Anaerobic transformation of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) by ovine rumen microorganisms

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<ul><li><p>om</p><p>am</p><p>Stat</p><p>n S</p><p>12;</p><p>ne 4</p><p>tetranitro-1,3,5,7-tetrazocine (HMX) have been widely</p><p>focal atrophy as well as the functions of these organs (U.S.</p><p>toward three vertebrate species showed differences in levels oftoxicity between animals when fed equal amounts of HMX.</p><p>et al., 2003) and cold marine sediments (Zhao et al., 2004).Several microorganisms have also been shown to co-metabolize HMX and RDX (Bhushan et al., 2003; Kittset al., 1994; Zhao et al., 2004). Both aerobic and anaerobic</p><p>* Corresponding author. Tel.: 1 541 737 6541; fax: 1 541 737 2730.E-mail address: sudeep.perumbakkam@gmail.com (S. Perumbakkam).</p><p>Research in Microbiologused in various military activities with worldwide impact.Structurally, HMX is similar to hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX: similar monomeric units ofCH2eNeNO2), but more resilient to biodegradation(Crocker et al., 2006).</p><p>While 2,4,6 trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) have adverse effects on thecentral nervous system of mammals and are potential carcin-ogens, HMX toxicity is still unclassified by the Agency forToxic Substances and Disease Registry (ATSDR) (Lynchet al., 2002). The US Environmental Protection Agency(EPA) states that HMX affects liver and kidney by changing</p><p>This study concluded that physiology and gastrointestinalstructure and function were responsible for the varying effectsof HMX among species (Johnson et al., 2010).</p><p>Depending on remediation needs, both in situ and ex situremediation have been used to detoxify polluted sites. Witha need for greener remediation technologies, an effort to usemicroorganisms to detoxify pollutants in contaminated sites ispresently a priority. This technology, referred to as bioreme-diation, can use either native or supplemented microbialpopulation based on remediation needs. Biotransformation ofHMX has been shown to occur in various habitats such assewage sludge (Hawari et al., 2001), soil (Monteil-RiveraHighly energetic explosives such as octahydro-1,3,5,7-</p><p>EPA, 2011). Recent evaluation of the toxicity of HMX1. IntroductionExplosives such as octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) provide a challenge in terms of bioremediation. In the presentstudy, sheep rumen was studied for its potential to detoxify HMX using analytical chemistry and molecular microbial ecology tools. Resultsindicated significant loss ( p &lt; 0.05) of HMX at 8 h post-incubation and complete disappearance of the parent molecule after 16 h. QualitativeLCeMS/MS analysis provided evidence for the formation of 1-NO-HMX and MEDINA metabolites. A total of 1006 16S rRNA-V3 clones weresequenced and the Classifier tool of the RDPII database was used to sort the sequences at their phylum level. Most sequences were associatedwith either the phylum Bacteroidetes or Firmicutes. Significant differences at the phylum level ( p &lt; 0.001) were found between 0 h and 8 hHMX treatments. Using LibCompare analysis, 8 h HMX treatment showed enrichment of clones ( p &lt; 0.01) belonging to the genus Prevotella.From these results, it could be concluded that members of the genus Prevotella are enriched in the rumen and are capable of detoxifying HMX. 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.</p><p>Keywords: HMX; Rumen; 16S rRNA; Phylogenetics; LCeMS/MSAnaerobic transformation of octahydr(HMX) by ovine ru</p><p>Sudeep PerumbakkaDepartment of Environmental and Molecular Toxicology, Oregon</p><p>bDepartment of Veterinary Medicine, Orego</p><p>Received 8 January 20</p><p>Available onli</p><p>Abstract0923-2508/$ - see front matter 2012 Institut Pasteur. Published by Elsevier Mahttp://dx.doi.org/10.1016/j.resmic.2012.08.001-1,3,5,7-tetranitro-1,3,5,7-tetrazocineen microorganisms</p><p>a,*, A.M. Craig b</p><p>e University, 139 Oak Creek Building, Corvallis, OR 97331, USA</p><p>tate University, Corvallis, OR 97331, USA</p><p>accepted 15 July 2012</p><p>August 2012</p><p>y 163 (2012) 567e575www.elsevier.com/locate/resmicsson SAS. All rights reserved.</p></li><li><p>supernatant was passed through a 13 mm (0.2 mm) PTFE filtervials</p><p>(VWR International). A 10 ml sample was injected into the</p><p>(PerkineElmer). Metabolite identification was based onwith</p><p>LCeMS/MS was used to qualitatively assess the presence</p><p>ch ibiodegradation of HMX has been reported (Crocker et al.,2006). Since HMX is structurally very similar to RDX, ithas a similar mechanism of degradation. Pure cultures ofaerobic white-rot fungus Phanerochaete chrysosporium(Fournier et al., 2004a), Methylobacterium sp. (Van Akenet al., 2004) and a few anaerobic strains of the genus Clos-tridium (Bhushan et al., 2004) have been reported to detoxifyHMX. Most studies on the anaerobic fate of HMX list nitrosoderivatives as intermediates formed from two-electronreduction of the nitro groups on the ring (Crocker et al.,2006).</p><p>Since the EPA has set drinking water guidelines to 2 ppmfor RDX, there is still a high prevalence of RDX in ground-water (36 ppm) in the United States (ATSDR, 1995). Thissuggests that the native microflora in soil and groundwater areincapable of detoxifying RDX and introduction of an externalmicrobial source is needed for detoxification. In light of thepresent general dislike for the use of genetically modifiedorganisms (GMOs), a safer and more natural source/bioreactoris needed. Ruminants are habitats to a large number ofmicroorganisms that can potentially be used as bioreactorswhen introduced into environments where anaerobic degra-dation is needed. Anaerobic microbial transformation of nitro-aromatics is also receiving increased attention due to theincreased susceptibility of polynitro-aromatics under anaer-obic conditions (Chunlong and George, 2005; Nishino andSpain, 2002). To study the effects of toxicity of munitionson ruminants, sheep were fed radiolabeled TNT and resultsindicate that the rumen converted most of the grass-boundTNT into organic matter (Smith et al., 2008). The sheep didnot experience any toxic side effects; thus, the combination ofphytoremediation with ruminal degradation can be helpful inbioremediation (Eaton et al., 2011).</p><p>The rumen has been relatively well characterized in termsof its physiology (Hess et al., 2011; Russell and Rychlik,2001; Tajima et al., 2001), microbial community structure(Edwards et al., 2004; Perumbakkam and Craig, 2011),detoxification of numerous plant toxins such as pyrrolizidinealkaloids (Rattray and Craig, 2007), oxolate (Allison et al.,1981), pyrindinediols (Allison et al., 1992), mimosine(Dominguez-Bello and Stewart, 1991), dihydroxypyridine(Dominguez-Bello et al., 1997) and nitroaromatic compounds(De Lorme and Craig, 2009; Perumbakkam et al., 2011).</p><p>Since HMX is more resilient to degradation compared toRDX, it could pose a significant problem in the near futureunless a more suitable mobile microbial source in a biore-actor form can be found to degrade HMX. With the future inmind, the aim of this study was to determine whether ovinewhole rumen fluid (WRF) detoxifies HMX and to understandchanges in the bacterial community structure during sucha transformation process. Analytical chemistry techniquessuch as high performance liquid chromatography (HPLC),LCeMS/MS were used to observe degradation of HMX overtime, and changes to the microbial community structure werecharacterized using established molecular ecology tech-</p><p>568 S. Perumbakkam, A.M. Craig / Researniques such as cloning of the 16S rRNA-V3 gene andphylogenetics.of the parent molecule and metabolites in the rumen samples.a comparison of retention times and UV/Vis spectrastandard curves of the parent compound.HPLC system and analyzed with suitable prepared standardsusing the parent HMX compound. Separations were performedusing a guard column hand packed with pellicular C8 materialthat was attached to an Acclaim explosives E1 column(4.6 250 mm, 5 mm particle size, Dionex, Sunnyvale, CA).The column was eluted under isocratic conditions withmethanol and water (43:57) at a flow rate of 1.0 ml min1 witha total run time of 45 min. A column heater (PerkineElmer,Waltham, MA, USA) was used to maintain the temperature at32 C. The HPLC system consisted of a solvent deliverysystem (PerkineElmer Series 200 Pump) equipped witha sample injector (PerkineElmer ISS 200 autosampler) andUV detector monitoring at 254 nm. Detector output wascaptured via an analog to a digital converter (PE Nelson 600Series LINK interface) connected to a computer equipped witha Turbochrom workstation and TurboScan 200 software(VWR International) and transferred to amber HPLC2. Materials and methods</p><p>2.1. Whole rumen fluid collection, sheep diet andexperimental design</p><p>WRF was collected from fistulated wethers (n 3) housedat the Oregon State University (OSU) sheep facility. The sheepdiet consisted of grass-hay. Rumen samples were collectedusing sterile equipment and transported to the laboratory ina CO2 flushed thermos. The experimental design consisted ofthree conditions: WRF with HMX (HMX-T), WRF withoutHMX (HMX-C) and autoclaved WRF biomass to which HMXwas added after sterilization (HMX-SC). Bulge tubes con-taining the samples were sealed with sterile butyl rubberstoppers and aluminum crimp caps. An HMX concentration of20 mg ml1 was used for all incubation experiments. Alltreatments were done in triplicate. Experiments were under-taken in an anaerobic glove box (Coy, Grass Lake, MI) witha mixed atmosphere of CO2 and H2 (9:1, respectively). TheBulge tubes were incubated at 39 C in the dark under constantrocking.</p><p>2.2. HPLC and LC MS/MS analysis</p><p>Time point samples were processed for both DNA andmetabolite identification. The samples were spun at10,000 g for 2 min to pellet the cells and the supernatantwas used for HPLC and LCeMS/MS analysis. To 250 ml ofsupernatant, an equal volume of acetonitrile (LCeMS/MSgrade, VWR International, West Chester, PA) was added andsamples were centrifuged at 10,000 g for 5 min. The</p><p>n Microbiology 163 (2012) 567e575Analysis was performed on an ABI/SCIEX QTRAP 3200LCeMS/MS system (Applied Biosystems, Foster City CA)</p></li><li><p>using a turbo spray interface in negative ion mode. Sampleswere separated on an HPLC system (Perkin Elmer Series 200Micropump) using an Ultracarb ODS 250 4.6 mm, 5 mmparticle size column (Phenomenex, Torrance CA). The methodused for separation of HMX involved a 15 ml injection volumefollowed by a mobile phase gradient program with A (meth-anol) and B (200 mM formic acid dissolved in ultrapure H2O)pumps. The HPLC was set to equilibrate at 100% B for 5 minfollowed by a linear increase to 100% A in 20 min. Thecolumn was re-equilibrated for 10 min with 100% B. The flowrate was set at 300 ml min1. The method was optimized by</p><p>, asgene</p><p>reaction contained approximately 75 ng of purified bacterialgenomic DNA, 200 mmol of each dNTP, 5 ml of 10 PCRbuffer, 5 ml of 25 mM MgCl, 20 ng of bovine serum albumin(BSA), primer concentration at 25 pmoles (each primer),0.25 U polymerase and the remaining volume was made upwith sterile water. All PCR reactions were set up in triplicateand products were visualized on a gel and pooled beforepurifying using the QIAquick PCR purification kit accordingto the manufacturers recommendations (Qiagen Inc., Valen-cia, CA, USA). PCR products were quantified, cloned andtransformed into competent Escherichia coli cells using the</p><p>SA)r for</p><p>cted</p><p>pre</p><p>Ent</p><p>pote</p><p>33331212</p><p>569S. Perumbakkam, A.M. Craig / Research in Microbiology 163 (2012) 567e575kit (Qiagen, Valencia, CA) combining the extraction procedurefor Gram-positive and Gram-negative bacteria. Tubes were leftat room temperature to hydrate overnight and run on a 1%agarose gel stained with ethidium bromide. Samples werequantified using a Nanodrop (Thermo Fisher, Waltham MA),stored at 20 C and used for all subsequent PCR reactions.</p><p>The hypervariable region V3 of the 16S rRNA was used asa gene marker. The primers and PCR amplification protocolused in this study have been described previously (Muyzeret al., 1993). PCR thermocycling was carried out usingrecombinant AmpliTaq Gold polymerase (Applied Bio-systems, Foster City, CA) in a PTC-200 thermocycler (MJResearch Inc., Watertown, MA, USA). Each 50 ml PCR</p><p>Table 1</p><p>MS/MS transition detection parameters and times for HMX metabolites dete</p><p>electrospray mode and multiple reaction monitoring (MRM). Detection time re</p><p>HMX in WRF.</p><p>Metabolite MS/MS transitions Detection</p><p>time (min)</p><p>Declustering</p><p>potential (V)</p><p>HMX 341.06/145.80 22 15.00341.06/146.60 22 15.00341.06/147.00 22 15.00</p><p>1-NO-HMX 325.10/146.90 21 15.00MEDINA 134.90/61.10 9.25 15.00</p><p>135.10/61.00 9.25 15.00Genomic DNA was extracted from cell pelletsmentioned in the previous section, using the Gentra Purerunning the LCeMS/MS in the infusion mode with the parentmolecule HMX and two important metabolites, methyl-enedinitramine (MEDINA) and 4-nitro-2,4-diazabutanal(NDAB). Data were acquired using multiple reaction moni-toring (MRM) as the survey scans to generate MS/MS spectrawithin the Analyst 1.4.2 software package (Applied Bio-systems). Based on these optimization runs, the final methodhad the following parameters: curtain gas (nitrogen) set at30 psi, temperature at 450 C, dwell time of 60 ms, gas 1(GS1) 45.00, gas 2 (GS2) 45.00 and a scan range of50e400 Da. Declustering potential, entrance potential andcollision energy were dependent upon the ion being scanned(Table 1).</p><p>2.3. Isolation of genomic DNA, V3 region amplification,PCR conditions, cloning and plasmid extraction135.00/61.10 9.25 15.00 12135.00/60.60 9.25 15.00 12Genomic Research and Biocomputing (CGRB) of OregonState University. Single reads utilizing the T7 promoter wereused to determine nucleotide sequences. The sequences wereimported into the Geneious computer program (Drummondet al., 2007) extracted, checked for chimeras and the result-ing FASTA file was used for further analysis. The RDPIIClassifier (Wang et al., 2007) was used to sort sequences intotheir respective operational taxonomic units (OTUs) ata confidence interval of 50% (Claesson et al., 2009). Lib-Compare software from the RDPII database was also used tocompare significance of community changes at a confidenceinterval of 50% (Wang et al., 2007). The Mothur softwarepackage (Schloss et al., 2009) was used for analysis of data</p><p>through LCeMS/MS analysis. Data were quantified using the negative ion</p><p>sents the time point at which metabolites were detected in a 4 h incubation of</p><p>rance</p><p>ntial (V)</p><p>Collision cell</p><p>entrance potential (V)</p><p>Collision</p><p>energy (V)</p><p>Collision cell</p><p>exit potential (V)</p><p>.50 10.00 12.00 4.00</p><p>.50 10.00 12.00 2.00</p><p>.50 10.00 12.00 3.00</p><p>.50 10.00 12.00 2.00</p><p>.00 16.65 10.00 2.00</p><p>.00 16.65 10.00 2.00v. 3.1 Cycle Sequencing Kit (Applied Biosystems, CA, Uusing an ABI Prism 3730 Genetic Analyzer at the CenteTOPO TA Cloning Kit for Sequencing (Invitrogen Corpora-tion, Carlsbad, CA, USA) according to the manufacturersrecommendations. Transformants were spread onto petridishes containing LB agar (EMD Chemicals Inc., Gibbstown,NJ, USA) supplemented with 50 mg ml1 kanamycin sulfate(EMD). Plates were incubated at 37 C overnight. Clones werepicked and grown for 36 h i...</p></li></ul>

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