Research in Microbiology 163 (2012) 567e575www.elsevier.com/locate/resmic
Anaerobic transformation of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine(HMX) by ovine rumen microorganisms
Sudeep Perumbakkam a,*, A.M. Craig b
aDepartment of Environmental and Molecular Toxicology, Oregon State University, 139 Oak Creek Building, Corvallis, OR 97331, USAbDepartment of Veterinary Medicine, Oregon State University, Corvallis, OR 97331, USA
Received 8 January 2012; accepted 15 July 2012
Available online 4 August 2012
Abstract
Explosives 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 < 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 < 0.001) were found between 0 h and 8 hHMX treatments. Using LibCompare analysis, 8 h HMX treatment showed enrichment of clones ( p < 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.
Keywords: HMX; Rumen; 16S rRNA; Phylogenetics; LCeMS/MS
1. Introduction
Highly energetic explosives such as octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) have been widelyused 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).
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
* Corresponding author. Tel.: þ1 541 737 6541; fax: þ1 541 737 2730.
E-mail address: [email protected] (S. Perumbakkam).
0923-2508/$ - see front matter � 2012 Institut Pasteur. Published by Elsevier Ma
http://dx.doi.org/10.1016/j.resmic.2012.08.001
focal atrophy as well as the functions of these organs (U.S.EPA, 2011). Recent evaluation of the toxicity of HMXtoward three vertebrate species showed differences in levels oftoxicity between animals when fed equal amounts of HMX.This study concluded that physiology and gastrointestinalstructure and function were responsible for the varying effectsof HMX among species (Johnson et al., 2010).
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-Riveraet 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
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568 S. Perumbakkam, A.M. Craig / Research in Microbiology 163 (2012) 567e575
biodegradation 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).
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).
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).
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-niques such as cloning of the 16S rRNA-V3 gene andphylogenetics.
2. Materials and methods
2.1. Whole rumen fluid collection, sheep diet andexperimental design
WRF was collected from fistulated wethers (n ¼ 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 ml�1 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.
2.2. HPLC and LC MS/MS analysis
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. Thesupernatant was passed through a 13 mm (0.2 mm) PTFE filter(VWR International) and transferred to amber HPLC vials(VWR International). A 10 ml sample was injected into theHPLC 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 min�1 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(PerkineElmer). Metabolite identification was based ona comparison of retention times and UV/Vis spectra withstandard curves of the parent compound.
LCeMS/MS was used to qualitatively assess the presenceof the parent molecule and metabolites in the rumen samples.Analysis was performed on an ABI/SCIEX QTRAP 3200LCeMS/MS system (Applied Biosystems, Foster City CA)
569S. Perumbakkam, A.M. Craig / Research in Microbiology 163 (2012) 567e575
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 min�1. The method was optimized byrunning 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) ¼ 45.00, gas 2 (GS2) ¼ 45.00 and a scan range of50e400 Da. Declustering potential, entrance potential andcollision energy were dependent upon the ion being scanned(Table 1).
2.3. Isolation of genomic DNA, V3 region amplification,PCR conditions, cloning and plasmid extraction
Genomic DNA was extracted from cell pellets, asmentioned in the previous section, using the Gentra Puregenekit (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.
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
Table 1
MS/MS transition detection parameters and times for HMX metabolites detected
electrospray mode and multiple reaction monitoring (MRM). Detection time repre
HMX in WRF.
Metabolite MS/MS transitions Detection
time (min)
Declustering
potential (V)
Ent
pote
HMX 341.06/145.80 22 �15.00 �3
341.06/146.60 22 �15.00 �3
341.06/147.00 22 �15.00 �3
1-NO-HMX 325.10/146.90 21 �15.00 �3
MEDINA 134.90/61.10 9.25 �15.00 �12
135.10/61.00 9.25 �15.00 �12
135.00/61.10 9.25 �15.00 �12
135.00/60.60 9.25 �15.00 �12
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 manufacturer’s recommendations (Qiagen Inc., Valen-cia, CA, USA). PCR products were quantified, cloned andtransformed into competent Escherichia coli cells using theTOPO� TA Cloning Kit for Sequencing (Invitrogen Corpora-tion, Carlsbad, CA, USA) according to the manufacturer’srecommendations. Transformants were spread onto petridishes containing LB agar (EMD Chemicals Inc., Gibbstown,NJ, USA) supplemented with 50 mg ml�1 kanamycin sulfate(EMD). Plates were incubated at 37 �C overnight. Clones werepicked and grown for 36 h in TYGPN (Elbing and Brent,2002) supplemented with 50 mg ml�1 kanamycin. Sterileglycerol was added to the colonies at a final concentration of40% and stored at �20 �C. The colonies were transferred toTYGPN media with 50 mg ml�1 kanamycin and plasmid DNAwas extracted as per previously published protocol(Engebrecht et al., 2000).
2.4. Sequencing, RDPII and phylogenetic analysis
Sequencing was performed using the BigDye� Terminatorv. 3.1 Cycle Sequencing Kit (Applied Biosystems, CA, USA)using an ABI Prism� 3730 Genetic Analyzer at the Center forGenomic 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
through LCeMS/MS analysis. Data were quantified using the negative ion
sents the time point at which metabolites were detected in a 4 h incubation of
rance
ntial (V)
Collision cell
entrance potential (V)
Collision
energy (V)
Collision cell
exit potential (V)
.50 �10.00 �12.00 �4.00
.50 �10.00 �12.00 �2.00
.50 �10.00 �12.00 �3.00
.50 �10.00 �12.00 �2.00
.00 �16.65 �10.00 �2.00
.00 �16.65 �10.00 �2.00
.00 �16.65 �10.00 �2.00
.00 �16.65 �10.00 �2.00
570 S. Perumbakkam, A.M. Craig / Research in Microbiology 163 (2012) 567e575
and estimation of collectors and rarefaction curves. Allsequence data from this study were submitted to the GenBankdatabase (Benson et al., 2010) under accession numbersHQ013359eHQ014364.
3. Results
3.1. Degradation of HMX by ovine bacteria determinedby HPLC analysis
Anaerobic Bulge tubes containing WRF were inoculatedwith HMX and sampled at 0, 4, and 8 h post inoculation. WRFsamples were processed through an HPLC. The treatment andcontrol tubes had an initial HMX concentration of 20 mg ml�1
at 0 h (Fig. 1A). The HMX concentration decreased in theexperimental tubes to 12 mg ml�1 at 4 h and were furtherreduced to 11 mg ml�1 at 8 h. Control tubes showed nodegradation throughout the experiment (Fig. 1A). LC-MS/MSanalysis of 0, 4 and 8 h treatment samples showed a reductionin the parent metabolite (Fig. 1B). ANOVA performedbetween treatments and control showed significant reductionof HMX in the treatment samples [(HMX-T (0) heHMX-T(4) h ( p � 0.01)) and (HMX-T (4) heHMX-T (8) h( p � 0.001))]. There was no background degradation of HMXin the control tubes.
3.2. LCeMS/MS analysis of HMX metabolites
Qualitative LCeMS/MS analysis identified the parentHMX ion at a m/z of 341.06 Da [M þ HCOO � H] with theadded formic acid adduct (Table 1). A second ion with a m/z of325.10 Da [M � O þ HCOO], previously identified as 1-NO-HMX in the degradation of HMX, was also identified as early
Fig. 1. Degradation of HMX by ovine rumen fluid. Panel A) HMX (20 mg ml�1)
HMX detoxification in WRF (C) and (,) an autoclaved WRF biomass control at
samples at sampled time-points. Error bars represent �1 standard deviation of three
points 0, 4 and 8 h post-incubation with HMX. Collected time-point samples wer
methods section. Peaks indicate the loss of parent molecules at 0, 4, and 8 h post
as 1 h post-incubation (Fournier et al., 2004b) (Fig. 1S,Supplemental data). Only MEDINA, one of two importantintermediates (MEDINA and NDAB) indicative of HMXdegradation, was found in the analysis. MEDINA[M þ HCOO � 206.10] was identified at 4, 6 and 8 h afterincubation with HMX at 135.00 Da (Fig. 2S, Supplementaldata). There was no metabolite that matched the molecularmass of NDAB in the analysis. Analysis was not performed toquantify CO2 in order to completely show mineralization ofHMX. The qualitative LC-MS/MS data suggested completedisappearance of HMX after 16 h of incubation in WRF (datanot shown).
3.3. Classification of 16S rRNA HMX clones
Time-point PCR products from HMX-T (0) h and HMX-T(8) h (treatment) and HMX-C (0) h and HMX-C (8) h (control)were cloned, sequenced and classified to their phylum levelusing the Classifier tool of the RDPII database (Wang et al.,2007) (Table 2). A total of 1006 clones were used in theclassification process of both the treatment and controlsamples. In all control and treatment samples, the predominantmicrobial communities belonged to either the phyla Firmi-cutes or Bacteroidetes (Table 2). The minor components of therumen consisted of clones associated with the phyla Actino-bacter, TM7 and Proteobacteria.
In HMX-T (0) h treatment, out of 273 sequenced clones,the RDPII Classifier tool placed 142 clones into the phylumFirmicutes and 32 clones into the phylum Bacteroidetes. Ofthe 142 clones associated with the phylum Firmicutes, 93sequences or 65.49% of the clones were classified only to theclass/order level (data not shown). For the phylum Bacter-oidetes, out of a total of 32 classified clones, 18 clones or
was incubated in Bulge tubes and time-point samples analyzed using HPLC.
times 0, 4 and 8 h. Values indicate the HMX concentration remaining in the
replicate analyses. Panel B) LCeMS/MS analysis of the parent HMX at time-
e injected into LCeMS/MS and analyzed as mentioned in the Materials and
-incubation.
Table 2
Tabulated classification of 16S rRNA-V3 clones associated with HMX (T) and HMX (C) samples at 0 and 8 h at the phylum level using the Naı̈ve Bayesian
classifier of the RDPII website at a confidence interval of 50%. Trimmed and chimera-checked DNA sequences from both HMX (T) and HMX (C) samples
submitted as a FASTA file to the Classifier tool of the RDPII database. The table also represents the percentage each phylum with respect to the total sampled
population and significant treatments ( p < 0.01) using Libshuff analysis.
Phylum Treatment
HMXeT (0)b HMXeC (0) HMXeT (8)b HMXeC (8)
No. of clones Total (%) No. of clones Total (%) No. of clones Total (%) No. of clones Total (%)
Actinobacteria 4 (1.46) 3 (1.15) 1 (0.38) 1 (0.40)
Bacteroidetes 32 (11.72) 119 (53.36) 60c (23.07) 126 (50.40)
Chloroflexi 2 (0.73)
Firmicutes 142 (52.01) 75 (33.63) 138 (53.07) 101 (40.40)
Lentisphaerea 1 (0.36)
Planctomycetes 2 (0.76)
Proteobacteria 5 (1.83) 3 (1.34) 9 (3.46) 1 (0.40)
Synergistetes 3 (1.09) 2 (0.76) 1 (0.40)
Tenericutes 1 (0.38)
TM7 28c (10.25) 3 (1.34) 11 (4.23) 4 (1.60)aUncla bacteria 50 (18.31) 18 (8.07) 30 (11.53) 14 (5.60)
Verrucomicrobia 5 (1.83) 6 (2.30) 2 (0.80)
Total 273 100.00 223 100.00 260 100.00 250 100.00
a Unclassified bacteria.b Indicates Libshuff significance between treatments ( p < 0.01).c Indicates LibCompare significance between treatments ( p < 0.01).
571S. Perumbakkam, A.M. Craig / Research in Microbiology 163 (2012) 567e575
56.25% were classified to the class/order level (data notshown). Unclassified clostridiales (51 clones) made up thesingle largest phylotype. A large percentage of the clones (34clones or 23.94%) were unable to be placed in any phyla andwere classified as unclassified bacteria by the RDPII Classifiertool. There was an abundance of clones that belonged to thephylum TM7 (29 clones or 10.62%) using only this treatment.
In HMX-C (0) h treatment, out of 223 clones, the RDPIIClassifier tool placed 119 clones in the phylum Bacteroidetesand 75 clones in the phylum Firmicutes. Of the 119 clonesassociated with the phylum Bacteroidetes, 90 sequences or75.63% of the clones were classified as belonging to the genusPrevotella, forming the most abundant clone population(Stevenson and Weimer, 2007). For the phylum Firmicutes,out of a total of 75 classified clones, 54 clones or 72.00% wereclassified to the class/order level (data not shown).
In HMX-T (8) h treatment, out of 260 clones, the RDPIIClassifier tool placed 138 clones in the phylum Firmicutes and60 clones in the phylum Bacteroidetes. Of the 138 clonesassociated with the phylum Firmicutes, 97 sequences or70.23% of the clones were classified only to the class/orderlevel (data not shown). The genus Prevotella (36 clones or
Table 3
Phylogenetic analyses of HMX (T) and HMX (C) treatments at sampling time poin
program. OTUs common to treatments are also tabulated. All data represent OTU
Treatment aObserved OTUs bCommon OTUs
HMXeC (0) HMXeT (8) HMXeC
HMXeT (0) 133 36 52 36
HMXeC (0) 131 50 47
HMXeT (8) 157 50
HMXeC (8) 127
a ChaoI observed OTUs at 0.03% cutoff.b ChaoI estimated OTUs at 0.03% cutoff.c Number of OTUs common to treatments.
60.00%), belonging to the phylum Bacteroidetes was the mostabundant clone. In HMX-C (8) h treatment, out of 250 clones,the RDPII Classifier tool placed 126 clones in the phylumBacteroidetes and 101 clones in the phylum Firmicutes. Of the126 clones associated with the phylum Bacteroidetes, 83sequences or 65.87% of the clones were classified asbelonging to the genus Prevotella forming the most abundantclone population. For the phylum Firmicutes, out of a total of103 classified clones, 68 clones or 65.87% were classified tothe class/order level (data not shown).
3.4. ChaoI, rarefaction, Libshuff and LibCompareanalyses of HMX treatments
The total observed and ChaoI estimated OTUs for allgroups (HMX-T (0), HMX-T (8), HMX-C (0) and HMX-C(8)) were 360 and 714.83 respectively (Table 3). Theobserved OTU estimate for HMX treatments between time-points 0 and 8 h were 133 and 157, with 52 OTUs commonto both samples. In control samples, the observed OTUs were141 and 134 OTUs for the 0 and 8 h time-point with 40 OTUscommon. ChaoI estimates placed 303.04 and 391.80 OTUs
ts 0 and 8 h. Observed and estimated OTUs were calculated using the Mothur
Classification at a cutoff at 3%.
cEstimated OTUs bCommon OTUs
(8) HMXeC (0) HMXeT (8) HMXeC (8)
303.04 175.35 178.72 179.07
441.40 246.37 91.10
391.80 129.69
239.85
572 S. Perumbakkam, A.M. Craig / Research in Microbiology 163 (2012) 567e575
common to the HMX treatment samples at time periods 0 and8 h. The control treatment had 441.40 and 239.85 OTUs forthe same time period. ACE, Simpson and Shannon-Weaverindex analyses of HMX samples were also performed (Table1S, Supplemental data).
A three-way comparison between the treatments also esti-mated the shared OTUs between treatments. The number ofspecies shared between groups HMX-T (0), HMX-T (8), andHMX-C (0) was 27 OTUs (data not shown). The number ofspecies shared between groups HMX-T (0), HMX-C (0) andHMX-C (8) was 21 OTUs. The number of species sharedbetween groups HMX-T (0), HMX-T (8) and HMX-C (8) was27 OTUs. The number of species shared between groupsHMX-C (0), HMX-T (8) and HMX-C (8) was 26 OTUs (datanot shown).
Libshuff analysis was performed to show significantdifferences between HMX treatments and controls as a func-tion of HMX detoxification (Table 2). There were significantdifferences ( p < 0.0001) between microbial populations atHMX-T (0) and HMX-T (8). To associate the phylum/class ofbacteria associated with the changes, LibCompare analysiswas performed (Table 4). Results indicated that the changes inpopulation principally involved clones associated with phylumTM7 and Bacteroidetes. Phylum TM7 had 28 clones associ-ated with it in HMX-T (0) treatment and 11 clones in HMX-T(8) treatment, indicating a significant reduction in clonesassociated with this phylum (Table 4). The clones associatedwith the phylum Bacteroidetes showed a significant increasebetween the initial and final treatment population. There were33 clones associated with HMX-T (0) treatment and thissignificantly increased ( p < 0.01) to 62 clones in HMX-T (8)treatment. OTUs that contributed to this significant increasewere associated with the genus Prevotella.
4. Discussion
In this study, we examined rumen for its potential todetoxify HMX, and we sought to understand associatedchanges in bacterial community structure. By combininganalytical chemistry and molecular microbial ecology tech-niques, we were able to show that whole rumen fluid (WRF) iscapable of biological transformation of a toxic molecule andwe identified the key genus involved in the enrichmentprocess.
Table 4
LibCompare analyses of HMX (T) 0 h and HMX (T) 8 h samples. 16S rRNA-
V3 clone library data was submitted to the RDPII database and results tabu-
lated using the LibCompare tool at a confidence interval of 50%.
Bacterial classification HMX (O h) HMX (8 h) aSignificance
Phylum TM7 28 11 7.58E�3
Genus TM7 genera
incertae sedis
28 11 7.58E�3
Phylum Bacteroidetes 27 58 3.84E�4
Class Bacteroidia 22 45 1.28E�3
Family Prevotellaceae 14 40 8.00E�5
Genus Prevotella 13 36 2.80E�4
a LibCompare significance at ( p < 0.01).
Based on results of HPLC analysis, we showed that WRFcompletely detoxified HMX (20 mg ml�1) in less than 16 h(data not shown). There was a significant reduction in theparent molecule in less than 4 h (w60%). This reduction wasqualitatively verified using LCeMS/MS. WRF detoxified theHMX relatively quickly compared to other previous mixedcommunity studies (Hawari et al., 2001; Monteil-Rivera et al.,2003; Zhao et al., 2004) and single species experiments(Bhushan et al., 2004; Fournier et al., 2004b).
The rumen is a reduced environment and has evolved todetoxify various plant toxins such as pyrrolizidine alkaloids(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 Lormeand Craig, 2009; Perumbakkam et al., 2011). Two indepen-dent mechanisms are responsible for the degradation of HMX(Hawari et al., 2001). The first step involves the familiarreduction of N-NO2 to form the corresponding nitroso deriv-atives. The other mechanism involves initial denitration fol-lowed by direct ring cleavage. The qualitative data from thepresent study have shown that HMX is detoxified to MEDINAwith the intermediate formation of 1-NO-HMX[M þ HCOO � HNO]. Based on our data, we propose twopathways in detoxification of the HMX molecule: A reductivemechanism combined with a hydrolysis-based pathway. Bothpathways act on the parent molecule as described previously(Hawari et al., 2001). The formation of mono-nitroso products(HMX-NO2) is indicative of the reductive mechanism,whereas the formation of MEDINA is characteristic ofhydrolysis (Hawari et al., 2001). Most researchers concludedthat 1-NO-HMX is not toxic to the microorganisms,andproposed two routes of breakdown. In the first method, 1-NO-HMX undergoes N-denitration, leading to the formation of anintermediate that is reactive with one molecule of water toproduce R-hydroxy-alkylnitramine intermediate (Fournieret al., 2004b). In the second mechanism, 1-NO-HMX mightbe subjected to R-hydroxylation, as frequently observedduring enzymatic degradation of di-alkylnitrosamines. The R-hydroxy-alkylnitramine intermediates, since they are unstable,decompose to produce the ring cleavage intermediates NDAB,N2O, and HCHO (Fournier et al., 2004b). In this study, we didnot find NBAD in our metabolic profile. The second metab-olite identified in our analysis was methylenedinitramine orMEDINA. It is known that MEDINA is unstable in aqueoussolution and eventually decomposes to produce HCHO andN2O (Halasz et al., 2002). These degradation products makethe intermediates harmless to the microorganisms.
The degradation pathways could act independently or inconjunction with one another. In the case of nitroaromaticcompounds, we assume that once the parent molecule hasentered a reductive pathway by losing the first oxygen(Fleischmann et al., 2004), it starts a cascade of reactions tooquick to be captured at intervals of even less than 15 min, andthus of a transient nature (Crocker et al., 2006). AlthoughWRF was sampled every 15 min for the first 2 h of incubationand 2 h thereafter for 16 h, we were unable to find nitroso
Fig. 2. Rarefaction plot of HMX (T) and HMX (C) conditions with the 16S
rRNA-V3 primers as calculated by the Mothur program. The estimates plotted
represent 3% cutoff numbers of observed OTUs in relation to the number of
sequences sampled. All filled symbols represent 3% cutoff. (C) represents the
observed OTUs at 3% difference in the HMX (T) 0 h sample, (-) at 3%
difference in the HMX (T) 8 h sample, (:) at 3% difference in the HMX (C)
0 h and (;) represents the 3% difference in the HMX (C) 0 h.
573S. Perumbakkam, A.M. Craig / Research in Microbiology 163 (2012) 567e575
intermediates other than the initial product (1-NO-HMX[M þ HCOO � HNO]) due to the transient nature, complexityof the matrix or amounts of the metabolites formed. Anotherreason for not identifying metabolites could be due toformation of conjugates, which have previously been shown tooccur with TNT (Smith et al., 2008). Such a mechanism isunlikely to occur due to the absence of forage particles in theWRF, but there might be residual molecules from theseorganic particles that could interact with the munitionintermediates.
On the microbial front, the rumen is very diverse, with anestimated 1000 OTUs (Hess et al., 2011). From availablesequence data, using the 16S rRNA-V3 gene marker, it is clearthat most sequences fall into the two major phyla, Firmicutesand Bacteroidetes. These OTU estimates (Table 3) arecomparable to other rumen studies using Sanger-basedsequencing (Edwards et al., 2004; Perumbakkam and Craig,2011). Due to the high diversity of microorganisms in therumen, the rumen has developed various biochemical strate-gies to derive nutrition from complex plant-based substrates.The munitions compounds are mostly cyclic in structure andmimic plant-based substrates. This leads to non-specificenzyme attack and reduction of munitions in the rumen.These products are broken down further and are either used upby the microbes or the host.
One observation from gut-microflora-associated studies isthe presence of either the phylum Firmicutes and Bacter-oidetes as a majority. Such data is seen in humans (Turnbaughet al., 2007), birds (Godoy-Vitorino et al., 2008) and ruminants(Edwards et al., 2004; Perumbakkam and Craig, 2011). Thisdata is interesting due to the diverse nutritional strategies andphysiologies of the associated examples. Ruminants such assheep and cows are foregut fermenters, whereas humans andhorses are hindgut fermenters. Although having completelydifferent physiology and anatomy, they still possess either thephylum Firmicutes and Bacteroidetes in the major gut flora. Isthis due to the general nature of physiology or to the lack totools for understanding differences between them?
Previously, it was shown that the phylum Proteobacteriaalso forms a large part of the rumen population (Perumbakkamand Craig, 2011). In this study, the number of clones associ-ated with this phylum was minimal (18 clones). One couldargue that the grazing nature of sheep would introduce a largepopulation of Proteobacteria; a large phylum is well repre-sented in soil-based ecosystems, in the rumen. We feel thatthis is due to some kind of bias involved either in PCR orcloning.
There were significant differences in bacterial communitiesin the rumen between the two HMX treatments (0 and 8 h).The number of clones associated with the phylum Bacteroidesincreased in 8 h HMX treatment compared to the 0 h timepoint (Table 4). Specifically, clones associated with the familyPrevotellaceae and the genus Prevotella showed a significantincrease. Members of the genus Prevotella are regarded as themost dominant bacterium in the rumen (Stevenson andWeimer, 2007). The significant increase in the number ofBacteroides OTUs in group 0 vs. 8 h suggests that the bacteria
from this genus were enriched during the breakdown of HMX.Clones associated with the genus TM7 were also abundant inthe 0 h HMX treatment, but not at 8 h. Such enrichment ofcertain microbes from a community was also seen with TNT-associated rumen degradation, where Ruminococcus sp. wasenriched (Perumbakkam et al., 2011). There is also thepossibility of a consortium-based reduction in these munitionproducts in the rumen due to the very large number ofmicrobes and the energy needed to sustain such populations.
In the present study, there were differences in the initial 0 htime-point between treatment and control HMX tubes (Table 2).There are two possible explanations for these results. The firstcould lie in the sampling limitation, as shown by the absenceof an asymptotic curve with rarefaction data (Fig. 2). Thesecond might be due to bias induced by cloning. Althoughthere were differences between control and treatment tubes,LibCompare analysis was only performed between HMXtreatments (0 h and 8 h), but not between control and treatmenttubes, making these observed changes in treatment valid. Boththese limitations could be circumvented with the use of next-generation sequencing (NGS) techniques like 454 or Illuminasequencing, that would do away with cloning sequences andincrease the length and number of reads. We are presentlyutilizing NGS technology for understanding population levelchanges and optimizing our analytical methods to extract otherpossible metabolites of HMX degradation from this verycomplex matrix, whole rumen fluid.
In conclusion, microbes in the rumen have been shown tobe responsible for breakdown munitions such as TNT andRDX in previous studies and HMX in this study, makingruminants “bio-reactors on hooves.” This bioreactor is pres-ently being used in field trials. To understand the microbiologyof such degradation, pure culture representatives of the genusPrevotella are presently been studied to establish the pathwayand understand the regulation of HMX degradation.
Acknowledgments
This research was supported by a jointly funded grant bythe Oregon Agricultural Experiment Station project
574 S. Perumbakkam, A.M. Craig / Research in Microbiology 163 (2012) 567e575
ORE00871 and by the US Department of Agriculture underproject number 6227-21310-007-00D agreement nos. 58-6227-8-044 and 58-1265-6-076. Any opinions, findings,conclusion and recommendations expressed in this publicationare those of the author(s) and do not necessarily reflect theview of the US Department of Agriculture. The authors wouldlike to thank Ms. Karen Walker for her help with HPLC, LiaMurty with LCeMS/MS assistance and Ms. Zelda Zimmer-man for editorial assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found athttp://dx.doi.org/10.1016/j.resmic.2012.08.001.
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