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Journal of Hazardous Materials 280 (2014) 662–670

Contents lists available at ScienceDirect

Journal of Hazardous Materials

jo ur nal ho me p ag e: www.elsev ier .com/ locate / jhazmat

ybrid bioreactor (HBR) of hollow fiber microfilter membrane andross-linked laccase aggregates eliminate aromatic pharmaceuticalsn wastewaters

idy Baa, J. Peter Jonesa, Hubert Cabanab,∗

Department of Chemical Engineering and Biotechnological Engineering, Université de Sherbrooke, 2500 Boulevard de l’Université, Sherbrooke, Québec J1KR1, CanadaDepartment of Civil Engineering, Université de Sherbrooke, 2500 Boulevard de l’Université, Sherbrooke, Québec J1K 2R1, Canada

i g h l i g h t s

CLEA-Lac was recycled by a MF mem-brane without a significant loss ofactivity.There was a clear synergistic effectbetween the CLEA-Lac and the MFmembrane.HBR achieved more than 90% elimi-nations of the three selected pharma-ceuticals.HBR could be used as an innovativebioprocess to eliminate micropollu-tants.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 10 March 2014eceived in revised form 27 August 2014ccepted 30 August 2014vailable online 6 September 2014

eywords:LEA-laccaseicrofilter membraneybrid bioreactor

a b s t r a c t

Widespread detection of numerous micropollutants including aromatic pharmaceuticals in effluents ofwastewater treatment plants has prompted much research aimed at efficiently eliminating these contam-inants of environmental concerns. In the present work, a novel hybrid bioreactor (HBR) of cross-linkedenzymes aggregates of laccase (CLEA-Lac) and polysulfone hollow fiber MF membrane was developedfor the elimination of acetaminophen (ACT), mefenamic acid (MFA) and carbamazepine (CBZ) as modelaromatic pharmaceuticals. The MF alone showed removals of the three drugs varying approximatelyfrom 50 to 90% over the course of 8 h in the filtrate of aqueous solution. Synergistic action of the MFand CLEA-Lac during operation achieved eliminations from aqueous solution of around 99%, nearly 100%and up to 85% for ACT, MFA and CBZ, respectively. Under continuous operation, the HBR demonstrated

harmaceuticalsastewater

elimination rates of the drugs from filtered wastewater up to 93% after 72 h for CBZ and near completeelimination of ACT and MFA was achieved within 24 h of treatment. Concomitantly to the drugs elimina-tions in the wastewater, the CLEA-Lac exhibited 25% residual activity while being continuously recycledwith no activity in the filtrate. Meanwhile, the filtrate flowrate showed only minor decline indicatinglimited fouling of the membrane.

© 2014 Elsevier B.V. All rights reserved.

∗ Corresponding author at: Environmental Engineering Laboratory, Department ofivil Engineering, Université de Sherbrooke 2500 boul. de l’Université, Sherbrooke,uébec J1K 2R1, Canada. Tel.: +1 819 821 8000x65457; fax: +1 819 821 7974.

E-mail address: Hubert.Cabana@USherbrooke.ca (H. Cabana).

ttp://dx.doi.org/10.1016/j.jhazmat.2014.08.062304-3894/© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Increasing findings of pharmaceutical micropollutants inwastewaters over the last two decades have raised many concernsabout the ecological and health hazards of these contaminants.Indeed, many pharmaceutical molecules have been detected in

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S. Ba et al. / Journal of Hazard

quatic systems throughout the world [1]. Effluents of wastewaterreatment plants (WWTPs) have been identified as the primaryource of entry of these micropollutants into aquatic systems [2].his is a consequence of the inability of conventional WWTPs toffectively remove many of these contaminants. In fact, WWTPsre not designed to remove these contaminants found at very lowoncentrations (ng/L to �g/L) [3].

Despite their low concentrations, various chemical micropol-utants including aromatic pharmaceuticals have been associated

ith disturbances of vital biological functions of aquatic orga-isms [1,3]. These authors reported reproductive impairments,ecreased fecundity, brain and lung defects as well as feminiza-ion of aquatic organisms caused by certain drugs and persistentntibiotic resistance of microorganisms to some of these micropol-utants. Regardless of their environmental ecotoxicity, the effects ofhese contaminants on humans remain disputed [2]. Yet, continu-us discharge of pharmaceuticals to aquatic systems should not beverlooked and these contaminants must effectively be removedrom wastewaters on the basis of the precautionary principle.

Various biological, physical and chemical treatment methodsave been attempted to remove micropollutants found in WWTPsffluents without total satisfaction [4]. Lately, much work hasocused on advanced oxidation techniques such as semiconductorhotocatalysis, UV/Fenton, UV/O3, UV/H2O2, O3/H2O2, O3/UV/TiO2,tc. [5]. Although in many instances these methods provided highlimination efficiency of the targeted recalcitrant compounds, theyoncurrently generate byproducts of unknown effects which mayenerate risks or toxicity higher than their parent compounds [6].ther methods involved membrane filtrations using ultrafiltrationnd nanofiltration (UF and NF). With their minuscule pore sizes,F and NF are effective in removing micropollutants includingharmaceuticals but the membranes foul rapidly under the highrganic matter content characteristic of wastewaters and the pro-ess requires a great deal of energy [7]. Consequently, the filtrateux of the membranes steadily declines in continuous operationhus requiring additional maintenance cost to their energy con-umption. It has been suggested that control of micropollutants inastewaters is urgent and necessary but cannot be circumscribed

o the removal efficiency only in a context of energy crisis andoncerns over greenhouse effect. And thus any treatment systemhould be considered using a holistic approach for the sake of sus-ainability [8].

There has been a growing interest in using enzymes to eliminateicropollutants [9,10]. Versatile peroxidase and glucose oxidaseere used in a study to eliminate five different aromatic endocrineisruptors [11]. The potential of oxidoreductive enzymes such as

accases to effectively eliminate aromatic micropollutants fromastewaters has been discussed [4,12]. Laccases (EC 1.10.3.2)

re non-toxic biodegradable oxidoreductases and are widely dis-ributed in nature and capable of transforming many varietiesf aromatic substrates including pharmaceutical molecules [13].nlike most enzymes, laccases catalyze the transformation of their

ubstrates by requiring readily available molecular oxygen as theole cofactor to produce water as a by-product and active radicalsf the substrates [14]. These radicals generally polymerize simi-arly to cross-coupling mechanisms known from other enzymesatalysis [15] and precipitate to become easier to separate fromhe reaction solution. Laccases have been shown to biotransformarious aromatic micropollutants into innocuous byproducts inastewaters treatment [16–18]. The use of enzymes in wastewa-

er treatment necessitates their insolubilization (i.e., cross-linkednzymes aggregates or CLEAs) and confinement in a reactor as

art of a treatment system operating continuously [19]. Indeed, thehortcomings of free enzymes in solution are well-known [20,21].

In this study, we proposed a novel approach of enzymaticreatment using a simple and efficient hybrid bioreactor (HBR)

aterials 280 (2014) 662–670 663

combining microfiltration membrane (MF) with CLEA-Lac operat-ing continuously. To our knowledge, similar studies have not beenpublished to date, although an approach combining UF and freelaccase was recently reported [22]. The proposed hybrid systemadvantageously associates insolubilized biocatalysts and relativelylow energy-consuming membranes, thus having a potential tobecome a cost-effective and sustainable technology for micropol-lutant elimination in wastewaters. Our objective was to confineCLEA-Lac within a hollow fiber MF to preclude it from pass-ing through the membrane filtrate, unlike free enzyme, so thatthe biocatalyst could be recycled back to the reaction solutionwhile retaining residual activity. Then, a continuous eliminationof three model aromatic pharmaceuticals was examined in munic-ipal wastewaters while operating the bioreactor over an extendedperiod of time. These three pharmaceuticals were acetaminophen,mefenamic acid and carbamazepine. Their selection was based ontheir diverse chemical structures, relatively high stability againstriver water microbes, relatively high consumption and/or potentialfor ecological risk [23].

2. Materials and methods

2.1. Materials

Trametes versicolor laccase (TvL) with a specific activ-ity of 22.4 U/mg-solid, chitosan from crab shells (65%deacetylation and molecular weight of 750 kDa), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride(EDAC), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS), acetaminophen (ACT), mefenamic acid (MFA), carba-mazepine (CBZ) were purchased from Sigma–Aldrich Corporation(Saint-Louis, MO, USA). All chemicals were of analytical gradewith purities superior to 99%. Relevant characteristics of thepharmaceuticals such as molecular weight (MW), water solubility(WS), octanol/water partition coefficient (Log Kow) are providedin Table S1 of the supplementary data.

Polysulfone hollow fiber MF membrane of 0.1 �m pore size waspurchased from GE Healthcare Bio-Sciences (AB Björkgatan 30, 75184 Uppsala, Sweden). The MF had a pH stability range of 2–13,a temperature range of up to 80 ◦C and its cartridges were auto-clavable. Other characteristics of the membrane are provided inTable S2 of the supplementary data.

2.2. Bioreactor scheme and setups

The bioreactor was setup in two versions as shown in Fig. 1.Setup 1 was aimed at determining the retention capacity of thebiocatalysts and the drugs by the MF on one hand, and the syn-ergistic effect of the MF and the CLEAs for the elimination of thedrugs on the other hand. Setup 2 was aimed at testing continuouselimination of the drugs from wastewater by the HBR.

Setup 1 consisted of a 1000 mL glass-beaker used as a vessel forthe reaction solution of the pharmaceuticals and/or the biocatalystscoupled with polysulfone hollow fiber MF. PTFE tubing preventingadsorption of the pharmaceuticals with biocatalysts on its inter-nal surface was used to connect the glass-beaker and the MF andalso to collect the filtrate and the retentate from the MF [24]. A peri-staltic pump (P1) was used to pump the reaction solution to the MF.Two manometers (M1 and M2) were mounted between the glass-beaker and the MF and on the retentate tubing to monitor pressurevariations. Two needle valves (V1 and V2) were also connected to

the filtrate and retentate tubing to control the filtrate flowrate andfor cleaning the membrane when needed. The reaction solution inthe glass-beaker was continuously mixed thoroughly with a mag-netic stirrer using a clean Teflon-coated stir bar. The second setup

664 S. Ba et al. / Journal of Hazardous Materials 280 (2014) 662–670

Fig. 1. Schematic representation of bioreactor experimental setups.

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(MW: 151.16 g/mol for ACT, 241.29 g/mol for MFA and 236.27 g/molfor CBZ) are in a range of molecules small enough to breakthroughthe MF membrane [28]. This may be due in part to the relatively highconcentration (100 �g/L for each drug) used, which is higher than

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S. Ba et al. / Journal of Hazard

as identical to the first with an additional 3500 mL of aqueousolution with pharmaceuticals in a glass-beaker used to feed theeaction solution by means of a second peristaltic pump (P2).

.3. Preparation of CLEA-Lac and activity assays

CLEA-Lac was prepared as previously described [25,26,26]. Thectivity assays of the biocatalysts were performed with a double-eam UV–Vis spectrophotometer (SpectraMax Plus 384, Molecularevices Corporation, Sunnyvale, CA). ABTS was used as substrates

o determine the activities of the biocatalysts according to previousrotocol [27].

.4. Pharmaceuticals elimination protocols

Application of the HBR for the elimination of the model aromaticharmaceuticals was carried out in five successive experimentsescribed below. Samples were drawn hourly from filtrate andnalyzed for pharmaceuticals removal according to an analyticalethod employed previously using an Acquity UPLC/MS/MS XEVO

Q mass spectrometer (Waters Corporation, Milford, MA) [18].Experiment 1: using setup 1, the MF was first flushed with a high

ux of Milli-Q ultrapure water (mLQW) (18.2 M� cm at 25 ◦C, TOC 10 ppb) according to startup procedures provided in the operat-

ng handbook by the supplier in order to establish optimum crossow of the membrane. Next, mixed solution of ACT, MFA and CBZas prepared with mLQW to a final concentration of 100 �g/L for

ach of the three drugs. In order to assess the removal effect of theF component of the HBR, 550 mL of the pharmaceuticals solution

including 50 mL accounting for tubing and MF system) was fedhrough the setup at a flow rate of 5.25 mL/min for 8 h. Membraneffluent (filtrate) flow rate was set at 0.9 mL/min and retentate wasot recirculated back to the feed solution. The HBR operated at roomemperature (∼20 ◦C) over the course of experiment.

Experiment 2: using setup 1, 550 mL of free laccase solutionrepared with mLQW at a final concentration of 5000 U/L was circu-

ated through the HBR to monitor the breakthrough and retention ofhe free biocatalyst through the MF. The HBR operated for 8 h a daya cycle) for 5 days. All other operation parameters and conditionsere identical to experiment 1.

Experiment 3: using setup 1, 550 mL of CLEA-Lac prepared withLQW to a final concentration of 50 U/L was circulated through theBR to monitor the retention of the insolubilized biocatalyst within

he MF similar to experiment 2. All other operation parameters andonditions were identical to experiment 1.

The biocatalysts activities in experiments 2 and 3 werexpressed in relative percent forms as the ratios of the actual bio-atalysts activities measured (in feed, retentate and filtrate) overhe input concentration (5000 U/L for free laccase or 50 U/L forLEA-Lac) multiplied by 100.

Experiment 4: using setup 1, 550 mL of reaction solution ofLQW containing the three pharmaceuticals to 100 �g/L of final

oncentration for each and CLEA-Lac of 50 U/L for final activityas thoroughly mixed and fed through the HBR. The retentate was

ecycled back to the reaction solution vessel and pharmaceuticallimination under the combined effect of CLEA-Lac and MF wasonitored hourly for 8 h identical to experiment 1.Experiment 5: using setup 2, the experiment was performed

sing municipal wastewater from the influent of WWTP (Magog,C) with characteristics provided in Table S2 of the supplementaryata. The wastewater had an initial pH of 7.5 and was acclimated to

pH of 2 for 48 h in order to inactivate all background microorgan-

sms susceptible to biodegrade the to-be-added pharmaceuticals.hen, the wastewater was readjusted back to its initial pH of 7.5nd filtered through 1 mm fiber glass after decantation to remove

bulk of the suspended matters. Next, 3500 mL of the filtered

aterials 280 (2014) 662–670 665

wastewater was contaminated with the pharmaceuticals to a finalconcentration of 100 �g/L and used as feed solution for the biore-actor system. The solution was continuously stirred at 300 rpm. Bymeans of P2, feed solution was pumped continuously at 0.9 mL/min(equaling the filtrate initial outflow rate) to the reaction solu-tion which operated as a continuous stirred-tank reactor (CSTR).At the start of the experiment, CSTR contained 550 mL includingCLEA-Lac at 50 U/L and operated for 0.95 h of hydraulic retentiontime (HRT). The HBR operated continuously over 120 h while theelimination of the pharmaceuticals from the filtrate, the filtrateoutflow rate and the CLEA-Lac activity in the CSTR were beingmonitored.

At the end of each experiment set, the MF membrane wascleaned by flushing it with a high flow (14 mL/min) of pure waterover an extended period of time (8–10 h). The cleaning processwas stopped only when no particles were coming out of the HBRand no visually apparent difference could be detected between theappearance (turbidity) of the inflow and the outflow of the cleaningwater.

2.5. Batch experiments

Batch experiments were systematically conducted parallel tothe HBR as control studies during experiments 4 and 5. A 1000 mLglass-beaker was used with a magnetic stirrer and solutions werethoroughly mixed with a Teflon-coated stir bar. The batch solutionswere all identical to the reaction solutions used in the HBR setups(see Section 2.4).

3. Results and discussion

3.1. Retention efficiency of pharmaceuticals by MF

The retention curves for the three drugs under the sole effect ofthe MF membrane of the HBR are presented in Fig. 2. The resultsshowed significant removal of the pharmaceuticals varying approx-imately between 11.3–51 �g/L, 11.3–33.1 �g/L and 15.6–44.5 �g/L(i.e. 50–90%, 60–90% and 55–80%) for ACT, MFA and CBZ, respec-tively, in the solution filtrates over the course of 8 h. These removalrates are high considering that the molecular weights of the drugs

Fig. 2. MF removal of the three pharmaceuticals in a mixed solution of mLQW atpH 7.0, 20 ◦C, and 12 psi in the absence of enzymatic activity using setup 1 with-out recycling the retentate. Each datum point represents the mean of triplicatevalues ± std dev.

666 S. Ba et al. / Journal of Hazardous Materials 280 (2014) 662–670

Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5

Free

Lac

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tive

activ

ity (A

/A0*

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, %

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Fig. 3. Free laccase activity variation in mLQW at pH 7.0, 20 ◦C, and 12 psi from HBRur

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120FeedRetentate

Fig. 4. CLEA-Lac activity variation in mLQW at pH 7.0, 20 ◦C, and 12 psi from HBR

sing setup 1 without recycling the retentate from experiment 2. Each datum barepresents the mean of triplicate values ± std dev.

ost concentrations of pharmaceuticals encountered in munici-al wastewaters for membrane treatment [29,30]. Also, the shapesf the retention curves followed similar patterns of successivelyncreasing removal of the drugs to a plateau followed by decliningemoval rates. These patterns are typical of breakthrough curves ofarious trace pharmaceuticals transported through porous media31] and appear to illustrate attachment and detachment processesf the drugs within the inner surface and pores of the mem-rane. The attachment may be associated with adsorption on andore constriction within the membrane favoring retention of therugs followed by possible breakup of the adsorbed buildup withubsequent release of the drugs in the filtrate (i.e. detachment).reakthroughs in the filtrate ranging from 10 to 50 �g/L are stillonsiderable for treated micropollutants, hence the necessity tomprove the removal performance. This could be achieved throughnzymatic supplementation [22].

.2. Retention efficiency of free laccase by MF

Results from experiment 2 are presented in Fig. 3. During therst cycle of the experiment, about 105% of relative activity of the

eed free laccase solution was recovered in the retentate (perhapsue to the enzyme microenvironment change induced by the flow)hile about 55% broke through the membrane in the filtrate. Dur-

ng the second cycle, the relative activity dropped steeply to aboutalf in the retentate and about 40% in the filtrate relative to the firstycle while the feed’s actual activity remained unchanged. All otheronditions being identical, activity reductions indicate that the freeiocatalyst was retained within the membrane. The relative activ-

ties of the laccase in the retentate increased steadily to peak atbout 90% in the fifth and last cycle of the experiment. Meanwhile,he relative activities in the filtrate stabilized to around its initialalue during the first cycle (i.e. more or less 55%). The experimenthowed that the free laccase could not be fully separated from thequeous solution in order to be confined within the MF membranend the retentate. The use of the free enzyme would result in a sig-ificant loss of the biocatalyst in the filtrate if it were to be used

n a continuous process. This drawback can be circumvented bysing either a smaller pore size membrane such as UF capable of

rapping the free enzyme as previously demonstrated [32] or insol-bilized enzyme which aggregates sizes are larger than the poresf the MF membrane [33]. The latter option appears more appeal-ng considering the high energy consumption of membranes withmaller pore sizes.

using setup 1 without recycling the retentate in experiment 3. Each datum barrepresents the mean of triplicate values ± std dev.

3.3. Retention efficiency of CLEA-Lac by MF

Results from experiment 3 are presented in Fig. 4. Contrary to theexperiment with free laccase, no CLEA-Lac activity was measuredin the filtrate. This supports our postulate that the insolubilizedenzymes can be confined within the MF membrane and recycledback to the feed vessel. On the other hand, the relative activity inthe retentate was very high from the first cycle (over 85%) to the last(nearly 100% recovery). Attachment of CLEA-Lac to the inner wallof the membrane was therefore not a significant factor. Limitedattachment of CLEA-Lac within the MF membrane provides twoadvantages to the bioreactor. Firstly, it indicates that most of thebiocatalyst was recycled for continuous elimination of the drugs.Secondly, it limited high concentrations inside the membrane andprevented rapid fouling of the membrane. Fouling is known as theprimary cause of short membrane lifespans [34].

3.4. HBR elimination of pharmaceuticals in Milli-Q water

Results from experiment 4 for the elimination of the drugs fromthe HBR and batch experiments are reported in Fig. 5A–C. Theconcentration reduction curve of ACT in batch mode was steepduring the first hour of reaction time between the drug and theCLEA-Lac followed by a gradual decrease down to 4 �g/L after8 h. This curve and the values are consistent with results of ACTtransformation under the effect of laccase activity in mLQW per-formed under similar conditions [18]. The curve of ACT elimination(elimination designating the combined removal from MF and trans-formation from CLEA-Lac) from HBR showed a rapid eliminationof the drug followed by a steady state elimination down to around1.8 �g/L after 2 h. In comparison to the results found from the batchmode and to that from experiment 1, there was a clear synergisticeffect between CLEA-Lac and MF membrane leading to the steadilyenhanced elimination of ACT from the bioreactor.

The elimination curves for MFA from both the batch and HBRstudies (Fig. 5B) showed similar trends to that of the ACT from theHBR with a steep initial increase followed by a gradual increase.The HBR treatment resulted in nearly complete elimination of thedrug from the sixth hour on (i.e. only 0.2 �g/L or less remainedin the effluent over that period) and was higher than that of the

batch treatment where significant transformation of the contami-nant was achieved. On average, nearly 90 �g/L (i.e. 90%) of the drugwas transformed between the third and eighth hour in the batchtreatment. This result is supported by a previous study in which 90%

S. Ba et al. / Journal of Hazardous Materials 280 (2014) 662–670 667

Time, h

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ocTn(

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ig. 5. HBR and batch eliminations of ACT (A), MFA (B) and CBZ (C) from mLQW and

n experiment 4. Each datum point represents the mean of triplicate values ± std de

f MFA was removed after 1 h 40 min of treatment with TvL indi-ating that MFA could be highly transformed by the enzyme [35].he high elimination of MFA from the HBR was expected given thatearly 90% removal from the sole effect of MF was already reachedcf. Fig. 2).

The curves for CBZ reductions from both batch and HBR stud-es (Fig. 5C) initially decreased to stabilize at around 40 �g/L and5 �g/L (60 and 85% reductions), respectively, after 3 h. Theseeduction rate patterns are consistent with those of ACT (from HBR)nd MFA studies although the elimination efficiencies are lower forBZ but still high for this drug. Previous research has shown CBZransformation up to 61% from its initial concentration of 50 �g/Ln batch mode using T. versicolor laccase pellets, although this waschieved after 7 days of treatment [36].

Fig. 5D provides CLEA-Lac activity variation monitored duringxperiment 4 alongside the pharmaceutical elimination. In the par-llel batch experiment, the insoluble biocatalyst appeared rathertable around its initial activity of 50 U/L over the course of thexperiment. The activity measurement of CLEA-Lac in the feed ofBR showed a gradual increase to nearly double the initial valuet the end of the experiment. This increase of CLEA-Lac activ-ty was due to volume loss of feed solution through the filtrate

hile recycling the CLEA-Lac, thus corresponding to a concentra-ion increase of the biocatalyst in the feed. Overall, these resultsemonstrated that insolubilization of the TvL made it possible toecycle the biocatalyst without significant loss of activity in the

Lac activity (D) at pH 7.0, 20 ◦C, and 12 psi using setup 1 with recycling the retentate

short run as previously found for other enzymes [37]. More impor-tant, the recovery and increase of activity of the biocatalyst in theexperiment constitute a favorable factor for the transformation ofthe contaminants during the experiment.

3.5. HBR continuous elimination of the pharmaceuticals inmunicipal wastewater

Continuous elimination of the three micropollutants was inves-tigated in municipal wastewater under conditions described in theprotocol for experiment 5. The results are presented in Fig. 6A–C.After 8 h of HBR operation with 50 U/L of CLEA-Lac activity in thereaction solution, ACT, MFA and CBZ eliminated at about 122, 80,and 84 �g/L, respectively, from their initial concentrations. Mean-while, the batch solution transformed about 121, 70 and 32 �g/Lof ACT, MFA and CBZ, respectively. Thereafter, the elimination ofACT and MFA reached nearly 100% within 24 h and remained therefor the remaining course of the HBR operation. The batch treat-ment for the two compounds exhibited similar trends with theirelimination levels slightly lower than those of the HBR at less than99%. The breakthrough curve of CBZ from the HBR followed a differ-ent pattern than those of ACT and MFA but similar to those already

observed and described in Fig. 2. The maximum elimination for CBZreached about 94 �g/L (i.e. about 94% elimination) over the courseof the 120 h of treatment. On average, this rate was lower thanthe maximum found for the two other drugs over the same period

668 S. Ba et al. / Journal of Hazardous Materials 280 (2014) 662–670

Time, h

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Fig. 6. HBR and batch eliminations of ACT (A), MFA (B) and CBZ (C) from wastewater, pH 7.5, 20 ◦C, and 12–15 psi using setup 2 in experiment 5. And CLEA-Lac activity andM ls andd

bt6efeociwrtrbCemtrs(oWom

F filtrate flowrate variations (D) during the same experiments. For pharmaceuticaev.

ut appears considerably high for this drug. From the batch test,he transformation curve of CBZ gradually decreased from about8 �g/L after 8 h of contact time to about 40 �g/L after 120 h (i.e. 60%limination at the end of the treatment). This batch result obtainedor CBZ elimination was significantly lower compared to the HBRlimination, on one hand, and to that of the batch transformationf ACT and MFA on the other hand. The latter indicating that theombination of MF with CLEA-Lac was more efficient in remov-ng CBZ than the CLEA-Lac alone. The result is, however, consistent

ith several studies for various enzymes including TvL under envi-onmentally relevant conditions in which CBZ was moderatelyransformed [36,38–40]. But the result does contrast with othereports in which CBZ was either not or only slightly transformedy enzymatic activities [36,38]. The concentration reductions ofBZ in the batch may be influenced by adsorption due to the pres-nce of chitosan as cross-linker for the CLEA-Lac or some sort ofediation or cross-coupling reactions induced by the mixture with

he other pharmaceuticals. Indeed, in the study by Li et al., theemoval efficiency of CBZ was ascribed to its adsorption on theawdust used to immobilize the enzyme from the white-rot fungusWRF) Phanerochaete chrysosporium [39]. Likewise, the adsorption

f the foam (the foam was used as growing surface for mycelia ofRF) was found to represent up to 40% of the 60–80% removals

f CBZ achieved from a non-sterile fungal bioreactor [40]. Effect oficropollutant mixtures in enzyme-mediated oxidation has also

CLEA-Lac activity, each datum point represents the mean of triplicate values ± std

been reported. For instance, TvL was found to transform 25% ofdiclofenac when it was the sole compound in a solution, but itstransformation increased to 60% in the presence of bisphenol A, andup to 95% when MFA was added to the solution mixture [35] sug-gesting mediation or cross-coupling reactions. Increased removalrate does not always result from multi-compounds solution medi-ation for some molecules as was the case for BPA and MFA in thelatter study.

During experiment 5, the residual CLEA-Lac activities were alsomonitored and the results are provided in Fig. 6D. The time coursemonitoring of the CLEA-Lac activity showed about 70 and 75% activ-ity loss in the batch and CSTR, respectively, over 96 h. The residualCLEA-Lac activities tended to stabilize for the next 24 h as sug-gested by the reduced rate of decrease of the curves in Fig. 6D.Steady decrease of CLEA activity followed by constant activity levelthereafter has been reported and was associated with leachate ofthe biocatalyst in water and mechanical stirring causing confor-mational changes which distort the active site [37]. Results fromthe activity measurements suggest that there was no direct cor-relation between the CLEA-Lac activities and the elimination ofthe compounds both from the batch and the HBR studies. While

the CLEA-Lac activities were decreasing, the elimination rates ofthe drugs kept rising or remained constant most of the time. Thisfinding of no correlation between the biocatalyst activity and theelimination rate of the drugs is supported in the study of CBZ

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S. Ba et al. / Journal of Hazard

limination by TvL in which decrease of the drug concentrationas observed while no laccase activity was detected [36]. Thending also corroborates earlier assumptions that adsorption maylay an important role in the removal of the drugs in batch reac-ors, through synergism with the CLEA-Lac. More important, itighlights the benefit of hybridizing the biocatalyst with the MFembrane for eliminating the micropollutants as the contribution

rom MF membrane in the HBR appeared significant. Indeed, morehan 90% to nearly 100% eliminations from the HBR for all threerugs most likely stemmed from enhancement caused by the MFembrane coupling with the CLEA-Lac. This is supported by results

rom experiment 1 where the removal rates of the drugs in mLQWrom the virgin membrane wavered between 50 and 90%.

The effectiveness of the HBR might have resulted from com-lex interactions between the drugs, wastewater, CLEA-Lac andF membrane based on their respective characteristics. It could

easonably be assumed that the mixture of the drugs with the CLEA-ac in the reaction solution vessel (CSTR) for an HRT of about 1 hesulted in a removal rate equivalent to that of the batch solu-ion. As the mixture is pumped to the MF membrane, the flowithin the tubing and the membrane provided additional con-

act time between the biocatalyst and the drugs in the solutionwith possible adsorption of the drugs on the membrane) trans-ated to an increased removal rate. It has been demonstrated thatigher removal rate of aromatic contaminants was achieved by

accase activity at longer contact time, especially during the firstours of reaction [16,32]. Within the membrane, it was established

rom Fig. 4 that, due to its macromolecular size, the CLEA-Lac waslocked from passing through and limited from adsorbing on theembrane due to size exclusion or solution pH [41]. These latter

uthors reported low protein adsorption on the surface of poly-ulfone UF membrane at pH greater (or lower) than the isoelectricoint (pI) of the protein as a result of electrostatic repulsion. Theheory was validated in a study of bovine serum albumin (BSA)ltration through a 0.1 �m glass membrane during which adsorp-ion of the protein diminished as a result of repulsion betweenhe BSA molecules and the pore surface of the membrane at pHbove the pI 4.9 of the BSA [42]. In our study, the MF membraneas made of polysulfone, the T. versicolor laccase had pI 3.6 and

he wastewater solution was at pH 7.5. Such interactions betweenLEA-Lac and the membrane would favor the floating and recyclingf the biocatalyst to continue reacting with the drugs in solution.t can be assumed that dissolved natural organic matters in the

astewater contributed to a gradual formation of a fine film layerver the inner surface of the membrane as part of a fouling processescribed by Wu and coworkers [43]. The film layer probably cre-ted secondary adsorption of the drugs and their removal as theyigrate through the film. Perhaps, the predominant factor that con-

ributed to eliminating the drugs resulted from direct interactionsetween the drugs and the membrane surface. As the pore sizef the MF polysulfone membrane (0.1 �m) is known to be largenough to not retain low MW compounds of small size as is thease for ACT, MFA and CBZ, size exclusion cannot be considered.he rejection of the micropollutants was therefore highly likely toave occurred via adsorption (as shown in Fig. 2). This assumption

s supported in a review of studies in which adsorption was foundo be the driving cause of micropollutant removal by larger poreize membranes [8]. Several other factors effecting micropollutantemoval from membranes and enzymatic reaction including molec-lar features of the compounds (e.g. chemical structure, chemicalomposition, hydrophobicity) in relation with the characteristicsf the physicochemical properties of the wastewaters have been

omprehensively discussed [44,45].

During experiment 5, the flowrate of MF filtrate and the pressurehange were also measured. Over the course of 5 days of con-inued operation of the HBR, there was nearly constant pressure

[

aterials 280 (2014) 662–670 669

(12–15 psi) and no significant decline was observed for the flowratethat varied from 25 mL/h initially to 23.2 mL/h as shown in Fig. 6D.This feature of the HBR operational is important as it indicates thatfouling of the membrane was rather negligible during that periodalthough wastewater was used. Low fouling may have been pro-moted by the fact that the wastewater was filtered before usageand to the MF character with large pore size of the membrane.

4. Conclusions

A hybrid bioreactor consisting of MF membrane and CLEA-Lacwas tested and operated continuously for the treatment of filteredwastewater. Synergistic effects of the biocatalyst and the mem-brane achieved elimination rates higher than 90% for CBZ and nearly100% for ACT and MFA from the wastewater. The CLEA-Lac wasrecycled over the course of the experiment while retaining signifi-cant residual activity. Negligible decline in the filtrate flowrate wasobserved from the initial value suggesting limited fouling of themembrane. The results indicate that a HBR of CLEA-Lac and MFmembrane could become a viable novel technology applicable tothe polishing of wastewaters to remove micropollutants. Furtherinvestigations are needed to evaluate hydraulic parameters andcosts associated with the functioning of the system.

Acknowledgements

This project was supported by grants from the National Scienceand Engineering Research Council of Canada (NSERC). The authorsare thankful to Olivier Savary, Serge Gagnon, Isabelle Arsenault andthe members of the Environmental Engineering Laboratory of theUniversité de Sherbrooke for their assistance.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2014.08.062.

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