RDX Degradation Research Articles

Comparative analysis of explosive RDX-induced proteomes in the Pseudomonas sp. HK-6 wild-type strain and its rpoH mutant strain

Pseudomonas sp. HK-6 can utilize the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as the sole nitrogen source under aerobic conditions. It is known that HK-6 is capable of completely degrading 50 μM RDX within 50 days, while the rpoH mutant degrades less than 10% of that amount in the same period of time. The proteomes of the HK-6 and the rpoH mutant strains grown under RDX stress conditions were compared using 2-dimensional electrophoresis (2-DE). A total of 14 upregulated and down-regulated unambiguous protein spots were analyzed using MALDI-TOF MS. Several down-regulated proteins connected with energy metabolism, including NirB, RimO, and NahH, and a transport and binding protein (AapJ) were less expressed in the rpoH genetic background than in the wild-type, and certain proteins connected with the cell envelope, including OprQ and Alg8, were more highly expressed in the rpoH mutant than in the wild-type. It was shown that certain proteins such as GroEL were not expressed in rpoH cells. These results provide insight into survival and the role of the rpoH gene for RDX degradation under RDX stress conditions. In addition to the proteome analysis, the 16S rRNA of HK-6 was cloned and sequenced to draw a phylogenetic tree for precise species identification. The 16S rRNA sequence of HK-6 is closely related to that of Pseudomonas putida.

RDX Degradation Potential in Soils Previously Unexposed to RDX and the Identification of RDX-Degrading Species in One Agricultural Soil Using Stable Isotope Probing

The aim of this work was to investigate the susceptibility of the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to biodegradation in a range of soils and to identify RDX-degrading organisms using stable isotope probing (SIP). RDX degradation was examined in ten soils, primarily from agricultural areas. RDX biodegradation was observed in six samples and only when the microcosms were not aerated. For one soil, 15N- and 13C-based DNA SIP was used to identify the microorganisms responsible for RDX degradation. Two RDX concentrations were examined (10 and 20 mg/L), however, only the higher concentration resulted in a significant SIP signal. In these ultracentrifugation fractions, one terminal restriction fragment length polymorphism (TRFLP) fragment (260 bp) showed a reliable trend of label uptake. This fragment was of higher relative abundance in the heavier fractions from labeled samples compared with the heavier fractions from the unlabeled control samples, indicating that the organism producing this fragment was responsible for label uptake (hence RDX degradation). Partial 16S rRNA gene sequencing indicated the organisms represented by fragment 260 bp belonged to either Sphingobacteria (phylum Bacteroidetes) or the phylum Acidobacteria. To date, these organisms have not previously been directly linked to RDX degradation. The 16S rRNA sequences were compared with the NCBI database and, in all cases, were most similar to uncultured bacteria. The results suggest SIP is a viable method for discovering novel, previously uncultured, RDX degraders.

Identification of Microbial Populations Assimilating Nitrogen from RDX in Munitions Contaminated Military Training Range Soils by High Sensitivity Stable Isotope Probing

The leaching of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) from particulates deposited in live-fire military training range soils contributes to significant pollution of groundwater. In situ microbial degradation has been proposed as a viable method for onsite containment of RDX. However, there is only a single report of RDX degradation in training range soils and the soil microbial communities involved in RDX degradation were not identified. Here we demonstrate aerobic RDX degradation in soils taken from a target area of an Eglin Air Force Base bombing range, C52N Cat's Eye, (Eglin, Florida U.S.A.). RDX-degradation activity was spatially heterogeneous (found in less than 30% of initial target area field samples) and dependent upon the addition of exogenous carbon sources to the soils. Therefore, biostimulation (with exogenous carbon sources) and bioaugmentation may be necessary to sustain timely and effective in situ microbial biodegradation of RDX. High sensitivity stable isotope probing analysis of extracted soils incubated with fully labeled (15)N-RDX revealed several organisms with (15)N-labeled DNA during RDX-degradation, including xplA-bearing organisms. Rhodococcus was the most prominent genus in the RDX-degrading soil slurries and was completely labeled with (15)N-nitrogen from the RDX. Rhodococcus and Williamsia species isolated from these soils were capable of using RDX as a sole nitrogen source and possessed the genes xplB and xplA associated with RDX-degradation, indicating these genes may be suitable genetic biomarkers for assessing RDX degradation potential in soils. Other highly labeled species were primarily Proteobacteria, including: Mesorhizobium sp., Variovorax sp., and Rhizobium sp.

Microbial Community Dynamics during Acetate Biostimulation of RDX-Contaminated Groundwater

Biostimulation of groundwater microbial communities (e.g., with carbon sources) is a common approach to achieving in situ bioremediation of organic pollutants (e.g., explosives). We monitored a field-scale approach to remediate the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) in an aquifer near the Iowa Army Ammunition Plant in Middletown, IA. The purpose of the study was to gain insight into the effect of biostimulation on the microbial community. Biostimulation with acetate led to the onset of RDX reduction at the site, which was most apparent in monitoring well MW309. Based on previous laboratory experiments, we hypothesized that RDX degradation and metabolite production would correspond to enrichment of one or more Fe(III)-reducing bacterial species. Community DNA from MW309 was analyzed with 454 pyrosequencing and terminal restriction fragment length polymorphism. Production of RDX metabolites corresponded to a microbial community shift from primarily Fe(III)-reducing Betaproteobacteria to a community dominated by Fe(III)-reducing Deltaproteobacteria (Geobacteraceae in particular) and Bacteroidetes taxa. This data provides a firsthand field-scale microbial ecology context to in situ RDX bioremediation using modern sequencing techniques that will inform future biostimulation applications.

Degradation of TNT, RDX, and HMX Explosive Wastewaters Using Zero‐Valent Iron Nanoparticles

AbstractThe high‐energy explosives 2,4,6‐trinitrotoluene (TNT), hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX), and the high melting explosive octahydro‐1,3,5,7‐tetranitro‐1,3,5,7‐tetrazocine (HMX) are common groundwater contaminants at active and abandoned munitions production facilities causing serious environmental problems. A highly efficient and environmentally friendly method was developed for the treatment of the explosives‐contaminated wastewaters using zero‐valent iron nanoparticles (ZVINs). ZVINs with diameters of 20–50 nm and specific surface areas of 42.56 m2 g−1 were synthesized by the co‐precipitation method. The explosives degradation reaction is expressed to be of pseudo first‐order and the kinetic reaction parameters are calculated based on different initial concentrations of TNT, RDX, and HMX. In addition, by comparison of the field emission scanning electron microscopy (FE‐SEM) images for the fresh and reacted ZVINs, it was apparent that the ZVINs were oxidized and aggregated to form Fe3O4 nanoparticles as a result of the chemical reaction. The X‐ray diffraction (XRD) and X‐ray absorption near edge structure (XANES) measurements confirmed that the ZVINs corrosion primarily occurred due to the formation of Fe3O4. Furthermore, the postulated reaction kinetics in different concentrations of TNT, RDX, and HMX, showed that the rate of TNT removal was higher than RDX and HMX. Furthermore, by‐products obtained after degradation of TNT (long‐chain alkanes/methylamine) and RDX/HMX (formaldehyde/methanol/hydrazine/dimethyl hydrazine) were determined by LC/MS/MS, respectively. The high reaction rate and significant removal efficiencies suggest that ZVINs might be suitable and powerful materials for an in‐situ degradation of explosive polluted wastewaters.

Push‐Pull Tests for Estimating RDX and TNT Degradation Rates in Groundwater

AbstractIn situ bioremediation is being considered to optimize an existing pump‐and‐treat remedy for treatment of explosives‐contaminated groundwater at the Umatilla Chemical Depot. Push‐pull tests were conducted using a phased approach to measure in situ hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) and 2,4,6‐trinitrotoluene (TNT) degradation rates associated with various carbon substrates. Phase I included short‐duration transport tests conducted in each well to determine dilution rates and retardation factors for RDX and TNT. Phase II included aquifer “feedings” conducted by injecting 150 gallons of treated site groundwater amended with ethanol, corn syrup, lactose or emulsified oil (concentrations 10, 25 and 27 mM, respectively; 12% by volume for emulsified oil). Wells received up to 6 substrate “feedings” over the course of 3 months followed by monitoring dissolved oxygen, nitrate, Fe(II), and sulfate to gauge in situ redox conditions as indicators of anaerobic microbial activity. Phase III included push‐pull tests conducted by injecting 150 gallons of site groundwater amended with approximately 1000 µg/L RDX, 350 µg/L TNT, carbon substrate and a conservative tracer, followed by sampling over 8 d. Corn syrup resulted in the best RDX removal (82% on average) and the largest RDX degradation rate coefficient (1.4 ± 1.1 d−1). Emulsified oil resulted in the best TNT removal (99%) and largest TNT degradation rate coefficient (5.7 × 10−2 d−1). These results will be used to simulate full‐scale in situ bioremediation scenarios at Umatilla and will support a go/no‐go decision to initiate full‐scale bioremediation remedy optimization.

Application of 13C-stable isotope probing to identify RDX-degrading microorganisms in groundwater

We employed stable isotope probing (SIP) with 13C-labeled hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) to identify active microorganisms responsible for RDX biodegradation in groundwater microcosms. Sixteen different 16S rRNA gene sequences were derived from microcosms receiving 13C-labeled RDX, suggesting the presence of microorganisms able to incorporate carbon from RDX or its breakdown products. The clones, residing in Bacteroidia, Clostridia, α-, β- and δ-Proteobacteria, and Spirochaetes, were different from previously described RDX degraders. A parallel set of microcosms was amended with cheese whey and RDX to evaluate the influence of this co-substrate on the RDX-degrading microbial community. Cheese whey stimulated RDX biotransformation, altered the types of RDX-degrading bacteria, and decreased microbial community diversity. Results of this study suggest that RDX-degrading microorganisms in groundwater are more phylogenetically diverse than what has been inferred from studies with RDX-degrading isolates.

Validation of a novel extraction method for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation by ruminal microbiota

A simple, fast liquid-liquid extraction method was developed for studying hexahydro-1,3,5-trinitro-1,3,5 triazine (RDX) biodegradation using small sample volumes. The method was tested in vitro with anaerobic incubations of RDX with whole rumen fluid (WRF) and a commercial Sporanaerobacter acetigenes strain in methanogenic media for RDX. Additionally, validation experiments were conducted in deionized water in order to show applicability toward various aqueous matrices. Conditions for extraction were as follows: 300 μL of sample were mixed with an equal volume of a 0.34 M ammonium hydroxide solution to reach a basic pH, extracted with a hexane/ethyl acetate 1:1 (v/v) solution (1 mL) and shaken vigorously for 10 s. The resulting organic phase was transferred, then dried under a constant flow of N2 and reconstituted with acetonitrile (300 μL) for HPLC-UV and LC-MS/MS analysis. Percent recovery values were obtained (83-101%) in all matrices for RDX. In WRF (n=3 animals), RDX degradation was observed with almost 100% elimination of RDX after 4 h. The five nitroso and ring cleavage metabolites were observed by mass spectrometry. Liquid cultures of S. acetigenes did not show significant RDX biodegradation activity. RDX extractions from deionized water samples indicated acceptable recoveries with low variability, suggesting suitability of the method for aqueous matrices. Overall, the new method demonstrated acceptable efficiency and reproducibility across three matrices, providing an advantageous alternative for studies where complex matrices and small volume samples are in use.

Investigation on Ultraviolet Absorption Properties, Migration, and Catalytic Performances of Ferrocene‐Modified Hyper‐Branched Polyesters

AbstractThe ultraviolet absorption properties of a series of ferrocene‐modified hyper‐branched polyesters (HBPE‐Fcs) were analyzed by Ultraviolet/visible spectrometry. HBPE‐Fcs were used as burning rate catalyst components added into hydroxyl‐terminated polybutadiene (HTPB) based elastomers to investigate their migration behavior. Migration inhibition effects of HBPE‐Fcs in different aging conditions were analyzed. The diffusion coefficients (D) of the migration components were calculated according to Fick’s law of diffusion. The catalytic performances of HBPE‐Fcs for the thermal decomposition of cyclotrimethylenetrinitramine (RDX) were also investigated by non isothermal measurements using Kissinger method. The incorporation of ferrocenes into hyper‐branched polyesters (HBPEs) endows HBPEs with new ultraviolet absorption properties. The migration of HBPE‐Fc was minimized by grafting ferrocene on the hyper‐branched structures compared to that of simple ferrocene derivatives. HBPE‐Fcs present efficient catalytic effects on the thermal degradation of RDX; and, the catalytic reactions were characterized by decreased activation energies and increased rate constants.

Role of Nitrogen Limitation in Transformation of RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine) by Gordonia sp. Strain KTR9

The transcriptome of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)-degrading strain Gordonia sp. strain KTR9 and its glnR mutant were studied as a function of nitrogen availability to further investigate the observed ammonium-mediated inhibition of RDX degradation. The results indicate that nitrogen availability is a major determinant of RDX degradation and xplA gene expression in KTR9.

Insight on RDX Degradation Mechanism by Rhodococcus Strains Using 13C and 15N Kinetic Isotope Effects

The explosive Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is known to be degraded aerobically by various isolates of the Rhodococcus species, with denitration being the key step, mediated by Cytochrome P450. Our study aimed at gaining insight into the RDX degradation mechanism by Rhodococcus species and comparing isotope effects associated with RDX degradation by distinct Rhodococcus strains. For these purposes, enrichment in (13)C and (15)N isotopes throughout RDX denitration was studied for three distinct Rhodococcus strains, isolated from soil and groundwater in an RDX-contaminated site. The observable (15)N enrichment throughout the reaction, together with minor (13)C enrichment, suggests that N-N bond cleavage is likely to be the key rate-limiting step in the reaction. The similarity in the kinetic (15)N isotope effect between the three tested strains suggests that either isotope-masking effects are negligible, or are of a similar extent for all tested strains. The lack of variability in the kinetic (15)N isotope effect allows the interpretation of environmental studies with greater confidence.

Microbial Degradation and Toxicity of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine

In the present work, current knowledge on the potential fate, microbial degradation, and toxicity of hexahydro- 1,3,5-trinitro-1,3,5-triazine (RDX) was thoroughly reviewed, focusing on the toxicological assessment of a variety of potential RDX degradation pathways in bacteria and fungi. The present review on microbial degradation pathways and toxicities of degradation intermediates suggests that, among aerobic RDX degradation pathways, the one via denitration may be preferred in a toxicological perspective, and that among anaerobic pathways, those forming 4- nitro-2,4-diazabutanal (NDAB) via ring cleavage of 1-nitroso- 3,5-dinitro-1,3,5-triazinane (MNX) may be toxicologically advantageous owing to its potential mineralization under partial or complete anoxic conditions. These findings provide important information on RDX-degrading microbial pathways, toxicologically most suitable to be stimulated in contaminated fields.

Assessment of aged biodegradable polymer-coated nano-zero-valent iron for degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)

BACKGROUND: This study reports on the effects of aging on suspension behavior of biodegradable polymer-coated nano-zero-valent iron (nZVI) and its degradation rates of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) under reductive conditions. The polymers investigated included guar gum, potato starch, alginic acid (AA), and carboxymethyl cellulose (CMC). Polymer coating was used to mitigate nZVI delivery hindrance for in situ treatment of RDX-contaminated groundwater. RESULTS: The RDX degradation rates by bare nZVI and starch-coated nZVI suspensions were least affected by aging although these suspensions exhibited the least favorable dispersion behavior. CMC, AA, and guar gum coating improved nZVI rates of degradation of RDX but these rates decreased upon aging. The best suspension stability upon aging was achieved by CMC and AA. Guar gum with loadings rates one order of magnitude lower than that of CMC and AA achieved good iron stabilization but significantly higher RDX degradation rates. CONCLUSION: It is demonstrated that both migration and reactivity of polymer-stabilized nZVI should be explicitly evaluated over a long period before application in the field. Guar gum coated nZVI appeared best suited for in situ application because it maintained good suspension stability, with RDX degradation rates least affected by aging compared with the other polymers tested. © 2012 Society of Chemical Industry

Anaerobic bioremediation of RDX by ovine whole rumen fluid and pure culture isolates

The ability of ruminal microbes to degrade the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in ovine whole rumen fluid (WRF) and as 24 bacterial isolates was examined under anaerobic conditions. Compound degradation was monitored by high-performance liquid chromatography analysis, followed by liquid chromatography-tandem mass spectrometry identification of metabolites. Organisms in WRF microcosms degraded 180 μM RDX within 4 h. Nitroso-intermediates 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) were present as early as 0.25 h and were detected throughout the 24-h incubation period, representing one reductive pathway of ring cleavage. Following reduction to MNX, peaks consistent with m/z 193 and 174 were also produced, which were unstable and resulted in rapid ring cleavage to a common metabolite consistent with an m/z of 149. These represent two additional reductive pathways for RDX degradation in ovine WRF, which have not been previously reported. The 24 ruminal isolates degraded RDX with varying efficiencies (0-96 %) over 120 h. Of the most efficient degraders identified, Clostridium polysaccharolyticum and Desulfovibrio desulfuricans subsp. desulfuricans degraded RDX when medium was supplemented with both nitrogen and carbon, while Anaerovibrio lipolyticus, Prevotella ruminicola, and Streptococcus bovis IFO utilized RDX as a sole source of nitrogen. This study showed that organisms in whole rumen fluid, as well as several ruminal isolates, have the ability to degrade RDX in vitro and, for the first time, delineated the metabolic pathway for its biodegradation.

Photocatalytic and Photoelectrocatalytic Degradation of the Explosive RDX by TiO2 Thin Films Prepared by CVD and Anodic Oxidation of Ti

AbstractTwo kinds of TiO2 films prepared by CVD and by anodization of titanium mesh are compared for the photocatalytic (PC) and photoelectrocatalytic (PEC) degradation of RDX aqueous solutions illuminated under a 200 W UV light. The PC activity of anodically formed TiO2 is lower than that with CVD‐produced TiO2. The PC efficiencies of degradation of RDX with both anodized and CVD films are improved by applying a potential of 1 V versus saturated calomel electrode (SCE), with up to 95% of RDX being destroyed in two hours by a CVD‐produced TiO2 film, and 81% by anodized TiO2. Chemical analysis shows that during RDX degradation, nitrite is the first mineralized product which is further oxidized to nitrate.

Enhanced Degradation of TNT and RDX by Bio-reduced Iron Bearing Soil Minerals

We demonstrated that reductive degradation of 2,4,6-Trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (Royal Demolition Explosive, RDX) can be enhanced by bio-reduced iron-bearing soil minerals (IBSMs) using Shewanella putrefaciens CN32 (CN32). The degradation kinetic rate constant of TNT by bio-reduced magnetite was the highest (0.0039 <TEX>$h^{-1}$</TEX>), followed by green rust (0.0022 <TEX>$h^{-1}$</TEX>), goethite (0.0017 <TEX>$h^{-1}$</TEX>), lepidocrocite (0.0016 <TEX>$h^{-1}$</TEX>), and hematite (0.0006 <TEX>$h^{-1}$</TEX>). The highest rate constant was obtained by bio-reduced lepidocrocite (0.1811 <TEX>$h^{-1}$</TEX>) during RDX degradation, followed by magnetite (0.1700 <TEX>$h^{-1}$</TEX>), green rust (0.0757 <TEX>$h^{-1}$</TEX>), hematite (0.0495 <TEX>$h^{-1}$</TEX>), and goethite (0.0394 <TEX>$h^{-1}$</TEX>). Significant increase of Fe(II) was observed during the reductive degradation of TNT and RDX by bio-reduced IBSMs. X-ray diffraction and electron microscope analyses were conducted for identification of degradation mechanism of TNT and RDX in this study. 4-amino-dinitrotoluene were detected as products during TNT degradation, while Hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine, Hexahydro-1,3-dinitroso-5-nitro-1,3,5triazine, and Hexahydro-1,3,5-trinitroso-1,3,5-triazine were observed during RDX degradation.

Involvement of cytochrome c CymA in the anaerobic metabolism of RDX by Shewanella oneidensis MR-1

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitramine explosive commonly used for military applications that is responsible for severe soil and groundwater contamination. In this study, Shewanella oneidensis MR-1 was shown to efficiently degrade RDX anaerobically (3.5µmol·h(-1)·(g protein)(-1)) via two initial routes: (1) sequential N-NO(2) reductions to the corresponding nitroso (N-NO) derivatives (94% of initial RDX degradation) and (2) denitration followed by ring cleavage. To identify genes involved in the anaerobic metabolism of RDX, a library of ~2500 mutants of MR-1 was constructed by random transposon mutagenesis and screened for mutants with a reduced ability to degrade RDX compared with the wild type. An RDX-defective mutant (C9) was isolated that had the transposon inserted in the c-type cytochrome gene cymA. C9 transformed RDX at ~10% of the wild-type rate, with degradation occurring mostly via early ring cleavage caused by initial denitration leading to the formation of methylenedinitramine, 4-nitro-2,4-diazabutanal, formaldehyde, nitrous oxide, and ammonia. Genetic complementation of mutant C9 restored the wild-type phenotype, providing evidence that electron transport components have a role in the anaerobic reduction of RDX by MR-1.

Biodegradation of explosives mixture in soil under different water-content conditions

Soil redox potential plays a key role in the rates and pathways of explosives degradation, and is highly influenced by water content and microbial activity. Soil redox potential can vary significantly both temporally and spatially in micro-sites. In this study, when soil water content increased, the redox potential decreased, and there was significant enhancement in the biodegradation of a mixture of three explosives. Whereas TNT degradation occurred under both aerobic and anaerobic conditions, RDX and HMX degradation occurred only when water content conditions resulted in a prolonged period of negative redox potential. Moreover, under unsaturated conditions, which are more representative of real environmental conditions, the low redox potential, even when measured for temporary periods, was sufficient to facilitate anaerobic degradation. Our results clearly indicate a negative influence of TNT on the biodegradation of RDX and HMX, but this effect was less pronounced than that found in previous slurry batch experiments: this can be explained by a masking effect of the soil in the canisters. Fully or partially saturated soils can promote the existence of micro-niches that differ considerably in their explosives concentration, microbial community and redox conditions.

Electrochemical Degradation of RDX in Aqueous Solution at Constructed Sb-Doped SnO&lt;sub&gt;2&lt;/sub&gt; Anode

The constructed Sb-doped SnO2 electrode was obtained for electrochemical degradation of RDX. The influences of current density and initial concentration of RDX on electrochemical degradation of RDX were studied. Kinetics analysis shows that the electrochemical degradation of RDX follows the pseudo first-order reaction. The mechanism of electrochemical degradation of RDX was also discussed.

Geochemical and microbiological processes contributing to the transformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in contaminated aquifer material

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a potential human carcinogen, and its contamination of subsurface environments is a significant threat to public health. This study investigated abiotic and biological degradation of RDX in contaminated aquifer material. Anoxic batch systems were started with and without pre-aeration of aquifer material to distinguish initial biological RDX reduction from abiotic RDX reduction. Aerating the sediment eliminated chemical reductants in the native aquifer sediment, primarily Fe(II) sorbed to mineral surfaces. RDX (50 μM) was completely reduced and transformed to ring cleavage products when excess concentrations (2 mM) of acetate or lactate were provided as the electron donor for aerated sediment. RDX was reduced concurrently with Fe(III) when acetate was provided, while RDX, Fe(III), and sulfate were reduced simultaneously with lactate amendment. Betaproteobacteria were the dominant microorganisms associated with RDX and Fe(III)/sulfate reduction. In particular, Rhodoferax spp. increased from 21% to 35% and from 28% to 60% after biostimulation by acetate and lactate, respectively. Rarefaction analyses demonstrated that microbial diversity decreased in electron-donor-amended systems with active RDX degradation. Although significant amounts of Fe(III) and/or sulfate were reduced after biostimulation, solid-phase reactive minerals such as magnetite or ferrous sulfides were not observed, suggesting that RDX reduction in the aquifer sediment is due to Fe(II) adsorbed to solid surfaces as a result of Fe(III)-reducing microbial activity. These results suggest that both biotic and abiotic processes play an important role in RDX reduction under in situ conditions.