Biodegradation of the Nitramine Explosive CL-20

The cyclic nitramine explosive CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane)was examined in soil microcosms to determine whether it is biodegradable.CL-20 was incubated with a variety of soils. The explosive disappearedin all microcosms except the controls in which microbial activityhad been inhibited. CL-20 was degraded most rapidly in gardensoil. After 2 days of incubation, about 80% of the initial CL-20had disappeared. A CL-20-degrading bacterial strain, Agrobacteriumsp. strain JS71, was isolated from enrichment cultures containinggarden soil as an inoculum, succinate as a carbon source, andCL-20 as a nitrogen source. Growth experiments revealed thatstrain JS71 used 3 mol of nitrogen per mol of CL-20.

INTRODUCTION

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Introduction

CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane)is a new, highly energetic explosive related to the explosivesRDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) and HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine)(Fig. 1). Due to its higher energy and its moderate sensitivity,CL-20 is expected to replace earlier explosives (19, 20, 22,25). The production of CL-20 and its use in munitions and propellantscan be expected to lead to environmental contamination. RDXand HMX, common contaminants in soil and groundwater at munitionmanufacturing sites and firing ranges, are toxic and possiblycarcinogenic (7, 8, 11, 12, 16, 23, 27). Because of the structuralsimilarity of CL-20 to RDX and HMX, it is likely that CL-20has similar effects. So far, there is little information availableabout the properties of CL-20. In contrast to RDX and HMX, CL-20is a caged molecule (Fig. 1). The water solubility of CL-20is 4.8 mg/liter at 25°C (Stevens Institute of Technology[http://www.cee.stevens-tech.edu/ResProj.html]), which is lowerthan the solubility of the nitramines RDX and HMX (38.4 and6.6 mg/liter at 20°C, respectively) (27).

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FIG. 1. Molecular structures of the cyclic nitramine explosives RDX, HMX, and CL-20.

The microbial degradation of RDX and HMX under aerobic and anaerobicconditions has been extensively investigated (1, 2, 4-6, 9,13-15, 17, 21). Under anaerobic conditions, three degradationpathways have been proposed. The first includes the productionof nitroso derivatives of RDX and HMX, which are subject tofurther degradation (15, 21). Hawari et al. suggested a seconddegradation pathway which includes the direct ring cleavageof RDX and HMX without the initial reduction of the nitro groups(14, 15). The third proposed degradation pathway is based onN denitration prior to ring cleavage (1).

Under aerobic conditions, RDX is degraded by a yet-unknown mechanism.The initial attack seems to be catalyzed by a cytochrome P450(1a, 6, 24). RDX degradation by Rhodococcus sp. strain DN22leads to the formation of nitrite, nitrous oxide, ammonia, formaldehyde,and a dead-end product with a molecular weight of 119 whichwas recently identified as 4-nitro-2,4-diazabutanal (1a, 5,9). Fournier et al. proposed a degradation pathway which includesan initial denitration of RDX followed by ring cleavage to formaldehydeand the dead-end product (9).

To the best of our knowledge, no biodegradation of CL-20 hasbeen reported to date. It is, therefore, important to determinewhether biodegradation might affect the fate and transport ofCL-20 in terrestrial and aquatic ecosystems. To that end weevaluated the biodegradation of CL-20 in laboratory microcosmsand isolated a bacterial strain that is able to grow on CL-20as the sole nitrogen source.

CL-20 biodegradation in soil.

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Cl-20 biodegradation in soil.

Microcosms with dried and sieved garden and agricultural soilswere incubated with CL-20 provided by the High Explosives Researchand Development Facility at Eglin Air Force Base, Fla.

Microcosm experiments were conducted in 5-ml amber vials containingsoil (2 g), sterile water (1.0 to 1.2 ml), and CL-20 (100 nmol).CL-20 was added from a 5 mM stock solution in methanol. Theamounts of added water corresponded to the water absorptivecapacity of the soils. For inhibiting microbial activity insoils, the added water contained glutaraldehyde (1%) and mercuricchloride (90 mg/liter). The microcosms were vigorously mixedby vortexing them and incubated in the dark at 30°C. Fortime course studies, one microcosm was sacrificed at each timepoint. The CL-20 concentrations in the microcosms in acetonitrileextracts were determined by high-pressure liquid chromatography(HPLC) analysis. The mobile phase (65% [vol/vol] acetonitrile,0.1% [vol/vol] trifluoroacetic acid) was pumped at a flow rateof 1.0 ml/min over a Spherisorb C₈ column (250 by 4.6 mm; particlesize, 5 µm; Alltech, Deerfield, Ill.) or a Synergi Polar-RPcolumn (150 by 4.6 mm; particle size, 4 µm; Phenomenex,Torrance, Calif.). Absorbance was measured at a wavelength of230 nm.

CL-20 was degraded in all three soils (Fig. 2). The persistenceof CL-20 in sterile controls indicated that the degradationprocess in active soil was biological. CL-20 degradation wasfastest in the microcosms containing garden soil. CL-20 didnot disappear completely in any of the soils. The incompletedegradation of CL-20 might be due to limited bioavailability.Residual amounts of CL-20 may be sorbed to the soil and thereforebe unavailable for the microorganisms. In general, the availabilityof contaminants for microorganisms is dependent on the propertiesof the soil and the chemical compound, as well as on mass transport(3, 29).

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FIG. 2. CL-20 biodegradation in microcosms. Soils from Florida (A), Iowa (B), and Texas (C) were incubated with CL-20. CL-20 amounts are in nanomoles per microcosm. ${blacksquare}$ , active soil; ${blacktriangleup}$ , soil with inhibited microbial activity. Degradation experiments were performed in triplicate. d, days.

During CL-20 degradation in the microcosms with garden soil,traces of a transient metabolite with a molecular weight of247 could be detected by HPLC-mass spectrometry in the negativeelectrospray ionization mode. Further investigations are underway to identify the putative CL-20 degradation product.

It is not clear whether CL-20 degradation in the soil microcosmsoccurred aerobically or anaerobically. No precautions were takento maintain aerobic conditions. Degradation started relativelyquickly, so it can be assumed that CL-20 was degraded underaerobic conditions. On the other hand, it is possible that smallanaerobic zones existed in the soil, where microbes could havedegraded CL-20 anaerobically. In preliminary experiments CL-20was also degraded under anaerobic conditions in sewage sludge(data not shown).

Enrichment and isolation of the Agrobacterium strain JS71.

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Enrichment and Isolation of...

Enrichment cultures were carried out in 10 ml of M medium (pH7.0) without sodium chloride (5). The medium was supplementedwith CL-20 (100 µM) in methanol (5 mM stock solution)as the sole nitrogen source and succinate (20 mM) as the solecarbon source and inoculated with 1 g of Florida garden soilin 50-ml shake flasks. The cultures were incubated on a shakerat 30°C and 250 rpm. Microbial activity in the controlswas inhibited by the addition of glutaraldehyde (1%) and mercuricchloride (90 mg/liter). The concentrations of CL-20 in the enrichmentswere determined by HPLC. Samples were mixed with 1.5 volumesof acetonitrile, and suspended material was removed by centrifugationprior to HPLC analysis.

CL-20 degradation in the first enrichment culture started afterabout 3 days (Fig. 3). The disappearance of CL-20 verified thatCL-20 was degraded under aerobic conditions. CL-20 degradationin the enrichments was slower than its degradation in the soilmicrocosms, probably because of the smaller inoculum and thevery low water solubility of CL-20. In contrast to the partialdegradation of CL-20 in the soil microcosms, CL-20 was completelydegraded in the enrichment cultures. The results support thehypothesis that some of the CL-20 in the microcosms (Fig. 2)was unavailable because of sorption to the soil.

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FIG. 3. CL-20 biodegradation in enrichments with Florida soil. ${blacksquare}$ , active soil; ${blacktriangleup}$ , soil with inhibited microbial activity. Minimal medium was supplemented with succinate (20 mM) and CL-20 (100 µM). Enrichments were performed in triplicate. d, days.

After three subcultures of the enrichment, appropriate dilutionswere plated on the above-named enrichment medium solidifiedwith 1.5% (wt/vol) agarose (molecular biology grade). Singlecolonies were tested for the ability to grow on CL-20 as thesole nitrogen source. The selective enrichment yielded a bacterialstrain that was able to grow with CL-20 as the sole nitrogensource. The 16S rRNA gene sequence from the isolate was determinedand evaluated by Midi Labs (Newark, Del.). The 16S rRNA genesequence indicated that the strain was most similar to Agrobacteriumrubi (0.64% difference) and Agrobacterium tumefaciens (0.84%difference). The isolate was gram negative, motile, and rodshaped. These properties correspond to the characteristics ofthe genus Agrobacterium (18). The isolate was, therefore, namedAgrobacterium sp. strain JS71.

The transformation of explosives by Agrobacterium strains hasbeen described previously. Agrobacterium sp. strain 2PC wasable to biotransform 2,4,6-trinitrotoluene to monoaminodinitrotoluenes(10). White et al. isolated an Agrobacterium radiobacter strainthat transforms glycerol trinitrate to glycerol dinitrates andfinally to glycerol mononitrates. The strain used two nitrogensfrom glycerol trinitrate as the nitrogen source for growth (28).An NADH-dependent reductase able to remove one nitro group fromglycerol trinitrate in the form of nitrite was isolated fromthe strain (26). The enzyme also denitrated pentaerythrol tetranitrate,isosorbide dinitrate, and ethyleneglycol dinitrate but did notdenitrate isopropyl nitrate, 2,4,6-trinitrotoluene, or RDX.Thus, the enzyme was able to reduce nitrate esters but not nitrogroups connected to a carbon or nitrogen atom.

Growth studies with Agrobacterium strain JS71.

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Growth studies with...

The Agrobacterium strain JS71 was grown under the same nitrogen-limitedconditions as those used for the enrichment cultures but withthe addition of 250 µM CL-20. CL-20 was added from a stocksolution in methanol (20 mM). The optical densities of the culturescould not be determined because the undissolved CL-20 interferedwith the absorption. Therefore, bacterial growth was estimatedby determining the protein concentration. Cell suspensions weremixed 1:1 with acetone, and the cells were collected by centrifugationand washed with 100 µl of acetone. The pelleted cellswere suspended in 0.1 M NaOH and incubated at 80°C for 5min to lyse the cells. Protein concentrations were determinedwith a bicinchoninic acid protein assay kit (Pierce, Rockford,Ill.). CL-20 concentrations in the cultures were measured byHPLC after the addition of 4 volumes of acetonitrile to thesamples and centrifugation as described above.

Strain JS71 grew in minimal medium containing succinate andCL-20 at a growth rate of 0.14 per day. The isolate did notgrow with CL-20 as the sole nitrogen and carbon source. Theslow growth of strain JS71 was probably due to the very lowwater solubility of CL-20. When the surfactant Tween 80 (0.1%[vol/vol]) was added to the cultures to increase the solubilityand therefore the availability of CL-20, the growth rate was0.59 per day (Fig. 4). Strain JS71 could not grow in media withouta nitrogen source (Fig. 4) or in media containing RDX or HMXas the sole nitrogen source (data not shown).

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FIG. 4. Growth of Agrobacterium strain JS71. Aerobic cultures were grown in medium with 20 mM succinate-0.1% (vol/vol) Tween 80 with ( ${blacksquare}$ ) and without ( ${square}$ ) 250 µM CL-20 as the sole nitrogen source. ${blacktriangleup}$ , CL-20 concentration. The growth studies were performed in triplicate ( ${blacksquare}$ ) or duplicate ( ${square}$ ). d, days.

The addition of Tween 80 to cultures containing CL-20 facilitatedthe determination of CL-20 concentrations in addition to increasingthe growth rate. The disappearance of CL-20 and the growth ofstrain JS71 correlated directly, which suggested that initialmetabolites of CL-20 were used as the nitrogen source.

To determine how many moles of nitrogen are assimilated from1 mol of CL-20 by the Agrobacterium isolate, the strain wasgrown in various concentrations of CL-20 (25, 50, 100, and 250µM) or NaNO₂ (100, 200, 400, and 600 µM) as thesole nitrogen source. The growth yield of strain JS71 in liquidmedium was 213 ± 13 g of protein per mol of CL-20 (Fig.5). The growth yield in medium with NaNO₂ as the sole nitrogensource was 65 ± 3 g of protein per mol of NaNO₂ (Fig.5). Comparison of the growth yields suggests that the Agrobacteriumstrain JS71 uses 3 of the 12 nitrogen atoms from the CL-20 molecule.

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FIG. 5. Growth yield of Agrobacterium strain JS71 with 20 mM succinate and different concentrations of CL-20 ( ${blacksquare}$ ) or NaNO₂ ( ${blacktriangleup}$ ). The CL-20 cultures were supplemented with 0.1% (vol/vol) Tween 80. All growth studies were performed in duplicate.

Preliminary HPLC-mass spectrometry negative electrospray ionizationmass analysis results indicated the formation of a metabolitewith a molecular weight of 88 in cultures degrading CL-20 (datanot shown). Further investigations are necessary to identifythe structure of the intermediate.

The results indicate clearly that CL-20 is biodegraded readilyin soil. Therefore, CL-20 might be less persistent in the environmentthan RDX and HMX, which accumulate at contaminated sites. Thelower water solubility of CL-20 will be a major determinantof its availability for biodegradation. Further investigationis required to elucidate the pathway and final products of CL-20degradation.

Nucleotide sequence accession number.

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Nucleotide sequence accession...

The 16S rRNA gene sequence of isolate JS71 was deposited atGenBank under accession number AY174112.

ACKNOWLEDGMENTS

This work was funded by the U.S. Air Force Office of ResearchScience and the U.S. Strategic Environmental Research and DevelopmentProgram and supported in part by the Oak Ridge Institute forScience and Education (U.S. Department of Energy).

We thank Joe Hughes, Bill Wallace, and Dan Lessner for providingsoil samples.

FOOTNOTES

* Corresponding author. Mailing address: Air Force Research Laboratory—MLQL, Building 1117, 139 Barnes Dr., Tyndall AFB, FL 32403. Phone: (850) 283-6058. Fax: (850) 283-6090. E-mail: Jim.Spain{at}Tyndall.af.mil.

REFERENCES

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