Biodegradation of RDX nitroso products MNX and TNX by cytochrome P450 XplA.

Anaerobic transformation of the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) by microorganisms involves sequential reduction of N-NO(2) 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 NO(2)(-), NO(3)(-), N(2)O, 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 ONNHCH(2)NHCHO (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-NO(2) 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-NO(2) over the N-NO linkages, but could nevertheless degrade all three nitroso derivatives, demonstrating the potential for complete RDX removal in explosives-contaminated sites.

Aerobic mineralization of nitroguanidine by Variovorax strain VC1 isolated from soil.

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 NH(3) (50.0%), nitrous oxide (N(2)O) (48.5%) and CO(2) (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 NH(3), N(2)O, and CO(2). However, we were unable to detect urea. Based on products distribution and reaction stoichiometry, we suggested that degradation of NQ, O(2)NN═C(NH(2))(2), might involve initial enzymatic hydroxylation of the imine, -C═N- bond, leading first to the formation of the unstable α-hydroxynitroamine intermediate, O(2)NNHC(OH)(NH(2))(2), whose decomposition in water should lead to the formation of NH(3), N(2)O, and CO(2). 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.

Aerobic biotransformation of 2,4-dinitroanisole in soil and soil Bacillus sp.

2,4-Dinitroanisole (DNAN) is a low sensitive melt-cast chemical being tested by the Military Industry as a replacement for 2,4,6-trinitrotoluene (TNT) in explosive formulations. Little is known about the fate of DNAN and its transformation products in the natural environment. Here we report aerobic biotransformation of DNAN in artificially contaminated soil microcosms. DNAN was completely transformed in 8 days in soil slurries supplemented with carbon and nitrogen sources. DNAN was completely transformed in 34 days in slurries supplemented with carbons alone and persisted in unamended microcosms. A strain of Bacillus (named 13G) that transformed DNAN by co-metabolism was isolated from the soil. HPLC and LC-MS analyses of cell-free and resting cell assays of Bacillus 13G with DNAN showed the formation of 2-amino-4-nitroanisole as the major end-product via the intermediary formation of the arylnitroso (ArNO) and arylhydroxylamino (ArNHOH) derivatives, indicating regioselective reduction of the ortho-nitro group. A series of secondary reactions involving ArNO and ArNHOH gave the corresponding azoxy- and azo-dimers. Acetylated and demethylated products were identified. Overall, this paper provides the evidence of fast DNAN transformation by the indigenous microbial populations of an amended soil with no history of contamination with explosives and a first insight into the aerobic metabolism of DNAN by the soil isolate Bacillus 13G.

Selenite and selenate removal in a permeable flow-through bioelectrochemical barrier.

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.

Biodegradation of RDX and MNX with Rhodococcus sp. strain DN22: new insights into the degradation pathway.

Previously we demonstrated that Rhodococcus sp. strain DN22 can degrade RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) aerobically via initial denitration. The present study describes the role of oxygen and water in the key denitration step leading to RDX decomposition using (18)O(2) and H(2)(18)O 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 NO(2)(-), NO(3)(-), NDAB (4-nitro-diazabutanal), NH(3), N(2)O, and HCHO with NO(2)(-)/NO(3)(-) molar ratio reaching 17 and ca. 2, respectively. In the presence of (18)O(2), DN22 degraded RDX and produced NO(2)(-) with m/z at 46 Da that subsequently oxidized to NO(3)(-) containing one (18)O atom, but in the presence of H(2)(18)O we detected NO(3)(-) without (18)O. A control containing NO(2)(-), DN22, and (18)O(2) gave NO(3)(-) with one (18)O, confirming biotic oxidation of NO(2)(-) to NO(3)(-). Treatment of MNX with DN22 and (18)O(2) produced NO(3)(-) with two mass ions, one (66 Da) incorporating two (18)O atoms and another (64 Da) incorporating only one (18)O atom and we attributed their formation to bio-oxidation of the initially formed NO and NO(2)(-), respectively. In the presence of H(2)(18)O we detected NO(2)(-) with two different masses, one representing NO(2)(-) (46 Da) and another representing NO(2)(-) (48 Da) with the inclusion of one (18)O atom suggesting auto-oxidation of NO to NO(2)(-). 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 NO(2)(-).

Manipulating redox conditions to enhance in situ bioremediation of RDX in groundwater at a contaminated site.

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.

In situ pilot test for bioremediation of energetic compound-contaminated soil at a former military demolition range site.

Bioremediation was performed in situ at a former military range site to assess the performance of native bacteria in degrading hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and 2,4-dinitrotoluene (2,4-DNT). The fate of these pollutants in soil and soil pore water was investigated as influenced by waste glycerol amendment to the soil. Following waste glycerol application, there was an accumulation of organic carbon that promoted microbial activity, converting organic carbon into acetate and propionate, which are intermediate compounds in anaerobic processes. This augmentation of anaerobic activity strongly correlated to a noticeable reduction in RDX concentrations in the amended soil. Changes in concentrations of RDX in pore water were similar to those observed in the soil suggesting that RDX leaching from the soil matrix, and treatment with waste glycerol, contributed to the enhanced removal of RDX from the water and soil. This was not the case with 2,4-DNT, which was neither found in pore water nor affected by the waste glycerol treatment. Results from saturated conditions and Synthetic Precipitation Leaching Procedure testing, to investigate the environmental fate of 2,4-DNT, indicated that 2,4-DNT found on site was relatively inert and was likely to remain in its current state on the site.

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-(14)C]-HMX, group IV isolates were found to mineralize HMX (26.8% in 308 d) as determined by liberated (14)CO(2), but negligible mineralization was observed in groups I-III. Resting cells of isolates metabolized HMX to N(2)O 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-NO(2) group(s) to the corresponding N-NO bond(s) prior to ring cleavage.

Metabolism of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine by Clostridium bifermentans strain HAW-1 and several other H2-producing fermentative anaerobic bacteria.

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 microM) 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).

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.

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-EB21(T), 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-EB21(T) was only distantly related to the genera Propionigenium and Ilyobacter with 6.6-7.5 % and 8.2-10.5 % dissimilarity as measured by 16S rRNA and 23S rRNA gene sequence analyses, respectively. Strain HAW-EB21(T) displayed unique properties in being psychrotrophic (18.5 degrees C 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-EB21(T) utilized glucose, fructose, maltose, N-acetyl-d-glucosamine, citrate, pyruvate, fumarate and Casitone as carbon sources and produced H(2) and acetate as the major fermentation products. Cells grown at 10 degrees C produced C(15 : 1) (30 %), C(16 : 1)omega7 (15 %) and C(16 : 0) (16 %) as major membrane fatty acids. The novel strain had a genomic DNA G+C content of 28.1 mol%, 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-EB21(T) (=DSM 19335(T)=JCM 14977(T)).

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 CH(2)=N-C(=N-NO(2))-CH=N-CHO or its isomer N(NO(2))=CH-CH=N-CO-CH=NH. Due to their chemical specificity, these two intermediates may be considered as markers of in situ attenuation of CL-20 in soil.

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, O(2), 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.

Shewanella canadensis sp. nov. and Shewanella atlantica sp. nov., manganese dioxide- and hexahydro-1,3,5-trinitro-1,3,5-triazine-reducing, psychrophilic marine bacteria.

Two strains belonging to the genus Shewanella, HAW-EB2(T) and HAW-EB5(T), were isolated previously from marine sediment sampled from the Atlantic Ocean, near Halifax harbour in Canada, for their potential to degrade explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). In the present study, strains HAW-EB2(T) and HAW-EB5(T) were found to display high 16S rRNA gene sequence similarity (90-99.5 %) to species of Shewanella, but their gyrB sequences were significantly different from each other and from species of Shewanella (79-87.6 %). Furthermore, DNA-DNA hybridization showed that the genomic DNA of the two strains was only 22 % related and showed less than 41 % relatedness to closely related species of Shewanella. In comparison to other species of Shewanella, strains HAW-EB2(T) and HAW-EB5(T) were also unique in some phenotypic properties such as activities of beta-galactosidase and tyrosine arylamidase and the ability to metabolize certain organic acids and sugars. Both strains HAW-EB2(T) and HAW-EB5(T) utilize malate, valerate, peptone and yeast extract as sole carbon and energy sources. The major membrane fatty acids of the two strains were C(14 : 0), iso-C(15 : 0), C(16 : 0), C(16 : 1)omega7, C(18 : 1)omega7 and C(20 : 5)omega3 and their major quinones were Q-7, Q-8 and MK-7. On the basis of these results, strain HAW-EB2(T) (=NCIMB 14238(T) =CCUG 54553(T)) is proposed as the type strain of Shewanella canadensis sp. nov. and strain HAW-EB5(T) (=NCIMB 14239(T) =CCUG 54554(T)) is proposed as the type strain of Shewanella atlantica sp. nov.

Shewanella halifaxensis sp. nov., a novel obligately respiratory and denitrifying psychrophile.

Indigenous bacteria found in the sediment of the Emerald Basin (depth of 215 m, Atlantic Ocean) located offshore of Halifax Harbour (Nova Scotia, Canada) were previously found to be able to degrade the explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). In the present study, a novel obligately respiratory, denitrifying and RDX-mineralizing bacterium, designated strain HAW-EB4(T), was isolated from the marine sediment. This bacterium utilized peptone, yeast extract, Casamino acids, esters (Tweens 20, 40 and 80), sugars (N-acetyl-D-glucosamine, ribose), several C2 and C3 acids (acetate, pyruvate, lactate, propionate) and amino acids (serine, proline) as sole carbon and energy sources. Aerobically grown cells (in marine broth 2216 at 10 degrees C) contained C(14 : 0) (6 %), iso-C(15 : 0) (12 %), C(16 : 0) (20 %), C(16 : 1)omega7 (37 %), C(18 : 1)omega7 (7 %) and C(20 : 5)omega3 (7 %) as major membrane fatty acids, and Q7 (28.1 %) and MK-7 (60.9 %) as dominant respiratory quinones, consistent with deep-sea species of Shewanella. The novel bacterium had a DNA G+C content of 45 mol% and showed similarity to Shewanella species in terms of 16S rRNA and gyrB gene sequences (93-99 and 67.3-88.4 % similarity, respectively), with Shewanella pealeana being the most closely related species. Genomic DNA-DNA hybridization between strain HAW-EB4T and S. pealeana revealed a level of relatedness of 17.9 %, lower than the 70 % species cut-off value, indicating that strain HAW-EB4T (= NCIMB 14093T = DSM 17350T) is the type strain of a novel species of Shewanella, for which the name Shewanella halifaxensis sp. nov. is proposed.

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-EB3(T)) 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-EB3(T) represents a novel species of Shewanella. Strain HAW-EB3(T) 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-EB3(T) 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-EB3(T) belongs to the Na(+)-requiring group of Shewanella species. The HAW-EB3(T) 16S rRNA gene sequence displayed < or =97.4 % 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-EB3(T) 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-EB3(T) (=NCIMB 14036(T)=DSM 17055(T)) as the type strain.

Biodegradation of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by Phanerochaete chrysosporium: new insight into the degradation pathway.

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 alpha-hydroxylation prior to ring cleavage. The degradation of 1-NO-HMX gave the ring-cleavage product 4-nitro-2,4-diazabutanal (NDAB), 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 micromol kg(-1)) (military base soil) or HMX (3057 micromol kg(-1)), and RDX (342 micromol 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.