RDX used in experiments was obtained from the Holston Army Ammunition Plant (93.8 to 99.6 percent pure). RDX analytical standards were obtained from a commercial vendor (AccuStandard, Inc., New Haven, CT). All other chemicals were obtained from major suppliers of chemicals and were of the highest purity obtainable.
Biodegradation studies were carried out in serum bottles using sludge and wastewater obtained from an industrial wastewater treatment plant located at the Holston Army Ammunition Plant. The treatment plant receives wastewater contaminated with RDX and HMX. Samples were collected from the beginning segment of the treatment plant known as the anoxic filter and stored at 4 °C until use.
Biodegradation of RDX was evaluated by comparing substrate disappearance in the experimental bottles to that in sterile controls. Microcosms were prepared by making a stock solution of RDX in acetonitrile and adding it to sterile 160 ml serum bottles. The acetonitrile was allowed to evaporate overnight, leaving behind a thin layer of RDX. Eighty ml of a sterile (steam sterilization, 121 °C, 15 min) basal salts medium containing resazurin (0.0002 percent) was added to the serum bottles, followed by the addition of 20 ml of a wastewater-sludge slurry containing 5 grams wet sludge. The basal salts solution consisted of the following quantity per liter: NaCl, 0.8 g; NH4Cl, 1.0 g; KCl, 0.1 g; MgSO4_7H2O, 0.02 g; KH2PO4, 1.35; K2HPO4, 1.75 g; NaHCO3, 1.0 g; trace metal solution, 10 ml; vitamins, 10 ml. Trace metal and vitamin solutions were made as previously described (Tanner, McInerney, and Nagle 1989). The pH of the medium was adjusted to 7.2. The basal salts medium refers to the basal salts containing trace metals and vitamins. The medium was prepared and dispensed using strict anoxic techniques as previously described (Shelton and Tiedje 1984). After addition of the slurry, the bottles were sealed with black butyl stoppers and aluminum crimp seals. The headspace of the bottles was evacuated and replaced with a mixture of N2:CO2 (80:20) three times, and then pressurized to 1.3 ATM. Sterile controls were prepared by taking serum bottles prepared as described above, with the exception that they were not amended with RDX. They were autoclaved on 3 successive days (121 °C, 15 min). Then the contents were transferred to a sterile serum bottle containing RDX. The bottles were then sealed as previously described. The study was conducted in triplicate.
A methanogenic enrichment culture was obtained by re-amending serum bottles with RDX after its degradation and adding ethanol or butyrate as an electron donor. After several additions of RDX, the enrichment was periodically transferred (20 to 40 percent) to fresh basal salts medium. Studies with the enrichment culture were done in smaller serum bottles (35 ml volume). A 200 _M RDX stock solution in deionized water was made and shaken overnight at 35 °C. Approximately 17 ml of the enrichment culture was added to sterile, nitrogen flushed serum bottles. Three ml of the RDX stock solution were added to the serum bottles to reach a target concentration of approximately 30 _M RDX. Studies were conducted in triplicate at room temperature. Strict anaerobic techniques were used during media preparation, culturing, and sampling.
RDX and biodegradation intermediates were analyzed by high pressure liquid chromatography (HPLC) using a Waters Module 1 HPLC System outfitted with a Lichrosphere C-18 reverse phase column (250 mm x 4.6 mm, 5 _m; Alltech Associates, Inc., Deerfield, IL). The following HPLC conditions were used: mobile phase, 55:45 (methanol:50 mM acetate buffer, pH 4.5); injection volume, 20 ul; flow rate, 1.1 ml/minute; wavelength, 254 nm. Later analyses employed an acetonitrile:acetate buffer (45:55%, v/v) mobile phase. Identification of unknown biodegradation intermediates was carried out by comparing their retention time with those of authentic standards.
The headspace of the serum bottles was monitored for the formation of CH4 by gas chromatography. Methane produced from unamended controls was subtracted from that produced in substrate-amended bottles. This amount was compared to the theoretically expected amount of CH4 (Gottschalk 1986; McInerney 1986). Gas samples were injected into a SRI gas chromatograph equipped with a flame ionization detector and a Porapak-Q packed column (Alltech Associates, Inc., Deerfield, IL). The GC conditions were: helium flow rate, 30 ml/min; injector, oven, and detector temperatures, 75 °C.
Liquid samples were taken periodically from the serum bottles using a syringe and needle and were stored at -20 °C until use. Samples were centrifuged at 12,000 x g for 4 minutes in a bench top microcentrifuge before analyzing by HPLC. Methane concentrations were determined by taking samples of the headspace gas (0.2 ml) using a 1 ml disposable syringe with a 21 ga needle, and then injecting the samples directly into the GC as described above.