MICROBIAL DEGRADATION OF TNT BY PSEUDOMONAS SAVASTANOI

J.L. Martin, T.A. Kokjohn, S.D. Comfort and P.J. Shea

ABSTRACT

Soils at the former Nebraska Ordnance Plant (NOP) are grossly contaminated with TNT (2,4,6-trinitrotoluene). We found that this TNT-contaminated soil contains a variety of Pseudomonads. Our objective was to determine the potential of a single microbial species (Pseudomonas savastanoi) to degrade and mineralize TNT. All experiments were performed in Pseudomonas minimal medium (PMM) containing TNT (60 to 65 mg L^-1) as the sole C source. Cells were prepared as inocula by growing to approximately mid-log phase in PMM (with glucose) for 9.5 h. Cells were then washed twice with saline and suspended in PMM (TNT sole C source). The inoculum was added to a concurrent set of experimental units containing PMM with either labeled (¹⁴C-TNT) or unlabeled TNT. Unlabeled medium was used for chemical (TNT, aminodinitrotoluenes, NO₂ and NO₃) and viable cell count determinations; samples were taken every 5 to 10 d for 72 d. Daily emissions of ¹⁴CO₂ and ¹⁴C-volatiles were determined from labeled medium. Results indicate that ¹⁴CO₂ emissions mimic viable cell populations with highest emissions within 24 h after inoculation. When we reinoculated the medium with a second and third inoculum (day 36 and 55), ¹⁴CO₂ production was similar to the first inoculum. Inoculating the medium with killed cells did not produce ¹⁴CO₂. Although ¹⁴CO₂ production was related to cell density, overall mineralization was limited and less than 1% of total ¹⁴C applied. Chemical analysis revealed TNT concentrations declined to approximately 5 mg TNT L^-1 within 72 d; the largest decrease in TNT concentration occurred within 24 h after inoculation. The production of aminodinitrotoluene was minimal and can not solely account for the loss of TNT. We also observed a steadily increasing NO₂ concentration with time. These results indicate that Pseudomonas savastanoi can readily degrade but may have a limited potential to mineralize TNT.

KEY WORDS

TNT, degradation, mineralization

This paper is from the Proceedings of the 10th Annual Conference on Hazardous Waste Research 1995, published in hard copy and on the Web by the Great Plains/Rocky Mountain Hazardous Substance Research Center.