Origin of p-cresol in the anaerobic degradation of trinitrotoluene C.F. Shen, J.A. Hawari, G. Ampleman, S. Thiboutot, and S.R. Guiot Abstract: p-Cresol was repeatedly detected as a trace metabolite in anaerobic slurry reactors treating 2,4,6- trinitrotoluene (TNT)-contaminated soils. This study shows that p-cresol was not a metabolite of the anaerobic degrada- tion of TNT, by using a combination of analytical techniques and 13 C-labelled TNT. Instead, p-cresol, an intermediate in the degradation pathway of some amino acids, was shown to be inhibited by TNT and its metabolites. The range and persistence of inhibition to p-cresol microbial degradation decreased with the level of amino-substitution of the de- rivatives. This explains why p-cresol accumulated within the TNT-treating anaerobic bioslurry, as it could not be fur- ther biodegraded in the presence of TNT. Key words: p-cresol, bioremediation, trinitrotoluene, inhibition, metabolites. Résumé : Le p-crésol est régulièrement détecté en quantité infime dans les réacteurs anaérobies en phase boueuse, trai- tant des sols contaminés par le 2,4,6-trinitrotoluène (TNT). En combinant des techniques analytiques et l’utilisation de TNT marqué au 13 C, la présente étude a démontré que le p-crésol n’était pas un métabolite de la dégradation anaérobie du TNT. Par contre, la formation de p-crésol, un intermédiaire des voies de dégradation de certains acides aminés, était inhibée par le TNT et ses métabolites. Le niveau et la durée de l’inhibition de la dégradation microbienne du p-crésol diminuaient selon le niveau de substitution aminée des dérivés. Cela explique pourquoi le p-crésol s’accumule dans la boue lors du traitement biologique de sols contaminés avec du TNT, puisque ce produit ne peut pas être dégradé de fa- çon plus complète en présence de TNT. Mots clés : p-crésol, biorestauration, trinitrotoluène, inhibition, métabolites. [Traduit par la Rédaction] Shen et al. 124 Introduction High concentrations of explosives such as 2,4,6-trinitro- toluene (TNT) are commonly found at military sites where explosives loading, handling, and packing activities were conducted (Kaplan and Kaplan 1982). TNT is an environ- mental concern because of its toxicity (Sunhara et al. 1998; Tan et al. 1992). Feasibility of bioremediation of TNT and other explosive-contaminated soils by anaerobic soil slurry processes have been recently demonstrated (Funk et al. 1993; Manning et al. 1996; Shen et al. 1998). During the an- aerobic treatment process, TNT was transformed in a step- wise fashion into its corresponding amino derivatives (Funk et al. 1993; Roberts and Pendharkar 1995; Shen et al. 1998). In addition, a further metabolite, p-cresol, was detected from the anaerobic degradation of TNT, and hypothesized as an intermediate of TNT degradation (Funk et al. 1993; Roberts and Pendharkar 1995; Crawford et al. 1995). Results from our bioslurry process remediating TNT-contaminated soil re- peatedly confirmed appearance of p-cresol during the anaer- obic bioremediation process (Shen et al. 1998). p-Cresol is known to be catabolized under both aerobic and anaerobic conditions by a variety of microorganisms (Bisaillon et al. 1994; Evans and Fuchs 1988; Hopper and Taylor 1975). Hence, if TNT was anaerobically transformed into p-cresol, it should be finally mineralized to CO 2 . In contrast, exten- sive mineralization of TNT was not observed in anaerobic microcosm tests spiked with 14 C-labelled TNT (Funk et al. 1993; Shen et al. 1998). Furthermore, recent literature (Hawari et al. 1998) mentioned that 13 C-labelled p-cresol was not detected in anaerobic microcosms using 13 CH 3 -la- belled TNT. The apparent contradiction of the above reports needs to be clarified. The objectives of the present study were to confirm comprehensively that p-cresol was not a metabolite from TNT degradation, and to investigate the rea- sons for its recurrence during TNT bioremediation. Materials and methods Sources of microorganisms and chemicals This study used anaerobic granular biomass from a food indus- try wastewater treatment plant (Cornwall, Ontario). [Methyl- 13 C]- TNT was synthesized and purified to over 99%, according to the method described by Ampleman et al. (1995). 2- And 4-amino- dinitrotoluene (ADNT), and 2,4- and 2,6-diaminonitrotoluene (DANT) were purchased from AccuStandard (New Haven, Conn.). The 2,4,6-triaminotoluene HCI salt was bought from Chem Service (West Chester, Pa.). p-Cresol with a purity of over 99% was Can. J. Microbiol. 46: 119–124 (2000) © 2000 NRC Canada 119 Received July 13, 1999. Revision received October 7, 1999. Accepted October 8, 1999. C.F. Shen, J.A. Hawari, and S.R. Guiot. 1 Biotechnology Research Institute, National Research Council of Canada, 6100 Royalmount Avenue, Montréal, QC H4P 2R2, Canada. G. Ampleman, S. Thiboutot. Defense Research Establishment Valcartier, 2459 Pie-XI Blvd. North, Val- Bélair, QC G3J 1X5, Canada. 1 Author to whom all correspondence should be addressed (e-mail: serge.guiot@nrc.ca).