Biochemical and Microbial Analysis of Ovine Rumen Fluid Incubated with 1,3,5-Trinitro-1,3,5-triazacyclohexane (RDX)
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- Perumbakkam, S. & Craig, A.M. Curr Microbiol (2012) 65: 195. doi:10.1007/s00284-012-0144-1
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In this study, the rumen was assessed for its potential to detoxify RDX using molecular microbial ecology as well as analytical chemistry techniques. Results indicated significant loss (P < 0.05) of RDX in <8-h post incubation, and qualitative LC-MS/MS analysis showed evidence for the formation of 1-NO-RDX (M–O + HCOO) and methylenedinitramine metabolites. A total of 1106 16S rRNA-V3 clones were sequenced, and most sequences associated with either the phyla Bacteroidetes or Firmicutes. A LibCompare analysis for the RDX treatment showed an enrichment (P < 0.01) of the genus Prevotella. From these results, it can be concluded that the rumen is capable of detoxifying RDX, and the members of the genus Prevotella are linked to this detoxification.
Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX; royal demolition explosive) is a high-energy trinitrated cyclic compound developed during World War II. RDX is also used with other munition mixtures and formulations with worldwide impact . Owing to its extensive use, RDX is present as a contaminant in several military sites as munition waste and unexploded ordnance. The munition runoff causes potential harm to man and the environment. In humans, accidental ingestion of RDX-containing compounds has occurred several times and induces rash, nausea, as well as neurological effects including headaches, dizziness, and seizures .
Structurally, RDX is unabsorbed in the soil making it easy for its transportation into ground water aquifers [29, 44]. The present US Environmental Protection Agency (EPA) guideline for RDX quantity in drinking water is set at 2 ppm, but actual data suggest RDX prevalence as high as 36 ppm in the United States . This suggests that the native microflora in soil and ground water do not have the capacity of detoxifying RDX and an introduction of an external microbial source is needed to complete remediation. With the use of genetically modified organisms (GMOs) debatable, and with the expense of ex situ remediation, many researchers are looking to develop affordable, practical, and safe technologies for remediation .
Anaerobic microbial transformation of nitroaromatics is also receiving increased attention because of the increased susceptibility of nitroaromatics molecules to degrade under anaerobic conditions [1, 8]. The animal rumen has been relatively well characterized in terms of its physiology . Ruminants have tremendous potential in degrading or detoxifying numerous substrates ranging from grass-based carbohydrates to munitions [12, 15, 31] due to their anaerobic ecosystem and strong metabolic potential due to the diversity of microbes. Moreover, ruminants could be used as “bio-reactors on hooves” when combined with phytoremediation, using cool season grasses, thus presenting an easy accesses to in situ-based remediation . The grasses would help in the transport of munitions from the soil to be available for the sheep to graze. Munitions also seem to have less toxic effect on ruminants. In a previous study , sheep were fed radiolabeled TNT, and the results indicate that the rumen converted most of the grass-bound TNT into organic matter without any toxic effect to the sheep. Tissue samples from the sheep showed no signs of radioactive damage suggesting that the microbes in the rumen play an important part in the detoxification. Such a novel idea of using phytoremediation combined with ruminal degradation can be helpful in bioremediation of nitroaromatic compounds .
In this study, the rumen was assessed for its potential to detoxify RDX by using classic molecular microbial techniques such as cloning of the 16S rRNA gene and phylogenetics. LibCompare and Libshuff analysis  tested differences between the samples (treatment and control) at the community level. Further, the metabolites of RDX detoxification were identified using LC-MS/MS analysis.
Materials and Methods
Whole Rumen Fluid Collection, Sheep Diet, and Experimental Design
Ovine whole rumen fluid (WRF) was collected as described before. Sheep diet consisted of grass-hay. The experimental design consisted of three conditions: WRF with RDX (RDX-T), WRF without RDX (RDX-C), and autoclaved WRF biomass to which RDX was added after sterilization (RDX-SC). Balch tubes containing the WRF samples were sealed with sterile butyl rubber stoppers and aluminum crimp caps. RDX concentration of 25 μg mL−1 was used for all incubation experiments. All treatments were done in triplicate. Experiments were undertaken in an anaerobic glove box (Coy, Grass Lake, MI) with a mixed atmosphere of CO2 and H2 (9:1). The Balch tubes were incubated at 39 °C in the dark under constant rocking.
High-Performance Liquid Chromatography (HPLC) and LC-MS/MS Analyses
Time point samples were processed for both DNA and metabolite identification. The samples were spun at 10,000×g for 3 min to pellet the cells, and the supernatant was used for the HPLC and LC-MS/MS analyses. HPLC analyses were carried out using the US Environmental Protection Agency (EPA) method 8330 .
LC-MS/MS was used to qualitatively assess the presence of the parent molecule and metabolites in the rumen samples. Analysis was performed on an ABI/SCIEX QTRAP 3200 LC-MS/MS system (Applied Biosystems, Foster City CA) using a turbo spray interface in negative ion mode. Samples were separated on an HPLC system (Perkin Elmer Series 200 Micropump) using an Ultracarb ODS 250 × 4.6 mm, 5-μm particle size column (Phenomenex, Torrance CA). The method used for the separation of HMX involved a 15-μL injection volume followed by a mobile phase gradient program with A (methanol) and B (200 mM formic acid dissolved in ultrapure H2O) pumps. The HPLC was set to equilibrate at 100 % B for 5 min followed by a linear increase to 100 % A in 20 min. The column was re-equilibrated for 10 min with 100 % B. The flow rate was set at 300 μL min−1. The method was optimized by running the LC-MS/MS in the infusion mode with the parent molecule HMX and two important metabolites: methylenedinitramine (MEDINA) and 4-nitro-2,4-diazabutanal (NDAB). Data were acquired using multiple reaction monitoring (MRM) as the survey scans to generate MS/MS spectra within the Analyst 1.4.2 software package (Applied Biosystems). Based on these optimization runs, the final method had the following parameters: curtain gas (nitrogen) set at 30 psi, temperature at 450 °C, dwell time of 60 ms, gas 1 (GS1) = 45.00, gas 2 (GS2) = 45.00, and a scan range of 50–400 Da. Declustering potential, entrance potential, and collision energy were dependent on the ion being scanned (Supplemental data, Table S1).
Isolation of Genomic DNA, V3 Region Amplification, PCR Conditions, Cloning, and Plasmid Extraction
Genomic DNA was extracted from the cell pellets, as mentioned in the previous section, using the Gentra puregene kit (Qiagen, Valencia, CA) combining the extraction procedure for Gram-positive and Gram-negative bacteria. Tubes were left at room temperature to hydrate overnight and run on a 1 % agarose gel stained with ethidium bromide. Samples were quantified using a Nanodrop (Thermo Fisher, Waltham MA), stored at −20 °C and used for all subsequent PCR reactions.
The hypervariable region, V3, of the 16S rRNA was used as a gene marker. The primers and PCR amplification protocol used in this study have been described previously . PCR thermocycling was carried out using recombinant AmpliTaq Gold polymerase (Applied Biosystems, Foster City, CA) in a PTC-200 thermocycler (MJ Research Inc., Watertown, MA). Each 50-μL PCR reaction contained approximately 75 ng of purified bacterial genomic DNA, 200 μmol of each dNTP, 5 μL of 10× PCR Buffer, 5 μL of 25 mM MgCl, 20 ng of bovine serum albumin (BSA), primer concentration at 25 pmol (each primer), and 0.25 U polymerase; the remaining volume was made up with sterile water. All PCR reactions were setup in triplicate, and the products were visualized on a gel and pooled before purifying using the QIAquick PCR purification kit, according to the manufacturer’s recommendations (Qiagen Inc., Valencia, CA). PCR products were quantified, cloned, and transformed into competent E. coli cells using the TOPO® TA Cloning Kit for Sequencing (Invitrogen Corporation, Carlsbad, CA), according to the manufacturer’s recommendations. Transformants were spread onto petri dishes containing Luria–Bertani (LB) agar (EMD Chemicals Inc., Gibbstown, NJ) supplemented with 50 μg mL−1 kanamycin sulfate (EMD). Plates were incubated at 37 °C overnight. Clones were picked and grown for 36 h in TYGPN  supplemented with 50 μg mL−1 kanamycin. Sterile glycerol was added to the colonies at a final concentration of 40 % and stored at −20 °C. The colonies were transferred into TYGPN media with 50 μg mL−1 kanamycin, and plasmid DNA was extracted as per previously published protocol .
Sequencing, RDPII, and Phylogenetic Analyses
Sequencing was performed using the BigDye® Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems, CA) using an ABI Prism® 3730 Genetic Analyzer at the Center for Genomic Research and Biocomputing (CGRB) at Oregon State University. Single reads utilizing the T7 promoter were used to determine the nucleotide sequences. The sequences were imported into the Geneious computer program  extracted and checked for chimeras, and the resulting FASTA file was used for further analysis. The RDP II Classifier  was used to sort sequences into their respective operational taxonomic units (OTUs) at a confidence interval of 50 % . The LibCompare software of the RDPII database was also used to compare significance of community changes at a confidence interval of 50 % . The MOTHUR software package  was used for the analysis of the data and estimation of collectors and rarefaction curves. All sequence data from this study were submitted to the GenBank database  under accession numbers HQ159874–HQ161053.
Results and Discussion
Although ruminants consume forage as primary mode of nutrition, they possess diverse microflora that has been used to help detoxify several plant toxins such as pyrrolizidine alkaloid (PA) , oxalate , nitropropanol , dihydroxypyridine , and munition such as TNT [12, 31] and RDX .
Tabulated classification of the 16S rRNA-V3 clones associated with RDX (T) and RDX (C) sampled at 0-, 4-, and 8-h at the phylum level using the Naïve Bayesian classifier of the RDPII website at a confidence interval of 50 %
No of clones
No of clones
No of clones
No of clones
No of clones
Despite having high-quality sequences with which to search RDPII database, 42.35 % or 499.73 of the 1,180 sequenced clones did not associate to any well-characterized bacterial groups in the RDPII database. Neither of the cultured representatives is available or the region chosen for amplification, and in this case, V3 of the 16S rRNA gene is not appropriate for rumen-associated studies. Most researchers have either used the V6 region [23, 36] or V3 region, with a few researchers claiming both are equally good in differentiating communities .
Phylogenetic analyses of RDX (T) and RDX (C) treatments at sampling time point’s 0-, 4-, and 8-h. Observed and estimated OTUs were calculated using the MOTHUR program. The OTUs common between treatments are also tabulated. All data represented OTU classification at a cutoff at 3 %
Libcompare comparison of the RDX clonal libraries at 0-, 4-, and 8-h post incubation. The sequences were submitted to LibCompare software of the RDPII database and significant shifts in populations among time points were estimated
RDX (O hr)
RDX (4 h)
RDX (8 h)
TM7 genera Incertae sedis
Prevotella strains are Gram-negative, non-motile, rod-shaped, and singular cells that thrive in anaerobic growth conditions. Members of the genus Prevotella are regarded as the most dominant bacterium in the rumen (Stevenson and Weimer 2007). Prevotella sp. are among the most numerous microbes cultivable from the rumen and hind gut of cattle and sheep, where they help in the breakdown of protein and carbohydrate derived from plant material . They are also present in humans as opportunistic pathogens. Two strains of Prevotella sp. (Prevotella intermedia 17 and Prevotella ruminicola 23) have been completely sequenced by The Institute for Genomic Research (TIGR). In silico analysis (data not shown) of sequenced Prevotella genomes indicated the presence of a nitroreductase enzyme system that is responsible for the breakdown of TNT. Such an enzyme system has also been shown to be active with RDX .
In conclusion, this study shows the potential of rumen fluid to degrade RDX. Further research has presently been undertaken in establishing the microbial population using stable isotope probing (SIP) in combination with next generation sequencing (NGS). We are also fine tuning our analytic procedure to tease apart this complex matrix to better understand the metabolites. Finally, we are incubating pure cultures of Prevotella sp. with RDX to decipher the metabolites and regulation of detoxification.
The research was supported by a jointly funded grant by the Oregon Agricultural Experiment Station project ORE00871 and by the U.S. Department of Agriculture under project number 6227-21310-007-00D agreement nos. 58-6227-8-044 and 58-1265-6-076. Any opinions, findings, conclusion, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. The authors would like to thank Ms. Karen Walker for her help with HPLC, Lia Murty during the LC-MS/MS analysis, and Ms. Zelda Zimmerman for editorial assistance.