DNAN (2,4-dinitroanisole) is one of the insensitive nitroaromatic ingredients increasingly used as a replacement for 2,4,6-trinitrotoluene (TNT) in munitions. DNAN or its metabolites can be toxic to earthworms, bacteria, algae, and plants (1, 2). Therefore, the release of DNAN to the environment could pose ecological and health risks. There is little information about the environmental behavior of DNAN (2), and no bacteria capable of complete biodegradation have been reported.The initial reaction in the biotransformation of DNAN by bacteria and in abiotic transformation with zero valent iron is the reduction of the nitro group in the ortho position to yield 2-amino-4-nitroanisole (3-5). Under anoxic conditions, DNAN is biotransformed to toxic metabolites such as diaminoanisole (3,(5)(6)(7)(8). A Bacillus strain was reported to transform DNAN slowly under aerobic conditions to 2-amino-4-nitroanisole as a predominant dead-end product (4). A recent investigation revealed substantial aerobic biodegradation of DNAN by enrichment cultures derived from activated sludge, but the responsible bacteria were not isolated (9). During alkaline hydrolysis, DNAN is converted to 2,4-dinitrophenolate via an unstable hydride-Meisenheimer complex (10). Phototransformation of DNAN resulted in 2-hydroxy-4-nitroanisole and 2,4-dinitrophenol (2,4-DNP) as major and minor products, respectively (5, 11). The pathway of 2,4-DNP biodegradation under aerobic conditions is well-known, and the genes involved have been characterized for Rhodococcus erythropolis and Nocardia (12, 13). A Rhodococcus sp. has been reported to degrade 4-nitroanisole by a pathway involving removal of the methyl group and subsequent degradation of the resulting 4-nitrophenol via 4-nitrocatechol and 1,2,4-trihydroxybenzene (14). Biotransformation of DNAN to 2,4-DNP has been reported in mammals (15).We isolated bacteria able to grow on DNAN as the sole carbon source under aerobic conditions and elucidated the catabolic pathway. An initial O-demethylation catalyzed by a hydrolase was followed by degradation of the resultant 2,4-dinitrophenol by a pathway involving formation of a hydride-Meisenheimer complex (16,17). MATERIALS AND METHODSIsolation of DNAN-degrading bacteria. An activated sludge sample from Holston Army Ammunition Plant was inoculated (20% [vol/vol]) into 1/4-strength minimal salts medium (MSB) (18) (pH 6.5) containing 2,4-dinitroanisole (DNAN) (100 M), and the suspension was incubated at 30°C with shaking. Following the disappearance of DNAN as monitored by high-performance liquid chromatography (HPLC) (see below), samples (20% [vol/vol]) were repeatedly transferred into fresh medium and then spread on MSB agar (1.5%) plates containing DNAN (100 M). Individual colonies that appeared after 4 days of incubation were tested for the ability to degrade DNAN in carbon-and nitrogen-free MSB. Isolates that used DNAN as the sole source of carbon, nitrogen, and energy were selected for further study. 16S rRNA gene analysis was used for identification of the strains...

Lignin is a complex aromatic polymer found in plant cell walls that makes up 15 to 40% of plant biomass. The degradation of lignin substructures by bacteria is of emerging interest because it could provide renewable alternative feedstocks and intermediates for chemical manufacturing industries. We have isolated a bacterium, strain SG61-1L, that rapidly degrades all of the stereoisomers of one lignin substructure, guaiacylglycerol-β-guaiacyl ether (GGE), which contains a key β-O-4 linkage found in most intermonomer linkages in lignin. In an effort to understand the rapid degradation of GGE by this bacterium, we heterologously expressed and kinetically characterized a suite of dehydrogenase candidates for the first known step of GGE degradation. We identified a clade of active GGE dehydrogenases and also several other dehydrogenases outside this clade that were all able to oxidize GGE. Several candidates exhibited stereoselectivity toward the GGE stereoisomers, while others had higher levels of catalytic performance than previously described GGE dehydrogenases for all four stereoisomers, indicating a variety of potential applications for these enzymes in the manufacture of lignin-derived commodities.

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