DTIC ADA588658: Soil Column Evaluation of Factors Controlling Biodegradation of DNT in the Vadose Zone

11 Pages·0.21 MB·English

by  Defense Technical Information Center

Environ.Sci.Technol.2003,37,3382-3391
Soil Column Evaluation of Factors BecausebothDNTisomerscanbebiodegradedbyaerobic
bacteria,bioremediationofcontaminatedmediahasbeen
Controlling Biodegradation of DNT in
anareaofresearchanddevelopmentforsometime.Theuse
ofvariousex-situprocessestobiodegradeDNTinsoiland
the Vadose Zone
groundwater has been reported (3-9), but little attention
hasbeengiventoin-situprocesses(eithernaturalattenuation
oracceleratedbioremediation)despitetheabilityofspecific
JOHN D. FORTNER,† CHUNLONG ZHANG,‡
bacteriatouseboth2,4-DNTand2,6-DNTascarbon,energy,
JIM C. SPAIN,§ AND andnitrogensources(3,10-15).
JOSEPH B. HUGHES*,†
During aerobic biodegradation, DNT depletion and
DepartmentofCivilandEnvironmentalEngineering,
oxygenconsumptionarecoupledwithnitriteevolution,CO
RiceUniversity,6100MainStreet,Houston,Texas77005, 2
production,andbiomassgrowth(10-12).Dapratoetal.(16)
SchoolofNaturalandAppliedSciences,Universityof
HoustonsClearLake,Houston,Texas77058,andAirForce describedthestoichiometryoftheoverallprocess(eq1)using
a simple empirical formula for biomass composition
ResearchLaboratory/MLQR,139BarnesDrive,Suite2,
TyndallAFB,Florida32403-5323 (C5H7O2N):
C H N O +5.61O f1.63NO -+1.63H++
7 6 2 4 2 2
5.17CO +0.89H O+0.37C H O N (1)
High concentrations of 2,4-dinitrotoluene (2,4-DNT) and 2 2 5 7 2
2,6-dinitrotoluene(2,6-DNT)arepresentinvadosezonesoils
On the basis of this stoichiometry and observations in
at many facilities where explosives manufacturing has
laboratorystudies,itwouldseemasifoxygenandappropriate
taken place. Both DNT isomers can be biodegraded under
microbeswouldbesufficientfortheonsetofDNTdegrada-
aerobic conditions, but rates of intrinsic biodegradation
tion.Bothisomers,however,oftendisplayanunexpectedly
observedinvadosezonesoilsarenotappreciable.Studies
highdegreeofrecalcitranceinaerobicenvironments.This
presented herein demonstrate that nutrient limitations isparticularlytrueincontaminatedvadosezonesoilswhere
control the onset of rapid 2,4-DNT biodegradation in such muchoftheexistingDNTcontaminationresides.
soils. In column studies conducted at field capacity, TheobservedpersistenceofDNTisomersinvadosezone
highlevelsof2,4-DNTbiodegradationwererapidlystimulated soilsraisesquestionsregardingthefactor(s)thatlimitnatural
by the addition of a complete mineral medium but not by biodegradation processes, the extent to which natural
bicarbonate-buffered distilled deionized water or by bioattenuationmaybeloweringcontaminantlevels,andthe
phosphate-amended tap water. Biodegradation of 2,6-DNT abilitytoinduceactivityin-situ.Whereastheavailabilityof
was not observed under any conditions. Microcosm carbon or nitrogen is not a concern, several other factors
mayresultintherecalcitrancenatureofDNTinvadosezone
studiesusingaDNT-degradingculturefromcolumneffluent
soils.FirstisthepotentialabsenceofDNT-degradingstrains;
suggest that, after the onset of 2,4-DNT degradation,
however,wehavebeenabletocultureDNTdegradersfrom
nitrite evolution will eventually control the extent of
all historically contaminated sites examined to date (17).
degradation achieved by two mechanisms. First, high
SecondisthepotentialforhighconcentrationsofDNTtobe
levels of nitrite (40 mM) were found to strongly inhibit 2,4- inhibitory or toxic to bacteria. Previous studies in ex-situ
DNT degradation. Second, nitrite production reduces the reactorsdemonstratedthathighconcentrationsof2,4-DNT
solutionpH,andatpHlevelsbelow6.0,2,4-DNTdegradation willinhibit2,6-DNTdegradationbutnot2,4-DNTdegrada-
slows rapidly. Under conditions evaluated in laboratory- tion. Other possible factors that may contribute to the
scalestudies,2,4-DNTbiodegradationenhancedtherateof recalcitranceofDNTincludedemandsforphosphorus(15,
contaminant loss from the vadose zone by a factor of 10 16, 18) (or other essential nutrients), low pH from nitrite
productionduringDNTbiodegradation(4,15,18),andthe
when compared to the washout due to leaching.
accumulationofinhibitorynitritelevelswhenthereplenish-
mentofwaterdoesnotallowforsignificantnitritedilution
(17).
Introduction
Inthispaper,wepresenttheresultsofalaboratory-scale
evaluationoffactorsthatcontroltheaerobicbiodegradation
Dinitrotoluenes (DNT) are formed in the second step of
ofDNTinasimulatedvadosezone.Agedvadosezonesoils
toluenenitrationduring2,4,6-trinitrotoluene(TNT)synthesis.
containing a high concentration of 2,4-DNT and a low
Inparticular,twoDNTisomers[2,4-dinitrotoluene(2,4-DNT)
concentrationof2,6-DNTwereobtainedfromBadgerArmy
and 2,6-dinitrotoluene (2,6-DNT)] are formed at yields of
AmmunitionPlant(BAAP)inBaraboo,WI.Initialexperiments
76% and 19%, respectively (a small percentage of other
were conducted in column systems to assess the factors
isomers are produced) (1). Poor handling and disposal
requiredtoinitiaterapidbiodegradationofDNT.Additional
practicesassociatedwiththeproductionofTNThaveledto
column studies were conducted to investigate how the
substantial DNT contamination problems at ammunition
operationalparametersofinterestinvadosezonebioreme-
productionandhandlingfacilitiesworldwide(2).2,4-DNT
diation,includingaerationfrequencyandwaterrecycling,
and2,6-DNTarebothlistedasprioritypollutantsbytheU.S.
willimpactthedegradationactivity.Aseriesofrespirometer
EPA, thus requiring remediation at contaminated sites.
studieswereconductedusingculturesobtainedfromcolumn
systems to further investigate the factors that control the
*Correspondingauthorphone: [phone];fax: [phone];
extentofdegradationachievablebeforenitriteaccumulation,
e-mail: [email protected].
pHdrop,ornutrientlimitationsslowdegradationactivity.
†RiceUniversity.
‡UniversityofHoustonsClearLake. Results from these studies suggest that, at field capacity,
§AirForceResearchLaboratory/MLQR. nutrientavailabilityisthecontrollingfactorintheonsetof
33829ENVIRONMENTALSCIENCE&TECHNOLOGY/VOL.37,NO.15,2003 10.1021/es021066sCCC:$25.00 ª 2003AmericanChemicalSociety
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1. REPORT DATE 3. DATES COVERED
2003 2. REPORT TYPE 00-00-2003 to 00-00-2003
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Soil Column Evaluation of Factors Controlling Biodegradation of DNT in
5b. GRANT NUMBER
the Vadose Zone
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Air Force Research Laboratory/MLQR,139 Barnes Drive, Suite REPORT NUMBER
2,Tyndall AFB,FL,32403
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
11. SPONSOR/MONITOR’S REPORT
NUMBER(S)
12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release; distribution unlimited
13. SUPPLEMENTARY NOTES
Environ. Sci. Technol. 2003, 37, 3382-3391,NO. 15, 2003
14. ABSTRACT

15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
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a. REPORT b. ABSTRACT c. THIS PAGE Same as 11
unclassified unclassified unclassified Report (SAR)
Standard Form 298 (Rev. 8-98)
Prescribed by ANSI Std Z39-18
Column Studies. BAAP soil was placed into custom-
TABLE 1.BAAP Soil Characteristics fabricatedplexiglasscolumns(10.2cmi.d.(cid:2)30.5cm)de-
parameter value method signedtosupportthesoilabovethecolumnbasetoallow
forsamplecollectionandtoprovideanevenflowofairinto
PhysicalParameters thebottomofthesoilpack.Tosupportthesoil,wiremesh
pH 7.4
cloth(0.145cm)wasusedandheldinplaceabovethebottom
satpastemoisture(%) 17.7
ofthecolumn(3.8cm)byfoursmallplexiglassrods(0.64cm
moisture(%) 0.4
diameter).Alayerofgravel(7.6cm)wasplacedonthewire
SPEC(m¿/cm) 2.0
texture(%) 3.4.3.5a meshtopreventsoilwashout.Soil(2kg)wasplacedinthe
sand 90.0 columns at even intervals (5 intervals, 400 g each) with
silt 8.0 consistentpackingtominimizevariationbetweencolumns.
clay 2.0 Alayerofgravel(7.6cm)wasthenplacedontopofthepacking
soilclass sand tostabilizethesoil.Samplingportswerefittedwitha0.64-
bulkdensity 1.99g/cm3 14-3b cmballvalve(Swagelok,NiagaraFalls,ON,Canada)centrally
moistureretention(0.33bar) 4.2%
placedatthebottomofthecolumntodraintheeffluent.Air
PlantAvailableNutrients(mg/kg) inletswereplacedapproximately1.9cmfromthebottomof
ammonia-N 3.0 350.3c thecolumnandfittedwitha0.95-cmquick-connectvalve
nitrate-N 4.0 353.2c (Swagelok,NiagaraFalls,ON,Canada).
nitrite-N <1.0 353.2c
Aqueousmedium(50mL)wasaddeddailytothetopof
o-phosphate 30.0 365.4c
copper 2.1 200.7c thecolumnandallowedtoinfiltrateintothesoils.Effluent
iron 8.1 200.7c wascollectedfromthebottomofthecolumnsasiteluted.
manganese 6.4 200.7c Thenominalhydraulicretentiontime(basedontheinfiltra-
zinc 2.8 200.7c tionrateandsoilfieldcapacity)wasapproximately5d.All
molybdenum <0.05 200.7c columnstudieswereoperatedwiththewatercontentofthe
soil at field capacity (0.15 g of water/g of soil) and under
aEPA600/4-79-020.bBlake,G.R.;Hartage,K.H.InMethodsofSoil
AnalysisPart1:PhysicalandMineralogicalMethods.Klute,A.,Ed.; aerobic conditions. Humidified air was delivered to the
AmericanSocietyofAgronomy: Madison,WI,1986;pp363-375.cEPA columnbasewithaperistalticpumpandallowedtoexitto
600/2-78-054. theatmosphereatthetop.
Mediumcompositionswerechangedperiodicallyinthe
activityandthattheextentofdegradationthatfollowswill column experiments but can be grouped into three cate-
becontrolledbynitriteaccumulation. gories.Firstwasdistilled,deionized(DI)waterwithbicar-
bonatebuffer.Secondwastapwaterwithbicarbonatebuf-
Materials and Methods
ferandpolyphosphate.Lastwasamineralmediumcontain-
Chemicals.2,4-Dinitrotoluene(97%)and2,6-dinitrotoluene ing phosphate buffer and in some cases additional buf-
(98%) were obtained from Aldrich (Milwaukee, WI). The fering capacity as bicarbonate. Mineral medium was pre-
following reagent grade chemicals were used as media pared in DI water with the following: MgSO4(cid:226)7H2O (50
constituents: CaSO4(cid:226)5H2O,MgSO4(cid:226)7H2O,FeSO4(cid:226)7H2O,CaCl2(cid:226) mg/L), FeSO4(cid:226)7H2O (3 mg/L), CaCl2(cid:226)7H2O (100 mg/L),
7H2O,NaCl,ZnSO4(cid:226)7H2O,Na2MoO4(cid:226)2H2O,andH3BO3(Fisher NaCl (500 mg/L), H3BO3 (100 mg/L), CaSO4(cid:226)5H2O (50
Scientific,J.T.BakerChemicalCo.,andMallinckrodtInc.). mg/L), ZnSO4(cid:226)7H2O (50 mg/L), Na2MoO4(cid:226)2H2O (50 mg/L)
All reagent grade buffers for media, KHPO, KHPO, (11),andvariablebufferstrength.Table2lists,inchrono-
2 4 2 4
NaHCO,andNaPO wereobtainedfromFisherScientific. logical order, the media composition used in all column
3 5 3 10
SoilPreparationandAnalysis.AgedDNT-ladensoilwas experiments.
collectedpreviouslyfromBAAP(Baraboo,WI)atanaverage An initial column study was conducted to determine
boringdepthof30ftandhomogenizedasdescribedbyZhang whetheritwaspossibletostimulateaerobicDNTdegradation
et al. (15). Initial DNT concentrations in the soil were throughthedailyadditionofmedium(50mL)andair(532
determinedasdescribedbyZhangetal.(15).Soilcontained mLat26.6mL/minfor20min).Inthis175-dstudy,duplicate
8700((420)mgof2,4-DNTand148((14)mgof2,6-DNT/ columnsservedastheexperimentalsystemswhileduplicate
kgofsoil(drybasis).Thiscompositesoilwasusedforallsoil abioticcontrolcolumnswereoperatedwithsodiumazide
columnexperiments.Additionalsoilanalysiswasperformed added to the medium. Subsequent column tests involved
by Soil Analytical Services Inc. (College Station, TX). The threesetsofcolumns,operatedinduplicate.Thethreesets
analyticalmethodsusedforsoilcharacterizationandcor- ofcolumnswereemployedtoassessdifferentstrategiesto
respondingresultsarereportedinTable1. sustainandenhanceDNTbioremediationoftheBAAPsoil.
AnalyticalMethods.DNTanalysiswasperformedona The first of the three column sets served as a baseline
WatersMillenniumIIHPLCsystemequippedwithadiode (repeating the operation of initial experimental systems),
arraydetectorwithcompoundsquantifiedatA .Initially, beingfedmineralmedium(50mL)andair(532mLat26.6
230
separationwasachievedatambienttemperaturewithaNova- mL/min for 20 min) on a daily basis. The second set of
PakC 60Å4-(cid:237)msilica-basedHPLCcolumn(3.9(cid:2)150mm, columns(enhancedaeration)wasoperatedsimilarly,except
18
Waters, USA) with a mobile phase of 2-propanol/water thatairwasaddedcontinuously(24h/dat27mL/min).The
(19:81) at 1 mL/min. Subsequently, HPLC was performed thirdsetofcolumnswasoperatedinarecyclemode.The
withaHypercarbporousgraphitecolumn(5(cid:237)m(cid:2)150mm, recyclecolumnswereaeratedascarriedoutinthebaseline
Thermohypersil,U.K.)withamobilephaseofacetonitrile/ columns (532 mL at 26.6 mL/min for 20 min), but the
water(90:10)containingtrifluoroaceticacid(0.55mL/L)(3). compositionofthemediumaddedwasprimarilythatofthe
Nitrite analysis was performed using a colorimetric effluent from the previous day. Of the roughly 50 mL of
method,couplingdiazotizedsulfanilamidewithN-(1-naph- effluent taken each day, 5 mL was retained for various
thyl)ethylenediamineproducinganazodye,andmeasured analyses;therest((cid:24)45mL)wasstoredat4(cid:176) Candreusedthe
usingaspectrophotometer(TurnerSP-830;Dubuque,IA)at followingdayastheinfluent.Beforethestoredliquidwas
543nm(19).Soilnitriteextractionwasperformedwith2M addedtothecolumninfluent,thepHwasadjustedto8.3
KClsolutiondisplacingsoil-boundNO-intosolution(20). withNaOH,andfreshmediumwasadded((cid:24)5mL)tobring
2
Nitrate analysis was performed with ion chromatography the final volume to 50 mL. The three-column system was
(DionexIC20,Sunnyvale,CA)(21). operatedcontinuouslyfor90d.
VOL.37,NO.15,2003/ENVIRONMENTALSCIENCE&TECHNOLOGY93383
TABLE 2.Soil Column Studies: Influent Composition Time Line TABLE 3.Respirometer Studies: Design Matrix
Julian parameter rangetested
columna days mediumcomposition
baseline
A,B1 0-45 DIwater carbon, 10mM2,4-DNTasTOC
10mMbuffer(NaHCO ) nitrogen
3
pH7.5 source
46-56 tapwater medium mineralmedium(100mLperflask)
20mMbuffer(Na5P3O10and buffer 20mM(10mMK2HPO4,10mM
NaHCO3,1:1) KH2PO4)
pH7.5 pH 8.0,maintainedwithadditionofdilute
57-69 tapwater NaOH
15mMbuffer(Na5P3O10and bacteria 2,4-DNTdegradingculture,enriched
NaHCO3,2:1) fromcolumneffluent(approximately
pH7.5 80mgofwashedbiomass
70-85 mineralmedium addedin1mLofmedium)
10mMbuffer(K2HPO4and pH baselineconditionswithinitialpH
KH2PO4,1:1) rangingfrom5.5to9.0atan
pH8.3 incrementof0.5;pHwasnot
86-138 mineralmedium maintainedoverthecourse
20mMbuffer(K2HPO4and ofthestudy
KH2PO4,1:1) media baselineconditionswithmedium
pH8.3 strengthat1(cid:2),2(cid:2),and4(cid:2)
139-171 mineralmedium nitrite baselineconditionswithinitialnitrite
40mMbuffer(K2HPO4,KH2PO4, concentrationsof0,10,15,20,
andNaHCO3,1:1:2) and40mM
pH8.3 bufferstrength baselineconditions(20mMphosphate
B2,O,R 0-4 mineralmedium buffer)withadditionalbuffer
10mMbuffer(K2HPO4and strengthof0,20,40,60,and80mM
KH2PO4,1:1) NaHCO3;pHwasnotmaintainedover
pH8.3 thecourseofthestudy
5-61 mineralmedium phosphorus baselineconditionswithphosphate(1:1
20mMbuffer(K2HPO4and K2HPO4andKH2PO4)
KH2PO4,1:1) concentrationsvariedat0,0.4,0.8,
pH8.3 1.6,and8.0mM;initialconcentration
61-94 mineralmedium of2,4-DNTwas5mM
40mMbuffer(K HPO ,KH PO ,
2 4 2 4
andNaHCO 1:1:2)
3
pH8.3 wereestablishedwithcolumneffluentandmonitoreddaily
aColumnid: A,abioticcontrol;B1,baseline1;B2,baseline2;O, forDNTdisappearanceandnitriteproduction.Shakeflask
enhancedaeration;R,recycle. studieswereconductedusingonlycolumneffluent((cid:24)40mL)
directlytransferredtoserumvials(120mL),stirredcontinu-
Biodegradationinthecolumnswasevaluatedbytheuse ously,fittedwithfoamstopperstopreventevaporation,and
ofseveralindicators.FirstwasthedepletionofeffluentDNT analyzed for DNT and corresponding end products for a
concentrationsincomparisontoabioticcontrols.Second, periodof9-10d.ThepHwasadjustedto8.0withNaOHas
nitriteproductionwasquantified.Last,shakeflaskstudies needed.
FIGURE1. Initialsoilcolumnstudies.Averageeffluentnitriteconcentrations: baseline1(O)andabioticcontrol(b).
33849ENVIRONMENTALSCIENCE&TECHNOLOGY/VOL.37,NO.15,2003
FIGURE2. Columneffluentshakeflaskstudies.(A)Results(averaged)fromabioticcontroleffluent.(B)Results(averaged)frombiologically
activecolumn(baseline1)effluent.Forbothstudies,(b)2,4-DNTconcentration,(O)2,6-DNTconcentration,and(2)aschangeinnitrite
concentration.
Toensureaccuratequantificationofnitrite,shakeflask 20mMphosphatebufferandDNTastheonlycarbonand
studieswereconductedtodetermineifbiologicalnitrification, nitrogensourceatconcentrationsroughlyequaltothosein
oxidizing nitrite to nitrate, would occur with the nitrite columneffluents(1000(cid:237)M2,4-DNTand400(cid:237)M2,6-DNT).
evolved from DNT biodegradation as shown in previous Theculturewasstirredcontinuouslyatroomtemperature,
studies(5).Theprocedurewasidenticaltothatdescribed andnitriteandpHweremeasureddaily.ThepHwasadjusted
above.Nitriteandnitrateanalyseswereperformedoverthe each day to 7.5-8.0. When the release of nitrite from the
courseof10d. biodegradationofDNTneared70-80%oftheoretical,10%
Withtheexceptionofabioticcontrols,afterallcolumns ofthesuspensionwastransferredtofreshmedium.
hadexhibitedapproximately90dofactive2,4-DNTdegra- RespirometerStudies.2,4-DNTbiodegradationexperi-
dation,allsystemsweredestructivelysampledbycontinuous mentswereconductedwiththeDNT-enrichmentculturein
coringthroughthesoil.Azerocontaminationsoilsampler a10-chamberO/CO Micro-Oxymaxrespirometer(Colum-
2 2
(2.06cmdiameter;Cole-PalmerP-99025-40)wasmanually busInstrumentsCorp.,Columbus,OH)atroomtemperature.
driven through the soil, and the resulting soil cores were Aspreviouslydescribed,bothoxygenuptake(5,10-12)and
extrudedintoplasticsleeves.Thecoreswerethenfrozen(-4 carbondioxideproduction(3,22)wereusedtomeasureDNT
(cid:176) C)andcutintoquartersafteranygravelfromcolumnpacking biodegradationrates.Aconfoundingfactorinsomeinstances
was discarded. These subsamples were analyzed for pore whererapidDNTbiodegradationtookplacewastheupper
waternitritelevels. quantificationlimitforCO evolutionbythedetector(i.e.,
2
DNTEnrichmentCulture.ADNT-degradingmixedcul- CO levelsexceededthedynamicrange)andCO production
2 2
turewasdevelopedfromtheoriginalbioticcolumneffluent. rates were underestimated in those instances. No such
Asinoculum,approximately50mLofeffluentwastransferred difficultywasencounteredduringO uptakemeasurements.
2
intoaflask(2L)containingmineralmedium(900mL)with Inallrespirometerexperiments,2,4-DNTcomprisedtheonly
VOL.37,NO.15,2003/ENVIRONMENTALSCIENCE&TECHNOLOGY93385
FIGURE3. Initialsoilcolumnstudies.AverageeffluentDNTconcentrations: Baseline1,(1)2,4-DNTand(3)2,6-DNT.Abioticcontrol,
(b)2,4-DNTand(O)2,6-DNT.
organiccarbonandnitrogensource.Foreachexperiment, production,thepHofeffluentbegantodecrease.Afternitrite
inoculumwastakenfromtheenrichmentculturedescribed levelshadstabilized,thepHofeffluentmediumwastypically
above.Preparationoftheinoculuminvolvedaliquotsofthe 6.75-7.25.Appreciablenitrateproductionwasnotobserved
enrichment culture centrifuged (4 (cid:176) C, 10000g for 10 min) duringthestudy.
withthesupernatantbeingcarefullypouredoffleavingonly Asnitriteconcentrationsstabilized,itwasnecessaryto
thebacterialpellet.Thepelletwasthenresuspended(through confirmthatnitriteproductionwasanaccurateindicatorof
vigorous shaking) in DNT-free mineral medium and cen- the DNT degradation activity in the column systems.
trifugedagain.Thisprocesswasrepeatedforatotalofthree Therefore, shake flask studies using only column effluent
washeswithDNT-freemedia.Inaddition,aminimalvolume wereinitiatedasdescribedabove.Resultsofthesestudies
(1mL)ofsuspendedinoculumwasusedforeachrespirometer (Figure 2) show that 2,4-DNT was immediately depleted
flaskcontaining100mLofmineralmedium.Respirometer within 24 h to levels below the detection limit with a
experimentsevaluatedtheeffectsofpH,mediumconcen- correspondingreleaseofnitrite(approximately1.7molof
trations,initialnitriteconcentration,phosphorusconcentra- nitritereleased/molofDNT).Undernocircumstanceswas
tion,andbufferstrengthontherateandextentof2,4-DNT 2,6-DNT depleted. Identical shake flask studies were con-
degradation. No carbon dioxide production or oxygen ductedperiodicallyasdescribedthroughouttheremainder
consumptionwasseeninarespirometercontrolcontaining of the experimental period with similar results (data not
mineralmedia,2,4-DNT,butnobacterialinoculum.Table shown).Also,nonitrification(conversionofnitritetonitrate)
3describestheexperimentalprotocolsanddetailsthematrix waseverobservedinthesestudies.
designofvariedparameters. Theeffluent2,4-DNTconcentrationsinexperimentaland
control systems were similar before the addition of the
Results and Discussion
mineralmedium(Figure3).Asconcentrationsofnitritebegan
ColumnStudies.Preliminarycolumnstudieswereconducted to increase in the active systems, effluent 2,4-DNT levels
to evaluate the stimulation of DNT biodegradation by dropped. No such drop occurred in the controls. Effluent
mediumadditionandaeration.Nitriteproductionwasused 2,6-DNTconcentrationsalsodroppedafterapproximately
asanindicatorofDNTbiodegradation(3,5,10,11,23)(Figure 60dofoperation,butthedecreaseinconcentrationcould
1).Theinitialmediumwasbicarbonatebuffer.Overthefirst notbeduetobiologicalactivitybecausesimilardecreases
10dofoperation,therewasanelevatedbutdecreasinglevel were observed in both active and control systems. In
ofnitriteintheeffluentfromexperimentalsystems.After20 accordance,periodiceffluentshakeflaskstudiesshowedno
dofoperation,however,nitritelevelsinthebioticcolumns lossof2,6-DNT.
weresimilartothoseinthecontrolcolumns.Onthebasis Subsequent column studies were conducted to test
of phosphate limitations cited in previous studies (15, 16, whether high levels of 2,4-DNT biodegradation could be
18),thefeedmediumwaschangedonday45substituting induced and sustained by the use of a mineral medium
phosphateforcarbonateasabufferandalsoasanutrient. throughout the experimental period. We also sought to
Therewasnosignificantincreaseinnitriteproduction. evaluatehowchangesincolumnoperationmightinfluence
Onday70,theinfluentwaschangedtoacompletemineral the extent of degradation achieved. 2,4-DNT degradation
medium,whichresultedintheimmediateelevationofnitrite began immediately in all systems (Figure 4). Nitrite con-
levels in the column effluents. After stabilizing, nitrite centrationsincreasedsteadilyforapproximatelythefirst20
concentrationsaveraged8500(cid:237)M((1460)fortheremainder d and then stabilized. Nitrite concentrations were slightly
of the study. Commensurate with the onset of nitrite higherincolumnsthatwerecontinuouslyaeratedoroperated
33869ENVIRONMENTALSCIENCE&TECHNOLOGY/VOL.37,NO.15,2003
FIGURE4. Columnstudiesinvestigatingparameterdesign.Averageeffluentnitriteconcentrations: baseline2(b),extendedaeration(O),
andrecycle(1).
in recycle. Interestingly, the lack of continued nitrite ac- TABLE 4.Terminal Soil Column Dissections: Pore Water
cumulationintherecyclecolumnsindicatesalossofrapid Nitrite Concentrationsa
DNT biodegradation after an initial period of biological
activity.Shakeflaskstudiesconducted,asdescribedprevi- nitrite(pore
column section water)((cid:237)M)
ously,confirmed2,4-DNTbiodegradationwithno2,6-DNT
biodegradationobserved. abioticcontrolb 1 0.27
The pH in all active columns dropped from 8.3 in the 2 0.34
influenttoapproximately6.75-7.25intheeffluentdueto 3 0.34
nitrite production. After bicarbonate buffer was added to 4 0.6
the medium (day 61), the average nitrite concentration baseline1b 1 179
2 763
increased slightly in the baseline columns. In the recycle
3 819
columnsabriefperiodofincreasednitriteproductionwas
4 2440
followedbyadeclinetoalowlevel. baseline2c 1 320
Atthecompletionofthecolumnstudies,soilcoreswere 2 1760
analyzed for pore water nitrite concentrations to better 3 4260
understandhow2,4-DNTdegradationactivitywasdistributed 4 8250
throughoutthelengthofcolumns.Incolumnswhereactive enhancedaerationc 1 530
2,4-DNTdegradationwasoccurring,nitriteconcentrations 2 1630
3 3340
increasedwiththeincreaseincolumndepth.Theseresults
4 5600
indicatethattherewasactivitythroughoutthelengthofthe
recyclec 1 39900
columnandsuggestthatifcolumnshadalongerresidence 2 40800
time,additionalactivitycouldbeexpected.Conversely,in 3 36300
abioticcontrolsandrecyclesystems,nitriteconcentrations 4 34900
remained nearly constant throughout the length of the
aSectionstakenin3.8-cmincrements.Spatialdistributionisshown
columns (Table 4). It is important to note that high assections1-4,withsection1representingthetopofthecolumn.
concentrationofnitriteintherecyclecolumns(Table4)does bCorestakenafter175dofoperationwiththelast90dbeingfedmineral
not imply continued activity of DNT biodegradation. The media.cCorestakenafter90dofoperation,beingfedmineralmedia
pore water nitrite concentration in the recycle columns forthedurationofthestudy.
approachedthesumofthetotalnitriteproductionthrough-
outthedurationofstudy.Asindicatedearlierinthispaper, 2,4-DNT mass that had been degraded based on the
90%ofthecolumneffluentintherecyclemodewasretained stoichiometrypresentedabovewherethedegradationof1
andreusedinthefollowingdayastheinfluentdilutedonly molofDNTyields1.63molofnitrite.Ascanbeseenfrom
bythe10%addition(vol/vol)offreshmineralmedium. Table 5, the measured 2,6-DNT washout was high for all
EstimatesofDNTmassremovalfromthecolumnsystems columnsotherthanthoseoperatedinrecycle,as90%2,6-
wereconductedasdescribedbyOrtega-Calvoetal.(23)using DNTintheeffluentfromthosecolumnswasreintroduced
effluentDNTconcentrationsandnitriteanalysis.Toestimate asinfluent.Thepercentageof2,4-DNTthatwashedoutwas
the washout of both 2,4-DNT and 2,6-DNT, effluent con- much less than that of 2,6-DNT due to the higher initial
centrationsweresummedovertheexperimentalperiodto concentrations of 2,4-DNT. Biodegradation (22-30%) of
provide the cumulative mass of washout that took place initial2,4-DNTmass(Table5)indicatesthatthebiologically
(whenasamplewasnottakenonagivenday,theconcen- activesystemshavethepotentialtorapidlyreducethemass
trationwasextrapolatedusingacentraldifferencingmethod). of2,4-DNTpresentinavadosezonebioremediationsystem.
Similarly,nitriteconcentrationswereusedtocalculatethe In the columns fed mineral medium without recycle, 2,4-
VOL.37,NO.15,2003/ENVIRONMENTALSCIENCE&TECHNOLOGY93387
TABLE 5.Average Column DNT Mass Loss as a Percentage of Initial Concentrationa
%washout %biodegradation total(%washout+%biodegradation)
column 2,4-DNT 2,6-DNT 2,4-DNT 2,6-DNTb 2,4-DNT 2,6-DNT
abioticcontrol 5.7 81 0.11 5.8 81
baseline1 3.4 82 19 22 82
baseline2 1.8 91 25 27 91
enhancedaeration 1.9 85 28 30 85
recycle 0.21 13 5.1 5.3 13
aNumbersaretheaverageoftheduplicatecolumns.b2,6-DNTwasnotfoundtodegradeunderanycircumstances,thustheonlymasswas
fromwashout.
DNTdepletionfrombiodegradationwasapproximately10 onlylossof2,6-DNTwasthroughdissolutionandwashout.
timesthatofdissolutionandwashout. Intherecyclecolumns,2,6-DNTconcentrationsremained
Several common conclusions can be drawn from the highbecausetheremovalofeffluentwasonly10%ofthat
comparisonofthetwosetsofcolumnstudies.First,thesoils inothersystems.Similarobservationsregardingtheinability
were quickly depleted of a required nutrient(s) because toactivelystimulate2,6-DNTdegradingactivityinBAAPsoil
phosphate addition alone was not sufficient to support hasbeenreportedbyNishinoandSpain(18).
activity. In previous studies investigating rapid DNT bio- Athirdconclusionfromthesestudies(enhancedaeration,
degradation, 2,4-DNT biodegradation rates were clearly Table5)isthatoxygenavailabilitydidnotstronglyaffectthe
subjecttophosphatelimitations(16,18).Similardepletion levelofbiodegradationachievedwiththewatercontentat
ofphosphateorothernutrientmayhaveoccurredincolumns fieldcapacityandwithperiodicaeration.Highlevelsofnitrite
afteractivitywasestablished,butitwasnotthebasisoflimited productionwereobservedinallsystems,andthecontinuous
activitypriortotheadditionofthemineralmedium.High aerationofcolumnsledtoonlysmallincreasesinactivity.
ratesof2,4-DNTbiodegradationwereinitiatedbyproviding Itcouldnotbeestablishedfromthestudieswhatfactor-
moistureandmineralnutrients.Aftertheonsetofactivity, (s)control2,4-DNTbiodegradationaftertheonsetofactivity.
theintermittentadditionofmineralmediumthenbecame Insystemsnotoperatedinrecycle,alongerresidencetime
criticaltosustainactivityinthecolumnsasnutrientcon- should result in additional production of nitrite that may
centrationsweremaintainedandnitritewasflushedfrom resultineitherdirectinhibitionorcauseapHdrop,which
interstitialporewater. can also become inhibitory. If not inhibited by nitrite
A second conclusion from these studies was that 2,6- accumulation,thedevelopmentofnutrientlimitationswould
DNTwasnotreadilybiodegraded.Whereasconcentrations be expected to develop as has been observed in ex-situ
ofbothisomersdecreasedintheeffluentsofcolumns,the systems(15).Inrecyclecolumns,withlongeffectivecontact
decreaseof2,4-DNTinthecolumneffluentwaslikelythe times,eithertheslowreplenishmentofmedium,thehigh
resultofboththedepletionof2,4-DNTinthesoilandthe nitrite concentrations, or both appear to have caused the
continueddegradationinthecolumndrainagesystem(i.e., inabilitytosustaindegradationactivity.Becauseofthelimited
gravelandsamplereservoir).Shakeflaskstudieswitheffluent sizeofthecolumnsystems,sucheffectscouldnotbeassessed
samplesrevealedacontinuedrapiddegradationof2,4-DNT, directly, thus a series of batch assays were conducted to
whichisconsistentwiththeabovehypothesis.Decreasesin evaluatehowthesevariousfactorswouldeventuallyaffect
2,6-DNT effluent concentrations could not be explained theextentofdegradationwithlongercontacttimes.
similarly.Thefactthateffluentshakeflaskstudiesdidnot Batch Respirometer Studies. The first set of batch
demonstrate2,6-DNTdegradationactivitysuggeststhatthe respirometerstudiesinvestigatedthedirecteffectofnitrite
FIGURE5. Effectofinitialnitriteconcentrationon2,4-DNTmineralizationasmeasuredbyoxygenconsumption(5.6molofO consumed
2
duringthemineralizationof1molof2,4-DNT(16)).
33889ENVIRONMENTALSCIENCE&TECHNOLOGY/VOL.37,NO.15,2003
FIGURE6. EffectofinitialpHon2,4-DNTmineralizationasmeasuredbyoxygenconsumption(5.6molofO consumedduringthemineralization
2
of1molof2,4-DNT(16)).
FIGURE7. Effectofbufferingstrengthon2,4-DNTmineralizationasmeasuredbyoxygenconsumption(5.6molofO consumedduring
2
themineralizationof1molof2,4-DNT(16)).
accumulation due to 2,4-DNT biodegradation and the limitthebiodegradationof2,4-DNT.MediawithinitialpH
indirecteffectofpHdropfromnitriterelease.Theimpact valuesof6.0and6.5supportedimmediaterapidinitialrates
ofnitriteaccumulationwasmeasuredusingoxygenuptake ofbiodegradationthatdecreasedsharplyasthepHfellto
andproductionofcarbondioxidefromtheaerobicbiodeg- inhibitory levels. When the initial pH was 7.5 and above,
radationof2,4-DNTatvariousinitialnitritelevelsbutwith DNT biodegradation was rapid and sustained. Similar
theinitialpHof8.5.Increasingnitriteconcentrationsclearly observationsmadebyNishinoandSpainstronglysupport
exert a negative effect on biodegradation rate and extent, theapparenteffectofpHonDNTbiodegradationrates(18).
withasevereinhibitionobservedat40mM(Figure5). Based on the nitrite production and pH drop in column
Asecondaryeffectofnitriteproductionisthereduction studies,itappearsthatconditionsinthelowerportionsof
of pH in the medium. To investigate the impact of pH, thecolumnswereapproachingthosewherereducedrates
respirometerstudieswereconductedatarangeofinitialpH of2,4-DNTdegradationwouldbeexpected.Inthecaseof
values with no nitrite added to the medium. The results columns operated in recycle, the accumulation of nitrite
(Figure6)indicatethatpHvaluesof6.0andbelowseverely probablycausedcessationofactivity.Incolumnsoperated
VOL.37,NO.15,2003/ENVIRONMENTALSCIENCE&TECHNOLOGY93389
FIGURE8. Effectofinitialphosphateconcentrationon2,4-DNTmineralizationasmeasuredbyoxygenconsumption(5.6molofO consumed
2
duringthemineralizationof1molof2,4-DNT(16)).Notethattheinitialconcentrationof2,4-DNTis5mM;normallyitis10mM2,4-DNT
forbaselineconditions.
FIGURE9. Effectofmediumstrengthon2,4-DNTmineralizationasmeasuredbycarbondioxideproduction(5.2molofCO producedduring
2
themineralizationof1molof2,4-DNT(16)).
without recycle, charge balance calculations indicate that studiescontained20mMphosphatebuffer.Additionalbuffer
accumulationof15mMnitritewouldhavecausedthepH wasaddedintheformofsodiumbicarbonate(NaHCO)at
3
drop to the range of 6.0-6.5 assuming 20 mM phosphate concentrationsof20,40,60,and80mM.TheinitialpHwas
buffer and an initial pH of 8.5. At that pH and nitrite 8.5withnofurtheradjustment.Atallbufferconcentrations,
concentration,bothvariableswouldbeexpectedtoreduce 2,4-DNTwasdegradedimmediately(Figure7).Respiration
ratesofbiodegradationwithpHeffectsbeingthemostacute. ratesweresustainedinaccordancewithbufferconcentration.
Itisofnotethatthebiologicaloxidationofnitritetonitrate Theseresultsindicatethatbyincreasingbuffercapacityof
through nitrification did not occur in columns or effluent the medium and maintaining a favorable pH range, it is
shakeflaskstudies.Thus,maintaininghighratesofbiodeg- possibletosustainrapidratesofbiodegradationforlonger
radationwill,inpart,becontrolledbytheabilitytodilute periodsoftime.
nitriteconcentrationsatfieldcapacity(i.e.,flushingevolved Incolumnstudiesitbecameapparentthatnutrientavail-
nitritefromporewater). abilitywasalsoanimportantcontrollingfactorin2,4-DNT
A further study investigated the role of the buffering biodegradationactivity.Previousstudiesrevealedphospho-
capacityofthemediuminmaintainingpHandminimizing rusasalimitingnutrientinthebiodegradationofDNTin
inhibitory effects. The mineral medium used in column soilslurries(4,16).Tofurtherinvestigatethelimitingeffects
33909ENVIRONMENTALSCIENCE&TECHNOLOGY/VOL.37,NO.15,2003