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Surficial application of waste glycerol (WG) for enhanced bioremediation was tested in situ at an old military range site to address hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) contaminated groundwater. This treatment was effective in inducing strong reducing conditions (range: −4 to −205 mV) and increasing the concentrations of organic carbon (from 10 to 729 mg/L) and fatty acids (from 0 to 940 mg/L) concomitantly with a decrease in RDX concentrations (range: 17 to 143 μg/L) to below detection limits (0.1 μg/L) in 2 of the 3 monitoring wells (MWs) evaluated. None of these changes were observed in the control MW. RDX disappeared without the detection of any common anaerobic nitroso degradation intermediates, with the exception of one MW where the concentration of organics did not significantly increase (range: 10 to 20 mg/L), suggesting the conditions were not favourable for biodegradation. Ecotoxicological analysis suggested that the use of WG may have some dose-related deleterious effects on different soil and aquatic receptors. Analysis of the microbial community composition, using 16S rRNA gene amplicon sequences, which provided insight into whether the process design had selected for and stimulated the optimal microbial populations, indicated co-existence of numerous Operational Taxonomic Units (OTUs) belonging to groups known to be capable of RDX degradation under anaerobic conditions, with a positive link between Geobacter spp. enrichment and the presence of RDX nitroso metabolites. Overall, the results from this field test show that this treatment process can provide an effective long-term, semi-passive remediation option for RDX contaminated groundwater. Unlabelled Image • Waste glycerol (WG) was used in situ to treat RDX contaminated groundwater. • Surficially applied WG reached groundwater and induced strong reducing conditions. • Organic carbon and fatty acid concentrations increased while that of RDX decreased. • Dose-related deleterious effects of WG observed on soil and aquatic receptors • Amplicon sequence analysis revealed known RDX degrading taxa enriched by treatment. [ABSTRACT FROM AUTHOR]

Anaerobic transformation of the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) by microorganisms involves sequential reduction of N-NO2 to the corresponding N-NO groups resulting in the initial formation of MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine). MNX is further reduced to the dinitroso (DNX) and trinitroso (TNX) derivatives. In this paper, we describe the degradation of MNX and TNX by the unusual cytochrome P450 XplA that mediates metabolism of RDX in Rhodococcus rhodochrous strain 11Y. XplA is known to degrade RDX under aerobic and anaerobic conditions, and, in the present study, was found able to degrade MNX to give similar products distribution including NO2-, NO3-, N2O, and HCHO but with varying stoichiometric ratio, that is, 2.06, 0.33, 0.33, 1.18, and 1.52, 0.15, 1.04, 2.06, respectively. In addition, the ring cleavage product 4-nitro-2,4,-diazabutanal (NDAB) and a trace amount of another intermediate with a [M-H]- at 102 Da, identified as ONNHCH2NHCHO (NO-NDAB), were detected mostly under aerobic conditions. Interestingly, degradation of TNX was observed only under anaerobic conditions in the presence of RDX and/or MNX. When we incubated RDX and its nitroso derivatives with XplA, we found that successive replacement of N-NO2 by N-NO slowed the removal rate of the chemicals with degradation rates in the order RDX > MNX > DNX, suggesting that denitration was mainly responsible for initiating cyclic nitroamines degradation by XplA. This study revealed that XplA preferentially cleaved the N-NO2 over the N-NO linkages, but could nevertheless degrade all three nitroso derivatives, demonstrating the potential for complete RDX removal in explosives-contaminated sites. [ABSTRACT FROM AUTHOR]

Previously we demonstrated that Rhodococcus sp. strain DN22 can degrade RDX (hexahydro-1,3,5-trinitro-l,3,5-triazine) aerobically via initial denization. The present study describes the role of oxygen and water in the key denitration step leading to RDX decomposition using 18O2 and H2 18O labeling experiments. We also investigated degradation of MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine) with DN22 under similar conditions. DN22 degraded RDX and MNX giving N02-, NO3-. NDAB (4-nitro-diazabutanal), NH3, N2O, and HCHO with NO2-/NO3- molar ratio reaching 17 and ca. 2, respectively. In the presence of 18O2, DN22 degraded RDX and produced NO2- with m/z at 46 Da that subsequently oxidized to NO3- containing one 18O atom, but in the presence of H2 18O we detected NO3- without 18O. A control containing NO2-, DN22, and 18O2 gave NO3- with one 18O, confirming biotic oxidation of NO2- to NO3-. Treatment of MNX with DN22 and 18O2 produced NO3- with two mass ions, one (66 Da) incorporating two 18O atoms and another (64 Da) incorporating only one 18O atom and we attributed their formation to bio-oxidation of the initially formed NO and NO2-, respectively. In the presence of H218O we detected NO2- with two different masses, one representing NO2- (46 Da) and another representing NO2-. (48 Da) with the inclusion of one 18O atom suggesting auto-oxidation of NO to NO2-. Results indicated that denitration of either RDX or MNX and denitrosation of MNX by DN22 did not involve direct participation of either oxygen or water, but both played major roles in subsequent secondary chemical and biochemical reactions of NO and NO2-. [ABSTRACT FROM AUTHOR]

The article reports on the study of terminal electron acceptors (TEA). It aims to determine the effect of TEA on the development of strain Shewanella halefaxensis HAW-EB4. It also examines the role of TEA in the enzymic processes involved in the metabolism of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). It shows that HAW-EB4 used periplasmic proteins in transforming RDX to nitroso and ring cleavage products. It also demonstrates that HAW-EB4 creates different sets of c-type chromosomes in different TEA and trimethylamine N-oxide (TMAO). The study also indicates that cytochrome and TMAO-reducing conditions are involved in RDX metabolism.

Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) is a toxic explosive known to be resistant to biodegradation. In this study, we found that sediment collected from two unexploded ordnance (UXO) disposal sites (UXO-3, UXO-5) and one nearby reference site (midref) in Hawaii contained anaerobic bacteria capable of removing HMX. Two groups of HMX-removing bacteria were found in UXO-5: group I contained aerotolerant anaerobes and microaerophiles, and group II contained facultative anaerobes. In UXO-3 and midref sediments, HMX-metabolizing bacteria were strictly anaerobic (group III and group IV). Using 16S rRNA sequencing, group I was assigned to a novel phylogenetic cluster of Clostridiales, and groups II and III were related to Paenibacillus and Tepidibacter of Firmicutes, respectively. Group IV bacteria were identified as Desulfovibrio of Deltaproteobacteria. Using [UL-14C]-HMX, group IV isolates were found to mineralize HMX (26.8% in 308 d) as determined by liberated 14CO2, but negligible mineralization was observed in groups I–III. Resting cells of isolates metabolized HMX to N2O and HCHO via the intermediary formation of 1-nitroso-octahydro-3,5,7-trinitro-1,3,5,7-tetrazocine together with methylenedinitramine. These experimental findings suggest that HMX biotransformation occurred either via initial denitration followed by ring cleavage or via reduction of one or more of the N-NO2 group(s) to the corresponding N-NO bond(s) prior to ring cleavage. [ABSTRACT FROM AUTHOR]

This study demonstrated the removal of selenite and selenate in flow-through permeable bioelectrochemical barriers (microbial electrolysis cells, MECs). The bioelectrochemical barriers consisted of cathode and anode electrode compartments filled with granular carbon or metallurgical coke. A voltage of 1.4 V was applied to the electrodes to enable the bioelectrochemical removal of selenium species. For comparison, a similarly designed permeable anaerobic biobarrier filled with granular carbon was operated without voltage. All biobarrier setups were fed with water containing up to 5,000 µg L−1 of either selenite or selenate and 70 mg L−1 of acetate as a source of organic carbon. Significant removal of selenite and selenate was observed in MEC experimental setups, reaching 99.5–99.8% over the course of the experiment, while in the anaerobic biobarrier the removal efficiency did not exceed 88%. By simultaneously operating several setups and changing operating parameters (selenium species, influent Se and acetate concentrations, etc.) we demonstrated enhanced removal of Se species under bioelectrochemical conditions. ga1 • Selenate and selenite removal in a flow-through bioelectrochemical barrier was demonstrated. • Anaerobic treatment resulted in 85% Se removal efficiency. • A 1.4 V applied voltage coupled with acetate addition enhanced selenate and selenite removal. • Up to 99.5% selenate and selenite removal achieved in the bioelectrochemical system. [ABSTRACT FROM AUTHOR]

Nitroguanidine (NQ) is an energetic material that is used as a key ingredient of triple-base propellants and is currently being considered as a TNT replacement in explosive formulations. NQ was efficiently degraded in aerobic microcosms when a carbon source was added. NQ persisted in unamended microcosms or under anaerobic conditions. An aerobic NQ-degrading bacterium, Variovorax strain VC1, was isolated from soil microcosms containing NQ as the sole nitrogen source. NQ degradation was inhibited in the presence of a more favorable source of nitrogen. Resting cells of VC1 degraded NQ effectively (54 μmol h-1 g-1 protein) giving NH3 (50.0%), nitrous oxide (N2O) (48.5%) and CO2 (100%). Disappearance of NQ was accompanied by the formation of a key intermediate product that we identified as nitrourea by comparison with a reference material. Nitrourea is unstable in water and suffered both biotic and abiotic decomposition to eventually give NH3, N2O, and CO2. However, we were unable to detect urea. Based on products distribution and reaction stoichiometry, we suggested that degradation of NQ, O2NN═C(NH2)2, might involve initial enzymatic hydroxylation of the imine, -C═N- bond, leading first to the formation of the unstable α-hydroxynitroamine intermediate, O2NNHC(OH)(NH2)2, whose decomposition in water should lead to the formation of NH3, N2O, and CO2. NQ biodegradation was induced by nitroguanidine itself, l-arginine, and creatinine, all being iminic compounds containing a guanidine group. This first description of NQ mineralization by a bacterial isolate demonstrates the potential for efficient microbial remediation of NQ in soil. [ABSTRACT FROM AUTHOR]

Shewanella halifaxensis and Shewanella sediminis were among a few aquatic γ-proteobacteria that were psychrophiles and the first anaerobic bacteria that degraded hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Although many mesophilic or psychrophilic strains of Shewanella and γ-proteobacteria were sequenced for their genomes, the genomic evolution pathways for temperature adaptation were poorly understood. On the other hand, the genes responsible for anaerobic RDX mineralization pathways remain unknown. To determine the unique genomic properties of bacteria responsible for both cold-adaptation and RDX degradation, the genomes of S. halifaxensis and S. sediminis were sequenced and compared with 108 other γ-proteobacteria including Shewanella that differ in temperature and NaP+ requirements, as well as RDX degradation capability. Results showed that for coping with marine environments their genomes had extensively exchanged with deep sea bacterial genomes. Many genes for NaP+-dependent nutrient transporters were recruited to use the high NaP+ content as an energy source. For coping with low temperatures, these two strains as well as other psychrophilic strains of Shewanella and γ-proteobacteria were found to decrease their genome G+C content and proteome alanine, proline and arginine content (p-value <0.01) to increase protein structural flexibility. Compared to poorer RDX-degrading strains, S. halifaxensis and S. sediminis have more number of genes for cytochromes and other enzymes related to RDX metabolic pathways. Experimentally, one cytochrome was found induced in S. halifaxensis by RDX when the chemical was the sole terminal electron acceptor. The isolated protein degraded RDX by mono-denitration and was identified as a multiheme 52 kDa cytochrome using a proteomic approach. The present analyses provided the first insight into divergent genomic evolution of bacterial strains for adaptation to the specific cold marine conditions and to the degradation of the pollutant RDX. The present study also provided the first evidence for the involvement of a specific c-type cytochrome in anaerobic RDX metabolism. [ABSTRACT FROM AUTHOR]

Octahydro- 1,3,5,7-tetranitro- 1,3,5,7-tetrazocine (HMX) is a recalcitrant energetic chemical that tends to accumulate in soil, close to the surface. The present study describes the aerobic biodegradability of HMX using Phanerochaete chrysosporium. When added to 7 day old static P. chrysosporium liquid cultures, HMX (600 nmol) degraded within 25 days of incubation. The removal of HMX was concomitant with the formation of transient amounts of its mono-nitroso derivative (1-NO-HMX). The latter apparently degraded via two potential routes: the first involved N-denitration followed by hydrolytic ring cleavage, and the second involved α-hydroxylation prior to ring cleavage. The degradation of 1-NO-HMX gave the ring-cleavage product 4-nitro-2,4-diazabutanal (NOAB), nitrite (NO2-), nitrous oxide (N2O), and formaldehyde (HCHO). Using [14C]-HMX, we obtained 14CO2 (70% in 50 days), representing three C atoms of HMX. Incubation of real soils, contaminated with either HMX (403 μmol kg-1) (military base soil) or HMX (3057 μmol kg-1), and RDX (342 μmol kg-1) (ammunition soil) with the fungus led to 75 and 19.8% mineralization of HMX (liberated 14CO2), respectively, also via the intermediary formation of 1-NO-HMX. Mineralization in the latter soil increased to 35% after the addition of glucose, indicating that a fungus-based remediation process for heavily contaminated soils is promising. The present findings improve our understanding about the degradation pathway of HMX and demonstrate the utility of using the robust and versatile fungus P. chrysosporium to develop effective remediation processes for the removal of HMX. [ABSTRACT FROM AUTHOR]

Several H2-producing fermentative anaerobic bacteria including Clostridium, Klebsiella and Fusobacteria degraded octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (36 μM) to formaldehyde (HCHO) and nitrous oxide (N2O) with rates ranging from 5 to 190 nmol h-1 g [dry weight] of cells-1. Among these strains, C. bifermentans strain HAW-1 grew and transformed HMX rapidly with the detection of the two key intermediates the mononitroso product and methylenedinitramine. Its cellular extract alone did not seem to degrade HMX appreciably, but degraded much faster in the presence of H2, NADH or NADPH. The disappearance of HMX was concurrent with the release of nitrite without the formation of the nitroso derivative(s). Results suggest that two types of enzymes were involved in HMX metabolism: one for denitration and the second for reduction to the nitroso derivative(s). [Copyright &y& Elsevier]