Removal of Antibiotics in
Wastewater: Effect of Hydraulic
and Solid Retention Times on the
Fate of Tetracycline in the Activated
Sludge Process
SUNGPYO KIM,† PETER EICHHORN,‡
JAMES N. JENSEN,†
A . S C O T T W E B E R , † A N D D I A N A S . A G A * ,‡
Department of Civil, Structural, and Environmental
Engineering, State University of New York at Buffalo, 207
Jarvis Hall, Buffalo, New York 14260, and Department of
Chemistry, State University of New York at Buffalo, 611
Natural Science Complex, Buffalo, New York 14260
A study was conducted to examine the influence of
hydraulic retention time (HRT) and solid retention time
(SRT) on the removal of tetracycline in the activated sludge
processes. Two lab-scale sequencing batch reactors
(SBRs) were operated to simulate the activated sludge
process. One SBR was spiked with 250 µg/L tetracycline,
while the other SBR was evaluated at tetracycline
concentrations found in the influent of the wastewater
treatment plant (WWTP) where the activated sludge was
obtained. The concentrations of tetracyclines in the influent
of the WWTP ranged from 0.1 to 0.6 µg/L. Three different
operating conditions were applied during the study
(phase 1sHRT: 24 h and SRT: 10 days; phase 2sHRT:
7.4 h and SRT: 10 days; and phase 3sHRT: 7.4 h and SRT:
3 days). The removal efficiency of tetracycline in phase
3 (78.4 ( 7.1%) was significantly lower than that observed
in phase 1 (86.4 ( 8.7%) and phase 2 (85.1 ( 5.4%) at
the 95% confidence level. The reduction of SRT in phase
3 while maintaining a constant HRT decreased tetracycline
removal efficiency. Sorption kinetics reached equilibrium
within 24 h. Batch equilibrium experiments yielded an
adsorption coefficient (Kads) of 8400 ( 500 mL/g and a
desorption coefficient (Kdes) of 22 600 ( 2200 mL/g. No
evidence of biodegradation for tetracycline was observed
during the biodegradability test, and sorption was found
to be the principal removal mechanism of tetracycline in
activated sludge.
Introduction
The tetracycline group of antibiotics is the second most widely
used antimicrobial in the world, with applications in human
therapy and the livestock industry (1). Only small portions
of tetracycline administered to the treated species are
metabolized or absorbed in the body, with most of the
unchanged form of the drug being eliminated in feces and
urine (2). Residues of tetracyclines have been detected in
* Corresponding author phone: (716)645-6800 x2226; fax: (716)645-6963; e-mail: [email protected].
† Department of Civil, Structural, and Environmental Engineering.
‡ Department of Chemistry.
many surface water resources that receive discharges from
municipal wastewater treatment plants (WWTPs) and agricultural runoff (3-6). A recent study showed that as high
as 4 µg/L tetracycline and 1.2 µg/L chlortetracycline have
been detected in municipal wastewater (7). Further, a
reconnaissance study by the United States Geological Survey
(USGS) reported detectable levels of tetracyclines in several
rivers and streams from many parts of the U.S. (4). Although
tetracyclines are known to be highly sorbed to clay materials,
soil, and sediments (8, 9), their occurrence in surface waters
suggests that their sorption to solids is not irreversible and
that there are conditions that could favor their mobility in
the environment.
The presence of low levels of antibiotics and their
transformation products in the environment could provide
conditions for the transfer and spread of antibiotic resistant
determinants among microorganisms, an emerging issue in
public health (10). There is an increased interest in improving
the removal efficiency of microcontaminants, such as
antibiotics and other pharmaceuticals, in WWTPs (11, 12).
While existing treatment technologies produce water that
satisfies current regulatory standards, it has been demonstrated that the removal of many emerging contaminants,
including antibiotics, personal care products, and hormones,
is incomplete (13). Because of the need to provide sustainable
water supplies to meet the escalating water consumption
associated with population growth and increased agriculture
and industrialization (14), the ability to recover water from
wastewater for reuse is critical. In this regard, it is crucial to
understand the fate of currently unregulated chemicals
introduced into the wastewater.
The activated sludge process is the most common form
of secondary treatment employed in the U.S. (15). It is wellknown that activated sludge process operating conditions
such as solid retention time (SRT) can have a significant
effect on the biodegradation and adsorption of contaminants
during the treatment process (16). To date, only limited
studies have investigated the influence of operating conditions on the removal efficiency of emerging contaminants
such as tetracycline. The study presented in this paper aimed
to (i) estimate the tetracycline concentration in wastewater
and the removal efficiencies in a laboratory-scale activated
sludge process under various operating conditions and to
(ii) determine the extent of tetracycline removal resulting
from adsorption and biodegradation. Two sequencing batch
reactors (SBR) operated at different SRT and hydraulic
retention times (HRT) were employed to simulate a typical
activated sludge process in the laboratory. On the basis of
literature review, two major study hypotheses were tested:
(i) the removal of tetracycline in biological WWTPs is a
function of operational conditions such as HRT or SRT and
(ii) the fate of tetracycline in biological WWTPs is largely
influenced by adsorption processes rather than biodegradation.
Although the persistence of tetracyclines has been demonstrated in agricultural soils that received antibioticcontaining manure (17, 18), there is little literature on the
biodegradation of tetracyclines in secondary biological
wastewater treatment plants. Recently, the mechanisms of
tetracycline adsorption on clays and the factors that affect
their sorption in soil have been described (19, 20). However,
the sorption behavior of tetracyclines in sludge may differ
significantly from that in clay or soil due to the high organic
matter content and complex nature of the mixed liquor
present in biological wastewater treatment plants. To address
the fate of tetracycline, additional experiments were per-
day at the end of the aeration period. The biomass concentration, determined by the Standard Methods 2540D and
2540E (21), and the pH of the wastewater in the SBR were
measured frequently. The ranges and mean values are
compiled in Table 2.
The concentration of dissolved oxygen (DO) in each
reactor was always >2 mg/L, as measured three times a week
during the aeration time using a DO meter (Model 54A, Yellow
Springs Instrument, Yellow Springs, OH).
FIGURE 1. Schematic diagram of the sequencing batch reactor
(SBR). [TC: tetracycline].
formed to evaluate the relative contribution of adsorption/
desorption and biodegradation on the observed removal of
tetracycline.
Materials and Methods
Design and Operation of Sequencing Batch Reactors. To
evaluate the tetracycline fate in the activated sludge process,
two identical sequencing batch reactors (SBR-1 and SBR-2)
were built. The operation of SBR-2 differed from SBR-1 in
that the wastewater was amended with tetracycline. The
schematic diagram of the experimental setup is presented
in Figure 1.
Each SBR consisted of an open 5 L Plexiglass cylinder
loaded with 4 L of wastewater. Liquid agitation of the SBR
contents was achieved through a Fisher direct-drive stirrer
(Fisher Scientific Co, Pittsburgh, PA). Humidified compressed
air was used for aeration, introduced into the water through
a Pyrex glass-fritted diffuser ensuring enhanced oxygen
transfer efficiency. Influent wastewater of the reactor was
stored in a 20 L Nalgene carboy, which was continuously
stirred to maintain a mixed feed. Influent was pumped from
the storage carboy through Tygon tubing (0.5 in i.d.) into the
5 L SBR via a Masterflex pump (no. 17 head, 6-600 rpm,
Cole-parmer, Chicago, IL). The decanting procedure from
the SBR was controlled by a Masterflex pump (no. 14 head
6-600 rpm) connected to Nalgene 890 Teflon FEP tubing
(3/16 in. i.d). All pumping and mixing cycles in the SBR were
controlled by a Chrontrol programmable timer (Model CD,
Lindburg Enterprises, San Diego, CA). A Masterflex pump
(no. 13 head, 1-100 rpm) was used to deliver an aqueous
tetracycline solution (100 mg/L, freshly prepared each day)
to achieve a SBR-2 influent concentration of 250 µg/L. The
aluminum foiled 125 mL Kimax Elenmeyer flask was used
for tetracycline stock solution storage. Activated sludge for
initial inoculation of both reactors was collected from the
Amherst, NY first stage activated sludge aeration tanks, while
the effluent from the primary clarifier at the same plant was
used as influent wastewater for both SBRs. Collection of the
primary clarifier effluent for use as an SBR influent was
conducted twice a week in 30 L carboys and stored at 4 °C
until used. The two SBRs were subjected to three different
operating conditions during the course of the study according
to the schedule listed in Table 1.
In phase 1, each SBR was operated with an SRT of 10 days
and a hydraulic retention time (HRT) of 24 h. These conditions
are similar to those of an oxidation ditch (HRT: 8-36 h and
SRT: 10-30 days) (15). In phase 2, the reactors were operated
under the same SRT as in phase 1 but with a shorter HRT
of 7.4 h. These operating conditions are consistent with a
conventional activated sludge process (HRT: 4-8 h and
SRT: 5-15 days) (15). In phase 3, the SRT was set at 3 days
while keeping the HRT at 7.4 h. This SRT is at the low end
of a conventional biological wastewater treatment. The target
SRT was maintained by manually wasting SBR biomass every
Determination of Tetracyclines in SBR Influent and
Effluent using ELISA. To monitor the tetracycline concentration in SBR influent and effluent, a commercially available
96-well microtiter plate tetracycline enzyme-linked immunosorbent assay (ELISA) (R-Biopharm GmbH, Darmstadt,
Germany) was employed. The ELISA procedure provided in
the instruction manual (RIDASCREEN Tetracycline, RBiopharm GmbH, Darmstadt, Germany) was followed.
Briefly, samples or standards (50 µL) were added to the BSAtetracycline coated microwells, followed by a solution of antitetracycline antibodies (50 µL). The mixture was gently mixed
in a plate shaker for 1 h at room temperature. After washing
the wells with phosphate buffered saline and with Tween 20,
a solution of a peroxidase-conjugated secondary antibody
(100 µL) against the anti-tetracycline antibodies was added
into each well and incubated for 15 min at room temperature.
The wells were washed again, then, a 1:1-mixture (100 µL per
well) of substrate (urea peroxide) and chromogen (tetramethylbenzidine) was added and incubated for another 15
min. Finally, the reaction was stopped by adding 100 µL of
1 M sulfuric acid into each well. The absorbance was
measured at 450 nm in a plate reader, and the amount of
tetracycline present in the samples was calculated based on
the four-parameter fit calibration curve using the KC4
software (Bio-Tek instruments, Winooski, VT).
Sorption Kinetics of Tetracycline onto Activated Sludge.
Biomass from the first stage activated sludge aeration tanks
of the Amherst WWTP was collected for sorption experiments.
Amherst WWTP activated sludge was used in these studies
because it has a low natural tetracycline loading. In addition,
it was the source of the inocula for these studies and would
be similar to the lab biomass because it has been developed
with the same wastewater. One experiment was carried out
with unwashed activated sludge (biomass concentration:
3600 mg/L). This concentration is typical of Amherst WWTP
operation and was used to represent adsorption phenomenon
in a full-scale activated sludge process. In a second adsorption
experiment, washed activated sludge at a biomass concentration of 1000 mg/L was used to mimic the biomass
concentrations in the studied SBRs (see Table 2). The washing
procedure with 10 mM phosphate buffer (pH 7.2) was
performed three times as follows: biomass was separated
from the water by centrifugation at 2000g for 3 min, the
supernatant was discarded, and the biomass was resuspended in 10 mM phosphate buffer. In both experiments,
150 mL aliquots of the sludge were inoculated in duplicate
in 250 mL Erlenmeyer flasks wrapped in aluminum foil to
prevent possible photodegradation of tetracycline. As a
control sample, a 10 mM phosphate buffer was used. To
minimize any tetracycline elimination due to biotic processes,
0.15 g (0.1%, w/v) of sodium azide was added into each flask.
The flasks were shaken for 30 min at 150 revolutions per
minute (rpm) on an orbital shaking table and then a 100 µL
aliquot of a tetracycline stock solution was added to each
flask to achieve a concentration of 250 µg/L. Aliquots of 1500
µL were withdrawn from each solution after 0.1, 1, 2, 4, 7,
and 24 h. After centrifugation at 2000g for 3 min, the
supernatants were transferred into 2 mL vials for analysis by
liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS), which was done immediately. The
TABLE 1. Operating Schedule of Sequencing Batch Reactorsa
operating times in each cycle
phase
duration
(days)b
HRT
(h)
SRT
(days)
feeding
(min)
aeration
(min)
settling
(min)
decanting
(min)
total
(h)
# of cycles
(days-1)
treated wastewater
(L/day)
1
2
3
56
37
48
24
7.4
7.4
10
10
3
2
2
2
598
298
298
90
45
45
30
15
15
12
6
6
2
4
4
4
13
13
a HRT: hydraulic retention time and SRT: solid retention time.
between phases.
b
Actual sampling collecting periods. These periods exclude any transition period
TABLE 2. Mean Biomass Concentration and pH in Sequencing Batch Reactors (SBR) during the Three Operational Phases
phase 1
SBR-1
biomass concentration
[mg/L] (range)
pH (range)
phase 2
SBR-2
SBR-1
SBR-1
SBR-2
509
514
1246
1191
950
933
(440-620)
7.38
(7.00-7.52)
(360-640)
7.27
(6.93-7.57)
(950-1375)
7.17
(6.70-7.54)
(890-1345)
7.24
(6.71-7.60)
(620-1460)
7.26
(6.59-7.72)
(700-1220)
7.27
(7.00-7.67)
tetracycline concentrations were measured using external
calibration and plotted against equilibration time.
Activated Sludge Tetracycline Adsorption and Desorption Coefficients. On the basis of the results of the kinetic
experiments, 24 h proved sufficient to reach the adsorption
equilibrium of tetracycline onto activated sludge. For the
equilibrium experiments, a total of ten 40-mL conical
polyethylene tubes were prepared. Of these tubes, eight were
filled in duplicate at biomass concentrations of 500, 1000,
1500, and 2000 mg/L (biomass prewashed and resuspended
in 10 mM phosphate buffer pH 7.2 as described previously)
and spiked with an aqueous tetracycline stock solution to
achieve a final concentration of 250 µg/L. One of the
remaining tubes, containing 10 mM phosphate buffer, was
used as a control to monitor the tetracycline stability during
the experiment, while another tube was amended with
biomass at a concentration of 2000 mg/L but without adding
tetracycline to account for any desorption of tetracycline
from the native sludge. As in the kinetic sorption test, 0.1%
sodium azide was added into each tube to minimize any
tetracycline elimination due to biotic processes. All test
mixtures were agitated using an orbital shaker for 24 h and
were protected from light to prevent possible photodegradation of tetracycline. After a 24 h equilibration period, the
supernatant was quantitatively separated from the biomass
by centrifugation at 2000g for 5 min. An aliquot was used for
the determination of the tetracycline concentration in the
dissolved phase employing LC-ESI-MS. For the desorption
experiment, the biomass in each tube from the adsorption
work was resuspended with 40 mL of 10 mM phosphate buffer
and agitated for another 24 h. After centrifugation, an aliquot
of the supernatant was subjected to LC-ESI-MS analysis.
For the determination of the adsorption coefficient (Kads),
the adsorbed tetracycline concentration in the biomass, Cs,
was calculated using eq 1
Cs )
phase 3
SBR-2
X (C0 - Ce)V C0 - Ce
)
)
M
CB V
CB
(1)
where X is the total mass of tetracycline in the biomass, M
is the total dried weight of the biomass, CB is the biomass
concentration, V is the solution volume, C0 is the initial
tetracycline concentration, and Ce is the final tetracycline
concentration in the liquid phase after 24 h of equilibration.
The change in volume of the test mixture in the bioreactors
due to the added tetracycline stock solution is negligible
(<0.001%) and is therefore ignored in the calculations. The
final concentration in the liquid phase, Ce, was determined
using LC-ESI-MS. The initial concentration used in the
calculations was the nominal amount (250 µg/L) of tetracycline added to the solution. The tetracycline Kads, defined
as the ratio of the equilibrium concentration of tetracycline
in the sludge relative to the concentration remaining in the
liquid phase (as expressed in eq 2) can be derived from the
slope of the plot of Cs (in mg/g) versus Ce (in mg/mL).
Kads ) Cs/Ce
(2)
The desorption coefficient (Kdes) was determined by reequilibrating the biomass with a known solid-phase concentration of tetracycline (from the earlier adsorption tests)
in 40 mL of 10 mM phosphate buffer for 24 h. After
reequilibrating the sample and determining the liquid-phase
concentration (Ce), a mass balance was used to determine
the new solid-phase concentration (Cs). Kdes was then
calculated using the same equation used to obtain Kads.
Tetracycline Biodegradability. An additional batch experiment was carried out to investigate the biodegradability
of tetracycline and the possible formation of microbial
metabolites using biomass collected from SBR-2 in phase 3
(SRT: 3 days). The procedure for the setup and operation of
the batch reactor was as follows (in duplicate): a 4 L amber
glass bottle was amended with a 200 mL aliquot of biomass
from SBR-2 and diluted with 3800 mL of distilled water. Air
was introduced continuously into the test medium to
maintain aerobic conditions, and continuous mixing using
a 6 mm Teflon tubing with perforations at the bottom outlet
was performed. An aliquot of a freshly prepared aqueous
tetracycline solution (1000 mg/L) was spiked into the reactor
to achieve a test concentration of 200 µg/L. All tests were
conducted under dark conditions to prevent possible photodegradation of tetracycline. Two types of duplicate batch
reactors were used for this additional experiment: biodegradation and control. The two control batch reactors were
amended with 0.1% sodium azide to minimize any tetracycline elimination by microbial activity. The first sample of
4500 µL was taken 5 min after spiking the tetracycline to
these reactors and was transferred into an amber vial
containing 500 µL of McIlvaine buffer (pH 4.0; added 0.1 M
EDTA-Na2). Prior to analysis by LC-ESI-MS, a sample aliquot
was centrifuged at 2000g for 4 min, and the supernatant was
transferred into an amber autosampler vial.
FIGURE 2. Time profile of tetracycline concentration in influent and effluent of SBR-1 and SBR-2.
TABLE 3. Tetracycline Concentrations (Mean Value ( Standard Deviation) and Removal Efficiencies during the Three Operational
Phases
phase 1
SBR-1
influent (µg/L)
effluent (µg/L)
removal efficiencies (%)
a
0.2 ( 0.1
0.2 ( 0.1
n.d.b
Spiked tetracycline concentration;
b
phase 2
SBR-2
250a
33.9 ( 21.8
86.4 ( 8.7
phase 3
SBR-1
SBR-2
SBR-1
SBR-2
0.4 ( 0.1
0.1 ( 0.1
n.d.
250
37.2 ( 13.6
85.1 ( 5.4
0.4 ( 0.3
0.2 ( 0.1
n.d.
250
53.9 ( 17.7
78.4 ( 7.1
n.d.) not determined.
Liquid Chromatography-Electrospray Ionization-Mass
Spectrometry. The liquid chromatograph used was an Agilent
Series 1100 comprising the following modular components:
a quaternary pump, a microvacuum solvent degasser, and
an autosampler with thermostated 100-well tray, set to 4 °C.
Separations were achieved on a Thermo Hypersil-Keystone
BetaBasic-18 100 mm × 2.1 mm (5 µm) column equipped
with a 10 mm × 2.1 mm guard column of the same packing
material. The mobile phases were (A) water acidified with
0.3% formic acid and (B) acetonitrile. The gradient program
started from 90% A to 10% B (1 min). The portion of A was
linearly decreased to 45% within 11.6 min and further to 5%
within 0.1 min. These conditions were held for 3.5 min. The
initial mobile phase composition was restored within 0.1
min and maintained for column regeneration for another
6.7 min resulting in a total run time of 23 min. The flow rate
was 250 µL/min, and the injection volume was 20 µL. During
the first 2 min and the last 6.7 min of each chromatographic
run, the LC stream exiting the analytical column was directed
to the waste via a programmable switching valve integrated
in the mass spectrometer. The mass spectrometric analysis
was performed on an Agilent Series 1100 SL single-quadrupole
instrument equipped with an electrospray ionization (ESI)
source. A capillary voltage of +4000 V was applied to the
nebulizer needle tip to generate protonated molecular ions
[M + H]+ of the target analytes. Nitrogen was used as nebulizer
gas (35 psi) as well as a drying gas at a temperature of 350
°C and a flow rate of 10 L/min. Molecular ions of the target
analytes were recorded at m/z 445 for tetracycline using
fragmentor values of 140. For confirmation purposes, the
m/z 410 for the tetracycline fragment ion was included.
Quantification was done by external calibration using
standard solutions in the range of 2-250 µg/L. The detection
limit for tetracycline based on a signal-to-noise ratio of 3
was between 0.2 and 0.8 µg/L.
Statistical Analysis. To test the significance of the
differences in the mean values of the results from the various
experimental conditions, a t-test with one tail was performed
at the 95% confidence level. This test determines if the mean
value of detected tetracycline concentrations in the effluent
of the SBR in one phase is significantly higher or lower (one
tail) than with another phase tested. Before conducting the
t-test, data were tested for their normality by the ShapiroWilk method provided by the Origin pro 7.0 (Origin) software
program and showed to follow a normal distribution under
95% confidence level.
Results and Discussion
Behavior of Tetracycline at Different SBR Operating
Conditions. The tetracycline concentrations in the influent
and effluent of the SBRs were measured using ELISA. This
technique detects all tetracycline derivatives including
tetracycline, chlortetracycline, doxycycline, oxytetracycline,
and their transformation products (22). The results are
therefore more appropriately reported as total tetracyclines.
The detection limit of the ELISA in wastewater is 0.1 µg/L
total tetracyclines (Instruction Manual: RIDASCREEN Tetracycline, R-Biopharm GmbH, Darmstadt, Germany). The
time profiles of total tetracycline concentrations in the
influent and effluent of SBR-1 and SBR-2 are presented in
Figure 2. Calculated average concentrations in the influent
and effluents of the two reactors are given along with the
removal efficiencies in Table 3. The background total
tetracycline concentrations in the influent of the SBR (i.e.,
in the effluent from the primary clarifier of the Amherst
FIGURE 3. Time profile of tetracycline residue percentages under two different biomass concentrations. (Error bars correspond to one
standard deviation.)
WWTP) were below 1 µg/L throughout the operation time of
the SBR (Figure 2).
Photodegradation is known as one of the main transformation reactions of tetracyclines in the environment.
However, the focus of this study was to determine the role
of biomass for removing tetracycline in biological wastewater
treatment plants; therefore, potential photodegradation was
eliminated by protecting the test liquor from light. Other
known abiotic transformations of tetracyclines are isomerization and epimerization, which are highly pH dependent
and reversible. The ELISA method measures total tetracyclines, which include all the isomers and epimers of
tetracyclines. The tetracycline concentrations in Amherst
WWTP are similar to those previously reported in the
literature. In monitoring studies conducted at six U.S.
treatment plants, which applied different treatment technologies, the tetracycline concentrations were between 0.27
and 4 µg/L in the untreated sewage and between 0.23 and
1.2 µg/L in the treated effluent samples (7, 23). In our study,
the total tetracycline concentrations in the SBR-1 effluent
appear to be generally lower as compared to the influent
wastewater, but it is difficult to assess if this difference was
due to elimination in the bioreactor or due to the intra-assay
variability typical of ELISA analysis. Therefore, the removal
efficiency in SBR-1 was not determined. In the case of SBR2, a substantial difference between the initial total tetracycline
concentration (spiking level 250 µg/L) and the final concentration in the effluent was obtained, as presented in Table
3. Total tetracycline concentrations determined in the SBR-2
effluent ranged from 10 to 84 µg/L. On the basis of the average
concentrations given in Table 3 and an initial concentration
of 250 µg/L (background concentration neglected), the
removal efficiencies for SBR-2 amounted to 86% in phase 1,
85% in phase 2, and 78% in phase 3. Statistical evaluation
of these data using t-tests showed that there was no significant
differences at a 95% confidence level between phase 1 and
phase 2 mean total effluent tetracycline concentrations (p )
0.366). These results suggest that lowering the hydraulic
retention time from 24 to 7.4 h, which also resulted in an
increase in the mean SBR biomass concentration from 514
to 1191 mg/L, did not influence tetracycline removal.
However, decreasing the SRT of SBR-2 to 3 days in phase 3
resulted in a significant reduction in tetracycline removal
when compared to the 10 days SRT used in phase 1 (p )
0.029) and phase 2 (p ) 0.032).
Adsorption and Desorption of Tetracycline. Removal of
tetracycline from the dissolved phase in SBR-2 as shown in
Figure 2 may be achieved either through adsorption and/or
biodegradation. Partitioning onto the suspended matter is
expected to play a key role since tetracyclines, despite their
high water solubility and low n-octanol/water partition
coefficients, are reported to sorb strongly onto soil (24). Ionic
interactions and the metal-complexing properties of tetracyclines have been found to largely govern its adsorption
behavior.
To investigate the adsorption behavior of tetracycline, a
kinetic study was carried out at two different biomass
concentrations. Figure 3 shows the time profile of tetracycline
at biomass concentrations of 1000 and 3600 mg/L. As can be
seen, more than 75 and 95%, respectively, of the tetracycline
initially present at 250 µg/L was removed from the dissolved
phase after an equilibration time of only 1 h, indicating a
very fast sorption onto the sludge. Equilibrium concentrations
were achieved quickly at 3600 mg/L biomass and stayed
virtually unchanged over the 24 h study. On the basis of this
adsorption kinetic test, it was assumed that 24 h was sufficient
time to reach equilibrium for both adsorption and desorption
tests. Other researchers (9, 25) also used 24 h as an
equilibration time for tetracycline adsorption/desorption
tests in soil. The sorption isotherm of tetracycline on activated
sludge is presented in Figure 4. The calculated Kads was 8400
( 500 mL/g (standard error of slope). This is about three
times that reported for the more polar oxytetracycline on
activated sludge (3020 mL/g) (26).
This value of Kads is substantially higher than has been
reported for tetracycline in soils (400 and 1140 mL/g) (24).
The calculated desorption isotherm of tetracycline from
activated sludge is presented in Figure 4. The calculated
desorption coefficient (Kdes) was 22 600 ( 2200 mL/g and is
more than three times higher than Kads. The difference
between Kads and Kdes suggests that a portion of adsorbed
tetracycline does not readily desorb from activated sludge,
thereby displaying adsorption/desorption hysteresis. Adsortion/desorption hysteresis of trace chemicals such as
proteins and metals on activated sludge is well-documented
(27, 28). The sludge adsorption experiments indicated that
elimination of tetracycline from the sewage in SBR is
influenced strongly by the sorption onto the biomass.
Biodegradability of Tetracycline. What is unclear from
the data presented thus far is the role of biodegradation in
FIGURE 4. Adsorption and desorption isotherms for tetracycline on sludge. [Kads: sorption coefficient coefficient and Kdes: desorption
coefficient (error bars correspond to one standard deviation)].
difficult to rule out the potential competing effects of HRT
and biomass concentration on tetracycline removal. In phase
1, longer HRT resulted in low biomass concentrations, while
in phase 2, shorter HRT resulted in an increase in the biomass
concentration. The longer HRT could have promoted equilibrium or near equilibrium conditions (more complete) in
phase 1, while the higher biomass concentrations in phase
2 could have compensated for shorter reaction times.
FIGURE 5. Time profile of tetracycline in a batch reactor spiked
with 200 µg/L.
the observed removal of tetracycline. To examine whether
the exposure of the sludge bacteria to elevated tetracycline
concentrations over an extended period of time led to an
acclimation to the substrate, a biodegradability assay was
conducted using sludge from SBR-2 in phase 3. To this end,
batch reactors containing 20-fold diluted sludge were spiked
with 200 µg/L tetracycline, and the concentration profile was
determined by LC-ESI-MS analysis. Control reactors,
amended with 0.1% sodium azide to inhibit microbial activity,
were used to account for sorptive effects. The profiles shown
in Figure 5 reveal a decrease in concentration of tetracycline
in both reactor types. LC-ESI-MS analysis of the liquid phase
for known degradation products such as 4-epi-tetracycline
and anhydrotetracycline (29) as well as for novel metabolites
did not show the production of new compounds. From the
tetracycline biodegradability test (Figure 5), the strong
similarity between inhibited and noninhibited biomass and
the lack of tetracycline metabolites strongly suggests that
sorption is the primary mechanism for tetracycline removal
observed in phase 3 instead of biodegradation.
From the tetracycline removal data, it is tempting to
conclude that SRT is a more important variable than HRT.
However, even though tetracycline removal efficiencies are
not statistically different between phase 1 and phase 2, it is
The reduction of SRT from 10 to 3 days in phase 3, while
maintaining a constant HRT of 7.4 h, did result in a significant
reduction in tetracycline removal. There are a number of
plausible explanations for this reduction in tetracycline
removal that are driven by changes in biomass physiology
and/or biomass quantity. It is well-documented that changes
in SRT reduce biodegradation efficiency, and this loss of
efficiency is most notable for difficult to degrade compounds
that support low biomass growth rates (15, 16, 30). In this
study, it was determined that under phase 3 SRT conditions,
no biodegradation of tetracycline was observed. Tetracycline
biodegradation was not assessed directly in phases 1 and 2,
which operated at a longer SRT. To date, there is little evidence
in the literature to suggest biodegradation as a likely removal
mechanism. If biodegradation was not responsible for the
reduced tetracycline removal efficiency in phase 3, then SRT
influence on sorption is of interest. Reducing the SRT in
phase 3 did reduce the biomass concentration as shown in
Table 2, and this reduction would favor less sorption and
less removal assuming that the biomass sorption characteristics were unchanged between phases. Sorption characteristics of the biomass may have changed with SRT. Several
researchers have observed increased biomass hydrophobicity
at higher SRTs (31, 32). In fact, recent work by Harper and
Yi (33) has shown that a bioreactor configuration can have
a significant influence on biomass hydrohobicity and particle
size, which can affect the bioavailability and fate of pharmaceuticals in WWTPs because of their impact on particle
floc characteristics. Even though tetracycline has a low
n-octanol/water partition coefficient, at certain pH values,
hydrophobic interactions still play a role for the sorption of
tetracycline on soil or clay (20). At the pH values observed
for the SBR in this study (Table 2), tetracycline is zwitterionic
(no net charge); therefore, hydrophobic interactions with
sludge become a relatively important sorption mechanism.
Finally, reductions in bacterially produced dissolved organic
matter (DOM) concentrations at lower SRTs (34) may have
reduced sorption at the lower SRT of phase 3. Which of the
previous factors was most important in the reduction of
tetracycline removal in this study is unclear and deserves
further study.
Implications in Wastewater Treatment. It is clear from
the data obtained in this study that biomass is an important
sink for tetracycline antibiotics in wastewater treatment
systems. This study provides an important basis for optimizing operational parameters in WWTPs to improve removal
of other micropollutants in wastewater. For example, identification of the critical SRT values and temperature could
be crucial to effectively remove recalcitrant micropollutants
from wastewater. It is highly likely that many of the
pharmaceutical pollutants that are resistant to biodegradation in WWTPs are also eliminated in the effluent by sorption
on sludge. For instance, the ciprofloxacin antibiotic, which
has similar sorption characteristics to tetracycline (24), was
reported to be 95% adsorbed on the sludge or effluent solids
from a Swiss wastewater treatment plant (35). On the basis
of these results, membrane bioreactor (MBR) technology
could become more popular for treating municipal wastewater soon because the MBR process can be operated under
conditions with very long SRT and high biomass concentrations as compared to a conventional activated sludge process
(31). Therefore, the influence of SRT and biosorption on the
removal of micropollutants in the WWTPs should be examined for important micropollutants, particularly those that
have already been shown to be persistent in surface waters
and are of ecological concern.
Finally, it is important to recognize that nonbiodegraded
tetracycline that sorb to biomass can become bioavailable
when localized conditions are changed such that desorption
becomes favorable. In such a case, the amount of tetracycline
sorbed in the biomass will become an issue during the land
application of biosolids because of the potential ecological
risks posed by antibiotics in the environment. Therefore, it
is important to measure not only the overall percent removal
of pharmaceuticals in WWTPs but also the quantities of the
sorbed portion in the biosolids. Finally, it is obvious from
this study that different activated sludge operational strategies
play a key role in defining the removal efficiency of
tetracycline in wastewater. Engineering design and operational decisions can influence the ultimate fate of pharmaceuticals in WWTPs and deserve greater attention in pharmaceutical fate studies.
Acknowledgments
The authors thank the Town of Amherst WWTP No. 16 facility
for allowing us to access the plant and obtain samples from
the treatment tanks. Part of this work was funded by the
National Science Foundation Grant 023700. Any opinions,
findings, and conclusions or recommendations expressed
in this material are those of the authors and do not necessarily
reflect the views of the NSF.
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Received for review January 2, 2005. Revised manuscript
received May 7, 2005. Accepted May 10, 2005.
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