APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, p. 450-454 Vol. 57, No. 20099-2240/91/020450-05$02.00/0Copyright ©) 1991, American Society for Microbiology
Degradation of Toluene and m-Xylene and Transformation ofo-Xylene by Denitrifying Enrichment Cultures
PATRICK J. EVANS,' DZUNG T. MANG,' AND L. Y. YOUNG12*Departments of Microbiology' and Environmental Medicine,2 New York University Medical Center,
New York, New York 10016
Received 12 October 1990/Accepted 4 December 1990
Seven different sources of inocula that included sediments, contaminated soils, groundwater, processeffluent, and sludge were used to establish enrichment cultures of denitrifying bacteria on benzene, toluene, andxylenes in the absence of molecular oxygen. All of the enrichment cultures demonstrated complete depletion oftoluene and partial depletion of o-xylene within 3 months of incubation. The depletion of o-xylene wascorrelated to and dependent on the metabolism of toluene. No losses of benzene, p-xylene, or m-xylene wereobserved in these initial enrichment cultures. However, m-xylene was degraded by a subculture that wasincubated on m-xylene alone. Complete carbon, nitrogen, and electron balances were determined for thedegradation of toluene and m-xylene. These balances showed that these compounds were mineralized with greaterthan 50% conversion to CO2 and significant assimilation into biomass. Additionally, the oxidation of thesecompounds was shown to be dependent on nitrate reduction and denitrification. These microbial degradativecapabilities appear to be widespread, since the widely varied inoculum sources all yielded similar results.
Groundwater can become contaminated and undrinkableupon leakage of gasoline from underground storage tanks(13). Contamination is primarily due to the presence ofbenzene, toluene, and xylenes (BTX), these compoundsbeing more soluble than other components of gasoline suchas alkanes and polyaromatic hydrocarbons. Benzene is ofthe most concern because of its association with the devel-opment of leukemia in humans (4).Much research on the biodegradation of BTX has been
initiated in the hope of developing bioremediation technolo-gies to purify BTX-contaminated groundwater. All five BTXcompounds (including the three xylene isomers) have beenfound to be biodegradable under aerobic (1, 6, 14, 23) andanaerobic (7, 9-13, 22, 25) conditions. Aerobic degradationof toluene, p-xylene, and m-xylene has been shown to begenetically encoded by the TOL plasmid (23). Benzene (6,14) and only recently o-xylene (1) have been shown to bedegraded by pure cultures of aerobic bacteria. Anaerobicstudies have been completed in soil columns and micro-cosms under different reducing conditions, including iron-reducing, denitrifying, sulfidogenic, and methanogenic con-ditions. Anaerobic degradation of toluene has beendefinitively shown in pure culture under iron-reducing (12)and denitrifying (24) conditions. The toluene-degrading den-itrifier (24) was also shown to degrade m-xylene. On theother hand, anaerobic degradation of benzene, p-xylene, ando-xylene has not been observed in pure culture.Although studies of the anaerobic transformation of BTX
have consistently shown toluene and m-xylene to be biode-gradable, benzene, p-xylene, and o-xylene have been shownto be biodegradable under mixed culture conditions in cer-tain studies and not in others (5, 8-11, 13, 17, 22). No onefactor, i.e., substrate concentration or composition, temper-ature, terminal electron acceptor, or medium composition(i.e., mineral salts or microcosms prepared without media),appears to account for these different results. Interestingly,although losses of benzene under denitrifying conditions
* Corresponding author.
have been observed in microcosms, it is uncertain whetherthis activity is sustainable under these conditions. Theorganisms that are responsible for the anaerobic degradationof benzene, p-xylene, and o-xylene may be exceptionallyfastidious in light of these results and the lack of success intheir isolation.
In this study, a variety of sources of inocula were utilizedto enrich for denitrifying bacteria that are potentially capableof anaerobically oxidizing a BTX mixture. These sourcesincluded river sediment, soil, groundwater, anaerobic di-gester sludge, and process effluent. Degradation of individ-ual BTX compounds was analyzed with complete balanceson carbon, nitrogen, and electrons to assess the extent oftheir transformation.
MATERIALS AND METHODS
Sources of inocula. Table 1 lists the seven sources ofinocula that were used to start the enrichment cultures.Samples from the soil and sediment sources (ER, NC, CA2,and CA3) were diluted 1:1 or 1:2 (wt/wt) with water. Samplesfrom source CAl were centrifuged, and the solids were thensuspended in a small volume of the supernatant so that thesample solids were concentrated by a factor of 5. Samplesfrom the remaining sources (BH and KC) were used asobtained.Growth medium and initiation of enrichment cultures.
Inocula were added in 5-ml aliquots in triplicate to 60-mlserum bottles with 45 ml of a mineral salts medium (amodified version of that described by Taylor et al. [19]). Aliter of this medium contained the following (unless other-wise noted): 7.9 g of Na2HPO4 7H20, 1.5 g of KH2PO4, 0.3g of NH4Cl, 2.02 g of KNO3 (20 mM), 0.1 g ofMgSO4 7H20, 5 ml of trace elements solution (20), 10 ml ofvitamins solution, and 0.01 g of yeast extract. The traceelements solution contained the following (per liter): 50 gof EDTA, 22 g of ZnSO4 7H20, 5.54 g of CaCl2, 5.06 g ofMnCl2 .4H20, 4.99 g of FeSO4 7H20, 1.1 g of(NH4)6Mo7024 4H20, 1.57 g of CuSO4 5H20, and 1.61 gof CoCl2. This solution was adjusted to a pH of 6.0 with
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DEGRADATION OF BTX BY DENITRIFYING CULTURES
TABLE 1. Sources of inocula used to start the denitrifyingenrichment cultures
Name Type Origin
ER Sediment East River boat marina at 26thStreet in New York City, N.Y.
BH Anaerobic digester Second digester at Berkeleysludge Heights, N.J.
NC Soil Lagoon repository for bottoms froma naphtha cracking process
KC Effluent Activated carbon process forremediation of BTX-contaminatedgroundwater in Michigan
CAl Groundwater, Partially remediated gasoline spillsediments site in California
CA2 Soil Untreated gasoline spill site inCalifornia
CA3 Soil Site of a Stoddard solvent (apetroleum distillate) spill
KOH. The vitamins solution contained the following (perliter): 0.002 g of biotin, 0.002 g of folic acid, 0.01 g ofpyridoxine hydrochloride, 0.005 g of riboflavin, 0.005 g ofthiamine, 0.005 g of nicotinic acid, 0.005 g of pantothenicacid, 0.0001 g of B12, 0.005 g of p-aminobenzoic acid, and0.005 g of thioctic acid. The pH of the medium was adjustedto 7.5 with NaOH. The inoculated media were sparged for 30min with argon that had passed through a column of reducedR3-11 catalyst (Chemical Dynamics, South Plainfield, N.J.)to remove traces of oxygen. A mixture of benzene, toluene,p-xylene, m-xylene, and o-xylene (200 mM each) in metha-nol was added (25 ,ul) to the medium after sparging was
complete. The bottles were sealed with Teflon-coated butylrubber stoppers (West Co., Lancaster, Pa.) and aluminumcrimps. The resultant methanol concentration was 12 mM,and the resultant concentrations for BTX compounds were100 puM each. Stoichiometric calculations, assuming com-
plete mineralization of methanol and BTX, showed that 19mM N03- is required if N03 is reduced to N2. Althoughmethanol was present initially in the enrichment cultures asa carrier for BTX and as a cosubstrate, it was determined tobe unnecessary for toluene and m-xylene degradation andwas not used further. Initial samples were taken, and thecultures were incubated at 30°C in a stationary position. Thecultures were supplemented with additional KNO3 or indi-vidual BTX compounds upon depletion. All samples andadditions were via sterile syringes that were flushed withargon. This procedure resulted in puncturing the Tefloncoating on the stoppers. Therefore, the stoppers were re-placed with new ones after sampling to minimize sorptiveBTX losses through the stoppers. Anaerobic conditionswere maintained during stopper replacement by gently flush-ing the headspace with argon.Medium preparation for subcultures. Mineral salts medium
without yeast extract was added to serum bottles, whichwere then sealed with Teflon-coated butyl rubber stoppersand crimps. The bottles were then degassed by evacuatingand then pressurizing the headspace with 67 kPa of argon.The bottles were shaken vigorously to ensure effectivegas-liquid mass transfer of oxygen. The evacuation andfilling procedure was repeated three times. The BTX com-
pounds were added after deoxygenation, and then the mediawere inoculated.
Analytical methods. Volumetric gas production data andheadspace composition data were utilized to calculate theproduction of N20 and N2. The volume of gas produced was
measured with a water-lubricated glass syringe that wasflushed with argon. The bottle to be sampled was shaken andthen pierced with the syringe. The gas volume was recordedafter the headspace gas had flowed into the syringe and thepressure had equilibrated. The composition of the gas wasmeasured chromatographically with a gas partitioner (model1200; Fisher Scientific, Pittsburgh, Pa.) equipped with a3.35-m by 4.76-mm column packed with 60/80 mesh 13Xmolecular sieves (Supelco, Bellefonte, Pa.) in series with a1.98-m by 3.18-mm column packed with 80/100 mesh Pora-pak Q (Supelco). The total N2O (which includes gaseous anddissolved N20) was determined from the N20 measured inthe headspace and Henry's constant at 25°C (1.71 x 106 mmHg [21]).CO2 was measured by the addition of 1 ml of culture
medium to a 14-ml sealed tube that contained 100 ,ul of 10 NH2SO4 to convert HCO3- and C032- to CO2. The tube wasthen shaken to transfer the CO2 to its headspace, which wasthen assayed for CO2 by gas chromatography.
Nitrate and nitrite were measured spectrophotometrically(Hach Co., Loveland, Colo.). The method employs cad-mium reduction of nitrate to nitrite, formation of the diazo-nium salt with sulfanilic acid, and formation of a coloredcomplex with chromotropic acid.The concentrations of the BTX compounds were mea-
sured on a gas chromatograph (model 3700; Varian, Sunny-vale, Calif.) with flame ionization detection. A 1.75% Ben-tone 34-5% SP1200 on Supelcoport (100/120) column (1.83 mby 3.18 mm) was used. The flow rate was 20 ml of nitrogenper min. The temperatures of the injector, detector, andcolumn were 120, 200, and 70°C (isothermal), respectively.Samples (1 ml) were taken anaerobically from the enrich-ment cultures and extracted in 2-ml vials with 0.4 ml ofpentane that contained 1 mM nonane as an internal standard.Aliquots of 1 ,lI of the extracts were injected. Any aromatichydrocarbon that was adsorbed to sample solids was mea-sured as well as dissolved hydrocarbon, since the sampleswere not centrifuged or filtered before extraction.Dry cell weight was determined by centrifugation of a
culture and washing of the sedimented cells with distilledwater. After a second centrifugation, the cells were trans-ferred to a tared aluminum weighing dish with a minimalamount of distilled water and dried at 120°C overnight. Thedish that contained the cells was allowed to cool in adesiccator before measurement of the gross weight.
RESULTS
Fate of BTX in the initial enrichment cultures. All of theenrichment cultures demonstrated losses of 90 to 100% oftoluene within 1 to 3 months of incubation, with the excep-tion of one of the ER replicates. A typical example is shownin Fig. 1. Toluene was nearly depleted in the BH cultures bythe time the first sample was taken. A second addition oftoluene was also effectively depleted by these cultures.Substrate loss was accompanied by loss of nitrate andproduction of N2. Electron balance determinations were notpossible with these initial cultures because of the metabolismof methanol.The decreases in benzene, p-xylene, and m-xylene con-
centrations (Fig. 1) were largely due to volatilization into theheadspace and subsequent loss during stopper replacement.On the other hand, o-xylene depletion, although incomplete,appeared to be greater than that of benzene, p-xylene, andm-xylene. These losses also appeared to occur simulta-neously with that of toluene and were repeated upon a
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0 20 40 60 80 100 120
Time (d)FIG. 1. Fates of benzene (0), toluene (O), p-xylene (A), in-xy-
lene (V), and o-xylene (-) in the BH enrichment culture. Theconcentrations are averages for the replicates. Toluene (100 ,uM),methanol (12 mM), and KNO3 (10 mM) were added on day 39. Onday 69 the cultures were found to be depleted of N03 and were fed20 mM KNO3.
second addition of toluene (Fig. 1). Partial depletion ofo-xylene concomitant to that of toluene was observed withall inocula.A statistical comparison was made to (i) substantiate a
correlation between the o-xylene and toluene losses and (ii)determine that the losses of o-xylene were significantlygreater than the losses of benzene, p-xylene, and m-xylene.The loss of compound (i.e., benzene, p-xylene, m-xylene, oro-xylene) between two sample points was calculated foreach instance in which the toluene loss was greater than 50p.M. These data were averaged for each compound andinclude data from all of the enrichment cultures and repli-cates (n = 23) (Table 2). Averages were also calculated forthe ratio of the loss of benzene or xylene to the loss oftoluene for each period of toluene loss greater than 50 ,uM.An analysis of variance (3) of the average losses of benzene,p-xylene, m-xylene, and o-xylene (Table 2) demonstratedthat these average losses were not all equal (P < 0.001).Therefore, the loss of o-xylene (Table 2) was greater thanany loss of benzene, p-xylene, or m-xylene during a periodof toluene depletion. The same conclusion can be drawn forthe ratio of compound loss to toluene loss.Metabolism of toluene and o-xylene in a subculture of the
CA2 enrichment culture. One of the CA2 culture replicateswas diluted into fresh mineral salts medium without yeastextract and twice fed 100 ,uM toluene alone. Toluene wasdepleted after each addition. This culture was split into 25-mlsamples in 27-ml anaerobic tubes with or without toluene
TABLE 2. Comparison of losses of benzene and xylenes duringperiods of substantial toluene loss
BTX Avg loss" (,uM) Loss ratio'compound + SD ± SD
Toluene 86.4 ± 13.1Benzene 10.9 ± 9.1 0.12 ± 0.07p-Xylene 13.0 ± 9.7 0.15 + 0.07m-Xylene 12.4 ± 9.6 0.14 ± 0.05o-Xylene 22.7 ± 9.2 0.26 ± 0.05
" Average losses of benzene or xylene and the concomitant average loss oftoluene. Averages are for all inoculum sources.
b Average ratios of benzene or xylene loss to the concomitant toluene loss.
0 20 40 60 80 100 120
Time (h)FIG. 2. Losses of o-xylene in the absence (O) and presence (U)
of toluene (0) in a subculture of the CA2 enrichment culturepreviously grown on toluene. Error bars are shown for data after theinitial sample and are + 1 standard deviation. The standard devia-tion is less than half the width of the symbol in cases that show noerror bar.
and supplemented with benzene, p-xylene, m-xylene, oro-xylene, in duplicate for each condition. A sterile controlthat contained all five BTX compounds was autoclaved afteraddition of the compounds. The loss of o-xylene was greaterin the presence of toluene than that in its absence (Fig. 2).This enhancement of o-xylene depletion appeared to occurduring the active metabolism of toluene. In the absence oftoluene, negligible depletion of o-xylene occurred relative tothat in the sterile control, which contained all five BTXcompounds (data not shown). No enhancement of the lossesof benzene, p-xylene, or m-xylene was observed in thepresence of toluene even though toluene was completelymetabolized. The concentrations of these compounds re-mained the same as those in the sterile control.
Toluene metabolism coupled to nitrate reduction. CultureCA2 was further enriched by dilution and subculture ontoluene as the sole carbon source. This culture was thenused to inoculate (1%, vol/vol) duplicate 150-ml quantities ofmineral salts medium without yeast extract, nitrate, ormethanol. These cultures were amended as shown in Table 3and incubated for 7 days. Toluene metabolism was depen-dent on the presence of nitrate, and denitrification wasdependent on the presence of toluene (Table 3). Growth wasobserved only in the presence of toluene and nitrate. Nitritecan also serve as the sole terminal electron acceptor fortoluene oxidation, as evidenced by complete metabolism oftoluene and production of nitrous oxide and dinitrogen (datanot shown).Carbon, nitrogen, and electron balances for toluene degra-
dation. Table 4 shows balances on carbon, nitrogen, and
TABLE 3. Dependence of toluene loss and associated growth onnitrate of a subculture of enrichment CA2
Initial toluene Initial KNO3 Growth" Final toluene N2 concnconcn (,uM) concn (mM) concn (tiM) (1iM)
300 0 - 320 120 2 - NAb 12
330 2 + 0 54
"Determined by visual inspection of turbidity."NA, Not applicable.
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DEGRADATION OF BTX BY DENITRIFYING CULTURES
TABLE 4. Balances on carbon, nitrogen, and electrons for aCA2 subculture grown on 300 ,uM toluene
Balance Source Sink Amt or e- %balancea Difference
Carbon Toluene (4.11) CO2 2.34 17.0Cells 2.46
Total 4.80
Nitrogen N03- (0.282) NO2- 0.227 22.0N20 0.0334N2 0.0845
Total 0.344
Electron Toluene -* CO2 + NO3- NO2 0.453 -13.7cells (1.17) NO3- N20 0.134
NO3-* N2 0.423Total 1.01
a Numbers are milligrams of carbon, millimoles of nitrogen (N), andmillimoles of electrons for carbon, nitrogen, and electron balances, respec-tively.
electrons after the degradation of 300 ,uM toluene as the solecarbon source by the subcultured CA2 enrichment in thepresence of 2 mM N03 . CO2 accounted for 57% of thetoluene loss. The remainder was accounted for as cell masson the basis of an assumption that approximately 50% of thecell dry weight is carbon (2). The nitrogen balance includesonly nitrogen species involved in electron transport and notthose involved in assimilation. The electron balance wasbased on the following equation to account for assimilationof carbon: C7H8 + 4;8NO3 + 4.8H+ + 0.6NH3 -- 4CO2 +0.6C5H702N + 2.4N2 + 5.2H20. The stoichiometry ofconversion of C7H8 to CO2 in the above equation wasestablished from the carbon balance data in Table 4. Theelemental formula for cell mass was taken from McCarty etal. (15). Thus the oxidation of 1 mmol of toluene yielded 24mmol of electrons. The above equation is based upon noaccumulation of N02 or N20; however, the nitrogen bal-ance showed that this basis is incorrect. Thus the millimolesof electrons used for the reduction of NO3- were calculatedfrom the nitrogen balance data in Table 4 and the followingstoichiometric factors: 2 mmol of electrons per mmol ofN02 produced, 8 mmol of electrons per mmol of N20produced, and 10 mmol of electrons per mmol of N2 pro-duced. The carbon and nitrogen balances in Table 4 did notcompletely close, possibly because of inaccuracies inherentto the measurement of the dry cell weight after growth onlow substrate concentrations, error in the assumption that50% of the dry cell weight is carbon, and inaccuraciesassociated with the assays used for nitrate and nitrite. Theseerrors in turn affected the outcome of the electron balance.m-Xylene degradation. The CA2 subculture was used as an
inoculum to generate new cultures with individual BTXcompounds as sole sources of carbon rather than a BTXmixture in methanol. The mineral salts medium withoutyeast extract was used, and the individual BTX compounds(excluding toluene) were added neat at 100 p.M. After 1 weekof incubation 50% of the m-xylene was depleted, and after 2weeks of incubation all of the m-xylene was gone. Nitrousoxide production was also observed in the m-xylene culture.No microbially mediated losses of benzene, p-xylene, oro-xylene or associated production of nitrous oxide wereobserved. The m-xylene culture was subcultured and grewon m-xylene upon a 10-6 dilution into fresh medium. Asubculture was used for a carbon, nitrogen, and electronbalance analogous to that for toluene. The balances shown in
TABLE 5. Balances on carbon, nitrogen, and electrons for aCA2 subculture grown on 500 ,uM m-xylene
Balance Source Sink Amt or e- %balancea Difference
Carbon m-Xylene (14.4) CO2 8.14 -16.0Cells 3.98
Total 12.1
Nitrogen N03- (1.53) NO2- 1.47 13.7N20 0.19N2 0.076
Total 1.74
Electron m-Xylene -- CO2 N03 - N02 2.94 -2.9+ cells (4.20) NO3- N20 0.76
N03 - N2 0.38Total 4.08
aSee footnote a of Table 4.
Table 5 are for the degradation of 500 ,uM m-xylene in thepresence of 5 mM N03. Carbon dioxide accounted for 57%of the m-xylene consumed. The balance equation that waswritten to account for assimilation is: C8H10 + 5.6NO3 +5.6H+ + 0.7NH3 -- 4.5CO2 + 0.7C5H702N + 2.8N2 +6.4H20. The stoichiometry of conversion of C8H10 to CO2was established from the data in Table 5, and the electronbalance was calculated similarly to that for toluene. Thecarbon and nitrogen balances possibly did not completelyclose for the same reasons outlined above for the toluenebalance.
DISCUSSION
The samples obtained for this study encompass a varietyof environments obtained from different parts of the UnitedStates (Table 1). Most of the samples were from sites knownto have been contaminated by specific petroleum products,whereas others (ER, BH) did not have a specific input ofcontaminant but are considered to have a general input offuels or industrial effluents. A relatively rapid degradation oftoluene was observed with all of these sources (Fig. 1).Furthermore, the toluene-associated partial metabolism ofo-xylene was also observed in all of the cultures (Table 2).This suggests that denitrifiers that metabolize toluene ando-xylene may be widespread and can be readily found in theenvironment.No transformation of benzene, p-xylene, or m-xylene was
noted in any of these initial cultures. Although m-xylenedegradation was not observed in the original enrichmentcultures (Fig. 1), it was observed after subculture of the CA2enrichment culture on toluene. Thus the m-xylene-degradingbacteria were present in the CA2 inoculum source, but theinitial enrichment conditions did not seem to be favorable.m-Xylene was the sole source of carbon when its degrada-tion was observed. Whether the other alkylbenzenes inhib-ited the m-xylene degraders or whether these microorgan-isms were unable to compete effectively for nutrients withthe toluene degraders is not known at this time. Addition-ally, the toluene degraders may have m-xylene degradationcapabilities that were inactive during the initial incubations.Nevertheless, these results demonstrate the importance ofthe environmental conditions with respect to the observationof metabolic activities, and may help to explain the variedresults on the biodegradability of benzene, p-xylene, ando-xylene found in the literature.The degradation of toluene and m-xylene was quantita-
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tively analyzed by means of balances on carbon, nitrogen,and electrons. This is the first demonstration in which all ofthe substrates and products of oxidation and reductionreactions, including biomass, have been accounted for in theanaerobic biodegradation of aromatic hydrocarbons. Bio-mass production in denitrifying systems can be considerableand not negligible as in methanogenic or sulfidogenic sys-tems. For example, the cell yield of Paracoccus denitrificanson succinate under denitrifying conditions was 80% of thatunder aerobic conditions (18).These balances provided evidence that (i) the main prod-
ucts of toluene and m-xylene degradation were CO2 andbiomass (Tables 4 and 5); (ii) aromatic ring fission takesplace, since more than 50% of the toluene and m-xylenecarbon was found as CO2 (Tables 4 and 5); and (iii) theanaerobic oxidation of toluene and m-xylene was stoichio-metrically dependent on nitrate reduction and denitrification(Tables 3, 4, and 5). These conclusions are valid and are
supported by the data, notwithstanding the fact that some ofthe balances did not completely close. An accumulation ofnitrite was observed during the degradation of toluene andm-xylene. This can be circumvented by decreasing thenitrate concentration to levels that require complete reduc-tion to N2 to account for the complete degradation of tolueneor m-xylene (unpublished results).
o-Xylene was not metabolized when it was present as thesole source of carbon in denitrifying mixed cultures. Themetabolism of o-xylene was dependent on the metabolism oftoluene and was biologically mediated (Table 2; Fig. 2). Thissynergistic effect of toluene was specific to o-xylene, sinceno analogous losses of benzene or of the other two xyleneswere observed. The dependence of o-xylene loss on toluenewas not unique to the CA2 enrichment culture, since it wasobserved in all of the enrichments (Table 2). Transformationof o-xylene in the presence of another degradable substratehas been previously observed (16). In this case Nocardiacorallina A-6 transformed o-xylene to o-toluic acid duringaerobic growth on hexadecane. This culture also trans-
formed p-xylene to p-toluic acid and 2,3-dihydroxy-4-meth-ylbenzoic acid. The o-xylene transformation that we ob-served is probably mechanistically different, since no
transformation of p-xylene was observed. Studies of pure
cultures that degrade toluene and m-xylene are currentlyunderway to facilitate the further characterization of theirmetabolism and identification of the o-xylene metaboliteunder these anaerobic conditions.
ACKNOWLEDGMENTS
We kindly thank Timothy Vogel for supplying the KC source
material, David Kossen and Gene Bolen for the NC and BH source
material, and Margaret Findlay at ABB Bioremediation Systems forthe CA1, CA2, and CA3 source material.
This research was partially supported by NIEHS ES 04895 andthe Hazardous Substances Management Research Center of NewJersey. Partial support for P.J.E. was from a National ResearchService award (5 T32 AI-07180) from the National Institute ofAllergy and Infectious Diseases.
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