Dye-decolorizing activity in isolated yeasts from the ecoregion of Las Yungas (Tucumán, Argentina

Dye-decolorizing activity in isolated yeasts from the ecoregion of Las Yungas (Tucumán, Argentina

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  This article was srcinally published in a journal published byElsevier, and the attached copy is provided by Elsevier for theauthor’s benefit and for the benefit of the author’s institution, fornon-commercial research and educational use including withoutlimitation use in instruction at your institution, sending it to specificcolleagues that you know, and providing a copy to your institution’sadministrator.All other uses, reproduction and distribution, including withoutlimitation commercial reprints, selling or licensing copies or access,or posting on open internet sites, your personal or institution’swebsite or repository, are prohibited. For exceptions, permissionmay be sought for such use through Elsevier’s permissions site at:http://www.elsevier.com/locate/permissionusematerial     A   u    t    h   o   r    '   s    p   e   r   s   o   n  a    l    c   o   p   y Enzyme and Microbial Technology 40 (2007) 1503–1511 Dye-decolorizing activity in isolated yeasts from the ecoregionof Las Yungas (Tucum´an, Argentina) Hip´olito F. Pajot a , Luc´ıa I.C. de Figueroa a , b , Julia I. Fari˜na a , ∗ a PROIMI-CONICET (Planta Piloto de Procesos Industriales Microbiol´ ogicos), Av. Belgrano y Caseros, T4001MVB Tucum´ an, Argentina b C´ atedra de Microbiolog´ıa Superior, Facultad de Bioqu´ımica, Qu´ımica y Farmacia, Universidad Nacional de Tucum´ an, Tucum´ an, Argentina Received 27 July 2006; received in revised form 20 October 2006; accepted 20 October 2006 Abstract Sixty-threeyeastisolatesobtainedfromsamplesofLaureldelmonte( Phoebeporphyria )andunderlyingsoilsfromLasYungasforests(Tucum´an,Argentina) were compared on their ability for textile dye decolorization. Seventeen isolates showed the highest decolorization ability on agar platescontaining Vilmafix ® Yellow 4R-HE, Vilmafix ® Red 7B-HE (Vi–R), Vilmafix ® Blue RR-BB (Vi–B) and Vilmafix ® Green RR-4B, either alone oras a mixture. Screening in liquid media supplemented with dyes led to the five outstanding isolates: 2023, 2184, 2030, 2118 and 2014 showing dyeremoval values as high as 87.9–98.7% for Vi–B and 89.8–94.5% for Vi–R after 72h of cultivation under aerobic conditions. Highest specific (3.06and 2.43mgg − 1 h − 1 for Vi–B and Vi–R, respectively) and volumetric (5.39 and 4.14mgl − 1 h − 1 for Vi–B and Vi–R, respectively) decolorizationrates were achieved with isolate 2023. Almost colorless biomass recovered after decolorization, supernatant UV–vis spectra and the presenceof extracellular oxidative factors suggested that biodegradation mechanisms may be involved in decolorization. Selected isolates were identifiedaccording to classical and molecular procedures and were clumped into group 2118-2014 corresponding to  Trichosporon multisporum  (100%similarity) and group 2023-2184-2030, closely related to  T. laibachii  (98.9% similarity).© 2006 Elsevier Inc. All rights reserved. Keywords:  Textile dyes; Biodecolorization; Wild yeasts; Trichosporon 1. Introduction Azo dyes, characterized by the presence of one or more azogroups (–N N–), are the most commonly used dyes in textile,paper, printing and cosmetic industries. They are an importantgroup of synthetic colorants, considered as xenobiotic com-poundsandhighlyrecalcitrantagainstbiodegradativeprocesses.Effluents of dyeing industries are markedly colored and the dis-posalofthesewastesintoreceivingwaterscausesdamagetotheenvironment. Since color reduces light penetration, they maysignificantly affect photosynthetic activity in aquatic life. Inaddition, due to their own toxicity as well as because of thepresence of metals, chlorides, etc., they are harmful to aquaticlife and also to living organisms drinking from these waters.Nevertheless, during the last few years it has been demonstratedthat several microorganisms are able to transform azo dyes tonon-colored products or even mineralize them [1–3]. ∗ Corresponding author. Tel.: +54 381 4344888; fax: +54 381 4344887.  E-mailaddresses:  jifarina@yahoo.com, jifarina@proimi.org.ar(J.I.Fari˜na). Reactive azo dyes are not totally degraded by conven-tional wastewater treatment processes due to their stabilityand xenobiotic nature. Current non-biological methods includecoagulation/flocculation, oxidation or adsorption, electro-chemical destruction and photocatalysis. Such methods maysuccessfully accomplish dye removal but could be very expen-sive because of the high chemical usage, costly infrastructureand high operating expenses [4]. Moreover, accumulation of concentrated sludge becomes a new disposal problem [5].Fungal biodecolorization ability has been widely reported[1,6] and it is commonly associated to lignin-degrading exoen-zymessuchaslignin-peroxidase(EC1.11.1.14),Mn-peroxidase(EC1.11.1.13)orlaccase(EC1.10.3.2).Thenon-specificnatureof these enzymes makes them able to transform, and eventu-ally mineralize, a variety of persistent environmental pollutants,including dyes. However, growth of filamentous fungi is slowcomparedwithmostsingle-cellmicroorganisms.Infact,theyarepoorly adapted to wastewater treatments because an exuberantmycelium growth generally occurs [7].Compared to bacteria and filamentous fungi, yeasts exhibitattractive features. Though not as fast as bacteria, yeasts can 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.enzmictec.2006.10.038     A   u    t    h   o   r    '   s    p   e   r   s   o   n  a    l    c   o   p   y 1504  H.F. Pajot et al. / Enzyme and Microbial Technology 40 (2007) 1503–1511 growfasterthanmostfilamentousfungi,andlikethem,theyhavethe ability to resist unfavorable environments. Up to present,however, the use of yeast strains in dye wastewater treatmentshas been limited [8].Theregionof“LasYungasAndinas”isahumidforestlocatedin mountainous sectors linked to the Andes. It discontinuouslyextends from Venezuela, through Ecuador, crossing Per´u andBolivia, reaching the northwest of Argentina with extreme rem-nants in the provinces of Salta, Jujuy, Tucum´an and Catamarca.This ecoregion represents one of the most valuable biodiversityreservoirs in Argentina.The present research has been focused on the Montane For-est (between 600 and 1200m a.s.l.), in Tucum´an, Argentina.This stratum plays a key ecological role not only because of its biodiversity but also, as it serves as refuge for many otherspecies arriving from different altitudinal strata during seasonalmigrations.DespitetheYungasfloraandfaunabiodiversityhavereceivedgreatattentionduringlastyears,microbialdiversityhasbeen scarcely explored. From a biotechnological point of viewandasanecofriendlyproposal,wehavetriedtodemonstratethepotential of yeasts isolated from this region for textile azo dyedecolorization. 2. Materials and methods 2.1. Samples Samples were aseptically collected from live and dead sections of “Laureldel monte” ( Phoebe porphyria ) trees and underlying soil, from “Las Yungas”,Tucum´an, Argentina (26 ◦ 43  S.L. and 65 ◦ 17  W.L.). Samples of 10–15g wereobtained and transported in sterile plastic bags and kept at 4 ◦ C until they wereprocessed. 2.2. Yeast isolation and maintenance One gram of sample was resuspended in 20ml of solution containing (ingl − 1 ): glucose, 1; yeast extract, 0.5 and Tween 80, 0.01. Suspensions wereincubated at 26 ◦ C and 200rpm for 2h and thereafter, 50  l of suspensionwere plated onto YM-agar (Difco) plates pH 4.5 and incubated at 26 ◦ C untildevelopment of colonies. From every sample, yeast isolates corresponding todifferent colony morphotypes were selected and maintained on YM agar slantsat 4 ◦ C. 2.3. Dyestuff  Four commercial dyes were used: Vilmafix ® Red 7B-HE (C.I. Name:Reactive Red 141), Vilmafix ® Blue RR-BB (C.I. Name: Reactive Blue 221),Vilmafix ® Green RR-4B (C.I. Name: Reactive Green, no number in the pub-lic domain) and Vilmafix ® Yellow 4R-HE (C.I. Name: Reactive Yellow 84)(Fig. 1). Vilmafix ® dyes were kindly provided by Vilmax S.A. Stock solutionswere prepared by dissolving powdered dyestuff, without prior purification, indistilled water up to a concentration of 2gl − 1 and filter sterilized (Milliporefilter, 0.22  m, Millipore Corp., Bedford, USA). 2.4. Decolorization screening on solid media ItwasperformedonPetridishescontaining20mlofoneofthefivefollowingsolid media: •  YEPD (in gl − 1 ): glucose, 20; peptone, 10; yeast extract, 5. •  Yeast extract medium (YE) (in gl − 1 ): yeast extract, 3 [9]. •  Yeastcarbonbase(YCB)-NH 4 NO 3 :filter-sterilizedyeastcarbonbase(Difco)plus 1gl − 1 NH 4 NO 3 . •  Malt extract-glucose (MEG) (in gl − 1 ): glucose, 10; malt extract, 5 [10]. •  Normal decolorization media (NDM) (in gl − 1 ): glucose, 20; (NH 4 ) 2 SO 4 ,2.5; yeast extract, 2.5; KH 2 PO 4 , 5, MgSO 4 · 7H 2 O, 0.5; CaCl 2 , 0.13 [11].Dye stock solutions were added to each culture media up to 200mgl − 1 (ppm) final concentration and this was the concentration systematically usedin subsequent experiments A mixture of all dyes (200ppm final concentration)was used in order to simulate a textile effluent composition. All media weresolidified with 15gl − 1 of agar.PlateswereinoculatedwithactivelygrowingyeastfromYM-agar,incubatedat 26 ◦ C and examined for decolorization along 72h of cultivation. As control,plates without dye were also inoculated.Fig. 1. Dye chemical structures of: (A) Vilmafix ® Red 7B-HE (C.I. Name: Reactive Red 141) and (B) Vilmafix ® Blue RR-BB (C.I. Name: Reactive Blue 221).     A   u    t    h   o   r    '   s    p   e   r   s   o   n  a    l    c   o   p   y  H.F. Pajot et al. / Enzyme and Microbial Technology 40 (2007) 1503–1511  1505 2.5. Decolorization in liquid cultures Dye(Vilmafix ® BlueRR-BBandVilmafix ® Red7B-HE)decolorizationwasevaluated in 20-ml tubes containing 7ml of NDM medium. From the previousagar-plate screening, 17 yeast isolates were selected and subsequently assessedin liquid medium. A total of 300  l yeast suspension (OD 550  =0.8) preparedfrom a fresh YM culture was used to inoculate each test tube. Incubations werecarried out at 200rpm and 26 ◦ C for 72h. Controls (biotic or abiotic) were per-formedusingthesamemediumwithoutdyesoryeasts,respectively.Tubeswerewithdrawn along cultivation and subsequently sacrificed for analyses. Sampleswere centrifuged for 10min at 4000 × g . Pellets were washed twice with steriledistilled water and dried at 80 ◦ C to constant weight for biomass dry weightdetermination. Supernatants were kept for estimating dye removal. 2.6. Dye monitoring DyedecolorizationwasmonitoredwithaBeckmanDU640spectrophotome-ter at each dye  λ opt  by using culture supernatants obtained as above described.Color removal was reported as percent decolorization=(  A 0  −  A t  )/   A 0  × 100,where  A 0  and  A t   were the absorbance of dye-amended medium at the start point(0) and at a cultivation time ( t  ), respectively. Additionally, culture supernatantsweresubjectedtospectralscanningbetween200and800nminordertoanalyzedye disappearance. 2.7. Agar-plate screening for oxidation of ABTS, SYR and Mn 2+ Oxidative factors (e.g. ligninolytic activities) potentially involved in biode-colorization were screened on Petri dishes containing NDM medium plus eitherABTS [2,2  -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)], syringaldazine(3,5-dimethoxy-4-hydroxybenzaldehydazyn; SYR) or MnCl 2 · 4H 2 O, as previ-ously described [12,13]. Plates were inoculated with previously selected yeasts,grownonYM-agar.ABTS,SYRandMnCl 2 · 4H 2 Owereaddedtoafinalconcen-tration of 100ppm. Stock solutions were sterilized by filtration and asepticallyadded to NDM. 2.7.1. Effect of pre-incubation with dyes and/or Mn 2+ Screening assay was repeated but using yeasts previously grown onplates supplemented with either Vilmafix ® Blue RR-BB and/or MnCl 2 · 4H 2 O(100ppm). 2.7.2. Effect of redox mediators Violuric acid (10mM) and 3-methyl-2-benzothiazolinone hydrazone,MBTH (0.07mM) were added to the corresponding ABTS-, SYR- or Mn 2+ -plates.All values and data points presented in this work are the means of at leasttriplicate determinations of independent assays. The statistical significance of differencesamongvalueswasassessedbyusingtheone-wayANOVAtest.Datawere analyzed using the GraphPad InStat Instant Biostatistics package version3.0. 2.8. Identification of dye-decolorizing yeasts Fortheidentificationofisolatedyeastswithhighestdecolorizationabilitiesapolyphasicapproachwasapplied.Itinvolvedmorphologicalexaminationaswellas classical and non-conventional biochemical and physiological tests [14–16].Further molecular characterization was carried out by sequencing the 26SrDNA D1/D2 domain using NL1 and NL4 primers for amplification [17]. DNAsequencing on both strands of each isolate was performed by the dideoxy chainterminationmethodwithanABI3730XLautomaticDNAsequencer(MACRO-GEN, Korea), according to the manufacturer’s protocol. The D1/D2 domainsequences were registered in the GenBank Data Library under accession num-bers DQ288848 for 2023 isolate, DQ288849 for 2030, DQ288850 for 2118,DQ288851 for 2184 and DQ288852 for 2014.Phylogenetic and molecular evolutionary analyses were conducted usingMEGA3.0package[18]byusingneighbor-joininganalysis[19].Distanceswere estimated according to Jukes and Cantor [20]. Gaps were handled as missingdata. Bootstrap values were based on 1000 replications; values <90% were notrecorded. For phylogenetic tree, sequences from type strains of closely relatedspecieswhosenameshavebeenvalidlypublishedinpublicdatabases(GenBank,11/05) were only taken into account. 3. Results and discussion The present work aimed to study the potential decoloriz-ing ability of wild yeasts isolated from Las Yungas, Tucum´an(Argentina).Attheendoftheisolationscheme,63isolateswereobtained as pure cultures from different samples of Laurel delmonte ( P. porphyria ) trees and underlying soils and maintainedon YM agar slants at 4 ◦ C by periodic subculturing. 3.1. Decolorization screening on solid media The63isolatedyeastswerefirstevaluatedontheirdecoloriza-tionabilitybyusingaqualitativemethod,asdescribedinSection2. First assays were attempted to establish the optimal condi-tions required for the evaluation of yeast decolorizing ability.Results showed a significant influence of culture medium com-position on the detection level attained during decolorizationexperiments.When using Vilmafix ® Blue RR-BB, YCB medium did notallow detecting decolorization haloes even after 72h of cul-tivation. On the other hand, MEG and YEPD media alloweddetecting decolorization in 13% and 47% of the evaluated yeastisolates, respectively. Better results (73%) were obtained withNDM medium and it was found to be the most suitable culturemedium for the screening purposes. Moreover, to be cautiouslytaken into account, the use of medium YE supplemented withthe tested dyes, and prior to inoculation, showed a spontaneousnon-microbial decolorization, and for that reason its use shouldbe ruled out. In accordance to previous findings by Nam et al.[21] who described for the first time the non-enzymatic azodye reduction by NADH, abiotic decolorization caused by thepresence of high NADH concentrations in yeast extract may bespeculated. Similarly to the foregoing results, when VilmafixRed ® 7B-HE was added, NDM led the best detection levels.Based on these results, NDM medium was selected for subse-quent screening.Accordingly, NDM medium supplemented with the differ-ent dyes, either alone or as a mixture, was used for evaluatingtheisolatedyeasts.Dye-amendedplateswereperiodicallyexam-inedbyvisualinspection;colorlessorlesscoloredhaloesaroundyeastcolonieswereassumedasaqualitativeparameterofdecol-orization ability and colony dying as a qualitative parameter of bioaccumulation (Fig. 2).As previously pointed out, these observations would be notenough to have any insight into the precise color removal rate.However, they at least provide partial information on the decol-orizing ability of the yeast strains tested [8]. Thus, a usefulassociation of high dye removal (according to the halo size) andlowcolonydyeingwasafeatureexpectedtoguidetowardyeastswith higher biodegradation than bioaccumulation potential.Following the above criterion, from the initial 63 isolatedyeasts,only17werechosenasthebeststrainsfordyedecoloriza-     A   u    t    h   o   r    '   s    p   e   r   s   o   n  a    l    c   o   p   y 1506  H.F. Pajot et al. / Enzyme and Microbial Technology 40 (2007) 1503–1511 Fig. 2. Decolorization screening on solid medium for some isolated yeasts withNDM medium plus Vilmafix ® Blue RR-BB. Differences on colony dyeing andthe presence of haloes are indicative of decolorization ability. tion and were then subjected to further studies. These selectedisolates were able to decolorize each of the dyes assayed as wellas their mixture. This ability would be particularly valuable atindustrial level, since the inapplicability to a variety of dyes hasbeen one of the main reasons for the lack of implementation of several biological decolorization treatments.Different levels of decolorization were however notedaccording to the nature of tested dye. Formazan dye Vilmafix ® Blue RR-BB was more easily decolorized than the other azodyes assayed. Among azo dyes, Vilmafix ® Red 7B-HE behavedas the most recalcitrant one. Such differences on dye suscep-tibility to decolorization may be explained on the basis of dyechemicalstructures.Reinforcingthisidea,anthraquinonic,triph-enylmethane and phthalocyanine dyes were previously found tobe more easily decolorized than mono- or disazo dyes by  Irpex lacteus  and  Pleurotus ostreatus  [10].Despite the interest on yeast decolorizing ability against dyemixtures, simulated-effluent composition was not further usedin liquid cultures because of the spectral complexity in thecorresponding aqueous solutions. Similarly, Vilmafix ® GreenRR-4B was also excluded as its spectrum showed a combina-tion of absorbance maxima (blue and yellow  λ opt ). Two dyemodels, formazan dye Vilmafix ® Blue RR-BB, most suscep-tible to decolorization, and azo dye Vilmafix ® Red 7B-HE,moderately recalcitrant, were chosen for further liquid mediumdecolorization assays. 3.2. Decolorization in liquid culture Dye removal ratios between Vilmafix ® Blue RR-BB andVilmafix ® Red7B-HEundersubmergedcultureconditionswereclose to the unit for most of the 17 selected isolates, givingevidence of similar decolorization rates for both tested dyes(Table 1). At 72h of cultivation, those isolates showing per-centages of decolorization significantly higher (  p <0.001 and  p <0.01), for one or both tested dyes, were selected. Thus, nineisolates were selected at this stage (Table 1, isolated in bold). Table 1Percentage of decolorization after 72h of cultivation in NDM mediumYeast isolates Dye removal (%)Vilmafix ® Blue RR-BB Vilmafix ® Red 7B-HE Ratio a 2023 b 98.7 89.8 1.12184 b 95.4 92.8 1.02136 91.6 90.0 1.02036 90.4 89.2 1.02118 b 87.9 90.2 1.02014 b 80.5 94.5 0.82030 b 83.1 91.2 0.92147 82.5 90.8 0.92006 85.4 81.6 1.0 2028 73.2 91.0 0.82056 72.1 90.2 0.82025 70.7 82.9 0.82178 67.2 82.4 0.82019 73.6 74.2 1.02021 62.7 84.8 0.72048 63.7 70.7 0.92088 36.4 54.5 0.7 a Ratio estimated as % removal blue  /% removal red . b Selected isolates at the end of the screening scheme. The second selection criterion in liquid media took intoaccount the decolorization kinetics for both dyes. Percentagesof decolorization at 36 and 72h were statistically compared andonlythoseyeastsshowingvaluessignificantlyhigher(  p <0.001)than those of at least three other tested isolates were chosen.Consequently, at the end of the screening process the selectedisolates were 2023, 2184, 2030, 2118 and 2014, and these werekept for further analysis.High kinetic parameter values such as volumetric decol-orization rate ( η , mgl − 1 h − 1 ) and specific decolorization rate( ν , mgg − 1 h − 1 ) were found for the selected isolates, and theywere well positioned with respect to those reported in theliterature. It should be however noted that, in some otherreports, higher initial dye concentrations (500–700ppm) [22]or dyes bearing simpler chemical structures [11,23] were used(Table 2).Results from abiotic controls indicated that both tested dyesremained almost intact (99 ± 1%) throughout incubation inNDM. Accordingly, any reduction on the initial dye concentra-tion for inoculated media could be securely assigned to yeastactivity. Comparing to the growth in dye-amended medium,biotic controls showed that yeast growth was not restricted intheirpresence(Fig.3A–E).Evenmore,forisolate2014,biomassreachedhighervalues(  p <0.001)whengrowingwithVilmafix ® Red 7B-HE (Fig. 3 A). Similar observations were previouslyreported for  Candida zeylanoides  [11].Additionally, when yeast cells were recovered by cen-trifugation from liquid cultures, it was noted that biomasspellets, usually following the exponential growth phase, werealmost colorless. Based on this observation, the involvement of biodegradationeventsratherthanbioaccumulationmaybespec-ulated, though this latter would certainly occur during the firststages of dye removal.
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