NOTE / NOTE High TNT-transforming activity by a mixed culture acclimated and maintained on crude-oil-containing media 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-containing medium for 29 h resulted in almost complete transformation of 100 ppm TNT. TNT transformation was observed with both growing and resting cells. With subculturing, it was found that TNT could support growth of the mixed culture when supplied as sole carbon source, sole nitrogen source, or sole carbon and nitrogen source. The finding that a mixed microbial culture maintained on crude oil exhibited high TNT transformation activity without prior subculture on 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 a démontré une activité élevée de transformation du 2,4,6-trinitrotoluène (TNT). Une incubation de la culture mixte dans un milieu renfermant du glucose pendant 29 h a entraîné une transformation quasi complète de 100 ppm de TNT. La transformation du TNT a été observé avec des cellules en croissance ou au repos. Lors des repiquages, nous avons constaté que le TNT pouvait soutenir la croissance de la culture mixte lorsqu’il était fourni comme seule source de carbone, 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 un milieu contenant du TNT, représente une découverte notoire. Elle aurait des applications pratiques potentielles dans l’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 because of their very low solubility. Thus far, a biological pathway for the complete mineralization of 2,4,6-trinitrotoluene (TNT) has not been described. Individual aerobic strains of bacteria 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 sole nitrogen source (French et al. 1998). Several attempts to cul- ture bacterial strains on TNT as the sole carbon source were not successful (Boopathy et al. 1994; Khachatryan et al. 2000). Microbial communities from landfills (Tharakan and Gordon 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 of at least partially degrading TNT. Rates of TNT biotrans- formation have been shown to increase in soil containing particulate 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 the biodegradation of recalcitrant compounds, including the above-cited cases for TNT, the microorganisms are obtained from sites containing the contaminant. Consortia or pure cul- tures are typically isolated by incorporating the contaminant into selective isolation media, and cultures prior to being used 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 Research Press Web site at http://cjm.nrc.ca on 17 July 2003. J.T. Popesku, A. Singh, and O.P. Ward. 1 Department of Biology, 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. 1 Corresponding author (e-mail: opward@sciborg.uwaterloo.ca).