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TABLE 2. Mean Biomass Concentration and pH in Sequencing Batch Reactors (SBR) during the Three Operational Phases
phase 1 phase 2 phase 3
SBR-1 SBR-2 SBR-1 SBR-2 SBR-1 SBR-2
biomass concentration 509 514 1246 1191 950 933
[mg/L] (range)
(440-620) (360-640) (950-1375) (890-1345) (620-1460) (700-1220)
pH (range) 7.38 7.27 7.17 7.24 7.26 7.27
(7.00-7.52) (6.93-7.57) (6.70-7.54) (6.71-7.60) (6.59-7.72) (7.00-7.67)
tetracycline concentrations were measured using external (<0.001%) and is therefore ignored in the calculations. The
calibration and plotted against equilibration time. final concentration in the liquid phase, Ce, was determined
Activated Sludge Tetracycline Adsorption and Desorp- using LC-ESI-MS. The initial concentration used in the
tion Coefficients. On the basis of the results of the kinetic calculations was the nominal amount (250 µg/L) of tetra-
experiments, 24 h proved sufficient to reach the adsorption cycline added to the solution. The tetracycline Kads, defined
equilibrium of tetracycline onto activated sludge. For the as the ratio of the equilibrium concentration of tetracycline
equilibrium experiments, a total of ten 40-mL conical in the sludge relative to the concentration remaining in the
polyethylene tubes were prepared. Of these tubes, eight were liquid phase (as expressed in eq 2) can be derived from the
filled in duplicate at biomass concentrations of 500, 1000, slope of the plot of Cs (in mg/g) versus Ce (in mg/mL).
1500, and 2000 mg/L (biomass prewashed and resuspended
in 10 mM phosphate buffer pH 7.2 as described previously) Kads ) Cs/Ce (2)
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 The desorption coefficient (Kdes) was determined by re-
used as a control to monitor the tetracycline stability during equilibrating the biomass with a known solid-phase con-
the experiment, while another tube was amended with centration of tetracycline (from the earlier adsorption tests)
biomass at a concentration of 2000 mg/L but without adding in 40 mL of 10 mM phosphate buffer for 24 h. After
tetracycline to account for any desorption of tetracycline reequilibrating the sample and determining the liquid-phase
from the native sludge. As in the kinetic sorption test, 0.1% concentration (Ce), a mass balance was used to determine
sodium azide was added into each tube to minimize any the new solid-phase concentration (Cs). Kdes was then
tetracycline elimination due to biotic processes. All test calculated using the same equation used to obtain Kads.
mixtures were agitated using an orbital shaker for 24 h and Tetracycline Biodegradability. An additional batch ex-
were protected from light to prevent possible photodegra- periment was carried out to investigate the biodegradability
dation of tetracycline. After a 24 h equilibration period, the of tetracycline and the possible formation of microbial
supernatant was quantitatively separated from the biomass metabolites using biomass collected from SBR-2 in phase 3
by centrifugation at 2000g for 5 min. An aliquot was used for (SRT: 3 days). The procedure for the setup and operation of
the determination of the tetracycline concentration in the the batch reactor was as follows (in duplicate): a 4 L amber
dissolved phase employing LC-ESI-MS. For the desorption glass bottle was amended with a 200 mL aliquot of biomass
experiment, the biomass in each tube from the adsorption from SBR-2 and diluted with 3800 mL of distilled water. Air
work was resuspended with 40 mL of 10 mM phosphate buffer was introduced continuously into the test medium to
and agitated for another 24 h. After centrifugation, an aliquot maintain aerobic conditions, and continuous mixing using
of the supernatant was subjected to LC-ESI-MS analysis. a 6 mm Teflon tubing with perforations at the bottom outlet
For the determination of the adsorption coefficient (Kads), was performed. An aliquot of a freshly prepared aqueous
the adsorbed tetracycline concentration in the biomass, Cs, tetracycline solution (1000 mg/L) was spiked into the reactor
was calculated using eq 1 to achieve a test concentration of 200 µg/L. All tests were
conducted under dark conditions to prevent possible pho-
todegradation of tetracycline. Two types of duplicate batch
X (C0 - Ce)V C0 - Ce
Cs ) ) ) (1) reactors were used for this additional experiment: biodeg-
M CB V CB radation and control. The two control batch reactors were
amended with 0.1% sodium azide to minimize any tetra-
where X is the total mass of tetracycline in the biomass, M cycline elimination by microbial activity. The first sample of
is the total dried weight of the biomass, CB is the biomass 4500 µL was taken 5 min after spiking the tetracycline to
concentration, V is the solution volume, C0 is the initial these reactors and was transferred into an amber vial
tetracycline concentration, and Ce is the final tetracycline containing 500 µL of McIlvaine buffer (pH 4.0; added 0.1 M
concentration in the liquid phase after 24 h of equilibration. EDTA-Na2). Prior to analysis by LC-ESI-MS, a sample aliquot
The change in volume of the test mixture in the bioreactors was centrifuged at 2000g for 4 min, and the supernatant was
due to the added tetracycline stock solution is negligible 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 phase 2 phase 3
SBR-1 SBR-2 SBR-1 SBR-2 SBR-1 SBR-2
influent (µg/L) 0.2 ( 0.1 250a 0.4 ( 0.1 250 0.4 ( 0.3 250
effluent (µg/L) 0.2 ( 0.1 33.9 ( 21.8 0.1 ( 0.1 37.2 ( 13.6 0.2 ( 0.1 53.9 ( 17.7
removal efficiencies (%) n.d.b 86.4 ( 8.7 n.d. 85.1 ( 5.4 n.d. 78.4 ( 7.1
a Spiked tetracycline concentration; b n.d.) not determined.
WWTP) were below 1 µg/L throughout the operation time of Adsorption and Desorption of Tetracycline. Removal of
the SBR (Figure 2). tetracycline from the dissolved phase in SBR-2 as shown in
Photodegradation is known as one of the main trans- Figure 2 may be achieved either through adsorption and/or
formation reactions of tetracyclines in the environment. biodegradation. Partitioning onto the suspended matter is
However, the focus of this study was to determine the role expected to play a key role since tetracyclines, despite their
of biomass for removing tetracycline in biological wastewater high water solubility and low n-octanol/water partition
treatment plants; therefore, potential photodegradation was coefficients, are reported to sorb strongly onto soil (24). Ionic
eliminated by protecting the test liquor from light. Other interactions and the metal-complexing properties of tetra-
known abiotic transformations of tetracyclines are isomer- cyclines have been found to largely govern its adsorption
ization and epimerization, which are highly pH dependent behavior.
and reversible. The ELISA method measures total tetracy- To investigate the adsorption behavior of tetracycline, a
clines, which include all the isomers and epimers of kinetic study was carried out at two different biomass
tetracyclines. The tetracycline concentrations in Amherst concentrations. Figure 3 shows the time profile of tetracycline
WWTP are similar to those previously reported in the at biomass concentrations of 1000 and 3600 mg/L. As can be
literature. In monitoring studies conducted at six U.S. seen, more than 75 and 95%, respectively, of the tetracycline
treatment plants, which applied different treatment tech- initially present at 250 µg/L was removed from the dissolved
nologies, the tetracycline concentrations were between 0.27 phase after an equilibration time of only 1 h, indicating a
and 4 µg/L in the untreated sewage and between 0.23 and very fast sorption onto the sludge. Equilibrium concentrations
1.2 µg/L in the treated effluent samples (7, 23). In our study, were achieved quickly at 3600 mg/L biomass and stayed
the total tetracycline concentrations in the SBR-1 effluent virtually unchanged over the 24 h study. On the basis of this
appear to be generally lower as compared to the influent adsorption kinetic test, it was assumed that 24 h was sufficient
wastewater, but it is difficult to assess if this difference was time to reach equilibrium for both adsorption and desorption
due to elimination in the bioreactor or due to the intra-assay tests. Other researchers (9, 25) also used 24 h as an
variability typical of ELISA analysis. Therefore, the removal equilibration time for tetracycline adsorption/desorption
efficiency in SBR-1 was not determined. In the case of SBR- tests in soil. The sorption isotherm of tetracycline on activated
2, a substantial difference between the initial total tetracycline sludge is presented in Figure 4. The calculated Kads was 8400
concentration (spiking level 250 µg/L) and the final con- ( 500 mL/g (standard error of slope). This is about three
centration in the effluent was obtained, as presented in Table times that reported for the more polar oxytetracycline on
3. Total tetracycline concentrations determined in the SBR-2 activated sludge (3020 mL/g) (26).
effluent ranged from 10 to 84 µg/L. On the basis of the average This value of Kads is substantially higher than has been
concentrations given in Table 3 and an initial concentration reported for tetracycline in soils (400 and 1140 mL/g) (24).
of 250 µg/L (background concentration neglected), the The calculated desorption isotherm of tetracycline from
removal efficiencies for SBR-2 amounted to 86% in phase 1, activated sludge is presented in Figure 4. The calculated
85% in phase 2, and 78% in phase 3. Statistical evaluation desorption coefficient (Kdes) was 22 600 ( 2200 mL/g and is
of these data using t-tests showed that there was no significant more than three times higher than Kads. The difference
differences at a 95% confidence level between phase 1 and between Kads and Kdes suggests that a portion of adsorbed
phase 2 mean total effluent tetracycline concentrations (p ) tetracycline does not readily desorb from activated sludge,
0.366). These results suggest that lowering the hydraulic thereby displaying adsorption/desorption hysteresis. Ad-
retention time from 24 to 7.4 h, which also resulted in an sortion/desorption hysteresis of trace chemicals such as
increase in the mean SBR biomass concentration from 514 proteins and metals on activated sludge is well-documented
to 1191 mg/L, did not influence tetracycline removal. (27, 28). The sludge adsorption experiments indicated that
However, decreasing the SRT of SBR-2 to 3 days in phase 3 elimination of tetracycline from the sewage in SBR is
resulted in a significant reduction in tetracycline removal influenced strongly by the sorption onto the biomass.
when compared to the 10 days SRT used in phase 1 (p ) Biodegradability of Tetracycline. What is unclear from
0.029) and phase 2 (p ) 0.032). 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)].