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Research ArticleReceived: 26 November 2008 Revised: 7 January 2009 Accepted: 7 January 2009 Published online in Wiley Interscience: 12 March 2009

(www.interscience.wiley.com) DOI 10.1002/jctb.2140

The effect of three chemical oxidantson subsequent biodegradation of2,4-dinitrotoluene (DNT) in batch slurryreactorsDaniel Cassidy,∗ Abraham Northup and Duane Hampton

Abstract

BACKGROUND: Recent studies indicate that chemical oxidation may be compatible with subsequent biodegradationin contaminated soils. To test this, soil contaminated with 2,4-dinintrotoluene (2,4-DNT) was treated in batch slurryreactors with (1) ozone, (2) modified Fenton chemistry (MFC), and (3) iron-activated sodium persulfate (SPS). Chemical andsubsequent biological oxidation were monitored, and compared with biodegradation alone. Release of nitrite and nitratedistinguished biological from chemical oxidation of 2,4-DNT, respectively. DNT-degrading microorganisms were enumerated.The disappearance of volatile fatty acids (VFAs) accumulated during chemical oxidation was also monitored.

RESULTS: In the biological reactor 66% of the 2,4-DNT was degraded, but further biodegradation was inhibited by nitriteconcentrations approaching 18 mmol L−1. At the doses tested, all oxidants achieved chemical oxidation followed bybiodegradation, resulting in 98% DNT removal overall. Ozone achieved the greatest DNT removal (70%), but also causedthe greatest reduction in DNT degraders and the longest rebound time (60 days) before biodegradation of the remaining DNTand VFAs. SPS resulted in the least DNT removal by chemical oxidation (37%), but showed no obvious rebound period for DNTdegraders, and even signs of co-existing chemical and biological oxidation. By releasing nitrate, which is less toxic than nitrite,the oxidants kept nitrite levels below 18 mmol L−1, enabling the follow-on biodegradation step to attain lower concentrationsof 2,4-DNT than biodegradation alone.

CONCLUSIONS: All three chemical oxidants were compatible with biodegradation of residual 2,4-DNT. Post-oxidationbioremediation should be included in remedial designs.c© 2009 Society of Chemical Industry

Keywords: 2,4-dinitrotoluene (DNT); bioremediation; in situ chemical oxidation (ISCO); modified Fenton; ozone; persulfate

INTRODUCTIONDinitrotoluenes (DNT) are intermediates in the production of2,4,6-trinitrotoluene (TNT), and are also used in the manufactureof polyurethane foams, dyes, plasticizers, and air bags forautomobiles. Improper handling and disposal of DNT has causedwidespread contamination in soil and groundwater at chemicaland munition production facilities.1 By far the most abundantisomer is 2,4-dinitrotoluene (2,4-DNT), which is acutely toxic andis a low-level carcinogen.2 Because of its toxicity and abundanceas an environmental contaminant, 2,4-DNT has been classifiedas a priority pollutant by the US EPA3 and remediation of DNT-contaminated sites is required. At 20 ◦C, the solubility of 2,4-DNT is240 mg L−1, and the vapor pressure and Henry’s Law constant for2,4-DNT are 5.1×10−3 mm Hg and 5.0×10−6 atm m−3 mol−1.4 Assuch, 2,4-DNT has an extremely low tendency to volatilize. Whenpresent in soils above its aqueous solubility 2,4-DNT occurs as afine, yellowish powder.

Bioremediation and chemical oxidation are two of the mostpromising remediation methods for 2,4-DNT-contaminated sites.5

Anaerobic biodegradation of DNT is problematic because ofthe accumulation of aminonitrotoluenes, which are also priority

pollutants.6 A number of aerobic bacteria have been identifiedfrom contaminated soils worldwide that are capable of using2,4-DNT as a sole source of carbon, energy, and nitrogen.7,8

Aerobic biodegradation of 2,4-DNT has been demonstrated inlaboratory slurry reactors,9,10 soil columns,11 and fluidized bedbiofilm reactors.12,13 These studies also show that nitrite releasedduring DNT biodegradation becomes inhibitory to further DNTbiodegradation at levels ranging from 10 mmol L−1 to 40 mmolL−. Nitrite inhibition limits the use of bioremediation at DNT-contaminated sites.

Chemical oxidation of 2,4-DNT has also been demonstratedusing ozone11,14 and Fenton chemistry.5,15,16 In contrast tobiodegradation, chemical oxidation of 2,4-DNT releases nitrate,due to oxidation of the nitro groups after being removed from the

∗ Correspondence to: Daniel Cassidy, Western Michigan University, Departmentof Geosciences, 1187 Rood Hall, Kalamazoo, Michigan, USA.E-mail: daniel.cassidy@wmich.edu

Western Michigan University, Department of Geosciences, 1187 Rood Hall,Kalamazoo, Michigan, USA

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toluene. Therefore, nitrite accumulation after chemical oxidationis complete is a surrogate measure of biodegradation of 2,4-DNT. Ozone (O3) can oxidize 2,4-DNT directly, or indirectly viathe hydroxyl radical (•OH), which is produced through a seriesof reactions between ozone and water.17 Fenton chemistry usesa well-known reaction between hydrogen peroxide (H2O2) andFe2+ at low pH to form the hydroxyl radical (•OH) (see Reaction 1),which then oxidizes 2,4-DNT.5 Modified Fenton chemistry (MFC)promotes Reaction 1 at quasi-neutral pH and with a greaterratio of hydrogen peroxide to iron, which is much more suitablefor in situ applications.18 Sodium persulfate (SPS) (or sodiumperoxydisulfate) is a relatively new oxidant used in remediation,and is promising because it has a longer half-life in the subsurfacethan ozone or hydrogen peroxide. When activated the persulfateanion (S2O8

2−) forms sulfate radicals (SO4•−), which are nearly

as strong an oxidant as the hydroxyl radical.19 Reaction 2 showsthe activation of persulfate with Fe2+. Other activation methodsinclude high or low pH, high temperature, and hydrogen peroxide.Once formed, the sulfate radical can also react with water toform the hydroxyl radical (Reaction 3). SPS has not yet beenshown to degrade 2,4-DNT. However, SPS is known to oxidizemany contaminants, including monoaromatics,20 polychlorinatedbiphenyls,21 and 2,4-dichlorophenol and polycyclic aromatichydrocarbons (PAH).22

H2O2 + Fe2+ → •OH + OH− + Fe3+ (Reaction 1)

S2O82− + Fe2+ → SO4

•− + SO42− + Fe3+ (Reaction 2)

SO4•− + H2O → •OH + HSO4

− (Reaction 3)

The use of chemical oxidants (particularly ozone and hydroxylradicals) to kill microorganisms in aqueous systems is wellknown. However, recent studies in contaminated soils show thatactivity and numbers of indigenous soil microorganisms are oftenunaffected, and in some cases even enhanced by exposure tochemical oxidants.23 This counter-intuitive phenomenon can beattributed to the large diversity of microorganisms in soils, thephysical and chemical shelter provided by soil particles, and theintroduction of new microorganisms from incoming groundwater.Several studies showed treatment with Fenton’s chemistry provedbeneficial to subsequent biological activity and biodegradation ofPAH in soil.24 – 26 Possible explanations for this include reduction ofPAH concentrations by chemical oxidation to below the thresholdtoxicity levels for the soil microorganisms, and/or production ofvolatile fatty acids (VFAs) and other partial oxidation products thatare readily biodegradable. Co-existing chemical and biologicaloxidation in soil were observed using low doses of MFC, andcomplete recovery of microbial activity was observed within 24 h,even at the highest doses.27 Recent studies on heat-activated SPSshowed that persulfate concentrations up to 10 g L−1 did not affectthe activity or numbers of indigenous soil microorganisms.28 Thesestudies indicate that biological polishing should be included in thedesign of in situ chemical oxidation (ISCO) remediation systems,even if weeks to months are required for the soil microorganismsto recover after being exposed to chemical oxidants.

The overall purpose of this study was to determine the com-patibility of chemical oxidation using ozone, MFC, and SPS withsubsequent bioremediation by the indigenous microorganismsin soil with aged 2,4-DNT contamination. The specific goals ofthis study were: (1) to compare aerobic bioremediation of a soilwith aged 2,4-DNT contamination using chemical oxidation withozone, MFC, and SPS; (2) to distinguish chemical from biological

oxidation of 2,4-DNT by the accompanying release of nitrite vsnitrate; (3) to quantify the effect of these three chemical oxidantson the indigenous soil microorganisms.

MATERIALS AND METHODSChemicalsAll chemicals used were reagent grade. Sodium persulfate, ferroussulfate, tetrasodium ethylenediaminetetraacetic acid(EDTA) anddisodium EDTA were purchased from Merck (Gibbstown, NewJersey). Hydrogen peroxide (50%) was provided gratis by Solvay(Houston, Texas). 2,4-DNT (97% pure), sodium nitrite, sodiumnitrate, and acetic acid were purchased from Aldrich (Wilwaukee,Wisconsin). The nitrification inhibitor, 2-chloro-6-(trichloromethyl)pyridine (TCMP), was obtained from Hach (Loveland, Colorado).

Contaminated soilThe soil used in these studies was obtained from a site nearSarnia, Ontario (Canada), where polyurethane foam and otherplastics are manufactured. It is estimated that the soil hasbeen contaminated with DNT for at least 25 years, and previousexperiments had demonstrated that the soil contained nativeaerobic DNT-degrading microorganisms. Approximately 50 L ofsoil was obtained from the site, which was first washed with DIwater to remove any nitrite that may have been released fromin situ biodegradation of DNT, and then homogenized in a 150 Lportable cement mixer.

Slurry and reactorsThe reactors were 20 L, covered glass vessels, with an overheadmechanical mixer set at 400 rpm, and multiple ports in the cover.The maximum slurry volume in each reactor was 14 L. The slurrywas made by mixing 2.8 kg of the homogenized test soil withapproximately 13 L of tap water collected from the site until the14 L mark was reached, resulting in a 20% solids concentration(i.e. 0.2 kg dry soil L−1 slurry). Because 1 L of hydrogen peroxidesolution was added to the MFC reactor, 1 L less of tap water wasused to make this slurry.

Table 1 shows some of properties of the contaminated slurryused in the reactors. All analyses of the soil properties wereconducted according to standard methods.29 The test soil wasa sandy loam, high in carbonate content which buffered the pHnear 8, and low in native organic content. The only significant

Table 1. Selected properties of the contaminated slurry used in thestudies

Characteristic Value

pH 8.1

Native organic carbon content (%) <0.2

Sand (%) 72

Silt (%) 22

Clay (%) 6

Total carbonates (%) 4.5

2,4-DNT-degrading microorganisms (log CFU g−1) 3.72 ± (0.39)a

2,4-DNT concentration (mg kg−1) 11,450

2,4-DNT concentration (mmol L−1) 13.5

a Arithmetic mean ± (standard deviation).

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isomer at the site was 2,4-DNT, with a concentration in the slurry of11 450 mg kg−1, or 13.5 mmol L−1. The background concentrationof 2,4 DNT-degrading microorganisms was 3.72 log CFU g−1.

The following soluble solids were also added to the slurry, de-pending on the reactors. Using the average 2,4-DNT concentrationof 11 450 mg kg−1 (Table 1) each reactor contained approximately32.5 g of 2,4-DNT (C7H6N2O4), or 15 g of carbon. All reactors re-ceived 11.45 g of NH4Cl, resulting in a C : N ratio of 5 : 1. An excessof ammonia was added because microorganisms prefer this as anitrogen source to nitrate or nitrite, and these two were intendedto serve as indicators of chemical and biological oxidation of DNT,respectively. The pH of the slurry in each reactor was buffered at 8by adding a phosphate buffer, consisting of 1 g of NaH2PO4 • H2O,and 20 g of Na2HPO4 • 7H2O to each reactor, which also providedexcess phosphorus for biodegradation. To inhibit microbial nitrifi-cation, 500 mg of TCMP were added to each reactor. All reactorswere kept at 15 ◦C.

One of the ports of each reactor also housed a stainless steel tubeattached to a fine-bubble air diffuser stone placed near the bottomof the reactor. The reactors were sparged with air throughoutthe studies to keep the dissolved oxygen (DO) concentrationsufficiently high to promote aerobic biodegradation, and to avoiddenitrification of the nitrate and nitrite released from the DNT. TheDO in the slurry reactors was monitored daily, and never droppedbelow 7 mg L−1. The ozone reactor was sparged with ozone forthe first 2 days followed by air for the remaining time. Volatilelosses of DNT from the slurry were quantified by keeping unusedports closed, sealing the ports housing the mechanical mixer andthe sparger with gaskets and vacuum grease, and mounting twoTenax-GC resin tubes in series to the only venting port.

Oxidant dosesOzone was sparged continuously for 48 h at an air flow rate of 50 Lh−1, at an ozone concentration of 1.0% (v/v). This resulted in anozone flow rate of 10.5 mol h−1 and a total ozone dose of 504 molover 2 days. Ozone was generated using an Ozone Services ModelOL-100 generator (Burton, British Columbia, Canada). Effluentozone was destroyed with an ozone destructor. MFC was promotedby adding 600 mg EDTA (200 g as tetrasodium-EDTA, and 400 gas disodium-EDTA) to serve as a chelating agent for iron. Then3 g Fe2+ (as ferrous sulfate) were added, followed by 1 L of50% hydrogen peroxide. The persulfate reactor received 200 gof sodium persulfate powder, resulting in a concentration ofpersulfate anion (S2O8

2−) of approximately 12 g L−1. Then 3 gFe2+ (as ferrous sulfate) were added with 600 mg EDTA (200 g astetrasodium-EDTA, and 400 g as disodium-EDTA) as a chelant.

Enumeration of DNT-degrading microorganismsQuadruplicate 20 mL samples of slurry were used for microbialenumeration. Cells were extracted with sodium pyrophosphateaccording to Van Elsas and Smalla.30 Serial dilutions were platedon Noble agar (Difco Laboratories, Detroit, MI), prepared with 2,4-DNT as the sole carbon source. Plates were incubated for 2 weeksat 25 ◦C and colony forming units (CFUs) were counted.

Chemical analysesFor 2,4 DNT, samples of whole slurry from the reactors wereextracted with acetonitrile and analyzed using high performanceliquid chromatography (HPLC), using the method described byNishino et al.9 An Agilent 1000 HPLC was used, equipped with adiode array detector, a Hypersil porous graphite column (5 µm ×

150 mm), and using a mobile phase of acetonitrile/water (90 : 10)containing trifluoroacetic acid (0.5 mL L−1). The detection limit of2,4-DNT was approximately 10 mg kg−1. The concentrations of2,4-DNT in Figs 1–4 are expressed as mmol L−1, in order to allow asimple mass balance for the NOx in 2,4-DNT, nitrate and nitrite. TheTenax-GC resin tubes were extracted with acetone and 2,4-DNT inthe extract was quantified by gas chromatography using a Thermal

0

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ol L

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02468101214161820

NO

2- & N

O3- (

mm

ol L

-1)

2,4-DNT VFAs NO2 NO3

Figure 1. Concentrations of 2,4-DNT, VFAs, nitrite, and nitrate with time inthe biological reactor.

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O3- (

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2,4-DNT VFAs NO2 NO3

Figure 2. Concentrations of 2,4-DNT, VFAs, nitrite, and nitrate with time inthe reactor treated with ozone.

00 4 8 12 16 20 24 28 32

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Figure 3. Concentrations of 2,4-DNT, VFAs, nitrite, and nitrate with time inthe reactor treated with MFC.

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00 2 4 6 8 10 12 14

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2,4-DNT VFAs Persulfate NO2 NO3

Figure 4. Concentrations of 2,4-DNT, VFAs, persulfate (S2O82−), nitrite, and

nitrate with time in the reactor treated with SPS.

Energy Analyzer (TEA) using the US Department of Labor-OSHAMethod 44 (2001).31

Nitrite, nitrate, and VFAs were quantified in filtrate (i.e. afterpassing slurry samples through a 0.45 µm filter) using StandardMethods.32 VFA concentrations are expressed as mg L−1 as acetate.The persulfate anion was also quantified in filtrate samples usingthe iodometric titration method.33 Ozone concentrations weremeasured in the effluent gas of the ozonated reactor usinga photometer (Anseros Ozonomat GP, Tubingen, Germany).Hydrogen peroxide concentrations in filtrate samples weremeasured spectrophotometrically.34 DO and pH in the slurrieswere measured by inserting an Orion DO/pH probe directly intothe slurry reactors.

RESULTS AND DISCUSSIONControlling nitrification and denitrificationOne goal of these studies was to take advantage of the unusualproperties of 2,4-DNT, which allows biological from chemicaloxidation of 2,4-DNT to be distinguished by tracking the releaseof nitrite and nitrate, respectively. This required that nitrate andnitrite released not be used for dissimilatory denitrification in thereactors, which was accomplished by air sparging to keep theDO above 7 mg L−1. It was also necessary to prevent assimilatorydenitrification in the reactors, in which microorganisms use nitriteor nitrate as a nitrogen source for cell growth in the absenceof ammonia nitrogen. To avoid this, sufficient ammonia nitrogenwas provided for biodegradation of DNT, since practically allheterotrophs prefer ammonia nitrogen over NOx.35 TCMP wasadded to prevent microbial nitrification of added ammoniaand nitrite. It is possible that some nitrite released duringbiodegradation of DNT was chemically oxidized afterwards, butthis would have been possible only with persulfate, which was stillpresent as biological DNT oxidation began (see discussion belowand Fig. 4).

Despite continual air sparging and gases released duringchemical oxidation, no DNT volatilization was detected in anyof the reactors. This is because of the low volatility of 2,4-DNT.2

Measurements of pH are not included in the discussion belowbecause pH values remained near 8 in all the reactors throughoutthe studies. The pH of the slurry was 8 (Table 1), and additionalphosphate buffer was added to maintain a pH of 8.

Time profiles of DNT removal in reactorsFigure 1 shows time profiles of concentrations of 2,4-DNT, VFAs,nitrite (NO2

−) and nitrate (NO3−) in the reactor promoting only

biodegradation by the indigenous soil microorganisms. After a2 week lag period, 2,4-DNT was readily biodegraded to levelsof approximately 4.6 mmol L−1 within 14 days. Biodegradationreleased nitrite from the DNT, which accumulated in theslurry to concentrations of nearly 18 mmol L−1. There was nomeasurable accumulation of VFAs or nitrate. Biodegradationstopped because nitrite had reached levels inhibitory to theindigenous microorganisms. Nitrite inhibition of DNT degradationat concentrations ranging from 10 mmol L−1 to 40 mmol L−1

has been reported previously.10,11 Previous studies on this soilshowed nitrite inhibition near 18 mmol L−1, and also showed thatDNT biodegradation was re-established if the nitrite-rich slurrywater was removed and replaced with fresh water, even withoutadditional nutrients.

Figure 2 shows the time profiles of concentrations of 2,4-DNT,VFAs, nitrite and nitrate in the reactor sparged continuously withozone for the first 2 days, and with air thereafter. Ozone treatmentreduced concentrations of 2,4-DNT to nearly 4 mmol L−1, witha concomitant release of nitrate to concentrations that reached19 mmol L−1. Removal of 2,4-DNT stopped once ozone spargingwas stopped. Ozone concentrations in the effluent gas from thereactor dropped to below detection within 2 h of the ozone beingturned off, which is consistent with the short half-life of ozonein the presence of soils.18 Ozone caused VFA concentrations toincrease to over 2 mmol L−1 (as acetate). Chemical oxidation oforganic contaminants with ozone and MFC commonly resultsin the accumulation of VFAs (e.g. oxalate, acetate),18,36 whichare resistant to further chemical oxidation.27 Concentrations of2,4-DNT remained near 4 mmol L−1 until day 60, after whichthey dropped to 0.22 mmol L−1 by day 90. The second phaseof DNT removal can be attributed to biodegradation, becausenitrite was released instead of nitrate, and because VFAs, whichare readily biodegradable by heterotrophs, also dropped to levelsbelow detection during this period. It is noteworthy that nitratelevels above 18 mmol L−1 did not appear to inhibit microbial DNTdegradation.

Figure 3 shows concentrations with time of 2,4-DNT, VFAs,nitrite and nitrate in the slurry treated with MFC. Treatmentwith MFC reduced 2,4-DNT concentrations to nearly 8 mmol L−1

within 1 to 2 days, with a simultaneous increase in concentrationsof VFAs and nitrate to approximately 3.5 mmol L−1 (as acetate)and 11.5 mmol L−1, respectively. Concentrations of hydrogenperoxide in samples of slurry filtrate were below detection after48 h, which explains why MFC stopped oxidizing DNT at that time.Hydrogen peroxide, like ozone, is quite unstable in the presenceof soils.18 After day 20, the remaining 2,4-DNT was reduced bybiodegradation to 0.21 mmol L−1, with a resulting release of nitrite.VFAs that accumulated during chemical oxidation with MFC werealso metabolized by the indigenous soil microorganisms duringthis period of DNT biodegradation.

Figure 4 shows the time profiles of concentrations of 2,4-DNT,VFAs, persulfate anion (S2O8

2−), nitrite and nitrate in the reactortreated with SPS. Persulfate oxidized 2,4-DNT, as evidenced by thereduction in DNT concentrations and the accompanying increasein nitrate concentrations. However, persulfate oxidized DNT moreslowly than ozone (Fig. 2) or MFC (Fig. 3), which is expectedbecause persulfate is more stable in soil than ozone and hydrogenperoxide. It is also noteworthy that there was no marked lagbetween the end of chemical oxidation of DNT with persulfateand the onset of biodegradation, as observed with ozone andMFC. On day 6 nitrite concentrations began to rise, indicating that2,4-DNT removal after that time was due to biodegradation. The

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concentration of persulfate ion also dropped to below detectionon day 6. Nitrate release ended after day 4, when there was stillpersulfate ion present, suggesting that some biodegradation ofDNT may even have co-existed with persulfate oxidation. VFAsaccumulated during the first few days of persulfate oxidation werebiodegraded well before persulfate was exhausted, which is a clearindication of simultaneous chemical oxidation and heterotrophicmetabolism.

Figures 1 to 4 clearly show that nitrite is released when DNT isbiodegraded, while chemical oxidation of DNT releases nitrate. Thereason for this is that nitrite released during chemical oxidationof DNT is further oxidized to nitrate by the oxidant. Figures 2,3 and 4 show that pretreatment with all three oxidants alloweda secondary biodegradation step to reach lower concentrationsof 2,4-DNT than could be obtained with biodegradation alone(Fig. 1). The reason for this is that chemical oxidation releasednitrate, which would otherwise have been released as nitrite,thereby avoiding the high nitrite concentrations that causedinhibition. In general, nitrite has a greater specific toxicity tomost microorganisms than nitrate.35,37 – 40 Chemical oxidationfollowed by biodegradation resulted in a sum of nitrate andnitrite concentrations between 26 and 27 mmol L−1 (Figs 2 to 4),but the nitrite concentration never approached the inhibitoryconcentration of 18 mmol L−1 (Fig. 1). These results demonstratethat combining chemical oxidation and biodegradation of 2,4-DNT provided greater overall removal than biodegradation alonecould achieve.

VFA concentrations in Figs 2 to 4 also provide an indirectindication of aerobic metabolic activity by the indigenous soilmicroorganisms. VFAs accumulate during chemical oxidationbecause microorganisms are inhibited and/or killed. Later removalof VFAs marks the rebound of aerobic heterotrophic metabolism.In the case of persulfate, VFAs were actually degraded whilethe oxidant was still present (Fig. 4), indicating that persulfateinhibited microorganisms to a much lesser degree than ozone orMFC, at the doses used.

Overall DNT removalTable 2 lists the overall removal of 2,4-DNT in the slurries and thetime required. At the doses tested, all three chemical oxidantsalong with follow-on biodegradation achieved an overall removalof 2,4-DNT of 98% (i.e. both chemical oxidation and subsequentbiodegradation). In contrast, biodegradation alone removed only66% of the 2,4-DNT in 30 days. The difference in time requiredfor the chemical oxidants to achieve the same overall DNTremoval was predominantly a function of the time requiredfor rebound of biological activity after chemical oxidation (cf.Figures 2, 3 and 4). The data clearly show that SPS achieved the

best overall performance, with 98% DNT removal in 10 days dueto combined chemical and biological oxidation. MFC and ozonecombined with subsequent biological polishing, required 30 and90 days, respectively, to reduce DNT concentrations by 98%. Allthree oxidants resulted in greater overall DNT removal than didbiodegradation alone, for reasons of nitrite toxicity.

Table 2 also lists the percentage recovery of NOx (i.e. nitrite andnitrate) from the 2,4-DNT degraded in the reactors, and the finalcounts of DNT-degraders after the time periods given. Recoveryof NOx was calculated as the sum of the moles of nitrite (NO2

−)and nitrate (NO3

−) released, divided by the moles of 2,4-DNTdegraded multiplied by one-half (since each mole of DNT contains2 moles of NOx). Recoveries of NOx ranged from approximately97% to 104%. Given the experimental error inherent in measuringconcentrations of 2,4-DNT, nitrite and nitrate, the calculated NOx

recoveries near 100% are quite good. The NOx recoveries alsoindicate that the measures taken to prevent nitrification anddenitrification in the reactors were effective, which lends credenceto the approach of using release of nitrite vs nitrate to distinguishbiological from chemical oxidation of 2,4-DNT. Table 2 shows thatcounts (CFUs) of DNT-degrading microorganisms at the end of thevarious treatment times were not statistically different from oneanother, or from counts measured in the slurry before treatment(Table 1). Although counts of DNT degraders were reducedsignificantly immediately after chemical oxidation with ozoneand MFC (see Table 3 and discussion below), the results in Table 2show that subsequent biological growth restored counts of DNT-degrading microorganisms to their background number levels.

Chemical vs biological oxidationTable 3 lists the breakdown of chemical vs biological oxidation of2,4-DNT for the doses of the three oxidants tested, and the effect ofchemical oxidation on counts of DNT degraders and the reboundtime for biological activity. Ozone achieved the greatest extentof chemical oxidation of DNT (70%), leaving the least amount ofDNT to be biodegraded (28%). However, ozone treatment alsoreduced counts of DNT-degrading microorganisms by roughly 2log units CFUs g−1 soil (two orders of magnitude). Ozone hadthe greatest negative impact on DNT degraders, which explainswhy the time required for rebound of biological activity was alsothe longest of all the oxidants (60 days). With MFC, 41% of theoverall DNT removal was due to chemical oxidation, leaving 57%due to biodegradation. MFC reduced counts of DNT degraders byroughly 1 log unit (one order of magnitude), and approximately20 days were required before DNT biodegradation was observed.Treatment with SPS chemically oxidized only 37% of the DNT,leaving 61% to be biodegraded. There was only a very slightreduction in counts of DNT degraders caused by the dose of

Table 2. Overall removal of 2,4-DNT and the time required, recovery of NOx from 2,4-DNT degradation, and final counts of DNT-degraders afterbiodegradation

Treatment scenarioFinal DNT conc.

(mmol L−1)DNT removed

(%)Time required

(d)NOx recovered fromDNT degraded (%)a

Final counts of DNTdegraders (log CFU g−1)

Bio 4.6 66 30 96.7 4.26 ± (0.29)b

Ozone + Bio 0.22 98 90 104.3 3.86 ± (0.34)

MFC + Bio 0.21 98 30 101.9 3.71 ± (0.27)

SPS + Bio 0.23 98 10 98.8 4.13 ± (0.21)

a =(mol NO2− released + mol NO3

− released)/(mol 2,4-DNT degraded)(0.5).b Arithmetic mean ± (standard deviation).

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Table 3. Breakdown of chemical vs biological oxidation of 2,4-DNT, and the effect of chemical oxidation on counts of DNT degraders and reboundtime

Treatment scenarioDNT chemically

oxidized (%)DNT biodegraded

(%)Reduction in DNT degraders caused by chemical

oxidation (log CFU g−1)Rebound time for DNT

degraders (d)

Ozone + Bio 70 28 2.03 60

MFC + Bio 41 57 1.17 20

SPS + Bio 37 61 0.14 0-2

SPS used, and as a result there was only a very short time (0to 2 days) required for the indigenous microorganisms to begindegrading DNT after persulfate was exhausted. Biodegradationof VFAs actually co-existed with SPS oxidation of 2,4-DNT (Fig. 4).Regardless of the death of significant numbers of DNT degraderscaused by ozone and MFC, microbial counts returned to theirpre-treatment values within 90 days and 30 days, respectively(cf. Tables 1, 2 and 3). These results show that post-oxidationbioremediation should be factored into ISCO designs.

It should also be noted that the results in this study cannot beused to choose one oxidant over another, insofar as their ability toachieve chemical vs biological oxidation. Different doses and/orapplication rates of the three oxidants tested would also haveproduced quite different distributions of chemical vs biologicaloxidation of 2,4-DNT from those obtained in this study. Forexample, had ozone been applied at a lower rate and for alonger period of time, it could have resulted in the greatest ratioof biological to chemical oxidation, instead of SPS. A higher doseof SPS would likely have resulted in more chemical oxidation ofDNT, and in greater death of DNT degraders and longer reboundtimes. Likewise, a lower dose of MFC would likely have resultedin a shorter rebound time and in a greater ratio of biological tochemical oxidation of 2,4-DNT. In fact, co-existing chemical andbiological oxidation of contaminants in soil with low doses of MFChas recently been demonstrated.27

Moreover, no attempt was made in this study to optimizechemical vs biological oxidation of 2,4-DNT for any of the threeoxidants tested. The doses of each oxidant were chosen arbitrarily,with the sole criterion of leaving some residual 2,4-DNT afterchemical oxidation so that follow-on biodegradation could beinvestigated. All three oxidants could have been applied at doseshigh enough to achieve complete chemical oxidation of 2,4-DNT,making follow-on biodegradation impossible. During ISCO, theseconditions are likely to prevail only adjacent to the injectionwell. However, the purpose of these studies was to evaluate theeffect of these three oxidants on subsequent bioremediation,under conditions in which there is insufficient oxidant to achievecomplete chemical oxidation. Such conditions are the rule inthe subsurface during ISCO, for example, at a distance from theinjection wells. Experience has shown that one injection of oxidantis rarely sufficient to achieve site cleanup. These studies show that,if nutrients are injected with the oxidants, bioremediation can beused in ISCO as a polishing step to lower costs compared withre-injection of chemical oxidants.

Cost is perhaps the most important consideration is choosinga technology for site cleanup. While bioremediation is usuallythe least expensive treatment option, the apparent inhibitionassociated with nitrite accumulation during biodegradation ofthis soil would discourage choosing bioremediation. In general,capital and operation/maintenance (O/M) costs are greater forchemical oxidation than bioremediation. However, chemical

oxidation can reduce long-term sampling and monitoring costsrelative to bioremediation because it destroys contaminants faster.This is the primary reason for increased interest in chemicaloxidation technologies for site cleanup in the last decade.The results from this study do not distinguish one chemicaloxidation technology as the most cost-effective. All three ofthe chemical technologies tested were effective at destroyingthe DNT, using some combination of chemical oxidation andbioremediation. All three chemical oxidation technologies areconsidered to be cost-effective. To the authors’ knowledge thereare no publications directly comparing the cost-effectiveness ofdifferent chemical oxidation technologies in pilot- or full-scaletreatment at contaminated sites. This is due to the high costsinvolved in demonstrating multiple technologies in the field. Asa result, the choice of oxidant is often based on the personalpreference of the consultants and/or responsible parties involved.In general, ozone tends to have higher capital costs than theother two technologies because ozone must be generated on-site using an ozone generator. However, these costs may berecovered if ozone results in shorter treatment times, as was thecase in these studies. Liquid hydrogen peroxide is approximately5–6 times less expensive than SPS. While both MFC and SPSperformed well in this study, SPS had a lower impact on DNT-degrading microorganisms, which can reduce overall treatmenttimes when residual contaminants are left after chemical oxidantsare exhausted. SPS is also more stable in the subsurface thanhydrogen peroxide.

CONCLUSIONSOzone, MFC and SPS were all effective at oxidizing 2,4-DNT, andwere all compatible with bioremediation of residual 2,4-DNT.At the doses tested, SPS showed the best overall performance,promoting combined chemical and biological oxidation of 98% ofthe 2,4-DNT within 10 days. In general, the oxidant doses resultingin the most effective chemical oxidation step also killed the mostDNT-degrading microorganisms and delayed the onset of DNTbiodegradation. However, 60 days was the longest rebound periodobserved (with ozone), which is insignificant in terms of in situapplications. The results from this study show that bioremediationof residual contaminants and partial oxidation products should beincluded in ISCO designs in order to provide optimal treatmentand reduce costs.

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