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Applied and Environmental Microbiology, March 2003, p . 1871-1874, Vol . 69, No . 3

Biodegradation of the Nitramine Explosive CL-20

Sandra Trott,1 Shirley F . Nishino,1 Jalal Hawari,2 and Jim C . Spain1*

Air Force Research Laboratory, Tyndall Air Force Base, Florida 32403,1 Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec H4P 2R2, Canada2

Received 16 September 2002/ Accepted 17 December 2002


   ABSTRACT

 
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 disappeared in all microcosms except the controls in which microbial activity had been inhibited . CL-20 was degraded most rapidly in garden soil . After 2 days of incubation, about 80% of the initial CL-20 had disappeared . A CL-20-degrading bacterial strain, Agrobacterium sp . strain JS71, was isolated from enrichment cultures containing garden soil as an inoculum, succinate as a carbon source, and CL-20 as a nitrogen source . Growth experiments revealed that strain JS71 used 3 mol of nitrogen per mol of CL-20 .


   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 explosives RDX (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 propellants can be expected to lead to environmental contamination . RDX and HMX, common contaminants in soil and groundwater at munition manufacturing sites and firing ranges, are toxic and possibly carcinogenic (7, 8, 11, 12, 16, 23, 27) . Because of the structural similarity of CL-20 to RDX and HMX, it is likely that CL-20 has similar effects . So far, there is little information available about the properties of CL-20 . In contrast to RDX and HMX, CL-20 is a caged molecule (Fig . 1) . The water solubility of CL-20 is 4.8 mg/liter at 25°C (Stevens Institute of Technology [http://www.cee.stevens-tech.edu/ResProj.html]), which is lower than the solubility of the nitramines RDX and HMX (38.4 and 6.6 mg/liter at 20°C, respectively) (27) .


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

 
The microbial degradation of RDX and HMX under aerobic and anaerobic conditions has been extensively investigated (1, 2, 4-6, 9, 13-15, 17, 21) . Under anaerobic conditions, three degradation pathways have been proposed . The first includes the production of nitroso derivatives of RDX and HMX, which are subject to further degradation (15, 21) . Hawari et al . suggested a second degradation pathway which includes the direct ring cleavage of RDX and HMX without the initial reduction of the nitro groups (14, 15) . The third proposed degradation pathway is based on N 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 DN22 leads to the formation of nitrite, nitrous oxide, ammonia, formaldehyde, and a dead-end product with a molecular weight of 119 which was recently identified as 4-nitro-2,4-diazabutanal (1a, 5, 9) . Fournier et al . proposed a degradation pathway which includes an initial denitration of RDX followed by ring cleavage to formaldehyde and the dead-end product (9) .

To the best of our knowledge, no biodegradation of CL-20 has been reported to date . It is, therefore, important to determine whether biodegradation might affect the fate and transport of CL-20 in terrestrial and aquatic ecosystems . To that end we evaluated the biodegradation of CL-20 in laboratory microcosms and isolated a bacterial strain that is able to grow on CL-20 as the sole nitrogen source .


   CL-20 biodegradation in soil.

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

Microcosm experiments were conducted in 5-ml amber vials containing soil (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 . The amounts of added water corresponded to the water absorptive capacity of the soils . For inhibiting microbial activity in soils, the added water contained glutaraldehyde (1%) and mercuric chloride (90 mg/liter) . The microcosms were vigorously mixed by vortexing them and incubated in the dark at 30°C . For time course studies, one microcosm was sacrificed at each time point . The CL-20 concentrations in the microcosms in acetonitrile extracts 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 rate of 1.0 ml/min over a Spherisorb C8 column (250 by 4.6 mm; particle size, 5 µm; Alltech, Deerfield, Ill.) or a Synergi Polar-RP column (150 by 4.6 mm; particle size, 4 µm; Phenomenex, Torrance, Calif.) . Absorbance was measured at a wavelength of 230 nm .

CL-20 was degraded in all three soils (Fig . 2) . The persistence of CL-20 in sterile controls indicated that the degradation process in active soil was biological . CL-20 degradation was fastest in the microcosms containing garden soil . CL-20 did not disappear completely in any of the soils . The incomplete degradation of CL-20 might be due to limited bioavailability . Residual amounts of CL-20 may be sorbed to the soil and therefore be unavailable for the microorganisms . In general, the availability of contaminants for microorganisms is dependent on the properties of the soil and the chemical compound, as well as on mass transport (3, 29) .


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 of 247 could be detected by HPLC-mass spectrometry in the negative electrospray ionization mode . Further investigations are under way to identify the putative CL-20 degradation product .

It is not clear whether CL-20 degradation in the soil microcosms occurred aerobically or anaerobically . No precautions were taken to maintain aerobic conditions . Degradation started relatively quickly, so it can be assumed that CL-20 was degraded under aerobic conditions . On the other hand, it is possible that small anaerobic zones existed in the soil, where microbes could have degraded CL-20 anaerobically . In preliminary experiments CL-20 was also degraded under anaerobic conditions in sewage sludge (data not shown) .


   Enrichment and isolation of the Agrobacterium strain JS71.

 
Enrichment cultures were carried out in 10 ml of M medium (pH 7.0) without sodium chloride (5) . The medium was supplemented with CL-20 (100 µM) in methanol (5 mM stock solution) as the sole nitrogen source and succinate (20 mM) as the sole carbon source and inoculated with 1 g of Florida garden soil in 50-ml shake flasks . The cultures were incubated on a shaker at 30°C and 250 rpm . Microbial activity in the controls was inhibited by the addition of glutaraldehyde (1%) and mercuric chloride (90 mg/liter) . The concentrations of CL-20 in the enrichments were determined by HPLC . Samples were mixed with 1.5 volumes of acetonitrile, and suspended material was removed by centrifugation prior to HPLC analysis .

CL-20 degradation in the first enrichment culture started after about 3 days (Fig . 3) . The disappearance of CL-20 verified that CL-20 was degraded under aerobic conditions . CL-20 degradation in the enrichments was slower than its degradation in the soil microcosms, probably because of the smaller inoculum and the very low water solubility of CL-20 . In contrast to the partial degradation of CL-20 in the soil microcosms, CL-20 was completely degraded in the enrichment cultures . The results support the hypothesis that some of the CL-20 in the microcosms (Fig . 2) was unavailable because of sorption to the soil .


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 dilutions were plated on the above-named enrichment medium solidified with 1.5% (wt/vol) agarose (molecular biology grade) . Single colonies were tested for the ability to grow on CL-20 as the sole nitrogen source . The selective enrichment yielded a bacterial strain that was able to grow with CL-20 as the sole nitrogen source . The 16S rRNA gene sequence from the isolate was determined and evaluated by Midi Labs (Newark, Del.) . The 16S rRNA gene sequence indicated that the strain was most similar to Agrobacterium rubi (0.64% difference) and Agrobacterium tumefaciens (0.84% difference) . The isolate was gram negative, motile, and rod shaped . These properties correspond to the characteristics of the genus Agrobacterium (18) . The isolate was, therefore, named Agrobacterium sp . strain JS71 .

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


   Growth studies with Agrobacterium strain JS71.

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

Strain JS71 grew in minimal medium containing succinate and CL-20 at a growth rate of 0.14 per day . The isolate did not grow with CL-20 as the sole nitrogen and carbon source . The slow growth of strain JS71 was probably due to the very low water solubility of CL-20 . When the surfactant Tween 80 (0.1% [vol/vol]) was added to the cultures to increase the solubility and therefore the availability of CL-20, the growth rate was 0.59 per day (Fig . 4) . Strain JS71 could not grow in media without a nitrogen source (Fig . 4) or in media containing RDX or HMX as the sole nitrogen source (data not shown) .


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 facilitated the determination of CL-20 concentrations in addition to increasing the growth rate . The disappearance of CL-20 and the growth of strain JS71 correlated directly, which suggested that initial metabolites of CL-20 were used as the nitrogen source .

To determine how many moles of nitrogen are assimilated from 1 mol of CL-20 by the Agrobacterium isolate, the strain was grown in various concentrations of CL-20 (25, 50, 100, and 250 µM) or NaNO2 (100, 200, 400, and 600 µM) as the sole nitrogen source . The growth yield of strain JS71 in liquid medium was 213 ± 13 g of protein per mol of CL-20 (Fig . 5) . The growth yield in medium with NaNO2 as the sole nitrogen source was 65 ± 3 g of protein per mol of NaNO2 (Fig . 5) . Comparison of the growth yields suggests that the Agrobacterium strain JS71 uses 3 of the 12 nitrogen atoms from the CL-20 molecule .


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 ionization mass analysis results indicated the formation of a metabolite with a molecular weight of 88 in cultures degrading CL-20 (data not shown) . Further investigations are necessary to identify the structure of the intermediate .

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


   Nucleotide sequence accession number.

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

 


   ACKNOWLEDGMENTS

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

We thank Joe Hughes, Bill Wallace, and Dan Lessner for providing soil 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 .


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