105 C for 1 h) before
and after removal of biofilm. Removal of biofilm solids were made in H
2
SO
4
(4 M) through
mechanical shaking and ultrasonication, followed by thorough brushing.

3. Results and discussion
3.1. Nitrification capacity and reactor conditions
The nitrification capacity per unit biomass was higher for the activated sludges than for the
carriers (Table 2). All reactors with biomass from full-scale plants were run at similar sludge
concentration or carrier filling ratio as at the plant at the time of sampling. Higher biomass
concentration in the carrier systems than in the activated sludge systems could, consequently,
compensate for part of the observed differences in nitrification capacity per unit biomass. The
difference in the nitrification capacity between the first (Nykping 1) and the second
subsequent (Nykping 2) aerated compartment at Nykping WWTP, indicates that ammonia
oxidizing bacteria benefit from the lower organic load in the second aerated compartment.
Continuous formation of NO
x
-
-N through hydrolysis and subsequent nitrification was
observed in the reactors.

For the reactor with carriers from the partial nitritation/anammox sludge liquor treatment, the
initial and final NO
2
-
-N concentrations were 42 and 0.2 mg/L, respectively. Based on the
stoichiometry of the two-step partial nitritation/ anammox process (Gut et al., 2007) and the
initial NO
2
-
-N concentration of 42 mg/L, the nitritation and anammox processes each
contribute to approximately 50% of the NH
4
+
-N removal in the reactor.

Table 2 - Nitrification capacities and reactor conditions
Nitrificationrate
(max)
(mgNO
x

N/(gBS*h))
Biomass
(g/L)

Surface
area
(m
2
/L)
COD
(mg/L)
NH
4
+
N
(mg/L)
NO
x

N
(mg/l)

pH

Initial End Initial End Initial End Initial End
CarriersKlippan 3.1 7.1 0.6* 247 69 <2 <2 56 77 7.2 7.6
CarriersNykping1 0.4 13.4 0.38* 270
E
71 <2 <2 18 34 7.5 7.1
CarriersNykping2 1.2 10.2 0.38* 270
E
69 <2 <2 16 37 7.5 7.1
CarriersSaleboda 1.1 5.4 0.38* 262 66 3.6 <2 8.8 24 8.2 7.7
CarriersHimmerfjrdsverket 13.0 0.36* 278 268 251 <2 173 131 8.0 8.5
CarriersControl 0 0.31* 8.8 8.2
SludgeKllby 4.6 2.0 295 77 <2 <2 7.1 13 8.6 8.6
SludgeKlagshamn 7.1 2.4 291 75 <2 <2 8.6 19 8.1 8.3
Sludgeresundsverket 4.1 4.7 281 75 <2 <2 0.6 6.3 7.8 7.8
*Surfaceareasarecalculatedfromtheprotectedsurfaceareaofnewcarriers,numberofcarriersandliquidvolumeinthereactor
E
EstimatedinitialCODconcentrationbasedontheadditionofpharmaceuticalstogetherwiththesolvent


3.2. Pharmaceutical removal
Figure 1 shows profiles of dissolved pharmaceuticals in six of the experiments, including the
experiments with the highest volumetric nitrification capacities, the experiment with carriers
from the sludge liquor treatment and the carrier control experiment. The carrier control
without biomass confirms that the observed pharmaceutical removal is due to interactions
with the biomass and not to photolysis, stripping or adsorption onto the carrier. As the
reactors with carriers or sludge from the full-scale plants were operated at the same sludge
concentration or carrier filling ratio as at the plant, the results presented in Figure 1 give a
plant related indication of the aerobic pharmaceutical removal at different HRTs. Based on
typical aerobic HRTs for nitrifying activated sludge processes (5-10 h) and MBBR processes
(2-4 h), Figure 1 also indicates that considerably higher removal of several pharmaceuticals
can be expected for MBBR processes compared to nitrifying activated sludge processes.

The first-order removal rate constants presented in Figure 2 were calculated by applying least
square regression to the dissolved pharmaceutical concentrations in the batch reactors, with
subsequent normalization by the biomass concentration. Thus, the rate coefficients
incorporate sorption, desorption, and biological degradation. Sorption to activated sludge is
generally agreed to be a rapid process and Ternes et al. (2004) reported that solid-water
equilibriums were reached within 30 minutes for many pharmaceuticals, including diclofenac,
ibuprofen and clofibric acid. This rapid partitioning of pharmaceuticals between solids and
water suggests that sorption contributes little to the observed pharmaceutical removal in the
sludge reactors.

Figure 1 - Dissolved pharmaceutical concentrations in six reactors.

Low transport rates within the carrier biofilm prolong the time for solid-water equilibriums to
be reached and sorption may contribute to the observed pharmaceutical removal in the biofilm
reactors. For the reactor with carriers from the sludge liquor treatment with a biomass
concentration of 13 g/L, the removal observed between 10 min and 24 h in the water phase
was, however, negligible for all pharmaceuticals except mefenamic acid, which showed a
slight decrease (Figure 1). This indicates that sorption has little direct effect on the observed
removal and that the observed removal is primarily caused by biological
degradation/transformation. Sorption before the initial measurement at 10 min may, however,
induce subsequent desorption, if biological degradation/transformation causes decreasing
concentrations in the water phase.

3.2.1. Removal with carriers and sludges from full-scale plants
Diclofenac and clofibric acid were not removed in the activated sludge reactors, while the
carrier reactors demonstrated clear diclofenac and clofibric acid removal. The rate constants
previously reported for diclofenac and activated sludge show great variability and range from
0.01 to 1.2 L/g SS*d (Suarez et al., 2010; Tran et al., 2009; Urase and Kikuta, 2005) (Table
3). The carrier biomass investigated in the present study fell into that range. In lab scale
biofilm systems, Zwiener and Frimmel (2003) observed no removal of diclofenac under oxic
conditions and slight removal under anoxic conditions in a submerged biofilm reactor. On the
other hand, Grning et al. (2007) observed clear removal of diclofenac by biofilms formed on
river sediments. For clofibric acid, Joss et al. (2006) reported a removal rate constant for
activated sludge that was 3 to 8 times higher compared to the rate constants for carrier
biomass in this study, while the rate constant they presented for MBR sludge was more
similar to those found for carrier biomass in this study. Zwiener and Frimmel (2003) reported,
however, hardly any removal of clofibric acid in reactors with submerged biofilm or activated
sludge and Winkler et al. (2000) observed no removal of clofibric acid in biofilms formed in
river water.

Figure 2 - Relationship between the nitrification capacity and the first order removal rate
constants for reactors with sludge or carriers from the main processes. Note differences in
scaling of y-axis. Tbars indicate 95% confidence intervals.

Mefenamic acid showed a minor removal tendency or no removal at all in the sludge reactors,
while clear removal was observed for the carrier reactors. No rate constant is available in the
literature for mefenamic acid, but the average removal of approximately 30% in activated
sludge treatment plants (Mige et al., 2009) indicates that mefenamic acid is barely removed
in activated sludge.

A more rapid removal of ketoprofen and gemfibrozil was observed for the carrier reactors
than for the sludge reactors. Furthermore, the normalized removal rates confirm a distinct
difference in the ability of the sludge and carrier biomass to remove these substances (Figure
2). For ketoprofen and gemfibrozil, Urase and Kikuta (2005) presented rate constants for
activated sludge that were lower or similar to the rate constants observed for the sludges in
this study, whereas Tran et al. (2009) presented rate constants for activated sludge that were
comparable or lower than those observed for the carrier biomass. In a pilot scale moving bed
biofilm reactor for tertiary treatment, Lundstrm et al. (2010) observed some removal of
ketoprofen, but no removal of gemfibrozil.

For ibuprofen and naproxen, the observed differences in removal between the reactors with
sludge and carriers (Figure 1) were not primarily caused by a disparity in the removal capacity
per unit biomass, but by a higher biomass concentration in the carrier reactors than in the
sludge reactors. The removal rate constants for ibuprofen and naproxen were generally higher
or similar to the rate constants presented for activated sludge by Tran et al. (2009) and Urase
and Kikuta (2005), but lower or similar to the rate constants presented for aerated activated
sludge by Joss et al. (2006) and Suarez et al. (2010). For submerged biofilm reactors,
considerable removal of ibuprofen and naproxen has been observed in a full scale (Joss et al.,
2005) and high removal of ibuprofen has also been confirmed in lab-scale biofilm reactors
(Zwiener and Frimmel, 2003).


Table 3 Removal rate constants


Ibuprofen
(L/gBiomassd)
Naproxen
(L/gBiomassd)
Gemfibrozil
(L/gBiomassd)
Ketoprofen
(L/gBiomassd)
Clofibricacid
(L/gBiomassd)
Mefenamicacid
(L/gBiomassd)
Diclofenac
(L/gBiomassd)
CarriersKlippan 0*15.5 0.91.5 1.72.1 1.63.6 0.120.17 0.290.48 0.260.38
CarriersNykping 1 4.7 0.60.9 0.81.2 0.91.2 0.050.11 0.080.41 0.060.25
CarriersNykping 2 5.3 0.71.2 11.1 1.22.3 0.090.14 0.140.44 0.090.23
CarriersSaleboda 2.23.4 0.10.2 0.61.1 1.21.8 0.060.13 0.170.44 0.130.37
SludgeKllby 2.43.0 0.30.5 0.010.12 0.010.13 0*0.04 0*0.05 0*0.001
SludgeKlagshamn 3.04.8 0.51.0 0.200.27 0.190.32 0*0.02 0*0.05 0*0.01
Sludgeresundsverket 2.64.1 0.61.1 0.120.18 0.080.17 0*0.02 0.010.06 0*0.02
UraseandKikuta, 2005
A
0.209 0.005 0.062 0.031 n.a. n.a. 0.01
Tranetal.,2009
B
3.244.01 0.390.93 1.011.84 0.681.59 n.a. n.a. 0.310.52
Suarezetal.,2010
C
20 9 n.a. n.a. n.a. n.a. 1.2
Jossetal.,2006
D
2135 1.01.9 6.49.6 n.a. 0.30.8 n.a. $0.1
Jossetal.,2006
E
922 0.40.8 0.51.8 n.a. 0.10.23 n.a. $0.1

Rangesindicate95%confidenceintervals,*indicatethatnegativevaluesarecoveredbytheconfidenceinterval,and n.a.indicatesthatnodataisavailable.
A
Activatedsludgefromafullscaleprocess
B
Activatedsludge enrichedin nitrifyingbacteria
C
Activatedsludgefromanaerobiclabscalereactorfeedwithsyntheticwastewater
D
Activatedsludgefromafullscaleprocess

E
Sludgefromapilotscale membranebioreactor

As indicated, the removal rates presented for the target substances in literature are not
completely consistent (Table 3). Joss et al. (2006) suggested that some inconsistent results in
literature may be partly due to: i) the applied pharmaceutical concentration, ii) the conditions
in the treatment process originally sampled (including sludge age, wastewater composition
and other process conditions); and iii) the handling of biomass between sampling and
experiment (including storage and feeding with new substrates). This variability may also be
explained by different conditions in experimental reactors. Based on the experimental setup
for the carriers and sludges from the full-scale plants, it is therefore most likely that the
observed differences in the removal rate constants are due to the biomass origin. Thus, the
carrier biomass in this study have a much higher capacity to remove diclofenac, clofibric acid,
mefenamic acid, gemfibrozil, and ketoprofen than nitrifying activated sludge.

3.2.2. Dependency on autotrophic bacteria
The removal rate constants for ibuprofen and naproxen showed no dependency on the
nitrification capacity, while a clearly positive trend between the nitrification capacity and the
rate constants was observed for ketoprofen, clofibric acid and mefenamic acid in the carrier
reactors (Figure 2). This correlation was, however, restricted to the carrier biomass and the
low rate constants found for these substances in activated sludge with high nitrification
capacities suggest that there is no link between the nitrification capacity and the removal of
ketoprofen, clofibric acid, and mefenamic acid at low NH
4
+
-N concentrations. Furthermore,
the low or negligible removal of pharmaceuticals in the reactor with carriers from the partial
nitritation/anammox sludge liquor treatment indicates that ammonia oxidizing bacteria have
very limited ability to degrade/transform the target pharmaceuticals even at high NH
4
+
-N
concentrations and realistic nitritation rates. Tran et al. (2009) on the other hand observed
decreased removal of diclofenac, ketoprofen, gemfibrozil, naproxen, and ibuprofen when
allylthiourea was added to inhibit nitritation in batch experiments with activated sludge
enriched in ammonia oxidizing bacteria and an initial NH
4
+
-N concentration of 100 mg/L.
Allylthiourea targets ammonia monooxygenase in ammonia oxidizing bacteria, but inhibition
of other bacterial activity have been observed (Hamamura et al., 1999) which might explain
the reduced removal of pharmaceuticals in the presence of this inhibitor. Taken together, the
experiments in this study indicate that nitrifying bacteria contribute very little or not at all to
the removal of the target pharmaceuticals in nitrifying biological treatment systems, and that
the higher removal rates observed for the carrier biomass compared to the sludge biomass
most likely are due to a difference in heterotrophic degradation/transformation of the
pharmaceuticals.

The low or negligible removal of pharmaceuticals in the reactor with carriers from the partial
nitritation/anammox sludge liquor treatment does not only confirm that ammonia oxidizing
bacteria contribute insignificantly to the pharmaceutical removal, but also that anammox
bacteria have a very limited or no ability at all to degrade the target pharmaceuticals.
Furthermore, the experiment indicates that the high sludge ages, required by ammonia
oxidizing bacteria and anammox bacteria, on their own are insufficient to maintain high
pharmaceutical removal.

The causes for the higher pharmaceutical removal with carrier biomass compared to activated
sludge biomass are not well understood. The authors will, however, give two potential
explanations to the observed difference in removal: i) slow growing pharmaceutical
degrading/transforming microorganisms may benefit from a higher biomass retention time in
the carrier biofilm compared to activated sludge, and ii) substrate and redox gradients within
the biofilm may induce highly stratified microbial communities, with microorganisms adapted
to easily degradable organic substrates in the outer part of the biofilm and microorganisms
adapted to the remaining and hardly degradable organic substrates in the inner part of the
biofilm. Microbial degradation/transformation of hardly degradable organic substrates, in the
inner part of the biofilm, may require specific enzymes that can enhance
degradation/transformation of pharmaceuticals and other organic micropollutants.

4. Conclusions
Compared to activated sludge, the biofilm carriers demonstrated considerably higher removal
rates per unit biomass for diclofenac, ketoprofen, gemfibrozil, clofibric acid and mefenamic
acid, whereas similar removal rates were observed for ibuprofen and naproxen.

The three nitrifying sludges did not degrade the primary structure diclofenac and clofibric
acid to a detectable level while the biofilm carriers from the nitrifying plants exhibited a
significant degradation/transformation of these two pharmaceuticals.

Although all treatment plants were run with full nitrification the activated sludge biomass
showed significantly higher nitrification rates than the carrier biomass, which indicates that
the observed difference in pharmaceutical removal is not due to the ammonia oxidizing
bacteria or nitrite oxidizing bacteria, but to a difference in the heterotrophic microbial
community.

The low ability of ammonia oxidizing bacteria to degrade or transform the target
pharmaceuticals was further demonstrated by the limited pharmaceutical removal in an
experiment with continuous nitritation and biofilm carriers from a partial nitritation/anammox
sludge liquor treatment process.

The results demonstrate that moving bed biofilm carriers have a pharmaceutical reduction
potential superior to activated sludge and that further research on micropollutant removal with
biofilm carriers is needed to find the underlying mechanism of the enhanced removal and to
fully explore the potential.

Acknowledgements

The staff at Klagshamn WWTP, Kllby WWTP, resundsverket WWTP, Klippan WWTP,
Nykping WWTP, Saleboda WWTP, and Himmerfjrdsverket WWTP is acknowledged for
providing sludge or carriers for the experiments. This work is part of the MistraPharma
project, which is funded by Mistra, the Foundation for Strategic Environmental Research.

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Supplementary information:
Suspended biofilm carrier and activated
sludge removal of acidic pharmaceuticals

S1. Characteristics of sampled WWTPs

Nykping WWTP receives mainly domestic wastewater from 35,000 people. Incoming
wastewater, with annual average concentrations of approximately 190 mg/L BOD
7
and 40
mg/L N
tot
, is treated mechanically (screening, grit removal and sedimentation). The
subsequent moving bed biofilm reactor, with carriers (K1, AnoxKaldnes) and a filing ratio of
50-60%, is operated in a pre-denitrification mode and the aerated part is compartmentalized in
two sections. The first aerated compartment is intended for BOD removal, whereas the second
aerated compartment is intended for nitrification. Post-precipitation is used for phosphorous
removal. At the time of sampling, the volumetric ratio between anoxic and aerobic zones was
approximately 1:2. The average HRT in the line of sampling is 6-7 h.

Saleboda WWTP receives domestic wastewater from 180 people. Incoming wastewater, with
annual average concentrations of approximately 270 mg/L BOD
7
and 40 mg/L N
tot
, is treated
mechanically (screening and sedimentation). The subsequent moving bed biofilm reactor,
with carriers (K1, AnoxKaldnes) and a filing ratio of 40-60%, is aerated over its full length
and compartmentalized in two equally sized sections. Post-precipitation is used for
phosphorous removal. The average HRT in the Moving bed biofilm reactor is 11-12 h.

Klippan WWTP receives mainly domestic wastewater from 13,000 people. Incoming
wastewater, with annual average concentrations of approximately 100 mg/L BOD
7
and 40
mg/L N
tot
, is treated mechanically (grit removal, screening and sedimentation).The subsequent
biological treatment is divided in two equally sized pre-denitrifying lines with simultaneous
precipitation and industrial potato residues as additional carbon source. The first line is
operated solely with activated sludge. The second line is operated with activated sludge in the
anoxic part and the first half of the oxic zone whereas the second half of the oxic zone is
operated with both activated sludge and carriers (Bio-film Chip M, AnoxKaldnes), 30-40%
filling ratio. At the time of sampling, the volumetric ratio between anoxic and aerobic zones
was approximately 1:1. The average HRT in the line of sampling is approximately 12 h.

Himmerfjrdsverket WWTP with the only sampled sludge liquor treatment process, receives
domestic wastewater from 270,000 people and further a wide range of industries. Reject water
from the mesophilic anaerobic digestion, with annual average concentrations of 300 mg/L
TOC and 900 mg/L NH
4
+
-N is treated with a single reactor partial nitritation/anammox
process with carriers (K1, AnoxKaldnes) and a filing ratio of approximately 30%. The reactor
is compartmentalized, in three sections with intermittent aeration. The oxygen level is kept at
2 mg/L or less during aeration, to enable simultaneous nitritation and anammox activity in the
biofilm. The average HRT in the line of sampling is approximately 35 h.

Kllby WWTP in Lund receives mainly domestic wastewater from 80,000 people. Incoming
wastewater, with annual average concentrations of approximately 180 mg/L BOD
7
and 40
mg/L N
tot
is treated mechanically (screening, grit removal, and sedimentation).The subsequent
low loaded activated sludge process is operated with enhanced biological phosphorous
removal, pre-denitrification with side stream hydrolysis to provide additional carbon source
and with nitrification in the aerated compartments. Post-precipitation is used as a complement
to the enhanced biological phosphorous removal. At the time of sampling, the total sludge age
was 17-18 days, and the volumetric ratio between anaerobic, anoxic, and aerobic zones was
approximately 1:1:7. The average HRT in the line of sampling is approximately 18 h.
Klagshamn WWTP in Malm receives mainly domestic wastewater from 70,000 people.
Incoming wastewater, with annual average concentrations of BOD
7
130 mg/L and N
tot
30
mg/L, is treated mechanically (screening, grit removal, and sedimentation with chemical
precipitation). The subsequent, low loaded activated sludge process is mainly designed for
BOD-removal and nitrification, but it can also be operated with partial pre-denitrification.
Denitrifications occur mainly in a MBBR-process, which is separate from the activated sludge
treatment. At the time of sampling, the total sludge age was approximately 7 days and the
activated sludge basin was aerated over its full length. The average HRT in the line of
sampling is 5-6 h.
resundsverket WWTP in Helsingborg receives wastewater from 120,000 people and further
a wide range of industries. Incoming wastewater, with annual average concentrations of
approximately BOD
7
180 mg/L and N
tot
30 mg/L, is treated mechanically (screening, grit
removal, and sedimentation with primary sludge hydrolysis). The subsequent, low loaded
activated sludge process is operated with enhanced biological phosphorous removal, pre-
denitrification with hydrolysed primary sludge as additional carbon source and nitrification in
the aerated zones. No chemicals for phosphorus removal are used at the plant. At the time of
sampling, the total sludge age was 25-30 days and the volumetric ratio between anaerobic,
anoxic, and aerobic zones was approximately 1:4:5. The average HRT in the line of sampling
is approximately 14 h.