Electron shuttle-stimulated RDX mineralization and biological production of 4-nitro-2,4-diazabutanal (NDAB) in RDX-contaminated aquifer material

The potential for extracellular electron shuttles to stimulate RDX biodegradation was investigated with RDX-contaminated aquifer material. Electron shuttling compounds including anthraquinone-2,6-disulfonate (AQDS) and soluble humic substances stimulated RDX mineralization in aquifer sediment. RDX mass-loss was similar in electron shuttle amended and donor-alone treatments; however, the concentrations of nitroso metabolites, in particular TNX, and ring cleavage products (e.g., HCHO, MEDINA, NDAB, and NH₄ ⁺) were different in shuttle-amended incubations. Nitroso metabolites accumulated in the absence of electron shuttles (i.e., acetate alone). Most notably, 40–50% of [¹⁴C]-RDX was mineralized to ¹⁴CO₂ in shuttle-amended incubations. Mineralization in acetate amended or unamended incubations was less than 12% within the same time frame. The primary differences in the presence of electron shuttles were the increased production of NDAB and formaldehyde. NDAB did not further degrade, but formaldehyde was not present at final time points, suggesting that it was the mineralization precursor for Fe(III)-reducing microorganisms. RDX was reduced concurrently with Fe(III) reduction rather than nitrate or sulfate reduction. Amplified 16S rDNA restriction analysis (ARDRA) indicated that unique Fe(III)-reducing microbial communities (β- and γ-proteobacteria) predominated in shuttle-amended incubations. These results demonstrate that indigenous Fe(III)-reducing microorganisms in RDX-contaminated environments utilize extracellular electron shuttles to enhance RDX mineralization. Electron shuttle-mediated RDX mineralization may become an effective in situ option for contaminated environments.     

Microbially mediated biodegradation of hexahydro-1,3,5-trinitro-1,3,5- triazine by extracellular electron shuttling compounds

The potential for humic substances to stimulate the reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was investigated. This study describes a novel approach for the remediation of RDX-contaminated environments using microbially mediated electron shuttling. Incubations without cells demonstrated that reduced AQDS transfers electrons directly to RDX, which was reduced without significant accumulation of the nitroso intermediates. Three times as much reduced AQDS (molar basis) was needed to completely reduce RDX. The rate and extent of RDX reduction differed greatly among electron shuttle/acceptor amendments for resting cell suspensions of Geobacter metallireducens and G. sulfurreducens with acetate as the sole electron donor. AQDS and purified humic substances stimulated the fastest rate of RDX reduction. The nitroso metabolites did not significantly accumulate in the presence of AQDS or humic substances. RDX reduction in the presence of poorly crystalline Fe(III) was relatively slow and metabolites transiently accumulated. However, adding humic substances or AQDS to Fe(III)-containing incubations increased the reduction rates. Cells of G. metallireducens alone reduced RDX; however, the rate of RDX reduction was slow relative to AQDS-amended incubations. These data suggest that extracellular electron shuttle-mediated RDX transformation is not organism specific but rather is catalyzed by multiple Fe(III)- and humic-reducing species. Electron shuttle-mediated RDX reduction may eventually become a rapid and effective cleanup strategy in both Fe(III)-rich and Fe(III)-poor environments.     

Microbially Mediated Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5- Triazine by Extracellular Electron Shuttling Compounds

The potential for humic substances to stimulate the reduction of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was investigated. This study describes a novel approach for the remediation of RDX-contaminated environments using microbially mediated electron shuttling. Incubations without cells demonstrated that reduced AQDS transfers electrons directly to RDX, which was reduced without significant accumulation of the nitroso intermediates. Three times as much reduced AQDS (molar basis) was needed to completely reduce RDX. The rate and extent of RDX reduction differed greatly among electron shuttle/acceptor amendments for resting cell suspensions of Geobacter metallireducens and G. sulfurreducens with acetate as the sole electron donor. AQDS and purified humic substances stimulated the fastest rate of RDX reduction. The nitroso metabolites did not significantly accumulate in the presence of AQDS or humic substances. RDX reduction in the presence of poorly crystalline Fe(III) was relatively slow and metabolites transiently accumulated. However, adding humic substances or AQDS to Fe(III)-containing incubations increased the reduction rates. Cells of G. metallireducens alone reduced RDX; however, the rate of RDX reduction was slow relative to AQDS-amended incubations. These data suggest that extracellular electron shuttle-mediated RDX transformation is not organism specific but rather is catalyzed by multiple Fe(III)- and humic-reducing species. Electron shuttle-mediated RDX reduction may eventually become a rapid and effective cleanup strategy in both Fe(III)-rich and Fe(III)-poor environments.     

Effect of iron(III), humic acids and anthraquinone-2,6-disulfonate on biodegradation of cyclic nitramines by Clostridium sp. EDB2

AIMS: To determine the biodegradation of cyclic nitramines by an anaerobic marine bacterium, Clostridium sp. EDB2, in the presence of Fe(III), humic acids (HA) and anthraquinone-2,6-disulfonate (AQDS). METHODS AND RESULTS: An obligate anaerobic bacterium, Clostridium sp. EDB2, degraded RDX and HMX, and produced similar product distribution including nitrite, methylenedinitramine, nitrous oxide, ammonium, formaldehyde, formic acid and carbon dioxide. Carbon (C) and nitrogen (N) mass balance for RDX products were 87% and 82%, respectively, and for HMX were 88% and 74%, respectively. Bacterial growth and biodegradation of RDX and HMX were stimulated in the presence of Fe(III), HA and AQDS suggesting that strain EDB2 utilized Fe(III), HA and AQDS as redox mediators to transfer electrons to cyclic nitramines. CONCLUSIONS: Strain EDB2 demonstrated a multidimensional approach to degrade RDX and HMX: first, direct degradation of the chemicals; second, indirect degradation by reducing Fe(III) to produce reactive-Fe(II); third, indirect degradation by reducing HA and AQDS which act as electron shuttles to transfer electrons to the cyclic nitramines. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study could be helpful in determining the fate of cyclic nitramine energetic chemicals in the environments rich in Fe(III) and HA.     

The periplasmic 9.6-kilodalton c-type cytochrome of Geobacter sulfurreducens is not an electron shuttle to Fe(III)

Geobacter sulfurreducens contains a 9.6-kDa c-type cytochrome that was previously proposed to serve as an extracellular electron shuttle to insoluble Fe(III) oxides. However, when the cytochrome was added to washed-cell suspensions of G. sulfurreducens it did not enhance Fe(III) oxide reduction, whereas similar concentrations of the known electron shuttle, anthraquinone-2,6-disulfonate, greatly stimulated Fe(III) oxide reduction. Furthermore, analysis of the extracellular c-type cytochromes in cultures of G. sulfurreducens demonstrated that the dominant c-type cytochrome was not the 9.6-kDa cytochrome, but rather a 41-kDa cytochrome. These results and other considerations suggest that the 9.6-kDa cytochrome is not an important extracellular electron shuttle to Fe(III) oxides.     

Determination of key metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine with Rhodococcus sp. strain DN22.

Rhodococcus sp. strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known. The present study describes aerobic biodegradation of RDX (175 microM) when used as an N source for strain DN22. RDX was converted to nitrite (NO(2)(-)) (30%), nitrous oxide (N(2)O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [(15)N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N(2)O with two molecular mass ions: one at 44 Da, corresponding to (14)N(14)NO, and the second at 45 Da, corresponding to (15)N(14)NO. The nonlabeled N(2)O could be formed only from -NO(2), whereas the (15)N-labeled one was presumed to originate from a nitramine group ((15)N-(14)NO(2)) in RDX. Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion [M-H] at 118 Da. High-resolution MS indicated a molecular formula of C(2)H(5)N(3)O(3). When the experiment was repeated with ring-labeled [(15)N]-RDX, the [M-H] appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX. When [U-(14)C]-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in (14)CO(2) (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO(2) and the other two were incorporated in the ring cleavage product with an MW of 119. Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119.     

Influence of Reduced Electron Shuttling Compounds on Biological H<Subscript>2</Subscript> Production in the Fermentative Pure Culture <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

Several reports suggest that extracellular electron shuttles influence fermentative metabolism in a beneficial manner for bioremediation and biotechnology strategies. The focus of this research was to characterize the effects of reduced electron shuttling molecules on fermentative H₂ production. Reduced electron shuttles may provide reducing equivalents to generate H₂, which influences alternate cellular processes. Electron shuttling compounds cycle between reduced-oxidized states and influence fermentative physiology. Clostridium beijerinckii fermentation was altered using a physiological approach that resulted in H₂ production with the reduced extracellular electron shuttle anthrahydroquinone-2,6,-disulfonate (AH₂QDS) and biologically reduced humic substances as the primary electron donors. Cells were suspended in a buffer with an excess of the biological electron transfer molecule NAD⁺, with AH₂QDS (100–1000 μM) or biologically reduced humic substances (0.01–0.025 g/L) as the sole electron source. Increasing concentrations of AH₂QDS and reduced humics increased H₂ production, while H₂ production was suppressed by Fe(III) hydroxides, which outcompeted the cells for electrons from the reduced shuttles, suggesting that the shuttles are in fact electron donors for H₂ production. Oxidized AQDS/humics did not increase H₂ production. Organic acid production shifted toward butyric acid in the presence of reduced electron shuttles, particularly with growing cells. Growth and hydrogen production rates in growing cells were initially faster in the presence of the reduced electron shuttles; however, the final biomass yield was inversely proportional to the starting AH₂QDS concentration, which suggests that reduced shuttles may compete with anabolic cell processes for available energetic resources or that the shift to excess butyrate becomes toxic to the cells.     

Protective role of tolC in efflux of the electron shuttle anthraquinone-2,6-disulfonate

Extracellular electron transfer can play an important role in microbial respiration on insoluble minerals. The humic acid analog anthraquinone-2,6-disulfonate (AQDS) is commonly used as an electron shuttle during studies of extracellular electron transfer. Here we provide genetic evidence that AQDS enters Shewanella oneidensis strain MR-1 and causes cell death if it accumulates past a critical concentration. A tolC homolog protects the cell from toxicity by mediating the efflux of AQDS. Electron transfer to AQDS appears to be independent of the tolC pathway, however, and requires the outer membrane protein encoded by mtrB. We suggest that there may be structural and functional relationships between quinone-containing electron shuttles and antibiotics.     

Metabolism of hexahydro-1,3,5-trinitro-1,3,5-triazine through initial reduction to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine followed by denitration in<Emphasis Type="Italic"> Clostridium bifermentans</Emphasis> HAW-1

A fast hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-degrading [28.1 mol h^–1 g (dry weight) cells^–1; biomass, 0.16 g (dry weight) cells^–1] and strictly anaerobic bacterial strain, HAW-1, was isolated and identified as Clostridium bifermentans using a 16S-rRNA-based method. Based on initial rates, strain HAW-1 transformed RDX to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) with yields of 56, 7.3 and 0.2%, respectively. Complete removal of RDX and its nitroso metabolites produced (%, of total C or N) methanol (MeOH, 23%), formaldehyde (HCHO, 7.4%), carbon dioxide (CO₂, 3.0%) and nitrous oxide (N₂O, 29.5%) as end products. Under the same conditions, strain HAW-1 transformed MNX separately at a rate of 16.9 mol h^–1 g (dry weight) cells^–1 and produced DNX (25%) and TNX (0.4%) as transient products. Final MNX transformation products were (%, of total C or N) MeOH (21%), HCHO (2.9%), and N₂O (17%). Likewise strain HAW-1 degraded TNX at a rate of 7.5 mol h^–1 g (dry weight) cells^–1 to MeOH and HCHO. Furthermore, removal of both RDX and MNX produced nitrite (NO₂^–) as a transient product, but the nitrite release rate from MNX was quicker than from RDX. Thus, the predominant pathway for RDX degradation is based on initial reduction to MNX followed by denitration and decomposition. The continued sequential reduction to DNX and TNX is only a minor route.     

Determination of Key Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine with Rhodococcus sp. Strain DN22

Rhodococcus sp. strain DN22 can convert hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to nitrite, but information on degradation products or the fate of carbon is not known. The present study describes aerobic biodegradation of RDX (175 μM) when used as an N source for strain DN22. RDX was converted to nitrite (NO₂⁻) (30%), nitrous oxide (N₂O) (3.2%), ammonia (10%), and formaldehyde (HCHO) (27%), which later converted to carbon dioxide. In experiments with ring-labeled [¹⁵N]-RDX, gas chromatographic/mass spectrophotometric (GC/MS) analysis revealed N₂O with two molecular mass ions: one at 44 Da, corresponding to ¹⁴N¹⁴NO, and the second at 45 Da, corresponding to ¹⁵N¹⁴NO. The nonlabeled N₂O could be formed only from -NO₂, whereas the ¹⁵N-labeled one was presumed to originate from a nitramine group (¹⁵N-¹⁴NO₂) in RDX. Liquid chromatographic (LC)-MS electrospray analyses indicated the formation of a dead end product with a deprotonated molecular mass ion [M-H] at 118 Da. High-resolution MS indicated a molecular formula of C₂H₅N₃O₃. When the experiment was repeated with ring-labeled [¹⁵N]-RDX, the [M-H] appeared at 120 Da, indicating that two of the three N atoms in the metabolite originated from the ring in RDX. When [U-¹⁴C]-RDX was used in the experiment, 64% of the original radioactivity in RDX incorporated into the metabolite with a molecular weight (MW) of 119 (high-pressure LC/radioactivity) and 30% in ¹⁴CO₂ (mineralization) after 4 days of incubation, suggesting that one of the carbon atoms in RDX was converted to CO₂ and the other two were incorporated in the ring cleavage product with an MW of 119. Based on the above stoichiometry, we propose a degradation pathway for RDX based on initial denitration followed by ring cleavage to formaldehyde and the dead end product with an MW of 119.     

Characterization of Fe(III) Reduction by Chlororespiring Anaeromxyobacter dehalogenans

Anaeromyxobacter dehalogenans strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of ortho-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [¹⁴C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.     

Characterization of Fe(III) reduction by chlororespiring Anaeromyxobacter dehalogenans

Anaeromyxobacter dehalogenans strain 2CP-C has been shown to grow by coupling the oxidation of acetate to the reduction of ortho-substituted halophenols, oxygen, nitrate, nitrite, or fumarate. In this study, strain 2CP-C was also found to grow by coupling Fe(III) reduction to the oxidation of acetate, making it one of the few isolates capable of growth by both metal reduction and chlororespiration. Doubling times for growth of 9.2 and 10.2 h were determined for Fe(III) and 2-chlorophenol reduction, respectively. These were determined by using the rate of [(14)C]acetate uptake into biomass. Fe(III) compounds used by strain 2CP-C include ferric citrate, ferric pyrophosphate, and amorphous ferric oxyhydroxide. The addition of the humic acid analog anthraquinone 2,6-disulfonate (AQDS) increased the reduction rate of amorphous ferric iron oxide, suggesting AQDS was used as an electron shuttle by strain 2CP-C. The addition of chloramphenicol to fumarate-grown cells did not inhibit Fe(III) reduction, indicating that the latter activity is constitutive. In contrast, the addition of chloramphenicol inhibited dechlorination activity, indicating that chlororespiration is inducible. The presence of insoluble Fe(III) oxyhydroxide did not significantly affect dechlorination, whereas the presence of soluble ferric pyrophosphate inhibited dechlorination. With its ability to respire chlorinated organic compounds and metals such as Fe(III), strain 2CP-C is a promising model organism for the study of the interaction of these potentially competing processes in contaminated environments.     

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation by <Emphasis Type="Italic">Acetobacterium paludosum</Emphasis>

Substrates and nutrients are often added to contaminated soil or groundwater to enhance bioremediation. Nevertheless, this practice may be counterproductive in some cases where nutrient addition might relieve selective pressure for pollutant biodegradation. Batch experiments with a homoacetogenic pure culture of Acetobacterium paludosum showed that anaerobic RDX degradation is the fastest when auxiliary growth substrates (yeast extract plus fructose) and nitrogen sources (ammonium) are not added. This bacterium degraded RDX faster under autotrophic (H₂-fed) than under heterotrophic conditions, even though heterotrophic growth was faster. The inhibitory effect of ammonium is postulated to be due to the repression of enzymes that initiate RDX degradation by reducing its nitro groups, based on the known fact that ammonia represses nitrate and nitrite reductases. This observation suggests that the absence of easily assimilated nitrogen sources, such as ammonium, enhances RDX degradation. Although specific end products of RDX degradation were not determined, the production of nitrous oxide (N₂O) suggests that A. paludosum cleaved the triazine ring.     

Biodegradation of the nitramine explosives hexahydro-1,3,5-trinitro-1,3,5-triazine and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine in cold marine sediment under anaerobic and oligotrophic conditions

The in situ degradation of the two nitramine explosives, hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), was evaluated using a mixture of RDX and HMX, incubated anaerobically at 10 degrees C with marine sediment from a previous military dumping site of unexploded ordnance (UXO) in Halifax Harbor, Nova Scotia, Canada. The RDX concentration (14.7 mg.L-1) in the aqueous phase was reduced by half in 4 days, while reduction of HMX concentration (1.2 mg.L-1) by half required 50 days. Supplementation with the carbon sources glucose, acetate, or citrate did not affect the removal rate of RDX but improved removal of HMX. Optimal mineralization of RDX and HMX was obtained in the presence of glucose. Using universally labeled (UL)-[14C]RDX, we obtained a carbon mass balance distributed as follows: CO2, 48%-58%; water soluble products, 27%-31%; acetonitrile extractable products, 2.0%-3.4%; and products covalently bound to the sediments and biomass, 8.9% (in the presence of glucose). The disappearance of RDX was accompanied by the formation of the mononitroso derivative hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and formaldehyde (HCHO) that subsequently disappeared. In the case of HMX, mineralization reached only 13%-27% after 115 days of incubation in the presence or absence of the carbon sources. The disappearance of HMX was also accompanied by the formation of the mononitroso derivative. The total population of psychrotrophic anaerobes that grew at 10 degrees C was 2.6 x 10(3) colony-forming units.(g sediment dry mass)-1, and some psychrotrophic sediment isolates were capable of degrading RDX under conditions similar to those used for sediments. Based on the distribution of products, we suggest that the sediment microorganisms degrade RDX and HMX via an initial reduction to the corresponding mononitroso derivative, followed by denitration and ring cleavage.     

Enhanced Biodegradation and Fate of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) and Octahydro-1,3,5,7-Tetranitro-1,3,5,7-Tetrazocine (HMX) in Anaerobic Soil Slurry Bioprocess

Native soil microbial populations and unadapted municipal anaerobic sludges were compared for nitramine explosive degradation in microcosm assays under various conditions. Microbial populations from an explosive-contaminated soil were only able to mineralize 12% hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (at a concentration of 800 mg/kg slurry) or 4% octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (at a concentration of 267 mg/kg slurry). In contrast, municipal anaerobic sludges were able to mineralize them to carbon dioxide, with efficiencies of up to 65%. Reduction of RDX and HMX into their corresponding nitroso-derivatives was notably faster than their mineralization. The biodegradation of HMX was typically delayed by the presence of RDX in the microcosm, confirming RDX is used as an electron acceptor preferentially to HMX. The laboratory-scale bioslurry reactor reproduced the results of the microcosm assays, yet with much higher RDX and HMX degradation rates. A radiolabel-based mass balance in the soil slurry indicated that, besides a significant mineralization to carbon dioxide, 25% and 31% of RDX and HMX, respectively, appeared as acetonitrile-extractable metabolites, while the remaining part was incorporated into biomass and irreversibly bound to the soil matrix. About 10% of the HMX derivatives were estimated to be chemically bound to the soil matrix, while for RDX the estimation was nil.     

Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine

Biodegradation of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) occurs under anaerobic conditions, yielding a number of products, including: hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine, hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine, hexahydro-1,3,5-trinitroso-1,3,5-triazine, hydrazine, 1,1-dimethyl-hydrazine, 1,2-dimethylhydrazine, formaldehyde, and methanol. A scheme for the biodegradation of RDX is proposed which proceeds via successive reduction of the nitro groups to a point where destabilization and fragmentation of the ring occurs. The noncyclic degradation products arise via subsequent reduction and rearrangement reactions of the fragments. The scheme suggests the presence of several additional compounds, not yet identified. Several of the products are mutagenic or carcinogenic or both. Anaerobic treatment of RDX wastewaters, which also contain high nitrate levels, would permit the denitrification to occur, with concurrent degradation of RDX ultimately to a mixture of hydrazines and methanol. The feasibility of using an aerobic mode in the further degradation of these products is discussed.     

Evaluating Biodegradation as a Primary and Secondary Treatment for Removing RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) from a Perched Aquifer

Ground water beneath the U.S. Department of Energy (USDOE) Pantex Plant is contaminated with the high explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). The authors evaluated biodegradation as a remedial option by measuring RDX mineralization in Pantex aquifer microcosms spiked with ¹⁴C-labeled RDX (75 g soil, 15 ml of 5 mg RDX/L). Under anaerobic conditions and constant temperature (16°C), cumulative ¹⁴CO₂ production ranged between 52% and 70% after 49 days, with nutrient-amended (C, N, P) microcosms yielding the greatest mineralization (70%). The authors also evaluated biodegradation as a secondary treatment for removing RDX degradates following oxidation by permanganate (KMnO₄) or reduction by dithionite-reduced aquifer solids (i.e., redox barriers). Under this coupled abiotic/biotic scenario, we found that although unconsumed permanganate initially inhibited biodegradation, > 48% of the initial ¹⁴C-RDX was recovered as ¹⁴CO₂ within 77 days. Following exposure to dithionite-reduced solids, RDX transformation products were also readily mineralized (> 47% in 98 days). When we seeded Pantex aquifer material into Ottawa Sand that had no prior exposure to RDX, mineralization increased 100%, indicating that the Pantex aquifer may have an adapted microbial community that could be exploited for remediation purposes. These results indicate that biodegradation effectively transformed and mineralized RDX in Pantex aquifer microcosms. Additionally, biodegradation may be an excellent secondary treatment for RDX degradates produced from in situ treatment with permanganate or redox barriers.     

Cloning,sequencing, and characterization of the hexahydro-1,3,5-Trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.     

Cloning, Sequencing, and Characterization of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Degradation Gene Cluster from Rhodococcus rhodochrous

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.     

电子穿梭体对菌株Clostridium butyricum LQ25异化铁还原性质影响

【目的】在异化铁还原细菌培养体系中,通过外加电子穿梭体,分析电子穿梭体种类与浓度对细菌异化铁还原性质的影响。【方法】以一株发酵型异化铁还原细菌Clostridium butyricum LQ25为研究对象,设置水溶性介体蒽醌-2-磺酸钠和核黄素作为外加电子穿梭体。【结果】在氢氧化铁为电子受体、葡萄糖为电子供体培养条件下,不同浓度蒽醌-2-磺酸钠和核黄素对菌株LQ25异化铁还原效率影响具有显著性差异。外加蒽醌-2-磺酸钠浓度为0.5 mmol/L时,菌株累积产生Fe(Ⅱ)浓度最高,为12.95±0.08 mg/L,相比对照组提高88%。核黄素浓度为100mg/L时,菌株累积产生Fe(Ⅱ)浓度是11.06±0.04mg/L,相比对照组提高61%。外加电子穿梭体能够改变菌株LQ25发酵产物中丁酸和乙酸浓度,提高乙酸相对含量。【结论】蒽醌-2-磺酸钠和核黄素作为外加电子穿梭体能显著促进细菌异化铁还原效率,为揭示发酵型异化铁还原细菌胞外电子传递机制提供实验支持。