high tnt-transforming activity by a mixed culture acclimated and maintained on crude-oil-containing...

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High TNT-transforming activity by a mixed cultureacclimated and maintained on crude-oil-containingmedia

Jason T. Popesku, Ajay Singh, Jian-Shen Zhao, Jalal Hawari, and Owen P. Ward

Abstract: A mixed microbial culture originating from a petroleum-contaminated site and maintained on crude oil ex-hibited high 2,4,6-trinitrotoluene (TNT) transformation activity. Cultivation of the mixed culture in glucose-containingmedium for 29 h resulted in almost complete transformation of 100 ppm TNT. TNT transformation was observed withboth growing and resting cells. With subculturing, it was found that TNT could support growth of the mixed culturewhen supplied as sole carbon source, sole nitrogen source, or sole carbon and nitrogen source. The finding that amixed microbial culture maintained on crude oil exhibited high TNT transformation activity without prior subcultureon TNT-containing media is novel and may have potential practical applications in the bioremediation of munitions-contaminated soil and wastewater.

Key words: 2,4,6-trinitrotoluene, mixed culture, transformation, co-metabolism, surfactant.

Résumé : Une culture microbienne mixte provenant d’un site contenant du pétrole et maintenue dans du pétrole brut adémontré une activité élevée de transformation du 2,4,6-trinitrotoluène (TNT). Une incubation de la culture mixte dansun milieu renfermant du glucose pendant 29 h a entraîné une transformation quasi complète de 100 ppm de TNT. Latransformation du TNT a été observé avec des cellules en croissance ou au repos. Lors des repiquages, nous avonsconstaté que le TNT pouvait soutenir la croissance de la culture mixte lorsqu’il était fourni comme seule source decarbone, seule source d’azote, ou seule source de carbone et d’azote. Le fait qu’une culture microbienne mixte main-tenue dans du pétrole brut démontre une activité élevée de transformation du TNT, sans repiquage préalable dans unmilieu contenant du TNT, représente une découverte notoire. Elle aurait des applications pratiques potentielles dansl’assainissement biologique de sols et d’eaux usées contaminés aux munitions.

Mots clés : 2,4,6-trinitrotoluène, culture mixte, transformation, cométabolisme, surfactant.

[Traduit par la Rédaction] Popesku et al. 366

Barriers to the biodegradation of nitroaromatic com-pounds include the toxicity of nitroaromatics to microorgan-isms and the low bioavailability of the compounds becauseof their very low solubility. Thus far, a biological pathwayfor the complete mineralization of 2,4,6-trinitrotoluene(TNT) has not been described. Individual aerobic strains ofbacteria converted TNT mainly to various aminonitrotoluenes,but these compounds could not be further degraded(Boopathy et al. 1994; Alvarez et al. 1995; Montpas et al.1997; Hawari et al. 2000; Khachatryan et al. 2000). Some

bacterial strains have been found to grow on TNT as the solenitrogen source (French et al. 1998). Several attempts to cul-ture bacterial strains on TNT as the sole carbon source werenot successful (Boopathy et al. 1994; Khachatryan et al.2000).

Microbial communities from landfills (Tharakan andGordon 1999), anaerobic sludge (Hawari et al. 1998), soil(Radtke et al. 2000), or lakes (Wikström et al. 2000) con-taminated with munitions have been shown to be capable ofat least partially degrading TNT. Rates of TNT biotrans-formation have been shown to increase in soil containingparticulate TNT pretreated with acetone (Radtke et al. 2000)or in liquid media containing a surfactant, such as Tween 80(Montpas et al. 1997).

In most of the examples where microbes are used for thebiodegradation of recalcitrant compounds, including theabove-cited cases for TNT, the microorganisms are obtainedfrom sites containing the contaminant. Consortia or pure cul-tures are typically isolated by incorporating the contaminantinto selective isolation media, and cultures prior to beingused for contaminant biotransformation or bioremediation

Can. J. Microbiol. 49: 362–366 (2003) doi: 10.1139/W03-049 © 2003 NRC Canada

362

Received 19 February 2003. Revision received 9 June 2003.Accepted 11 June 2003. Published on the NRC ResearchPress Web site at http://cjm.nrc.ca on 17 July 2003.

J.T. Popesku, A. Singh, and O.P. Ward.1 Department ofBiology, University of Waterloo, Waterloo, ON N2L 3G1,Canada.J.-S. Zhao and J. Hawari. Biotechnology Research Institute,National Research Council, Montréal, QC H4P 2R2, Canada.1Corresponding author (e-mail: [email protected]).

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are usually pre-acclimated in media containing the contami-nant. From a safety and environmental perspective, havingto use hazardous chemicals in pre-acclimation media com-plicates bioremediation processes. Where the hazardouschemicals are explosives, such as TNT, which have addi-tional use restrictions, incorporation of TNT into pre-acclimation media is even more onerous.

In this paper, we report on the ability of a culture, whichneither originated from a munitions-contaminated site norwas preselected nor acclimated in TNT-containing media, torapidly transform TNT. Investigations were initiated by us-ing structurally similar aromatic compounds as cosubstrates,which might induce enzymes that are involved in the trans-formation of nitroaromatic compounds (Hawari et al. 2000;Zhao et al. 2000; Zhao and Ward 2000). We further reportthat the rates of transformation observed are higher than thehighest rates obtained from cultures originating from and ac-climated in TNT-containing environments. In addition, wefound that TNT could be utilized as a growth substrate.

Despite widely reported TNT-degrading activity, mineral-ization of TNT by a single bacterial culture is rarely reported.Duque et al. (1993) described that the toluene-degrading oxy-genase system coded on the TOL plasmid seemed to helpdegrade TNT. Thus in this study, we tested a petroleum-degrading mixed bacterial culture for its ability to degradeTNT. A mixed microbial population isolated from petro-leum-hydrocarbon-contaminated soil and maintained for sev-eral years on crude oil, motor oil, or diesel in cyclonefermentors (Ward and Singh 1997; Van Hamme et al. 2000)was used in the present investigation. Microbial character-ization of this oil-degrading mixed culture (Van Hamme etal. 2000) indicated that the predominant strains includedPseudomonas/Flavomonas, Acinetobacter, Alcaligenes, Ochra-bactrum, and Stenotrophomonas. Pseudomonas, Alcaligenes,and Enterobacter have been shown to be capable of trans-forming TNT into various intermediates (Duque et al. 1993).About 50% of the reactor volume (1 L) was replaced everyweek with fresh medium and 5000 ppm of the appropriatepetroleum source. For inoculum preparation, we combined a5-mL culture from each of the cyclones and disgarded thesupernatant after centrifugation at 5000 × g for 10 min. Thepellet was washed twice with and resuspended in 0.2 Mphosphate buffer (pH 7) to a volume of 15 mL.

Growing-cell experiments were carried out with 5 mL ofmodified mineral salts medium (Van Hamme et al. 2000) con-taining 100 ppm TNT in 25-mL Erlenmeyer flasks. TNT(98% pure) was provided by Defence Research and Develop-ment Canada, Valcartier, Quebec. TNT was added as a20 000 ppm stock solution in acetone. The mineral salts me-dium contained the following (per litre): 1.4 g KH2PO4, 2.7 gNa2HPO4·7H2O, 0.2 g MgSO4·7H2O, 0.1 g Na2CO3, 0.05 gCaCl2·2H2O, 0.005 g FeSO4, 1.0 g NH4Cl, and 3.0 mL oftrace metals solution. The trace metals solution containedthe following (per litre): 0.162 g FeCl3·6H2O, 0.0144 gZnCl3·4H2O, 0.012 g CoCl2·6H2O, 0.012 g Na2MoO4·2H2O,0.006 g CaCl2·2H2O, 1.9 g CuSO4·5H2O, 0.05 g H3BO4, and35 mL HCl. Culture flasks were inoculated with 2%inoculum and incubated at 30°C on an orbital shaker(Labline Instruments Inc., Melrose Park, Ill.) set at 200 rpm.

For resting-cell experiments, the mineral salts medium inthe culture flasks was replaced with 0.2 M sodium phos-

phate (pH 7.0) and inoculated with washed cells to achieve afinal cell OD of 2.0 at 600 nm.

For TNT analysis an equal volume of acetonitrile wasadded to each flask, and the flasks were placed on an orbitalshaker at 150 rpm for 10 min. After centrifugation at 5000 × gfor 10 min, the supernatant was stored in Teflon-lined screw-cap glass vials. Samples were analyzed by HPLC (ShimadzuLC-600, Kyoto, Japan) on a 4.6 mm × 250 mm Zorbax SB-C18 column (Chromatographic Specialties Inc., Brockville,Ont., Canada), using a 50:50 methanol/water mobile phasecontaining 0.1% trifluoroacetic acid at 1.0 mL/min with de-tection at λ = 254 nm. Microbial growth was monitored bymeasuring culture absorbance at 600 nm in a 1-cm lightpath, using a Shimadzu UV-120-02 spectrophotometer.

Degradation experiments were carried out in duplicate,and data reported are the averages of duplicate tests. Varia-tions between duplicates were typically less than 5%.

Since it is difficult for bacteria to utilize TNT as sole car-bon source, co-metabolic transformation of TNT has beeninvestigated using simple sugars and organic acids ascosubstrates (Collie and Donnely 1995; Kalafut et al. 1998;Hawari et al. 2000). The ability of this petroleum-hydrocarbon-degrading mixed culture to grow and transform TNT in themineral salts medium in the presence of aromatic or non-aromatic cosubstrates was evaluated. Aromatic supplementswere added at a concentration of 50 ppm because of their in-herent toxicity, and nonaromatic substrates were used at aconcentration of 3000 ppm. The control contained nocosubstrate. A HgCl2-poisoned control was also included toobserve any abiotic degradation of TNT.

Absorbance, due to biomass growth and TNT disappear-ance, data are presented in Table 1. Growth on glucose,yeast extract, and citric acid was excellent, and TNT re-moval after 72 h was almost 100%. While good growth wasobserved with catechol, TNT degradation was poor. Al-though good growth was observed with surfactants ascosubstrate, TNT removal ranged from only 53 to 75%. Incontrast, growth was relatively poor with 4-nitrocatechol;57% of the TNT was removed. Growth was negligible withother aromatic cosubstrates and TNT removal (0–9.2%) wasless than that of the control without a cosubstrate. The aboveresults indicate TNT transformation during cell growth.

The effect of supplementing media containing TNT andan aromatic cosubstrate with glucose was investigated to de-termine if substrates with structural relationships to TNTcould augment the high transformation rates observed withglucose alone. The results indicated that these compoundsdid not have an inhibitory effect on growth (Table 2). How-ever, TNT removal, ranging from 55% for catechol to 96%for 2-nitrophenol, was less than that observed with glucosealone.

The ability of 2% inocula to degrade 100 ppm TNT inglucose-supplemented minerals salts medium in 48 h alsovaried with the nature of the hydrocarbon in the maintenancemedium. Percent TNT transformation for cultures main-tained on motor oil, diesel oil, crude oil, and a combinationof the three was 88.1, 89.1, 94.9, and 92.2%, respectively.

Supplementation of the glucose – TNT – mineral salts me-dium with surfactant Tween 80 reduced the time for completeTNT removal from 45 to 29 h (Fig. 1). When almost 100% ofthe TNT was transformed, the metabolites 2-amino-4,6-

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Popesku et al. 363



dinitrotoluene (2-ADNT) and 4-amino-2,6- dinitrotoluene(4-ADNT) were detected in the culture. The concentrationsof 2-ADNT and 4-ADNT were less than 1 mg/L. Hence,only about 2% of the TNT was converted to these metabo-lites.

Growth at 30°C of the oil-degrading culture in the liquidmodified mineral salts medium containing TNT as (i) solesource of carbon and nitrogen, (ii) carbon only, and (iii) ni-trogen only was investigated over a 35-day period. In thecase of (i) and (iii) NH4Cl was deleted from the mineralsalts medium. In the case of (iii), the medium contained1 g/L of glucose. After every 2–4 day intervals, a 1-mL sam-ple from each flask was used to inoculate a correspondingflask with fresh medium with TNT. For the first 17 days,TNT was added to give a final concentration of 100 ppm,and increases in growth were relatively low. Thereafter, TNTwas added at the equivalent of a final concentration of500 ppm, and this boosted the growth with an increase ofOD600 values by up to 0.14 (TNT as sole carbon and nitro-gen source), 0.13 (TNT as sole source of carbon), or 0.36(TNT as a sole source of nitrogen) (Fig. 2). Precautionswere taken in determining OD600 biomass data when TNT

was added at concentrations in excess of its aqueous solubil-ity. The excess TNT forms crystals, which quickly sedimentin aqueous media on standing for 1 min and do not interferewith OD600 readings.

It has been noted that the biological transformation rate ofTNT is in part a function of TNT bioavailability, and Tween80 may enhance bioavailability through micelle formation(Hawari et al. 2000). Van Hamme and co-workers (2000,2001) observed enhancement of petroleum hydrocarbondegradation by this mixed culture and showed that two iso-lates from the culture appeared to have a contrasting mecha-nism of hydrocarbon degradation. Tharakan and Gordon(1999) observed complete removal of 100 ppm TNT frommedia in 72 h, using a mixed culture isolated from a TNT-contaminated site and maintained in media containing TNT.They suggested that the TNT transformation in aerobic liq-uid cultures supplemented with glucose or citrate wasgrowth associated.

To prove that the culture maintained on petroleum hydro-carbons had TNT-transforming ability, we took cells directlyfrom the maintenance culture, washed them twice with0.2 M phosphate buffer (pH 6.8), and resuspended them in

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CosubstrateMicrobial growth∆ OD600

% TNT transformedin 72 h

Control (HgCl2 poisoned) 0 0No cosubstrate 0 17.1Glucose 1.15 99.3Citric acid 0.85 98.3Yeast extract 0.80 99.8Monawet MO65-150 0.03 57.1Sodium dodecyl sulfate 0.52 52.8Tween 80 0.25 74.8Benzene 0 0Toluene 0.01 1.8Catechol 1.06 19.54-Nitrocatechol 0.28 57.02-Nitrophenol 0.00 9.22,4-Dinitrophenol 0.02 7.72,4-Dinitrotoluene 0 2.82,6-Dinitrotoluene 0 0

Note: Initial TNT concentration was 100 mg/L.

Table 1. Effect of cosubstrates on growth of and 2,4,6-trinitrotoluene (TNT) transformationby a mixed culture.

CosubstrateMicrobial growth∆ OD600

% TNT transformedin 48 h

No cosubstrate 0.02 14.0Glucose only 1.20 99.0Catechol + glucose 0.61 54.64-Nitrocatechol + glucose 1.55 83.42-Nitrophenol + glucose 1.48 96.3Benzene + glucose 1.37 81.62,4-Dinitrotoluene + glucose 1.36 89.72,6-Dinitrotoluene + glucose 1.51 75.2

Note: Initial TNT concentration was 100 mg/L.

Table 2. Effect of addition of glucose to aromatic cosubstrates on culture growth and2,4,6-trinitrotoluene (TNT) transformation.



phosphate buffer or mineral salts medium to an OD600 of 2.0(1-cm light path). The resting cells were equilibrated at 30°Cfor 1 h, and TNT was added to the flasks with or withoutglucose or Tween 80. The cells were incubated for up to 8 hand analyzed for TNT removal. All flasks showed TNTtransformation (Table 3). Under these resting-cell condi-tions, highest TNT transformation was observed in buffersupplemented with Tween 80 (82.6 mg/L TNT) or glucose(64.0 mg/L TNT).

The finding that a mixed microbial culture originatingfrom a petroleum-contaminated site and maintained on crudeoil exhibited high TNT transformation activity is novel andhas potential practical applications in bioremediation. Thelack of a catabolic system to degrade nitroaromatics wasconsidered a barrier to nitroaromatic degradation (Taha et al.1997; French et al. 1998). Crude oil, which contains a vastarray of aliphatic, aromatic, and other hydrocarbons also,contains only low concentrations of nitroaromatic com-pounds (Speight 1991). The results suggest that some of the

enzymes and (or) metabolic pathways present in crude-oil-degrading consortia have the capacity to transform TNT. ATNT-degrading Pseudomonas sp. has been found to mineral-ize TNT after recruiting a TOL plasmid coding the tolueneoxygenase system (Duque et al. 1993). This TOL plasmidmay be present in the mixed culture used in the present in-vestigation, because toluene is a component of petroleumand the aerobic bacteria used was from a petroleum- degrad-ing culture. It seems that a constitutive TNT-degrading en-zyme system is present in this culture and is capable ofbreaking the benzene ring to provide carbon for bacterialgrowth, as evidenced from the results in Fig. 2.

The quantitative relationship between TNT transforma-tion, metabolite appearance, and evidence of TNT mineral-

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Popesku et al. 365

Fig. 1. Time course for the transformation of 2,4,6-trinitrotoluene(TNT) by the mixed culture grown and maintained on crude oil.Mineral salts medium containing 100 ppm TNT was supple-mented with 3000 ppm of glucose, inoculated with 2% mixedculture, and incubated on a rotary shaker (200 rpm) at 30°C.

Fig. 2. Growth of mixed bacterial culture on 2,4,6-trinitrotoluene(TNT) as (a) sole source of carbon, (b) sole source of nitrogen,and (c) sole source of carbon and nitrogen. Mineral salts me-dium containing 100 ppm TNT was supplemented with3000 ppm of glucose or 250 ppm of ammonium chloride whereappropriate. Arrows indicate a transfer of 1 mL of culture to thefresh medium.

Medium*

% TNT transformed attime (h)2 4 8

Buffer only 22.4 21.1 27.2Mineral salts medium only 25.6 28.3 38.1Buffer + glucose 39.2 56.7 64.0Buffer + Tween 80 41.9 55.8 82.6Mineral salts medium + glucose 23.6 45.1 57.2

*Cells taken directly from the maintenance culture were washed withand resuspended in 0.2 M phosphate buffer (pH 6.8) to an OD600 of 2.0and were used for transformation in TNT-containing buffer or mineralsalts medium with or without supplements. Initial TNT concentration was100 mg/L.

Table 3. 2,4,6-Trinitrotoluene (TNT) transformation by restingcells from the crude-oil-supplemented maintenance medium.



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366 Can. J. Microbiol. Vol. 49, 2003

ization will now be evaluated. Studies on the use of thisculture to degrade TNT in soil are being pursued. In addi-tion, the degradative properties towards TNT and other haz-ardous chemicals of some of the predominant isolates fromthe mixed culture when maintained on crude oil and non-hydrocarbon media are also being examined. The capacity ofpure culture isolates to grow on TNT as sole carbon sourcewill also be characterized.

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high tnt-transforming activity by a mixed culture acclimated and maintained on crude-oil-containing...

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