Cloning,sequencing, and characterization of the hexahydro-1,3,5-Trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.     

Cloning, Sequencing, and Characterization of the Hexahydro-1,3,5-Trinitro-1,3,5-Triazine Degradation Gene Cluster from Rhodococcus rhodochrous

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a high explosive which presents an environmental hazard as a major land and groundwater contaminant. Rhodococcus rhodochrous strain 11Y was isolated from explosive contaminated land and is capable of degrading RDX when provided as the sole source of nitrogen for growth. Products of RDX degradation in resting-cell incubations were analyzed and found to include nitrite, formaldehyde, and formate. No ammonium was excreted into the medium, and no dead-end metabolites were observed. The gene responsible for the degradation of RDX in strain 11Y is a constitutively expressed cytochrome P450-like gene, xplA, which is found in a gene cluster with an adrenodoxin reductase homologue, xplB. The cytochrome P450 also has a flavodoxin domain at the N terminus. This study is the first to present a gene which has been identified as being responsible for RDX biodegradation. The mechanism of action of XplA on RDX is thought to involve initial denitration followed by spontaneous ring cleavage and mineralization.     

Isolation and characterization of RDX-degrading <Emphasis Type="Italic">Rhodococcus</Emphasis> species from a contaminated aquifer

Groundwater contamination by the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a global problem. Israel’s coastal aquifer was contaminated with RDX. This aquifer is mostly aerobic and we therefore sought aerobic bacteria that might be involved in natural attenuation of the compound in the aquifer. RDX-degrading bacteria were captured by passively sampling the indigenous bacteria onto sterile sediments placed within sampling boreholes. Aerobic RDX biodegradation potential was detected in the sediments sampled from different locations along the plume. RDX degradation with the native sampled consortium was accompanied by 4-nitro-2,4-diazabutanal formation. Two bacterial strains of the genus Rhodococcus were isolated from the sediments and identified as aerobic RDX degraders. The xplA gene encoding the cytochrome P450 enzyme was partially (~500 bp) sequenced from both isolates. The obtained DNA sequences had 99% identity with corresponding gene fragments of previously isolated RDX-degrading Rhodococcus strains. RDX degradation by both strains was prevented by 200 μM of the cytochrome P450 inhibitor metyrapone, suggesting that cytochrome P450 indeed mediates the initial step in RDX degradation. RDX biodegradation activity by the T7 isolate was inhibited in the presence of nitrate or ammonium concentrations above 1.6 and 5.5 mM, respectively (100 mg l⁻¹) while the T9N isolate’s activity was retarded only by ammonium concentrations above 5.5 mM. This study shows that bacteria from the genus Rhodococcus, potentially degrade RDX in the saturated zone as well, following the same aerobic degradation pathway defined for other Rhodococcus species. RDX-degrading activity by the Rhodococcus species isolate T9N may have important implications for the bioremediation of nitrate-rich RDX-contaminated aquifers.     

Sequential anaerobic–aerobic degradation of munitions waste

A sequential anaerobic–aerobic biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) was studied. The results demonstrated that: (i) a complete degradation of RDX was achieved within 20 days using a consortium of bacteria from a wastewater activated sludge, (ii) RDX degradation did not occur under aerobic conditions alone, (iii) RDX-degrading bacterial strain that was isolated from the activated sludge completely degraded RDX within 2 days, and (iv) RDX- induced protein expressions were observed in the RDX-degrading bacterial strain. Based on fatty acid composition and a confirmation with a 16S rRNA analysis, the RDX-degrading bacterial strain was identified as a Bacillus pumilus—GC subgroup B.     

Evidence that RDX biodegradation by Rhodococcus strain DN22 is plasmid-borne and involves a cytochrome p-450

AIMS: To investigate the biodegradation of the explosive compound RDX in Rhodococcus strain DN22, a bacterium previously isolated for its ability to grow on RDX as sole nitrogen source. METHODS AND RESULTS: Analysis of the rates of RDX degradation and nitrite production indicated that 2 mol nitrite were produced per mole RDX degraded. Cells of strain DN22 had the highest activity against RDX during the exponential phase and low activity in the stationary phase. Nitrite production from RDX was inhibited by metyrapone, menadione, piperonyl butoxide, n-octylamine and carbon monoxide and inducible by pyrrolidine, pyridine and atrazine. Acridine orange treatment yielded RDX-minus derivatives of strain DN22 at a curing rate of 1.5% and all of the cured derivatives had lost a large plasmid. CONCLUSIONS: RDX biodegradation in strain DN22 appears to involve a plasmid-encoded cytochrome p-450 enzyme. SIGNIFICANCE AND IMPACT OF THE STUDY: Plasmid-borne RDX degradation genes could potentially be transferred between bacteria. Our research into RDX metabolism in strain DN22 will facilitate future applications of this bacterium for bioremediation.     

Mineralization of the cyclic nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine by Gordonia and Williamsia spp

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitroamine explosive that is a major component in many military high-explosive formulations. In this study, two aerobic bacteria that are capable of using RDX as the sole source of carbon and nitrogen to support their growth were isolated from surface soil. These bacterial strains were identified by their fatty acid profiles and 16S ribosomal gene sequences as Williamsia sp. KTR4 and Gordonia sp. KTR9. The physiology of each strain was characterized with respect to the rates of RDX degradation and [U-14C]RDX mineralization when RDX was supplied as a sole carbon and nitrogen source in the presence and absence of competing carbon and nitrogen sources. Strains KTR4 and KTR9 degraded 180 microM RDX within 72 h when RDX served as the only added carbon and nitrogen source while growing to total protein concentrations of 18.6 and 16.5 microg/ml, respectively. Mineralization of [U-14C]RDX to 14CO2 was 30% by strain KTR4 and 27% by KTR9 when RDX was the only added source of carbon and nitrogen. The addition of (NH4)2SO4- greatly inhibited KTR9's degradation of RDX but had little effect on that of KTR4. These are the first two pure bacterial cultures isolated that are able to use RDX as a sole carbon and nitrogen source. These two genera possess different physiologies with respect to RDX mineralization, and each can serve as a useful microbiological model for the study of RDX biodegradation with regard to physiology, biochemistry, and genetics.     

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) degradation by <Emphasis Type="Italic">Acetobacterium paludosum</Emphasis>

Substrates and nutrients are often added to contaminated soil or groundwater to enhance bioremediation. Nevertheless, this practice may be counterproductive in some cases where nutrient addition might relieve selective pressure for pollutant biodegradation. Batch experiments with a homoacetogenic pure culture of Acetobacterium paludosum showed that anaerobic RDX degradation is the fastest when auxiliary growth substrates (yeast extract plus fructose) and nitrogen sources (ammonium) are not added. This bacterium degraded RDX faster under autotrophic (H₂-fed) than under heterotrophic conditions, even though heterotrophic growth was faster. The inhibitory effect of ammonium is postulated to be due to the repression of enzymes that initiate RDX degradation by reducing its nitro groups, based on the known fact that ammonia represses nitrate and nitrite reductases. This observation suggests that the absence of easily assimilated nitrogen sources, such as ammonium, enhances RDX degradation. Although specific end products of RDX degradation were not determined, the production of nitrous oxide (N₂O) suggests that A. paludosum cleaved the triazine ring.     

Mineralization of the Cyclic Nitramine Explosive Hexahydro-1,3,5-Trinitro-1,3,5-Triazine by Gordonia and Williamsia spp.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a cyclic nitroamine explosive that is a major component in many military high-explosive formulations. In this study, two aerobic bacteria that are capable of using RDX as the sole source of carbon and nitrogen to support their growth were isolated from surface soil. These bacterial strains were identified by their fatty acid profiles and 16S ribosomal gene sequences as Williamsia sp. KTR4 and Gordonia sp. KTR9. The physiology of each strain was characterized with respect to the rates of RDX degradation and [U-¹⁴C]RDX mineralization when RDX was supplied as a sole carbon and nitrogen source in the presence and absence of competing carbon and nitrogen sources. Strains KTR4 and KTR9 degraded 180 μM RDX within 72 h when RDX served as the only added carbon and nitrogen source while growing to total protein concentrations of 18.6 and 16.5 μg/ml, respectively. Mineralization of [U-¹⁴C]RDX to ¹⁴CO₂ was 30% by strain KTR4 and 27% by KTR9 when RDX was the only added source of carbon and nitrogen. The addition of (NH₄)₂SO₄ greatly inhibited KTR9's degradation of RDX but had little effect on that of KTR4. These are the first two pure bacterial cultures isolated that are able to use RDX as a sole carbon and nitrogen source. These two genera possess different physiologies with respect to RDX mineralization, and each can serve as a useful microbiological model for the study of RDX biodegradation with regard to physiology, biochemistry, and genetics.     

Metabolism of the Aliphatic Nitramine 4-Nitro-2,4-Diazabutanal by Methylobacterium sp. Strain JS178

The aliphatic nitramine 4-nitro-2,4-diazabutanal (NDAB; C₂H₅N₃O₃) is a ring cleavage metabolite that accumulates during the aerobic degradation of the energetic compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by various Rhodococcus spp. NDAB is also produced during the alkaline hydrolysis of either RDX or octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) and during the photolysis of RDX. Traces of NDAB were observed in a soil sampled from an ammunition-manufacturing facility contaminated with both HMX and RDX, suggesting natural attenuation. In this study, we report the isolation of a soil bacterium that is able to degrade NDAB under aerobic conditions. The isolate is a pink-pigmented facultative methylotroph affiliated with the genus Methylobacterium. The strain, named Methylobacterium sp. strain JS178, degrades NDAB as a sole nitrogen source, with concomitant growth and formation of 1 molar equivalent of nitrous oxide (N₂O). Comparison of the growth yield of strain JS178 grown on NDAB, nitrite (NO₂⁻), or ammonium (NH₄⁺) as a nitrogen source revealed that 1 N equivalent is assimilated from each mole of NDAB, which completes the nitrogen mass balance. In radiotracer experiments, strain JS178 mineralized 1 C of the [¹⁴C]NDAB produced in situ from [¹⁴C]RDX by Rhodococcus sp. strain DN22. Studies on the regulation of NDAB degradation indicated that allantoin, an intermediate in the purine catabolic pathway and a central molecule in the storage and transport of nitrogen in plants, up-regulated the enzyme(s) involved in the degradation of the nitramine. The results reveal the potential for the sequential participation of rhodococci and methylobacteria to effect the complete degradation of RDX.     

Biotransformation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) by a Rabbit Liver Cytochrome P450: Insight into the Mechanism of RDX Biodegradation by Rhodococcus sp. Strain DN22

A unique metabolite with a molecular mass of 119 Da (C₂H₅N₃O₃) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.     

Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22

A unique metabolite with a molecular mass of 119 Da (C(2)H(5)N(3)O(3)) accumulated during biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by Rhodococcus sp. strain DN22 (D. Fournier, A. Halasz, J. C. Spain, P. Fiurasek, and J. Hawari, Appl. Environ. Microbiol. 68:166-172, 2002). The structure of the molecule and the reactions that led to its synthesis were not known. In the present study, we produced and purified the unknown metabolite by biotransformation of RDX with Rhodococcus sp. strain DN22 and identified the molecule as 4-nitro-2,4-diazabutanal using nuclear magnetic resonance and elemental analyses. Furthermore, we tested the hypothesis that a cytochrome P450 enzyme was responsible for RDX biotransformation by strain DN22. A cytochrome P450 2B4 from rabbit liver catalyzed a very similar biotransformation of RDX to 4-nitro-2,4-diazabutanal. Both the cytochrome P450 2B4 and intact cells of Rhodococcus sp. strain DN22 catalyzed the release of two nitrite ions from each reacted RDX molecule. A comparative study of cytochrome P450 2B4 and Rhodococcus sp. strain DN22 revealed substantial similarities in the product distribution and inhibition by cytochrome P450 inhibitors. The experimental evidence led us to propose that cytochrome P450 2B4 can catalyze two single electron transfers to RDX, thereby causing double denitration, which leads to spontaneous hydrolytic ring cleavage and decomposition to produce 4-nitro-2,4-diazabutanal. Our results provide strong evidence that a cytochrome P450 enzyme is the key enzyme responsible for RDX biotransformation by Rhodococcus sp. strain DN22.     

Microaerophilic degradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) by three Rhodococcus strains

Aim: The goal of this study was to compare the degradation of hexahydro‐1,3,5‐trinitro‐1,3,5‐triazine (RDX) by three Rhodococcus strains under anaerobic, microaerophilic (<0·04 mg l^?1 dissolved oxygen) and aerobic (dissolved oxygen (DO) maintained at 8 mg l^?1) conditions. Methods and Results: Three Rhodococcus strains were incubated with no, low and ambient concentrations of oxygen in minimal media with succinate as the carbon source and RDX as the sole nitrogen source. RDX and RDX metabolite concentrations were measured over time. Under microaerophilic conditions, the bacteria degraded RDX, albeit about 60‐fold slower than under fully aerobic conditions. Only the breakdown product, 4‐nitro‐2,4‐diazabutanal (NDAB) accumulated to measurable concentrations under microaerophilic conditions. RDX degraded quickly under both aerated and static aerobic conditions (DO allowed to drop below 1 mg l^?1) with the accumulation of both NDAB and methylenedinitramine (MEDINA). No RDX degradation was observed under strict anaerobic conditions. Conclusions: The Rhodococcus strains did not degrade RDX under strict anaerobic conditions, while slow degradation was observed under microaerophilic conditions. The RDX metabolite NDAB was detected under both microaerophilic and aerobic conditions, while MEDINA was detected only under aerobic conditions. Impact and Significance of the Study: This work confirmed the production of MEDINA under aerobic conditions, which has not been previously associated with aerobic RDX degradation by these organisms. More importantly, it demonstrated that aerobic rhodococci are able to degrade RDX under a broader range of oxygen concentrations than previously reported.     

Cosubstrate independent mineralization of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a <Emphasis Type="Italic">Desulfovibrio</Emphasis> species under anaerobic conditions

Past handling practices associated with the manufacturing and processing of the high explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has resulted in extensive environmental contamination. In-situ biodegradation is a promising technology for remediating RDX contaminated sites but often relies on the addition of a cosubstrate. A sulfate-reducing bacterium isolated from an RDX-degrading enrichment culture was studied for its ability to grow on RDX as a sole source of carbon and nitrogen and for its ability to mineralize RDX in the absence of a cosubstrate. The results showed the isolate degraded 140 μM RDX in 63 days when grown on RDX as a carbon source. Biomass within the carbon limited culture increased 9-fold compared to the RDX unamended controls. When the isolate was incubated with RDX as sole source of nitrogen it degraded 160 μM RDX in 41 days and exhibited a 4-fold increase in biomass compared to RDX unamended controls. Radiolabeled studies under carbon limiting conditions with ¹⁴C-hexahydro-1,3,5-trinitro-1,3,5-triazine confirmed mineralization of the cyclic nitramine. After 60 days incubation 26% of the radiolabel was recovered as ¹⁴CO₂, while in the control bottles less than 1% of the radiolabel was recovered as ¹⁴CO₂. Additionally, ~2% of the radiolabeled carbon was found to be associated with the biomass. The 16S rDNA gene was sequenced and identified the isolate as a novel species of Desulfovibrio, having a 95.1% sequence similarity to Desulfovibrio desulfuricans. This is the first known anaerobic bacterium capable of mineralizing RDX when using it as a carbon and energy source for growth.     

Type I nitroreductases in soil enterobacteria reduce TNT (2,4,6,-trinitrotoluene) and RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)

Many enteric bacteria express a type I oxygen-insensitive nitroreductase, which reduces nitro groups on many different nitroaromatic compounds under aerobic conditions. Enzymatic reduction of nitramines was also documented in enteric bacteria under anaerobic conditions. This study indicates that nitramine reduction in enteric bacteria is carried out by the type I, or oxygen-insensitive nitroreductase, rather than a type II enzyme. The enteric bacterium Morganella morganii strain B2 with documented hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) nitroreductase activity, and Enterobacter cloacae strain 96-3 with documented 2,4,6-trinitrotoluene (TNT) nitroreductase activity, were used here to show that the explosives TNT and RDX were both reduced by a type I nitroreductase. Morganella morganii and E. cloacae exhibited RDX and TNT nitroreductase activities in whole cell assays. Type I nitroreductase, purified from E. cloacae, oxidized NADPH with TNT or RDX as substrate. When expression of the E. cloacae type I nitroreductase gene was induced in an Escherichia coli strain carrying a plasmid, a simultaneous increase in TNT and RDX nitroreductase activities was observed. In addition, neither TNT nor RDX nitroreductase activity was detected in nitrofurazone-resistant mutants of M. morganii. We conclude that a type I nitroreductase present in these two enteric bacteria was responsible for the nitroreduction of both types of explosive.     

Degradation of 2,5-dimethylpyrazine by <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> strain DP-45 isolated from a waste gas treatment plant of a fishmeal processing company

A bacterium, strain DP-45, capable of degrading 2,5-dimethylpyrazine (2,5-DMP) was isolated and identified as Rhodococcus erythropolis. The strain also grew on many other pyrazines found in the waste gases of food industries, like 2,3-dimethylpyrazine (2,3-DMP), 2,6-dimethylpyrazine (2,6-DMP), 2-ethyl-5(6)-dimethylpyrazine (EMP), 2-ethylpyrazine (EP), 2-methylpyrazine (MP), and 2,3,5-trimethylpyrazine (TMP). The strain utilized 2,5-DMP as sole source of carbon and nitrogen and grew optimally at 25°C with a doubling time of 7.6 h. The degradation of 2,5-DMP was accompanied by the growth of the strain and by the accumulation of a first intermediate, identified as 2-hydroxy-3,6-dimethylpyrazine (HDMP). The disappearance of HDMP was accompanied by the release of ammonium into the medium. No other metabolite was detected. The degradation of 2,5-DMP and HDMP by strain DP-45 required molecular oxygen. The expression of the first enzyme in the pathway was induced by 2,5-DMP and HDMP whereas the second enzyme was constitutively expressed. The activity of the first enzyme was inhibited by diphenyliodonium (DPI), a flavoprotein inhibitor, methimazole, a competitive inhibitor of flavin-containing monooxygenases, and by cytochrome P450 inhibitors, 1-aminobenzotriazole (ABT) and phenylhydrazine (PHZ). The activity of the second enzyme was inhibited by DPI, ABT, and PHZ. Sodium tungstate, a specific antagonist of molybdate, had no influence on growth and consumption of 2,5-DMP by strain DP-45. These results led us to propose that a flavin-dependent monooxygenase or a cytochrome P450-dependent monooxygenase rather than a molybdenum hydroxylase catalyzed the initial hydroxylation step and that a cytochrome P450 enzyme is responsible for the transformation of HDMP in the second step.     

Influence of ammonium,nitrate and nitrite on the performance of the pure culture of <Emphasis Type="Italic">Acinetobacter junii</Emphasis>

The influence of different concentration ranges (0–500 mg/L) of ammonium, nitrate and nitrite presence in the wastewater, on the performance of the pure culture of phosphate-accumulating bacterium Acinetobacter junii in the anaerobic and aerobic conditions, was investigated. A. junii was able to use ammonium and nitrate salts as the source of nitrogen, unlike in the case of nitrite salt. Comparing to the control reactors with the peptone and yeast extract as the sources of nitrogen, at the lowest tested concentration of ammonium and nitrate the performance of the system was inhibited due to the nitrogen deficit in the wastewater, while at the highest concentration it was positively influenced. Nitrite in all concentrations detrimentally affected the phosphate release and uptake rates, chemical oxygen demand uptake rates, nitrogen uptake rates, as well as multiplication of A. junii. The higher the nitrite concentration, the more pronounced was the effect. At the highest nitrite concentration tested a complete failure of the system was observed.     

Degradation of Morpholine by an Environmental Mycobacterium Strain Involves a Cytochrome P-450

A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ ¹H nuclear magnetic resonance (¹H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and ¹H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. ¹H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C—N bond of the morpholine ring.     

Effective biodegradation of 2,4,6-trinitrotoluene using a novel bacterial strain isolated from TNT-contaminated soil

In this environmental-sample based study, rapid microbial-mediated degradation of 2,4,6-trinitrotoluene (TNT) contaminated soils is demonstrated by a novel strain, Achromobacter spanius STE 11. Complete removal of 100 mg L⁻¹ TNT is achieved within only 20 h under aerobic conditions by the isolate. In this bio-conversion process, TNT is transformed to 2,4-dinitrotoluene (7 mg L⁻¹), 2,6-dinitrotoluene (3 mg L⁻¹), 4-aminodinitrotoluene (49 mg L⁻¹) and 2-aminodinitrotoluene (16 mg L⁻¹) as the key metabolites. A. spanius STE 11 has the ability to denitrate TNT in aerobic conditions as suggested by the dinitrotoluene and NO₃ productions during the growth period. Elemental analysis results indicate that 24.77 mg L⁻¹ nitrogen from TNT was accumulated in the cell biomass, showing that STE 11 can use TNT as its sole nitrogen source. TNT degradation was observed between pH 4.0–8.0 and 4–43 °C; however, the most efficient degradation was at pH 6.0–7.0 and 30 °C.     

Biodegradation of RDX in Unsaturated Soil

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a military explosive that is a common soil and groundwater contaminant at facilities that manufacture, handle, and dispose of munitions. One such facility is the U.S. Department of Energy Pantex Plant, the focus of this research in which the feasibility of in situ bioremediation of contaminated soil in the vadose zone was assessed. A batch technique using ¹⁴C-RDX was developed to investigate the degradation of RDX under aerobic, microaerobic, and anaerobic conditions. In addition, the effect of nutrients (organic carbon and phosphorus) on biodegradation rates was studied. The extent of mineralization was quantified by monitoring the production of ¹⁴CO₂, and RDX biodegradation rates were estimated for each environmental condition. The results showed that RDX degraders were indigenous to the contaminated soil and degraded RDX to a significant extent under anaerobic conditions. Little biotransformation was observed under aerobic conditions. The addition of a biodegradable organic carbon source significantly increased the RDX biodegradation rate. Under appropriate environmental conditions, significant mineralization of RDX also was observed. The half-lives for the degradation of RDX under anaerobic conditions were approximately 60 days and decreased to approximately 40 days with nutrient addition. In contrast, the half-life for aerobic degradation was on the order of 1000 days, with an upper 95% confidence interval approaching infinity.