Weissenfels W.D., H.J. Klewer, and J. Langhoff (1992) Adsorption of ...... (Andelman & Snodgrass 1974) by PAHs has been reported and several PAHs have.
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BIOAVAILABILITY ANDBIODEGRADATIONOF POLYCYCLICAROMATIC HYDROCARBONS
FrankVolkering
CENTRALE LANDBOUWCATALOGUS
0000 0664 9277
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Promotor: Copromotor:
dr. ir.W.H.Rulkens hoogleraar indemilieutechnologie dr.A.M.Breure onderzoeker aanhetRijksinstituut voorVolksgezondheidenMilieuhygiene(RIVM)
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1 De beweringvan Keuth &Rehmdatdeexponentiele groeisnelheid van bacterien toeneemtbijsubstraatconcentratieswelkebovendemaximaleoplosbaarheidliggen, isonjuist. Keuth S. and H.J. Rehm (1991) Biodegradation of phenanthrene by Arthrobacterpolychromogenes isolatedfromacontaminated soil.Appl. Microbiol.Biotechnol.34:804-808.
2 Het kunstmatig verontreinigen van grond door de verontreiniging in een kleine hoeveelheid vluchtig oplosmiddel aan de grond te voegen en het oplosmiddel vervolgens snelafte dampen levert een produkt opdatslechts inbeperkte mate kanwordenvergeleken metvervuildegrondzoalsdeze indepraktijkvoorkomt. Ditproefschrift 3 Hetideedatdeverhogingvandesnelheidvanbiologischeafbraak van organische verontreinigingen indebodemdiebereiktwordtdoorhettoepassenvansurfactantoplossingen, kan worden gekoppeld aan de mate van solubilisatie van de verontreiniging indesurfactant-oplossing, berust opeen misvatting. Dit proefschrift
4 Het in de bodemwetgeving gebruiken van verontreinigingsconcentraties die zijn bepaald via chemische extractie gaat goeddeels voorbij aan de biologische beschikbaarheid van de verontreinigingen, waardoor de ecotoxicologische onderbouwing vandezewetgeving discutabelis. Dit proefschrift
5 Het bezuinigen op (experimenteel) wetenschappelijk onderzoek binnen het Rijksinstituut voor Volksgezondheid en Milieuhygiene getuigt van een beperkte toekomstvisie,dienietpastbijeeninstituutdatzichwilprofilerenalstoonaangevend ophetgebiedvan milieu-en volksgezondheids-toekomstverkenningen. 6 Literatuurbesprekingen geven vaak een nieuwe kijk op de waarde van wetenschappelijke publikaties en zijn bijzonder nuttig bij het zelf schrijven van manuscripten.Hetisdaaromvoor(universitaire)onderzoeksgroepenaantebevelen regelmatig dergelijke besprekingen tehouden. 7 Een uitgebreide lunch is een goede voedingsbodem voor wetenschappelijk onderzoek. 8 Ineentijdwaarinhetkrijgenvankinderen isveranderd inhet nemenvan kinderen is,geziendehogebevolkingsdichtheid inditland,hethuidigeNederlandsesysteem voor kinderbijslag eenanachronisme.
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A.N.;'.
ISBN 90-5485-575-4
VOORWOORD
Ditproefschrift ishet resultaatvaneenprojectdat inhetkadervan het Speerpuntprogramma Bodemonderzoekisuitgevoerd bijdeAfdeling BiotechnologischOnderzoek (BTO)van het LaboratoriumvoorAfvalstoffen en Emissies(LAE) vanhetRijksinstituut voorVolksgezondheid enMilieuhygiene (RIVM) inde periode 1989-1994. Ineerste instantie betrof heteendriejarig project, maardooreen verlenging vananderhalfjaar, waarbijdevakgroep Milieutechnologie vande LandbouwuniversiteitWageningen bijhet projectwerdbetrokken,konhet onderzoek uiteindelijk totdit proefschrift leiden.Datdesamenwerkingtussendeafdeling BTO endevakgroep Milieutechnologie goed bevielblijktondermeer uithetfeiter inmiddelseenvervolg opditonderzoekwordt uitgevoerd,waarbij eenpromovendus vandeLUWbijhetRIVMisgedetacheerd. Achteraf gezienvalt mijnperiode bijdeafdeling BTOinde bloeiperiode vande afdelingenmijndankgaatdanookindeeerste plaats uitnaarJohanvanAndeldie het,mededoorzijnmensenkennis,voorelkaar heeftgekregen een universitair aandoendeonderzoeksgroep bijhet RIVMoptezetten.Alsafdelingshoofd was hij deprettigste "baas"diejeje kuntwensen. Dedagelijkse begeleiding vanhetonderzoek was inhandenvanTon Breure. Naastco-promotor,kamergenootenonverbeterlijke sloddervoswas hijtochvooral deenthousiaste,stimulerendeenkritischewetenschapper diemeoverde moeilijke puntenheenhielp.Zonder hemwasdit proefschrift hoogstwaarschijnlijk niettot standgekomen. Verderwilikallemedewerkersenstudentenvandeafdeling BTObedankenvoor deprimasfeer, metnamede"lunchclub". Ikmisdegeweldige engezellige lunches met inspirerende discussies overzowelonderzoek als meerwereldsezaken nog iederedag.KlaasDoesburg,Wilfredvander Sterrenen RiekevanderWielhebben inhetkadervaneenstageofafstudeervak belangrijke bijdragen aan het onderzoek geleverd. WimRulkens,als promotor pasde laatstejaren bijhet project betrokken, heeft hetvaak nietmakkelijkgehad met"diemicrobiologen", maarwistdoorzijngrondige fysisch-chemische kennis hetonderzoek zeker meerdiepgangtegeven.
Decorrectiesvan het Engels inditproefschrift door RuthdeWijsvan hetRIVM komenmijook nunoggoedvanpasbijhetschrijven van Engelseteksten.Marian Vermuewilikgraag bedankenvoorhetgebruikvanhaarcomputer bij Proceskunde. Ikhoopdat mijngezwoeg indeavondurenenweekenden nietvooralteveel overlast heeftgezorgd. Tenslottegaatmijndank natuurlijk noguitnaar imke;haarhebikzekerveel overlast bezorgd,maarbinnenkort krijg ikdekans haar netzogoedtesteunenals zijmijgedaan heeft!
CONTENTS
Chapter1
GENERAL INTRODUCTION
1
Chapter2
ISOLATIONAND CHARACTERIZATION OF PAH-DEGRADING BACTERIA
Chapter3
21
BIODEGRADATION OFCRYSTALLINE POLYCYCLIC AROMATIC HYDROCARBONS
Chapter4
39
BIODEGRADATION OFSORBED POLYCYCLIC AROMATIC HYDROCARBONS
Chapter5
63
MICROBIOLOGICAL ASPECTS OFTHE USEOF SURFACTANTS INBIOLOGICAL SOIL REMEDIATION . . . 91
Chapter6
INFLUENCE OF NONIONIC SURFACTANTS ON BIOAVAILABILITY AND BIODEGRADATION OF POLYCYCLIC AROMATIC HYDROCARBONS 117
Chapter7
DESORPTIONAND BIODEGRADATION OF SORBED NAPHTHALENE INTHE PRESENCE OF NONIONIC
Chapter8
SURFACTANTS
137
CONCLUDING REMARKS
159
SUMMARY
165
SAMENVATTING
169
CHAPTER 1
GENERAL INTRODUCTION
Abstract Polycyclicaromatic hydrocarbons (PAHs) are hydrophobic organic pollutants abundantly present inthe environment. Thisfact,alongwiththeirtoxicityand mutagenicity, makesthem priority pollutants. Experimentswith PAHs under laboratory conditions haveshownthatthese compounds canbedegraded byvarious microorganisms. Underfieldconditions however, PAHsareknownas persistent pollutants,forming animportant problemforthe biological remediationofPAHpolluted soil.Thischapterwilloutlinethe physicochemicalcharacteristics ofPAHs, theenvironmental problemscaused bythepollutionofsoilwith PAHs,andthe different aspectsthat playarole inthe biologicalclean-up ofthesecompounds.
Chapter1
Soil pollution Thecontamination ofsoilwith polluting compounds isoneofthe major environmental problems inboth Europeandthe United States.Onthe basisofhow thepollution ispresent inthe soil,twocategories canbedistinguished.Thefirstis diffuse pollution,wherethe polluting compound ispresent at lowconcentrations in largeareas.Atmospheric deposition isthe major sourceofdiffuse pollution.The second isspot pollution.Thecontaminated area isrelativelysmallandvery high concentrations ofthe pollutant mayoccur.Thesource ofthecontamination isusually aspecific industrialactivitywhich has beenorisstillbeingcarriedoutatthespot. Sincethe 1970s,many polluted areas,causing vastsocialandeconomic problems, havebeendiscovered.Thenumberofknown pollutedsitesinthe Netherlands is approximately 120,000andthecostsfortheclean-up ofthesesites has been estimated atmorethan 50billion U.S. dollars (Stoop&Hesselink 1993). Anotherdistinction isbasedonthetypeofpollution:pollutionwithheavy metallic compounds andwithorganic compounds. Pollutionwithheavy metals isbeyondthe scopeofthisthesis andwillnotbedealtwith. Governments ofseveralcountries haveset up rulesconcerning soilpollution.The EPA list(USA)andthe Dutchstandards (The Netherlands) areexamplesof guidelinesgiving regulations on pollutants insoiland groundwater.
Polycyclic aromatic hydrocarbons Structures and properties of polycyclic aromatic hydrocarbons Polycyclic aromatic hydrocarbons areorganic moleculesthat consist oftwoor morefused benzene rings inlinear, angular orcluster arrangement. The chemical structure ofthe mostfrequently occuring PAHs,allEPA priority pollutants,is presented inFigure 1.Thesimplest PAHisthetwo-ring compound naphthalene;a well-knownexampleofahigh molecularweight PAHisthefive-ring compound benz[a]pyrene.
General
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Chapter1 Table 1givesanoverview ofsomephysical andchemical properties ofthemost frequently occurring PAHs.Generally,the hydrophobicity issaidto increase,andthe aqueous solubilitytodecreasewithanincreasing number ofaromatic rings. Table1:Physicaland chemicalproperties ofthemost frequentlyoccurringPAHs (Sims& Overcash1983). Molecular weight
Aqueous solubility at 30°C[mg-L1]
Vapour pressure [N-nr2at20°C]
Logoctanol/ water partition coefficient
naphthalene*
128
31.7
6.56
3.37
acenaphthylene
152
3.93
3.87
4.07
acenaphthene
154
3.47
2.67
4.33
fluorene
166
1.98
1.73
PAH
phenanthrene* anthracene* pyrene
178 178 202
1.29
4.18 2
4.46
2
4.45
4
5.32
5
9.07-10" 2
7.3-10"
2.61-101
1.35-10"
1
8.00-10"
fluoranthene*
202
2.60-10"
9.11-10'
5.33
benz[a]anthracene*
228
4.0-10"2
6.67-10"7
5.61
228
3
5
5.61
5
6.04
5
6.57
5
6.84
8
7.66
8
7.23
8
5.97
chrysene* benz[a]pyrene* benzo[k]fluoranthene* benzo[b]fluoranthene indeno[123cd]pyrene* benzo[ghi]perylene* dibenz[ah]anthracene
2.0-10"
3
252
4.0-10"
252
3
252 276 276 278
1.2-10
4
5.5-10"
2
6.2-10"
4
2.6-10"
4
5.0-10"
8.40-10"
6.67-10" 6.67-10" 6.67-10
1.33-10"
1.33-10" 1.33-10"
*:referencePAHsinDutchlist Sources andenvironmental concern PAHsoccur naturally intheenvironment and asaresult ofhumanactivities. Naturalsourcesincludethe productsfromforestandbushfires,thermalgeochemical processes (e.g.vulcanic activity) and synthesis bymicroorganisms and plants (Blumer 1976). Background concentrations of PAHs inpristinesamples can range from50to 1100ug-kg"1insoil (Fritz &Engst 1975),0.001to0.1 ug-L1in groundwater (Suess 1976),and 0.01to 88ug-kg"1inplants (Sims&Overcash 1983).
Generalintroduction Theanthropogenic sources ofPAHemissionscanbedivided intothreemain categories: 1- the incomplete combustionoforganic compounds (e.g.fossilfuels),resulting in theatmospheric deposition ofPAHs 2- thedeposition ofliquid/solid PAH-containing industrial products andwaste,such asoilspillage anddeposit ofcoal-tar ongas productionsites 3- the useof PAH-containing products,suchasasphalt, coatingsforships,the wood-preserving creosote andanthracene oil,etc. Concentrations insoilof PAHsoriginatingfromatmospheric deposition arevariable, butmeasurements inthe Netherlands haveshownanaverage depositionof2.5-105 kgperyear (Sloofefal.1989). PAHconcentrations at heavily polluted sitesmaybe ashighas 12g k g 1 (Fritz &Engst 1975).Anoverview ofPAHconcentrations at different polluted industrialsites isgiven inTable2.Oneofthe bestexamples ofthe problemswith PAH-contaminatedsites isthe pollution atsitesofformer gas production plants.Thepollution consists mainlyof highconcentrations ofPAHs, cyanides,and heavy metals.Ofthesecontaminated sites234are knowninthe Netherlands,the costs ofcleaningthem uphavebeenestablished at400 million U.S. dollars(RIVM1991). Theabundance of PAHs intheenvironment gives risetoconcern becauseofthe riskthesecompounds pose,bothfrom anecotoxicological andahuman-health point ofview. PAHsmayaccumulate infattytissues ofmammals andspecific PAHshave provedtobemutagenic, carcinogenic, andteratogenic (LaVoieetal. 1979). Because ofthis,the UnitedStates Environmental ProtectionAgency hasplaced 16PAHson their priority pollutant list(Keith &Telliard 1979).The Dutchstandardsfor PAHsin soilandsludge are based onecotoxicological research.Whentheconcentration is belowthe referencevalue,thesoil isconsidered cleanand multifunctional.At concentrations higherthanthe intervention value,thesoil istoo pollutedforfurther useand hastobetreated.At intermediate pollution levelsotherfactors,suchasthe function ofthe polluted site,areconsidered indecidingwhetherthe soil istobe cleaned.The reference and interventionvaluesfor PAHsarebasedonthe concentration oftheten PAHsonthe Dutch list(seeTable 1).Thevalues are dependent onthe amount oforganic material present inthesoilandcanbe calculated accordingto: - referencevalue oftotal PAHs =0.1 x%humic material[mg PAHskg"1dryweight] - interventionvalue oftotal PAHs=4.0x%humicmaterial[mg PAHskg"1dryweight] Forasoilwith 10%organic matterthe referencevalue is 1mg.kg1 andthe
Chapter 1 intervention value 40 mg.kg'1. Comparing these values withthe concentrations in Table 2 , itcan be seen that all ofthese soils are to betreated and that a reduction of the PAH concentration by a factor of 100-10,000 isnecessary to obtain soilthat can be usedwithout restrictions. Table2: ConcentrationsofPAHs inmg-kgr1dryweightatselectedcontaminatedsites (Wilson&Jones1993). Wood preserving PAH
topsoil
Creosote production
subsoil mean
Wood Coking Coking Treatment plant plant
range
naphthalene*
1
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Isolationandcharacterizationofbacteria Substrate specificity Mostofthe strains isolatedwerecapableofgrowthonseveraldifferent PAHs(see Table2).To investigatethe substrate specificity ofthe bacteria,experiments have beenperformedwithconcentrated cellsuspensions ofthestrains 8909Nand8803F. Strain8909Ndid notshowgrowthonphenanthrene.Whenthisstrainwas precultured on naphthalene,additionof phenanthrene resulted inan intenseyellow colouring ofthe medium.The UVspectrumofthesupernatant showedgood agreementwiththe spectrumof3,4-dihydroxyphenanthrene. Incubation ofstrain 8909Ncells,precultured onsuccinate, using phenanthrene assubstratedidnot result indetectableformationof intermediates. Incontrastwithstrain8909N,which couldnotgrowonphenanthrene, strain8803Fwascapableof growthonboth naphthalene and phenanthrene. However,whenstrain8803Fcells,precultured on phenanthrene,were incubatedwithnaphthalene,alagphaseof2-4 daysoccurred; initiallytheproductionof 1,2dihydroxy-naphthalene could beobserved, indicating thattheconversionof both PAHstodihydroxylated compounds ismediated bythe sameenzymes andthatdifferent enzymes areinvolved inthe ringfission,the next step inthedegradation pathway. Effectofco-occurring substrates Toassesstheeffect ofthe presence ofdifferenttypes ofcosubstrates onthe biodegradation of naphthalene, phenanthrene,andanthracene,the C0 2 production duringgrowth oncrystalline PAHwasfollowed inthepresenceandabsenceof cosubstrates.Anexample isgiveninFigure 1,whichshowsthe C0 2 productionby strain8803Fgrowing on naphthalene,phenanthrene,andonamixture ofthetwo substrates.Adding upthe C0 2 concentrations ofthedegradation curves ofthetwo separate PAHsgives acurvethat isalmost identicaltothatofthe C0 2 production in thepresenceof bothsubstrates.Thus,itwasconcludedthat both PAHsare degradedsimultaneously. Thistypeofexperiment hasbeenperformedfor mixtures ofseveralsubstrates.The resultsoftheseexperiments aresummarized inTable5. Succinate canbeconcludedtobedegraded preferentially overtheother substrates.Whentwoaromatic substrates are present,theyare usually degraded simultaneously. The results ofthese experiments areconsistentwiththe observations of Bauer &Capone (1988) andofTiehm&Fritsche (1995),whofound thatthesimultaneous presence ofthe PAHs naphthalene,phenanthrene,and anthracenedid notalterthe degradation patterns.
29
Chapter2
5-104s"1for naphthaleneto 103s_1 for anthracene. Using avalue of 10"for phenanthrene, itispossibletogiveaglobal estimationforthe maximalexit ratesatdifferent residualconcentrations.These rates aremuch higherthanthe bacterial uptake rates,ascan beseen inTable7. Itcan therefore beassumedthat theconcentration ofphenanthrene inthe micelles isin equilibriumwiththe aqueous phenanthrene concentration,making itpossibleto calculatethe aqueous concentration accordingto Equation 2(Volkering etal.1995): tot aq
V + m •V
(2)
where Caq isthe aqueous concentration [mg-L1],Ctotisthetotal concentration [mg-L1], Vtbe volume ofthe aqueous phase[L],m the micelle-water partition coefficient, previously determinedto be3.3-104for phenanthrene (Volkering etal. 1995),andVmic the micellarvolume [L].Althoughthisequation has beenvalidated 35
Chapter 2 for high PAH concentrations only, deviations atlow PAH concentrations will be small for non-ionic surfactants (Dougherty & Berg 1974). The calculated residual aqueous concentrations atthe different steady states are shown in the last column ofTable8. Although these values are not very accurate, itispossible toestimate the Ksof strain 8803F on phenanthrene at3-12 ug-L"1, a value of the same order ofmagnitude as the value for Ks for the growth ofstrain 8909N on naphthalene. NOMENCLATURE Ca„ Ctot Ks m ms V Vmjc Ym Vxs [i Wmax
aqueous concentration [mg-L 1 ] total concentration [mg-L 1 ] bacterial affinity constant [kg-nrr3] micelle-water partition coefficient [-] maintenance coefficient [kg substratekg b i o m a s s 1 h 1 ] volume o faqueous phase [L 1 ] micellar volume [L"1] overall growth yield [kg biomass-kg substrate' 1 ] maximal growth yield [kg biomass-kg substrate 1 ] growth rate [h 1 ] maximum growth rate [h 1 ]
REFERENCES Almgren M., F.Grieser, and J.K. Thomas (1979) Dynamic and static aspects of solubilization of neutral arenes in ionic micellar solutions. J.Am. Chem. Soc. 101:279-291. Bauer J.E., and D.G. Capone (1988) Effects of co-occurring aromatic hydrocarbons on degradation ofindividual polycyclic aromatic hydrocarbons in marine sediment slurries. Appl. Environ. Microbiol. 54:1649-1655. Boldrin B., A. Tiehm, and C. Fritzsche (1993) Degradation of phenanthrene, fluorene, fluoranthene, and pyrene by aMycobacterium sp.Appl. Environ. Microbiol. 59: 1927-1930. Cerniglia C.E. (1984) Microbial metabolism of polycyclic aromatic hydrocarbons. Adv. Appl. Microbiol. 30:31-71. Dougherty S.J., and J.C. Berg (1974) Distribution equilibria in micellar solutions. J. Colloid. Interface Sci. 48:110-121. Davies J.I.,and W.C. Evans (1964) Oxidative metabolism ofnaphthalene by soil pseudomonads: the ring-fission mechanism. Biochem. J. 91:251-261. 36
Isolationandcharacterizationofbacteria Evans C.G.T., D. Herbert, and D.W. Tempest (1970) The continuous cultivation of microorganisms. 2.Construction ofachemostat. In: NorrisJ.R., and D.W. Ribbons (eds), Methods of Microbiology,Vol2.AP London,p277-327. EvansW.C., H.N. Fernley, and E. Griffith (1965) Oxidative metabolism of phenanthrene and anthracene bysoil pseudomonads:the ring-fission mechanism. Biochem. J.95:819831. GuerinW.F.,andG.E.Jones (1988) Mineralization of phenanthrene bya Mycobacteriumsp. Appl. Environ.Microbiol.54:937-944. Herbert D., P.J. Phipps, and R.E. Strange (1971)Chemical analysis ofmicrobial cells.In: NorrisJ.R., and D.W. Ribbons (eds), Methods inmicrobiology vol5BAP London,p 209-343. Keuth S.,and H.J. Rehm (1991) Biodegradation of phenanthrene byArthrobacter polychromogenesisolatedfrom acontaminated soil.Appl.Microbiol.Biotechnol. 34:804-808. Perry R.H., C.H. Chilton,and S.D. Kirkpatrick (1963) Chemical engineers' handbook, McGraw-Hill BookCo., NewYork,U.S.A. Peterson G.W. (1977)A simplification ofthe proteinassay of Lowry efal. which ismore generally applicable.Anal.Biochem.83:346-356. Sanseverino J.,B.M.Applegate, J.M.H.King,andG.S. Sayler (1993) Plasmid-mediated mineralization of naphthalene, phenanthrene, andanthracene.Appl Environ.Microbiol. 59:1931-1937 Tempest D.W., and R.E. Strange (1966)Variation incontent anddistribution ofmagnesium, andits influence onsurvival,inAerobacter aerogenesgrown inachemostat. J.Gen. Microbiol.44:273-279. TiehmA., and C. Fritzsche (1995) Utilization ofsolubilized andcrystalline mixtures of polycyclic aromatic hydrocarbons by aMycobacteriumsp.Appl.Microbiol.Biotechnol. 42:964-968. Van't Riet K., and J.Tramper (1991) Basic bioreactor design.Marcel Dekker Inc, NewYork, USA. Volkering F.,A.M. Breure,J.G.vanAndel, andW. H. Rulkens (1995) Influenceof nonionic surfactants onthe bioavailability and biodegradation of polycyclic aromatic hydrocarbons. Appl. Environ.Microbiol.61:1699-1705. Walter, U., M. Beyer, J. Klein,and H.J. Rehm (1991) Degradation of pyrene by Rhodococcussp UW1. Appl.Microbiol. Biotechnol.34:671-676. Weber F., L.P. Ooijkaas, R.W. Schemen, S. Hartmans, andJ.A.M.de Bont (1993) Adaptation of Pseudomonasputida S12to highconcentrations of styreneand other organic solvents.Appl. Environ.Microbiol.59:3502-3504.
37
CHAPTER 3
BIODEGRADATION OFCRYSTALLINE POLYCYCLIC AROMATIC
HYDROCARBONS
Abstract Bacterialgrowth oncrystalline naphthalene andphenanthrene hasbeen demonstrated to berelatedtothedissolution rateofthesecompounds. Noevidence forenhancement ofsubstrateavailability duetobacterial influence couldbe detected. Using amodelbasedondissolution kineticsforsubstrate availability and Monodkineticsfor bacterialgrowth,itwas possibletosimulate bacterialgrowthon crystalline naphthalene.This modeliswidely applicabletogrowthof microorganisms onpoorlywater-soluble substrates andcanbeeasily adaptedtoamorecomplex systemsuchasmicrobialgrowth onsubstrates adsorbedtomatrices.
Thischapteris arevised formof two publications: VolkeringF.,A.M. Breure,A. Sterkenburg, andJ.G. vanAndel (1992)Microbialdegradation of polycyclic aromatic hydrocarbons: effect ofsubstrate availability onbacterial growth kinetics.Appl. Microbiol.Biotechnol. 36:548-552. VolkeringF.,A.M. Breure, andJ.G. vanAndel (1993) Effectofmicro-organisms on the bioavailability andbiodegradation of crystalline naphthalene. Appl.Microbiol. Biotechnol. 40:535-540.
39
Chapter3
Introduction Polycyclic aromatic hydrocarbons (PAHs)arehazardous compounds originating fromoil,tar,woodpreserving creosote,orfrom incompletecombustion offossilfuels. Thecontamination ofair(Daisey era/. 1979),soil(Bossert &Bartha 1984) andwater (Andelman&Snodgrass 1974) by PAHshasbeenreported andseveral PAHs have showntobemutagenic and/or carcinogenic (LaVoieetal.1979).Since ithasbeen shownthat PAHsarebiodegradable compounds (e.g.Davies&Evans 1964; Evans etal,1965),biotechnologicaltechniques might beapplicablefor remediationofPAHpolluted soil. However, application ofbiologicalsoilremediation has demonstrated that PAHs(especially theoneswiththe higher molecularweights) aredegradedvery slowly,andthatthe residual PAHconcentration isoftentoo highto permit unrestricted application ofthetreated soilaccordingtothe Netherlands governmental guidelines (Ministryof Housing,Physical Planningand Environment 1987;Soczo& Staps 1988;Staps 1990). Becauseoftheir hydrophobicity PAHspresent insoilwilloccur mainly innonaqueous phases.Theycanbeabsorbed ontosoilparticles,present intar particlesor inanoily phase,oroccurascrystals. Ithasbeenshownthatsome microorganisms cantake upnon-aqueous hydrocarbons directly (Reddy etal.1982). For PAHs,only onestudy reportsthecolonization ofphenanthrenecrystalsbyaMycobacteriumsp., indicatingdirect substrate uptake (Guerin &Jones 1988). Inallother documentation ithasbeenfoundthat purecultures ofbacteria can use PAHs inthedissolvedstate only (e.g.Thomas era/. 1986,Wodzinski&Bertolini 1972,Wodzinski &Coyle 1974). Thereforethedissolution ofsolid PAHs isaprerequisiteforgrowthtooccur. This impliesthateven insystemswithout intra-particlemasstransfer limitation,likepure insoluble substrates inshaking cultures, masstransferfromthe solidphasetothe aqueous phasemight be ratelimiting.Thiscanexplaintheoften observed linear growthofbacteria andyeasts onslightlysolublesubstrates (e.g.McLee&Davies 1972,Prokop etal. 1971, Stucki&Alexander 1987;Thomas efal.1986). Thisstudywas performedto investigatethe kinetics of PAHdegradation in relationtothesolubilization rateof PAHs. Insight intothese kinetics could leadtoa better understanding ofthefactors involved inthe incomplete removalof PAHsin biologicalsoilremediation processes.
40
Biodegradationofcrystalline PAHs
Theory Theratesof biomassformation andsubstrate uptakecanbedescribed by followingequation (Tempest, 1970): (IX, t
dt
dSt -Y , • — ov.t
(1)
d f
whereX,isthe biomass concentration [kg.nr3], S,thetotalamountofsubstrate per unitofvolume [kgm 3 ],Ymt theoverall bacterialgrowthyield [kg biomassformed-kg substrate used"1],and fthetime [h].Theoverall bacterialgrowthyieldcanbe expressed as(Van'tRiet&Tramper 1991):
Y , ov,t
Y xs
, "Vyxs 7+
(2)
withYxs beingthe maximalyield [kgbiomassformed-kg substrate used"1],msthe maintenance coefficient [kgsubstrate-rr'-kgbiomass1],andJU, thespecific growth rate[h 1 ]. Whentheyieldchangesduringtheexperiment,themicrobialcellmassis notagood measureforthe number ofcells perunitofvolumeN,[m 3 ],andis therefore notsuitedforcalculatingthegrowth rate. Because ofthis,the actualweight ofonecellW,[kg]hasto beincorporated inthemodel:
^ - W0-£*-
(3)
ov,t
withW0 beingthe initialweightofonecell[kg].Thechange inthe biomass concentration canthen beexpressedas: dX, dNt — I =i v . — l
dt
(4)
' dt
The relation betweenthegrowth ratey,andtheactualsubstrate concentration C,can beexpressed usingthe Monodequation:
41
Chapter3
" ' " Nt' dt ~ **""*'C, r
+
(5)
K t
s
where^mgx isthe maximalspecificgrowth rate[h 1 ]andKsthesaturation constant [kgnr 3 ]. Whenthesubstrate ispresent inasolidandaliquidphase,the substrate concentration cannot bewrittenas S ( , but hastobedivided inthe aqueous substrateconcentration C, [kg-nr3]andtheamountofsolidsubstrate Q, [kg]. Equation (1)can then bewrittenas: dX. 1 dQ. dC. — - =- v ,-(—•—- + — - ; 0VJ dt v dt dt'
(6)
w
where Vistheaqueousvolume [m3].The rateofmasstransferfromthesolidphase totheaqueous phase (dissolution) ofspherical particles canbedescribed as (Perry era/. 1963): dQ
- ^ =W < w -c,;
(?)
wherek,isthe masstransfer coefficent [mh 1 ], Cmaxthemaximalsolubility ofthe substrate [kg.m3], andA, the actualcontactsurfacearea [m2]. Themasstransfer coefficient canbeexpressedsemi-empiricallyas(Perry etal.1963):
k, -^(2 d
+0.6-(^^-r-(^-n
n
P,O
inwhichDistheeffective diffusion coefficient [m 2 h 1 ]; cfthe particle diameter [m];p, theliquiddensity [kgm 3 ];vdthe relativevelocity oftheparticles [mh 1 ], and n the liquidviscosity [kg-h"1-nr3].Thisequation cannot beusedforcalculatingk,inshaking culturessincethe relativevelocity oftheparticles isunknownandprobably different forparticleswithdifferent diameters. However, itshowsthatk:isdependent onthe particlediameter andthe liquid-mixing properties.Withtheassumptionthatthe particles arespherical,the contact surface area canbeexpressedas:
42
(8)
BiodegradationofcrystallinePAHs 3Qt A =
>
fn
')3*
p'"-L
w
4-n p p s -n wherenp isthe number ofparticles [-] andpsthespecificdensity ofthesolid substrate [kg-rrr3]. Inmostoftheexperiments presented herethechangein diameter, andthereforethechanges ink,and inAt, aresmall.Byexpressing dX,/dt anddQf/dtasafunction of Ct,aswasdone inthe Equations4,5,and7,Equation6 becomesalineardifferential equationthatcanbesolved numerically. Inbatchexperiments at lowercelldensities (whenC , » 0)masstransfer isnonlimitingandexponentialgrowthcanoccur.At highcelldensities ingrowing cultures, however, onemayassumethatC, isnegligiblecomparedtoCmax and constant (dC,/dt=0;C,=0)and,therefore,thatthedissolution approaches its'maximal rate • U (kg-m-3-h-1):
j ..ift max
v
df
.^L5=L 'max
(10,
y
Inthissituation,the rateofsubstrate uptake bythe biomasscannot exceedthe dissolution rateand,byconsequence,the biomassformation rate islimitedtothe masstransfer rateaccordingto: dXt
Y , dQt
t _ _
dt
=
ov,t i
" V
11
dt
max
Y
,-k,A-C
ov.t I
t
max
(111
V
Equation 11describesthe linear increaseofthebiomass concentration intime when theparticle diameter andtheyieldareconsidered constant. Thecomputer program PSI/c(BOZA automatisering, Pijnacker, The Netherlands) wasusedtosimulate microbialgrowthoncrystalline naphthalene.Thevaluesfor Yxs andms, determined inchemostatexperiments (Chapter 2),were 1.2kgbiomasskg substrate1and0.015kgsubstrate-h'1kg biomass1 respectively.Thespecific density psofnaphthalene is 1200 kgm 3 . Anexample ofagraphical representation ofthemodelisgiven in Figure 1; inthis simulaton itisassumedthatthedecrease inparticlediameter isnegligible,allowing calculationwith aconstantvaluefork,.
43
Chapter3 o in
c o
• * •
"••-•
CO
Q
O
c CD O
c o o 4-*
CD
CO
(0 CO .Q
5 o
CO
time [h] Figure 1:Graphicalrepresentation ofthemodelforbatchgrowth onsolidsubstrate: — biomassconcentrationXt; — substrateconcentrationCt; specificgrowth rate/J, calculatedwiththemodel describedabove.
Materialsand methods Bacterial cultures Mixed bacterial populations capable ofgrowth onnaphthalene, phenanthrene, or anthracene asthe single source ofcarbonandenergy were isolated byselective enrichment from adomestic waste-water treatment plant inDordrecht (The Netherlands). Theywere all Gram-negative motile rods andcould be identified as Pseudomonasspec. Strains8909N, 8803F, and 8902Awere usedfor growthexperiments on naphthalene, phenanthrene, and anthracene, respectively. Thestrains 8805N,8806N,and8909Nwere usedto investigate the influence of bacterial excretion products onthe dissolution of naphthalene; strains 8803F and8804Fwere usedforthesametypeofexperiments with phenanthrene. Growth conditions Bacteriawere grown at 30°C inmineral medium, essentially composed asdescribed by Evans efal.(1970),with 1mM EDTA asachelating agent andthe concentrations ofthe other medium components being halfthose described.The medium consisted of50mM NH4CI, 5mM NaH2P04, 5mM KCI, 1mM Na2S04, 0.625mM MgCI2, 0.01 mM CaCI 2 , 0.05 uM Na2Mo04, and2.5 ml-L"1of aspore solution containing 0.12 mM HCI, 5mMZnO,20mM FeCI3, 10mM MnCI2, 1mM CuCI2, 2 mMCoCI2, and0.8 mMH3B03.When usedfor batch 44
BiodegradationofcrystallinePAHs cultures the mediumwas buffered atpH7.0 with 50 mMsodium phosphate. Pure cultures were maintained on agar slants containing 0.1% (w/v) ofthe PAHrequired (storage at4°C). Mixedcultures degradingsingly dosed PAHwere maintained insequential batchcultures in 300-mlconicalflasks suppliedwith 100mlmedium.Crystalline PAHswere addedas sole sources ofcarbon andenergy. Strain8909Nwasgrown inanaphthalene-limited continuous culture usingastirred (1000rpm)fermentor (Applicon,Schiedam,TheNetherlands) witha workingvolume of 1.2Lat adilution rate of0.2 h"1.The mineral medium usedwasthe same asdescribed above.The pHwas maintained at7.0 +0.1with 0.5 NNaOH,the temperature set at30°C.Aeration and substrate supply were accomplished by leading naphthalene-saturated air at 180 L-h"\ =4.84-10"4molnaphthaleneh1throughtheculture. Dissolution experiments Dissolutionexperiments were performed at30°C inarotary shaker (200 rpm).The same type offlasks andaqueous volume were used asinthe batch-growth experiments: 500-ml flaskswith 150mlsterile mineral mediumfor naphthalene and250-mlconicalflaskswith 100 mlsterile mineral medium for phenanthrene and anthracene.The appropriate amount of PAHcrystals was addedtotheflasks and samplesweretakento determinethe aqueous PAHconcentration.Totestthe possible influence ofbacterial excretion products onthe dissolution ofthesubstrate,experiments were performed inwhichsupernatant of bacterial cultures,obtained bycentrifugation (15 min, 12,000g),was used insteadof mineralmedium. Samplesweretakento determine the PAHconcentration insolution.The non-linear fitting program Enzfitter (Biosoft, NewJersey, USA)was usedto calculateCmaxandthefactork^A, fromthedissolution curves using Equation 12:
k;A{t
c,-c„.,
, • \
^
\ *' i
o o (D
>
0.20
~"~~~A •
i i
»
>f.
*
10
•-»
>
* *"*
1
i i
i
5
0.10
i
• •
' 0.00
particle diameter [mm] Figure8:Theparametersk,(*•) andvp(•), calculatedfrom theresultofdissolution experiments, asafunction oftheparticlediameter. Effectof bacteria onsubstrate availability Bycomparingthe maximaldissolution rates(fromresultsofthe dissolution experiments) withthe slopes of linear biomassincreasefound inthe batch-growth experiments described above,itispossibletodeterminewhetherthe bacterial growth islimited bythe availability ofthesubstrate.Themaximaldissolution rates Umax =krA,-Cmax/V) forthedifferent sievefractions ofnaphthalene andfor phenanthrene aregiveninTable 1. InFigure9the measured maximaldissolution ratesofnaphthalene areshownas afunctionofthe particlediameter andcomparedwiththedissolution rates calculated fromthe lineargrowthcurves. Ifthe bacteria have noinfluence onthe substrate availability,thereshould benodifference betweenthe measured andthe calculated rates.Thedifferencethatcanbeobservedcouldsuggestasmallinfluenceofthe bacteria onthe bioavailability. Forphenanthrene themaximaldissolution rate calculatedfromthe C0 2 production rateoftheexperiment shown inFigure4Awas 7.8 kg-nr3.h"1,whereasthedissolution ratecalculatedfromthe dissolution experimentswas6.9 kgm 3 .h 1 . 52
Biodegradation ofcrystallinePAHs 0.60
\ \
0.40 •
O)
\ \
0.20
\ ^ ^ •
0.00
A~. • • - — ^. ft
1
particle diameter [mm] Figure9:Themaximaldissolutionrate, Jmgxcalculatedfromgrowth experiments(A) determinedindissolutionexperiments(•) asafunctionoftheparticlediameter. Totest ifthis influence onthe bioavailability iscaused by solubility-enhancing compounds produced bythe bacteria,dissolutionexperimentswith naphthalene and phenanthrene have been performed insupematantsofdifferent bacterial strains growing inbatch under conditions inwhich masstransferwas limiting.Fromthe resultsofthese experiments shown inFigure 10Aand Bitcanbeconcludedthat there isnoenhancement ofthesolubility andsolubilization ratesofnaphthalene and phenanthrene inthesupernatant ofthePseudomonasstrains usedandthereforethe effect ofthebacteria onthe substrate availability cannot beattributedtothe excretion ofsolubility-enhancing compounds. Sincemicroscopical examination revealedthat nogrowthofbacteriaonthesurfaceofthecrystalsoccurred,excluding direct uptakeof PAH,thesmallenhancement ofsubstrate availability islikelytobe duetodeviations inthe calculationfactorsused.
53
Chapter3
£ c o
« o o X
< 0.
3
4
0
time [h] Figure 10:Dissolutionofnaphthalene (A) andphenanthrene (B) inmineralmedium or supernatantsofdifferentbacterialstrainsgrowingon thePAHinvolved. A: • mineral medium, + sup. 8805N, o sup. 8806N, -A sup. 8909N. B: • mineralmedium,+ sup. 8803F, 0 sup. 8804F, -•-sup 8808F, o deadcells8803F. Modellingofmicrobial growth Intheexperiments described above, PAHcrystalswere present inabundance Hence,thesurface areaandthediameter ofthe naphthalene crystals couldbe considered constant, resulting inconstantdissolution rates. Under naturalconditions, however, substrate limitations are usually caused bydesorption processes. Desorption ratesaregenerally notconstantforprolongedtimes,making it impossible tofindalinearsection inthegrowthcurve. Estimationofthesubstrate availability during batchgrowthexperiments canthen bedone bysimulation ofthe process.As anexample ofasystemwithout aconstant masstransfer rate,dissolution experiments andgrowthexperimentswithasmallamount ofnaphthalene havebeen performed andsimulatedwiththe modelpresented intheTheory section.Duetothe smallamountofsubstratethechange inthesurface areaduring bacterialgrowthis considerable and itishardtofindalineargrowth rateandthustoestimateJmax. However, bysimulation itispossibletoestimatethe important process parameters. The resultsoftwogrowthexperiments andtheir simulations areshown in Figure 11. The resultsofthedissolutionexperiments andthevalues oftheparameters usedfor thesimulations areshown inTable 2.Themodeldescribesthegrowthwellandthe valuesfork,found inthegrowthexperiments andinthedissolutionexperiments are inthesamerange. 54
BiodegradationofcrystallinePAHs
0.4
1.2 ^ • y ^
n '•'
0.8
0.4
OD 540 |
^ ^ " 0.3
0.2
/[s.
growth rate
0.1
U^^i — o w Q O
0.0
0.0
10
20
30
40 0.4
1 2
"
growth rate
B
0.8
0.3
CO -1
o 3"
3 CD
1?
V
OD540 sA
0.2 ^
0.4 0.1
0.0
0.0 5
10
15
time [h] Figure 11:Growthkinetics ofstrain 8909Nonnaphthalene(A:0.12gandB:0.25g)in100 mlmineralmedium shaking (150rpm)at30°Cin500-mlconicalflasks: measured values; calculated values.Theparameters usedforsimulationaregivenin Table 2. Table2:Parameters usedfor simulatingthegrowthofstrain8909Nonnaphthalene presented inFigure 11AandB.
n„
h'max 1
Ks [kgm-3]
No [L-1]
k, [m-rr1]
V
[-]
Qo [g]
Cmax
[h- ]
[kg-nr3]
[L]
Dissolution 0.25 gr
-
-
-
0.21
18
0.250
-
0.15
Growth 10A
0.33
1-10'5
1.25-10"
0.18
8
0.117
3.2-10"2
0.1
Growth 10B
0.33
1-10'5
1.25-10"
0.19
17
0.245
3.2-102
0.1
Parameter
55
Chapter3
Discussion Intheexperiments described inFigures 1-4, thehypothesiswasconfirmedthatthe rateofsubstratedissolution could restrict bacterialgrowthonpolycyclic aromatic hydrocarbons. Bycomparingthe maximaldissolution rate(measured indissolution experiments) withthegrowth rate inthedissolution-limited phase,itwasshownthat thepresence ofbacteria and bacterialexcretion products had nosignificant effect on themaximaldissolution rateof naphthalene andphenanthrene.Theestimation of specific bacterialgrowth ratesonthese poorly solublesubstrates inbatch isoften not very reliable.Theobservation of Keuth &Rehm(1991),thatthespecific growth rate increaseswith increasingamountsof(solid) substrate,canbeexplained bythe mechanismproposed inthis paper. Themodelpresented here,inwhichdissolution kinetics and Monod kineticsare coupled,canbeconcludedtobesuitablefordescribing bacterialgrowth inasystem wherethesubstrate supply ismasstransfer limited.Thus itcanformabasisfora morecomplex modeldescribing bacterialgrowth onsubstrates adsorbedto heterogeneous surfaces,suchassoil. Furthermore, itallows insight intotheeffect of thephysicaland biological parameters inasystem inwhichthe biodegradation is limitedbymasstransfer.Awayofenhancingthemicrobialclean-upofpollutedsoils isaugmentation with bacteriathat havethecapability ofquickly degradingthe polluting compound and haveahighaffinity for it.Sincethe important microbial parameters[imaxand Ks andthe initialnumber ofbacteriaN0are incorporated into the modelpresented above,itispossibletoshowtheeffect ofthese parameters on thecourseofthebiomassandsubstrate curves.Figure 12represents simulations in which\smm, Ks, andN0arevariedoverawide range. Itcan beseenthatthegrowth rateandtheaffinity constant ofthe bacteria have little influenceontheamount of remainingsubstrateafter 150hours.Augmentationwithextra bacteria biodegradation isof nouse unlessthe initialconcentration ofactive biomass isvery low.Concluding,itcanbesaidthat applicationofthis kindofspecialized bacteria in thebiotechnologicaltreatment ofpollutedsoils isnoteffective whenthe mass transfer ofthe polluting compound isthe rate-limiting factor.
56
Biodegradation of crystalline PAHs
0.70 0.60 0.50 0.40 0.30
_ m
E O) XL
0.20 0.10 0.00 150 0
50
100
CD
•—*
C
o
0.60
*—» 0)
0.50
4-»
CO
0.70
c d> o r o o
0.10
CO CO
0.00
p: effect of Ks\
o
J2
c w to c oto
0.40 0.30 0.20
/ /
I—*
V
f» CD 50
CO
E
CX.
r^-
0.70
150 0
X
|E: effect of N0 |
0.60
50
^-^^^"T • „^ss-" ==*"~
0.50
100
effect of N„ \
80
\
60
0.40 0.30 0.20 0.10
100
40
•A?
20
' • • / /
0.00 50
100
150 0
50
100
150
time [h]
Figure 12: Effect of varying \}max (A,B), Ks(C,D) and inoculation size (E,F) on biomass and residual substrate concentration during growth on solid substrate, as calculated using the model presented; other parameters the same as in Figure 10A . ••Umax = 0.4; A,B: • »n~, = 0.1 (h1) Pmax = 10; — M m . = 0-2;3 4 5 —K=1-1(f ; • KS = 1-1CT2(kg-m3) ••K.^1-10 ; C,D: Ks= 1-10- ; • N0= 1-1010 (L1) N0= 1.25-10"; —N0 = 2.5-1010; Nn=5-1011; E,F:
57
Chapter3 Whenthemasstransfer rate islimiting,thedegradation ratecanonly be increased by increasingthis masstransfer rate,ascanbeseen inFigure 13.The linesinthisfigure represent modelsimulation asdescribed above,with different initialvaluesforkt. Asexpected,the initialstage inthefigure isverysimilarto Figure 5,inwhichthefactorkrA,wasvaried bychangingthecontactarea.Enhancingthe masstransfer canbedoneby improving mixingcharacteristics, byenlargingthe contactsurfacearea,orby increasingtheaqueous solubility.
1.00 0.80 0.60 0.40 0.20 0.00
Figure13:EffectofvaryingtheinitialvalueofK,onthegrowthonsolidsubstratecalculated withtothemodelpresented; otherparametersthesameasinFigure 4A : k,=0.4;— k, =0.2;•~k,= 0.1; •kt =0.05. Awayofincreasingthe aqueous solubilitythat iswidely under investigationat present istheaddition ofsurface-active compounds. However, introductionof syntheticsurfactants canbe introduction ofanother pollutant and istherefore debatable.Asanalternative,applicationof bacteriathat produce surface-active compoundscouldenhancebiodegradation rates(e.g.Bury&Miller 1992, Reddyet al.1982).Intheexperiments described here noenhancement ofdissolution and biodegradationwasfound.This isnotsurprising sincethesolubilization effect of surfactants isattributed mainlytotheformation ofmicelles (Laha &Luthy 1991), whichformostofthe biosurfactants takes placeat highconcentrations (~20mg-L"1) forsurfactant produced byPseudomonasaeruginosaUG1,Jain etal.1992). Moreover,whenbacteria arepresenttokeeptheaqueous substrate concentration low,thedissolution rateisat itsmaximum.Thepresenceofmicelles underthese 58
Biodegradation ofcrystalline PAHs conditions will probably have very little ornoinfluence ontheaqueous substrate concentration and will therefore not have asignificant effect on thedissolution rate of crystalline substrates. Inthe case ofsubstrate adsorbed tosoil, however, surface-active compounds may have a more pronounced effect on thesubstrate availability. This can be a positive effect, like mobilizing substrate inganglia (Vigon & Rubin 1989), aswell asa negative effect, like clogging ofpores bydispersion ofclay particles (Abdul etal. 1990). Literature onthe effect ofsurfactants onbiodegradation also shows varying results, ranging from enhancement (e.g.Aronstein etal. 1991, Oberbremer etal. 1990) toinhibition (e.g. Laha & Luthy 1992) ofbiodegradation.Wearenow investigating microbial growth onsorbed PAHs and thepossible influenceof (bio)surfactants togain a better insight inthis process. Inthe experiments described here, conversion ofsubstrates was coupled directly to bacterial growth, asthe substrate tobeconverted was the only carbon and energy source. Therefore, theconversion rate ofthe substrate isdirectly coupled tothe microbial growth rate. However, ifthe pollutant isnot the only carbon and energy source, andisconverted bycometabolic processes, the conversion rate can stillbe limited bythe mass transfer rate and themodel can beeasily adapted tothis situation.
Nomenclature A, Cmgx C, Jmgx k, Ks ms np Ng N, OD540 Q, vp
surface area [m2] maximal aqueous concentration [kg-nrr3] actual aqueous substrate concentration [kg-rrr3] maximal dissolution rate [kgnrr 3 h" 1 ] mass transfer coefficient [ m h 1 ] bacterial affinity constant [kg-nrr3] bacterial maintenance coefficient [kg substrateh'kg biomass 1 ] number of PAH crystals [-] initial number ofcells per m 3 [nr 3 ] number ofcells perm 3 [m 3 ] optical density at540 nm [-] amount ofsolid substrate[kg] realtive particle velocity [m.h 1 ]
59
Chapter3 V
volume of the aqueous phase [m3]
W0
initialweight of one cell [kg]
W,
weight of one cell [kg]
Xt
biomass concentration [kg-nr3]
Yovt
overall bacterial growth yield [kg biomasskg substrate 1 ]
YX5
maximal bacterial growth yield [kg biomasskg substrate' 1 ]
n
viscosity [ k g h 1 m 3 ]
\i,\it
bacterial growth rate [h 1 ]
[smax
maximal bacterial growth rate [h 1 ]
ps
specific density of the substrate [kg-m 3 ]
p,
specific density of the liquid phase [kgm 3 ]
References AbdulA.S.,T.L Gibson.,and D.N. Rai(1990) Selection ofsurfactants forthe removal of petroleum products from shallow sandy aquifiers. GroundWater 28:920-926. AndelmanJ.B.,andJ.E. Snodgrass (1974) Incidence andsignificance of polynuclear aromatic hydrocarbons inthewater environment. Crit Rev Environ.Control 5:69-83. Aronstein B.N..Y.M.Calvillo, M.Alexander (1991) Effect ofsurfactants at low concentrations onthedesorption and biodegradation ofsorbedaromatic compounds insoil. Environ. Sci. Technol.25:127-133. Bossert I.,and R. Bartha (1984) Thefate ofpetroleum insoilecosystems. In:Atlas R.M. (ed) Petroleum microbiology, Macmillan Publ. Co., NewYork, USA pp434-476. Bury S.J.,andC.A. Miller (1992) Effect of micellarsolubilization on biodegradation rates of hydrocarbons. Environ.Sci.Technol.27:104-110. DaiseyJ.M.,M.A. Leyko,andT.J. Kneip (1979)Source identification andallocation of polynuclear aromatic hydrocarbon compounds inthe NewYork City aerosol: methods andapplications. In: Jones P.W.,andP. Leber (eds) Polynuclear aromatic hydrocarbons. AnnArbor Science Publishers,AnnArbor, Mich,USA, pp201-215. Davies J.I., andW.C. Evans (1964) Oxidative metabolism ofnaphthalene bysoil pseudomonads:the ring-fission mechanism. Biochem. J.91:251-261. EvansW.C, H.N. Fernley, and E. Griffith (1965) Oxidative metabolism of phenanthrene and anthracene bysoil pseudomonads: the ring-fission mechanism. Biochem J.95:819-831. Evans C.G.T.D. Herbert ,andD.W. Tempest (1970)The continuous cultivation of microorganisms. 2.Construction of achemostat. In: NorrisJ.R, and D.W. Ribbons (eds) Methods of Microbiology,Vol 2.AP London, p277-327. GuerinW.F.,andG.E. JJones (1988) Mineralization of phenanthrene byaMycobacterium sp.Appl. Environ.Microbiol.54:937-944. 60
BiodegradationofcrystallinePAHs Jain D.K., H. Lee,andJ.T. Trevors (1992) Effect ofaddition of Pseudomonasaeruginosa UG2 inocula or biosurfactants on biodegradation ofselected hydrocarbons insoil. J. Ind. Microbiol. 10:87-93. KeuthS.,and H.-J. Rehm(1991) Biodegradation of phenanthrene by arthrobacter polychromogenesisolatedfrom acontaminated soil.Appl. Microbiol.Biotechnol. 34:804-808. LahaS.,and R.G. Luthy (1991) Inhibition of phenanthrene mineralization by nonionic surfactants insoilwater systems. Environ.Sci.Technol.25:1920-1930. Laha S.,and R.G. Luthy (1992) Effect of nonionic surfactants onthe solubilization and mineralization of phenanthrene insoil-water systems. Biotechnol. Bioeng.40:13671380. LaVoie E.J.,V. Bendenko, N. Hirota,S.S. Hecht, and D. Hoffmann (1979)A comparison of the mutagenicity, tumor-initiating activity andcomplete carcinogenicity of polynuclear aromatic hydrocarbons. In: Jones R.W., and P. Leber (eds.) Polynuclear aromatic hydrocarbons,AnnArbor Sci. Publ.,AnnArbor, USA p. 705-721. McLeeA.G., and S.L. Davies (1972) Linear growthof a Torulopsissp. onn-alkanes.Can.J. Microbiol. 18:315-319. Ministry of Housing, Physical Planning,and Environment (1987) Environmental Programmof the Netherlands 1988-1991. Staatsuitgeverij,The Hague,The Netherlands. OberbremerA., R. Miiller-Hurtig,and F.Wagner (1990) Effect ofthe addition of microbial surfactants on hydrocarbon degradation inasoil population inastirred reactor.Appl. Microbiol. Biotechnol.32:485-489. Perry R.H., C.H.Chilton,and S.D. Kirkpatrick (eds) (1963) Chemical engineers' handbook, McGraw-Hill Book Co., NewYork, USA. ProkopA., L.E. Erickson,and O. Paredes-Lopez (1971) Growth models ofcultureswithtwo liquid phases.V. Substrate dissolved indisperse phase-experimental observations. Biotechnol. Bioeng. 13:241-256. Reddy P.G., H.D. Singh, P.K. Roy, andJ.N.Baruah (1982) Predominant role of hydrocarbon solubilization inthe microbial uptake of hydrocarbons. Biotechnol. Bioeng. 24:12411269. Soczo E.R., andJ.J.M.Staps (1988) Review of biological soiltreatment techniques inThe Netherlands. InWolf K., W.J.Vanden Brink, and F.J. Colon (eds) Contaminated Soil '88. KluwerAcademic Publishers, Dordrecht, The Netherlands pp.663-670,. StapsJ.J.M. (1990) International evaluation of in-situ biorestauration ofcontaminated soil andgroundwater. RIVM Reportnr. 738708006. Stucki G.,and M.Alexander (1987) Roleofdissolution rate andsolubility in biodegradation ofaromatic compounds.Appl. Environ.Microbiol.53:292-297.
61
Chapter3 Tempest D.W. (1970)The continuous cultivation of micro-organisms. 1. Theory ofa chemostat. In: Norris J.R., and D.W. Ribbons (eds) Methods in microbiology, Vol 2AP London pp259-276. Thomas J.M.,J.R. Yordy, J.A. Amador, and M.Alexander (1986) Rates ofdissolution and biodegradation ofwater-insoluble organic compounds.Appl. Environ.Microbiol. 52:290-296. Van't Riet K., andJ.Tramper (1991) Basic bioreactor design. Marcel Dekker Inc, NewYork, USA. Vigon B.W., andA.J. Rubin (1989) Practicalconsiderations inthe surfactant-aided mobilization of contaminants inaquifiers.J.Water Pollut. Control Fed.61:1233-1240. Wodzinski R.S., and D. Bertolini (1972) Physicalstate inwhich naphthalene and bibenzylare utilized by bacteria.Appl.Microbiol. 23:1077-1081. Wodzinski R.S., andJ.E.Coyle (1974) Physical state ofphenanthrene for utilization by bacteria.Appl.Microbiol.27:1081-1084.
62
CHAPTER4
BIODEGRADATION OFSORBED POLYCYCLIC AROMATIC HYDROCARBONS
Abstract Thisstudydescribestheeffectofsorptiononthebioavailability and biodegradation ofpolycyclic aromatic hydrocarbons. Desorptionand biodegradation experiments havebeenperformedwithactivated carbon,synthetic carrier materials, anddifferent soils,loadedwith naphthalene orphenanthrene.Withallofthese matricessorption isshownto reducethedegradation rates,andtherefore the bioavailability ofthe PAHs.Thedesorption ofnaphthalene andphenanthrene from thesynthetic matricesAmberliteXAD-4andXAD-7andfromthesoilscouldbe describedwithatwo-compartment model,but notwitharadialdiffusion model.For soilsthe physical interpretation ofthe parameters obtainedwas limited.When comparing desorption and biodegradation ofsorbed PAHs,itwasfoundthatforthe poroussynthetic matricesthe biodegradation proceededfasterthatcouldbe expectedfromdesorption alone. Incontrast,thebiodegradation ofsoil-sorbed naphthalene and phenanthrene could beexplained bydegradation of aqueous phase PAHonly.
63
Chapter4
Introduction Theslowdesorption ofhydrophobic organic compounds (HOCs)fromsoilhas beenidentifiedasoneofthe mostimportant reasonsfortheslow removalofthese compoundsfrom contaminated sites inbiologicalsoilremediation processes (Mihelcic etal.1993).Sorption processesaregoverned bythetype and physical stateofthe pollutant, bythetype andstructure ofthe soil,andbyotherfactors such astemperature,pH,presenceofoxygen,etc. Especiallythe heterogeneity ofsoils makesprediction ofsorption processes insituverydifficult. The processesthatplay a role inbiological soilremediationareevenmorecomplicated because ofthe interactions of microorganisms withthesoiland pollutants. Someevidence has beenfoundthatdirect uptakeofsorbedsubstrate bymicroorganisms is possible (Guerin&Boyd 1992,Hermannson&Marshall 1985).Moststudies, however, showedthatdegradation islimited bydesorptionofthesubstrate andthat therefore onlyaqueous-phase substrate isdirectly availablefor uptake.Asyet, nodefinite answertothisquestion hasbeenfound.However,evenwhensorbed substrate coulddirectly bedegraded by microorganisms, itwasfoundto beless bioavailable thanfreesolutesubstrate (Gordon &Millero 1985).Desorptiontherefore isacrucial process inthe biodegradation ofsorbedsubstrates. Inthisstudythe relation betweendesorption and biodegradation willbe investigatedusingthreedifferenttypesofsorbent:activatedcarbon,syntheticcarrier materials,andsoils.Theactivated carbon isusedasanexample ofasystemwith sorbedsubstrate.Thesynthetic materials havebeen used asmodelsystems in whichdesorptionandbiodegradation canbemeasuredaccurately.Thesoils used wereareasandy soil,ananthropogenic sandy soilandapeat soil.
Theoryand modeling Modelsthat couple biodegradation anddesorption kinetics can bea powerful tool inpredicting biodegradation rates.These models generally consist ofonepart describing desorption kinetics andtheother partdescribing degradation kinetics. Duetothecomplexity ofsoil, models describingthedesorption kineticswill always beasimplification. Animportant parameter inallofthese models istheequilibrium partitioning of the HOCoverthe soilandtheaqueous phase. Depending onthetypeand 64
BiodegradationofsorbedPAHs concentration of HOCandonthetypeofsoil,different equations canbe usedto describethis partitioning (Weber 1972).At lowpollutantconcentrations the sorption isotherm isoftenfoundto beproportional,inwhichcasethepartitioning canbe characterized byaconstant soil-water partitioncoefficient (Equation 1): eq
P
eq
\'l
where Qeq isthe equilibrium sorbedpollutant concentration [mg-g"1],kpthe soil-water partition coefficient [L-g1], and C^ theequilibrium pollutantconcentration insolution [mg-L1]. Inattemptstopredictthe partition coefficient, itisoftendescribed asa functionof parameters suchastheorganiccarboncontent, pH,salinity,etc.(e.g. Dzombak&Luthy 1984, Hegemanetal. 1995,Karickhoffetal.1979,McCarthy etal. 1989,Meansetal. 1980).At higher concentrations isotherms are usually not linear. Thetwomost used non-linear models arethe Langmuirmodelandthe Freundlich model(Voice&Weber 1983).The Langmuir model(Equation 2) isamodelfor adsorption,inwhich itisassumedthatthe maximumsorptioncapacity is reached whenthetotaladsorbing surface iscoveredwithamonomolecular layerofthe adsorbingcompound: k -C e
"
max
y
u k C a
'
eq
where Qmax isthe maximumsorbedpolltuant concentration [mg-g"1]andkathe Langmuir constant [L-g-1]. The Freundlich modelisamodelthatcan beusedfordescribing bothabsorption andadsorption andcan bedescribed asin Equation3: Q . , =Kf(Ceq)Un
0)
whereKfisthe Freundlichsorptioncapacityconstant[mg11/nL1"ng1]andn(>1)the Freundlich sorption energy constant [-].The Freundlich model islargelyempirical, although attempts have beenmadetogiveathermodynamicalbasisfor it(Weber 1972). Todescribethedesorption kineticsofpollutantsfromsoil,different models have beendeveloped.Thetwo most usedtypes aremulti-compartment models(e.g. Brusseauetal. 1991,Karickhoff 1980,MarcaSchrap 1990,Peel&Benedek 1980) and radialdiffusion models (e.g.Carrolletal.1994, Rijnaarts etal.1990,Scow& Alexander 1992,Scow &Hutson 1992,Wu&Gschwend 1986). Inmulticompartment modelsthesoil isdivided intoseparatecompartments (usuallytwo) 65
Chapter4 withdifferent sorption characteristics.Althoughthistypeofmodelislargelyempirical, itusuallyfitsthedesorption datawell(Carroll etal.1994). Inradialdiffusion models thesoilisconsidered homogeneous andthedesorption islimitedbythediffusion of thepollutantthroughthe matrix. Inthis study asimpletwo-compartment modelanda radialdiffusion modelarecompared.Thetwo-compartment modelusedisbasedon thefollowing assumptions: thesolid phase isaporous materialwithtwodifferenttypes ofpores;shallow anddeep pores,each havingequaladsorption properties; theporesare indirect contactwiththeaqueous bulk phase, notinterconnected, andstraight; the pollutant canoccur intheaqueous bulk phase,adsorbed ontothe porewall orintheporeliquid; theamount ofpollutant inthepore liquid isnegligible comparedtothe amountof adsorbed pollutant; transport ofthepollutant inthe poresoccursviaporewalls (surface diffusion); transport ofthe pollutantfromthe porestotheaqueous bulk phase occursvia thecontact length betweentheporesandtheaqueous bulkphase; masstransfer limitation outsidethesolid phasecanbeneglected. Aschematic illustration ofthe modelisgiven inFigure1.
Figure1:Schematicalillustration ofthe two-compartmentmodel; forexplanationofthe symbolsseetext. 66
BiodegradationofsortedPAHs Themassbalanceoverthedifferent phases isgiven in Equation4 below: V&V-q^
+d -f)-qd0) •V-C0- Vx-Sx-(f-qst+(1-f) -qJ +V-C,
(4)
whereVx isthe amount ofsolid phase[g],Sxthespecific surface areaofthe porous particles [m2g~1],f thefraction ofthesurface areathat consistsofshallow pores[-], qs theaverage loading ofthe shallow pores [mg-m2],qd theaverage loadingofthe deep pores [mgm 2 ], Vthe aqueousvolume [L],and Ctheconcentration inthe aqueous bulk phase [mg-L1]. Thesubscripts 0anfreferto initalandactualvalues, respectively. Thetransportfromadeep poretotheaqueous phase isdependent onthe lengthofthe poreld [m],theeffective surfacediffusion coefficient Dse„ [m2h~1],the lengthofthecontact betweentheporewallandtheaqueous phase (u-dd, dd=pore diameter [m])andthedrivingforce,asshown inEquation5:
— * - ^ = -5tr'n^(q"'-q^
(5)
Theequilibrium adsorbed concentration q^ , [mgm 2 ] canbecalculatedfromthe aqueous bulk concentration usingtheappropriate isotherm model.Rearranging this equationyields: dt
- WO*,,- 1eq,
»)
inwhichkd =Dseff/0.5-ld2 [h 1 ]. Likewise,thetransportfromashallow poretothe aqueous bulk phasecanbedescribedas:
~
= ks(%r W
(7)
withks=Dseff/0.5 ls2 [h 1 ], where/ s istheshallow pore length[m]. Bydifferentiating Equation4andcombining itwith Equations6and7,the changeintheaqueousbulkconcentration canbeexpressedas: dC, V
dq.. dq. = -(1- f)VS—^ - f-V-S—— x x dt * dt * dt
(8)
67
Chapter4 Atypicalexample ofmodeling batchdesorptionwiththetwo-compartment modelis showninFigure2.Thedesorption rateinthefirstphase ismainlydependent onthe valueofks, the rate inthesecond phaseisgoverned bythevalueofkd. Thefraction ofshallow pores,f,determines the positionwherethecurvebends.This isconsistant withthesetupofthemodel.
time (h)
k„ - 0.25
K - 0.01
Figure2:Graphicalrepresentation oftheeffectofvaryingtheparametersf(A),ks (B), andkd (C)on the resultsofmodelingofbatch desorption withthetwo-compartmentmodel. 0
2
4
6
time (h)
Inthe radialdiffusion modelforthedesorption used,diffusion ofthe pollutant through homogeneous particles isassumedtobethe rate-limiting process.The diffusionfrom homogeneously loadedspherical particles intoawell-stirred solutionof limitedvolumethat isinitiallyfreeofpollutantwasmodeled according to Crank (1975).Themass balanceoverthetwophases isgiven inEquation 9below:
68
BiodegradationofsorbedPAHs VxQ0 = VxQ( • V.Ct
(9)
where Q0 isthe initialloading [mg-g"1],and Q, theactualaverage concentration in theparticles [mg-g"1].Thechange intheconcentration inthe particles isgivenby: dQ, d2Q. 2 3Q. — 1 = D-( ^2 + -•—!) dt dr r dr
(10)
inwhichDisthe apparentdiffusion coefficient [m 2 h 1 ]which isconsidered constant, andrthe radialdistance [m].Undertheassumptionthatthe isotherm isproportional, andthatthe particledensity isequaltothedensity ofwater,thisequationcanbe solvedforbatchdesorptionaccordingto Crank (1975):
V Q, s1_ j > « - r j + « ; V - D ^ Q
o~
Q
eq
n=1
(11)
2 2
9+ 9 - a +
qn a
where Q^ istheequilibrium sorbed pollutantconcentration [mg-g"1],atheparticle radius [m],a, aparameterthatdescribesthefinalfractionalamountdesorbed,can beexpressed as inEquation 12,and inwhichqnarethe non-zero rootsof Equation 13. 1
% -Q
tan ( a )
eq
Q
1 + 1/OL
(12)
3 In 3+
1mm.The initial loadingofthesoilswas determined byextraction (seebelow). Batchdesorption experiments The batchdesorption experiments were performed in250-mlserumflasks onarotary shaker (200 rpm) at 30°C.The experiments were started byadding 0.025to 0.1 gof loaded synthetic matrix or soiltotheflasks containing 100or 150mlof sterile buffered mineral medium.At regular intervalsfiltered samples of0.75 mlwere removedto determine the PAH concentration inthe liquid phase. Intheexperimentswiththe synthetic matricesthe liquid wasdecanted after approximately oneweek andtheequilibrium naphthalene concentration of both liquidand solid phaseswasdetermined.Totestforthe influence of bacterial excretion products onthedesorption of naphthalene and phenanthrene fromthe synthetic
71
Chapter4 matrices, similarexperiments have been performed inwhichfiltered (0.2 urnrotrandfilter, Schleicher &Schuell,Germany) supernatantsof batchcultures growing on naphthalene (strain8909N) orphenanthrene (strain8803F)were used instead ofmineral medium. Continuous desorption experiments Thecontinuous desorption experiments or leaching experiments with naphthaleneloadedXAD-4andXAD-7were performed inamixed system. Mineral mediumwas pumped into35-mlserum bottles containing 12to 15mlofliquid and0.1 gof naphthalene-loaded resin. Sinteredglassfilterswerefixed intothe outlet ofthe bottlesto protect the resins from washing out.The serum bottleswere placed inarotary shaker (230 rpm) at 30°C.The naphthalene concentration inthe leachateswas determined by measuringthe absorbance at 275nm (A275)every 12minwithaspectrophotometer usingaflow-through cuvette.At regular intervals samplesweretakento determine the effluent naphthalene concentrations with HPLC.The pumpflow,set at± 30 ml.h 1 , was measured atthe beginning andattheend oftheexperiment. The leachateswerecollected in5-Ljarswhich contained 25 mlofa5M NaOHsolution to prevent biodegradation ofthedesorbed naphthalene.Atthe end ofthe experiment,thevolumes ofthe leachate,thenaphthalene concentration inthejars andthe residualloadings ofthe matricesweredetermined. Biodegradation experiments Biodegradation experiments were performed in250-mlserumflasks onarotary shaker (150or200 rpm) at 30°C.Theflasks contained 100mlof mineral medium and were supplementedwith 0.1 to 0.5 gof loaded material.Intheexperiments withXAD-4 and XAD-7the headspace oftheflasks wasfilledwithoxygengastoeliminatethe possibility of oxygen limitation.Theexperiments were started byinoculation with 1mlofactivebatchgrowncells ofstrain8909N orstrain 8803Ffor experiments with naphthalene or phenanthrene, respectively. Inthe experimentswithsoils andsynthetic matrices the biodegradation of naphthalene and phenanthrene wasfollowed by measurement ofthe percentage of C0 2 inthe headspace gas ofthe bottles.Atthe endofthe experiments 1mlof a 12MHCIsolution was addedtotheflasksto removethedissolved C0 2,andthe C0 2 concentration inthe headspace gaswas measured once more.The residual naphthalene concentrations ofthe liquidandthe solid phasesweredetermined. Intheexperiments withactivated carbonthe biodegradation of PAHswas followedby measurement ofthe proteincontent ofthe liquidphase. Analytical procedures Proteinwas assayed after alkalitreatment of cellswiththe methodof Lowry efal.(1951) using bovine serum albumin asastandard.C0 2 inthe headspace gasofserum flaskswas determined using agas chromatograph (Hewlett Packardtype 5890) equippedwitha
72
Biodegradation ofsorbedPAHs thermalconductivitydetectorandaHayesepQpackedstainlesssteelcolumn(diameter 1/8 inch,length2m,Chrompack,TheNetherlands).Heliumwasusedascarriergaswithaflow rateof30ml-min*1.Theinjectortemperaturewas150°C,theoventemperature80°C,andthe detectortemperature200°C.Theinjectionvolumewas250ulwithsplitlessinjection. Aqueousnaphthaleneconcentrationsweredeterminedbyinjectionoffiltered(0.2urn), with acetonitrilediluted(1:1)samplesonaHPLCwithanChromspherC18 (PAH)column (Chrompack,TheNetherlands).Eluentwasan85/15mixtureofacetonitrile/water.Peakswere detectedwithaUVdetectorbymeasurementoftheA275fornaphthaleneandoftheA254for phenanthrene.Extractionsamplesweremeasuredlikewise,butweredilutedtoa naphthaleneconcentrationlowerthan100mg-L1.Extractionofnaphthalenefromthesolid phaseswasperformedbyadding50or100mlofacetonitriletothematrixandincubatingfor oneweek,afterwhichthenaphthaleneconcentrationintheacetonitrilewasmeasured. Whentheextractionwasperformedwithhighlyloadedmaterial(determinationofinitial loadings),theacetonitrilewasreplacedbythesamevolumeofcleanacetonitrileand the procedurewasrepeated.Forthesyntheticmatrices,thedryweightofthematrixwas determinedbydryingaknownamountofwetmaterialat80°Ctoconstantweight.
Results and discussion Experimentswithactivated carbon Biodegradation experiments withamixture ofPAHsoriginatingfrom pollutedsoil havebeenperformedwith activated carbonasthesorbate.Batchcultureswith250 and500mgof loadedactivated carbon in 100mlmineral mediumwere inoculated withamixed PAH-degrading culture andthe increase inbiomasswasfollowedby determination ofcellular protein.Figure 3showsthatwith PAHsadsorbed onto activated carbon a linear increase inbiomasswasfound,similartothe lineargrowth oncrystalline PAHs (Chapter 3). Moreover itcanbeseenthat for adsorbed substrategrowthwasproportionaltotheamount of PAHonactivated carbonadded, i.e.,thetotaldesorbing surface.Thisshowsthatthe biodegradation of PAHssorbed ontoactivatedcarbonwas limited bythedesorption process.
73
Chapter4 50
+ /
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10
,c «
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.
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12
.
16
20
time [days] Figure3:Batchgrowth ofamixedbacterialculture on amixture ofpolycyclicaromatic hydrocarbons, adsorbedonto250(u)or500(+)mgloadedactive carbon.
Experimentswithsynthetic matrices Loadingofthematrices. Withthe method used itwas possibletoobtain loaded carrier materialthat remained stablefor longer periods.Themaximal loadingofthe XAD-4resinwas 140-150 mg-g"1,whichisequivalentto310-330 mgg"1dryweight. Sincethis resinhasaspecific surfaceareaof727 m 2 g 1andthesurface areaof1 moleculeofnaphthalene is4.29-1019m2(Radt 1948),itcanbecalculatedthatabout 87%ofthe resinsurface areawascoveredwith naphthalene.Thisindicatesthatthe naphthalenewasevenly distributed overthe resin.ForXAD-7themaximal loading was68mg-g"1,which amountstoacoverage of89%.Thesurface areaofthe coated polymerwas unknown;the maximal loadingobtainedwas6.1 mgg 1 . Batchdesorption.Batchdesorptionexperimentswere performed byadding different amountsof matrixtoaknownvolumeofmineral mediumandfollowingthe aqueous 74
BiodegradationofsorbedPAHs naphthalene concentration.Theequilibrium concentrations thatwerefound atthe endofthedesorption experimentswere usedtoconstructthedesorption isotherms, whichforthethree materialscould bedescribed usingthe Freundlich equation (Equation 1).The results ofthe measurements andthefitting areshown inFigure4; the Freundlichconstants thatwerefound aregiven inTable 1.
Table 1:Freundlich constantsfordesorption of naphthalene fromthesyntheticmatrices. material
n
K, [mg1-1/n-L1-r •g1]
[-]
polymer
0.44
1.59
XAD-7
5.11
1.47
XAD-4
39.5
2.50
150
XAD-4
Q
< X
.
*
m
100 •
E _>. o
s
Q
'polymer
< X 50 • /
Q.
XAD-7
E O
-
• 1
* * - *
•*.++ 10
20
30
C eq [mg.L 1 ] Figure4:DesorptionisothermofnaphthaleneloadedontoXAD-4 (m), XAD-7(A),orthe coatedpolymer(*). 75
Chapter4 Inthedesorptionexperiments withthecoated polymer,equilibriumwas reached within 1h.Thisprovidestoo littledatatofollowthedesorption kinetics.The resultsof thedesorptionexperimentswithXAD-4andXAD-7werefittedwithboththetwocompartment andthe radialdiffusion models.Thenon-proportinalityofthe isotherms forms aproblemfor usingthe radialdiffusion model.Theanalytical solution presented byCrank (1975) isonlyvalidwhenpartitioning isproportional. For nonlinearisotherms noanalyticalsolutionswereavailable andthereforethe isothermsof XAD-4andXAD-7were linearized.Thisresulted inpartition coefficients of 6.96 and 1.74 L-g"1 forXAD-4 andXAD-7,respectively.Thebest results offittingthe desorption of0.1gofnaphthalene-loaded XAD-4 in 100mlofmineral mediumwith the radialdiffusion modelareshown inFigure5asanexample (solid line).Ascanbe seen,itwas notpossibletoproduceacurvethat gaveagooddescriptionofthe experimental data.Althoughthis may bepartlycaused bythe linearization ofthe isotherm,the radialdiffusion modelcannotexplainthebiphasic character ofthe desorption curveobserved. Itislikelythat better resultscan beobtainedwithamore complicated model,combining adsorption anddiffusion process. However, the construction ofsuchamodelwas notwithinthescopeofthisstudy.
time [h] Figure5:BatchdesorptionkineticsofnaphthaleneloadedontoXAD-4 (+).Linesrepresent fittingwith thetwo-compartment (dottedlines) andradialdiffusionmodel(solidlines). 76
Biodegradation ofsorbedPAHs Usingthetwo-compartment modelitwas possibletofittheexperimental results well,ascanbeseen in Figure5too(dotted line). However, sincethecurves couldbe fittedwithdifferent setsofvaluesforthedesorptionvariables f,kd,andks, the accuracy bywhichthesevariablescould bedeterminedwas low(Table2). Therefore,theresultsofthebatchdesorptionexperimentscouldnotbeusedto makeareliable estimationofthethreeparameters. Table2:Desorptionparametersfoundbyfittingbatch andcontinuousdesorptionof naphthalene fromXAD-4 andXAD-7with thetwo-compartmentmodel. resin
batch desorption
continuous desorption f
f 1
1
[-]
[h- ]
In" )
H
[h-1]
[rr1]
XAD-4
0.20 - 0.45
0.02-0.06
0.5-1.5
0.26-0.29
0.048-0.052
0.69-0.75
XAD-7
0.50 - 0.80
0.01-0.05
0.35-0.55
0.63-0.67
0.024-0.028
0.39-0.43
Continuous desorption.Continuous desorptionexperiments wereperformed ina mixedsystem.This hasthe advantage overcolumnexperiments inthatabetter comparisonwiththe biodegradation experiments ispossible.The results ofthe measurements ofthe naphthaleneconcentration inthe leachatesareshowninfor XAD-4andXAD-7 in Figure6A. InFigure6Bthesedata havebeenconvertedtothe desorbedfractions ofthe napthalene initially present desorbed. Itcan beseenthat thedesorptionfromXAD-7 proceeds muchfasterthanfromXAD-4.Thedashed lines inthefiguresshow curveswhichwerefitted usingthetwo-compartment model, assuming idealmixing inthe bulkphase.Thevaluesfor f,kd, andksthat havebeen obtained byfitting the measured data arepresented inTable2.Thefactthatthese values couldalso beusedtodescribethe resultsofthe batch desorption experiments showsthatthetwo-compartment modelcanbeusedtodescribethe desorption of naphthalene fromtheXADresins.
77
Chapter4
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Figure5:Oxygenuptakerateofstrain8909Nonnaphthaleneasafunctionoftheapparent napthaleneconcentration with andwithoutsurfactants TritonX-100(A) orTergitolNPX(B). Symbols•: nosurfactant, m: 5g-L1surfactant. ThecurvesrepresentfittedMichaelis-Menten kinetics.
Discussion Effectofsurfactants ondissolution kinetics Theincrease inthe levelofapparent solubilityduetothepresence of surfactant micelleswhichweobserved isconsistentwiththe resultsofEdwards etal.(1991),
128
SurfactantsandbiodegradationofcrystallinePAHs whoobtained slightly lowervaluesfor logKmw, probably because oftheir useof 1% methanol inthe aqueous phase.Althoughthewater-micelle partition coefficient isa usefulparameter, itprovides noinformation aboutdissolution kinetics.Therefore,a dissolution modelwasconstructed inwhich itwasassumedthatthe increase inthe apparent levelofsolubility wastheonlyeffect ofthe micelles.As anexample,the resultsofmodeling fordissolutionofnaphthalene whenTergitol NPXwasthe surfactant areshown i,. Fig.6.Thefactor kls-At,ameasureofthe maximum dissolution rate,wascalculatedfromtheexperiment performedwithoutsurfactant as described previously (Volkeringefal.1992);thesamevaluewas usedfor modeling theexperimentswithsurfactant (Fig.6andTable2). 400
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Figure6:Modelingofdissolutionkineticsinthepresenceofdifferentconcentrationsof TergitolNPX. Thesymbolsrepresentmeasureddata, dashedlinesrepresentmodel predictions. Themodelpredictionsfordissolution kinetics clearly underestimatethe dissolution ratesof PAHsatsurfactant concentrations higherthanthe CMC.Byincreasing the factorkls-A,, however, itwas possibletoobtainagooddescription ofthe dissolution kinetics.Themaximaldissolution rates{Jmax=krArCmax / V[kg-m'3-h1]),foundby increasingthe kls-At valuesforthedifferent surfactants,aregiven inTable2.
129
Chapter 6 Table 2: Maximum dissolution rates (Jmax) calculated from dissolution experimental data obtained with crystalline naphthalene and phenanthrene in the presence of different concentrations of nonionic Surfactant
surfactants. 1
Jmaxnaphthalene [g-L'1- r ]
Jmax
phenanth rene [g-L"1 •h"1]
concentration Triton
Tergitol
Igepal
X-100
NPX
CA720
Triton
Tergitol
Igepal
X-100
NPX
CA720
0
0.013
0.013
0.013
0.012
0.001
0.001
0.001
0.001
0.1
0.014
0.013
0.014
0.011
-
-
-
-
0.5
0.019
0.015
0.017
0.013
0.010
0.010
0.009
0.008
1
0.025
0.016
0.022
0.015
0.017
0.016
0.017
0.013
2
0.029
0.019
0.026
0.017
0.031
0.027
0.028
0.022
5
0.035
0.034
0.037
0.024
0.068
0.060
0.072
0.047
[g-L 1 ]
Brij 35
Brij 35
-: not determined
T h e effects o f t h e surfactants w e r e m o r e p r o n o u n c e d for p h e n a n t h r e n e t h a n for naphthalene; t h e r e a s o n for this difference is not clear. T h e high v a l u e s for kls-A, cannot be e x p l a i n e d by d e c r e a s e s in viscosity, as viscosity m e a s u r e m e n t s revealed that the viscosity of a surfactant solution increased slightly as the surfactant concentration i n c r e a s e d ; this is consistent with the observations of V i g o n & Rubin (1989). T h e r e f o r e , t h e p r e s e n c e of micelles affects the dissolution rate, a n d the a s s u m p t i o n that t h e micelles only act as a s e p a r a t e p h a s e is not valid. T w o possible m e c h a n i s m s for t h e effect of surfactant micelles o n the dissolution rate c a n be put forward. In the first m e c h a n i s m t h e micelles are present not only in t h e a q u e o u s bulk p h a s e , but also in t h e s t a g n a n t layer s u r r o u n d i n g t h e P A H crystals. T h i s s h o u l d d e c r e a s e the P A H c o n c e n t r a t i o n in the layer a n d thus increase the kls v a l u e . More likely, however, is t h e possibility that a hemicellular layer of surfactant molecules is f o r m e d a r o u n d t h e crystals. This allows a d y n a m i c e x c h a n g e of the micellar P A H to occur. T h e latter m e c h a n i s m is very similar to the interactions of surfactant molecules with soil interfaces ( N a y y a r et al. 1994) a n d has also b e e n p r o p o s e d by G e r s o n (1993), a l t h o u g h G e r s o n presented no e v i d e n c e . O n t h e basis of t h e results of the experiments d e s c r i b e d a b o v e , no distinction b e t w e e n t h e t w o m e c h a n i s m s c a n be made.
130
SurfactantsandbiodegradationofcrystallinePAHs Surfactanttoxicity Itiswellknownthatsurfactants canbetoxictobacteria.Although nonionic surfactants aregenerally lesstoxicthan ionicsurfactants andalthoughgramnegativebacteriaaregenerally lesssensitivethangram-positive bacteria (Swisher 1987),itseemed likelythatthe presenceofsurfactants atthe high concentrations used inthisstudy (upto 10g-L1)could haveanegativeeffect onthe bacterial strains used. However, inallofthegrowthandactivityexperimentsthatwereperformed,no toxiceffect ofthesurfactants onthe microorganismswasfound. Biodegradation experiments Addition ofsurfactant tobatchcultures growing oncrystalline naphthalene or phenanthrene duringthedissolution-limited phase results inan increase inthe linear growthrate.Theeffect ismorepronouncedfor phenanthrene thanfor naphthalene. Thiseffect cannot becaused byan increase intheapparent PAHsolubility sincethe aqueous PAHconcentration inthedissolution-limited phase isvirtuallyzero.This impliesthatthe maximaldissolution rate increaseswhensurfactant is present (facilitatedtransport).These resultsconfirmtheincrease inthedissolution ratewhich weobserved inthe dissolution experiments. However,the increase inthegrowth rate was lessthanthe increase inthe maximaldissolution ratefound inthe dissolution experiments.Thiscan beexplained bythefactthattherewas less PAHpresent in the biodegradation experiments thaninthedissolutionexperiments.An additional explanation involvessorption ofthesurfactant ontothe bacteria,acommon phenomenon (Swisher 1987)which results inloweraqueous surfactant concentrations. Whensurfactantwasaddedatthe beginning ofbatchgrowth experiments with naphthalene,theexponential growth phasewas longer, butthe maximalgrowth rate onnaphthalenedidnotincrease. Incontrast, BuryandMiller (1993)observed higher maximalgrowth ratesonn-decaneand n-tetradecane inthepresence of surfactants than inthe absenceofsurfactants.Thesurfactantswhichthese authorsused, however,weredegradedalongwiththealkanes,andthe increasecouldhavebeen caused bydirect bacterial uptakeofmicellesfilledwithalkanes. Bioavailability ofmicellarPAHs Inseveralstudies micellar solubilization hasbeen usedtoenhancethe availability ofpoorly soluble substrates (Bury&Miller 1993,Guerin&Jones 1988,Thiem 1994), andthe roleofsolubilization bymicrobialexcretion products inthegrowthonalkanes
131
Chapter6 hasbeenstudied extensively (Reddy etal.1982).The highexit ratesofmicellar substrate allowexponential growth athighcelldensities. Itshouldbe notedthatthis isnoevidencethatthesubstrate inthemicelles isreadily availabletothe microorganisms.Theoxygen uptake rateofstrain8909Nonnaphthalenewas affected bythe presenceofsurfactants,asshown inFig.5forTritonX-100and Tergitol NPX.Thiseffect cannot beattributedtotoxicity ofthesurfactants. One explanationforthisisthe possibilitythatthe PAHswerepartitionedbetweenthe water phaseandthe micellar phase.Figures 5and7showthe results ofthesame experiments,butinFig.7thex-axisshowstheaqueousnaphthalene concentration whichwascalculatedfromthetotal naphthalene concentration withthe partition coefficients inTable 1. TheKmvalues obtained byfittingthese resultswith MichaelisMentenkineticsarealso shown inFigures 5and7.Fromthecorresponding lines andvaluesforKm, itisclearthatthe naphthalene concentration inthewater phase controlsthebacterialactivity. Moreevidenceforthiswasobtainedfrom growth experiments performedwithstrain8909Ncells,whichwere notadaptedto high concentrations ofnaphthalene.Weobservedthatinbatchgrowthexperimentswith naphthalene (data notshown) inoculationwithactivecellsoriginatingfrom batch culturescontaining low naphthalene concentrations (lineargrowth phase) resulted in alagphaseof 10-24 h,whereas after inoculationwithactive cellsoriginating from culturescontaining high naphthalene concentrations (exponentialgrowth phase) therewas nolag phase.This indicatesthat highconcentrations ofnaphthalene can betoxictothebacteria andthatthebacteria canadapttothese high concentrations. Thisisnotanuncommonphenomenonforgram-negative bacteria (Weberetal. 1993).Growthexperiments performedwith naphthalene and unadapted strain8809N cellsshowedthat inculturestowhich 1 to5g-L"1TritonX-100wasadded before inoculation nolag phaseoccurred.This illustratesthatthetoxicity of naphthalene is reducedwhen itispresent inmicelles.This isagoodexplanationforthefactthat adaptationtimes areshorter inthe presence ofbiosurfactants than intheabsenceof biosurfactants, asdescribed byOberbremeretal.(1990).
132
Surfactants andbiodegradation ofcrystalline PAHs
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aqueous naphthalene concentration [mg.L 1] Figure 7: Oxygen uptake rate ofstrain 8909N onnaphthalene asa function of the aqueous napthalene concentration with and without the surfactants Triton X-100 (A)or Tergitol NPX (B) Symbols: •: no surfactant, m:5g-L1 surfactant. Thecurves represent fitted MichaelisMenten kinetics.
Themost important conclusionthatcanbedrawnfromthedatadescribed above isthat PAHs inthe micellarphaseare not readily availabletomicroorganisms.Thus, micellar PAHisaprotected reservoirthat may replenishaqueous-phase PAHwhenit isdepleted bybiodegradation.This hasbeenstated previously byZhang and Miller (1992), butthedatatheseauthors presented do notjustify their conclusion,asthey comparedequilibrium solubility withgrowthkinetics.Thefactthat micellar substrate isnot readily bioavailable has importantconsequencesfortheapplication of surfactants inbioremediation. First,the presenceofmicelles may lowerthe concentration ofcontaminant inthewater phase,thereby reducingthe bacterial activity orgrowth.Thiswasseen intheoxygen uptakeexperimentswhose results areshown inFig.5.Atthe sametotal naphthalene concentration,theactivity ofcells isreduced bythepresenceofsurfactant.Thiseffect,combinedwiththetoxicityof thesurfactants,couldexplainthe inhibition ofphenanthrene mineralization by micellar surfactant, asdescribed byLahaand Luthy (1991, 1992).Second,ininsitu processes,the nonavailable substrate may bewashed outandthus cause unwanted contamination ofgroundwater. Forthe reasonsdescribed above,carefulstudy is neededbeforethe useofsurfactants inbiologicalsoiltreatment canbe recommended.
133
Chapter 6
Nomenclature Amic A, Cmax Cmict C( Ctot Jmax klmic kls Km Kmv/ m Q, t V Vmic
micellar surface area [m 2 ] crystal surface area [m 2 ] maximum aqueous PAH concentration [kg-rrr 3 ] micellar PAH concentration [kg-nr 3 ] aqueous PAH concentration [ k g n r 3 ] overall PAH concentration insolution [kg-nr 3 ] maximum dissolution rate [kg-nr 3 -h 1 ] liquid-micelle mass transfer coefficient [ m h 1 ] liquid-solid mass transfer coefficient [mh' 1 ] Michaelis-Menten affinity constant [mg-L 1 ] molar water partition coefficient [-] water-micelle partition coefficient [-] amount ofsolid PAH [kg] time[h] aqueous volume [m 3 ] micellar volume [m 3 ]
References Abdul A. S., T.L. Gibson, and D.N. Rai (1990) Selection of surfactants for the removalof petroleum products from shallow sandy aquifers. Ground Water 28:920-926. Aronstein B.N., and M. Alexander (1992) Surfactants atlow concentrations stimulate the biodegradation of sorbed hydrocarbons in samples of aquifer sands and soil slurries. Environ. Toxicol. Chem. 11: 1227-1233. Aronstein B.N., Y.M. Calvillo, and M.Alexander (1991) Effect of surfactants atlow concentrations on the desorption and biodegradation of sorbed aromatic compoundsin soil. Environ. Sci. Technol.25:1728-1731. Aronstein B.N., and M. Alexander (1993) Effect ofa nonionic surfactant added to the soil surface on the biodegradation of aromatic hydrocarbons withinthe soil. Appl. Microbiol. Biotechnol. 39:386-390. Bury S.J., and C.A. Miller (1993) Effect of micellar solubilization on biodegradation ratesof hydrocarbons. Environ. Sci.Technol. 27:104-110. Cerniglia C.E. (1992) Biodegradation ofpolycyclic aromatic hydrocarbons. Biodegradation 3:351-368.
134
SurfactantsandbiodegradationofcrystallinePAHs Dubinsky Z., P.G. Falkowski,A.F. Post, and U.M.van Hes(1987)A system for measuring phytoplankton photosynthesis inadefined lightfieldwithanoxygenelectrode.J. Plankton Res. 9:607-612. Edwards D.A., Z.Liu, and R.G. Luthy (1992) Interactions between nonionic surfactant monomers, hydrophobic organic compounds, andsoil.Water Sci. Technol.26:147-158. Edwards D.A., R.G. Luthy, andZ. Liu(1991) Solubilization of polycyclic aromatic hydrocarbons in micellar nonionic surfactant solutions. Environ.Sci.Technol. 25:127-133. Evans C.G.T., D. Herbert, and D.W. Tempest (1970)The continuous cultivation of microorganisms. 2.Construction of achemostat. pp.277-327. In: Norris J.R., and D.W. Ribbons (eds.), Methods of microbiology vol.2.Academic Press, London,UK. Gerson D.F (1993) The biophysics ofmicrobial surfactants: growthon insoluble substrate. In: Kosaric N.(ed.), Biosurfactants, p. 269-286, Marcel Dekker, Inc., NewYork, USA. GuerinW.F.,andG.E. Jones (1988) Mineralization of phenanthrene bya Mycobacteriumsp. Appl. Environ.Microbiol.54:937-944. Laha S.,and R.G. Luthy (1991) Inhibition of phenanthrene mineralization by nonionic surfactants insoil-water systems. Environ.Sci.Technol. 25:1920-1930. Laha S.,and R.G. Luthy (1992) Effects of nonionic surfactants onthe mineralization of phenanthrene insoil-water systems. Biotechnol. Bioeng.40:1367-1380. LiuZ., D.A. Edwards, and R.G. Luthy (1992) Sorption of non-ionic surfactant onto soil. Water Res. 26:1337-1345. MihelcicJ.R., D.R. Lueking, R.J. Mitzell,andJ.M.Stapleton (1993) Bioavailability ofsorbedandseparate-phase chemicals. Biodegradation 4:141-153. NayyarS.P ,D.A. Sabatini, andJ.H. Harwell (1994) Surfactant adsolubilization and modified admicellarsorption of nonpolar, polar, and ionizable organic contaminants. Environ.Sci. Technol.28:1874-1881. Oberbremer A., R. Mueller-Hurtig, and F.Wagner (1990) Effect ofthe addition of microbial surfactants on hydrocarbon degradation inasoil population inastirred reactor.Appl. Microbiol. Biotechnol. 32:485-489. Reddy P.G., D. Singh,P.K. Roy, andJ.N. Baruah (1982) Predominant role of hydrocarbon solubilization inthe microbial uptake of hydrocarbons. Biotechnol. Bioeng. 24:12411269. Swisher R.D. (1987) Surfactant biodegradation; Surfactant science series 18,Marcel Dekker, Inc., NewYork, USA. TiehmA. (1994) Degradation of polycyclic aromatic hydrocarbons inthe presence of synthetic surfactants.Appl. Environ.Microbiol.60:258-263. Van Dyke M.I., S.L. Gulley, H. Lee,andJ.T. Trevors (1993) Evaluation of microbial surfactants for recovery of hydrophobic pollutants from soil. J. Ind. Microbiol. 11:163-170.
135
Chapter6 Vigon B.W., andA.J.Rubin (1989) Practical considerations inthe surfactant-aided mobilization ofcontaminants inaquifers.J.Wat. Pollut. Control.Fed.61:1233-1240. Volkering F.,A.M. Breure,A. Sterkenburg, andJ.G.vanAndel (1992) Microbial degradation of polycyclic aromatic hydrocarbons: effect of substrate availability on bacterial growth kinetics.Appl. Microbiol. Biotechnol.36:548-552. Volkering F.,A.M.Breure,andJ.G.vanAndel (1993) Effect of micro-organisms onthe bioavailability and biodegradation ofcrystalline naphthalene.Appl.Microbiol.Biotechnol. 40:535-540. Weber F., L.P., Ooijkaas, R.W. Schemen, S. Hartmans, andJ.A.M.de Bont (1993) Adaptation of Pseudomonasputida S12to highconcentrations ofstyrene andother organic solvents.Appl.Environ.Microbiol.59:3502-3504. Wilkinson T.G.,and D.E.F. Harrison (1973)The affinity for methane and methanol of mixed cultures grown on methane incontinuous culture.J.Appl. Bacteriol.36:309-313. ZhangY.M.,and R.M. Miller (1992) Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipidsurfactant (biosurfactant).Appl.Environ.Microbiol. 58:3276-3282.
136
CHAPTER 7
DESORPTION AND BIODEGRADATION OF SORBED NAPHTHALENE INTHE PRESENCE OF NONIONIC SURFACTANTS
Abstract Theeffect ofthe nonionic surfactants TritonX-100and Brij35onthe desorption andbiodegradation ofthe polycyclic aromatic hydrocarbon naphthalene sorbed onto twowelldefined synthetic matriceswas investigated. Inbatch desorption experiments itwasfoundthatthepresenceofsurfactant loweredthe solid-liquid partition coefficient, evenatconcentrations lowerthanthecritical micelle concentration.Theeffect ofsurfactant ondesorption kineticswasinvestigated in continuous desorption experiments.At concentrations below andabovethecritical micelleconcentration,the presenceofsurfactant wasfoundto increasethe desorption rates.Therefore micellarsolubilization wasconcludedtobe nottheonly mechanism bywhichthe surfactants stimulatedesorption.Biodegradation ofsorbed naphthalene wasstimulatedwhensurfactant wasadded inthedesorption-limited phase,supportingthe resultsfound inthedesorptionexperiments.Whendesorption andbiodegradation were compared,itwasfoundthattheextentofthestimulationby surfactantwas lessfor biodegradation thanfordesorption.Moreover itwas observed that biodegradation proceededfasterthancould beexplained bydesorptionalone.
ThischapterhasbeensubmittedforpublicationinEnvironmentalScience &Technologyas "Desorptionandbiodegradationofsorbednaphthaleneinthepresence ofnonionic surfactants"bytheauthorsF. Volkering, A.M. Breure, J.G. vanAndel, andW.H. Rulkens.
137
Chapter7
Introduction Hydrophobic organicpollutants insoilsusuallyoccurattachedtothesoilmatrix. Someresearchers havefoundthat microorganisms cantake upsorbed substrates directly (Guerin &Boyd 1992, Hermannson&Marshall 1985),but inmoststudiesit hasbeendemonstratedthat onlyaqueous phasesubstrateisdirectly availablefor biodegradation (e.g.Mihelcic &Luthy 1991, Robinsonetal. 1990,Scow &Alexander 1992).This impliesthat sorbedpollutant hastobedesorbed before itcanbe degraded.Theslowdesorptionof hydrophobic organic compounds (HOCs) isthe main reasonforthe slow biodegradation ofthese compounds inthe biologicalcleanupofsoil(Mihelcicetal. 1993,Providenti etal. 1994).Thisphenomenon iscalled limited bioavailability. Theuseofsurfactantstoenhancethemobility ofthe pollutant isonepossibleway ofsolvingthe problemoflimitedbioavailability. Surfactants aremoleculesthat usuallyconsistofahydrophobic andahydrophilic part. Becauseofthistheytendto concentrate atsurfaces and interfaces.Thepresence ofsurfactants lowersthe surfacetensionofaqueoussolutionsandthe interfacialtension at liquid-liquid and solid-liquid interfaces.Atconcentrations higherthanthe critical micelle concentration (CMC),surfactant moleculesformsmallaggregates,micelles,which increasethe apparent aqueous solubility of HOCs(solubilization). Micellarsolubilization may enhancethedesorption ofsorbedpollutants aswasfound inmostofthe literature describing studies ontheeffect ofsurfactants onthedesorption ofHOCsfromsoil (e.g. Edwards efal.1992, Liuetal.1992,Van Dykeetal.1993).Thepresenceof surfactants mayalsocausefacilitatedtransport ofthesorbedpollutant.Thisterm covers severalprocesses bywhichthedesorption ofpollutants isstimulated,suchas mobilizationofpollutantstrapped insoilganglia (Vigon&Rubin 1989),interactionof pollutantswithsinglesurfactants molecules (Edwardsetal.1992),and solubilization inadmicelles, micelle-like structuresthat areformed atsolidsurfaces (Nayyar efal. 1994). Surfactants mayalsodecreasethebioavailability ofHOCsby promoting sorption ofthe pollutantonto soil(Wagner etal.1994),byinterference withthe natural interactions among microorganisms and pollutants (Efroymson&Alexander 1994), orbyclogging ofsoilpores (Abduletal.1990).Other negative effects ofapplying surfactants insoilbioremediationmay becaused bysurfactanttoxicity orby preferentialdegradation ofthe surfactant.
138
EffectofsurfactantsonthebiodegradationofsorbedPAHs Studyingtheeffect ofsurfactants onthe biodegradation of HOCs insoilis complexduetotheinteractions amongsoil, bacteria,pollutant, andsurfactant. This complexity isreflected inthedifferent resultsfound inthestudies ontheeffect of surfactants onthe biodegradation of HOCssorbedontosoil.Someauthorsfound thatsurfactants had noeffect onthedesorption,butthattheystimulated bacterial mineralization (Aronstein etal. 1991,Aronstein & Alexander 1992).Othersfound thatwhilethedesorptionwasstimulated,thebiodegradationwas notaffected (Dohse & Lion 1994)oreven inhibited (Laha&Luthy 1991,1992). Inallthesestudiesthere weretoomany unknownvariablestodrawgeneralconclusions.Thereforewe have chosentostudytheeffect ofsurfactants ondesorption and biodegradation ina systemthat isbetterdefinedthansoil.Thepolycyclicaromatic hydrocarbon (PAH) naphthalene, usedasmodel HOC,wassorbedontoinert,synthetic matrices.The two nonionicsurfactants selected,TritonX-100and Brij35,were used inprevious researchconcerningthe interactions among naphthalene,surfactants,andthe naphthalene-degrading bacterialstrain(Volkering etal. 1995).Thissetupthusallows ustoassesstheaction ofthe surfactants onthedesorptionofnaphthalene,andthe effect ofthis actiononthe biodegradation ofnaphthalene.
Materials and methods Bacterialcultures Theisolationofstrain8909N,growingonnaphthalene,hasbeendescribedpreviously (Volkeringefal. 1992).Theorganism,agram-negativePseudomonasspecies,hasalso beenusedinpreviousstudiesonthebiodegradationofnaphthalene(Volkeringefal. 1992, 1993,1995). Mediaandculture conditions Organismsweregrownat30°Cinmineralmedium, withessentiallythesamecomposition asdescribedbyEvansetal. (1970),withthemediumcomponentsbeingone-tenthofthose described.Themediumconsistedof10mMNH4CI,1 mMNaH2P04,1 mMKCI,0.2mM Na2S04,0.125mMMgCI2,2uMCaCI2,0.01uMNa2Mo04and0.5mIL"1ofasporesolution containing0.12mMHCI,5mM ZnO,20mMFeCI3,10mMMnCI2,1mMCuCI2,2mM CoCI2 and0.8mMH3B03.Nochelatingagentwasadded.ThemediumwasbufferedatpH7.0with 50mM sodiumphosphate.Pureculturesweremaintainedonmineralmediumagarslants containing1.5% agarand0.1%(w/v)ofnaphthalene(storageat4°C).
139
Chapter7 Loading of naphthalene ontothe matrices The matrices usedinthis study werethe resinsAmberliteXAD-4andXAD-7 (Supleco, Bellefonte, USA). Some physical properties ofXAD-4andXAD-7 areshown inTable1; these resins have beendesignedfortheadsorption oforganic compounds from aqueous solutions. Beforeloading,theresinswerewashedwithde-ionizedwaterto remove contaminations. Thiswas repeated untilthe UVspectrum ofthewashing water, after being incontact withthe resinfor24 h,showed nodifference withthespectrum ofclean de-ionized water.Thewet resinswere storedat4°C inaclosedglassjarto prevent dehydration.To loadthe matrices, 20-30gofthe materialwastransferred into a500-mlserum bottlewith 250 mlofde-ionizedwater.After pasteurization at 80°Cfor 3h,aclosedtubular dialysis membrane (cutoff 1000 Dalton),filledwith 15gof naphthalene crystals and25 mlofwater was added.Thereuponthe bottlewas closedwith ascrewcapwithteflon liningandwas incubated horizontally inarotary shaker (90rpm)at30°Cfor one month.After this period the bottlewas opened,the membranewiththe naphthalene crystals was removed,andthe bottlewasclosedand incubated for one moreday. Next,asample ofthe aqueous phase wastakenfor measurement ofthe naphthalene concentration, andthewaterwasdecanted. The materialwasdriedfor 5minonfiltration paper andtransferred into aglassjar. Nitrogen gaswas ledthroughthejarto removeoxygen,after whichthejarwas closed andstored at 4°C.A smallamount ofthefreshly loaded materialwasdriedto determinethedry weight; another smallamount was extractedto determine the initial naphthalene loading.After a periodofthree monthstheloadingofthe resinswas againdetermined. Table1: Physicalproperties oftheresinsused. resin
chemical nature
porosity [vol. %]
density [kgm-3]
surface area [m2-g-1]
Av. pore diameter [A]
XAD-4
polyaromatic
45
1080
725
40
XAD-7
acrylic ester
55
1240
450
90
Adsorption of surfactant Toassesstheadsorptionofsurfactant ontothematrices,0.05to 0.5 gofunloaded matrix was addedto250-mlserum flasks containing 100mlofasolution of0.1to 5g-L1TritonX100inmineralmedium.Theflaskswere placedonarotary shaker (200 rpm) at 30°C.After 3 to5daysthe aqueous surfactant concentration wasdetermined by measuringthe absorbance at254 nm(A264)with aspectrophotometer (Perkin Elmer Lambda 15,USA). In someexperiments theadsorption kinetics were monitored bycontinuously pumping asmall part ofthe liquid phasethrough the spectrophotometer.
140
Effect ofsurfactants onthebiodegradationofsorbed PAHs Batch desorption of naphthalene Batchdesorption experiments, performed in250-mlserumflasks on arotary shaker (200 rpm, 30°C),werestarted upbyadding 0.05 to0.5 gof loaded material totheflasks containing 150mlofsterile buffered mineral medium andtheappropriate amount of surfactant. At regular intervals samples of0.75 mlweretakentodetermine the naphthalene concentration inthe liquid phase.After approximately oneweekthe liquid phasewas decanted andthe naphthalene concentration ofboththe liquidandthe solid phases determined by HPLC analysis. Continuous desorption of naphthalene Thecontinuous desorption leaching experimentswere performed inamixedsystem. Sterile mineral medium withthe appropriate surfactant concentration was pumped into 35-ml serum bottleswithaworkingvolume of 12to 15mlthat contained 0.1 (XAD-4) or0.2 g (XAD-7) ofnaphthalene-loaded resin.Sintered glassfilters werefixed intothe outletofthe bottlesto protectthe resinsfrom washing out. The bottleswere placed inarotary shaker (230 rpm) at 30°C. The pumpflow, set at ±30 m l h \ was measured atthe beginning andat theendoftheexperiment. The little change inflowthatwas observedwas consideredto be linearwithtime.The naphthalene concentration inthe leachatesweredetermined by measuringtheA275every 12minwithaspectrophotometer, using flow-through cuvettes.At regular intervals samples ofthe leachates weretakento determine the naphthalene concentration with HPLC.The leachateswere collected in5-Ljars containing 25 mlofa5M NaOHsolutionto prevent biodegradation. To make mass balances,thefinalvolume ofthe leachates,thefinal naphthalene concentration inthejars,andthe residual naphthalene loadingofthe matricesweredetermined atthe endofthe experiment. Biodegradation of naphthalene Biodegradation experiments were performed at30°C inclosed 250-mlserum flasks ona rotary shaker (150or200 rpm).Theflasks contained 100mlof mineral mediumwiththe appropriate amount ofsurfactant andweresuppliedwith0.25 gofloaded matrix. To eliminatethe possibility ofoxygen limitation,the headspace oftheflasks wasfilledwith oxygen. Theexperiments were started by inoculationwith 1mlofactive batch-grown strain 8909N cells.The biodegradation of naphthalenewas monitored by measuring the percentage of C0 2 inthe headspace gas ofthe bottle.Asthe pHshift overthe experiment was negligible,this percentage couldbeused asameasureforthetotalamount of C0 2 produced andthus asameasure of biodegradation.At the endoftheexperiment 1mlof 12 M HCIwas addedtotheflasks to removethe dissolved C0 2 , andtheC0 2 concentration in the headspace gaswas measured.The residual naphthalene concentration ofthe liquidand thesolid phasewas determined.
141
Chapter7 Analyticalprocedures Aqueousnaphthaleneconcentrationsweredeterminedbyinjectionoffiltered(0.2urn rotrandfilter,Schleicher&Schuell,Germany),withacetonitrilediluted(1:1)sampleson an HPLCwithaChromSpherC18(PAH)column(Chrompack,Middelburg,TheNetherlands). Theeluentused wasamixtureofacetonitrileandwater(85:15).Peaksweredetectedwith a UVdetectorbymeasuringtheA275.Extractionsamplesweresimilarlymeasured,butdiluted toanaphthaleneconcentrationlowerthan100mg-L"1.Extractionofnaphthalenefromthe matrixwasperformedbyadding50or100mlofacetonitriletothematrix,shakingforone weekandmeasuringthenaphthaleneconcentrationintheacetonitrile.Forextractionof highlyloadedmatrices(determinationofstartingconcentrations),thisprocedurewas repeatedoncemore.Thedryweightofthematrixwasdeterminedbydryingaknown amountofwetmaterialat80°Cuntiltheweightwasconstant.TheC02intheheadspacegas ofserumflaskswasdeterminedusingagaschromatograph(HewlettPackardtype5890) equippedwithathermalconductivitydetectorandaHayesepQpackedstainlesssteel column(diameter 1/8inch,length2m,Chrompack).Heliumwasusedascarriergaswith a flowrateof30ml-min"1.Theinjectortemperaturewas150°C, theoventemperature80°C, andthedetectortemperature200°C.Theinjectionvolumewas250piwithsplitlessinjection. ThespecificareaofTritonX-100wasdeterminedbymeasuringthesurfacetensionof aqueoussolutionswithdifferentsurfactantconcentrations,asdescribedbyLyklema(1991).
Results Loadingofthe resinswith naphthalene Theloading method used madeitpossibletoobtain high loadingswithout using hydrophobic solvents,whichwould interfere inthedesorptionexperiments. The aqueous naphthalene concentrations, measured afterthe membranes were removed,wereequaltothe maximumconcentration.Thisindicatesthat equilibrium wasreached,andthereforethatthe materialswere homogeneously loaded.Thefinal naphthalene loadingsofthe matriceswere68 mgg' 1forXAD-7,and 145mgg"1for XAD-4. Extractions revealedthat underanoxic conditions at4°Cthese loadings remained stableforat leastthree months. Adsorption ofsurfactant Toassestheeffect ofsurfactant onthedesorptionof naphthalene, itis necessary toobtain insight intothesorption ofthesurfactant ontothe matrices.The adsorption isotherms ofTritonX-100ontoXAD-4andXAD-7 areshowninFigure1.
142
Effectofsurfactantson thebiodegradationofsorbedPAHs 1200 + +
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C s q [mg.L-1] Figure1:IsothermsforadsorptionofTritonX-100ontoXAD-7(n)andXAD-4 (*);lines representisothermsfittedusingtheLangmuirequation. Theisothermscould bedescribed accordingtothe Langmuirequationas: Q
=Q
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Csq [mg.L 1 ] Figure2:Isothermsfordesorption ofnaphthalenefromXAD-7(O) andXAD-4 (+); lines representfittedisothermsusingtheFreundlich equation. Sincetheloadingofthe resinsdid notapproach maximumvaluesat highaqueous naphthalene concentrations,these isotherms could notbedescribed usingthe Langmuirequation.As can beseen inFigure2thedatacould befitted usingthe Freundlichequation:
Q.„= Kf(°J 144
1/n
(2)
Effectofsurfactants on thebiodegradationofsorbed PAHs where K, isthe Freundlich sorption capacity constant [mg 11/n L1'ng1]and n isthe Freundlich sorption energy constant [-]. The values ofKfand nobtained byfittingthe measured data are presented inTable 3. Table3:Equilibriumdata onthesorption ofnaphthaleneontothedifferent resins.
resin
Q a [mgg-1]
n
K, [mg - -L1/n-g-1] 1 1/n
XAD-4
39.5
[-] 0.40
XAD-7
5.11
0.63
coverageb
145
[%] 87
68
89
a
;determinedinloadingexperiments; ":calculatedusingaspecificarea fornaphthalene of4.29-10~19 m2(Radt,1948). The effect ofTriton X-100 onthe desorption kinetics of naphthalene from XAD-4 is shown in Figure 3.
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145
Chapter7 Thepresence ofsurfactant changesthepartitioning ofnaphthalene overthesolid andtheliquid phase.Althoughthedesorption proceededfaster athigh surfactant concentrations, itwas notpossibletodeterminetheeffect ofthesurfactant onthe initialdesorption rateintheseexperiments. UsingXAD-7,similar resultswerefound, butequilibriumwas reached quicker. Theeffect ofTritonX-100onthepartitioning ofnaphthalene overthe liquidand thesolidphaseisshown inTable4forXAD-4andXAD-7. Table4: Effectof TritonX-100 onthepartitioningof naphthalene overthesolidandthe liquidphase in batch desorption experiments with0.1gresinin 100mlmineralmedium. XAD-4
surfactant concentration
XAD-7
[L-g- ]
Ceq [mg-L-1]
[mg-g-1]
[i-g-1]
118
6.38
20.3
37.9
1.87
31
105
3.38
20.5
37.7
1.84
0.25
31.7
104
3.28
20.8
27.3
1.31
0.5
38.8
97
2.50
23.1
22.8
0.98
1.0
51.7
84
1.62
28.4
19.2
0.67
2.0
69
67
0.97
34.4
15.2
0.44
[g-L1]
QeqICeq
[mg-L1]
[mg-g-1]
0
18.5
0.1
1
Continuous desorption of naphthalene Thebatchdesorption experimentsdid notprovideenoughdistinct information on thekinetics ofdesorptiontodeterminewhetherfacilitatedtransport playeda rolein theeffects thesurfactant hadonthedesorption process. Forthis reason continuous desorptionexperiments inthepresenceofsurfactantwereperformed.Inthe instrumentalsetup usedfortheseexperiments,the naphthalene concentration was determinedbymeasuringtheA275.SinceTritonX-100alsoabsorbsatthis wavelength (duetothe presenceofanaromatic ring)the linearalkylethoxy ether Brij 35was usedassurfactant intheexperiments inwhichtheeffluent concentration was continuously measured. Figure4A representsthe resultsofthe naphthalene concentration measurements inthe leachatefortheexperiments withXAD-7.These datacanbeusedtocalculatethepercentageofdesorbed naphthalene asshownin Figure4B.The results showarelatively quick completedesorption and littleeffect of theuseofthesurfactant Brij35,evenatconcentrations ashighas2g-L1.
146
Effectofsurfactantsonthebiodegradation ofsorbedPAHs
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time [h] Figure4:ContinuousdesorptionofnaphthalenefromXAD-7inthepresenceofdifferent concentrationsofBrij35. A:naphthalene concentrationintheleachate, B: % naphthalenedesorberd; surfactantconcentrations: ;0g-L'1, — : 0.1 g-L1, :0.5g-L1, -•-•.2.0gL1.
A morepronouncedeffectwasfoundfor naphthalene sorbedontoXAD-4ascanbe seeninthe Figures 5Aand5B.Duetotheformationofbubbles inthecuvette inthe experimentwith 0.1 g.L 1 Brij35,the measurement was unreliablefrom 54h onwards. Itcanbeseen, however, thatevenatthis sub-CMC concentration,the desorption ofthesorbed naphthalene wasenhanced markedly. 147
Chapter7 Theaboveresultsareconfirmedbythemeasurement oftheresidualloadingsand thenaphthaleneconcentrations intheleachateattheendoftheexperiments.These resultsandthemassbalance (expressedasthe%naphthalenerecovered) overthe continuousdesorptionexperimentswithXAD-4andBrij35arepresentedinTable5.
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time [h] Figure5:ContinuousdesorptionofnaphthalenefromXAD-4inthepresenceofdifferent concentrationsofBrij35. A:naphthalene concentrationoftheleachate, B: % naphthalenedesorberd;surfactantconcentrations: :0g-L1,—: 0.1 g-L\ :0.5g-L' -•-:2.0gL1. 148
Effectofsurfactantson thebiodegradationofsorbedPAHs Thistable alsoshowsthe resultsoftheexperiments withXAD-4andTritonX-100. In theseexperiments the naphthalene concentration inthe leachatecould notbe continuously monitored;therefore onlyoveralldata,very similartothe dataobtained with Brij35,arepresented. Table5:Massbalance overthecontinuousdesorption ofnaphthalenefromXAD-4in the presenceofdifferentconcentrationsofBrij35orTritonX-100. surfactant cone. [g-L1]
Brij35 naphthalene desorbed [mgg 1 ]
residual loading [mgg-1]
TritonX-100 recovery [%]
naphthalene desorbed [mg-g-1]a
residual loading [mgg 1 ] 3
recovery [%]a
*a
*°
55
83
63
71.1
40
76
107
5.1
>88
82
109
2.3
76
143
120
4.5
108
91
139
1.8
97
1.0
151
129
3.7
112
97
148
1.6
103
2.0
161
133
1.3
119
98
N.D.
N.D.
N.D.
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59
43.6
0.1
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0.5
a
:calculatedfromtheonlinemeasurementoftheeffluentconcentration :determinedbymeasurementoftheoverallconcentrationintheelutedsolution N.D.:notdetermined
b
Naphthalene biodegradation Bacterialattachment ontothe matrixwasobservedtooccurduring growthon XAD-sorbed naphthalene. Becauseofthisthesuspendedbiomass concentration was notagood measurefor bacterialgrowth.Biodegradationwastherefore monitored bymeasurement ofthe C0 2 concentration inthe headspacegasofbatch cultures.Typically, growthonsorbed naphthalenestartswithanexponential growth phase,followed byaphase inwhichthe bacterialgrowth islimited bythe desorption ofnaphthalene fromthe matrix.Theaqueous-phase naphthalene concentration in thisstagewillbevirtuallyzero(Volkering efal.1993).Toassesstheeffect of surfactant onthe biodegradation ofsorbed naphthalene,thesurfactant was added either atthe beginning oftheexperiment orduringthedesorption-limited growth phase. IntheexperimentswithXAD-7,ashortdesorption-limited growth phasewas observedwith only littleeffect ofthesurfactants onthebiodegradation. Inthenonexponentential growth stage ofexperimentswithXAD-4,nonaphthalene couldbe 149
Chapter7 detected intheaqueous phase (data notshown),anddesorption of naphthalene fromthe matrixwasconcludedtoberate-limiting.Adding TritonX-100or Brij35at thestartofexperimentswithXAD-4resulted inlongerexponentialgrowth phases than inexperiments without surfactant (Figure6). Duetothis prolongued exponential phaseitwas notpossibletocomparethedesorption-limitedphases oftheblank experiments withthose observed intheexperiments withsurfactant. Therefore experimentswere performed inwhichthesurfactantwasadded inthe limited growth phase.TheC0 2 productionratesincreased aftersurfactantwasaddedascanbe seen inFigure7.Atthe end oftheexperiments noresidual naphthalene couldbe detected inthe liquid phases.InTable6givesthe residual naphthalene loadingsof theXAD-4andthe massbalances overthe biodegradation experiments.To confirm thattheobserved increase inthe C0 2 production ratewascausedonly bythe degradation ofnaphthalene,severalblankexperiments havebeenperformed.No C0 2 productionwasfound ininoculated culturesthat contained onlysurfactant or unloaded matrixes,andsurfactantdid notaffectthecarbonate equilibria, aswas tested byadding 1mlofa 12MHCIsolutiontothecultures. Moreover, the C0 2 yield found isingoodagreement withtheyieldfoundforthe batchgrowth ofstrain8909N oncrystallinenaphthalene (Chapter3).
Table 6:Massbalance overbatch experiments with strain 8909Ngrowingon naphthalenesorbedontoXAD-4. surfactant concentration [g-L-1]
residual loading
C0 2 produced
C02-yielda
[mg-g-1]
[mmol]
[C-molC-mol1]
0
20.6
0.051
0.52
0.1
4.7
0.052
0.54
0.5
0.5
0.052
0.54
1.0
1.1
0.053
0.55
2.0
0.3
0.051
0.53
calculatedasC-molC02producedperC-molnaphthalenedisappeared.
150
Effect ofsurfactants onthebiodegradationofsorbed PAHs
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time [h] Figure 6:C02 production ofstrain 8909Nonnaphthalenesorbed ontoXAD-4, TritonX-100 addedatt =0;surfactant concentrations: -•-: 0g-L1,-O-: 0.1 g-L1, -T-: 0.5g-L1, -L-2.0 g-L1.
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151
Chapter7 DISCUSSION Batchsorption ofTritonX-100and naphthalene Adsorption ofsurfactants ontosolidsurfaces isacomplicated processthatmay involvetheformationofsurfactant aggregates calledadmicellesorhemicelles, dependingonwhether theaggregates consist of 1or2layers of surfactant molecules (West & Harwell 1992). Forthe adsorption ofnonionic surfactants froman aqueoussolution ontoa hydrophobic surface suchasthe resins used here, a monomolecularlayer canbeexpected.Thisappearstobeconfirmed inthe adsorptionexperiments usingTritonX-100,andXAD-4andXAD-7.Anestimation of thecoverage ofthe materials canbemade usingthespecific surfaceareas ofthe matricesandasurfaceareaof6.7-1019m2permoleculeofTritonX-100.These calculations revealedthatabout 90%ofthe resinsurface areawascoveredwith surfactant molecules (Table2).Moreover,theadsorption isothermscouldbe describedwiththe Langmuir model,which isbasedonthe assumptionthata monomolecular layerofmolecules isformedontheadsorbingsurface. Fortheadsorption of naphthalene ontothematrices similar resultswerefound (Table3).With naphthalene (molecular surfacearea4.29-1019m2,Radt 1948),a coverageofapproximately 90% wasfoundfor bothXAD-4andXAD-7. Incontrastto TritonX-100, thedesorption isotherms ofnaphthalene could notbedescribed with the Langmuir model.Thisdifference maybecaused bythe lowaqueous solubility of naphthalene. Fromthe batchdesorption experimentswith naphthalenethat have been performed inthis study, itcanbeconcludedthatthepresence ofsurfactants athigh concentrations results inadecrease inthesolid-liquid partition coefficient dueto solubilization ofnaphthalene insurfactant micelles.This isconsistentwiththe results ofmoststudies onsorptionof HOCs inthe presence ofsurfactants (e.g.Edwardset al.1991,1992,Liuera/. 1992,Park &Jaffe 1993,Sun&Boyd 1993,Vigon &Rubin 1989).More interesting isthedecrease inthepartitioncoefficient at low surfactant concentrations (0.1and0.25 g-L1). Sincethese arethe initialconcentrations,the aqueous surfactant concentrations atequilibriumwillbeconsiderably lowerthanthe CMC(approximately 0.1 g-L"1forTritonX-100,Volkering etal.1995),becauseof adsorptionofsurfactant moleculesontothe matrix.Therefore nosurfactant micelles willbepresent,eliminating solubilization asthe reasonforthe increased aqueous naphthalene concentrations. Competition between naphthalene andTritonX-100for theavailablesorbing surface isthe most likelyexplanationforthis phenomenon.
152
Effectofsurfactantson thebiodegradationofsorbedPAHs Theresultsofthebatchwisedesorptionexperimentsdonotallowdistinct conclusions abouttheeffect ofsurfactant onthe initialdesorption rateof naphthalene (facilitatedtransport).The reasonthedesorption proceedsfaster inthe experimentswith highsurfactant concentrations may bethe partitioning of naphthalene intothe micelles, ratherthanfacilitatedtransport. This partitioning will causeaslowerdecrease inthedifference betweentheequilibrium concentrationand theactualconcentration,the drivingforce behindthedesorption process,andwill thus leadtoahigherdesorption rate. Continuous desorption of naphthalene The leachingexperiments were performed atonesurfactant concentration below the CMCandthreeconcentrations above it.Themassbalances overthe experimentswithXAD-4,and Brij35andTritonX-100(Table5)showthatforthe sub-CMCsurfactantconcentations (0and0.1g-L"1)the recoveryofnaphthalenewas rather poor.The most likely explanation forthis isthat,although asmuchstainless steeltubing aspossiblewas used,liquid-phase naphthalenewas lostdueto absorptionorvolatization.This losswillbelessatsurfactant concentrations higher thantheCMC,sincethen alargeamount ofthesolubilized naphthalenewillbe present inthe micellarphase.This meansthatfortheexperiments without surfactant andwith0.1 g-L"1surfactant,thedesorbed amountsof naphthalene arelikelytobe higherthanthe amount recovered inthe leachates.Thisdoes not interferewiththe conclusionthatthepresenceof0.1g.L 1 Brij35andTritonX-100(sub-CMC) clearly stimulatesthedesorption of naphthalene.Thiscanalso beseenfromthe residual loadingsofthe matrices after leaching,whichwere afactor 10-20lowerwhen surfactantwas present (Table 5).Thesolubility enhancement ofnaphthalene in aqueoussolutions atthissurfactant concentration isnegligible (Volkeringetal. 1995).Therefore itcanbeconcludedthatthesurfactant stimulatesthedesorptionof naphthalenethroughfacilitatedtransport. However, sincethedesorptionwas even faster atsurfactant concentrations higherthantheCMC,solubilization mayalsoplay a roleintheseexperiments.Weinferredearlier (Volkering etal 1993, 1995)thatfor enhancingthe biodegradation ofsoil-sorbedpollutant (but notfor soilwashing), facilitatedtransport ofpollutant islikelytobethemost importanteffect ofsurfactants. Thus,the relatively largeeffect foundattheconcentration belowthe CMC is promising,alsofromaneconomic point ofview.
153
Chapter7 Biodegradationofsorbed naphthalene Bacterialgrowthonsorbed hydrocarbons inbatchcultures canbedescribed analoguetothegrowthonsolid hydrocarbons (Volkering 1992, 1993). Initially,the desorption rate ishigherthanthedegradation rate,andexponential growth and C0 2 productionoccurs (exponential stage).Thedegradation ratewillincreasewith increasingcellnumbers andwilleventuallyexceedthedesorption rate,causingthe aqueous hydrocarbon concentration todrop.This results inastage inwhichthe bacterialgrowth islimitedbythedesorptionofthe HOC(desorption-limitedstage). Thepresence ofsurfactant inthis system maystimulatethe biodegradation ofthe hydrocarbon intwoways. Firstly,atconcentrations higherthantheCMC,more hydrocarbonwillbe desorbed inthefirst stageduetomicellarsolubilization, resulting inalonger exponentialgrowthstage.Secondly itmaycausefacilitatedtransport ofsorbed hydrocarbontothe aqueous phase,which results inbothalongerexponential stage andan increasedgrowth rate inthedesorption-limited stage. Intheexperiments with XAD-4described here,theaddition ofsurfactant atthestart ofthe experiment resulted,asexpected,inaprolonguedexponentialstage (Figure6).Additionofthe surfactant inthedesorption-limited stage resultedalso inastimulationofthe C0 2 productiononthesorbedsubstrate,ascanbeseeninFigure7.Thiswas notcaused bydegradation ofthesurfactant orbyachange inthebacterialyieldon naphthalene asshowninTable6.Therefore,these resultsconfirmtheconclusionfromthe continuousdesorption experiments, i.e.thatthe presenceofsurfactants results in facilitatedtransport ofsorbed naphthalene.Thisisalsosupported bythe resultsof themeasurements ofthe residual naphthalene loadings,whichwere performed after theexperiment wasstopped byaddition of 1ml 12MHCI.The residual loadingsin thebottleswithout surfactant weresignificantly higherthan intheotherexperiments, showingthat more naphthalenewasdegradedwhensurfactantwaspresent.Asto themechanism ofthismobilization,littlecanbesaidfromthedatapresentedhere. Comparisonofdesorptionand biodegradation Usingayieldfactor asdescribed previously (Volkering etal. 1993)and carbonate equilibria (Lindsay 1979), itispossibleto relatethe C0 2 concentration inthe headspacegastototal amount ofC0 2 produced andthustotheamount of naphthalenethatwasdegraded.This isshown inFigure8fortheexperiments with andwithout2.0g-L'1surfactant (addition att=0).Tocompare biodegradation and desorption,theamounts ofnaphthalene desorbed inthe leaching experiments
154
Effectofsurfactantson thebiodegradation ofsorbedPAHs withoutsurfactant andwith 2.0g-L1 surfactant arealsogiven inthefigure.Although theexperimental setupwasdifferent inbothexperiments,severalobservations can bemadefromthedata presented inFigure8.
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time [h] Figure8:Effectofsurfactantonbiodegradation (symbols+lines) andoncontinuous desorption (linesonly) fornaphthalenesorbedontoXAD-4;— : nosurfactant, — : 2.0g-L1 surfactant Comparingthe solid lines itappearsthat intheabsence ofsurfactant the biodegradation proceedsfasterthanthedesorption.This phenomenon hasbeen discussed previously inChapter4. Whensurfactant ispresent itismoredifficulttocompare batchgrowth experiments withcontinuous desorptionexperiments, becausethe presenceof surfactantcausesan important difference inthetwotypes ofexperiments. Inthe batchexperiments, aknownamount ofsurfactant ispresent,whichwillpartly adsorb ontothe matrix. Inthe leachingexperiments, however,freshmediumwiththesame surfactant concentration iscontinuously supplied.Hence,moresurfactant willbe absorbed ontothe matrixwithequal(initial) aqueoussurfactant concentrations and theeffect ofthesurfactant molecules onthedesorptionwillbedifferent ineachcase. However, inboththedesorptionexperiments (Table5)andthe biodegradation
155
Chapter 7 experiments (Table 6) little difference was found between the results of the experiments with the highest surfactant concentrations (1.0 and 2.0 g-L 1 ). Therefore it is possible to draw two more conclusions from Figure 8: (i) the effect of surfactants is less pronounced inthe biodegradation experiments than in the desorption experiments, and (ii) the effect of the microorganisms is less pronounced when surfactant is present (dashed lines). It appears that both the presence of surfactant and of growing bacteria have a positive effect on the bioavailability, and that the effect of the one is decreased by the effect of the other. Nevertheless, it can be concluded that application of surfactants, under the right conditions, can stimulate the degradation of organic pollutants and can therefore be seen as an interesting tool for soil bioremediation. NOMENCLATURE Ce, ka K, n Qa, Q max
equilibrium surfactant concentration in solution [mg-L 1 ] Langmuir constant [Lmg 1 ] Freundlich sorption capacity constant [mg 1 1 / n L 1 / n g 1 ] Freundlich sorption energy constant [-] equilibrium sorbed surfactant concentration [mgg' 1 ] m a x i m u m sorbed surfactant concentration [ m g g 1 ]
REFERENCES AbdulS.A., T.L.Gibson,andD.N. Rai(1990)Selectionofsurfactantsfortheremovalof petroleum products from shallow sandy aquifers.GroundWater28:920-926. Aronstein B.N.,andM.Alexander (1992)Surfactants atlowconcentrations stimulatethe biodegradation ofsorbed hydrocarbons insamples of aquifersands andsoilslurry's. Environ. Toxicol.Chem. 11:1227-1233. Aronstein B.N.,Y.M.Calvillo,andM.Alexander (1991) Effect ofsurfactants at low concentrations onthedesorption and biodegradation ofsorbed aromatic compounds in soil. Environ.Sci.Technol.25:1728-1731. Dohse D.M., and L.W. Lion (1994) Effect of microbial polymersonthe sorptionandtransport ofphenanthrene inalow-carbon sand.Environ.Sci.Technol.28:541-548. Edwards D.A., R.G. Luthy, andZ. Liu (1991) Solubilization ofpolycyclic aromatic hydrocarbons inmicellarnonionic surfactant solutions.Environ.Sci.Technol. 25:127-133. EdwardsD.A., Z. Liu,andR.G. Luthy (1992) Interactions between nonionic surfactant 156
Effect ofsurfactants onthebiodegradationofsorbed PAHs monomers.hydrophobicorganic compounds.andsoil.Water Sci.Technol.26:147-158. EfroymsonR.A., and M.Alexander (1994) Role ofpartitioning inbiodegradation of phenanthrene dissolved innonaqueous-phase liquids. Environ.Sci.Technol.28: 1172-1179 EvansC.G.T., D. Herbert, andD.W. Tempest (1970)Thecontinuous cultivation of microorganisms. 2. Construction ofachemostat. p.277-327. In: NorrisJ.R., and D.W. Ribbons (eds.), Methods ofMicrobiology vol.2.Academic Press, London,UK. GuerinW.F.,and S.A. Boyd (1992) Differential bioavailability of soil-sorbednaphthalene for two bacterial species.Appl. Environ.Microbiol.58:1142-1152. HermannsonM., andK.C. Marshall (1985) Utilization ofsurface localised substrate bynonadhesive marine bacteria. Microb. Ecol. 11: 91-105. LahaS.,and R.G. Luthy (1991) Inhibition of phenanthrene mineralization by nonionic surfactants insoil-water systems. Environ.Sci.Technol.25:1920-1930. Laha S., and R.G. Luthy (1992) Effects of nonionic surfactants onthe mineralization of phenanthrene insoil-water systems. Biotechnol. Bioeng.40:1367-1380. LindsayW.L. (1979) Chemical equilibria insoils.Wiley J.&Sons, Inc. NewYork. LiuZ., D.A. Edwards, and R.G. Luthy (1992) Sorption of non-ionic surfactant onto soil.Water Res. 26:1337-1345. LyklemaJ.(1991) Fundamentals of interface andcolloid science,vol. 1,Academic Press, London. Mihelcic J.R., D.R. Luekin,R.J. Mitzell,and J.M.Stapleton (1993) Bioavailability ofsorbedandseparate-phase chemicals. Biodegradation 4:141-153. Mihelcic J.R., and R.G. Luthy (1991) Sorption and microbial degradation of naphthalene in soil-water suspensions under denitrifying conditions. Environ.Sci.Technol.25:169-177. Nayyar S.P., D.A. Sabatini, andJ.H.Harwell (1994) Surfactant adsolubilization and modified admicellarsorption nonpolar, polar, and ionizable organic contaminants. Environ.Sci. Technol.28:1874-1881. ParkJ.W., andP.R. Jaffe (1993) Partitioning ofthree nonionic organic compounds between adsorbed surfactants, micelles andwater. Environ.Sci.Technol.27:2559-2565. Providenti M.A., H. Lee,andJ.T. Trevors (1993) Selectedfactors limitingthe microbial degradation of recalcitrant compounds. J. Ind. Microbiol. 12:379-395. Radt F. (ed) (1948) Elseviers encyclopaedia oforganic chemistry. Robinson K.G.,W.S. Farmer, andJ.T. Novak (1990)Availability ofsorbedtoluene insoilsfor biodegradation byacclimated bacteria.Water Res.24:345-350. Scow K.M., and M.Alexander (1992) Effect ofdiffusion andsorption onthe kinetics of biodegradation: experimental resultswithsynthetic aggregates. Soil Sci.Soc.Amer.J. 56:128-134. SunS.,andS.A. Boyd(1993) Sorptionof nonionic organic compounds insoilwater systems containing petroleum sulfonate oilsurfactants. Environ.Sci.Technol.27:1340-1346.
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Chapter7 Van Dyke M.I.,S.L. Gulley, H. Lee,andJ.T.Trevors (1993) Evaluation of microbial surfactants for recovery ofhydrophobic pollutants from soil.J. Ind. Microbiol. 11:163-170. Vigon B.W., andA.J. Rubin (1989) Practicalconsiderations inthe surfactant-aided mobilization ofcontaminants inaquifers.J.Water Pollut. Control.Fed.61:1233-1240. Volkering F.,A.M.Breure,A. Sterkenburg,andJ.G.vanAndel (1992) Microbial degradation of polycyclic aromatic hydrocarbons: effect ofsubstrate availability on bacterial growth kinetics.Appl. Microbiol.Biotechnol.36:548-552. Volkering F.,A.M.Breure,andJ.G.vanAndel (1993) Effect of micro-organisms onthe bioavailability and biodegradation ofcrystalline naphthalene.Appl.Microbiol. Biotechnol.40:535-540. Volkering F., A.M.Breure,J.G.vanAndel,andW.H. Rulkens (1995) Influence of nonionic surfactants onthe bioavailability andbiodegradation ofcrystalline polycyclic aromatic hydrocarbons.Appl.Environ.Microbiol.61:1699-1705. Wagner J., H.Chen,B.J. Brownawell,andJ.C.Westall (1994) Useofcationic surfactantsto modify soilsurfaces to promote sorption and retard migration of hydrophobic organic compounds. Environ.Sci.Technol.28:231-237. West C.C., andJ.H.Harwell (1992) Surfactants andsubsurface remediation. Environ.Sci. Technol. 25:127-133.
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CHAPTER8 CONCLUDINGREMARKS Bioavailability and biodegradation of polycyclic aromatic hydrocarbons Thisthesis hasdiscussedthe research onthe biodegradation of polycyclic aromatic hydrocarbons.These compounds are representative ofagroup of hydrophobic organic pollutantswhichare biodegradable under laboratory conditions, butarepersistent insoil. In 1990,whenthisprojectstarted,the realizationthatthe occurenceof masstransfer limitation insoilcouldbethe maincauseforthis contradiction gradually becameevident. Themaingoalofthis researchtherefore wasto revealthe relation betweenthe bioavailability ofthe hydrophobic pollutants (PAHs)andtheir biodegradation rates. Thefirststep inthe researchwastheisolationandcharacterization ofPAHdegrading bacteria,whichwerethen usedforstudyingtheeffect ofmasstransfer limitationsonthedegradation of PAHs indifferent systems. Forcrystalline PAHsit wasshownthatthedegradation ratescould bedirectly coupledtothe dissolution ratesofthedifferent PAHs.For PAHssorbed ontoamatrix, morecomplex results werefound.Usingsynthetic porous materialsasthesorbing matrix, itwasfoundthat thebiodegradation rateof naphthalene was2-3times higherthanthe calculated maximaldesorption rate.Therefore itmust beconcludedthatwiththistypeof matricesthe bacteria must havesome meansofgaining better accesstothesorbed naphthalene. Incontrastwiththis,itwasfound inexperiments withsoil-sorbed naphthaleneandphenanthrenethatthesamemicroorganismsdegradedaqueousphase PAHsonly.These results showthatthe biodegradation of hydrophobic pollutants insoilisacomplicated matter involving both physicochemical and biological processes. Inallcases,however, itwasfoundthatthe biodegradation rate ofseparate-phase PAHswas lowerthanthatoffree-phase PAHs,showingthatthe bioavailability of PAHscanbeanimportantfactor limitingtheirbiodegradation.
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Chapter8
Consequences of bioavailability limitations for biological soil remediation Inrecent studies on biologicalsoilremediation,limited bioavailability isgenerally consideredtobeoneofthe most importantfactors determiningthe resultof bioremediationofpoorly soluble pollutantssuchasPAHs, PCBs,andweatheredoil (Blackburn &Hafker 1993,Mihelcic etal. 1993, Providenti etal. 1993,SchulzBerendt 1994,Thomas &Lester 1993,Wilson &Jones 1993).This isalso reflected bythefactthatatthe"Third InSituandOnSiteBioremediation Symposium"inSan Diego(april 1995),twofullsessionswerededicatedtothe phenomenon of bioavailability. Thisshowsthat bioavailability isa"hottopic" inthe research concerning biological clean-up ofsoils.Whencomparedtootherfactorsthat playa roleinsoil remediation,bioavailability ismostnotablefor itsvagueness. Its definitions,suchasthe onepresented inthe introduction,are usuallygeneric. Fora good understanding of bioavailability itisessentialto realizethat itisatime-based concept. Moreover, bioavailability can bedifferent fordifferent organisms andis dependent ona large numberofphysico-chemical parameters,asdiscussed inthe previouschapters. Itisthereforedifficult tomake predictions onthe ratesof biodegradation inbioremediation processes,andthustopredictthetime necessary forsoilclean-up.Thepoor predictability and reliability arethe main reasonsforthe present limitedapplication of biologicalsoilremediation. Itisthereforeessentialthatstandard methodsaredevelopedbywhichthe bioavailability ofpollutants insoilsandsediments can becharacterized.Thesemay bebiologicalmethods, inwhichtheeffect ofthe pollutant inthesoilorsediment on standardorganisms ismeasured,orphysicochemicalmethods, inwhichthe mass transferofthe pollutanttotheaqueousphase ismeasured.Biologicalmethodsare generally morelaboriousandtherefore moreexpensivethan physicochemical methods.Theproblemwith physicochemical methods isthatthedifferences in bioavailability fordifferent organisms are nottaken intoaccount. However, fromthe resultspresented inthisthesis andfrom mostother studies,itcanbeconcludedthat thebioavailability ofsorbed pollutants ismuch lowerthanthat of aqueous-phase pollutants.Thus,bychoosingthe rightconditions and usingaproper experimental setup, itshould bepossibleto predict biodegradation rates using physicochemical methods.Thisisconfirmed bythe resultswithsoilpresented inChapter4, aswas discussedabove.Thetypeofphysicochemicalexperiments bestsuitedtothisare
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Concludingremarks probably column-leaching experiments. Inadditiontothese,itmay be necessary to performsimple biological experiments.
Application ofsurfactants asasolutionto limited bioavailability Thepossibility forsolvingtheproblem oflimited bioavailability by technological means isdependent onthetype ofbiological soiltreatment that isused. Whenthe polluted soilisexcavated,severaldifferent techniques areavailable: (i)mechanicalreductionofthesizeofthesoilaggregatestoreducethe lengthofthe desorption pathways andthustoacceleratethedesorption;(ii) increasing the temperature tospeed updesorption rates,(ii)separation ofthe highly polluted fraction ofthe soil,and(iv)additionof bioavailability-enhancing compounds.These techniques canbeapplied both beforethe biological process andsimultanuoustoit Withinsitutreatmentthe number oftechnological solutions islimited. Improving bioavailability ofthe pollutants canbeestablished by(i) increasingthetemperature, which isinmostcasestooexpensive, (ii) usingacoustictechniques,amethodthat is still inthedeveloping stage andonlyapplicableto liquid-phase pollutants,or(iii) addingcompoundsthat mayenhancethe bioavailability ofthpollutants.These includeco-solvents,fungalenzymes, chemicaloxidants and surface-active compounds,the latter beingthesolutionthat hasbeenappliedthemost. Thetechnological solutionforthe problemoflimited availability of hydrophobic organicpollutantsdealtwith inmostdetail inthisthesisisthe useof surface-active agents.Asdiscussed inChapter 5,thethree main mechanism bywhich surfactants canenhancethe bioavailability ofhydrophobic pollutants are (1)solubilization,(2) emulsification,and (3)facilitatedtransport. Solubilization canonlyoccurwhen micelles,aggregates of20-20surfactant molecules,are present. Thepresenceof micelles results inincreased (apparent) solubilities of hydrophobic pollutants. Insoil however, micelleformation occurs at rather highsurfactant concentrations dueto adsorption ofsurfactant molecules.Theemulsifying actionofsurfactant iscausedby thedecrease inthesurfacetension betweentheaqueous phaseandthe pollutant paseandcanonly occurwhenthe pollutant ispresent asaliquid-phase. Facilitated transport isatermthat covers several processes,suchasthe releaseofpollutantin trappedsoilpores,interaction ofpollutantwithsinglesurfactant molecules,anddirect interactions ofmicellar structureswithsorbed pollutants (adsolubilization). Thereare,however, also negativeeffects ofthe useofsurfactants,suchastoxic behavior orpreferentialdegradation ofthesurfactants,clogging ofsoilpores,or
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Chapter8 interference ofthesurfactants withthe natural interactions amongthemicroorganisms andthe pollutant. Duetothecomplexityoftheseeffects insoil,present knowledge isnotenough topredicttheeffect ofthe application ofsurfactants insoilremediation. Nevertheless, surfactants arefrequently usedtoprevent bioavailability problems inbioremediation processes,bothwith exsitu(e.g.Joshi&Lee 1995,Schmidt &Hahn 1995)andin situprocesses (e.g.Ducreux era/. 1995,Guttman 1995,Rhodesetal. 1995, Rosset a/. 1995,VanVree etal.1993). Asthe amountofsurfactant used may beoneofthefactorsdetermining the costsofthetreatment, optimizingthesurfactant addition isanimportant prerequisite forits use.Severalconsiderations can betakento minimizethe useofsurfactant. Firstly,surfactant additionwillonly beusefulwhenthebioavailability ofthe pollutant islimitingthe biodegradation rateaswasdiscussed intheChapters 5-7. Therefore addition ofsurfactants atthe startofthe bioremediation process islikelytobeoflittle use.Secondly, itseems ineffective toapply surfactant inhighconcentrations, aswas alsodiscussed inthe Chapters 5-7. Finally, itmight beusefultoaddsurfactants on anintermittentbasis. Another promising optiontoreducingthecostsofsurfactants may bestimulation oftheinsituproduction ofbiosurfactants bythe naturalpopulationor by introduced micro-organisms.Althoughthe useofbiosurfactants insoilremediation hasreceived considerable attention (see Chapter 5)andalthough somestudiesonthe useof biosurfactant-producing bacteria have beenpublished (Jainetal. 1992,Providentiet al.1995),the understanding ofprocesses involved inbiosurfactant production during bioremediation stillrequiresfundamental microbologicalresearch.
Consequences of limited bioavailability forsoil-quality limits Althoughtechnological solutionsfor bioavailability limitationsarebeing studied asdiscussedabove,another sideofbioavailability isoftenforgotten.Theterm bioavailabilityoriginates fromthefield oftoxicology, and itiseasyto understandthat apollutedsoilinwhichthe pollution hasalowbioavailability willposealower ecotoxicological riskthan asoil inwhichthesamepollutant hasahigh bioavailability, evenwhenthe levels ofthe pollutant concentration arethesame.However, this insightis not reflected inmostofthe regulations concerning soilpollution.Allthese regulations arebasedon pollutant concentrations measured byextraction ofthesoil withorganic solvents,although insomeguidelinestheorganic carbon content ofthe
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Concludingremarks soils is incorporated. By not taking the bioavailability of the pollutant into account, these regulations may lead to an overestimation of the ecotoxicological risks (LaGoy & Quirck 1994). An alternative to measurement of pollutant concentrations by solvent extraction is measurement of the leaching behavior of the pollutant, as explained in the previous section. At the moment the Dutch legislation on the quality of building materials is in revision. The new rules are based on the results of leaching experiments and standard methods are now under development (Ministry of Housing, Spatial Planning, and the Environment 1994). Rules based on this type of experiments have much better scientific background than the present rules for soil pollution. A similar approach has recently been proposed by Beck etal. (1995). The authors defined kinetically constrained soil-quality limits (KCSQLs). These are based on the timecourse of the decrease of the pollutant concentration. The most important agreement between the KCSQL-concept and the one presented here isthat the risk of pollution does not mainly depend on its concentration in the soil, but rather on the rate atwhich it may come available for uptake by organisms.
References BeckA.J., S.C.Wilson, R.E. Alcock, and K.C. Jones (1995) Kinetic constraints onthe lossof organic chemicalsfrom contaminated soils: implications for soil-quality limits.CRC Critical Rev. Environ.Sci.Technol.25:1-43. Blackburn J.W., andW.R. Hafker (1993) The Impact of biochemistry, bioavailability and bioactivity ontheselection of bioremediationtechniques. Trends Biotechnol. 11:328-333. Ducreux J., M. Baviere, P. Seabra,O. Razakarisoa, G. Shafer, andC.Arnaud (1995) Surfactant-aided recovery/in situ bioremediation for oil-contaminated sites. In: R.E. Hinchee,J.A. Kittel, and H.J. Reisinger (eds.)Applied bioremediation of petroleum hydrocarbons, p.435-443, Battelle Press,Columbus, USA. Guttman R.M. (1995) Insitu bioreclamationof accumulated poultry-processing solids.In: R.E. Hinchee,J.A. Kittel,and H.J. Reisinger (eds.)Applied bioremediation of petroleum hydrocarbons, p. 505-510. Battelle Press, Columbus, USA. Jain D.K., H. Lee,and J.T. Trevors (1992) Effect ofaddition of Pseudomonasaeruginosa UG2 inocula or biosurfactants onbiodegradation ofselected hydrocarbons insoil. J. Ind. Microbiol. 10:87-93. Joshi M.M., and S.G. Lee (1995)A noveltreatment trainfor remediation of PAH contaminated soils. Fresenius Environ. Bull.4:617-623. LaGoy P.K., andT.C. Quirk (1994) Establishing generic remediation goalsforthe polycyclic aromatic hydrocarbons: critical issues. Environ.Health Persp. 102:348-352.
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Chapter8 MihelcicJ.R., D.R. Lueking, R.J. Mitzell, andJ.M.Stapleton (1993) Bioavailability ofsorbedandseparate-phase chemicals. Biodegradation 4:141-153. Ministry of Housing,Spatial Planning, andthe Environment (1994) Environmental quality objectives inthe Netherlands, Staatsdrukkerij,The Hague,The Netherlands Providenti M.A., C.A. Flemming, H. Lee,andJ.T.Trevors (1995) Effect ofaddition of rhamnolipid biosurfactants orrhamnolipid-producingPseudomonasaeruginosa on phenanthrene mineralization insoil slurries. FEMSMicrobiol. Ecol. 17:15-26. Providenti M.A., H. Lee,andJ.T. Trevors (1993) Selectedfactors limitingthe microbial degradation of recalcitrant compounds. J. Ind. Microbiol. 12:379-395. Rhodes D.K., G.K. Burke, N.Smith,and D.Clark (1995) Insitudieselfuel bioremediation:a case history. In: R.E. Hinchee, R.N. Miller, and P.C. Johnson (eds.) Insituaeration:air sparging, bioventing,and related processes, p.235-240, Battelle Press,Columbus, USA. RossA., C.Tremblay, and C. Boulanger (1995) Insitu remeditionof hydrocarbon contamination using aninjection-extraction process. In: R.E. Hinchee,J.A. Kittel,and H.J. Reisinger (eds.)Applied bioremediation of petroleum hydrocarbons, p.435-443, Battelle Press,Columbus, USA. Schmidt K, and H.H. Hahn (1995) reclamation ofthefine-particlefractioninhydrocarboncontaminated soils. In: R.E. Hinchee and R.S. Skeen (eds.) Biological unit processes for hazardous waste treatment, p. 161-170. Battelle Memorial Press,Columbus, USA. Schulz-Berendt V. (1994) Biological soiltreatment -stateofthe art. Proceedings ofthe2nd International Symposium on Environmental Biotechnology, Brighton,United Kingdom. ThomasA.O., andJ.N.Lester (1993) The microbial remediation offormer gasworks sites-a review. Environ.Technol. 14:1-24. VanVree H.B.R.J., L.G.C.M. Urlings,and P.Geldner (1993)Application of insitu bioremediation techniques concerning PAH:applying nitrate asanalternative oxygen sourcedemonstrated onlaboratory and pilot plantscale. In: H.J.P.Eijsackers andT. Hamers (eds.) Selected proceedings ofthefirst European conference on integrated researchfor soil andsediment protection and remediation (EUROSOL) p.653-657. KluwerAcademic Publishers, Dordrecht, The Netherlands. WilsonS.C., and K.C. Jones (1993) Bioremediation ofsoil contaminatedwith polynuclear aromatic hydrocarbons (PAHs):areview. Environ.Pollut. 81: 229-249.
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SUMMARY
Oneofthe main problems inbiological soil remediation istheslow or incomplete degradation of hydrophobic organic pollutants.The principal reasonforthis problem isthefactthatthesecompounds bindstronglytothesoilmatrixoroccur asa separate non-aqueous phase inthe soil.As mostmicrobiological processes take place inthewater phase,transport ofthe polluting compoundtothis phaseis essentialfor biodegradation tooccur.Whenthistransport isthe limitingfactor inthe biodegradation process,this istermed limiting bioavailability. Thisthesisdealswiththeeffect ofbioavailability onthe biodegradation of polycyclic aromatic hydrocarbons (PAHs). PAHsare hydrophobic organic pollutancs that areabundantly present incontaminated soilsandgive raiseto environmental concern because oftheirtoxicity and mutagenicity. Most PAHsaredegradableby microorganisms andthe important biochemicalaspects ofthe PAH-degradation have been revealed.PAHsarenevertheless considered persistent pollutants insoil,afact that isattributedtotheir limited bioavailability. Thefirst partofthe research consisted ofthe isolation ofbacteria capableof degradingthe PAHs,naphthalene, phenanthrene, andanthracene.Subsequentlya numberof isolated bacterialstrainsweregrown inbatchandcontinuous culturesto determinethe most important microbialgrowth parameters,suchasthe maximum growth rate,the Monodsaturationconstant, andthe bacterialgrowthyield. Theeffect ofbioavailability onthebiodegradation of PAHswasstudied intwo modelsystems: (i)crystalline PAHsand (ii) PAHs boundtoamatrix. Forstudying the bioavailability ofcrystalline PAHsthe results ofdissolutionand biodegradation experiments werecompared. Inthedegradation experiments itwas foundthattwo phases could beobserved during batchgrowth:an exponential growth phase,followed by alineargrowth phase, inwhich biomassformationwas limited bythe availability ofthe PAHs. Byusing amodelinwhich Monod kineticsfor bacterialgrowthwere coupledtodissolution kineticsforsubstrate availability, itwas shownthatthe observed degradation rateswere matched bythe ratesofdissolution ofthe PAHstothe aqueous phase.Therefore itwasconcluded that inthis system only aqueous phase PAHswere availablefor bacterial uptake andthatthe bioavailability ofthe PAHswas notdirectly stimulated bythe presence ofthe microorganisms. Withmatrix-bound PAHsdesorption and biodegradation experimentswere conducted.Thefirst matrices studiedwerethesynthetic porous resinsXAD-4and
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Summary XAD-7.Thedesorption ofnaphthalenefromthese materialswasstudied inbatch andcontinuous desorption experiments.The resultsfromtheseexperiments could bedescribed usingatwo-compartment modelinwhichthe matrix isdivided ina fractionwithshallow poresand onewithdeep pores. Inbiodegradation experiments withnaphthalene-loaded resinsthesametypeofbatch-growth kineticswas observed asdescribed aboveforcrystallinesubstrates:exponentialgrowth,followed bya phaseinwhichsubstrateavailability limitsthedegradation rate. Bycomparingthe resultsofthedesorption experiments andthe biodegradation experiments itwas shownthatthe biodegradation proceededfasterthan could beexplained by desorption alone.Therefore itwasconcludedthatthe bacteria hadapositive effect onthebioavailability ofnaphthalene thatwasadsorbed ontothe resins.This effect wasnotcaused bythe presence ofbacterialexcretion products. Incontrasttothis itwasfoundthatthe biodegradation ofsoil-bound naphthalene andphenanthrene could beexplained bydegradation of PAHs present inthe aqueous bulk phaseonly.Thus,the bioavailability ofsorbed PAHsdepends onthe typeofmatrixthe PAHsaresorbedonto. Thesecond part ofthisthesis dealswiththemostwidely applied solutionforthe problemof limited bioavailability: theapplication ofsurface-active agentsor surfactants. Surfactants are moleculesthat usually consist ofahydrophillic anda hydrophobic part. Duetothisthey haveatendency toconcentrate atsurfaces and interfaces andtoform new interfaces.Thereareseveraldifferentways bywhich surfactants may increasethe bioavailability of hydrophobic compounds insoil: - solubilization inthe aqueous phase bythepresence ofmicelles,aggregates of20200surfactant moleculeswithahydrophobic interior; - emulsificationof liquid hydrocarbons inthewaterphase; - facilitatedtransport, atermthat covers several processes, suchas mobilisation of pollutant present insoilporesor interaction pollutantwithsingle surfactant molecules. Surfactants mayalso haveanegative effect onpollutant bioavailability, for instance bythetoxic effect or preferential degradation ofthesurfactant, orby interferencewith the naturalinteractions among microorganisms and pollutant. Theeffect ofseveral nonionic surfactants onthe bioavailability of PAHswas studied inthesame modelsystems asdescribed above:crystalline PAHsandPAHs sorbedontoamatrix. Dissolution experimentswith crystalline naphthalene and phenanthrene showed thatthe presenceofsurfactants causedan increase intheapparentsolubility andin
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Summary themaximum dissolution rateofthese PAHs.Both phenomena haveaneffect onthe bioavailability of PAHs.Although itwasfoundthatmicellar PAHswere notreadily availablefor uptake bythe bacteria,thetransport of PAHsfromthe micelles is sufficiently fasttoallow almost complete exponentialgrowthonsolubilized PAHs. Theeffect onthe maximumdissolution rate isprobably more important becausethis isthemost relevantfactor underbioavailability-limitingconditions.Addition of surfactanttoculturesgrowing on PAHinthedissolution-limited phase resulted inan increase inthe linear growth rate.Thisshowsthatforcrystalline PAHs surfactants can beusedto increasethe bioavailabilty Forsorbed naphthalene similar resultswerefound.Indesorption experiments it wasshownthat inthe presence ofsurfactant,thepartitioning ofnaphthalene tothe waterphase aswellasthe maximumdesorption ratewas increased.Additionof surfactants toculturesgrowing onsorbed naphthalene inthe desorption-limited phase resulted inan increase inthedegradation rate.Thisshowsthat surfactants canbeusedforenhancing the bioavailability ofsorbed PAHs. Thefirst generalconclusionfromthisthesis isthatthe bioavailability of hydrophobic pollutants insoilisacomplex matterandtherefore difficult toquantify. Inmodelsystems under laboratory conditions, however, itwas possibleto simulate theessential processes.Thisexperimentalwork revealedthe most important mechanisms that playa role inbioavailability limtations. Because ofthe large impact ofbioavailability on boththe performance ofbiologicalsoilremediation andonthe risksposed bysoilcontamination, itisessentialthat standard methods bedeveloped which provide criteriafor bioavailability. These criteria maybeusedto predictthe resultsofbiological soil remediation processes and mayformabasisforsoilquality limits inwhichthe bioavailability ofthe pollutant isconsidered. Secondly,the application ofsurfactants can beconcludedtobea promising optionforenhancingthe bioavailability ofhydrophobic pollutants. Intwo model sytemsitwasshownthat addition ofsurfactants speeded upthe biological degradation of PAHsmarkedly andsomeexplanationsforthis phenomenon have beenfound.However, toallowthe useofsurfactants asastandardtechnique in biological soil remediation,more insight intothecomplex interactions involved inthe introduction ofsurfactants intosoil is necessary.
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Summary
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SAMENVATTING
Bijdebiologische reinigingvanmetorganische verbindingen verontreinigde grand wordt eenvandegrootste problemengevormddoorde langzameen slechts gedeeltelijke afbraak van hydrofobeverontreinigingen. Debelangrijkste redenvoor ditverschijnsel ishetfeitdatdezeverbindingen insterke mate hechten aande bodemmatrix ofalseenapartefase indegrandvoorkomen. Omdatde biologische afbraakvoor hetgrootste deelindewaterfase plaatsvindt, moetende verontreinigingen eerst naardezefasewordengetransporteerd,wat inveelgevallen debeperkende factor inhet reinigingsprocesvormt. Ditverschijnselwordt limiterende biobeschikbaarheid genoemdenstaat momenteelsterk inde wetenschappelijke belangstelling. Indit proefschrift wordt heteffect van biobeschikbaarheid opde biologische afbraakvan polycyclische aromatischekoolwaterstoffen (PAKs) beschreven. PAKs zijn hydrofobe organische verbindingen welkeopgrateschaal indebodem voorkomen enbekendstaanomhuntoxischeencarcinogeneeigenschappen. Vrijwelalle PAKszijn microbiologisch afbreekbaar ende biochemische aspectenvan dezeafbraakzijnvrijgoed bekend.Desondanks staan PAKs bekendals persistente bodemverontreinigingen. Devoornaamstereden hiervoor isdeslechte biobeschikbaarheid vandezeverbindingen indebodem. Deeerste stap inhetonderzoek was het isolerenvan bacterienwelke instaat warenomde PAKs naftaleen,fenanthreen enanthraceen afte breken.Eenaantal vandegei'soleerdestammenisvervolgens inbatch-encontinue-culturesgekweekt omde belangrijkste parameters voordegroeivandezemicro-organismen opPAKs vasttestellen,zoalsde maximalegroeisnelheid,de Monodaffiniteitsconstante ende yield. Vervolgens isde relatietussen biobeschikbaarheid enbiodegradatie van PAKs onderzocht intwee modelsystemen: (i) kristallijne PAKsen (ii)aaneen matrix gebonden PAKs. Heteerste en meesteenvoudigesysteemisdeafbraakvan kristallijne PAKs. Batchgroeiopslecht oplosbareverbindingen als PAKs bestaat meestaluittweefasen:eenexponentiele groeifaseeneenfasewaarindegroei wordt gelimiteerd doorde beschikbaarheid (oplossnelheid) van hetsubstraat. Voor deafbraak van kristallijn naftaleen enfenanthreen konworden aangetoond datde afbraak evensnelverliep alsdeoplossnelheid vande kristallen. Debacterienwaren blijkbaar instaat omalleenopgelost substraat afte brekenen haddendusgeen directe invloedopdebiobeschikbaarheid van kristallijn naftaleen enfenanthreen. 169
Samenvatting Bijdeafbraakvan aaneen matrix gebonden PAKs bleekdat verschillende effecten kondenwordenwaargenomen. Deafbraakvanaansynthetische harsachtige materialen gebonden naftaleen verliep sneller dankonworden verklaard doordesorptie. Daarommoetende bacterien opeenofandere manier instaatzijn debiobeschikbaarheid van hetgebonden naftaleente beinvloeden. Integenstelling hiermeebleekdatdeafbraakvan aanverschillende standaardgronden gebonden naftaleen enfenanthreen welkonwordenverklaarddoorervan uittegaandat alleen gedesorbeerde PAKs kunnenworden afgebroken. Inallegevallenwerd gevonden datsorptieaaneenvaste matrix de biobeschikbaarheid vande PAKs reduceert, maarhetwas nietmogelijkdematevandeze reductiete voorspellen Hettweededeelvandit proefschrft behandeltdemeesttoegepaste oplossing voor biobeschikbaarheidsproblemen: hetgebruik vanoppervlakte-actieve stoffen, ookwelsurfactants genaamd. Surfactants zijn moleculen die meestaluiteen hydrofoobeneen hydrofieldeelbestaan enzichdaardoor concentreren aan oppervlakken engrensvlakken. Dezeverbindingen zijnviaeenaantal verschillende mechanismeninstaatslecht inwater oplosbareverontreinigingen inde bodemte mobiliseren: - solubilisatie indewaterfase via micellen,kleineaggregaten van surfactant moleculenwaarin hydrofobeverbinding kunnenoplossen; - emulgering vanvloeibare verontreinigingen indewaterfase; - mobilisatievanverontreinigingen indeporienvan bodemdeeltjes door verlaging vandeoppervlaktespanning. Daarnaast kunnen ook negatieve effecten optreden,bijvoorbeeld doorde toxische werking ofpreferentedegradatie vandesurfactant, ofdoorverstoring vande natuurlijke interactiestussen de micro-organismenendeverontreiniging. Hetonderzoek naar heteffect vansurfactants opde biobeschikbaarheid en biodegradatie van PAKs isuitgevoerd metdezelfdetweetypen modelsysteem als bovenbeschreven:kristallijne PAKsenaaneen matrixgebonden PAKs.Bij experimentenmet kristallijn naftaleen enfenanthreen bleekdatdeaanwezigheid van verschillende niet-ionogene surfactants leiddetoteenverhoging vanzowelde schijnbare oplosbaarheid (door micelvorming) alsde maximaleoplossnelheid van deze PAKs. Beideverschijnselen hebbeneeneffect opde biobeschikbaarheid van de PAKs.Omdatdebiobeschikbaarheid van hydrofobeverontreinigingen indegrand wordt bepaalddoortransportsnelheden, is heteffect vandesurfactants opde oplossnelheid waarschijnlijk het meest belangrijk. Hettoevoegenvansurfactants aan batchcultures dieoplos-gelimiteerdopnaftaleen offenanthreen groeiden resulteerde
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Samenvatting ineen hogeregroeisnelheid. Hettoepassenvansurfactants verhoogdedusde biobeschikbaarheid vande kristallijne PAKs. Soortgelijke effecten werdenwaargenomeninexperimentenmetaan inerte matrices geadsorbeerd naftaleen. Indeaanwezigheid vansurfactants werdbij desorptie-experimenten meernaftaleen vande matrices gedesorbeerd enwasde maximaledesorptie-snelheid hogerdan indeafwezigheid vansurfactants. Degroei opgeadsorbeerd naftaleen tijdensdedesorptie-gelimiteerdefasewerddoor het toevoegen vansurfactants gestimuleerd. Hetblijktdusdatookde biobeschikbaarheid vangeadsorbeerde PAKsvergroot kanwordendoor hettoepassen van surfactants. Deeerste algemene conclusievandit proefschrift isdatde biobeschikbaarheid van hydrofobe organische verontreinigingen inde bodemeenfenomeenisdatwordt bepaalddooreencompexgeheelvanfactorenendaarommoeilijk kwantificeerbaar is. Hetisechterwelmogelijkgebleken inmodelsystemendeverschillende deelprocessen nate bootsen engrotendeels teverklaren,waardoor meer inzichtis verkregen indeachterliggende mechanismen. Door hetgrotebelangvan biobeschikbaarheid voorzoweldebiologische reiniging vanvervuildegrand alsvoor het risicodatdoor bodemverontreinigingen wordt gevormd ishetsterk aante raden datstandaardmethodenworden ontwikkeld waarmeecriteriavoorde biobeschikbaarheid vanbodemverontreinigingen kanworden vastgesteld.Deze criteria kunnengebruiktworden bijdevoorspelling vande resultaten diemet biologische bodemreiniging behaald kunnenworden enals basisdienenvoor bodemverontreinigingsnormen waarindebiobeschikbaarheid vande verontreinigingen wordtmeegewogen. Tentweede isgeblekendat hetgebruik vansurfactants een veelbelovende manierisomdebiobeschikbaarheid van hydrofobe verontreinigingen tevergroten. In eentweetal modelsystemen isgevonden dattoepassing vansurfactants de biologische afbraak van PAKsduidelijk kanbevorderen enzijneenaantal duidelijke aanwijzingen voor deverklaring voorditfenomeen gevonden. Echter, voordat de toepassing vansurfactants alsstandaardmethode bijbiologische reiniging van verontreinigde bodem kanwordentoegepast, ismeer inzicht nodig inde complexe interacties die bijde introductievansurfactants inde bodemeen rolspelen.
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CURRICULUMVITAE
FrankVolkeringwerdop26 november 1963geborente'sGravenhage. In1982 behaaldehijhetVWO-diploma aande Rijswijkse Openbare Scholengemeenschap. Indatzelfdejaar begon hijmetdestudie Levensmiddelentechnologie aande Landbouwuniversiteit Wageningen. In 1988studeerde hijafmetals afstudeervakken Proceskunde enTechnische Microbiologie. Hijheeft stagegedaan bijTNO-MTte Zeist. In 1988ishijwerkzaamgeweest alstoegevoegd onderzoeker bijde secties Proceskunde en Industriele Microbiologie vandevakgroep Levensmiddelentechnologievande LUW.Van oktober 1989toten metaugustus 1994was hijwerkzaam als projectmedewerker bijdeafdeling Biotechnologisch Onderzoek van het LaboratoriumvanAfvalstoffen en Emissiesvan het Rijksinstituut voor Volksgezondheid en Milieuhygiene te Bilthoven.Deresultatenvan hetdaar uitgevoerde onderzoek staan beschreven indit proefschrift. Sinds 15mei 1995 ishijwerkzaamals milieutechnoloogbijMTI Milieutechnologie C.V.te Nijmegen.
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