Optimization of an AerobicPolishing Stage To Complete theAnaerobic Treatment ofMunitions-Contaminated SoilsD E B O R A H J . R O B E R T S , *F A R R U K H A H M A D , † A N DS U H A S I N I P E N D H A R K A R ‡

Department of Civil and Environmental Engineering,University of Houston, Houston, Texas 77204-4791

The addition of an external carbon source to allowthe creation of anaerobic conditions for theremediation of soils contaminated with nitroaromaticcompounds has been successfully applied to soilscontaminated with Dinoseb (2-sec-butyl-3,4-dinitro-o-cresol), an herbicide, and 2,4,6-trinitrotoluene (TNT),an explosive. The addition of an aerobic stage to removeexcess external carbon after the anaerobic stageproduces a treated soil with a lower oxygen demandthan the soil, which is presently left after the anaerobicstage. The use of acetate, soluble starch, glucose, andinsoluble starch as sources of external carbon forthe creation and maintenance of anaerobic conditionswas examined. The addition of glucose to staticallyincubated soil reactors allowed for the fastestreduction in redox potential and produced cultureswith the lowest redox potentials (-400 mV). Theamount of glucose added was optimized resulting inthe use of 0.25% (w/v) glucose to treat a sandy soilcontaminated with 12 000 mg of TNT, 3000 mg of RDX(hexahydro-1,3,5-trinitro-1,3,5-triazine), and 30 mgof HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazo-cine)/kg of soil. In these treatments, the anaerobicstage was complete within 14 days, and an additional7-day aerobic stage resulted in TOC concentrationsof 30 mg/L remaining in the aqueous phase.

IntroductionCompounds such as 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetraazocine (HMX) are found as soilcontaminants at many facilities where munitions areproduced and disposed of (1-3). These compounds arepersistent in aerobic soils and leach into groundwater fromcontaminated soils (2, 4, 5). TNT or its reductive inter-

mediates are mutagenic (6, 7) and toxic to plants, (8), fish(9, 10), larvae (7), algae (7, 9), microorganisms (7, 11, 12),and fungi (13-15).

The remediation of soils contaminated with thesecompounds is important for the protection of criticalgroundwater resources as well as for demilitarization ofDepartment of Defense (DOD) facilities. Incineration ispresently the most often used method for treatment ofthese soils (16), but is expensive and not well accepted bythe public. The development of an effective, environmen-tally sound, and economical treatment method to remediatemunitions-contaminated soils is important to allow thecleanup of these numerous sites. The biological degrada-tion of TNT has been investigated by many research groupsand has been the subject of several reviews (3, 17-20).Aerobic treatment processes generally result in the conver-sion of TNT to aminodinitrotoluenes, diaminonitrotoluenes,or tetranitroazoxytoluenes. These compounds are morepersistent and in some cases more toxic than TNT and mayrepresent slow release reservoirs for the release of ni-troaromatic compounds. TNT is also metabolized underanaerobic conditions through reductive mechanisms, butdue to the absence of O2 and the rapid reduction todiaminonitrotoluenes or triaminotoluene, relatively lowconcentrations of the hydroxylamine intermediates arepresent, and the formation of tetranitroazoxytoluenes isvery low if it occurs at all. The anaerobic degradation ofTNT to TAT (21), toluene (22-25), or further throughp-cresol (26, 27) have been reported.

A soil remediation procedure developed for the anaero-bic biological remediation of soils contaminated with thenitroaromatic herbicide Dinoseb (28-30) has been appliedsuccessfully to the treatment of soils contaminated withTNT and RDX (31-33). In this procedure, an external easilydegradable carbon source (usually potato starch) is addedto stimulate the consumption of O2 by aerobic bacteria,generating anaerobic conditions in the soil slurry. Onceanaerobic conditions are established, the degradation ofthe nitroaromatic compounds takes place. The use ofpotato starch as the external carbon source results in alarge amount of starchy material remaining at the end ofthe treatment procedure. The resulting treated soil has ahigh oxygen demand due to the excess starchy material.This oxygen demand is detrimental to agricultural soils dueto the rapid development of anaerobic conditions whenthe soil is wetted, such as after irrigation or rainfall. Theexcess oxygen demand also causes the treated soil to failtoxicity tests when Daphnia magna is used as a toxicityindicator due to the inability to keep enough oxygen in thetest system for Daphnia magna to survive (unpublishedresults).

In order to remove the excess oxygen left in the soil atthe end of the treatment procedure, a second aerobic stagehas been added to the treatment procedure. This paperdescribes the optimization of the amounts and type ofexternal carbon added to minimize the time required forthe aerobic utilization of excess carbon after the anaerobicstage is complete.

Materials and MethodsSoils. Two soils were utilized in this research. The first isa sandy soil that was contaminated with TNT, RDX, and

* Corresponding author telephone: 713-743-4281; fax: 713-743-4260, e-mail address: djroberts@uh.edu.

† Present address: 800 Babcock No. S4, San Antonio, TX 78201.‡ Present address: 1586 West Maggio Way, Apartment 9-2082,

Chandler, Arizona 85224.

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HMX as result of the disposal of washings from munitionsloading, handling, and dismantling at the Umatilla ArmyAmmunition Depot in Umatilla, OR. This soil is contami-nated with 12 000 mg of TNT/kg of soil, 3000 mg of RDX/kgof soil, and 300 mg of HMX/kg of soil. The soil was sievedwith a 10-mm sieve to remove gravel and stones. The sievedsoil was stored at 4 °C until used. This soil is referred toas “Umatilla soil”. It contains no culturable organisms (32)and cannot be treated microbiologically without inoculationwith a culture containing starch-degrading aerobes andnitroaromatic-degrading anaerobes (31).

The second soil is a sandy loam from Ellensburg, WA.This soil was originally contaminated with Dinoseb andseveral other herbicides due to washing of crop-dustingequipment at an airfield. It was treated using the anaerobictreatment procedure until the Dinoseb and other herbicideswere removed (28-30, 34). The aqueous phase was thendecanted, and the soil was air-dried and stored at 4 °C untilfurther use as an inoculum. This soil is referred to as“treated soil” and has been used successfully as an inoculumfor the past 4 years. The air-drying and storage of the soilhas only slightly affected its use as an inoculum. Themicroorganisms suspected of having important roles in theanaerobic metabolism of munitions compounds are anaer-obic spore-forming organisms (Clostridia). These organ-isms survive desiccation and storage well. Preliminarystudies on the enrichment of TNT-degrading organismsfrom the treated soil have revealed that 1 g of soil containsa population sufficient to enrich TNT-degrading organismsmost of the time, while using 4 g of soil guarantees successfulenrichment if the correct medium is used (35). The batchof treated soil used as inoculum for the enrichmentsreported above and the experiments reported here is thesame and had been stored for 3-4 years.

Experimental Design. All experiments were carried outin triplicate and have been reproduced at least once. Thebiological experiments were carried out in 500-mL Erlen-meyer flasks that received 4 g of Umatilla soil and 400 mLof 50 mM phosphate buffer containing 25 mM NH4Cl. Aninoculum of 4 g of treated soil was added. External carbonwas supplied as 1% (w/v) sodium acetate (Sigma), 1% (w/v) insoluble starch (potato waste centrifuge cake, J. R.Simplot, Boise ID), 1% (w/v) soluble starch (Sigma), or 0.01,0.1, 0.25, 0.5, 0.75, or 1% (w/v) glucose (Sigma). All cultureswere incubated at 30 °C in the dark under static conditions.Aerobic incubations were carried out at room temperatureon a rotary shaker at 100 rpm.

Analytical Procedures. The concentrations of TNT,RDX, and the reduced TNT intermediates in the aqueousphase of the cultures were monitored as described previ-ously (36). Samples of 0.5 mL were removed from thecultures at each time point and were centrifuged to removesuspended solids. A total of 10 µL of the supernatant fluidwas injected onto an Alltech Alltima 5-µm RP C18, 250× 2.1mm i.d. column. The LC analyses was performed with aHewlett-Packard HP 1090 Series II/M equipped with a DR-5ternary solvent delivery system, variable-volume auto-injector, temperature-controlled autosampler, thermostati-cally controlled column compartment, and a diode arraydetection system. Separation of TNT, RDX, and the TNTreduction products was achieved using a gradient of 11mM phosphate buffer and acetonitrile (36). The detectorwas set at 210 nm, and all peaks were scanned from 210to 600 nm for compound identification and verification.All quantitation was performed using an external standard

curve for each compound. Analytical standards wereprepared from analytical grade chemicals. TNT waspurchased from Chem Service (West Chester, PA). RDXwas supplied by Darlene Bader (U.S. Athema). 2-Amino-4,6-dinitrotoluene (2A), 4-amino-2,6-dinitrotoluene (4A),2,4-diamino-6-nitrotoluene (24DA), 2,2′,6,6′-tetranitro-4,4′-azoxytoluene, and 4,4′,6,6′ -tetranitro-2,2′-azoxytoluenewere obtained through the generosity of Dr. R. J. Spanggordof SRI International, Menlo Park, CA. 2,6-Diamino-4-nitrotoluene (26DA) was purchased from Aldrich.

Redox measurements were performed using a Corningplatinum combination redox electrode. Redox measure-ments were taken at the soil/water interface of each replicateculture. There was no sulfate present in the soil nor addedto the reactors, so sulfate interference with the probe wasnot a problem. TOC measurements were performed usinga Dohrman DC-80 TOC analyzer. Samples of 1 mL of theaqueous phase were centrifuged to remove solids, acidifiedwith phosphoric acid and sparged with nitrogen to removeinorganic carbon before UV-promoted persulfate oxidationin the TOC analyzer. Desorption isotherms were carriedout by placing various amounts of Umatilla soil in 100 mLof water or phosphate buffer. The concentrations of TNTin the aqueous phase were measured at different timepoints. Once the concentration in the aqueous phase stayedrelatively constant the concentration of TNT in the soil wasalso determined. Statistical analyses were performed usingthe statistical software package SigmaStat (Jandel Scientific).One-way Anova, Dunnetts, and Student Newman-Keulstests were performed to compare multiple means to acontrol or to each other.

Results and DiscussionSorption isotherms performed with Umatilla soil controlflasks showed that the munitions compounds were notsorbed to the soil but were merely present as a part of thesoil matrix. When the amount of soil used containedconcentrations of TNT that produced concentrations ofTNT in the aqueous phase that were lower than the solubilitylimit of TNT, there was no detectable TNT remaining in thesoil. This suggests that the presence of TNT in the aqueousphase was governed by dissolution of the free product andthe solubility of TNT in the aqueous phase, as opposed toreflecting an equilibrium with TNT sorbed to the sand. Theamount of Umatilla soil used was set at 1% as this providedTNT in concentrations just at its solubility limit and allowedfor a straightforward interpretation of the results.

In controls where the inoculum or external carbon sourcewas omitted from the reactor vessels, TNT degradation didnot occur. The ability of cultures fed 1% (w/v) initialconcentrations of glucose, soluble starch, insoluble starch,or acetate added as external carbon sources to consumeoxygen and create anaerobic conditions necessary forremoval of TNT and its reduced intermediates fromcontaminated soil was examined. The addition of glucoseto the statically incubated soil cultures allowed the fastestreduction of the redox potential (Figure 1). The redoxpotential was measured at less than -400 mV after 4 daysof incubation. This redox potential was maintained in thesecultures for over 20 days. A complementary removal ofTNT was observed in these cultures (Figure 2). The initialincrease in TNT concentration over the first day ofincubation, seen in this and other figures, is due to thedissolution of TNT from the soil into the aqueous phase ofthe cultures.

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The redox potential in the cultures fed insoluble starchwas reduced to below 50 mV within 2 days and wasmaintained at this value for the entire incubation period.The presence of TNT and 4A in the cultures hold the redoxpotential at 50 mV due to their oxidized nature. In thecultures receiving insoluble starch as the external carbonsource, the TNT was never completely removed (Figure 2);therefore, the redox potential in the cultures did not gobelow 50 mV. The apparent lack of ability to completelyremove TNT in these cultures was investigated further andwas discovered to be due to an inhibitory effect of theaccumulation of 4-amino-2,6-dinitrotoluene (manuscriptin preparation). In all cases when TNT was degraded, theproduction of 4-amino-2,6-dinitrotoluene and then 2,4-diamino-6-nitrotoluene was observed. These intermediateswere eventually removed in all cultures except when theredox potential increased to above -100 mV.

The redox potential in the cultures fed acetate or solublestarch was not reduced to values much below 200 mV(Figure 1) nor was TNT removal accomplished in thesecultures (Figure 2). The data for the TNT concentration foracetate cultures are identical to the data from isothermand control studies. The TNT initially dissolves into the

aqueous phase, this is essentially complete by 2 days ofincubation. After this period, the TNT concentrationremains relatively constant when biological activity isabsent. In these cultures as well as in isotherm and controlcultures, the production and accumulation of the reductionintermediates were not detected.

The inability of acetate to act as an electron donor foroxygen removal was unexpected. It is possible that TNTacts as an inhibitor of respiration, which has been observedfor other nitroaromatic compounds such as Dinoseb anddinitro-o-cresol. Since acetate is a nonfermentable sub-strate, inhibition of respiration would prevent growth andoxygen utilization by these organisms. It is also possiblethat TNT inhibits either the TCA cycle or the glyoxylateshunt, which are the pathways required for organisms togrow on acetate. This inhibition may not effect growthand oxygen utilization in cultures fed glucose or starchbecause these substrates are fermentable, thus organismscan increase in number to the point where they canovercome the inhibition by TNT and utilize oxygen. Thelack of growth and oxygen utilization in the cultures fedsoluble starch may be due to a lack of starch-degradingorganisms in the inoculum used. In contrast, the insolublestarch contains large numbers of starch degrading aerobes(28, 37).

The concentrations of TNT and its metabolic interme-diates over the time course of the incubation in the culturesfed glucose at 1% (w/v) are presented in Figure 3. Thereduction of TNT to 4-amino-2,6-dinitrotoluene and 2,4-diamino-6-nitrotoluene as well as the removal of thesecompounds was complete by 14 days (in other experimentsthe anaerobic phase was complete in as little as 8 days).This is an improvement of 10 days in the time required forthe anaerobic stage over that presented by Funk et al. (31)in which the anaerobic stage took 24 days to reach thispoint. The initial amount of soil contained enough TNTto provide an aqueous phase concentration of approxi-mately 0.53 mM. This was never observed in biologicalcultures due to the degradation of TNT occurring as it wasdissolving into the aqueous phase. Higher concentrationsof the intermediates are observed than TNT because theyare degraded much more slowly than the TNT. The 2,4-diamino-6-nitrotoluene accumulates to a higher concen-tration than the 4-amino-2,6-dinitrotoluene because it isdegraded slower than 4-amino-2,6-dinitrotoluene. The4-amino-2,6-dinitrotoluene accumulates to higher con-centrations than the TNT because it is degraded more slowlythan the TNT.

A fungus was observed growing on the surface of thecultures reported in the work by Funk et al. (31). This wasnot observed in the present work. In the present work anaerobic stage was added after 25 days of static incubationin order to act as a polishing step to remove excess oxygendemand remaining from the glucose added and fermenta-tion products of TNT. TOC in the culture supernatantswas analyzed as an indication of the amount of organiccarbon remaining at each time period. This does notprovide information concerning the specific chemicalspresent in the cultures, but gives a general impression ofthe amount of organic carbon, which can be related tooxygen demand, remaining in the cultures at any time point.The initial TOC of 1% glucose solution should be 360 mM.The TOC left after the anaerobic stage was 175 mM, thus185 mM organic carbon was used to create and maintainthe anaerobic conditions in the reactor. The aerobic stage

FIGURE 1. Redox potential in cultures of munitions-contaminatedsoil inoculated with treated soil and fed to 1% (w/v) initialconcentrations of 2 insoluble starch, 9 soluble starch, 1 acetate,and b glucose in 50 mM phosphate buffer with 25 mM NH4Cl. Datapoints represent the average of redox potential measurements intriplicate reactors. The error bars indicate one standard deviation.

FIGURE 2. TNT concentration in cultures of munitions-contaminatedsoil inoculated with treated soil and fed to 1% (w/v) initialconcentrations of 2 insoluble starch, 9 soluble starch, 1 acetate,and b glucose in 50 mM phosphate buffer with 25 mM NH4Cl. Datapoints represent the average of TNT concentrations in the aqueousphase in triplicate reactors. The error bars indicate one standarddeviation.

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required an extra 50-55 days to remove the majority of thecarbon present. This renders the aerobic polishing step asan uneconomical addition to the treatment procedure.

The reduction of the redox potential and the removal ofTNT and its metabolic intermediates from the cultures fed1% (w/v) glucose occurred very rapidly. There was also2100 mg/L (175 mM) TOC left in these cultures at the endof the anaerobic incubation. This suggests that the glucosehad been added in excess. Experiments were conductedto determine whether smaller amounts of glucose could beadded and still provide enough carbon for creation andmaintenance of anaerobic conditions in the staticallyincubated soil slurry cultures. Figure 4 presents the redoxpotentials measured during the incubation of munitions-contaminated soil cultures fed various amounts of glucose.The cultures fed 1%, 0.75%, 0.5%, and 0.25% all reducedthe redox potential to levels near or below -400 mV andmaintained this level. Results from the cultures fed 0.5and 0.75% glucose were not statistically different (P ) 0.05)from those fed 1% glucose, so they were not included in thefigure. Redox potentials of -200 to -300 mV were observedin cultures fed 0.1% while the cultures fed 0.01% glucosereduced the redox potential only to -100 and could notmaintain this redox potential.

Figure 5 presents the TNT concentrations in thesecultures. The use of 1% and 0.25% glucose as an externalcarbon source allowed the rapid and complete removal ofTNT from the cultures. Results from cultures fed 0.75%and 0.5% glucose were not statistically different (P ) 0.05)from those presented for 1% and 0.25% glucose. In culturesfed 0.1% glucose, an initial rapid reduction in the TNTconcentration was followed by an increase in TNT con-centration. This resulted in significantly greater amountsof TNT in these cultures than in cultures fed greater amountsof glucose. The cultures fed 0.01% glucose were not capableof totally removing the TNT from the aqueous phase.

The results of TOC analyses of the cultures fed decreasinglevels of glucose are presented in Figure 6. TOC removaloccurred at about the same rate in all of the cultures,independent of the TOC level present until TOC levels of100 mg/L were observed. This indicates that the oxygen

FIGURE 3. Time course analysis of TNT, its metabolites, and TOC in cultures of munitions-contaminated soil inoculated with treated soiland fed 1% (w/v) of glucose in 50 mM phosphate buffer with 25 mM NH4Cl. Data points represent the average of triplicate analyses of 4TNT, 9 RDX, ( 4-amino-2,6-dinitrotoluene, " 2,4-diamino-6-nitrotoluene, and b TOC. An aerobic stage was initiated after 25 days of anaerobicstatic incubation. The error bars indicate one standard deviation.

FIGURE 4. Redox potential in cultures of munitions-contaminatedsoil inoculated with treated soil and fed glucose to initialconcentrations of 2 0.01% (w/v), 1 0.1% (w/v), 9 0.25% (w/v), and` 1% (w/v) in 50 mM phosphate buffer with 25 mM NH4Cl. Datapoints represent the average of redox potential measurements intriplicate reactors. The error bars indicate one standard deviation.

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supply was the limiting factor. Cultures fed 0.25% glucosehad removed the TOC present by day 21 (7 days aerobicincubation) leaving approximately 30 mg/L TOC residual.A concentration of 0.25% glucose was chosen as theoptimum amount of external glucose to add since this wasthe lowest concentration of glucose fed to cultures thatresulted in both a rapid reduction of the redox potentialand TNT removal and the minimal time required for theaerobic stage. The results of the HPLC and TOC analysis

of cultures fed 0.25% glucose are presented in Figure 7.TNT, RDX, and the TNT reduction products were removedby day 14, and an aerobic stage instituted on day 15 allowedthe reduction of the TOC from approximately 400 mg/L(33mM) to 30 mg/L (2.5 mM) in 7 days. The wholeprocedure required 21 days of incubation. Past optimiza-tion experiments had resulted in an estimated 24 days timefor completion of the anaerobic stage alone.

AcknowledgmentsThis research was funded through a cooperative agreementwith the U.S. Environmental Protection Agency’s Environ-mental Research Laboratory in Athens, GA.

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Water Compounds in SoilsTNT, RDX, HMX; Technical ReportNATICK/TR-85/046; United States Army Natick Research andDevelopment Center: Natick, MA, 1985.

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A. M. Effects of Environmental Factors on the Transformation of2,4,6-Trinitrotoluene in Soils; Technical Report NATICK/TR-85/052; United States Army Natick Research and DevelopmentCenter: Natick MA, 1985.

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FIGURE 5. TNT concentrations in cultures of munitions-contaminatedsoil inoculated with treated soil and fed glucose to initialconcentrations of 2 0.01% (w/v), 1 0.1% (w/v), 9 0.25% (w/v), and` 1% (w/v) in 50 mM phosphate buffer with 25 mM NH4Cl. Datapoints represent the average of TNT concentrations in the super-natants of triplicate reactors. The error bars indicate one standarddeviation.

FIGURE 6. TOC in cultures of munitions-contaminated soil inoculatedwith treated soil in a 50 mM phosphate buffer solution with 25 mMNH4Cl and fed glucose to initial concentrations as indicated in figure.Data points represent the average of TOC measurements in triplicatereactors. The error bars indicate one standard deviation.

FIGURE 7. Time course analysis of TNT, its metabolites, and TOCin cultures of munitions-contaminated soil inoculated with treatedsoil and fed 0.25% (w/v) of glucose in 50 mM phosphate buffer with25 mM NH4Cl. Data points represent the average of triplicate analysesof 4 TNT, 9 RDX, ( 4-amino-2,6-dinitrotoluene, " 2,4-diamino-6-nitrotoluene, and b TOC. An aerobic stage was initiated after 14days of anaerobic static incubation. The error bars indicate onestandard deviation.

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(19) Gorontzy, T.; Drzyzga, O.; Kahl, M. W.; Bruns-Nagel, D.; Breitung,J.; von Loew, E.; Blotevogel, K. H. Crit. Rev. Microbiol. 1994, 204,265.

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Bioremediation of Pollutants in Soil and Water; ASTM STP 1235;Schepart, B. S., Ed.; American Society for Testing and Materials:Philadelphia, 1994.

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Received for review October 31, 1995. Revised manuscriptreceived February 26, 1996. Accepted February 27, 1996.X

ES950814T

X Abstract published in Advance ACS Abstracts, May 1, 1996.

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