1 Anaerobic bioremediation of RDX by ovine whole rumen fluid and pure culture isolates 2 3 H.L. Eaton1, J.M. Duringer2, L.D. Murty3 and A.M. Craig4* 4 1 5 6 2 Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331 7 3 8 9 4 Department of Microbiology, Oregon State University, Corvallis, OR, 97331 Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR, 97331 Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR 97331 10 11 12 *Corresponding Author: A.M. Craig. Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, 101 Magruder Hall, Corvallis, OR 97331, USA 13 Phone: (541) 737-3036 14 Fax: (541) 737-2730 15 E-mail: A.Morrie.Craig@oregonstate.edu 16 17 18 19 20 21 22 23 24 25 ABSTRACT The ability of ruminal microbes to degrade the explosive compound hexahydro-1,3,5- 26 trinitro-1,3,5-triazine (RDX) in ovine whole rumen fluid (WRF) and as 24 bacterial isolates was 27 examined under anaerobic conditions. Compound degradation was monitored by high 28 performance liquid chromatography (HPLC) analysis, followed by liquid chromatography- 29 tandem mass spectrometry (LC-MS/MS) identification of metabolites. Organisms in WRF 30 microcosms degraded 180 µM RDX within 4 h. Nitroso-intermediates MNX (hexahydro-1- 31 nitroso-3,5-dinitro-1,3,5-triazine), DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine) and 32 TNX (hexahydro-1,3,5-trinitroso-1,3,5-triazine) were present as early as 0.25 hours and were 33 detected throughout the 24 h incubation period, representing one reductive pathway of ring 34 cleavage. Following reduction to MNX, peaks consistent with m/z 193 and 174 were also 35 produced which were unstable and resulted in rapid ring cleavage to a common metabolite 36 consistent with an m/z of 149. These represent two additional reductive pathways for RDX 37 degradation in ovine WRF which have not been previously reported. The 24 ruminal isolates 38 degraded RDX with varying efficiencies (0-96%) over 120 hours. Of the most efficient degraders 39 identified, Clostridium polysaccharolyticum and Desulfovibrio desulfuricans subsp. 40 desulfuricans degraded RDX when medium was supplemented with both nitrogen and carbon, 41 while Anaerovibrio lipolyticus, Prevotella ruminicola and Streptococcus bovis IFO utilized RDX 42 as a sole source of nitrogen. This study showed that organisms in whole rumen fluid, as well as 43 several ruminal isolates, have the ability to degrade RDX in vitro and, for the first time, 44 delineated the metabolic pathway for its biodegradation. 45 46 Key words: Bioremediation, RDX, rumen, ovine, anaerobic 47 48 49 INTRODUCTION Since World War II, the use of energetic compounds by military groups has resulted in 50 the deposition of explosives residues at sites across the globe (Agency for Toxic Substances and 51 Disease Registry 1995; Clausen et al. 2004). Munitions including TNT (2,4,6-trinitrotoluene), 52 RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine), HMX (octahydro-1,3,5,7-tetranitro-1,3,5,7- 53 tetrazocine) and CL-20 currently contaminate the soil around manufacturing and storage 54 facilities and bombing/training ranges at approximately 16,000 military bases in the United 55 States alone (Environmental Protection Agency 1993; U.S. Environmental Protection Agency 56 2009). The U.S. Environmental Protection Agency classifies RDX as a group C possible human 57 carcinogen and a priority pollutant (U.S. Environmental Protection Agency 2009). In humans, 58 the target organ of toxicity for RDX exposure is the central nervous system (Agency for Toxic 59 Substances and Disease Registry 1995; Brooks et al. 1997; Banerjee et al. 1999; U.S. 60 Environmental Protection Agency 2009), where convulsions, loss of consciousness, vomiting, 61 skin lesions, and even death have been observed (Hesselmann and Stenstrom 1994). 62 Neurological impacts of RDX have also been observed in a variety of other species including 63 birds (Gust et al. 2009), mice, rats, and rabbits (Agency for Toxic Substances and Disease 64 Registry 1995), in addition to lethality in fish and reproductive effects in invertebrates (Turley 65 and Burton 1995). 66 67 Cyclic nitramines such as RDX are resistant to degradation in soil and are highly mobile in the water column; thus, they threaten the health of humans and the surrounding ecosystem 68 when they leach into groundwater or offsite locations, such as farmland (Daniels and Knezovich 69 1994; Sunahara et al. 2009). The RDX molecule is a six-membered ring of alternating carbon 70 and nitrogen atoms, with a nitro group attached to each nitrogen atom (Figure 1). Although RDX 71 is not planar, all three nitro groups are directed towards the same side of the molecule (Qasim et 72 al. 2007). This particular geometric configuration makes it possible for positive ions to 73 electrostatically attract the oxygen atoms of the nitro assemblages, making them good leaving 74 groups for nucleophilic reactions (Qasim et al. 2007; Sunahara et al. 2009), such as those 75 catalyzed through enzymatic attacks by microorganisms (Prescott et al. 2005). 76 Recent research has demonstrated that cyclic nitramines can be degraded without oxygen; 77 anaerobic electron acceptors, such as NO3-, SO42- and Fe(III) are used by bacteria to degrade 78 these contaminants (Coleman et al. 1998; Hawari et al. 2000a; Hawari et al. 2000b; Crocker et al. 79 2006; Sunahara et al. 2009). Furthermore, anaerobic biodegradation appears to be the 80 preferential pathway of converting cyclic nitramines since the nitro groups must be reduced prior 81 to ring cleavage and mineralization to CO2 under either anaerobic or aerobic conditions (Kwon 82 and Finneran 2006). Aerobic biodegradation by indigenous microbes is simply not quick enough 83 to prevent the movement of unadulterated cyclic nitramines through the aerobic portions of soil 84 into the ground water and anaerobic sediment, where accumulation poses a threat to humans. 85 Of the anaerobic systems that have been studied, the most rapid remediation of RDX took 86 place in ovine whole rumen fluid (WRF), with biotransformation of 30 µg mL-1 RDX occurring 87 in 4 hours (Eaton et al. 2011). The rumen is a powerfully reductive, anaerobic and natural 88 bioreactor (Hobson and Stewart 1997), which we hypothesize could be used in the 89 bioremediation of energetic compounds. This remediation technique, termed phyto-ruminal 90 bioremediation, would be cost-effective, in addition to providing a “green” alternative to the 91 current methods of soil removal and ex situ incineration, which release unburned explosive 92 chemicals into the air, soil and water (Axtell et al. 2000; Qasim et al. 2007). This process would 93 first utilize cool-season grasses to accumulate explosives residues from soil into the shoots 94 (Duringer et al. 2010); the ruminant would then consume the munitions-laden plant material, 95 which would be utilized by ruminal microbes for energy and thereby degraded to innocuous by- 96 products (De Lorme and Craig 2008; Smith et al. 2008; Eaton et al. 2011). Over time, with 97 successive pasture growth and grazing, the soil would be remediated. 98 Our previous study examining ovine WRF as a means to bioremediate RDX was 99 successful in determining that microbial communities in WRF, and the resulting culturable 100 consortia, could biotransform RDX, but the metabolite pathway of neither the WRF nor the 101 cultured consortia were elucidated (Eaton et al. 2011). In this study, we determined the metabolic 102 fate of RDX, in both WRF and in 24 commonly isolated individual bacteria from the rumen. We 103 hypothesized from the results of our previous study that RDX would be degraded in rumen fluid 104 faster and more completely than by the isolates, but that by examining isolates, we would gain 105 insight into which organisms may be crucial in identifying genes responsible for RDX 106 breakdown. 107 These objectives were accomplished by high performance liquid chromatography 108 (HPLC) analysis of spent culture supernatants to identify possible degraders, followed by 109 quantification and identification of metabolites by liquid chromatography-tandem mass 110 spectrometry (LC-MS/MS). Organisms in WRF microcosms degraded RDX to nitroso- 111 intermediates and then to proposed ring cleavage metabolites. From these results, three pathways 112 for RDX degradation by ruminal microbes were put forth. This study showed that organisms in 113 WRF and several ruminal isolates have the ability to degrade RDX in vitro and, for the first time, 114 delineated the metabolic pathway for its biodegradation. This research supports application of 115 our proposed phyto-ruminal bioremediation process to in vivo field trials using live grasses and 116 sheep. 117 118 MATERIALS AND METHODS 119 120 Chemicals and reagents 121 122 Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) (99% purity) was purchased from ChemService 123 (West Chester, PA). Hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) (99% purity), 124 hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX) (55% purity +17% MNX + 23% TNX), 125 hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) (>99% purity), 4-nitro-2,4-diazabutanal (4- 126 NDAB) (98% purity) and methylenedinitramine (MEDINA) (98% purity) were provided by R.J. 127 Spanggord from SRI International (Menlo Park, CA). Solvents were of HPLC and LC-MS/MS 128 grade. Reagents were of analytical grade and were purchased from Sigma–Aldrich (St. Louis, 129 MO). An ELGA (Cary, NC) Ultra PureLab reverse osmosis water purification system was used 130 to generate Milli-Q quality water (resistance >18.2 MΩ/cm) for all aqueous solutions. 131 132 133 Organisms, media, and growth conditions 134 Pure cultures were maintained by our lab or obtained from the German Collection of 135 Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany) (Table 1). Some strains 136 required species-specific media instead of a general complex medium for optimal growth. These 137 included Desulfovibrio medium (DSMZ medium 63), Clostridium polysaccharolyticum (DSMZ 138 medium 140), Lactobacillus ruminus (DSMZ medium 232), and Wolinella succinogenes (DSMZ 139 medium 157). The remaining cultures were grown in a complex medium with 40% clarified 140 rumen fluid (per liter: 400 mL clarified rumen fluid; 2.0 g trypticase; 1.0 g yeast extract; 4.0 g 141 cellobiose; 4.0 g sodium carbonate; 1.0 mL 0.1% resazurin; 10.0 mL VFA solution 142 [concentration, µmol· mL-1: 67.2 glacial acetic acid, 40.0 propionic acid, 20.0 butyric acid, 5.0 143 isobutyric acid, 5.0 2-methylbutyric acid, 5.0 valeric acid, 5.0 isovaleric acid]; 0.3 g potassium 144 phosphate, dibasic; 0.6 g sodium chloride; 0.3 g ammonium sulfate; 0.3 g potassium phosphate, 145 monobasic; 0.08 g calcium chloride, dihydrate; 0.12 g magnesium sulfate, heptahydrate; and 1.1 146 g sodium citrate, dihydrate). All media was prepared anaerobically by combining media 147 components in a round bottom flask, adjusting to the appropriate pH, then boiling for 5 min and 148 purging of oxygen by sparging with a N2:CO2 (75:25) gas mixture for 30 min. The media were 149 then corked and immediately placed into an anaerobic glove box H2:CO2 (10:90). All media 150 were dispensed as 9.7 mL aliquots into Balch tubes, which were sealed with butyl rubber 151 stoppers and aluminum crimp caps. The sealed media was autoclaved for 35 min at 120° C and 152 stored until use. Anaerobically prepared and sterilized reducing agent (1.25% cysteine sulfide) 153 and B-vitamins solution (per 100 mL: 20 mg thiamin HCl, 20 mg D-pantothenic acid, 20 mg 154 nicotinamide, 20 mg riboflavin, 20 mg pyridoxine HCl, 1.0 mg p-aminobenzoic acid, 0.25 mg 155 biotin, 0.25 mg folic acid, and 0.1 mg cyanocobalamin) were added to media prior to inoculation. 156 Cultures were grown at 39°C with shaking (150 rpm) for 18–24 h between transfers. Cultures 157 were transferred at least three times before degradation experiments. 158 159 Whole rumen fluid microcosms with RDX and TNX 160 161 Ovine WRF was collected from three cannulated male sheep maintained at the Oregon State 162 University (OSU) Sheep Center (Corvallis, OR) in accordance with International Animal Care 163 and Use Committee (IACUC) regulations. WRF was pooled in a sterile, pre-warmed thermos, 164 which was filled to the top to maintain an anaerobic environment, followed by immediate 165 transport to the lab. The thermos was then placed in an anaerobic glove box (Coy, Grass Lake, 166 MI) with an atmosphere of CO2:H2 (9:1). The thermos was gently inverted and a 25 mL portion 167 was poured into a sterile reagent basin and used for all inoculation experiments. The sheep were 168 fed a high forage diet of alfalfa hay twice daily. 169 WRF (7 mL) was inoculated into sterile, anaerobically prepared screw-capped tubes. 170 RDX was added to each tube for a final concentration of 180 µM, which is the lower limit of 171 solubility (solubility is 180-270 µM, depending on the matrix (Hesselmann and Stenstrom 172 1994)). Tubes were incubated anaerobically in the dark at 39°C on a rotary shaker (150 rpm) for 173 24 hours. Autoclaved tubes of WRF were used as controls. This experiment was repeated with 174 one of the RDX metabolites, TNX, at a concentration of 82 µM. All experiments were performed 175 in triplicate. Samples were collected every hour for the first five hours, and then at 24 hours. 176 Samples were frozen at -20˚C until prepared for LC-MS/MS analysis, as described below. 177 178 Rumen isolate incubations with RDX 179 180 Each isolate (Table 1) was first incubated with 180 µM RDX in low nitrogen basal (LNB) and 181 low carbon basal (LCB) media (Eaton et al. 2011). This concentration of RDX was toxic to 22 182 organisms. We then determined the limit of toxicity for each organism by progressively 183 decreasing or increasing the amount of RDX until growth and elimination of RDX, as 184 determined by HPLC, was observed. The resulting starting RDX concentration defined for each 185 organism is listed in Table 2. Cultures were then incubated anaerobically, in the dark, at 39°C on 186 a rotary shaker (150 rpm) for 120 hours. A media control consisted of 256 µM RDX, as this was 187 the highest concentration used in both LNB and LCB, without the addition of test organism. A 188 solvent control consisted of both types of media with 1.0 mL of overnight culture of the test 189 organism and the addition of 0.1 mL acetonitrile. All controls and tests were repeated in 190 triplicate. 191 192 Sample preparation for chromatography 193 194 WRF samples were collected and then frozen at -20˚C until preparation for HPLC and LC- 195 MS/MS analyses through solid phase extraction using Waters Oasis HLB (3 mL/60 mg, 30 µm) 196 cartridges (Milford, MA) per the manufacturer’s instructions. In brief, WRF samples were 197 treated 1:1 (v/v) with a basified water diluent consisting of 40 µL of concentrated ammonium 198 hydroxide in 1 mL of water. Samples were vortexed and then centrifuged at 8,000 x g for 10 199 minutes. Extraction was completed by evaporation of the final eluent in a Savant ISS 100 200 Integrated Speed Vac System (GMI, Inc., Ramsey, MN) at 43˚C, followed by reconstitution in 201 methanol:water 55:45 (v/v). Isolate samples were not prepared via the solid phase extraction 202 procedure outlined above, but were centrifuged at 16,000 x g at 4°C for 3 min. Supernatant was 203 removed, filtered through a 0.2 µm PTFE membrane filter (VWR International, Brisbane, CA), 204 and diluted 1:1 (v/v) with acetonitrile for immediate analysis by HPLC or LC-MS/MS. 205 206 HPLC and LC-MS/MS analyses 207 208 HPLC analyses were carried out using Environmental Protection Agency method 8330A (U. S. 209 Environmental Protection Agency 2007). In brief, separations occurred on a Phenomenex 210 Ultracarb ODS (20) column (250 mm x 4.6 mm i.d., 5 μm particle size) (Torrance, CA), eluting 211 under isocratic conditions with water and methanol (55:45 v/v) at 28.3°C and a flow rate of 0.8 212 mL min-1, with a total run time of 30 min. The HPLC system consisted of a Perkin–Elmer 213 (Waltham, MA) Series 200 pump equipped with a Perkin–Elmer ISS 200 autosampler and 214 Perkin–Elmer Series 200 UV/VIS detector, monitoring at 254 nm. TotalChrom software 215 (Perkin–Elmer) was used to quantify HPLC data. 216 LC-MS/MS analyses were performed on an ABI/SCIEX 3200 QTRAP LC-MS/MS 217 system (Applied Biosystems, Foster City CA) using atmospheric pressure chemical ionization in 218 the negative ion mode, modified from Borton and Olson (2006). A Phenomenex Ultracarb ODS 219 (20) column (250 mm x 4.6 mm i.d., 5 μm particle size) was used to separate RDX and its 220 metabolites at a flow rate of 0.65 mL min-1 over 35 minutes using a mobile phase consisting of 221 0.6 mM ammonium acetate in water (A) and methanol (B) in a gradient program as follows: 10% 222 B from 0-5 min, increasing to 20% B at 8 min, 25% B at 10 min, 30% B at 12 min, 35% B at 14 223 min, 40% B at 20 min, 45% B from 25-30 min, and 58% B at 35 min. Data was acquired using 224 multiple reaction monitoring (MRM), using 281→46 and 281→59 (RDX + CH3COO-), 265→46 225 and 265→59 (MNX + CH3COO-), 249→46 and 249→59 (DNX + CH3COO-), 233→59 (TNX + 226 CH3COO-), 135→61 (MEDINA) and 118→61 (4-NDAB) as transitions. Curtain gas was set at 227 10 psi, gas 1 at 35 psi, gas 2 at 0 psi, temperature at 275°C, nebulizer current at -8 µA, and dwell 228 time to 75 msec. Declustering potential, entrance potential, collision entrance potential, collision 229 energy and collision exit potential were as follows for each compound: RDX (-20, -2, -22, -14, - 230 4) for the 281→46 transition and (-5, -2, -22 -48, -2) for the 281→59 transition; MNX (-5, -2, - 231 14, -16, 0) for the 265→46 transition and (-5, -2, -14, -38, 0) for the 265→59 transition; DNX (- 232 5, -2, -14, -16, 0) for the 249→46 transition and (-5, -2, -14, -38, 0) for the 249→59 transition; 233 TNX (-5, -3, -12, -22, 0); MEDINA (-10, -2.5, -10, -20, 0); and 4-NDAB (-5, -3.5, -6, -10, 0). 234 Quantitation was performed by establishing a calibration curve in Analyst 1.4.2 using a linear 235 regression from WRF or media samples spiked with RDX at concentrations of 5-50 ng· mL-1. R2 236 values for the transition 281/46 were 0.997, 0.993, and 0.973 for WRF, LCB and LNB medias, 237 respectively. 238 Data used to identify possible new metabolites was acquired using enhanced mass spectra 239 (EMS) and enhanced product ion (EPI) scans via information dependent acquisition (IDA) 240 experiments developed in Analyst 1.4.2 software (Applied Biosystems). RDX and metabolites 241 were separated using the same conditions as in the MRM method, except a different gradient was 242 used: 0-5 minutes at 20% B, 5-30 minutes at 50% B, 30-50 minutes at 100% B, and 50-70 243 minutes at 100% B. Curtain gas was set to 10 psi, nebulizer gas to 35 psi, nebulizing current to -8 244 µA, temperature to 275ºC, declustering potential to -20 V, entrance potential to -10 V, collision 245 energy to -10 V, scan rate to 1000 amu·s-1, dynamic fill time and a mass range of 50 to 400 amu. 246 Final EMS data was analyzed using LightSight 2.0 (Applied Biosystems) and ChemDraw Ultra 247 12.0 (CambridgeSoft, Cambridge, MA) software to capture and interpret data and possible 248 metabolites. 249 250 Results 251 252 Degradation of RDX and TNX in ovine whole rumen fluid 253 254 Separation and fragmentation of RDX (m/z 281), nitroso intermediates MNX (m/z 265), 255 DNX (m/z 248) and TNX (m/z 233), and ring cleavage products 4-NDAB (m/z 118) and 256 MEDINA (m/z 135) by MRM analysis are illustrated in Fig. 1. This modified LC-MS/MS 257 method (Borton and Olson 2006) was developed and optimized in order to detect RDX and its 258 five commonly known metabolites in one injection. LC-MS/MS analysis of ovine WRF samples 259 using this method showed anaerobic degradation from 180 µM RDX to 45 µM RDX within 4 h, 260 with almost complete elimination of RDX (4.5 µM remaining) by 24 h (Fig. 2). Within 1 h, the 261 reduction products MNX (28.5 µM) and DNX (0.37 µM) were visible. MNX increased in 262 concentration until 3 h (35.8 µM), after which it slowly declined to 8.4 µM at the 24 h sampling 263 point. DNX steadily increased to 4.2 µM at 24 h. The other nitroso-intermediate, TNX, appeared 264 at 5 h (9 µM) and was present at about the same concentration through 24 h. The ring cleavage 265 products MEDINA and 4-NDAB were not detected. 266 Not all of the initial RDX concentration could be accounted for by MRM analysis; hence 267 we performed an enhanced mass spectrometry (EMS) scan to identify other possible metabolic 268 products. EMS scans of RDX metabolism in WRF at 0.25 and 24 h are illustrated in Fig. 3A-B. 269 Analysis showed a decrease in a peak consistent with an m/z of 193 (LC-MS Fig. 3C) and an 270 increase in a peak consistent with an m/z of 149 (LC-MS Fig. 3D) over 24 hours which appear to 271 be metabolites, since they were not visible in the autoclaved controls and contain a fragment of 272 59 amu, which is common to RDX and its five known metabolites (Fig. 1). The peak of m/z 193 273 is consistent with one of the bracketed molecules in the pathway described in Zhang (2003), yet 274 we propose this second route of RDX degradation by ruminal microbes ends in a ring cleaved 275 metabolite of m/z 149, as shown in Figure 4 (pathway 1). A peak consistent with an m/z of 175 276 was seen in the same spectra as the m/z 193 peak, which showed an increase over 24 hours. This 277 may represent a daughter ion of the m/z 193 peak, forming from a loss of H2O, or, alternatively, a 278 third possible pathway which moves through the nitroso-intermediate MNX, ending in a ring 279 cleaved metabolite of m/z 149 (Fig. 4, pathway 3). Figure 4 provides an overview for the 280 pathways we propose for anaerobic bioremediation of RDX by WRF and how a metabolite of 281 m/z 149 could result from ring cleavage of MNX via three different degradation routes. Lastly, 282 peaks after 40 minutes were noted in the EMS scan (Fig. 3); however further method 283 development is needed to separate metabolites eluting at this end of the chromatogram and 284 should be the subject of future studies. 285 In the RDX incubations, TNX increased to 9 µM at 5 h, then plateaued until 24 hours 286 (Fig. 2). To investigate if TNX was a dead-end metabolite, we performed ovine WRF 287 incubations with 82 µM TNX for 24 h. When quantified via MRM analysis, autoclaved WRF 288 controls showed an average of 36.8 µM TNX at 0.25 h, while live WRF samples showed an 289 average of 15.5 µM (coefficient of variation = 21.1%). Degradation was seen in one replicate 290 from 22.4 µM to 11.5 µM over 24 h, but this was not consistent with the other two replicates. 291 LightSight analysis of the 0.25 h live WRF samples showed metabolites consistent with 292 dehydrogenation and glycine conjugation (m/z 228.2); tridemethylation and glutamine 293 conjugation (m/z 259); taurine conjugation (m/z 280); loss of NO, oxidation and glutamine 294 conjugation (m/z 287); and loss of the cyclohexyl ring, trioxidation and possible conjugation with 295 glucose (m/z 300), which may explain the large discrepancy between the nominal and quantified 296 concentrations of TNX observed. Thus, overall, the TNX concentration did not decrease 297 significantly from 0.25 to 24 h in WRF, but the complication of conjugated metabolite formation 298 prevents any definitive conclusions from being made. 299 300 Twenty-four bacterial isolates from the rumen were tested for their ability to degrade RDX over 301 120 h (Table 1). Of these, two were identified as both being able to thrive in an environment with 302 a high concentration of RDX and being the most proficient at degrading it (Table 2). 303 Desulfovibrio desulfuricans subsp. desulfuricans was able to degrade 88% of the 256 µM RDX 304 supplied in low carbon basal medium; C. polysachharolyticum was able to degrade 65% of the 305 186 µM RDX in the low carbon basal medium (Fig. 5). Of the other three bacterial species that 306 demonstrated significant degradation of RDX, A. lipolyticus was able to degrade 70% of the 85 307 µM RDX supplied in low nitrogen basal medium (Fig. 5). Also capable of degradation of RDX 308 in low nitrogen basal medium were S. bovis IFO and Prevotella ruminicola, with high RDX- 309 degrading abilities of 86% and 96% respectively, but with a lower initial concentration of RDX 310 (34 µM, Fig. 5). Nine more organisms exhibited low RDX-degrading ability (30-49%) in one or 311 both types of media (Table 2). Ten of the organisms appeared to be unable to grow when 312 incubated with RDX at a concentration of 34 µM in both types of media; no visible growth was 313 observed after 24 h and RDX degradation was similar to that in reduced media with RDX, which 314 was ≤ 25% (Table 2). In general, controls (reduced media without bacteria) resulted in a minor 315 decrease in RDX concentration after 24 h (Table 2). Solvent controls did not appear to inhibit 316 growth of any organism (data not shown). Trace amounts of MNX were detected at 25 minutes, 317 but below the limit of quantitation for the five isolates discussed above. For the isolates A. 318 lipolyticus, P. ruminicola, and S. bovis IFO in LNB media, MRM transitions were detected at 4.4 319 minutes for RDX, MNX, DNX, and TNX, with a second TNX peak detected at 3.0 minutes. 320 These retention times do not correspond to the retention times established for these compounds 321 using purified standards (Fig. 1). This data suggests that in source fragmentation of RDX 322 conjugated metabolites is occurring and/or interactions with the LNB media are resulting in the 323 formation of alternate, conjugated metabolic products, as these 4.4 and 3.0 minute peaks were 324 not seen in WRF nor isolates studied in LCB media. In conclusion, the five bacterial isolates 325 with moderate to high RDX-degrading ability processed the RDX molecule to trace amounts of 326 the nitroso-intermediate MNX and then to unidentified metabolites over 120 hours. 327 328 Discussion 329 330 The rumen is the first of four chambers in the stomach of a ruminant, which includes 331 species such as deer, goats, cows, and sheep. Continuous fermentation and mixing occur in the 332 rumen through contractions every few minutes, which would benefit the remediation of 333 xenobiotic compounds by maximizing bioavailability to the fungi, protozoa, bacteria, and 334 archaea inhabiting this niche. As an example, previous research has shown ruminal 335 bioremediation of TNT to be quite effective (Fleischmann et al. 2004; De Lorme and Craig 2008; 336 Smith et al. 2008). This study examined the possibility of using the rumen as a bioreactor for the 337 remediation of the energetic compound RDX. We utilized whole rumen fluid, the matrix in 338 which the remediation would occur, to define the fate of RDX and ensure it would be degraded 339 in the rumen within 16-20 hours, which would prevent absorption into the bloodstream of the 340 animal and resultant toxicity. We also examined the capability of 24 commonly isolated ruminal 341 bacteria to utilize RDX in low carbon and low nitrogen basal media to gain an understanding as 342 to which bacteria may be responsible for RDX degradation in the rumen, with the idea that 343 successful isolates could be used in future studies to examine the genes involved in RDX 344 metabolism. 345 Our data showed that ruminal microorganisms, as a community under anaerobic 346 conditions, readily transformed the nitro groups of RDX into the corresponding nitroso groups of 347 the commonly observed metabolites MNX, DNX and TNX (Fig. 2). MEDINA and 4-NDAB 348 were not detected using MRM analysis, suggesting that these metabolites are not the end 349 products of degradation; it is still unclear if these are possible intermediate metabolites that are 350 degraded further, as seen in Hawari et al., (2000). Based on previously defined anaerobic 351 pathways, summarized in Fuller et al., (2009), we were not expecting to see 4-NDAB as a 352 metabolite since it has only been detected in aerobic degradation pathways to date. The 353 concentrations of all nitroso intermediates amounted to approximately 20 µM at 24 hours (Fig. 354 2), accounting for only 11% of the total initial concentration of RDX. The pathway seen in 355 Hawari et al., (2000) suggests an additional pathway to explain the m/z 149 peak shown in Figure 356 4. A peak representing m/z 174 was not detected in that study, but we are proposing that 357 degradation of MNX through m/z 174 to MEDINA and finally to m/z 149 is a major pathway of 358 RDX breakdown in WRF. We acknowledge that MEDINA was not identified in our study; 359 however, we suggest that the transformation from MNX to the m/z 149 peak is occurring at such 360 a rate that the MEDINA intermediate is not produced in amounts significant enough to be 361 detected. Additional experiments, similar to those elucidating the degradation of RDX in WRF 362 need to be conducted with MNX and MEDINA to investigate if these metabolites are further 363 degraded to m/z 149 by utilizing high resolution time of flight (TOF) mass spectrometry to 364 identify accurate masses and obtain an accurate chemical formula of m/z 149, the proposed end 365 product of RDX degradation by WRF. Nonetheless, the two additional pathways described in 366 Figure 4 would account for the dramatic increase of m/z 149 from 0.25 to 24 hours, with three 367 possible routes for the same m/z 149 value. Our data indicates that most of the RDX was 368 degraded via pathways 1 and 3 (Fig. 4), with only small amounts being transformed to the 369 nitroso-intermediates (pathway 2), but further chemical analyses with heavy or radioisotopes are 370 needed to confirm this hypothesis. 371 The rate of disappearance of 180 µM RDX in whole rumen fluid within 4 h (Fig. 2) is 372 considerably faster than microbial species currently used to degrade RDX via composting, which 373 takes several weeks (Williams et al. 1992), and treatment by municipal sludge, in which 374 degradation of 45 µM RDX took 48 hours (Hawari et al. 2000b). When compared to 375 bioremediation techniques by organisms in contaminated soil or sludge digesters (Bayman et al. 376 1995; Boopathy et al. 1998; Axtell et al. 2000; Hawari et al. 2000b; Crocker et al. 2005; Fuller et 377 al. 2005), the rumen not only increases access to the compound through continuous mixing, but 378 the environment is more reductive (Hobson and Stewart 1997), which could aid in faster 379 degradation of RDX. 380 We also examined the ability of whole rumen fluid to degrade TNX, the last nitroso- 381 intermediate in published anaerobic pathways of RDX metabolism (Zhao et al. 2003; Crocker et 382 al. 2006). The amount of TNX we quantified in our RDX incubations with WRF was very small, 383 and previous research has hypothesized that TNX, if further reduced, becomes a highly unstable 384 molecule that spontaneously reacts or breaks down to ring cleavage products (McCormick et al. 385 1976; Zhao et al. 2003; Fuller et al. 2009). Perhaps the reductive environment in the WRF 386 microcosms was powerful enough to reduce TNX to the point where it degraded abiotically. 387 While it is possible that TNX breaks down in WRF because of the metabolite seen with the loss 388 of the cyclohexyl ring, its ability to form conjugates with various amino acids and phase two 389 metabolites has not been fully investigated. Our initial loss of 45% of the starting concentration 390 of TNX, even in the 0.25 hour autoclaved control, may be explained by the WRF conjugating a 391 certain amount of the TNX (65 µM) immediately, which induced a saturation limit and/or was 392 toxic to the microbes in the 24 hour time period we examined. In the TNX experiment, we used a 393 significantly higher level of TNX than was produced in the 24 hour degradation of RDX (82 µM 394 compared to 9 µM), so toxicity seems plausible. 395 In the TNX experiment, the main conjugates formed were with glutamine. Glutamine is a 396 compound of central importance in nitrogen metabolism (Krishnan et al. 1986). Glutamine 397 synthetase is an essential enzyme present in all bacteria that is responsible for modulating the 398 overall flow of NH4+ to organic nitrogen by assimilating ammonia and synthesizing glutamine 399 (Kumada et al. 1993). The glutamine conjugate formed from WRF incubations with TNX could 400 represent metabolism of the TNX by bacteria in WRF, but further studies would be necessary to 401 determine if this enzyme was responsible for the degradation of TNX; glutamine may be present 402 simply as a by-product of the metabolism of other nitrogen sources in the rumen. Other 403 conjugates formed with TNX were with glycine and taurine. Glycine is naturally present in 404 rumen fluid and metabolized very efficiently by ruminal microbes, while taurine is an essential 405 amino acid provided to ruminants in their diet because they are not able to synthesize adequate 406 amounts (Hobson and Stewart 1997; Sejrsen et al. 2006). Conjugates of TNX with glycine and 407 taurine were not seen with the low levels of TNX produced from RDX degradation, but only in 408 the rumen fluid samples with high amounts of TNX (82 µM). Therefore, we speculate that these 409 conjugates are the result of saturation and not metabolism of TNX. Because the concentration of 410 TNX in the WRF incubations quantified by LC-MS/MS was less than half of that initially added, 411 our study concurs with published literature that TNX lacks stability and reacts abiotically with its 412 surroundings (Zhao et al. 2003; Crocker et al. 2006; Fuller et al. 2009). 413 Of the four ruminal isolates that demonstrated significant RDX degradation ability (Table 414 2), the two most adept were Clostridium sp. and Desulfovibrio sp. which are both sulfate- 415 reducers found in low concentrations in the rumen (Hobson and Stewart 1997) that have 416 demonstrated RDX degradation previously (Zhao et al. 2003; Arnett and Adrian 2009). Third, 417 Streptococcus bovis IFO has been identified before as a member in a ruminal consortia in an 418 enrichment for RDX-degraders (Eaton et al. 2011) and has been classified as one of the highest 419 proteolytically active ruminal bacteria in digesting substrates for amino acids (Hobson and 420 Stewart 1997). Fourth, Anaerovibrio lipolyticus, a lipolytic organism, has been previously shown 421 to degrade TNT (De Lorme and Craig 2008), but not RDX. Future studies will involve 422 examining the active genes used to degrade RDX in these bacteria. 423 Ruminal isolates were able to degrade RDX between a range of 34 to 256 µM in 120 424 hours, compared to near complete degradation of 180 µM RDX within 24 hours in whole rumen 425 fluid. This data indicates that, while several rumen bacterial strains may have the individual 426 ability to degrade RDX, bioremediation in whole rumen fluid is more efficient. This is 427 undoubtedly due to the volume of culturable and unculturable organisms within the rumen fluid 428 that had access to RDX and its metabolites, as well as an abundance of other substrates, which 429 organisms could utilize in co-metabolism. Since the parent compound RDX was nearly 430 eliminated within 24 hours in whole rumen fluid, we propose this compound would be safe for 431 sheep to ingest since the passage time in the rumen is 16-20 hours (Hobson and Stewart 1997), as 432 long as the concentration of nitroso-intermediates generated are not toxic; this will require 433 further investigation through feeding trials to determine if and where the nitroso-intermediates 434 are translocated (i.e. fat, muscle, brain, liver, feces, etc.). 435 The reduction and mineralization of cyclic nitramines by microorganisms under 436 anaerobic conditions (Boopathy et al. 1998; Hawari et al. 2000a; Adrian et al. 2003; Fournier et 437 al. 2005; Crocker et al. 2006; Arnett and Adrian 2009) generally involves reductive or 438 substitutive removal of the nitro group from the ring (Sunahara et al. 2009). Results from this 439 study have led us to propose a pathway for the degradation of RDX by ovine ruminal microbes 440 under anaerobic conditions (Fig. 4). First, RDX is reduced to MNX. Trace amounts of MNX are 441 further reduced to DNX and TNX. However, the majority of MNX is further degraded via two 442 other pathways: (1) pentahydro-1,3-dinitro-1,3,5-triazine, which is an unstable molecule that 443 results in rapid ring cleavage and degradation to m/z 149 (Fuller et al. 2009) and (2) via m/z 193 444 (Zhang and Hughes 2003) to an end product of m/z 149 . 445 Environmental contamination by explosive compounds such as TNT and RDX is an 446 international concern; many countries lack the money and technology to remediate dangerous 447 sites. The bioremediation technique discussed in this paper, “phytoruminal-bioremediation,” 448 would provide a low maintenance, affordable, and “green” way to rid unwanted contamination, 449 provided grass can be grown to pull compounds out of the soil for grazing sheep (Duringer et al. 450 2010). RDX degradation is affected by environmental conditions, which must be considered 451 when proposing an effective remediation strategy. These include adsorption, binding capacity 452 and complexation of media; the presence of other explosives; reactivities of proliferated 453 transformation products (Qasim et al. 2007; Sunahara et al. 2009); air and light exposure; and the 454 ability of RDX to move throughout the water column (Best et al. 1999). Many techniques 455 succeed in the laboratory, but fail in larger field trials because the expected outcome is thwarted 456 by factors impossible to control, including weather, climate and geology (Talley and Sleeper 457 1997). The benefit of the rumen is that it is a natural, transportable bioreactor, which is contained 458 and unaffected by weather; it is the ultimate combination of in situ and ex situ treatments. 459 However, variation in the bioremediation of RDX as affected by the factors outlined above still 460 need to be determined for phytoruminal-bioremediation to be a realistic mitigation approach. 461 Further, if grass will translocate multiple explosive residues out of the soil for grazing sheep, 462 research aimed at understanding if degradation of one compound such as RDX, is affected by the 463 presence of others, such as TNT, HMX and various forms of dinitrotoluene (DNT), needs to be 464 carried out. Lastly, the efficiency of the ruminal microbes to extract munitions from the ingested 465 explosive-laden plant material as a delivery vehicle (Stenuit and Agathos 2010) needs to be 466 investigated. 467 468 Acknowledgements 469 470 The authors would like to thank Michael Wiens for his technical assistance. This research was 471 supported in part by a gift from Ruminant Solutions, LLC (New Mexico), the Oregon 472 Agricultural Experiment Station project #ORE00871 and the United States Department of 473 Agriculture, Agriculture Research Service project #50-1265-6-076. Any opinions, findings, 474 conclusions, or recommendations expressed in this publication are those of the author(s) and do 475 not necessarily reflect the view of the United States Department of Agriculture. 476 477 References 478 479 480 481 Adrian, N.R., Arnett, C.M. and Hickey, R.F. (2003) Stimulating the anaerobic biodegradation of explosives by the addition of hydrogen or electron donors that produce hydrogen. Water Res 37, 3499-3507. 482 483 Agency for Toxic Substances and Disease Registry (1995) Toxicological profile for RDX. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service. 484 485 486 Arnett, C.M. and Adrian, N.R. (2009) Cosubstrate independent mineralization of hexahydro1,3,5-trinitro-1,3,5-triazine (RDX) by a Desulfovibrio species under anaerobic conditions. Biodegradation 20, 15-26. 487 488 Axtell, C., Johnston, C.G. and Bumpus, J.A. (2000) Bioremediation of soil contaminated with explosives at the Naval Weapons Station Yorktown. Soil & sediment contamination 9, 537-548. 489 490 Banerjee, H.M., Verma, M., Hou, L.-H., Ashraf, M. and Dutta, S.K. (1999) Cytotoxicity of TNT and its metabolites. Yale J Biol Med 72, 1-4. 491 492 Bayman, P., Ritchey, S.D. and Bennett, J.W. (1995) Fungal interactions with the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine). J Ind Microbiol 15, 418-423. 493 494 495 496 Best, E.P.H., Sprecher, S.L., Larson, S.L., Fredrickson, H.L. and Bader, D.F. (1999) Environmental behavior of explosives in groundwater from the Milan army ammunition plant in aquatic and wetland plant treatments. Removal, mass balances and fate in groundwater of TNT and RDX. Chemosphere 38, 3383-3396. 497 498 Boopathy, R., Manning, J. and Kulpa, C.F. (1998) Biotransformation of explosives by anaerobic consortia in liquid culture and in soil slurry. Int Biodeterior Biodegrad 41, 67-74. 499 500 501 Brooks, L.R., Jacobson, R.W., Warren, S.H., Kohan, M.J., Donnelly, K.C. and George, S.E. (1997) Mutagenicity of HPLC-fractionated urinary metabolites from 2,4,6-trinitrotoluene-treated Fischer 344 rats. Environ Mol Mutag 30, 298-302. 502 503 Clausen, J., Robb, J., Curry, D. and Korte, N. (2004) A case study of contaminants on military ranges: Camp Edwards, Massachusetts, USA. Environ Pollut 129, 13-21. 504 505 506 Coleman, N.V., Nelson, D.R. and Duxbury, T. (1998) Aerobic biodegradation of hexahydro1,3,5-trinitro-1,3,5-triazine (RDX) as a nitrogen source by a Rhodococcus sp., strain DN22. Soil biology & biochemistry 30, 1159-1167. 507 508 Crocker, F.H., Indest, K.J. and Fredrickson, H.L. (2006) Biodegradation of the cyclic nitramine explosives RDX, HMX, and CL-20. Appl Microbiol Biotechnol 73, 274-290. 509 510 Crocker, F.H., Thompson, K.T., Szecsody, J.E. and Fredrickson, H.L. (2005) Biotic and abiotic degradation of CL-20 and RDX in soils. Journal of Environmental Quality 34, 2208-2216. 511 512 513 514 Daniels, J.I. and Knezovich, J.P. (1994) Human health risks from TNT, RDX, and HMX in environmental media and consideration of the U.S. Regulatory Environment. In International Symposium on the Rehabilitation of Former Military Sites and Demilitarization of Explosive Ordnance. Kirchberg, Luxembourg: Proceedings Demil '94. 515 516 De Lorme, M. and Craig, A.M. (2008) Biotransformation of 2,4,6-trinitrotoluene by pure culture ruminal bacteria. Curr Microbiol. 517 518 Duringer, J.M., Craig, A.M., Smith, D.J. and Chaney, R.L. (2010) Uptake and transformation of soil [14-C]-trinitrotoluene by cool-season grasses. Environ Sci Technol 44, 6325-6330. 519 520 Eaton, H.L., De Lorme, M., Chaney, R. and Craig, A.M. (2011) Ovine ruminal microbes are capable of biotransforming hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Microb Ecol, 1-13. 521 522 523 Environmental Protection Agency, U.S. (1993) Risk estimate for carcinogenicity and reference dose for oral exposure for 2, 4, 6-trinitrotoluene. . Washington, D.C.: Office of Health and Environmental Assessment. U.S. EPA. 524 525 526 Fleischmann, T.J., Walker, K.C., Spain, J.C., Hughes, J.B. and Morrie Craig, A. (2004) Anaerobic transformation of 2,4,6-TNT by bovine ruminal microbes. Biochem Biophys Res Commun 314, 957-963. 527 528 529 Fournier, D., Trott, S., Hawari, J. and Spain, J. (2005) Metabolism of the aliphatic nitramine 4nitro-2,4-diazabutanal by Methylobacterium sp. strain JS178. Appl Environ Microbiol 71, 41994202. 530 531 532 Fuller, M.E., Lowey, J.M., Schaefer, C.E. and Steffan, R.J. (2005) A peat moss-based technology for mitigating residues of the explosives TNT, RDX, and HMX in soil. Soil & sediment contamination 14, 373-385. 533 534 535 Fuller, M.E., McClay, K., Hawari, J., Paquet, L., Malone, T.E., Fox, B.G. and Steffan, R.J. (2009) Transformation of RDX and other energetic compounds by xenobiotic reductases XenA and XenB. Appl Microbiol Biotechnol 84, 535-544. 536 537 538 539 Gust, K.A., Pirooznia, M., Quinn, J., M.J., Johnson, M.S., Escalon, L., Indest, K.J., Guan, X., Clarke, J., Deng, Y., Gong, P. and Perkins, E.J. (2009) Neurotoxicogenomic investigations to assess mechanisms of action of the munitions constituents RDX and 2,6-DNT in Northern Bobwhite (Colinus virginianus). Toxicol Sci 110, 168-180. 540 541 542 Hawari, J., Beaudet, S., Halasz, A., Thiboutot, S. and Ampleman, G. (2000a) Microbial degradation of explosives: biotransformation versus mineralization. Appl Microbiol Biotechnol 54, 605-618. 543 544 545 546 Hawari, J., Halasz, A., Sheremata, T., Beaudet, S., Groom, C., Paquet, L., Rhofir, C., Ampleman, G. and Thiboutot, S. (2000b) Characterization of metabolites during biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) with municipal anaerobic sludge. Appl Environ Microbiol 66, 2652–2657. 547 548 549 Hesselmann, R.M.X. and Stenstrom, M.K. (1994) Treatment Concept for RDX-Containing Wastewaters Using Activated Carbon with Offline Solvent Biological Regeneration. Los Angeles, CA University of California, Los Angeles 550 551 Hobson, P.N. and Stewart, C.S. (1997) The rumen microbial ecosystem. New York, NY: Blackie Academic and Professional. 552 553 Krishnan, I.S., Singhal, R.K. and Dua, R.D. (1986) Purification and characterization of glutamine synthetase from Clostridium pasteurianum. Biochemistry (Mosc) 25, 1589-1599. 554 555 556 557 Kumada, Y., Benson, D.R., Hillemann, D., Hosted, T.J., Rochefort, D.A., Thompson, C.J., Wohlleben, W. and Tateno, Y. (1993) Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes. Proceedings of the National Academy of Sciences 90, 3009-3013. 558 559 McCormick, N.G., Feeherry, F.E. and Levinson, H.S. (1976) Microbial transformation of 2,4,6trinitrotoluene and other nitroaromatic compounds. Appl Environ Microbiol 31, 949-958. 560 561 Prescott, L.M., Harley, J.P. and Klein, D.A. (2005) Microbiology. New York, NY: McGrawHill. 562 563 564 565 Qasim, M.M., Moore, B., Taylor, L., Honea, P., Gorb, L. and Leszczynski, J. (2007) Structural characteristics and reactivity relationships of nitroaromatic and nitramine explosives – a review of our computational chemistry and spectroscopic research. International Journal of Molecular Sciences 8, 1234-1264. 566 567 568 Sejrsen, K., Hvelplund, T. and Nielsen, M.O. (2006) Ruminant physiology: digestion, metabolism and impact of nutrition on gene expression, immunology and stress. Wageningen, The Netherlands: Wageningen Academic Publishers. 569 570 571 Smith, D.J., Craig, A.M., Duringer, J.M. and Chaney, R.L. (2008) Absorption, tissue distribution, and elimination of residues after 2,4,6-trinitro[14C]toluene administration to sheep. Environ Sci Technol 42, 2563–2569. 572 573 574 Stenuit, B.A. and Agathos, S.N. (2010) Microbial 2,4,6-trinitrotoluene degradation: could we learn from (bio)chemistry for bioremediation and vice versa? Appl Microbiol Biotechnol 88, 1043-1064. 575 576 Sunahara, G.I., Guilherme, L., Kuperman, R.G. and Hawari, J. (2009) Ecotoxicology of explosives: CRC Press, Boca Raton, FL. 577 578 Talley, J.W. and Sleeper, P.M. (1997) Roadblocks to the implementation of biotreatment strategies. Annals New York Academy of Sciences 829, 16-29. 579 580 581 Turley, S.D. and Burton, D.T. (1995) Reduction of hexahydro-l,3,5-trinitro-l,3,5-triazine (RDX) toxicity to the Cladoceran Ceriodaphniadubia following photolysis in sunlight. Bull Environ Contam Toxicol 55, 89-95. 582 583 U. S. Environmental Protection Agency (2007) Method 8330A (SW-846): Nitroaromatics and nitramines by high performance liquid chromatography (HPLC) Washington, D.C.: EPA. 584 585 U.S. Environmental Protection Agency (2009) Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Washington, DC: Environmental Protection Agency. 586 587 Williams, R.T., Ziegenfuss, P.S. and Sisk, W.E. (1992) Composting of explosives and propellant contaminated soils under thermophilic and mesophilic conditions. J Ind Microbiol 9, 137-144. 588 589 Zhang, C. and Hughes, J.B. (2003) Biodegradation pathways of hexahydro-1,3,5-trinitro- 1,3,5triazine (RDX) by Clostridium acetobutylicum cell-free extract. Chemosphere 50, 665-671. 590 591 592 Zhao, J.S., Paquet, L., Halasz, A. and Hawari, J. (2003) Metabolism of hexahydro-1,3,5-trinitro1,3,5-triazine through initial reduction to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine followed by denitration in Clostridium bifermentans HAW-1. Appl Microbiol Biotechnol 63, 187–193. 593 594 595 Fig. 1. A: Extracted ion chromatogram overlay of RDX and metabolite standards (50 ng/mL) 596 obtained via LC-MS/MS (APCI-) (top). Extracted ions include RDX (46.0→281.0), MNX 597 (46.0→265), DNX (46.0→249.0), TNX (59.0→233.0), NDAB (61.1→117.9) and MEDINA 598 (60.9→134.9). Fragmentation was obtained using a Q1 scan, scanning from 50-600 amu. 599 600 Fig. 2. RDX and metabolite concentrations as determined by MRM quantification via LC- 601 MS/MS over 24 hours in whole rumen fluid microcosms. Error bars represent the standard 602 deviation of three replicate samples per time point. 603 604 Fig. 3. Total ion chromatograms (TIC) of WRF incubated with 180 µM RDX at 0.25 (A) and 24 605 hours (B), obtained using an enhanced mass spectra scan. C and D represent LC-MS (APCI-) 606 scans of metabolite peaks consistent with m/z of 193 and 149, respectively. 607 608 Fig. 4. Proposed biodegradation pathway for hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by 609 ruminal microbes under anaerobic conditions as determined by enhanced mass spectra and 610 LightSight analysis. Hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX); hexahydro-1,3- 611 dinitroso-5-nitro-1,3,5-triazine (DNX); hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX). 612 613 Fig. 5. RDX concentration as determined by MRM quantification via LC-MS/MS over 120 614 hours from incubations with ruminal isolates identified to be most efficient at RDX degradation. TNX 1.5e5 1.4e5 Intensity (cps) 1.2e5 1.0e5 8.0e4 6.0e4 MNX 4.0e4 DNX MEDINA 2.0e4 NDAB RDX 0.0 5 10 15 20 25 30 Time (min) 58.8 1.00e5 O RDX + O - 3.5e4 58.8 3.0e4 N 8.00e4 O 6.00e4 + N N + N N O O O O + 2.0e4 N 1.5e4 O - 4.00e4 + O - N N 2.5e4 - O MNX N N N O N 1.0e4 251.9 76.8 2.00e4 5000.0 118.8 50 2.4e4 61.0 75.1 280.9 100 150 200 250 300 58.8 DNX 1.5e4 O 4.0e4 O 2.0e4 + N N 3.5e4 N 3.0e4 102.0 129.0 100 264.9 150 N TNX 250 300 250 300 O N N N O N N 2.0e4 O 200 233.0 59.0 2.5e4 O N N 1.0e4 50 N O 1.5e4 77.1 143.6 1.0e4 5000.0 218.8 76.9 5000.0 236.1 60.9 75.0 83.9 50 248.9 103.0 100 150 200 250 300 50 117.8 7.0e5 NDAB 6.0e5 100 150 200 58.8 5.0e4 O NH O MEDINA 4.0e4 O NH + 5.0e5 N N 3.0e4 O 4.0e5 O 60.8 3.0e5 O NH + NH N - O - 2.0e4 2.0e5 1.0e4 76.8 1.0e5 58.8 50 74.8 87.9 102.8 100 150 200 250 300 50 100 150 200 250 300 3.3e6 3.5e6 3.0e6 m/z 149 m/z 193 3.0e6 Intensity (cps) Intensity (cps) A: 0.25 hours 4.0e6 2.5e6 2.0e6 1.5e6 B: 24 hours m/z 149 2.5e6 2.0e6 1.5e6 m/z 193 1.0e6 1.0e6 5.0e5 5.0e5 0.0 0.0 10 20 30 40 Time (minutes) 50 60 p ( pp ) C: LC/MS of m/z 193 metabolite p 10 70 20 30 40 50 Time (minutes) 60 70 D: LC/MS of m/z 149 metabolite p 149.2 193.1 2.4e5 5.0e5 4.5e5 4.0e5 Intensity (cps) Intensity (cps) 2.0e5 1.5e5 1.0e5 3.5e5 3.0e5 2.5e5 2.0e5 1.5e5 1.0e5 5.0e4 5.0e4 175.6 60 80 100 120 140 m/z (amu) 160 180 58.9 60 80 100 120 140 m/z (amu) 160 180 200 Anaerovibrio lipolyticus Table 1. Strains and sources of ruminal bacteria tested for RDX degradation ability. Organism Anaerovibrio lipolyticus Butyrivibrio fibriosolvens Clostridium bifermentans Clostridium pasteurianum Clostridium polysaccharolyticum Desulfovibrio desulfuricans subsp. desulfuricans Eubacterium ruminantium Fibrobacter succinogenes Lactobacillus ruminus Lactobacillus vitulinus Megasphaera elsdenii Peptococcus heliotrinreducens Prevotella albensis Prevotella bryantii Prevotella ruminicola Selenomonas ruminantium Streptococcus bovis Streptococcus caprinus Succinovibrio dextrinosolvens Veillonella parvula Wolinella succinogenes 1 Strain D1 nyx 5 B MB GA 195 S85 RF1 T185 T-81 M384 B14 HD4 PC18 IFO JB1 2.2 0554 TE3 FDC 602 W Source1 ATCC 33276 ATCC 19171 ATCC 51255 ATCC 17836 ATCC 6013 ATCC 33142 ATCC 27774 ATCC 17233 ATCC 19169 ATCC 27780 ATCC 27783 ATCC 17753 ATCC 29202 DSMZ 13370 DSMZ 11371 ATCC 19189 ATCC 27209 ATCC 19205 ATCC 15351 ATCC 700410 ATCC 700065 ATCC 19716 ATCC 10790 ATCC 29543 ATCC = American Type Culture Collection; DSMZ = Leibniz-Institut DSMZ-German Collection of Microorganisms and Cell Cultures Table 2. Percent degradation of RDX by ruminal isolates after 120 hours, where RDX was supplemented as either the sole source of nitrogen (LNB) or carbon (LCB).a Initial RDX %RDX %RDX concentration degradation in degradation in Organism (µM) LNB LCB Anaerovibrio lipolyticus 85 70 5 Butyrivibrio fibriosolvens D1 34 37 15 Butyrivibrio fibriosolvens nxy 34 7 18 Clostridium bifermentans 34 19 29 Clostridium pasteurianum 34 3 49 Clostridium polysaccharolyticum 186 40 65 Desulfovibrio desulfuricans 256 3 88 subsp. desulfuricans Eubacterium ruminantium 34 32 6 Fibrobacter succinogenes 34 0 3 Lactobacillus ruminus 34 6 0 Lactobacillus vitulinus 34 39 0 Megasphaera elsdenii 34 33 27 Peptococcus heliotrinreducens 34 0 14 Prevotella albensis 34 19 7 Prevotella bryantii 34 25 4 Prevotella ruminicola 34 96 3 Selenomonas ruminantium HD4 34 33 34 Selenomonas ruminantium PC18 34 41 25 Streptococcus bovis IFO 39 86 18 Streptococcus bovis JB1 34 12 20 Streptococcus caprinus 34 49 18 Succinovibrio dextrinosolvens 34 0 18 Veillonella parvula 34 2 21 Wolinella succinogenes 34 14 7 Controlb 256 19 20 a Data are expressed as average percent degradation of initial RDX concentration during 120 h incubations in low nitrogen basal (LNB) and low carbon basal (LCB) media, using three replicates for each organism. b Control consisted of anaerobically reduced medium with RDX and no bacteria.