EnvironSciPollutRes(2014)21:9578–9588DOI10.1007/s11356-014-3036-z
REVIEWARTICLE
Biodegradationoforganicpollutantsinsaline
wastewaterbyhalophilicmicroorganisms:areview
LauraC.Castillo-Carvajal&JoséLuisSanz-Martín&BlancaE.Barragán-Huerta
Received:11January2014/Accepted:12May2014/Publishedonline:27May2014#Springer-VerlagBerlinHeidelberg2014
AbstractAgro-food,petroleum,textile,andleatherindus-triesgeneratesalinewastewaterwithahighcontentoforganicpollutantssuchasaromatichydrocarbons,phenols,nitroaromatics,andazodyes.Halophilicmicroorganismsareofincreasinginterestinindustrialwastetreatment,duetotheirabilitytodegradehazardoussubstancesefficientlyunderhighsaltconditions.However,theirfullpotentialremainsunex-plored.Theisolationandidentificationofhalophilicandhalotolerantmicroorganismsfromgeographicallyunrelatedandgeologicallydiversehypersalinesitessupportstheirap-plicationinbioremediationprocesses.Pastinvestigationsinthisfieldhavemainlyfocusedontheeliminationofpolycyclicaromatichydrocarbonsandphenols,whereasfewstudieshaveinvestigatedN-aromaticcompounds,suchasnitro-substitutedcompounds,amines,andazodyes,insalinewastewater.Informationregardingthegrowthconditionsanddegradationmechanismsofhalophilicmicroorganismsisalsolimited.Inthisreview,wediscussrecentresearchontheremovaloforganicpollutantssuchasorganicmatter,intermsofchemicaloxygendemand(COD),dyes,hydrocarbons,N-aliphaticandN-aromaticcompounds,andphenols,inconditionsofhighsalinity.Inaddition,someproposalpathwaysforthedegrada-tionofaromaticcompoundsarepresented.
Responsibleeditor:RobertDuran
L.C.Castillo-Carvajal:B.E.Barragán-Huerta(*)DepartamentodeIngenieríaenSistemasAmbientales,
EscuelaNacionaldeCienciasBiológicas,InstitutoPolitécnicoNacional,Av.WilfridoMassieu,UnidadProfesionalAdolfoLópezMateos,D.F07738,Mexicoe-mail:bbarraganh@ipn.mxB.E.Barragán-Huerta
e-mail:bbarraga59@yahoo.com
J.L.Sanz-Martín
DepartamentodeBiologíaMolecular,FacultaddeCiencias,
UniversidadAutónomadeMadrid,c/Darwin2,28049Madrid,Spain
KeywordsHalophilic.Biodegradation.Hydrocarbons.Dyes.Pollutants.PAH.Phenols
Introduction
Halophilicmicroorganismsarecapableofgrowingandcarry-ingouttheirmetabolicfunctionsunderhypersalineconditions.Non-halophilicmicroorganismsshowoptimalgrowthbelow2%NaCl,whilehalotolerantandhalo-dependent(halophilicsensusstrictus)microorganismscangrowinupto30%NaCl.Halophilicmicroorganismscanbeclassifiedaccordingtothesaltconcentrationsthattheyneedtogrowinasslighthalophiles(2–5%NaCl),moderatehalophiles(5–20%NaCl),andex-tremehalophiles(20–30%NaCl).Halophileshavebeenfoundineachofthethreedomainsoflife:Archaea,Bacteria,andEucarya(Oren2002a).Halophilesalsoexhibitgreatmetabolicdiversity;theyincludeoxygenicandanoxygenicphototrophs,aerobicheterotrophs,fermenters,denitrifiers,sulfatereducers,andmethanogens(Oren2002a).TheimportantdistinctionbetweenBacteriaandArchaeainhypersalinesettingsishowtheyosmoregulate,ingeneralKClforArchaeaandcompatiblesolutesforBacteria(Oren2002b).Thisaffectsviabilityandlimitstheirmetaboliccapabilities.Currently,thehalophileshavegreatpotentialinbiotechnologicalprocesses,especiallyinbioremediationprocessesbecauseoftheirabilitytodegradeorganicpollutants.
Duetoindustrialactivities,salineandhypersalineenviron-mentsarefrequentlycontaminatedwithorganiccompounds(Orenetal.1992).Additionally,severalagro-food,petroleum,textile,andleatherindustriesgeneratehighlysalinewastewa-terwithahighorganicmatterandpollutantcontent(Lozach2001;Lefebvreetal.2005;Diazetal.2002).Spillageofthosewastewaterwithoutpriortreatmentintofreshwateraffectsaquaticlife,waterpotability,andagriculture(LefebvreandMoletta2006).
EnvironSciPollutRes(2014)21:9578–9588Itiswellknownthatthedegradativeefficiencyofmicro-organismstowardpollutantsdecreasesinconditionsofhighsalinity,buttheuseofhalophilicorhalotolerantmicroorgan-ismscanovercometheselimitations(Zhuangetal.2010).BiodegradationoforganiccompoundsinsalineenvironmentswasreportedearlyonbyOrenetal.(1992),butmostofthereportsonthisissuehaveconcentratedonpetroleumaliphatichydrocarboncomponents.Recently,therehasbeenarenewedinterestinpollutioncontrolusingextremophilicmicroorgan-isms,probablyduetotheincreasingproblemsofpollutionanditsimpactonhumanhealthworldwide.
Salinewastewateroriginatesfrommanyindustries,suchastheproductionoffertilizers,chemicals,pharma-ceuticals,dyestuffs,pesticides,herbicides,somefoods,andthemeatpackingindustry.Theeffluentsfromoilandgasproduction,andtheminingandmineralindustriesarealsosaline(Linetal.1998).
Thehalophilicmicroorganismshaveshowngoodremovalefficienciesofpollutantssuchashydrocarbons(ArulazhaganandVasudevan2009;Garcíaetal.2005),dyes(Balamuruganetal.2011;Chanetal.2012),andphenols(Moussavietal.2010),bothinwater(Fairleyetal.2002;KapdanandErten2007)andinsoil(Amoozegaretal.2008;Zhaoetal.2009),Moreover,somehalophiles(Lietal.2012)canmetabolizethepollutantsunderaerobicandanaerobicconditions,andalmostalwayswithouttheproductionoftoxicdegradationinterme-diates(HaddadiandShavandi2013;Leit?oetal.2007).Thus,halophilicorganismshavepromisingpotentialinthebiological,environmentallyfriendly,treatmentofpollutedwastewaterandsoils.
TheeffectofsaltconcentrationonbiodegradationSalinityaffectsbiodegradationprocessesinseveralways.First,highsaltconcentrations(>1%)cancausealossofmicrobialactivityinconventionalactivatedsludgeunits,duetocelldehydrationordisintegrationbyosmoticdifferencesacrossthecellmembranes.Asaresult,lowremovalperfor-manceofchemicalandbiologicaloxygencanoccurathighsaltconcentrations(>2%)(DincerandKargi2000).Thestrategytoovercomethisproblemistoadaptthebiomasstohighsalinityortousehalotolerantorhalophilicmicroorgan-isms(LefebvreandMoletta2006;Abou-Elelaetal.2010).Secondly,ahighsaltcontentcandecreaseorganiccompoundsolubilityinwaterbyasaltingouteffect,andthereforede-creasethebioavailabilityoftheorganiccompounds(Whitehouse1984).Thisproblemcouldbenaturallyover-comebythebiosurfactantproductionfromhalophilichydrocarbon-degradingbacteria(HaoandLu2009;Djeridietal.2013)orbyaddingsubstancestoincreasethepollutantsolubility,suchas2-hydroxypropylβ-cyclodextrin(Sohnetal.2004).Thirdly,highsalinitydecreasesthedissolved
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oxygencontentinwater,limitingtheactionofoxygenaseenzymes(VonWedaletal.1988),andthereforetheaerobicbiodegradationrate.However,ithasbeenreportedthatex-tremehalophilicarchaeaHaloferax,Halobacterium,andHalococcushavehigherbiodegradationratesat2.2mgL?1thanat5.3mgL?1dissolvedoxygen(Al-Mailemetal.2010).Finally,highsaltconcentrationcouldinhibitthebiodegrada-tionofsomeintermediates,causingtheiraccumulationinthemedium(AlvaandPeyton2003).
Thesaltcontentinthesalinewastewaterproducedbyvariousindustriesvariesfrom2to15%,althoughthepro-ducedwater(PW)obtainedfromtheproductionofoilandgascanhaveasaltcontentofupto40%(Bonfáetal.2011).Ithasbeenreportedthatthenegativeeffectofsaltonbiodegradationratescanbeminimizedwhenmicroorganismsareimmobilized(Diazetal.2002).
Thus,bioaugmentationwithfreeorimmobilizedmi-croorganismswithbroadorganicpollutantdegradationcapabilities,atsaltconcentrationsasgreatof400gL?1canbeausefulstrategyforthebioremediationofsalineenvironmentsandthetreatmentofsalineindustrialwastewater(Kargi2002;Oren2010).
Sincetheearly1990s,thebiodegradationofpollutantsathighsaltconcentrationsbyhalophilicmicroorganismhasbeenstudied(LeBorgneetal.2008;Orenetal.1991,1992).Aftertheseinitialsystematicworks,muchefforthasbeendirectedtowardsthestudyofphylogeneticdiversityandthephysiolo-gyofmicroorganismslivinginhighsaltconditions(Oren2002b).Recently,researchonthedegradationpathways,genes,andenzymesinvolvedinpollutantbiodegradationbyhalophilicandhalotolerantorganismshasbeenconducted.Theperformanceofthesemicroorganismsunderdifferentculturalandenvironmentalconditionshasalsobeeninvestigated.
Biologicalorganicmatterremovalfromsalinewastewaterbyhalophilicmicroorganisms
Thewastewaterfromthepickledvegetableandfishprocess-ingindustriesarecharacterizedbyahighorganicmattercontent,expressedaschemicaloxygendemand(COD)andhighsaltcontent.TheCODvaluesseeninthistypeofwaste-waterthathaveasaltcontentof7.2to10%,rangefrom3,400to8,160mgCODL?1(Abou-Elelaetal.2010;Alouietal.2009).ThesearemuchhigherthantheCODvaluesofun-treatedmunicipalwastewater(500mgCODL?1).
CODremovalinthebiologicaltreatmentofsalinewaste-watercanbecarriedoutusingactivatedsludgeadaptedtosalineconditions,butatmorethan3%salt,thoseinoculantsarenoteffectiveinthetreatmentofsalinewastewaterbecausetheyaresensitivetochangesinionicstrength,andthebiolog-icaldegradationratesoftheorganiccompoundsdecreasewith
9580increasingsaltconcentration(KargiandDincer1998).Theseproblemscouldbeovercomebyusingpureculturesandconsortiaisolatedfromhypersalineenvironments.
Morethan95%oftheCODofpicklingprocessingwaste-watercanberemovedusingbioaugmentationwithHalobacterhalobium(Kargietal.2000).TheCODremovalefficiencydecreaseswithincreasingfeedCOD,CODloadingrate,andthesaltconcentration(DincerandKargi2001).Inthesamemanner,theeffectofStaphylococcusxylosusaloneorcombinedwithactivatedsludgeasinoculuminthetreatmentofwastewaterfromthepickledvegetableindustrieshasbeenanalyzedatvariousNaClconcentrations(0.5–3%w/v)(Abou-Elelaetal.2010).TheresultsshowedthattheCODremovalefficiencydependedontheNaClconcentration.AtlowNaClconcentrations(0.5and1.5%w/v),theCODre-movalefficienciesforpureandmixedculturearecomparable(90%),andat3%NaCltheCODremovalefficienciesslightlyincreaseto>94%forbothcultures.Incontrast,theCODremovalefficiencyusingactivatedsludgealoneasin-oculumdecreasesfrom90to64%at3%NaCl.ThisfactshowsthatCODfromsalinewastewatercanbereducedbyhalophilicmicroorganismwithoutadditionaltreatment.
Theperformanceofhalophilicmicroorganismsinthere-movalofCODhasbeenalsotestedinanaerobicconditions.Theanaerobicsalt-tolerantbacteriaHalanaerobiumlacusroseihasbeenusedinanupflowanaerobicpacked-bedreactorusingsyntheticsalinewastewaterwithvariouscon-centrationsofsalt?1(0–5%NaCl)andCODcontent(1,900–3,400mgO2L).ThehighestCODremovalefficiency(94%)wasobtainedatlowsaltandCODcontent(KapdanandErten2007).
Treatmentofsalinewastewaterpollutedwithhydrocarbons
Petroleumandgasnaturalreservoirscontainsalinewater.Duringtheproductionofcrudeoilandnaturalgas,largeamountsofreservoirwateraretypicallyalsoextracted,thisreservoirwaterisknownasproducedwater(PW)(Speight2007).AlthoughoilsandgreasesaretheconstituentsofPWthathavereceivedthemostattention,PWalsocontainsor-ganicandinorganiccompoundsthatcanvarydependingontheextractionsite(Veiletal.2004).PWfromgasproductioncontainsaromatichydrocarbonssuchasbenzene,toluene,ethylbenzene,andxylene(BTEX),whicharecharacterizedbytheirlowmolecularweightsandhightoxicity.
ThePWfromoilproductionalsocontainspolycyclicaro-matichydrocarbons(PAHs)(Jacobsetal.1992),whichpersistinsoilsandsedimentsforalongtimeduetotheirhydrophobicnature.PAHsaretoxic,carcinogenic,andmutagenic,andthereforetheypresentproblemsforhumanhealth.Thearo-matichydrocarbonsoflowtomediummolecularweightare
EnvironSciPollutRes(2014)21:9578–9588
relativelysolubleinPWcomparedtothehigh-molecular-weightnon-aromaticfractionsoffossilfuels.PAHscontributetowatertoxicitybecausetheirtoxicitiesareadditive,i.e.,eventhoughtheindividualtoxicitiesmaybeinsignificant,thecombinationofthesechemicalsresultsinanincreasedcom-binedtoxicity(Veiletal.2004).
Thebiodegradationofhydrocarbonsundersalineconditionshasbeenextensivelystudied.Thestudiesontheseorganicpollutantsusinghalophileshavemainlyfocusedontheisolationandidentificationofhydrocarbon-degradinghalophilicmicroor-ganismsfromhypersalineenvironments.Insomecases(Kleinsteuberetal.2006;Erdogmusetal.2013),theproficiencyofhalophilicisolatesinthedegradationofseveraltypesofhydrocarbonsoraromaticmetabolitesunderseveralcultureconditionshasbeenreported.IthasbeenshownthatnutrientadditioncanimprovethereductioninCODandhydrocarbonbiodegradation(Piubellietal.2012;Bonfáetal.2011).Recently,studiesidentifyingthekeyenzymesinvariousmetabolicpath-wayshavebeenconducted(Seoetal.2007;Zhongetal.2011).Aliphaticandaromatichydrocarbonshavebeenremovedbybothhalobacteriaandhaloarchaeastrains,anddifferencesinthecultureconditionsandremovalefficiencieshavebeendescribed(Table1).Furthermore,somemembersoftheHaloferaxgenushavebeenabletogrowonamixtureofsomeoftheintermediatesofPAHbiodegradation,asthesolecarbonsource(Bonfáetal.2011).Theintermediatesaresimulta-neouslydegradedwiththemixtureofPAHs(naphthalene,anthracene,phenanthrene,pyrene,andbenz[a]anthracene;0.3mMeach)inthepresenceof20%NaCl.Thisdemon-stratesthecatabolicversatilityofhaloarchaeatomineralizearomaticcompounds.Inaddition,thegrowthandextentofPAHdegradationbyHaloferaximprovedinthepresenceof0.05%w/vofyeastextract(Bonfáetal.2011)
TheabilityofhalophilicmicroorganismstodegradePAHsdecreaseswithincreasingmolecularweight(ArulazhaganandVasudevan2009),duetotheirdecreasedsolubilityand,conse-quently,theirdecreasedbioavailability.AconsortiumcollectedinChennaidegraded?189and74%oftheinitialphenanthreneat50and100mgL,respectively,butonly89and88%oftheinitialpyreneat5and10mgL?1,respectively,inthepresenceof3%NaCl(ArulazhaganandVasudevan2011a?1).Additionofglucose,sodiumcitrate,andurea(25mmolLeach)enhancedthePAHutilizationduetotheincreasedmicrobialgrowth(ArulazhaganandVasudevan2011b).Theuseofchemicaldispersingorco-culturewithbiosurfactant-producingbacteriacouldovercometheproblemofbioavailability.ThecultureconditionsforhydrocarbonbiodegradationbyhalobacteriaandhaloarchaeaundersalineconditionsaresummarizedinTable1.Toimprovetheprocessofpollutantbiodegradation,variouscultureconditionsandcomponentsofthemediausedforthedegradationstudycanbemanipulated.Thestatisticallybasedexperimentaldesignsforscreeningthenutritionalandenviron-mentalfactorswhichaffectthepollutantbiodegradationprocess
Table1BiodegradationofhydrocarbonsbyhalotolerantandhalophilicstrainsPollutantsp-Hydroxybenzoicacid,Naphthalene,Phenanthrene,Pyrene90%,100mgL-112d,30°C,1-17%NaCl,120rpm12d,38°C,10%NaCl130rpm65-80%,2mMeach100 0mgL-11000-150mgL-1Dastgheibetal.2012Piubellietal.2012Notreported(Optimalconcentrationsforgrowth80-120mgL-1)10-15d,37°C,20%NaCl,150rpmErdogmusetal.2013RemovalConditionsReferenceStrainsEnvironSciPollutRes(2014)21:9578–9588
Haloferaxsp.,Halobacteriumpiscicalsi,H.salinarum,6d,30°C,3%totalsaltcontent,150rpmFengetal.20125d,30°C,5.8-20.3%NaClLietal.201220%ofNaCl,168h,40°C,150rpm.10%ofNaCl,40°C,168h.Bonfáetal.2011Haloarculasp.,H.hispanica,Halorubrumsp.,H.ezzemoulenseConsortiumQphe-SubIVPhenanthrene(Halomonassp.+Marinobactersp.)Halomonassp.Phenol,benzoicacid,andp-hydroxybenzoicacidinproducedwaterMartelellasp.AD-3PhenanthreneMarinobactersp.,ProlixibacterBTEXsp.,Balneolasp.,Zunongwangiasp.,Halobacilluss.Haloferaxsp.Naphthalene,Anthracene,Phenanthrene,Pyrene,andBenz[a]anthracene3%NaCl,37°C,48hOchrobactrumsp.VA1ArulazhaganandVasudevan2011aOchrobactrumsp.VA1AnthracenePhenanthreneNaphthaleneFluorenePyreneBenzo(k)fluorantheneBenzo(e)pureneAnthracene30-90%(dependingonthearomaticcompound;0.3mMeach)65%oftheCODinthePWobtainedfromanoilrefinery,1345mg-CODL-1)88%(3mgL-1)98%(3mgL-1)90%(3mgL-1)97%(3mgL-1)84%(3mgL-1)57%(1mgL-1)50%(1mgL-1)87%(3mgL-1)MediumwithglucoseMediumwithsodiumcitrateMediumwithurea37°C,150rpm,5d,3%NaClArulazhaganandVasudevan2011bHaloferaxsp.,Halobacteriumsp.,Halococcussp.HeptadecaneEicosanePyreneAnthracenePyreneAnthracenePyreneCrudeoiln-OctadecanePhenanthrene3weeks,40-45°C,23.4%NaCl,180rpmAl-Mailemetal.2010Haloarculasp.,Haloferaxsp.83%(3mgL-1)81%(3mgL-1)76%(3mgL-1)88%(3mgL-1)84%(3mgL-1)13-47(-67-30%2gL-1each32-95%0.5gL-1each30d,40°C,22.5%NaCl,120rpmTapilatuetal.20109581
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9002nave9d60u00s02Va2e9.l5ur0da0p0nt0e2ae2hunterl.eaLaacFgbdautndheentrenazsaíeaaolnicfiaueeelarRSHArKGdl5mC,pmraNp0r5C5,°61C.7,°d311lC74,,a38hs,kN,l8edC4e5a,sw%l,n8lNCo21Caiaoft,,NidCCNn°°%%o07\0333.711)1)-L-L1)gg-mmLg0000m110dd1nndaan00a555(((%%%1)-Ld488gdMe778etdddmtlaμornnn1orv%paaa(po5eerr02t%%%%%tem00-o99905oR12N88879Nd,id,ciacacicoilarszulnueteercabFeyltrosxd,dmboiiccusdraacycciiowihnllneoyicdanp-pilngeeoaasorneyrd,istlprotlneihrplecanehteynituenognne)uafcniemzeeeaeellnudnno(lizeohaoeluweorenszPp-PBToCrhulyreeniePFPBDB.psa1inaM-oitd)rseleTtucasnauRibc.,tnoc.poeopsstccss(rocma.piunpalaiiosn1mhtsenporaoiliosomzbanltomamllraaeiaTatGHoSCHDHEnvironSciPollutRes(2014)21:9578–9588
bynon-halophilicmicroorganismhavebeenperformed(FaragandSoliman2011;Xiaetal.2012),butinsalineconditionsthisprocedurehasbeenpoorlyexplored.Factorialdesigntechniqueshaveadvantagesovertheone-factor-at-a-timeapproachtopollutantremovalinordertocarryoutbiostimulationandbioaugmentationstrategies.Withthoseapproaches,itispossibletoknowthesignificantfactorsandtogettheiroptimizationinthebiodegradationprocess.Ghevariyaetal.(2011)increasedthechrysenedegradation(from40.79to85.96%)byhalotolerantAchromobacterxylosoxidansusingCentralCompositeDesign.
Knowledgeofthemetabolicpathwaysinpollutantbiodeg-radationisimportantasitallowspredictingthefateofthedegradationproductsintheenvironment(LeBorgneetal.2008).Incaseofhalophilicmicroorganisms,informationaboutthemetabolicmechanisms,enzymes,andgenesin-volvedinPAHbiodegradationisscarce.Similartothenon-halophilicmicroorganisms,thedegradationpathwayofPAHsinsalineconditionsbyhalophilicmicroorganismsinvolvestheiroxidationtosalicylatethatcanthenbefurtherconvertedtoeithercatecholorgentisicacid(Fengetal.2012).Thecatecholringiscleavedby1,2-dioxygenase(orthopathway)or2,3dioxygenase(metapathway).Biodegradationproductsproducedbytheseoxidativeenzymeshavebeenexaminedinseveralrecentbiodegradationstudies(Fig.1).
ThehalophilicbacteriumMartelellasp.degradesPAHthroughthegentisicacidratherthanthecatecholpathway(Fengetal.2012).Thecatechol1,2dioxygenaseenzymewasfoundtobeinvolvedinPAHbiodegradationbyHaloferaxsp.,Halorubrumsp.,andHalobacteriumpiscisalsi,whicharehalophilicarchaeaisolatedfrom?amaltiSalterninTurkey(Erdogmusetal.2013).
Catechol2,3-dioxygenaseratherthancatechol1,2-dioxygenasewasinvolvedinthedegradationofbenzeneandtolueneinthepresenceof29%NaClbyArhodomonassp.,isolatedfromGreatSaltLake,Utah,USA(SeiandFathepure2009;LeBorgneetal.2008).Thesefindingsdemonstratethathalophilicarchaeaandhalophilicbac-teriaareabletometabolizePAH,andthatsomeofthemsharethesamecatabolicpathways.Haloarchaeaproba-blyevolvedfrommethanogens,uponwhichtheyhadtoswitchfromastrictlyanaerobicchemolithoautotrophictoanaerobicphoto-organoheterotrophiclifestyle.ThisswitchwasaccompaniedbyamassivegenegainfromtheBacteriado-main(Khomyakovaetal.2011).
TreatmentofsalinewastewaterpollutedwithphenolAsaconsequenceofitsserioushealthandecologicalconcerns,phenolhasbeenincludedintheprioritylistofhazardoussubstancesoftheEnvironmentalProtectionAgency(EPA)andtheEuropeanUnion(EU)(Buscaetal.2008).