RDX Degradation Research Articles

Multi-Omics Strategies to Investigate the Biodegradation of Hexahydro-1,3,5-trinitro-1,3,5-triazine in Rhodococcus sp. Strain DN22.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is an energetic and persistent explosive with long-lasting properties. Rhodococcus sp. strain DN22 has been discovered to be a microbe capable of degrading RDX. Herein, the complete genome of Rhodococcus sp. strain DN22 was sequenced and analyzed. The entire sequences of genes that encoded the two proteins participating in RDX degradation in Rhodococcus sp. strain DN22 were obtained, and were validated through proteomic data. In addition, few studies have investigated the physiological changes and metabolic pathways occurring within Rhodococcus sp. cells when treated with RDX, particularly through mass spectrometry-based omics. Hence, proteomic and metabolomic analyses were carried out on Rhodococcus sp. strain DN22 with the existence or lack of RDX in the medium. A total of 3186 proteins were identified between the two groups, with 115 proteins being significantly differentially expressed proteins. There were 1056 metabolites identified in total, among which 130 metabolites were significantly different. Through the combined analysis of differential proteomics and metabolomics, KEGG pathways including two-component system, ABC transporters, alanine, aspartate and glutamate metabolism, arginine biosynthesis, purine metabolism, nitrogen metabolism, and phosphotransferase system (PTS), were observed to be significantly enriched. These findings provided ponderable perspectives on the physiological alterations and metabolic pathways in Rhodococcus sp. strain DN22, responding to the existence or lack of RDX. This study is anticipated to expand the knowledge of Rhodococcus sp. strain DN22, as well as advancing understanding of microbial degradation.

Inhibition effect of 2,4,6-trinitrotoluene (TNT) on RDX degradation by rhodococcus strains isolated from contaminated soil and water

2,4,6-trinitrotoluene (TNT) is a highly toxic explosive that contaminates soil and water and may interfere with the degradation of co-occurring compounds, such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). We proposed that TNT may influence RDX-degrading bacteria via either general toxicity or a specific effect on the |RDX degradation mechanisms. Thus, we examined the impact of TNT on RDX degradation by Rhodococcus strains YH1, T7, and YY1, which were isolated from an explosives-polluted environment. Although partly degraded, TNT did not support the growth of any of the strains when used as either sole carbon or sole nitrogen sources, or as carbon and nitrogen sources. The incubation of a mixture of TNT (25 mg/l) and RDX (20 mg/l) completely inhibited RDX degradation. The effect of TNT on the cytochrome P450, catalyzing RDX degradation, was tested in a resting cell experiment, proving that TNT inhibits XplA protein activity. A dose-response experiment showed that the IC50/trans values for YH1, T7, and YY1 were 7.272, 5.098, and 9.140 (mg/l of TNT), respectively, illustrating variable sensitivity to TNT among the strains. The expression of xplA was also strongly suppressed by TNT. Cells that were pre-grown with RDX (allowing xplA expression) and incubated with ammonium chloride, glucose, and TNT, completely transformed into their amino dinitrotoluene isomers and formed azoxy toluene isomers. The presence of oxygen-insensitive nitroreductase that enable reduction of the nitro group in the presence of O2 in the genomes of these strains suggests that they are responsible for TNT transformation in the cultures. The experimental results concluded that TNT has an adverse effect on RDX degradation by the examined strains. It inhibits RDX degradation due to the direct impact on cytochrome P450, xplA, or its expression. The tested strains can transform TNT independently of RDX. Thus, degradation of both compounds is possible if TNT concentrations are below their IC50 values.

Effects of Perchlorate and Other Groundwater Inorganic Co-Contaminants on Aerobic RDX Degradation

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) pollution is accompanied by other co-contaminants, such as perchlorate and chlorates, which can retard biodegradation. The effects of perchlorate and chlorate on aerobic RDX degradation remain unclear. We hypothesized that they have a negative or no impact on aerobic RDX-degrading bacteria. We used three aerobic RDX-degrading strains—Rhodococcus strains YH1 and T7 and Gordonia YY1—to examine this hypothesis. The strains were exposed to perchlorate, chlorate, and nitrate as single components or in a mixture. Their growth, degradation activity, and gene expression were monitored. Strain-specific responses to the co-contaminants were observed: enhanced growth of strain YH1 and inhibition of strain T7. Vmax and Km of cytochrome P450 (XplA) in the presence of the co-contaminants were not significantly different from the control, suggesting no direct influence on cytochrome P450. Surprisingly, xplA expression increased fourfold in cultures pre-grown on RDX and, after washing, transferred to a medium containing only perchlorate. This culture did not grow, but xplA was translated and active, albeit at lower levels than in the control. We explained this observation as being due to nitrogen limitation in the culture and not due to perchlorate induction. Our results suggest that the aerobic strain YH1 is effective for aerobic remediation of RDX in groundwater.

A sketch of microbiological remediation of explosives-contaminated soil focused on state of art and the impact of technological advancement on hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation

When high-energy explosives such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), 2,4,6-trinitrotoluene (TNT) are discharged into the surrounding soil and water during production, testing, open dumping, military, or civil activities, they leave a toxic footprint. The US Environmental Protection Agency has labeled RDX as a potential human carcinogen that must be degraded from contaminated sites quickly. Bioremediation of RDX is an exciting prospect that has received much attention in recent years. However, a lack of understanding of RDX biodegradation and the limitations of current approaches have hampered the widespread use of biodegradation-based strategies for RDX remediation at contamination sites. Consequently, new bioremediation technologies are required to enhance performance. In this review, we explore the requirements for in-silico analysis for producing biological models of microbial remediation of RDX in soil. On the other hand, potential gene editing methods for getting the host with target gene sequences responsible for the breakdown of RDX are also reported. Microbial formulations and biosensors for detection and bioremediation are also briefly described. The biodegradation of RDX offers an alternative remediation method that is both cost-effective and ecologically acceptable. It has the potential to be used in conjunction with other cutting-edge technologies to further increase the efficiency of RDX degradation.

Enhanced bioremediation of RDX and Co-Contaminants perchlorate and nitrate using an anaerobic dehalogenating consortium in a fractured rock aquifer

The potential neurotoxic and carcinogenic effects of the explosives compound RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) on human health requires groundwater remediation strategies to meet low cleanup goals. Bioremediation of RDX is feasible through biostimulation of native microbes with an organic carbon donor but may be less efficient, or not occur at all, in the presence of the common co-contaminants perchlorate and nitrate. Laboratory tests compared biostimulation with bioaugmentation to achieve anaerobic degradation of RDX, perchlorate, and nitrate; a field pilot test was then conducted in a fractured rock aquifer with the selected bioaugmentation approach. Insignificant reduction of RDX, perchlorate, or nitrate was observed by the native microbes in microcosms, with or without biostimulation by addition of lactate. Tests of the RDX-degrading ability of the microbial consortium WBC-2, originally developed for dehalogenation of chlorinated volatile organic compounds, showed first-order biodegradation rate constants ranging from 0.57 to 0.90 per day (half-lives 1.2 to 0.80 days). WBC-2 sustained degradation without daughter product accumulation when repeatedly amended with RDX and lactate for a year. In microcosms with groundwater containing perchlorate and nitrate, RDX degradation began without delay when bioaugmented with 10% WBC-2. Slower RDX degradation occurred with 3% or 5% WBC-2 amendment, indicating a direct relation with cell density. Transient RDX daughter compounds included methylene dinitramine, MNX, and DNX. With WBC-2 amendment, nitrate concentrations immediately decreased to near or below detection, and perchlorate degradation occurred with half-lives of 25–34 days. Single-well injection tests with WBC-2 and lactate showed that the onset of RDX degradation coincided with the onset of sulfide production, which was affected by the initial perchlorate concentration. Biodegradation rates in the pilot injection tests agreed well with those measured in the microcosms. These results support bioaugmentation with an anaerobic culture as a remedial strategy for sites contaminated with RDX, nitrate, and perchlorate.

Anti‐migration of Nitrogen‐rich N‐Heterocyclic Ferrocenes and Their Combustion Catalytic Properties in the Thermal Decomposition of Energetic Oxidizers

AbstractTo retard high migration tendency of alkylferrocene‐based burning rate catalysts and ameliorate their combustion catalytic activity, twenty new nitrogen‐rich N‐heterocyclic ferrocenyl compounds 1–20 were synthesized by click reaction and characterized by NMR, UV‐Vis, FT‐IR, ESI‐MS, etc. Six compounds were structurally confirmed by single crystal X‐ray diffraction additionally. TG studies showed that the new ferrocenyl compounds are highly thermal stable. Cyclic voltammetry results implied that some new ferrocenyl compounds exhibit reversible electrochemical behavior, which are favorable for their combustion catalytic performance in the composite solid propellants. The anti‐migration tests confirmed that the new ferrocenyl compounds exhibit neglectable migration trends after four‐week aging on comparison with ferrocene and catocene. The catalytic combustion tests of AP, RDX and HMX over the compounds 1–20 indicated that these N‐heterocyclic ferrocenes are all highly active for improving thermal degradation of AP and RDX and more active than the aniline‐derived counterparts prepared by click reaction.

Characteristics of RDX degradation and the mechanism of the RDX exposure response in a Klebsiella sp. strain

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high-energy explosive that degrades with difficulty in the natural environment. The use of microbial remediation can effectively restore environments suffering from RDX pollution. In this study, a bacterial strain capable of efficient degradation of RDX was isolated from sewage. The bacterium was identified as a Klebsiella sp. Exposure to RDX at 40 mg L-1 increased the five-day biochemical oxygen demand (BOD5) to 971 mg L-1 and accelerated the logarithmic growth phase to occur at the 7th hour after inoculation, Overall, 81.9% of the RDX was degraded by 24 h. The surface of the bacterial cell became grooved and slightly curved. Levels of intracellular carboxylates, methyl-containing metabolites, primary aromatic amines, PO4-3, and ClO4- were altered after exposure to RDX solution. 362 different metabolites (DEMs) were identified using LC-MS metabolome analysis. Among them, 193 DEMs were upregulated and 169 DEMs were downregulated. The galactose metabolism pathway (ko00052) and the synthesis of other substances (such as UDP-glucose, D-galactose, D-fructose-1,6-bisphosphate, and dihydroxyacetone phosphate) were disrupted when the concentration of RDX was 40 mg L-1. The metabolites of cell membrane and cell wall were destroyed, such as lysophosphatidic acid (LPA) (0:0/16:0), phosphatidylethanolamine, and phosphatidylcholine.

Diversity and abundance of the functional genes and bacteria associated with RDX degradation at a contaminated site pre- and post-biostimulation.

Bioremediation is becoming an increasingly popular approach for the remediation of sites contaminated with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Multiple lines of evidence are often needed to assess the success of such approaches, with molecular studies frequently providing important information on the abundance of key biodegrading species. Towards this goal, the current study utilized shotgun sequencing to determine the abundance and diversity of functional genes (xenA, xenB, xplA, diaA, pnrB, nfsI) and species previously associated with RDX biodegradation in groundwater before and after biostimulation at an RDX-contaminated Navy Site. For this, DNA was extracted from four and seven groundwater wells pre- and post-biostimulation, respectively. From a set of 65 previously identified RDX degraders, 31 were found within the groundwater samples, with the most abundant species being Variovorax sp. JS1663, Pseudomonas fluorescens, Pseudomonas putida, and Stenotrophomonas maltophilia. Further, 9 RDX-degrading species significantly (p<0.05) increased in abundance following biostimulation. Both the sequencing data and qPCR indicated that xenA and xenB exhibited the highest relative abundance among the six genes. Several genes (diaA, nsfI, xenA, and pnrB) exhibited higher relative abundance values in some wells following biostimulation. The study provides a comprehensive approach for assessing biomarkers during RDX bioremediation and provides evidence that biostimulation generated a positive impact on a set of key species and genes. KEY POINTS: • A co-occurrence network indicated diverse RDX degraders. • >30 RDX-degrading species were detected. • Nine RDX-degrading species increased following biostimulation. • Sequencing and high-throughput qPCR indicated that xenA and xenB were most abundant.

Enhancing remediation of RDX-contaminated soil by introducing microbial formulation technology coupled with biostimulation

The degradation of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) through bioaugmentation and biostimulation was investigated. Aqueous phase experiment established the potential of Kinneretia asachharophila strain 12853 in invigorating the RDX-degradation. The microbe denitrified 75.7% RDX from the minimal salt medium by utilizing it as a nitrogen source based on nitrite production. High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography-Mass Spectrometry (LCMS) analyses confirmed the presence of methylenedinitramine (MEDINA) and 4-nitro-2,4-diazabutanal (NDAB) as intermediate metabolites. The microbe formulated into powder and beads showed 29% and 14% loss in viability, respectively, after a storage period of 3 months at 25 °C. The third type of formulation, i.e., water-dispersible granules (WDG), retained 98.52% viability at similar experimental conditions. The microcosm studies revealed a 40% RDX degradation in soil using WDG, while unformulated fresh cultures showed 29% degradation. The process became 1.9–2.4 times more efficient when WDG coupled with stimulants, wheat straw, or sucrose (0.5% w/w). We recorded first-order degradation kinetics with an RDX soil half-life of 16.54–24.15 days. The current study demonstrates the enhanced RDX remediation from the contaminated soil using a robust microbial formulation compatible with wheat straw as an organic stimulant.

In-vessel composting of HMX and RDX contaminated sludge using microbes isolated from contaminated site

Current study was carried out with an objective to remediate highly contaminated sludge with HMX and RDX obtained from an explosive manufacturing facility in North India employing indigenous microbes, Arthrobacter subterraneus (isolate no. S2-TSB-17) and Bacillus sonorensis (isolate no. S8-TSB-4) which were isolated from the same contaminated site. In-vessel composting of the explosive contaminated sludge was performed in 12 different bioreactors using cow manure and garden waste as bulking agents. 78.5% degradation of HMX was observed in reactor no. 2 with Bacillus sonorensis having combination of 10% sludge, 70% cow manure and 20% garden waste on 80th day. Two secondary metabolites Bis(hydroxymethyl)nitramine and methylene dinitramine were identified while studying the degradation pathway. Similarly, degradation of 91.2% was observed for RDX in reactor no. 11 with consortia of Arthrobacter subterraneus and Bacillus sonorensis on 80th day. During the study, release of significant nitrate and nitrite ions were observed. It has already been established that RDX and HMX degradation leads to release of nitrite/nitrate ions. The highest nitrite (reactor no. 11) and nitrate (reactor no. 2) release observed were 24.02 ± 0.05 mg/kg and 30.65 ± 0.99 mg/kg on 50th and 70th day, respectively. Scanning electron microscopic studies confirmed the attachment and presence of microbes with solid surface and no deformation in structure was observed in the microbial cells due to contamination stress. Findings of the study concluded that in-vessel composting assisted with native bacterial species can be a potential technology for the treatment of explosive contaminated sludge at the contaminated sites.

Optimization and Degradation Studies on Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) with Selected Indigenous Microbes under Aerobic Conditions

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) a nitramine explosive, which has contaminated various military sites during its use, storage and manufacturing worldwide. As RDX is a recalcitrant, less soluble and toxic to human beings and other organisms, it is essential to remediate the contaminated sites. In the current investigation, authors have explored the potential of two indigenous microbes i.e., Bacillus toyonensis (isolate No. WS4-TSB-3, MTCC No. 12857) and Paenibacillus dendritiformis (isolate No. S10-TSA-3, MTCC No. 12859) isolated from an explosive manufacturing facility in north India, for the degradation of RDX in aqueous medium. Furthermore, RDX degradation has been optimized using response surface methodology (RSM) in a 15 days experiment at concentration of 20, 40, and 60 mg/L. It was found that various factors such as initial concentration of RDX, inoculum volume (2, 4 and 6%) and time (5, 10 and 15 days) had impact on transformation and degradation of contaminant. Samples were analyzed using high performance liquid chromatography (HPLC) and intermediate products were identified using LC-MS/MS. Maximum RDX removal of 81.6 ± 1.3 and 84.7 ± 0.9% for Bacillus toyonensis (isolate No. WS4-TSB-3) and Paenibacillus dendritiformis (isolate No. S10-TSA-3), respectively, was observed on 15th day at 40 mg/L initial concentration. During the degradation Hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), Hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), 4-Nitro-2,4-diazabutanal, Bis(hydroxymethyl)nitramine and nitrite were identified as intermediate products. The findings of the investigation suggest that both the microbes have the potential to degrade RDX in the aqueous medium and can be used for up-scaling the degradation of RDX on explosive contaminated sites.

Degradation of high energetic material hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a microbial consortium using response surface methodological approach

Soil and water get polluted with hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) during its manufacturing, storage and use for civil and military purposes. RDX has toxic effects on living and non-living environment and is a recalcitrant compound. Therefore, the remediation of this compound is necessary. Microbial degradation of RDX can be a suitable and sustainable option to reduce its deleterious impact on the environment. Therefore, the optimization for degradation of energetic munition compound RDX employing the consortium of native bacterial species, isolated from an actual contaminated site, was performed. The experiment was planned with three independent variables (initial RDX concentration, inoculum size of microbes, and duration of the experiment) and three dependent variables (percentage removal of RDX, optical density, and nitrite release). Both independent and dependent variables were analyzed by the response surface methodology (RSM) using the Box–Behnken design. The statistical analysis using analysis of variance (ANOVA) depicted a high regression coefficient, R2 = 0.9881 with the statistically significant p-value fitted into a quadratic regression model for percentage removal of RDX. Results showed an initial RDX concentration of 40 mg/L, inoculation size 6 mL and a time duration of 12 days was optimal for the reduction of RDX up to 80.4%.

Degradation of RDX (Hexahydro-1,3,5-trinitro-1,3,5-triazine) in contrasting coastal marine habitats: Subtidal non-vegetated (sand), subtidal vegetated (silt/eel grass), and intertidal marsh

Hundreds of explosive-contaminated marine sites exist globally, many of which contain the common munitions constituent hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Quantitative information about RDX transformation in coastal ecosystems is essential for management of many of these sites. Isotopically labelled RDX containing 15N in all 3 nitro groups was used to track the fate of RDX in three coastal ecosystem types. Flow-through mesocosms representing subtidal vegetated (silt/eel grass), subtidal non-vegetated (sand) and intertidal marsh ecosystems were continuously loaded with isotopically labelled RDX for 16–17 days. Sediment, pore-water and overlying surface water were analyzed to determine the distribution of RDX, nitroso-triazine transformation products (NXs) and nitrogen containing complete mineralization products, including ammonium, nitrate+nitrite, nitrous oxide and nitrogen gas. The marsh, silt, and sand ecotypes transformed 94%, 90% and 76% of supplied RDX, respectively. Total dissolved NXs accounted for 2%–4% of the transformed 15N-RDX. The majority of RDX transformation in the water column was by mineralization to inorganic N (dissolved and evaded; 64%–78% of transformed 15N-RDX). RDX was mineralized primarily to N2O (62–74% of transformed 15N-RDX) and secondarily to N2 (1–2% of transformed 15N-RDX) which exchanged with the atmosphere. Transformation of RDX was favored in carbon-rich lower redox potential sediments of the silt and marsh mesocosms where anaerobic processes of iron and sulfate reduction were most prevalent. RDX was most persistent in the carbon-poor sand mesocosm. Partitioning of 15N derived from RDX onto sediment and suspended particulates was negligible in the overall mass balance of RDX transformation (2%–3% of transformed 15N-RDX). The fraction of 15N derived from RDX that was sorbed or assimilated in sediment was largest in the marsh mesocosm (most organic carbon), and smallest in the sand mesocosm (largest grain size and least organic carbon). Sediment redox conditions and available organic carbon stores affect the fate of RDX in different coastal marine habitats.

RDX degradation by chemical oxidation using calcium peroxide in bench scale sludge systems

The ability of calcium peroxide (CaO2) to degrade hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in contaminated soil slurries using CaO2-based modified Fenton oxidation was investigated. Results showed that increasing the CaO2 dose increased degradation rates of RDX and pH. RDX concentrations decreased to below detection after 18 h with 2 M and 2.5 M CaO2, after 30 h with 1.5 M CaO2, after 54 h with 1 M CaO2, but 0.1 M CaO2 achieved no significant RDX removal. Increasing the soil organic matter content decreased the rate and extent of RDX degradation. RDX degradation products 4-nitro-2,4-diazabutanal (NDAB) and methylenedinitramine (MEDINA) were quantified, and the greater accumulation of NDAB than MEDINA suggests denitration of RDX was the most likely initial degradation step. Isotopic ratios for nitrogen and oxygen associated with RDX oxidation are also consistent with either nitrification of NH4+ from soil or precipitation. Existing technologies merely only extract energetics from soils for treatment ex situ, whereas the approach introduced herein destroys RDX in situ with a one-step application.

Aerobic biodegradation of high explosive hexahydro-1,3,5- trinitro-1,3,5-triazine by Janibacter cremeus isolated from contaminated soil.

To evaluate the ability of Janibacter cremeus a soil bacterium isolated from explosive contaminated site in degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and to study enzyme responsible for degradation. The isolate exhibited 88% degradation of RDX in 30days of incubation. The biodegradation process followed the first order kinetics. The half- life of RDX was calculated to be 11.088days. The RDX degradation process was complemented by concomitant release of nitrite ions with 0.78mol of nitrite released per mole of RDX. The metabolites; Trinitroso- RDX, diamino-RDX, trimino-RDX, bis- (hydroxymethyl) nitramine and methylenedintramine derivative, viz, methylene- N- (hydroxy- methyl)- hydroxylamine- N-(hydroxymethyl) nitroamine corresponding to the molecular weights 174, 162, 132, 122 and 167Da respectively were also detected. Nitroreductase enzyme was found to be responsible for RDX degradation. J. cremeus could degrade RDX as sole source of nitrogen, via three different pathways wherein, Nitroreductase enzyme was found to play a major role. The efficient degradation of RDX makes J. cremeus suitable in treatment of contaminated water and soil at field scale levels.

Draft Genome Sequences of Rhodococcus sp. Strains YH1 and T7, Isolated from Explosive-Contaminated Environments.

We report the draft genome sequences for Rhodococcus sp. strains YH1 and T7. These strains are both capable of degrading hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and were isolated from explosive-contaminated soil and groundwater, respectively. Further genomic analysis might facilitate an understanding of the degradation of RDX and will contribute to the development of bioremediation methods for polluted soil and groundwater.

A Sporolactobacillus-, Clostridium-, and Paenibacillus- Dominant Microbial Consortium Improved Anaerobic RDX Detoxification by Starch Addition.

In the present study, an anaerobic microbial consortium for the degradation of hexahydro-1,3,5- trinitro-1,3,5-triazine (RDX) was selectively enriched with the co-addition of RDX and starch under nitrogen-deficient conditions. Microbial growth and anaerobic RDX biodegradation were effectively enhanced by the co-addition of RDX and starch, which resulted in increased RDX biotransformation to nitroso derivatives at a greater specific degradation rate than those for previously reported anaerobic RDX-degrading bacteria (isolates). The accumulation of the most toxic RDX degradation intermediate (MNX [hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine]) was significantly reduced by starch addition, suggesting improved RDX detoxification by the co-addition of RDX and starch. The subsequent MiSeq sequencing that targeted the bacterial 16S rRNA gene revealed that the Sporolactobacillus, Clostridium, and Paenibacillus populations were involved in the enhanced anaerobic RDX degradation. These results suggest that these three bacterial populations are important for anaerobic RDX degradation and detoxification. The findings from this work imply that the Sporolactobacillus, Clostridium, and Paenibacillus dominant microbial consortium may be valuable for the development of bioremediation resources for RDX-contaminated environments.

Application of a multiple lines of evidence approach to document natural attenuation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in groundwater

This study utilized innovative analyses to develop multiple lines of evidence for natural attenuation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in groundwater at the U.S. Department of Energy’s Pantex Plant. RDX, as well as the degradation product 4-nitro-2,4-diazabutanal (NDAB; produced by aerobic biodegradation or alkaline hydrolysis) were detected in a large portion of the plume, with lower concentrations of the nitroso-containing metabolites produced during anaerobic biodegradation. 16S metagenomic sequencing detected the presence of bacteria known to aerobically degrade RDX (e.g., Gordonia, Rhodococcus) and NDAB (Methylobacterium), as well as the known anoxic RDX degrader Pseudomonas fluorescens I–C. Proteomic analysis detected both the aerobic RDX degradative enzyme XplA, and the anoxic RDX degradative enzyme XenB. Groundwater enrichment cultures supplied with low concentrations of labile carbon confirmed the potential of the extant groundwater community to aerobically degrade RDX and produce NDAB. Compound-specific isotope analysis (CSIA) of RDX collected at the site showed fractionation of nitrogen isotopes with δ15N values ranging from approximately −5‰ to +9‰, providing additional evidence of RDX degradation. Taken together, these results provide evidence of in situ RDX degradation in the Pantex Plant groundwater. Furthermore, they demonstrate the benefit of multiple lines of evidence in supporting natural attenuation assessments, especially with the application of innovative isotopic and –omic technologies.

Spatially-distinct redox conditions and degradation rates following field-scale bioaugmentation for RDX-contaminated groundwater remediation

In situ bioaugmentation for cleanup of an hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-contaminated groundwater plume was recently demonstrated. Results of a forced-gradient, field-scale cell transport test with Gordonia sp. KTR9 and Pseudomonas fluorescens strain I-C cells (henceforth “KTR9” and “Strain I-C”) showed these strains were transported 13 m downgradient over 1 month. Abundances of xplA and xenB genes, respective indicators of KTR9 and Strain I-C, approached injection well cell densities at 6 m downgradient, whereas gene abundances (and conservative tracer) had begun to increase at 13 m downgradient at test conclusion. In situ push-pull tests were subsequently completed to measure RDX degradation rates in the bioaugmented wells under ambient gradient conditions. Time-series monitoring of RDX, RDX end-products, conservative tracer, xplA and xenB gene copy numbers and XplA and XenB protein abundance were used to assess the efficacy of bioaugmentation and to estimate the apparent first-order RDX degradation rates during each test. A collective evaluation of redox conditions, RDX end-products, varied RDX degradation kinetics, and biomarkers indicated that Strain I-C and KTR9 rapidly degraded RDX. Results showed bioaugmentation is a viable technology for accelerating RDX cleanup in the demonstration site aquifer and may be applicable to other sites. Full-scale implementation considerations are discussed.

Meta-analysis of RDX biotransformation rate by bacteria and fungi

Royal Demolition Explosive (RDX; hexahydro-1,3,5-trinitro-1,3,5-triazine) is a highly oxidized explosive compound commonly used in military and industrial operations. RDX is known to cause neurological dysfunction in exposed human subjects. Several bacterial and fungal species have the metabolic capability to transform RDX into less toxic, non-cyclic and de-nitrated compounds. Although the biodegradation pathways used by bacteria and fungi to transform RDX are well studied, far less is known about the kinetics. This meta-analysis uses published data from 46 independent research studies in 31 publications to calculate first-order rate coefficients (k) and half-life (t1/2) values as common metrics for the comparison of RDX degradation rates by three groups of microorganisms: aerobic bacteria; anaerobic bacteria and aerobic fungi. Relative degradation rates are statistically fastest for aerobic bacteria and slowest for fungi among the three groups. This analysis is the first attempt to characterize and compare RDX degradation kinetics by bacteria and fungi using a meta-analytical approach. Meta-analysis offers a useful method for comparing findings from a large and diverse body of published literature.