Fate of CL-20 in sandy soils : Degradation products as potential markers of natural attenuation

Hexanitrohexaazaisowurtzitane (CL-20) is an emerging explosive that may replace the currently used explosives such as RDX and HMX, but little is known about its fate in soil. The present study was conducted to determine degradation products of CL-20 in two sandy soils under abiotic and biotic anaerobic conditions. Biotic degradation was prevalent in the slightly acidic VT soil, which contained a greater organic C content, while the slightly alkaline SAC soil favored hydrolysis. CL-20 degradation was accompanied by the formation of formate, glyoxal, nitrite, ammonium, and nitrous oxide. Biotic degradation of CL-20 occurred through the formation of its denitrohydrogenated derivative (m/z 393 Da) while hydrolysis occurred through the formation of a ring cleavage product (m/z 156 Da) that was tentatively identified as CH2 N\u2013C( N\u2013NO2)\u2013CH N\u2013CHO or its isomer N(NO2) CH\u2013CH N\u2013CO\u2013CH NH. Due to their chemical specificity, these two intermediates may be considered as markers of in situ attenuation of CL-20 in soil.NRC publication: Ye

Abundance and diversity of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)-metabolizing bacteria in UXO-contaminated marine sediments

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.NRC publication: Ye

Regulation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) metabolism in Shewanella halifaxensis HAW-EB4 by terminal electron acceptor and involvement of c-type cytochrome

Shewanella halifaxensis HAW-EB4 was previously isolated for its potential to mineralize hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) from a UXO (unexploded ordnance)-contaminated marine sediment site near Halifax Harbor. The present study was undertaken to determine the effect of terminal electron acceptors (TEA) on the growth of strain HAW-EB4 and on the enzymic processes involved in RDX metabolism. The results showed that aerobic conditions were optimal for bacterial growth, but that anaerobic conditions in the presence of trimethylamine N-oxide (TMAO) or in the absence of TEA favoured RDX metabolism. RDX as a substrate neither stimulated respiratory growth nor induced its own biotransformation. Strain HAW-EB4 used periplasmic proteins to transform RDX to both nitroso [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)] and ring cleavage products (such as methylenedinitramine), with more nitroso formation in cells grown on TMAO or pre-incubated in the absence of TEA. Using spectroscopy, SDS-PAGE and haem-staining analysis, strain HAW-EB4 was found to produce different sets of c-type cytochromes when grown on various TEA, with several more cytochromes produced in cells grown on TMAO. Crude cytochromes from total periplasmic proteins of TMAO-grown cells metabolized RDX to products similar to those found in assays using total periplasmic proteins and whole cells. To prove the involvement of cytochrome in RDX metabolism, we monitored dithionite- or NADH-reduced cytochromes by their absorbance at the {alpha} (551 nm) or {gamma} (418-420 nm) bands during anaerobic incubation with RDX. In both cases we found that RDX biotransformation was accompanied by oxidation of reduced cytochrome. Furthermore, O2, an oxidant of reduced cytochrome, inhibited RDX transformation. The present results demonstrate that S. halifaxensis HAW-EB4 metabolizes RDX optimally under TMAO-reducing conditions, and that c-type cytochromes are involved.NRC publication: Ye

Abundance and diversity of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)-metabolizing bacteria in UXO-contaminated marine sediments

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.NRC publication: Ye

Psychrilyobacter atlanticus gen. nov., sp. nov., a marine member of the phylum Fusobacteria that produces H2 and degrades nitramine explosives under low temperature conditions

A Gram-negative and obligately anaerobic marine bacterium, strain HAW-EB21T, was isolated in a previous study from marine sediment from the Atlantic Ocean, near Halifax Harbor, Canada, and found to have the potential to degrade both hexahydro-1,3,5-trinitro-1,3,5-triazine and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine. In the present study, phylogenetic analyses showed that strain HAW-EB21T was only distantly related to the genera Propionigenium and Ilyobacter with 6.6\u20137.5\u200a% and 8.2\u201310.5\u200a% dissimilarity as measured by 16S rRNA and 23S rRNA gene sequence analyses, respectively. Strain HAW-EB21T displayed unique properties in being psychrotrophic (18.5\u2005\ub0C optimum) and unable to utilize any of the carbon substrates (succinate, l-tartrate, 3-hydroxybutyrate, quinate or shikimate) used for isolating members of the genera Propionigenium and Ilyobacter. Strain HAW-EB21T utilized glucose, fructose, maltose, N-acetyl-d-glucosamine, citrate, pyruvate, fumarate and Casitone as carbon sources and produced H2 and acetate as the major fermentation products. Cells grown at 10\u2005\ub0C produced C15\u200a:\u200a1 (30\u200a%), C16\u200a:\u200a1\u3c97 (15\u200a%) and C16\u200a:\u200a0 (16\u200a%) as major membrane fatty acids. The novel strain had a genomic DNA G+C content of 28.1\u2005mol%, lower than the values of the genera Ilyobacter and Propionigenium. Based on the present results, the novel isolate is suggested to be a member of a new genus for which the name Psychrilyobacter atlanticus gen. nov., sp. nov. is proposed. The type strain of the type species is HAW-EB21T (=DSM 19335T=JCM 14977T).Peer reviewed: YesNRC publication: Ye

Shewanella spp. genomic evolution for a cold marine lifestyle and in-situ explosive biodegradation.

Shewanella halifaxensis and Shewanella sediminis were among a few aquatic gamma-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 gamma-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 gamma-proteobacteria including Shewanella that differ in temperature and Na+ 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 Na+-dependent nutrient transporters were recruited to use the high Na+ content as an energy source. For coping with low temperatures, these two strains as well as other psychrophilic strains of Shewanella and gamma-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

Shewanella sediminis sp. nov., a novel Na+ -requiring and hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading bacterium from marine sediment

Previously, a psychrophilic rod-shaped marine bacterium (strain HAW-EB3T) isolated from Halifax Harbour sediment was noted for its ability to degrade hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). In the present study phenotypic, chemotaxonomic and genotypic characterization showed that strain HAW-EB3T represents a novel species of Shewanella. Strain HAW-EB3T contained lysine decarboxylase, which is absent in other known Shewanella species, and distinguished itself from most other species of Shewanella by the presence of arginine dehydrolase, ornithine decarboxylase and chitinase, and by its ability to oxidize and ferment N-acetyl-D-glucosamine. Strain HAW-EB3T grew on several carbon sources (N-acetyl-D-glucosamine, Tween 40, Tween 80, acetate, succinate, butyrate and serine) and showed distinctive fatty acid and quinone compositions. Both phenotypic and 16S rRNA gene phylogenetic cluster analyses demonstrated that HAW-EB3T belongs to the Na+-requiring group of Shewanella species. The HAW-EB3T 16S rRNA gene sequence displayed <=97\ub74 % similarity to all known Shewanella species and was most similar to those of two bioluminescent species, Shewanella hanedai and Shewanella woodyi. However, gyrB of strain HAW-EB3T was significantly different from those of other Shewanella species, with similarities less than 85 %. DNA-DNA hybridization showed that its genomic DNA was less than 25 % related to that of S. hanedai or S. woodyi. Therefore we propose Shewanella sediminis sp. nov., with HAW-EB3T (=NCIMB 14036T=DSM 17055T) as the type strain.NRC publication: Ye

Shewanella sediminis sp. nov., a novel Na+ -requiring and hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading bacterium from marine sediment

Previously, a psychrophilic rod-shaped marine bacterium (strain HAW-EB3T) isolated from Halifax Harbour sediment was noted for its ability to degrade hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). In the present study phenotypic, chemotaxonomic and genotypic characterization showed that strain HAW-EB3T represents a novel species of Shewanella. Strain HAW-EB3T contained lysine decarboxylase, which is absent in other known Shewanella species, and distinguished itself from most other species of Shewanella by the presence of arginine dehydrolase, ornithine decarboxylase and chitinase, and by its ability to oxidize and ferment N-acetyl-D-glucosamine. Strain HAW-EB3T grew on several carbon sources (N-acetyl-D-glucosamine, Tween 40, Tween 80, acetate, succinate, butyrate and serine) and showed distinctive fatty acid and quinone compositions. Both phenotypic and 16S rRNA gene phylogenetic cluster analyses demonstrated that HAW-EB3T belongs to the Na+-requiring group of Shewanella species. The HAW-EB3T 16S rRNA gene sequence displayed <=97\ub74 % similarity to all known Shewanella species and was most similar to those of two bioluminescent species, Shewanella hanedai and Shewanella woodyi. However, gyrB of strain HAW-EB3T was significantly different from those of other Shewanella species, with similarities less than 85 %. DNA-DNA hybridization showed that its genomic DNA was less than 25 % related to that of S. hanedai or S. woodyi. Therefore we propose Shewanella sediminis sp. nov., with HAW-EB3T (=NCIMB 14036T=DSM 17055T) as the type strain.NRC publication: Ye