“This is a post-peer-review, pre-copyedit version of an article published in Water Science and Technology (ISSN: 0273-1223). The finalauthenticated version is available online at: https://doi.org/10.2166/wst.2014.508 "Acclimation of nitrifying biomass and its effect on 2-chlorophenol removalJ. E. Perez-Alfaro, G. Gonzalez-Blanco, E. Sierra-Palacios, J. Marcial-Quino and R. Beristain-CardosoABSTRACTThe metaboliceandekineticebehavioreofea nitrifyingesludgeeexposedetoe2-chlorophenol (2-CP)ewas evaluatedein batchecultures.eTWO kinds of nitrifyingecultureewereeused;eoneeacclimatedetoe4-methylphenole(4-mp},eandetheeothereunacclimated to 4-mp.eTheeunacclimated cultureewas affectedeadverselyebyethee2-CP'sepresence,esinceeneitherenitrificationenore2-CPeoxidationewaseobserved. Nonetheless,etheeacclimatedeculture showed metabolicecapacity to nitrify and mineralize 2-CP. Ammonium removalewas 100%, withea nitrifying yield of 0.92 ± 0.04 mg NOrN/mg NH!-N consumed. The consumption efficiency fore2-CP was 100% and the halogenated compound was mineralized to CO2. Denaturing gradient gel electrophoresis (DGGE)epatternseshowedetheeshiftein microbialecommunity structure,eindicatingethat microbialediversityewas dueeto the acclimation process. Thiseis the first evidenceewhere nitrifying culture acclimated to 4-mp completely removedeammonium ande2-CP.INTRODUCTION2-Chlorophenole(2-CP) isea persistentecontaminantethateoccurseduringeanthropogeniceactivitieseandeisereleasedetoedifferent ecosystems,esucheas water,eair andesoil (Vidal & Diez 2003). Wastewater from industries such asepolymeric resin production,eoil refining, petrochemicals, papereand coking plantsealsoecontainethisekind ofeCPe(Quaneeteal.2003). This compound is considered toebe a serious pollutant because of itsetoxicity and significanteenvironmental impact.eBiodegradation of CP by microorganisms is moreespecific, environmentalefriendly anderelatively inexpensive (Eker & Kargi 2006). Severalebiologicale processese haveebeenetestedeineordere toe removeeCPefromewastewatere eithereineanaerobiceor aerobiceconditionse(Bali & Sengue2003 Ziloueieeteal.e2006; Martinez-Hernandezeeteal.e20n; Silvaeeteal.e20n). Foreexample, Satoheeteal.e(2005) studied the effect of 2-CP onea nitrifying biofilm, observing a decrease in the ammonium consumption rate,ebut 2-CP was not con-sumed.eMajumdereeteal.e(2007)etestedea similar concentrationeof 2-CP using aneaerobic rotatingebiological contact, but thisepollutantewaseconsumed after 215edays ofeacclimation.eMartinez-Hernandez eteal.e(20n)estudied the effect of 5 mg/L ofe2-CP onenitrifyingesludge ine batchecultures,e observing ammoniumeremoval ofe10%,e and that theenitrifyinge sludge requiredea long time (arounde30edays)efore2-CPedepletion.eSilvaeeteal.e(2011) workedewitheaerobicecultures acclimatedetoea mixtureeofephenolic compounds,eobservingephenolicseandeammonium removal.eNonetheless,estudieseonetheeeffecteofeCPeon nitrificationeareestill scarce in the literature.Aerobic granular sludgesefrom domestic sewage treat-ment plantseare frequently used aseinoculumetoestart-upethe biologicale treatmenteprocess.eThus,eiteappearedeuseful toeinvestigateetheeprocesseofeacclimationefore2-CPeremovalewith-outelosingethe nitrification.eTheeprocedureeof sludgeeacclimationewould beedevelopedefor the planteoperators,eineorderetoeshortenetheestart-up and to provide an efficientepro-cess.eTherefore,ethe objective ofethis work was to determine the effectseof 2-CP on theenitrifying sludge acclimatedeandeunacclimatedetoe4-mpeusingeresponseevariablesesucheasespecificerateseandeproducteyieldseasewelleaseto obtaineevi-dence of theeleveletoewhichethis compound acts.eInetheepresentestudy denaturingegradientegeleelectrophoresis (DGGE) wasechosen as the fingerprintingemethod to provideea rapid visual indication of variations in microbial commu-nity structure.eKey words | 2-chlorophenol, acclimation, DGGE-pattern, nitrificationeJ. E. Perez-AlfaroG. Gonzalez-Blanco Departamento de Blotecnologia,Universidad Aut6noma Metropoelitana rztapalapa, Mexico, D.F.E.eSierra PalacioseAcademia de Biologfa, COlegio de Ciencias ye Humanidades,Universidad Aut6noma de la Ciudad de Mexico, MexicoJ.eMarcial Quinoecatedras CONAcyr, 1nstltuto Nacional de Pediatria, Lab. Bioquimica Genetica, secretaria de Salud, MexicoeR. Beristain Cardoso Departamento de Recursos de la Tierra, Universidad Aut6noma Metropolltana Lerma, Edo, Mexicoe E mail: berlstaln 3@yahoo.com.mxMATERIALS AND METHODSInoculum sourceNitrifying biomass cultivated in a continuous-flow reactor (R 1) for more than 1 year was used for inoculating a second continuous stirred tank reactor (R 2), which was operated for 6 months. R 1 was fed with ammonium (unacclimated culture),2and Rwith ammonium and 4-mp (acclimated culture). Bioreactors were continuously aerated and operated at 25 WC, pH 7.6± 0.3 with a hydrau-lic retention time of 2 days. The reactors were fed with mineral media divided into two tanks, (A) containing nitrogen source and (B) containing carbon source as follows (g/L), Medium A: (NH4)2SO4 (1.73), NH4Cl (1.4), KH2PO4 (2.73), MgSO4 (0.6), NaCl (1); and Medium B: NaHCO3 (9.3) and CaCl2 (0.04). The sludge was washed with a solution of NaCl (9 g/L) and centri-fuged (4,000 g, 10 minutes) before being used in the batch assays.Batch bioassaysBatch assays were undertaken in serological bottles of 160 mL. Sludge incubations contained 100 mL of basal medium and were inoculated with the previously stabil-ized nitrifying sludge taken from R 1 and R 2 (approximately 208 mg microbialprotein/L). The culture medium consisted of (g/L): (NH4)2SO4 (0.24), NH4Cl (0.19), KH2PO4 (0.028), MgSO4 (0.02), NaCl (0.02), NaHCO3 (1.75) and CaCl2 (0.01). The pH of the basal medium was controlled at 7.5 ± 0.3 by the addition of bicarbonate, which was corroborated by pH determi-nations during the incubation period. The headspace and liquid were flushed with pure oxygen for 4 minutes in order to provide oxygenated cultures. All experimental treatments were carried out in duplicate and incubated at 30 WC in a shaker at 200 rpm. Each incubation bottle was an independent experimental unit, which was sacrificed after sampling. The response variables used to evaluate the behavior of the nitrifying sludge were consumption efficiencies (ENHþ4 or Ep Cr, (mg substrate consumed/mg substrate fed) × 100), nitrate production yields3(YNO , mgNO3 -N/mg substrate consumed), and specific consump-tion rates of NH4þ-N (qNHþ4 ), and NO3 -N (qNO3 ). Average specific rates (q, mg substrate or product/g protein h) and lag phases (λ) were calculated by using the Gompertz model, as was described by Beristain-Cardoso et al. (2009).Microbiological analysisThe DNA was obtained from the continuous stirred tank reac-tors; extraction was performed with an AxyPrep Multisource Genomic DNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA). The polymerase chain reaction (PCR) was performedusing the primers Bac-968f and Bac-1401r (UNAM, Instituto de Biotecnología, México DF, Mexico) designed for van Leerdam et al. () by attaching a tail of 40 bp of G and C at the 50 primer forward. We used 3 mM MgCl, 0.2 mM dNTPs, 0.4 μM each of theprimers, 80 ng DNA and 1 unit of enzyme brand Vent DNA polymerase (New England BioLabs, Wilbury, Hitchin, UK). The reaction was subjected to the next program in the thermocycler at 94 WC for 5-minute denaturation, then 30 cycles of amplifica-tion, using 94 WC for 30 seconds for denaturation, 54 WC for 2 minutes for annealing and 72 WC for 1 minute for polymeriz-ation. The PCR products were run in an acrylamide gel denaturing gradient using team D-Code Universal Mutation Detection System (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). Ten micrograms were placed in each of the PCR products on a gel containing 6% acrylamide denaturing gradi-ent of 48–52% of urea and formamide at 60 WC with an initial voltage of 200 V for 10 minutes, and then 85 V for 16 hours using TAE running buffer to 0.5×. After the shift, the gel was stained with silver nitrate according to the protocol of Sanguinetty et al. (14).Analytical methodsAmmonium was analyzed by a selective electrode (Phoenix Electrode Company, Houston, TX, USA). Nitrite and nitrate were analyzed by high-performance liquid chromatography (HPLC) (PerkinElmer series 200 HPLC Systems, Shelton, CT), using an ion exchange column (IC-Pak Anion HC, 4.6 × 150 mm, Waters, Dionex Corporation, Sunnyvale, CA, USA) and a UV detector at 214 nm; 2-CP was quantified by HPLC (PerkinElmer, HPLC Systems, Shelton, CT, USA), using a C-18 reverse phase column (Phenomenex, Torrance, CA, USA) and a UV detector at 274 nm, being the mobile phase acetonitrile/water (60:40 v/v). Total organic carbon was measured by TOC analyzer (TOC-5000, Shimadzu, Kyoto, Japan). Microbial protein was determined by Lowry’s method (Lowry et al. 1951), and pH and dissolved oxygen were determined using specific electrodes. Scanning electron microscopy (SEM) was used to observe the micro-organisms inside the aerobic granules, according to Bozzola & Russell (1991).RESULTS AND DISCUSSIONReactor performance3Table 1 shows the response variables of both reactors atsteady-state. In R-1,ENH4þwas100%, with YNO0.96 ± 0.02. In R-2, ENHþ4 and EpCr were 100% ± 4.0 and 95% ± 5.0,3respectively, with YNO 0.95 ± 0.03. Soluble organic carbon in the output was not detected, suggesting that the p-cresolconsumed was mainly mineralized. At steady-state conditions, both kinds of sludge were taken from the reac-tors in order to evaluate the acclimation process for simultaneous removal of ammonium and 2-CP.Nitrifying activity as control assays3Nitrifying profiles with unacclimated and acclimated sludge are shown in Figures 1 and 2. The unacclimated culture consumed NHþ4 -N in 7.5 hours (Figure 1). A concomitant formation of NO3 with a transitory NO2 formation was observed. Figure 1 shows that nitrite consumption was made within 8 hours. After 8 hours, for both studies, the substrate consumption efficiencies and nitrate yields were very high (Table 2). The unacclimated culture removedammonium with qNHþ4 of 193 ± 8.0 mg NHþ4 -N/ g protein h, whileacclimated culture displayed a similar metabolictheqNOwas 94 ± 7.0NO3 -N/g protein h. Thebehavior to unacclimated culture, since ENHþ4 and YNO3 did not significantly change (Figure 2). However, qNHþ4 and qNO3 diminished at 45% and 27%, respectively, in compari-son with the unacclimated culture. These experimental results suggested that the biomass exposed to 4-mp did not affect the nitrifying metabolism. But the kinetic aspect was altered, displaying adiminishment on specific rates. Bio-mass exposed to 4-mp in continuous-flow culture might have induced changes in chemicalstructure at membrane level; that is, isomerization of cis-unsaturated fatty acids to the trans-configuration as was reported by Heipieper et al.() and van Schie & Young (). The modification of the chemical structure of the membrane might havealtered the substrate transport.Nitrifying activity in the presence of 2-CPThe effect of 2-CP on nitrifying activity with unacclimated and acclimated sludge is shown in Figures 3 and 4. The unacclimated culture was not able to nitrify in the presence of 2-CP, and the phenolic compound was not consumed in the evaluated period. Satoh et al. ( ) evaluated the effect of 10 mg/L of 2-CP on a nitrifying biofilm not exposed to phenolic compounds, finding that nitrifying consumption rates were inhibited, and 2-CP was not consumed. Martínez-Hernández et al. () worked with biomass not acclimated to phenolic compounds, showing that nitrifying activity was strongly affected by 5 mg/L of 2-CP, and halogenated com-pound consumption took 30 days.In the present work, the acclimated culture was able to nitrify and to oxidize 2-CP (Figures 3 and 4). The culture consumed ammonium in 120 hours (∼3 days). A concomi-tant formation of NO3 with a transitory NO2 formation was observed, and nitrite consumption was made within120 hours. The ENH4þ was 100%, with YNO3 0.92 ± 0.04. Ammonium was consumed at a specific rate of 6.3 ±1.0 mg NHþ4 -N/ g protein h, after a lag phase of 12 hours (Table 2). 2-CP consumption started around 75 hours, with a pronounced linear consumption. 2-CP was oxidized at a specific rate of 0.36 ± 0.03 mg C/ g protein h, withnitrifying controls (Table 2). These experimental results showed that the presence of 2-CP diminished the specific rates of nitrification; nonetheless, high removal efficiencies for ammonium and 2-CP were obtained. Martfnez-Heman-dez et aL (20n) reported a consumption of 5 mg/L of 2-CP in 30 days, whereas in the present work, 5 mg/L of 2-CP was removed in 2.5 days by using acclimated sludge. In the literature, there are microorganisms with an intrinsic tolerance to resist highly-toxic compound concentrations; this intrinsic property can be defined as genetic properties that allow cells to avoid biocidal action (McLellan etal.7200). However, gr am-negative bacteria such as Pseudomo-nas and Vibrio have presented the isomerization of the fatty acids cis to trans position (Heipieper et al. 1992; Weber et al. 1994), which seems to be a system of adaptation to high concentrations of toxicity. In the present work, the acclimation process was crucial for 2-CP removal without losing nitrification, and this information should be taken as a sludge acclimation procedure for plant operators, par-ticularly to treat wastewater polluted with nitrogen and halogenated compounds.Analysis of DGGE patternThe compositions of bacterial communities from the reac-tors in steady-state conditions were compared using nested PCR­ DGGE and the amplified fragments were derived from the ribosomal 16S region. Differences among lanes in both DGGEprofiles of bacterial communities were clearly observed (Figure 5). There are many visible bands showing diversity of microbial ecology in the sludge samples . During R. 1 operation at steady-state conditions, seven bands with high intensitywere identified. It is reason-able to suppose that those bands derive from nitrifiers, since the reactor was operated under strict nitrifying conditions for more than 1 year. In the DGGE column, for R.2, six bands with high intensity were identified. Three of them (marked with dashed arrows) remained, so that being 4-mp fed did not affect these microbial populations. This be­ havior might be attributed to the following reasons: (1) acclimation at membrane level, as described above, or (2) perhaps these populations had the enzymatic capacity to oxidize 4-mp. For example, Radniecki et al. (2008) showed changes at membrane level after the exposition of Nitroso-monas europae to toluene and benzene. On the otherhand, nitrifiers have been reported where ammonium mono-oxygenase (AMO) enzyme is able to oxidize either organic compounds or ammonium (McCarty 1999).In R.2, new bands were displayed (A-1, A-2, and A-3). This information clearly shows that 4-mp addition affected thesludge's microbial community by causing the prolifer-ation of heterotrophic bacteria owing to the heterotrophic conditions. Microbial populations involved in phenolic com-pound removal might be heterotrophic aerobic bacteria or heterotrophic nitrifiers. For instance, Moir et al. (1996) reported that the heterotroph (i.e., Paracoccus) also contains the AMO enzyme, thus contributing to the organic matter and to ammonium oxidation.When nitrifying sludge from R. 1 was exposed to 2-CP, nitri-fying activity was lost, suggesting that 2-CP exerted a toxic effecton thesemicrobial populations. Kenner & Arp (1994) reported that ammonium oxidation is the step most affectedwhen aro-matic compounds, such as benzene, alkyl benzenes, phenol, acetophenone, pyridine and benzoic acid, are present in the culture medium. However, in the present work, the same nitri-fying sludge (but acclimated to 4-mp) removed 2-CP and ammonium. The successful removal of both compounds might have been associated with the microflora diversity owing to the acclimation process. The microbial diversity was also confirmed by SEM images (Figure 6). In granular sludge not exposed to phenolic compounds, the predominant mor-phology was short straight rods with sizes ranging from 0.5 to 1.0 µm long (Figure 6(a)). However, the predominant mor-phology inside the aerobic granular sludge exposed to p-cresol was filamentous bacteria comprising long chains of small bacterial cells with sizes about 10 µm long (Figure 6(b)).Finally, nitrifying sludge previously exposed to 4-mp pre-served the nitrifying capacity and it also showed the metabolic capacity to mineralize 2-CP; this metabolic behavior might indicate that the sludge in contact with 4-mp acquired the ability to tolerate and consume similar compounds, andthe presence of 4-mp also promoted the proliferation of micro-flora involved in organic matter consumption.CONCLUSIONSThese experimental results showed that the acclimation pro-cess was crucial for removing 2-CP without losing the nitrification. 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