The development of in situ bioremediation processes for the treatment of TNT and other nitroaromatics would greatly improve the Department of Defense’s ability to restore contaminated sites in a cost-effective manner. The factor that has limited the development of in situ bioremediation processes for treatment of TNT is the inability of bacteria to use TNT as a growth substrate. For this reason, current ex situ treatment systems (i.e., composting and slurry reactors) focus on cometabolic transformations that lead to binding of polyamino metabolites to soil and amendments. This approach to TNT bioremediation does not result in extensive biodegradation, and because the products are difficult to characterize or monitor, the treatment endpoint remains controversial.

The objective of this project was to determine the biochemical mechanism of TNT transformation in novel degradation pathways and to use the fundamental information to develop strategies that harness the activity in remediation systems. Specific objectives were to (1) identify the products of novel TNT transformation pathways, (2) determine the mechanism of TNT transformation and identify the enzymes responsible, (3) characterize the properties of the enzymes and their regulation, (4) develop strategies to direct TNT metabolism to ring fission products, and (5) examine the potential for mineralization of novel TNT metabolites.

Independent studies by the researchers converged with the discovery of a novel metabolic pathway that yielded hydroxylated products from TNT. The central metabolite, 2,4-dihydroxylamino-6-nitrotoluene (2,4DHANT), is hydroxylated via rearrangement and then transformed to polar products. In the systems previously studied, the rearrangement of 2,4DHANT serves as a gateway to the extensive metabolism of TNT. What remained was to understand the mechanisms and control of the reactions so that they could be harnessed for practical application.

Extensive metabolism of TNT in both anaerobic and aerobic systems depends on the activity of non-specific nitroreductases. In C. acetobutylicum, an Fe-only hydrogenase is primarily responsible for TNT transformation to hydroxylamino compounds and hydroxylated intermediates. Subsequent exposure to oxic conditions leads to mineralization. Both biotic and abiotic activities are responsible for the mineralization. The fate of partially reduced TNT products was determined in soil and model systems. In the presence of soil, TNT nitroso- products become bound to protein components of humic acids most likely by the nitroso-thiol reaction.

P. pseudoalcaligenes contains multiple nitroreductases, including a unique constitutively expressed nitroreductase that converts TNT to 2-amino-4,6-dinitrotoluene (2ADNT). The existence of the enzyme is crucial to avoid accumulation of the much more reactive hydroxylamino compounds. Purification and characterization of the novel enzyme will reveal whether it is widespread and whether its activity is responsible for the often observed accumulation of monoamino derivatives of TNT under aerobic conditions. During aerobic growth on molasses, constitutive enzymes, including the nonspecific nitroreductases and the constitutively expressed HabA, transform TNT to polar metabolites that bind readily to soil. A variety of other systems based on cometabolism of TNT have been used in the past. The advantage of the two systems described here is that polyamines do not accumulate and additional bulking of the soil is not involved.

The lack of fundamental understanding in the area of nitroaromatic metabolism has been recognized as the primary limitation in the extension of bioremediation alternatives to contaminated sites. This project provided insight into novel forms of TNT metabolism and identified important considerations for the eventual use of novel metabolic processes for TNT bioremediation.