In previous work, we studied the anaerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a methanogenic mixed culture that biodegrades RDX by using H2 as the sole electron donor. Strain HAAP-1 was isolated after enriching for the homoacetogens in a mineral medium containing RDX and an H2-CO2 (80:20) headspace. Strain HAAP-1 degraded 29.0 microM RDX in <14 days and formed 13.0 mM acetate when grown in a mineral medium with an H2-CO2 headspace. Methylenedinitramine was observed as a transient intermediate, indicating ring cleavage had occurred. In live cultures containing an N2-CO2 headspace, RDX was not degraded, and no acetate was formed. The 16S rRNA gene sequence for strain HAAP-1, consisting of 1485 base pairs, had a 99.2% and 99.1% sequence similarity to Acetobacterium malicum and A. wieringae, respectively. This is the first report of RDX degradation by a homoacetogen growing autotrophically and extends the number of genera known to carry out this transformation.

Existing methods for perchlorate remediation are hampered by the common co-occurrence of nitrate, which is structurally similar and a preferred electron acceptor. In this work, the potential for perchlorate removal using cell-free bacterial enzymes as biocatalysts was investigated using crude cell lysates and soluble protein fractions of Azospira oryzae PS, as well as soluble protein fractions encapsulated in lipid and polymer vesicles. The crude lysates showed activities between 41 700 to 54 400 U L(-1) (2.49 to 3.06 U mg(-1) total protein). Soluble protein fractions had activities of 15 400 to 29 900 U L(-1) (1.70 to 1.97 U mg(-1)) and still retained an average of 58.2% of their original activity after 23 days of storage at 4 °C under aerobic conditions. Perchlorate was removed by the soluble protein fraction at higher rates than nitrate. Importantly, perchlorate reduction occurred even in the presence of 500-fold excess nitrate. The soluble protein fraction retained its function after encapsulation in lipid or polymer vesicles, with activities of 13.8 to 70.7 U L(-1), in agreement with theoretical calculations accounting for the volume limitation of the vesicles. Further, encapsulation mitigated enzyme inactivation by proteinase K. Enzyme-based technologies could prove effective at perchlorate removal from water cocontaminated with nitrate or sulfate.

The anaerobic biodegradation of 3,3, octahydrol,3,5,3,5,, and 2,4,6-trinitrotoluene (TNT) by a methanogenic mixed culture was investigated. Microcosms containing a basal medium and the mixed culture were amended with ethanol, propylene glycol (PG), butyrate or hydrogen gas as the electron donor and a mixture of TNT (50 \iM), RDX (25 |JM), and HMX (8 pM). After 29 days, TNT and RDX were completely transformed to unidentified end products in the bottles amended with ethanol, hydrogen, or PQ while 53%, 40%, and 22% of the HMX was transformed, respectively There was no loss of RDX or HMX in the electron donor unamended control bottles. The ethanol and PG were transformed to near stoichiometric amounts of acetate and propionate, suggesting the immediate electron donor supporting the transformation of the H2 or electron donors that produce H2 may be a useful strategy for enhancing the anaerobic biodegradation of explosives in contaminated groundwater and soils.DISCLAIMER: The contents of this report are not to be used for advertising, publication, or promotional purposes. Citation of trade names does not constitute an official endorsement or approval of the use of such commercial products. All product names and trademarks cited are the property of their respective owners. The findings of this report are not to be construed as an official Department of the Army position unless so designated by other authorized documents. DESTROY THIS REPORT WHEN IT IS NO LONGER NEEDED. DO NOT RETURN IT TO THE ORIGINATOR. AbstractThe anaerobic biodegradation of hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX), octahydro-l,3,5,7-tetranitro-l,3,5,7-tetrazocine (HMX), and 2,4,6-trinitrotoIuene (TNT) by a methanogenic mixed culttire was investigated. Microcosms containing a basal medium and the mixed culture were amended with ethanol, propylene glycol (PG), butyrate or hydrogen gas as the electron donor and a mixture of TNT (50 ^M), RDX (25 ^M), and HMX (8 ^M). After 29 days TNT and RDX were completely transformed to unidentified endproducts in the bottles amended with ethanol, hydrogen, or PG, while 53%, 40%, and 22% of the HMX was transformed, respectively. There was no loss of RDX or HMX in the electron donor unamended control bottles. The ethanol and PG were transformed to near stoichiometric amounts of acetate and propionate, suggesting the immediate electron donor supporting the transformation of the explosives was the H2 evolved during the metaboUsm of the parent substrate. Our findings suggest that the addition of H2 or electron donors that produce H2 may be a useful strategy for enhancing the anaerobic biodegradation of explosives in contaminated groundwater and soils. Pubhshed by Elsevier Science Ltd.

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