Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (Figure 1) is the most important military high explosive in the United States today (Gorontzy et al. 1994). RDX is manufactured at the Holston Army Ammunition Plant (AAP) in Kingsport, TN. Wastewater produced from the manufacture of RDX is treated on site in an industrial wastewater treatment plant and discharged to the Holston River. RDX is also a frequent component of pink water, a hazardous wastewater generated from: (1) load, assembly, and packaging (LAP) of conventional ammunition items, and (2) demilitarization operations where explosives are washed out of disassembled ammunition (Concurrent Technologies Corporation 1996). Pinkwater is typically treated using granular activated carbon, although potentially less costly alternatives are under investigation (Concurrent Technologies Corporation 1996). One such alternative for pink water and other wastewater contaminated with nitroaromatic compounds is biological treatment using an anaerobic fluidized-bed granular activated carbon bioreactor (Concurrent Technologies Corporation 1996).
In the past, improper disposal of wastewater has led to environmental contamination. Recent reports estimate that at least 28 U.S. Army installations (Funk et al. 1993) and 200 areas in Germany contain soils contaminated with high explosives, including RDX (Binks, Nicklin, and Bruce 1995). Many of these sites have the potential to contaminate groundwater.
Figure 1. Molecular structure of the nitramine explosive RDX.
Despite the Army's need for information on the anaerobic biodegradation of explosives, relatively little is known (Gorontzy et al. 1994). RDX is reported to be more easily biodegraded under anaerobic, rather than aerobic conditions (Funk et al. 1993; Kitts, Cunningham, and Unkefer 1994; McCormick, Cornell, and Kaplan 1981; Roberts, Ahmad, and Pendharkar 1996). The few exceptions include RDX biodegradation by a white rot fungus (Fernando and Aust 1991), by the bacterium Stenotrophomonas maltophilia PB1 when using RDX as the sole source of nitrogen (Binks, Nicklin, and Bruce 1995), and during composting of explosives contaminated soil (Williams, Ziegenfuss, and Sisk 1992).
Most of the studies demonstrating RDX biodegradation under anaerobic conditions were conducted in poorly defined environments where the electron donor and acceptors were not firmly established. For example, in three such studies, the bacterial cultures were grown in nutrient broth (McCormick, Cornell, and Kaplan 1981), yeast extract (Kitts, Cunningham, and Unkefer 1994), and Brain Hear Infusion media (Regan and Crawford 1994). In the latter two cases, RDX biodegradation was carried out by pure cultures of bacteria isolated from explosives-contaminated soil. In the former case, the nutrient broth was inoculated with organisms from activated sludge.
Several studies have been carried out under nitrate-reducing conditions (Bell, Burrows, and Carrazza 1987; McCormick, Cornell, and Kaplan 1984). The results, however, are inconclusive. While RDX degradation and nitrate removal occurred in the experimental systems studied, it is not clear whether RDX degradation and nitrate depletion occurred simultaneously. Further research is required to determine if RDX degradation is linked to nitrate-reduction.
The primary objectives of this part of the study were to investigate RDX biodegradation under rigorously controlled anaerobic conditions and to obtain a RDX biodegrading enrichment culture. This study focused on the biodegradation of RDX by a methanogenic enrichment culture derived from an explosives manufacturing wastewater treatment plant. This is the first report demonstrating RDX biodegradation under methanogenic conditions.
1. A literature review was conducted of laboratory and field studies involving the biodegradation of RDX. The analyzed information indicated that RDX was susceptible to biodegradation under anaerobic conditions, but recalcitrant under aerobic conditions. Under anaerobic conditions, however, little is known about the specific conditions, making it difficult to extrapolate these findings to the field. The literature review identified areas needing further research. These areas are detailed in this report.
2. RDX was obtained from Holston AAP.
3. Biodegradation studies were carried out in serum bottles using sludge and wastewater obtained from the treatment plant at Holston AAP.
4. Enrichment cultures were obtained by re-amending serum bottles with RDX after its degradation, and by adding ethanol or butyrate as an electron donor.
5. Liquid samples were taken periodically from the serum bottles and analyzed.
6. RDX and biodegradation intermediates were analyzed by high pressure liquid chromatography.
7. The results were analyzed and conclusions were drawn based on the results of this stage of work.
Findings from this research will be incorporated into ongoing Exploratory Development (6.2) work in treatment of munitions wastewater.