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A feasibility study of immobilized and free mixed culture bioaugmentation for treating atrazine in infiltrate

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A feasibility study of phosphorylated-polyvinyl alcohol immobilized and free mixed bacterial culture bioaugmentation for removing atrazine in agricultural infiltrate was conducted utilizing a sand column setup. The effects of bacterial cell loading
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   Journal of Hazardous Materials 168 (2009) 1373–1379 Contents lists available at ScienceDirect  Journal of Hazardous Materials  journal homepage: www.elsevier.com/locate/jhazmat A feasibility study of immobilized and free mixed culture bioaugmentationfor treating atrazine in infiltrate Sumana Siripattanakul a , b , Wanpen Wirojanagud c , John M. McEvoy d ,Francis X.M. Casey e , Eakalak Khan f  , ∗ a National Center of Excellence for Environmental and Hazardous Waste Management, Chulalongkorn University, Bangkok 10330, Thailand b Department of Chemical Engineering and National Center of Excellence for Environmental and Hazardous Waste Management,Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand c Department of Environmental Engineering and Research Center for Environmental and Hazardous Substance Management, Khon Kaen University, Khon Kaen 40002, Thailand d Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58105, USA e Department of Soil Sciences, North Dakota State University, Fargo, ND 58105, USA f  Department of Civil Engineering, North Dakota State University, 1410 14th Avenue North, Civil and Industrial Engineering Building (Room 201), Fargo, ND 58105, USA a r t i c l e i n f o  Article history: Received 15 December 2008Received in revised form 5 March 2009Accepted 5 March 2009Available online 18 March 2009 Keywords: Atrazine biodegradationBacterial community changeCell immobilizationCell loadingInfiltration rateSingle strand conformation polymorphism a b s t r a c t A feasibility study of phosphorylated-polyvinyl alcohol immobilized and free mixed bacterial culturebioaugmentation for removing atrazine in agricultural infiltrate was conducted utilizing a sand columnsetup.Theeffectsofbacterialcellloadingandinfiltrationrateonatrazinedegradationwereinvestigatedbyshort-termtestsinwhichtheamountofsyntheticinfiltratefedthroughwasfivetimesofthevoidvolume(fiveporevolumes)ofthesandcolumn.Inaddition,thelossoftheinoculatedatrazine-degradingculturesand the change of bacterial community were determined. Selected tests were continued for monitoringa long-term performance of the system (50 pore volumes of the sand column). The results indicated thatthe inoculated cells removed 42–80% of the atrazine. The infiltration rate and cell loading significantlyaffected the atrazine removal. In the short-term tests, the immobilized and free cells provided similaratrazine removal; however, leaching of the free cells was much greater than that of the immobilizedcells. For the long-term performance, only the immobilized cells provided consistent atrazine removalefficiency throughout the test. Both immobilized and free cell systems exhibited a significant change inbacterial community structure during the atrazine degradation experiments. The infiltration rate was asignificant factor for the change.© 2009 Elsevier B.V. All rights reserved. 1. Introduction There has been an increasing interest to develop new on-siteremediation techniques. Biodegradation has been known as aneffectivemethodforremovingorganiccontaminants.Insomecases,biodegradationbyindigenousspeciescannotcopewithallcontam-inants or takes a long time. Cell bioaugmentation, an addition of sufficient contaminant-degrading microorganisms, can potentiallybe used to solve this problem. There are many factors influencingthe survival of bioaugmented microorganisms and their contami-nant degradation efficiencies, such as predation and competitionwith indigenous microorganisms, and unsuitable growth environ-ments [1]. ∗ Corresponding author. Tel.: +1 701 2317717; fax: +1 701 2316185. E-mail addresses:  sumana.s@ubu.ac.th (S. Siripattanakul), wanpen@kku.ac.th(W. Wirojanagud), john.mcevoy@ndsu.edu (J.M. McEvoy), francis.casey@ndsu.edu (F.X.M. Casey), eakalak.khan@ndsu.edu (E. Khan). Immobilized cells (IC) in a polymeric hydrogel are a poten-tialalternativetoaddresstheseconcerns.Immobilizationmatricescan alleviate environmental stresses [2,3]. This technique also prevents cell wash-out off the contaminated sites resulting inhighbiomassconcentrationsandcontaminantremovalefficiencies.Several studies have reported successful applications of immo-bilized cell bioaugmentation for point source pollution control,especially wastewater treatment [4–8]. Thus far, no research has been conducted on its application for non-point source pollutioncontrol.Agricultural activities including the use of herbicides areone of the main contributors of non-point source pollu-tion. Atrazine (6-chloro- N  -ethyl- N  -(1-methylethyl)-1,3,5-triazine-2,4-diamine)isoneofthemostwidelyusedherbicidesandhasbeenapplied to control broad-leaf weeds for crop production. Atrazinedetections in groundwater and surface water above the allowablecontaminant levels for drinking water of 0.1  g/L and 3.0  g/L inEurope and the United States, respectively, have been frequentlyreported [9–11]. 0304-3894/$ – see front matter © 2009 Elsevier B.V. All rights reserved.doi:10.1016/j.jhazmat.2009.03.025  1374  S. Siripattanakul et al. / Journal of Hazardous Materials 168 (2009) 1373–1379 Fig. 1.  A diagram of potential field-scale application of immobilized cell bioaugmentation for atrazine removal from agricultural infiltrate. In our previous work, biodegradation of atrazine in water by aphosphorylated-polyvinyl alcohol (PPVA) immobilized mixed cul-ture(MC)wassuccessfullyperformed[12].Theaimofthisresearch was to examine the possibility of the use of the immobilized cellbioaugmentation for remediating agricultural infiltrate contam-inated with atrazine, which could lead to a novel groundwaterpollution prevention scheme as depicted in Fig. 1. In practice, the immobilizedcellscanbeappliedtotopsoilduringthesoilprepara-tion period before the growing season. To validate this potentialbioremediation concept, bench-scale sand column experimentswere conducted. The effects of cell loading and infiltration rate onatrazineremovalweretested.Inoculationbyfreecells(FC)waseval-uatedagainstimmobilizedcellsfortheiratrazineremovalcapacity.During the test, the loss of the inoculated atrazine-degrading cul-ture and the change of bacterial community were determined.Long-term performance of atrazine removal was also monitored. 2. Materials and methods  2.1. Bacterial strain and cultural condition Astableatrazine-degradingMCwasenrichedfromatrazinecon-taminated soil collected from a field site in Oakes, North Dakota,USA following the procedure of Siripattanakul et al. [12]. The cul- turewaspreviouslycharacterizedandfoundtocontainmainlyfourbacterial strains with two of them being atrazine-degraders ( Kleb-siella ornithinolytica ND2and  Agrobacterium tumefaciens ND4)[13].The MC was routinely subcultured every seven days in a sterilebufferedbacterialmedium(20mMsodiumphosphatebufferatpH6.8) which contained (per liter) 20mg of atrazine, 1.0g of glucose,0.5g of K 2 HPO 4 , 0.5g of MgSO 4 · 7H 2 O, 10mg of FeCl 3 · H 2 O, 10mgof CaCl 2 · H 2 O, 0.1mg of MnCl 2 , and 0.01mg of ZnSO 4 . For its usein atrazine biodegradation tests, MC was subcultured in nutrientbrothspikedwith20mg/Lofatrazinefor24handthencentrifugedat 4500 ×  g   for 10min. The concentrated cells were immediatelyused for immobilization and/or the biodegradation tests. For thepreparation of immobilized dead cells (ID), the concentrated cellswere autoclaved at 121 ◦ C prior to the immobilization.  2.2. PPVA cell immobilization procedure The MC was immobilized using a PPVA technique following theprocedure described elsewhere [12]. Polyvinyl alcohol (PVA) was chosen as an immobilization matrix since it is durable and has nonegativeeffectonmicroorganismsandtheenvironment[4,14].The concentrated cells (20mg dry cells/ml) were mixed with a PVAsolution. The mixture was slowly dropped into a saturated boricacid solution in a 1L cylinder for 30min to form 6mm sphericalbeads. The formed hydrogel beads were then submerged in a 1Msodiumphosphatesolution(pH7)for60minforhardening.Thegelbeads were washed in de-ionized (DI) water and then stored in a20mM sodium phosphate solution (pH 6.8) at 4 ◦ C. The final PVAconcentration and cell-to-matrix ratio were 10% (w/v) and 3.5mgdry cells/ml matrix, respectively.  2.3. Synthetic agricultural infiltrate, sand, and column preparations 2.3.1. Synthetic agricultural infiltrate and sand preparations Synthetic agricultural infiltrate was prepared in the same man-ner as the bacterial medium (described above) except the additionof 1.5mg/L atrazine. Silica-quartz sand from Le Sueur, MN, USA(UniminCorporation,CT,USA)wasused.Thesandwaswashedwithtap water and dried at 105 ◦ C for 24h. The cleaned sand was sievedto obtain the grain sizes between 0.25mm and 0.42mm (US stan-dard sieves number 60 and 40). The sieved sand was autoclaved at121 ◦ C for 30min three times within three consecutive days. Thevoid ratio (v/v) of sieved sand loosely packed in a 400ml graduatecylinder was 0.30 (a void volume of 120ml).  2.3.2. Column setup A sand column was 6.35cm in diameter and 23cm in length. Ithad an effluent sampling port at the bottom (Fig. 2). All columns wererinsedwith70%isopropanolandautoclavedDIwater,respec-tively before used. The sterile sand and the cells were asepticallymixedandthenusedtofillthecolumns.Threesetsofsandcolumnsincluding set ID, set IC, and set FC, were packed as described inTable1.Thepackingdepthwas14cm.Eachsetofthecolumnscom-prised three columns at the cell loadings of 300mg, 600mg, and900mg dry cells/L empty bed volume. Note that the test set ID wasconducted as a control to determine whether there was atrazineadsorption by the immobilization matrix and/or the cells. Fig. 2.  A diagram of sand column setup.  S. Siripattanakul et al. / Journal of Hazardous Materials 168 (2009) 1373–1379  1375  Table 1 Descriptions of sand columns and their components.Column set ColumnnumberColumndescription(cell type)Cell loading (mg dry cells/L emptybed volume)Cell mass(mg dry cells)Bulk inoculatedvolume * (ml)Bulk dry sandvolume (ml)Total empty bedvolume (ml)ID ID1 Immobilizeddead cells300 120 40 360 400ID2 Immobilizeddead cells600 240 80 320 400ID3 Immobilizeddead cells900 360 120 280 400IC IC1 Immobilizedcells300 120 40 360 400IC2 Immobilizedcells600 240 80 320 400IC3 Immobilizedcells900 360 120 280 400FC FC1 Free cells 300 120 N/A ** 400 400FC2 Free cells 600 240 N/A ** 400 400FC3 Free cells 900 360 N/A ** 400 400 * Volume of the cells and matrices. ** Volume of the cells was negligible.  2.4. Atrazine biodegradation test in column system Duplicateatrazinebiodegradationexperimentsinsandcolumnswereperformed.Thecolumnswereoperatedasfollows.Theywerefilled up with a 20mM sodium phosphate solution (pH 6.8). Thesynthetic agricultural infiltrate was continuously applied as a stepinputatan8hinterval(threetimes/d)undergravity-flowcondition.The flow rates studied were 30ml/d, 90ml/d, and 180ml/d corre-sponding to the infiltration rates of 1cm/d, 3cm/d, and 6cm/d forobtaining the actual, high, and critical (extremely high) infiltrationrates,respectively.Thetestwasrunforfiveporevolumes(PV)(fivetimes of the void volume of the column or total pore water volumeof 600ml), which was selected because it was the period that thebreakthrough was reached for all test conditions.Duringthetest,theeffluentwassampledevery0.25PVtomea-sure atrazine and intermediate metabolite concentrations for theinfiltration rates of 1cm/d and 3cm/d. However, for the infiltra-tionrateof6cm/d,thiseffluentsamplingwasconductedata0.5PVfrequency.Thesamplesatevery1PVweredeterminedforthenum-ber of viable MC cells using a plate count technique. At 5PV, theeffluent samples from all viable cell columns (column sets IC andFC) were taken for detecting the change of bacterial communitystructureusingasinglestrandconformationpolymorphism(SSCP)technique. After 5PV, flow was continued for selected columnsfor long-term monitoring, where the effluent was monitored foratrazine and intermediate metabolite concentrations every 5PVbetween 5PV and 50PV.  2.5. Analytical methods 2.5.1. Atrazine and metabolite analysis The analytical methods used for atrazine, desethylatrazine(DEA), deisopropylatrazine (DIA), and hydroxyatrazine (HA) weremodified from D’Archivio et al. [15]. A solid phase extraction technique using 200mg of polymeric sorbent in a 6ml cartridge(StrataX, Phenomenex, CA, USA) was applied for atrazine andmetabolite extraction. The cartridge was prewashed and condi-tionedusing6mlofethylacetateand6mlofmethanol,respectively.Thecartridgewasthenwashedwith6mlofDIwater.Afterloadinga sample (2ml) and drying the cartridge under vacuum condition,the cartridge was eluted with 6ml of acetonitrile: methanol (1:1,v/v) through gravity flow. Then, the extract was evaporated to dry-ness under a gentle stream of dry nitrogen. The dry residue wasdissolved in 0.5ml of water: acetonitrile (1:1, v/v). The extract wasanalyzedforatrazineanditsmetabolitesonaHewlettPackard1100series high-performance liquid chromatograph equipped with aC18 reverse phase column (Jupiter, Phenomenex, CA, USA) at anultraviolet wavelength of 220nm. The isocratic mobile phase of water:acetonitrile (1:1) at a flow rate of 1ml/min was used.  2.5.2. Viable plate count  The bacterial loss from the column system was determined bythenumberofviablebacteriaintheeffluentsamples.Eacheffluentsample was serially diluted and spread onto a selective bacterialmedium agar. The agar formulation was the same as the syntheticagricultural infiltrate but added with agar of 2% (w/v). Bacterialcolonies were counted after 48h incubation at 30 ◦ C.  2.6. Bacterial community change detection using SSCP technique 2.6.1. Deoxyribonucleic acid (DNA) extraction Total genomic DNA was extracted from both immobilized andfree cells which were collected before and after the 5PV atrazinebiodegradation test. For free cells, the concentrated cells (asdescribedpreviouslyinSection2.1)wereusedtorepresentthecells before the test while the column effluent samples at 5PV were thesources of cells after the test. Similarly, the cells immediately afterimmobilizationandtheeffluentsamplesfromtheimmobilizedcellcolumn after the 5PV test were collected.TheDNAextractionprocedureforimmobilizedcellsbeganwithseparatingthecellsfromthematrix.Tenbeadscontainingimmobi-lizedcellswerecutinhalfandmixedthoroughlyin10mlof20mMphosphate buffer (pH 6.8) at 3200rpm for 2min using a vortexmixer (VWR International, PA, USA). The immobilized cell samples(10mlofthesupernatantfromthecellseparationprocedure),con-centrated free cell samples, and effluent samples were centrifugedat 16,000 ×  g   for 2min and used for extracting DNA. The genomicDNA extraction procedure followed the instruction from the DNAextraction kit (Wizard Genomic DNA Purification Kit, Promega, CA,USA).  2.6.2. DNA amplification Polymerase chain reaction (PCR)-SSCP procedure was modifiedfrom Lin et al. [16]. The V3 region of the 16S ribosomal ribonucleic acid (rRNA) gene (nucleotide positions 334–514 of   Esherichia coli )was amplified with primers EUB1 (5  -CAG ACT CCT ACG GGA GGCAGC AG 3  ) and UNV2 (5  -GTA TTA CCG CGG CTG CTG GCA C 3  ). A25  L PCR reaction contained 1.5mM of MgCl 2 , 200  M of dNTP,5.0  L of Taq polymerase buffer 5 × (Promega, CA, USA), 50  M of eachprimer,1.25UofTaqPolymerase(Promega,CA,USA),and2  L   1376  S. Siripattanakul et al. / Journal of Hazardous Materials 168 (2009) 1373–1379 Fig. 3.  Breakthrough curves of atrazine at the infiltration rates of: (A) 1cm/d, (B) 3cm/d, and (C) 6cm/d. ID, IC, and FC were the columns of the immobilized dead cells,immobilized cells, and free cells, respectively. The numbers 1, 2, and 3 represent the results of the columns at the cell loadings of 300mg/L, 600mg/L, and 900mg/L,respectively. of DNA template. DNase/RNase-free water was used for making upthe volume of samples. The PCR conditions consisted of an initialdenaturation at 94 ◦ C for 5min, 30 cycles at 94 ◦ C for 30s, 55 ◦ C for30s, and 72 ◦ C for 30s, and a final extension at 72 ◦ C for 5min. Thepresence of PCR products (approximately 200bp) was confirmedby 1.5% agarose gel electrophoresis.  2.6.3. SSCP gel electrophoresis The SSCP test was performed in a horizontal electrophoresissetup (Origins, Elchrom Scientific, Switzerland). The SSCP pro-cedure followed the instruction from the manufacturer. Threemicroliters of PCR products were mixed with 7  L of a denatur-ing solution (1ml of formamide, 10  L of 1M NaOH, and 20  L of 0.02% (w/v) bromphenol blue). The mixtures were heated at 95 ◦ Cfor5minandimmediatelyplacedintoiceuntilloadingtotheSSCPgel. The 10  L denatured PCR products were loaded into a pre-castpolyacrylamide gel (GMA TM , Elchrom Scientific, Switzerland). Thegel was run at a constant voltage of 72V and 9 ◦ C for 10h. The gelthen was visualized with SYBR  ® Gold staining (Molecular probes,OR, USA).  2.6.4. SSCP gel data analysis The images of DNA profiles were analyzed using Bionumericsversion5(AppliedMaths,TX,USA).Thepair-wisesimilarityamongthe samples was calculated using Dice index and an unweightedpair-group method with arithmetic average.  2.7. Statistical analysis Foratrazinebiodegradationdataanalysis,massofatrazineintheeffluentsampleswasstatisticallyanalyzedusingJMPIN ® 5.1.2(SAS,NC,USA).Thedatawereanalyzedusingamultipleregressionmodeland examined for the significance of the atrazine mass differencebetween test conditions with analysis of variance and  t  -test. 3. Results and discussion  3.1. Atrazine biodegradation using immobilized dead,immobilized, and free cells Fig. 3 shows the breakthrough curves of relative atrazineconcentration ( C  / C  0 ) of the ID, IC, and FC within 5PV. In allcurves,  C  / C  0  increased after 0.5–1.0PV and reached a plateauat approximately 2.5–4.0PV. The immobilized dead cells didnot have degradation ability resulting in no atrazine removal( C  / C  0  of 1.0) after 3.0–4.0PV while the breakthrough curvesof immobilized and free cell columns were stable at  C  / C  0  of 0.2–0.6. At the same cell loading and infiltration rate, atrazinewas detected in the effluent of the free cell columns earlierthan the immobilized cell and immobilized dead cell columns,in which atrazine sorption to the immobilization matrix likelyoccurred [12]. Based on the breakthrough curves of the immo- bilized dead cells (stable at  C  / C  0  of 1.0), the PPVA matrix onlyretarded the atrazine breakthrough but did not permanentlyremove it.The immobilized and free cell columns at the infiltration ratesof 1cm/d, 3cm/d, and 6cm/d (Fig. 3) removed atrazine at 65–80%, 50–73%, and 42–58%, respectively. The atrazine removal efficiencydecreasedwithincreasinginfiltrationratewhereashighercellload-ingratesprovidedgreaterremovalefficiency.Thestatisticalanalysisshowed that both infiltration rate (  p <0.0001) and cell loading(  p =0.0002)significantlyinfluencedatrazineremoval.However,forall experiments, there were no significant differences between theimmobilizedandfreecellsystemsforatrazineremoval(  p =0.4493).Therefore, the atrazine-degrading mixed culture used in this studyin either immobilized or free cell forms was efficient for atrazineremoval. During the atrazine biodegradation test, the atrazineprimary intermediate metabolites (HA, DEA, and DIA) were notdetected in any effluent samples. The result suggests that atrazine  S. Siripattanakul et al. / Journal of Hazardous Materials 168 (2009) 1373–1379  1377 Fig.4.  Numberofviablecellsintheeffluentsamplesfromthecolumnsoftheimmo-bilized(IC1,IC2,andIC3)andfree(FC1,FC2,andFC3)cellsattheinfiltrationratesof (A) 1cm/d, (B) 3cm/d, and (C) 6cm/d. wasquicklydegradedtosomeotherintermediateproductsbeyondthe primary intermediate metabolites.  3.2. Cell leaching during atrazine biodegradation The results of cell losses are presented in Fig. 4. The bacteria leaching from the immobilized and free cell columns at all cellloading was about 6–7log and 6–9logCFU/ml, respectively. Thetrends of the cell losses in the immobilized and free cell columnswere different. The cell losses from the immobilized cell columns(columns IC1 to IC3) were stable while those from all free cellcolumns(columnsFC1toFC3)declinedwithdurationoftheexper-iment.Theinitialnumberofcellsinoculatedtoallcolumnswasapprox-imately9logCFU/ml.At0PVand1PV,thenumbersofcellsleachingfrom the free cell columns were very close to the initial num-ber of cells. This could be because the sand columns could noteffectively retain the free cells. Yolcubal et al. [17] reported 90% loss of bioaugmented free cells treating salicylate and polycyclicaromatichydrocarbonsinasandcolumnsetup.Eventhoughmicro-bial growth might take place during the atrazine biodegradationtest, it is likely that the growth was not as much as the micro-bial loss from the column leading to less numbers of cells left inthe columns. This inference is supported by less numbers of theleachingcellsatlaterPVs.At0PVand1PV,thenumbersofcelllos-ing from the immobilized cell columns were about hundred timesless compared to those of the free cell columns. This indicates thatthe immobilization matrix could effectively protect the cells fromleaching.  3.3. Bacterial community change during atrazine biodegradationtest  Fig. 5 presents the SSCP profiles of the 16S rRNA gene fragmentof the atrazine-degrading mixed culture. The DNA band numbers3 and 6, 4 and 6, 1 and 2, and 5 represented  Alcaligenes faecalis ND1 (ND1) (EU075145),  K. ornithinolytica  ND2 (ND2) (EU075144), Bacillusmegaterium ND3(ND3)(EU075147),and  A.tumefaciens ND4(ND4) (EU075146) as previously identified by Siripattanakul et al.[13]. The samples (initial FC and initial IC) of atrazine-degradingmixed culture before and after immobilization were analyzed fordetermining the effect that the immobilization process had on thebacterial community structure. The cluster analysis showed thatthe similarity of the culture before and after the immobilizationprocesswasapproximately60%.Thissuggeststhattheimmobiliza-tion process affected the bacterial community structure to someextent.Chemical or physical stresses during the immobilization pro-cess might be the cause. It was reported that the immobilizationprocess affected the bacterial viability [12]. Some bacterial species might be less tolerant than the others and were killed in theimmobilizationprocedureleadingtothedifferenceinthebacterialcommunity structure. However, ND2 and ND4 which are atrazine-degrading strains [13], are present in the SSCP profiles of both initial free and immobilized cell samples. This indicates that theimmobilizationprocessaffectedsomebacterialspeciesbutnottheatrazine-degrading strains resulting in no influence on atrazinebiodegradation. This inference is supported by the biodegradationresult presented earlier, which both free and immobilized cellsbiodegraded atrazine successfully.For the pair-wise comparison between before and after theatrazine biodegradation test, the immobilized and free cell sys-tems provided comparable results. The similarity of the bacterialcommunity structure before and after the test was approximately40–50% (Fig. 5). The effluent samples at the same infiltration rate but different cell loadings had the similarity of the bacterial com-munity structure of 80–100%. On the contrary, the samples at thesame cell loading but different infiltration rates had the similarityof the bacterial community structure of 50–60%. This suggests thatthe infiltration rate influenced the bacterial community structuremore than the cell loading.TheDNAbandnumber5representedND4,ispresentinallsam-ples after the 5PV test while ND2 (band number 4) was detectedonly from the columns tested at the infiltration rate of 1cm/d.This could be because ND2 did not retain and/or grow well in thecolumns at the higher infiltration rates (3cm/d and 6cm/d). Con-sequently, at these infiltration rates, lower atrazine removal wasobserved. As shown in the previous section, the infiltration ratealso affected the atrazine removal more than the cell loading. Thiscoincidentseemedreasonablebecauseintuitivelyatrazineremovaland bacterial community structure should be related.
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