TOLERANCE TO NITROGENOUS EXPLOSIVES AND METABOLISM OF TNT BY CELL SUSPENSIONS OF DATURA INNOXIA

Authors: Lucero, Mary; MUELLER, WOLFGANG - NEW MEXICO STATE UNIV; HUBSTENBERGER, JOHN - NEW MEXICO STATE UNIV; PHILLIPS, GREG - NEW MEXICO STATE UNIV; O'CONNELL, MARY - NEW MEXICO STATE UNIV

Submitted to: In Vitro Plant
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/4/1999
Publication Date: N/A
Citation: LUCERO, M.E., MUELLER, W., HUBSTENBERGER, J., PHILLIPS, G.C., O'CONNELL, M.A. TOLERANCE TO NITROGENOUS EXPLOSIVES AND METABOLISM OF TNT BY CELL SUSPENSIONS OF DATURA INNOXIA. IN VITRO PLANT. 1999. V. 35(6). P. 480-486.

Interpretive Summary: Trinitrotoluene (TNT) was heavily manufactured throughout the 20th century. It was common for TNT-laden wastewaters left over from manufacturing to be pumped into holding lagoons, ponds, or freshwater streams. Significant contamination now exists at these sites where TNT concentrations are often reported in parts per thousand. Excavation and restoration of these sites by conventional techniques are expensive. Phytoremediation (using plants to remove hazardous waste), therefore, has drawn a great deal of interest, and the ability of a handful of higher plants to absorb and detoxify TNT has been described. Many reports have been criticized for failing to test plants in a microbe-free environment since the presence of microbes makes it difficult to determine whether TNT removal is being carried out by the plant or by the associated microbes. In this study, microbe-free cell cultures of jimson weed (Datura innoxia) were grown in the presence of TNT. .The loss of TNT and the appearance and subsequent disappearance of the TNT byproducts, 2- and 4-aminodinitrotoluene (ADNTs) were monitored. Cell cultures removed TNT and the ADNT metabolites from the growth media in less than 12 h. This is faster than rates reported for any previously tested strains of TNT-degrading bacteria. These results support growing evidence plants can fill a critical role in bioremediation. Observing biotransformation in D. innoxia is important since previously studied species came from humid environments, while D. species are common in arid lands. Many TNT-contaminated soils exist in desert rangelands where military test sites have been located. If phytoremediation of these sites is to be economically feasible, the process will have to rely on plants that thrive in an arid environment.

Technical Abstract: Cell suspension cultures of Datura innoxia were incubated in the presence of nitro-substituted explosives 2,4,6-trinitrotoluene (TNT), 1,3, 5-trinitro-1,3,5-triazine (RDX), and 1,3,5,7-tetranitro-1,3,5, 7-tetraazocyclooctane (HMX). Cellular tolerance levels and TNT biotransformation kinetics were examined. Tolerance to TNT varied as cell suspensions aged. Concentrations of RDX or HMX in excess of reported solubility limits produced no observable changes in cell viability. GC/MS analysis of TNT-treated cell media and cell lysates revealed rapid removal of TNT. Within 12 h, less than 1% of the initial TNT remained in the growth medium. Aminodinitrotoluenes (ADNTs), known metabolites of TNT, accumulated transiently in cell lysates and to a lesser extent in cell media. ADNT concentrations started to decrease after 3 h. After 12 h, less than 5% of the initial TNT could be detected as ADNT. Total ADNTs never exceeded 26% of initial TNT, suggesting additional biotransformation steps also occurred. No other nitroaromatics were detected. A pseudo-first order rate constant for TNT clearance was calculated, k = 0.40 **h-1. D. innoxia cell suspension cultures demonstrated virtually complete clearance of TNT and of subsequent ADNT metabolites in less than 12 h. This rapid metabolism of nitroaromatics by the Datura cell suspension system indicates the utility of this system for further molecular and biochemical studies.