Bioremediation of Soils Contaminated with the Herbicide2-sec-Butyl-4,6-Dinitrophenol (Dinoseb)t
RUSSELL H. KAAKE, DEBORAH J. ROBERTS, TODD 0. STEVENS,: RONALD L. CRAWFORD,*AND DON L. CRAWFORD
Department ofBacteriology and Biochemistry and Center for Hazardous Waste Remediation Research,University of Idaho, Moscow, Idaho 83843
Received 2 December 1991/Accepted 27 February 1992
A novel soil treatment method for achieving the removal of dinoseb (2-sec-butyl-4,6-dinitrophenol) fromcontaminated soils was investigated. One soil contained dinoseb as the major contaminant, although severalother hazardous compounds were also present. A second soil was highly contaminated with dinoseb. Dinosebwas not degraded in these soils under the aerobic conditions at each site. Pretreatment of the soils by theaddition of a starchy potato-processing by-product and flooding with phosphate buffer stimulated theconsumption of oxygen and nitrate from the soils, thereby lowering the redox potential and creating anaerobicconditions. Anaerobiosis (Eh less than -200 mV) promoted the establishment of an anaerobic microbialconsortium that degraded dinoseb completely, without the formation of the polymerization products seen underaerobic or microaerophilic conditions. When dinoseb was present at low concentrations in a chronicallycontaminated soil, the natural microflora was capable of establishing anaerobic conditions and degradingdinoseb as a result of starch degradation. Inoculation of this soil with an aerobic starch-degradingmicroorganism and then an acclimated, anaerobic, dinoseb-degrading consortium did not improve dinosebdegradation. In a second acutely contaminated soil, these inoculations improved dinoseb degradation rates overthose of uninoculated controls.
Dinoseb is a teratogenic, nitrophenolic herbicide thatpersists as a soil contaminant at many sites in the PacificNorthwest of the United States. In some cases, the persis-tence of dinoseb in soils is thought to be due to the lack ofdinoseb-degrading microorganisms (24). Under well-aeratedconditions, dinoseb biodegradation apparently does not oc-
cur (15, 26). However, under microaerophilic or denitrifyingconditions, microorganisms transform dinoseb to its aminoand acetamido forms (6, 26, 29, 31), which apparently retainsignificant toxicity (6). These reactive amino derivatives canundergo enzymatic (19) and nonenzymatic (11, 27) oxidationto form polymeric materials. While such immobilization ofaromatic compounds is easily accomplished in soils (forexample, by composting), other microorganisms that arecommon in soils are capable of cleaving the azo linkages ofpolymerized aminoaromatic compounds (5, 8, 12, 33), lead-ing to the rerelease of toxic compounds. In some acutelydinoseb-contaminated sandy soils, dinoseb represents themajority of the soil organic carbon (24), exacerbating theproblems associated with polymerization reactions by mak-ing the soils "slow-release" reservoirs of toxic metabolitesfor decades.
It has been shown that dinoseb can be degraded anaero-
bically in aqueous cultures (26). Although several otheranaerobic microbiological systems have been described fordegrading aromatic compounds (2, 21), little information isavailable on practical means of using these cultures tobioremediate contaminated soils. Indigenous anaerobic bac-teria have been stimulated by the addition of nutrients andelectron acceptors (7, 22), and anaerobic microorganismshave been added to contaminated soil in the form of accli-
* Corresponding author.t Publication 92503 of the Idaho Agricultural Experiment Station.t Present address: K4-06, Battelle, Pacific Northwest Labora-
tory, Richland, WA 99352.
mated sewage sludge (13). The simple addition of strictlyanaerobic bacteria to soil, however, seems unpromising,because many anaerobes are inhibited or killed by exposureto even small amounts of oxygen. Although many anaerobicmicrosites exist in soil (23, 26), these generally are occupiedby native microbial strains. Exogenously added bacteriaoften are excluded from these sites. Moreover, to avoidpolymerization reactions and to achieve faster degradationrates for compounds such as dinoseb, the entire soil bulksolution must be anaerobic.Here we report the development of an inexpensive scheme
for the bioremediation of dinoseb-contaminated soil usinginoculation with an anaerobic consortium or a simple bioen-richment procedure. We have been able to determine how toefficiently establish anaerobic conditions in open containersof soil and to demonstrate the bioremediation of dinoseb-contaminated soil without specialized bioreactors or othersophisticated equipment.Our method should be useful for the biotreatment of other
nitroaromatic and aminoaromatic compounds in soils. Eventhough many of these compounds can be mineralized inwell-aerated cultures (8, 9, 17, 33), they are also subject topolymerization reactions under the microaerophilic or aero-bic conditions (19) that are almost certain to occur in anypractical soil treatment system. Our procedures avoid thesecomplications.
MATERIALS AND METHODS
Bacterial strains and culture media. The anaerobic din-oseb-degrading microbial consortium was enriched by ex-
tended chemostat selection as previously described (26). Theconsortium contained at least three morphologically distinctbacterial species and degraded dinoseb via a series ofreduced aromatic intermediates (see Results). Dinoseb was
not degraded unless the medium was prereduced to a redox
potential of less than -200 mV. The consortium was main-tained on a reduced anaerobic mineral medium (RAMM) (pH7.0) containing (per liter of distilled water) 0.27 g of KH2PO4,0.35 g of K2HPO4, 1.5 g of NH4Cl, 0.5 g of glucose, 0.1 g ofyeast extract, 15 mg of CaCl2. 2H20, 20 mg ofMgCl2 6H20, 4 mg of FeCl2- 2H20, 0.5 mg ofMnCl2 4H20, 0.05 mg of H3BO3, 0.05 mg of ZnC12, 0.05 mgof CaCl2. 2H20, 0.05 mg of NiCl2 6H20, 0.03 mg of CuCl2,0.01 mg of NaMoO4. 2H20, 2.4 g of NaHCO3, 1 mg ofresazurin, and 0.02 g of NaS204. Cultures were incubated insealed containers in the dark without shaking at 30°C.A 2.5-liter dinoseb-degrading anaerobic digester consist-
ing of the consortium described above amended with freshanaerobic sewage sludge and fed dinoseb at a concentrationof 100 mg/liter in RAMM on a semicontinuous basis wasused as a supply of dinoseb-acclimated inoculum. Strictanaerobic procedures (10) were used during all mediumpreparation and transfer operations.An aerobic, amylolytic, dinoseb-resistant bacterium des-
ignated DSA was isolated from soil enrichments containing100 mg of dinoseb per liter and 5,000 mg of a starchypotato-processing by-product per liter (see below). It wasmaintained on an aerobic mineral medium of the samecomposition as RAMM, except that glucose, resazurin,dithionite, and bicarbonate were omitted. NH4Cl was ad-justed to 0.1 g/liter, and 2 g of soluble starch per liter wasadded. Cultures were incubated at 25°C in the dark andagitated at 200 rpm on a rotary shaker.
Soils. Two dinoseb-contaminated soils were used. The firstwas a sandy loam from an airstrip near Ellensburg, Wash. Itwas contaminated with dinoseb, as well as other herbicidesand fertilizers as a result of crop-dusting equipment washingover a period of several decades.The second soil was a silt loam from an airstrip near
Hagerman, Idaho. Contamination of this soil resulted fromthe leakage of dinoseb from several storage barrels. Extract-able nitrate and ammonium (4, 30), available potassium andphosphorus (18), and sulfate (1) concentrations were deter-mined by the University of Idaho Analytical Laboratory.Table 1 lists the inorganic characteristics of the two soils.Soils were sieved through a 2-mm-pore-size screen andstored at 4°C until used.
Dinoseb-contaminated Ellensburg soil that had previouslyundergone the treatment described below was used as aninoculum for Hagerman soil. Our target cleanup concentra-tion after treatment was 2.5 ppm of dinoseb for the Hager-man site. Soil dinoseb analyses done by the proceduredescribed below indicated an undetectable amount of din-oseb in treated Ellensburg soil. For quality assurance, por-tions of the treated soil were sent to a State of Washington-contracted laboratory, which reported 74 ppb of dinoseb.Treated soil was air dried, crushed, and stored at 4°C untilused. Treated soil had been stored for approximately 6weeks before the experiments were begun.
Carbon substrate. A by-product "centrifuge cake" from apotato-processing plant was provided by the J. R. SimplotCo., Caldwell, Idaho. It was analyzed for solid content by
weighing after oven drying and for total nitrogen content bythe Kjeldahl procedure (16). Available starch was deter-mined by incubating sterilized 1-g samples with 300 U ofac-amylase and 100 ,ul of Diazyme L-100 (Miles Pharmaceu-ticals, Kankakee, Ill.) for 24 h in 10 ml of sterile 0.4 Mphosphate buffer (pH 7.0). After incubation, the sampleswere diluted to 100 ml and analyzed for reducing sugars bythe dinitrosalicylate assay (14) with similarly prepared glu-cose as a standard. Available starch was assayed as milli-grams of sugar released per gram (dry weight) of by-product.Bacterial numbers in the by-product were determined bystandard plate counts on aerobic mineral medium agar (pH7.0) containing 2 g of glucose, 0.4 g of yeast extract, 1 g ofNH4C1, and 1 g of NaNO3 per liter for total heterotrophiccounts or 2 g of soluble starch, 1 g of NH4C1, and 1 g ofNaNO3 per liter for amylolytic bacteria.
Prior to the experiments, the by-product was autoclavedfor 20 min to reduce the number of viable bacteria.
Experimental design. Experiments with Ellensburg soilwere performed in triplicate as follows. Soil (300 g) wasplaced in a sterile, 500-ml, wide-mouth Erlenmeyer flask andflooded with 300 ml of 50 mM phosphate buffer (pH 7.0) toform a slurry. Flasks were covered with foil and incubatedwithout shaking at 30°C in the dark. Culture variablesincluded the addition of 6 g of autoclaved potato by-productand inoculation with 10 ml of bacterial strain DSA (opticaldensity at 600 nm, >1.0) and then with 15 ml from the2.5-liter anaerobic digester after the redox potential wasbelow -200 mV.Experiments with contaminated Hagerman soil were per-
formed in duplicate as described above, except that eachculture contained 200 g of soil, 4 g of autoclaved potatoby-product, and 200 ml of phosphate buffer. In addition toinoculation with 10 ml of the DSA culture and 15 ml from the2.5-liter anaerobic digester, the effect of using dried, treatedsoil as an inoculum was also investigated. Triplicate culturescontaining a total of 200 g of soil were prepared with 10 and50% (by weight) previously treated soil from the Ellensburgsite, allowing examination of the effect of inoculation of thesoil (10%) versus the effect of dilution of the dinoseb inaddition to inoculation (50%).To show that organisms in the potato by-product were not
responsible for the observed dinoseb degradation, we ad-justed a no-soil control containing 300 ml of aerobicallyprepared RAMM (without sodium dithionite) to approxi-mately 100 mg of dinoseb per liter by using a stock solutioncontaining 10,000 mg of dinoseb per liter dissolved in diluteNaOH. Six grams of autoclaved potato by-product wasadded to the flask.Sampling procedures. In all experiments, pH and redox
potential were measured potentiometrically at the soil-aque-ous phase interface. The aqueous phase was usually 3 to 5cm deep and was quite homogeneous. A 3-ml sample of theaqueous phase was removed with as little disturbance to theculture as possible for high-performance liquid chromatog-raphy (HPLC), nitrate, and ammonium analyses as de-scribed below. Soil samples of approximately 5 g were taken
by use of a spoon from the initial soil before it was mixedwith the aqueous phase and from the soil phase of the cultureafter dinoseb was no longer detectable in the aqueous phase.The excess aqueous phase was allowed to drain from the soilsamples before they were weighed. The results from theseextractions were therefore used for comparative purposesand as an indication of the completeness of the treatment.When the treatment was determined to be complete, theaqueous phase was decanted. The soil was dried at roomtemperature and sent to Manchester Laboratories (Manches-ter, Wash.) for analyses following strict EnvironmentalProtection Agency protocol to confirm our results.
Analytical methods. Dinoseb concentrations were deter-mined by HPLC with a Phenomonex (Torrance, Calif.)Spherex 5-,um C18 reverse-phase column (250 by 2 mm). AHewlett-Packard model 1090A instrument equipped with adiode array UV/VIS detector and a computerized datasystem was used for the analyses. The column was run with10% acetonitrile and 90% 11 mM phosphate buffer (pH 4.0)for 2 min, and then the acetonitrile was increased to 85%over the next 15 min. The acetonitrile was then increased to100% over 1 min and sustained at that level for 2 min. Theacetonitrile was then decreased back to 10% over 2 min. Thesolvent flow rate was 0.4 ml/min, and the column tempera-ture was 40°C. Dinoseb and possible transformation prod-ucts were detected by use of the diode array UVNVISdetector, recording the A210 with continuous scanning of theabsorption spectrum of each peak from 190 to 600 nm. Thedetection limit for dinoseb was determined to be 1 mg/liter.Dinoseb was extracted from 5 g of soil by sonication for 5
min in 5 ml of acetonitrile at 5°C. Prior to extraction, eachsample was amended with 100 pl of a 5,000-mg/liter stocksolution of 4,6-dinitro-o-cresol (DNOC) in methanol (whichacted as an extraction standard for monitoring intermediateproduction), and the methanol was allowed to evaporate.Aliquots of 1 ml of the extracts were passed through aprecolumn filter containing C18 packing material, and 10 RIwas injected into the HPLC column. Extraction efficiencieswere determined to be at least 98% for DNOC in all of theextractions. Dinoseb was recovered at an efficiency of 100%from dinoseb-spiked samples.The redox potentials of the cultures were measured at the
soil-aqueous phase interface with a platinum electrode (Ori-on model 96-78).
Nitrate and ammonium concentrations were determinedwith an ion-selective electrode (Orion model 95-12).
RESULTS AND DISCUSSION
Carbon substrate. To sustain anaerobiosis in the soiltreatment process, we required a source of readily degrad-able carbon as a supplement. We evaluated a number ofstarchy by-products from local food processors for this use.The desired characteristics of the by-product included a highnutrient content and low numbers of heterotrophic bacteriathat might compete with the inoculum. We chose dewateredsolids from a potato-processing plant as the preferred sub-strate. The characteristics of the substrate included thefollowing: 42% solids, 215 mg of available starch per g, 6.7mg of total nitrogen per g, 2.6 x 104 culturable heterotrophicbacteria per g, and 8 x 103 culturable amylolytic bacteria perg. The substrate was autoclaved prior to use in experimentsto reduce the numbers of organisms present. This treatmenthydrolyzed the starch to some extent, releasing reducingsugars from the starch matrix.
In control cultures designed to determine the effect of the
starch inoculum on dinoseb present in cultures not contain-ing any soil, the redox potential was reduced to less than-200 mV within 8 days and was maintained throughout theincubation. Dinoseb was removed to below detectable levelsby day 10. However, intermediates were still present after 76days, and a brownish red precipitate consistent with theformation of polymerization products covered the surface ofthe soil. This result suggests that the surviving organisms inthe potato by-product were capable of carrying out onlysuperficial modifications of the dinoseb molecule. The com-plete removal of dinoseb and its detectable transformationproducts was only accomplished in cultures receiving soilcontaining the appropriate microflora.
Ellensburg soil treatments. Simply flooding soils with phos-phate buffer did not produce anaerobic conditions. Figure 1illustrates the necessity for the addition of starch to induceanaerobiosis within the bulk soil solution. Also, inoculationwith DSA and the anaerobic consortium did not significantlylower the redox potentials, in comparison with those in theuninoculated controls, for the Ellensburg soil. Culturesattained a redox potential of less than -200 mV within 5days and maintained this redox potential throughout theremainder of the experiment.
Figure 2 shows an initial desorption of dinoseb from thesoil into the aqueous phase of the cultures shown in Fig. 1.When starch was not present, desorption of dinoseb from thesoil into the aqueous phase occurred over the first 2 days ofincubation. An equilibrium was reached between 2 and 3days at about 215 mg of dinoseb per liter in the aqueousphase. In cultures containing starch, the concentration ofdinoseb in the aqueous phase did not reach the equilibriumseen in the no-starch cultures because of the biologicalactivity. The initial biological removal of dinoseb did notexceed the desorption rate, so an initial increase in thedinoseb concentration was observed. After 2 days of incu-bation, the degradation of dinoseb in cultures containingstarch exceeded the desorption from the soil. This timeperiod for the rapid removal of dinoseb from the aqueousphase correlated with the initial drop in the redox potential(Fig. 1). The rapid decrease in the redox potential and thedesorption of new dinoseb from the soil into the aqueousphase precluded an accurate determination of the maximumredox potential at which dinoseb degradation may occur. By15 days, dinoseb was not detected in the aqueous phase ofthe cultures. At this time, soil extractions were performed,and no dinoseb was detected. Inoculation of the soil with thepreacclimated anaerobic consortium did not significantlyaffect the rate of dinoseb degradation in these cultures.
Figure 3 shows the effect of starch addition on the utiliza-tion of nitrate and ammonium in the cultures shown in Fig. 1and 2. In cultures lacking starch, nitrate and ammoniumlevels remained relatively constant. When starch waspresent, nitrate was removed within 4 days. Ammoniumlevels decreased slowly and then remained low (=10 to 15ppm as N) for the remainder of the incubation. It is pre-sumed that nitrate was utilized as an alternate electronacceptor or as a nitrogen source by anaerobically respiringbacteria after oxygen had been consumed by amylolyticaerobes, as previously reported (3, 32). The removal ofnitrate corresponded to the rapid decrease in the redoxpotential (Fig. 1).These results indicate that the Ellensburg soil contained
an indigenous dinoseb-degrading population. Thus, only asimple enhancement procedure to establish the requiredenvironment for dinoseb degradation was necessary to treatthis soil. Nitrate was removed quickly, so that strictly
Time (d)FIG. 1. Redox potentials in Ellensburg soil cultures. Error bars indicate 1 standard deviation.
starch addition; 0, 2% starch addition and 10% sludge inoculum addition. d, days.Symbols: El, no starch addition; 0, 2%
fermentative anaerobic conditions were established within 4 the treatment procedure, all contaminants present prior todays. treatment were degraded to some extent (Table 2). The soilDinoseb degradation in this soil occurred even though from cultures used to provide the information for Fig. 1 to 3
other contaminants were present. As an additional bonus to was pooled, dried, extracted, and analyzed to determine
250
200
-J
150E%-O
.0
° 100._
50
0
; 13-f '00
0 2 4 6 8 10 12 14 16Time (d)
FIG. 2. Aqueous-phase dinoseb concentrations in Ellensburg soil cultures. Error bars indicate 1 standard deviation. Symbols are asdefined in the legend to Fig. 1. d, days.
Time (d)FIG. 3. Nitrate and ammonium analysis of Ellensburg soil. Error bars indicate 1 standard deviation. Symbols: El, nitrate in cultures
receiving no starch; *, ammonium in cultures receiving no starch; 0, nitrate in cultures receiving 2% starch; *, ammonium in culturesreceiving 2% starch. d, days.
total herbicide and pesticide concentrations. The chemicalformulas for the contaminants detected are as follows:MCPP, 2-(4-chloro-o-tolyl)oxypropionic acid; Ioxynil, 4-cy-ano-2,6-diiodophenol; 2,4-D, (2,4-dichlorophenoxy)aceticacid; and dicamba, 3,6-dichloro-2-methoxybenzoic acid.These are all halogen-substituted aromatic compounds andrepresent a sampling of the types of herbicides commonlyused in the Pacific Northwest of the United States. It is veryencouraging that the treatment procedure designed for ni-troaromatic compounds was effective against halogen- andcyano-substituted compounds. This procedure may be avaluable technique in the remediation of multiply contam-inated soils, which may be common at crop-dusting opera-tion sites.Hagerman soil treatments. The reduction of redox poten-
tials in Hagerman soil treatments (Fig. 4) occurred moreslowly than that in Ellensburg soil treatments (Fig. 1). Theaddition of starch alone to Hagerman soil cultures resulted in
TABLE 2. Herbicide remediation of Ellensburg soila
Herbicide concn, pug/kg (SD), in: %Compound Initial soil, Final aqueous Final soil, Removal
a Herbicide and pesticide analyses were performed by Manchester Labo-ratories. Environmental Protection Agency methods 8150 and 8080 (28) wereused. The compounds listed were the only ones detected. Triplicate sampleswere taken from pooled, dried soils after 46 days of incubation. ND, notdetected.
a lag period of 6 days prior to a drop in the redox potential.Inoculation with DSA and the preacclimated anaerobicconsortium shortened the lag period to 3 days. However, themost dramatic shortening of the lag period was seen whencultures were inoculated with previously treated soil. When50% (by weight) treated soil was added to the Hagerman soil,the lag period was virtually eliminated and the pattern ofdinoseb removal was very similar to that in the originalEllensburg soil (Fig. 1). In these cultures, the desorption ofdinoseb into the aqueous phase from the contaminated soilmay have been effected by the adsorption of dinoseb ontothe treated soil added to the cultures as well as the dilutionof the total amount of dinoseb present because less contam-inated soil was used. The extent of the effects of these twophysical parameters on the biological activity of the cultureswas not determined because of the difficulty in completelysterilizing the soil and the effects of the sterilization proce-dures on the contaminants present. A 10% inoculum, whichwould not affect the physical parameters of the cultures tothe same extent as a 50% inoculum, shortened the lag periodto about 1 day. This lag period was still shorter than the lagperiod of cultures inoculated with DSA and the preaccli-mated anaerobic consortium.
Differences in inocula also affected dinoseb degradation inthe cultures shown in Fig. 4. Initial extracts of the Hagermansoil showed a dinoseb concentration of about 700 mg/kg. Allcultures showed an initial period of dinoseb solubilizationfrom the soil into the aqueous phase, after which the rate ofdegradation exceeded the rate of solubilization. Initial lagperiods in which the rate of degradation exceeded the rate ofsolubilization for these cultures were approximately 10 daysfor the uninoculated cultures, 3 days for the cultures inocu-lated with DSA plus sludge, and 2 days for the culturesinoculated with 10 and 50% previously treated soil. In the
uninoculated cultures, significant dinoseb degradation didnot take place until well after the redox potential was in theanaerobic range, indicating that the natural flora of the soilrequired an adaptation period in addition to anaerobic con-ditions. The cultures inoculated with DSA plus sludge didnot require a long adaptation period but degraded dinoseb ata much slower rate than the cultures inoculated with previ-ously treated soil. This observation reflects the advantagethat a soil inoculum has over an aqueous inoculum whenused in soil treatments. Dinoseb was removed from theaqueous phase in the cultures inoculated with 50 and 10%previously treated soil by 9 and 33 days, respectively. Thecultures inoculated with DSA plus sludge and the uninocu-lated cultures showed no detectable dinoseb at days 41 and50, respectively.Evidence for the formation of dinoseb polymerization
products was seen in uninoculated cultures and, to someextent, in cultures inoculated with DSA plus sludge. Aniridescent brownish red precipitate formed at the aqueousphase surface and then sank to the soil surface. This precip-itate was still present at the end of the experiment in bothsets of cultures. From this observation, we concluded thatsome of the dinoseb was partially modified to its hydroxylamino derivatives, which were retained in the culture super-natant long enough to be polymerized at the aqueous phasesurface. This observation underscores the importance offacilitating the creation of anaerobic conditions rapidly sothat degradation is possible and the longevity of toxic andreactive intermediates is minimized. The use of previouslytreated soil as an inoculum accomplished this, even at thehigh dinoseb concentrations seen in the Hagerman soil.The pattern of the utilization of nitrate in Hagerman soil
cultures was similar to the pattern of the redox potential.Nitrate was removed quickly (within 2 days) in culturesinoculated with 50% previously treated Ellensburg soil and
by day 8 in uninoculated cultures. Inoculations with DSAplus sludge and 10% previously treated Ellensburg soilresulted in nitrate removal within 4 days of incubation.Nitrate removal corresponded to the apparent shift fromdinoseb desorption to degradation in cultures inoculatedwith 10 and 50% previously treated soil and DSA plus sludgebut preceded degradation by 2 days in uninoculated cultures.
In this heavily contaminated soil, the use of an acclimatedinoculum served to reestablish a flora of microorganismscapable of the rapid removal of oxygen and nitrate in thecultures, as seen by the rapid reduction of the redox poten-tials. The inoculum also provided microorganisms capable ofdegrading dinoseb once anaerobic conditions were estab-lished.
Biotransformation products. The disappearance of dinosebis not a sufficient measure of bioremediation, because toxicaminoaromatic intermediates may be formed during remedi-ation processes. With the spectra obtained from the diodearray UV/VIS detector of the HPLC, it was possible tomonitor the production and degradation of aromatic dinosebtransformation products. In the above-described experi-ments, intermediates were observed during the course of theincubations. Incubations were continued until our analysesshowed that no aromatic intermediates were present in theaqueous phase or in the soil. In cultures more competent atdegrading dinoseb, such as the Ellensburg soil, intermedi-ates did not accumulate significantly, whereas in less com-petent cultures, significant intermediate accumulation didoccur. The amount of dinoseb carbon contained in theseintermediates could not be determined, as the identities ofthe compounds are still unknown. Previous studies showedthat the majority of dinoseb carbon can be traced to acetateby use of U-'4C-labelled dinoseb (25).
Conclusions. These bench-scale experiments indicate thatthis process may be applicable to the bioremediation of
nitroaromatic compound-contaminated soils on a commer-cial scale. Because of the relatively inexpensive materialsinvolved, biological treatments such as this one may proveto be highly economical methods of soil cleanup. Suchmethods should be particularly advantageous to owners ofdinoseb-contaminated sites, since many of these sites areowned by small businesses (e.g., crop dusters), which maynot be able to afford current remediation technologies, suchas incineration.
This process may also be applicable to the biotreatment ofsoils contaminated with other nitroaromatic compounds,such as the explosive trinitrotoluene and related molecules.Preliminary work (20) indicates that trinitrotoluene is de-graded under strictly anaerobic conditions in similar soiltreatments.Experiments are currently in progress to show that this
process can be scaled up to a feasible commercial scale.Future experiments will focus on the identification of theintermediates detected in the cultures and the illumination ofthe pathway of degradation of dinoseb.
ACKNOWLEDGMENTS
This work was funded by the Competitive Research Grants Pro-gram of the Idaho State Board of Education, as administered by theHigher Education Research Council; by the J. R. Simplot Co., Boise,Idaho; and by the U.S. Environmental Protection Agency under theEmerging Technologies Program (USEPA award CR816818010).We thank Wendy Davis-Hoover of the U.S. Environmental
Protection Agency, Cincinnati, Ohio, for suggestions and assistanceand Mike Cochran of the Washington State Department of Ecologyfor arranging quality assurance analytical chemistry services.
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