This article was downloaded by: [Florida Atlantic University]On: 12 November 2014, At: 10:43Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Soil and Sediment Contamination: AnInternational JournalPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/bssc20

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX)Biodegradation Kinetics Amongst SeveralFe(III)-Reducing GeneraMan Jae Kwon a & Kevin T. Finneran aa Environmental Engineering and Sciences, Department of Civiland Environmental Engineering , University of Illinois at Urbana-Champaign , Urbana, IL, USAPublished online: 25 Feb 2008.

To cite this article: Man Jae Kwon & Kevin T. Finneran (2008) Hexahydro-1,3,5-trinitro-1,3,5-triazine(RDX) and Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) Biodegradation Kinetics AmongstSeveral Fe(III)-Reducing Genera, Soil and Sediment Contamination: An International Journal, 17:2,189-203

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Soil & Sediment Contamination, 17:189–203, 2008Copyright © Taylor & Francis Group, LLCISSN: 1532-0383 print / 1549-7887 onlineDOI: 10.1080/15320380701873132

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) andOctahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine

(HMX) Biodegradation Kinetics Amongst SeveralFe(III)-Reducing Genera

MAN JAE KWON AND KEVIN T. FINNERAN

Environmental Engineering and Sciences, Department of Civil andEnvironmental Engineering, University of Illinois at Urbana-Champaign,Urbana, IL, USA

Cyclic nitramine explosives biodegradation was investigated among four Fe(III)- andquinone-reducing bacterial genera. This strategy is an attractive option for RDX and/orHMX contamination because of their ubiquity; however, the biotransformation kineticsamong different microbial populations is not known. The organisms investigated in-cluded two species within the Geobacteraceae, and one species each within the generaAnaeromyxobacter, Desulfitobacterium, and Shewanella. All species directly reducedRDX; however, humic substances (HS) and the HS analog anthraquinone-2,6-disulfonate(AQDS) significantly increased the rate and extent of RDX reduction. Degradation ki-netics varied amongst the species tested, but extracellular electron shuttle mediateddegradation rates were the fastest for each organism. RDX reduction rates ranged from7.4 to 269.3 nmol RDX hr−1 mg cell protein−1 when AQDS was present. HMX wasreduced more slowly by G. metallireducens than RDX; however, electron shuttles alsostimulated HMX degradation. These data suggest that electron shuttle mediated cyclicnitramine transformation is ubiquitous among the keystone Fe(III)-reducing microbialgenera, and that bioremediation strategies predicated on their physiology may be areasonable approach in situ for both Fe(III)-rich and Fe(III)-poor environments.

Keywords Bioremediation, RDX, HMX, electron shuttling, Fe(III)-reducing microor-ganism

Introduction

The cyclic nitramine explosives hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) andoctahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) are contaminants at many militarysites and live-fire training installations (Haas et al., 1990; Adam et al., 2004; Hawari and

We thank Robert Sanford (University of Illinois, Geology) for providing cultures ofAnaeromyxobacter and Desulfitobacterium. We thank Kelly Nevin (University of Massachusetts, Mi-crobiology) for cultures of Geobacteraceae. We also thank Joseph W. Stucki (University of Illinois,Natural Resources and Environmental Sciences) for providing cultures ofShewanella. We thank ClintArnett (USACE) for cyclic nitramine explosives. This work was supported by the Department ofDefense Strategic Environmental Research and Development Program (SERDP) project numberER-1377.

Address correspondence to Kevin T. Finneran, 3221 Newmark Civil Engineering LaboratoryMC-250, 205 North Mathews Avenue, Urbana, IL 61801, USA. E-mail: finneran@uiuc.edu

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190 M. J. Kwon and K. T. Finneran

Halasz, 2002). RDX (60 mg/L [270 µM]) and HMX (5 mg/L [17 µM]) solubility in wateris relatively low (Talmage et al., 1999); however, once dissolved they can migrate rapidlythrough the vadose and saturated zones to contaminate down-gradient aquifers. Ground-water contaminated by these explosives is of growing environmental concern because ofhuman health effects of RDX and HMX. RDX is a possible human carcinogen (lifetimehealth advisory for exposure to RDX in drinking water: 2 µg/L [9 nM]), and HMX dam-ages the central nervous system (lifetime health advisory for exposure to HMX in drinkingwater: 0.4 mg/L [1.4 µM]) (Palmer et al., 1996; Lachance et al., 1999). Despite severalreports suggesting that these compounds are readily biodegraded, RDX and HMX persistin subsurface environments (Meyers et al., 2007).

Bioremediation is one alternative for attenuating explosives contamination and severalmechanisms have been proposed using pure and mixed cultures, or contaminated sedimentor soil (Adrian et al., 2003; Adrian and Arnett, 2004; Sherburne et al., 2005; Thompsonet al., 2005; Meyers et al., 2007). Anaerobic biodegradation in particular has been suggestedas an effective strategy for RDX and HMX because of the nitro → nitroso degradation path-way (initial reductive step) (Adrian et al., 2003) and the fact that subsurface RDX or HMXplumes can be anaerobic (Adrian and Arnett, 2004). Direct microbial RDX or HMX reduc-tion has been reported (Zhao et al., 2002; Zhao et al., 2003; Boopathy and Manning, 2000),and this process can be catalyzed by microorganisms that specifically transfer electronsto nitramine compounds; however, without these microorganisms, the overall biodegrada-tion kinetics will be limited. There is still general disagreement whether microorganismsinvolved reduce RDX via co-metabolic reactions (i.e. non-energy generating reactions) orthrough direct respiration. It is likely some combination of both pathways, but the referencesabove generally describe co-metabolic reactions. Alternate degradation pathways are pos-sible, but are less easily stimulated in subsurface environments (Pennington and Brannon,2002; Crocker et al., 2006).

Cyclic nitramine biodegradation mediated by Fe(III)-reducing microorganisms and theextracellular electron transfer reactions catalyzed by these organisms may be another rea-sonable approach based on their ubiquity in subsurface environments (Coates et al., 1998).Recently, the role of fermentative cultures in reductive transformation of explosive com-pounds was investigated with humic substances (HS) and anthraquinone-2,6-disulfonate(AQDS) (Borch et al., 2005; Bhushan et al., 2006). The degradation of TNT was stimulatedby adding AQDS to fermentative cultures (Borch et al., 2005); however, TNT reduction isdifferent than cyclic nitramine biodegradation as TNT interacts with more reduced com-pounds than RDX or HMX. A fermentative bacterium, Clostridium sp. EDB2, effectivelydegraded RDX and HMX (Bhushan et al., 2006). However, adding AQDS did not signif-icantly increase the explosives reduction rates. The same group recently isolated a novelShewanella species, but these reactions have yet to be reported with that culture (Zhao et al.,2005). Although these data contribute to the potential strategies for explosives residues, itis unclear whether the specific fermentative organisms used within these experiments areubiquitous in explosives contaminated environments or can be directly stimulated.

Recently, Kwon and Finneran (2006) reported that remediation strategies that manipu-late Fe(III) reducers via extracellular electron shuttling are reasonable because they do notrely on organisms that must directly transfer electrons to RDX (Lovley and Phillips, 1986;Coates et al., 1995; Kashefi and Lovley, 2003). Despite the successful reduction of RDXwith two Geobacter species, the rates and extent of RDX between these organisms weredifferent in the presence or absence of Fe(III) oxide and/or electron shuttles. In addition,since Geobacteraceae proliferate in strictly anoxic environments, RDX or HMX reductionat the fringe between aerobic and anaerobic conditions may be limited.

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Fe(III)-Reduction Mediated RDX Biodegradation 191

In this study, one Geobacter species (G. metallireducens) and three non-Geobacteraceae Fe(III)-reducing genera also capable of reducing extracellular electronshuttles were investigated to identify the kinetics of RDX and HMX degradation.Anaeromyxobacter and Shewanella, gram-negative facultative anaerobes, and Desulfito-bacterium, gram-positive strict anaerobes were used in addition to the Geobacteraceae todetermine differences in RDX reduction amongst the “model” Fe(III)-reducing cells andhow the electron shuttling strategy will work in the presence or absence of Fe(III).

Material and Methods

Chemicals

RDX (97% pure) and HMX (99.8% pure) were provided by the U.S. Army Corpsof Engineers, Construction Engineering Research Laboratory (CERL), Champaign, IL.Hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX; 99%), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX; 58% pure with 34% MNX and 8% TNX), hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX; >99.9%), and octahydro-1-nitroso-3,5,7-trinitro-1,3,5,7-tetrazocine (1-NO-HMX; >98%) were obtained from SRI International (Menlo Park,CA, USA). Purified humic acid and anthrquinone-2,6-disulfonate (AQDS) were obtainedfrom Sigma Aldrich (Milwaukee, WI, USA). HPLC grade methanol was obtained fromAldrich Chemicals. All other chemicals used were of reagent grade quality or higher.

Microorganisms

Geobacter metallireducens strain GS-15 (ATCC 53774) was originally obtained from theUniversity of Massachusetts at Amherst and maintained using the Ferric Citrate and AQDSmedia described below. Anaeromyxobacter dehalogenans strain K (no ATCC number) andDesulfitobacterium chlororespirans strain Co23 (ATCC 700175) were provided by Dr.Robert Sanford, University of Illinois at Urbana Champaign. Shewanella oneidensis strainMR1 (ATCC 700550) was obtained from Dr. Joseph W. Stucki, University of Illinois atUrbana Champaign. Geobacter metallireducens, Anaeromyxobacter dehalogenans, andDesulfitobacterium chlororespirans were maintained using the Ferric Citrate and/or AQDSmedia, while Shewanella oneidensis was maintained on aerobic LB medium. Cultureswere transferred to fresh medium prior to the onset of any experiments. All media aredescribed below.

Medium and Culturing Conditions

The basal anaerobic medium consisted of (g/L unless specified otherwise): NaHCO3 (2.5),NH4Cl (0.25), NaH2PO4·H2O (0.6), KCl (0.1), modified Wolfe’s vitamin and mineral mix-tures (each 10 ml/L) (Lovley et al., 1984) and 1 mL of 1 mM Na2SeO4. Electron acceptorsused with the anaerobic medium included soluble Fe(III) citrate (45 mM), poorly crys-talline Fe(III) oxide (50 mmol/L), or anthraquinone-2,6-disulfonate (AQDS −5 mM). Themedium was buffered using a 30 mM bicarbonate buffer equilibrated with 80:20 (vol/vol)N2:CO2, as previously described (Finneran et al., 2003). Yeast extract (0.01%) was added tothe culture of A. dehalogenans. Acetate (20 mM) or lactate (20 mM) was added as the soleelectron donor, depending on the specific culture. Standard anaerobic and aseptic culturingtechniques were used throughout (Lovley and Phillips, 1988). The aerobic medium was anLB medium that consisted of (g/L) Bacto tryptone (10.0), Bacto yeast extract (5.0), and

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192 M. J. Kwon and K. T. Finneran

NaCl (10.0). The medium was autoclaved prior to inoculation and cultures were transferredin 250–500 mL conical flasks. Individual experiments utilized different culture tubes orbottles, as described below.

Resting Cell Suspensions

G. metallireducens was grown anaerobically in freshwater medium with acetate as the soleelectron donor and Fe(III) citrate as the sole terminal electron acceptor. A. dehalogenans wasgrown anaerobically in freshwater medium with acetate as the sole electron donor, Fe(III)citrate as the sole terminal electron acceptor, and yeast extract (0.01%). D. chlororespiranswas grown anaerobically in freshwater medium with lactate as the sole electron donorand AQDS as the sole terminal electron acceptor. S. oneidensis was grown aerobically inLB medium. One liter of each individual cell culture was harvested during logarithmicgrowth phase and centrifuged at 4225 × gravity for 15 minutes to form a dense cell pellet.Each resultant cell pellet was resuspended in 30 mM bicarbonate buffer under a stream ofanaerobic gas. The washed cells were centrifuged again at 4225 × gravity for 15 minutesand the resultant biomass was resuspended in 4.0 mL of bicarbonate buffer. Cells were usedwithin 30 minutes of processing.

Experimental tubes were prepared by sparging approximately 5.0 mL of 30 mM bicar-bonate buffer with anaerobic N2:CO2 and sealing the buffer under an anaerobic headspace.Electron acceptors or shuttles incubated with the cells including humic acids (0.25 g/L),poorly crystalline Fe(III) oxide (45 mmol/L), AQDS (5 mM), humic acid plus poorly crys-talline Fe(III) oxide, and AQDS plus poorly crystalline Fe(III) oxide. The sterilized stocksolution of humic acids (10 g/L) was prepared by filtration through 0.2 µm PTFE filters(humic acid did not interfere with nitramine quantitation). Cells were incubated at 30◦C.An aliquot (0.3 ml) of the resting cells was added to the sealed pressure tubes to initiateeach experiment.

Samples (0.3 ml) were collected periodically via anaerobic syringe and needle, andsamples were filtered through sterile, 0.2 µm PTFE filters (PALL Life Sciences; filtersdid not interfere with nitramine quantitation) prior to analyses. 0.2 ml of each sample wasused to quantify RDX or HMX at each time point. 0.1 ml of samples was used to quantifyFe(II) concentration in poorly crystalline Fe(III) amended incubations. No more than sevensamples were taken from any incubation; therefore, final volume of each incubation wasapproximately 8 mL at the end of the experiments (80% volume remaining). Total cellularprotein was determined by using the DC Protein Assay (Bio-RAD) and a modified Lowryprotein assay (Lowry et al., 1951). Cell protein normalized decay rates were calculated basedon pseudo first-order degradation coefficients. All experiments were performed in triplicate.

Analytical Techniques

RDX and its metabolites, MNX, DNX and TNX, and HMX and its metabolite, 1-NO-HMX,were analyzed using high-performance liquid chromatography (HPLC) with a variablewavelength photodiode array (PDA) detector (HPLC/UV, Dionex) at 254 nm as describedpreviously (Fournier et al., 2002). The filtered samples were manually injected into a Su-pelcosil LC-CN column (25 cm × 4.6 mm, 5 µm ID) at ambient temperature. A mobilephase consisting of 50% water and 50% methanol was used at a flow rate of 1 mL/min.RDX, MNX, DNX, TNX, HMX, and 1-NO-HMX were compared to certified analyticalstandards in acetonitrile at known concentrations. Aqueous Fe(II) and total solid phase ironconcentrations were quantified by the Ferrozine assay as described previously (Lovley and

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Fe(III)-Reduction Mediated RDX Biodegradation 193

Phillips, 1987). Each 0.1 mL aliquot from the incubations was diluted in 0.5 N HCl and 0.1mL aliquot of the diluted solution was added to 4.9 mL of 1 mM Ferrozine solution, andthen quantified at 562 nm.

Results

RDX Degradation by Anaeromyxobacter dehalogenans Strain K

A. dehalogenans reduced RDX most rapidly in the presence of AQDS (Figure 1A); however,RDX reduction rates by A. dehalogenans were relatively slow compared to the other cultures

Figure 1. RDX reduction in resting cell suspensions of Anaeromyxobacter dehalogenans strain Kwith or without extracellular electron shuttling compounds. Acetate was the sole electron donor. Cellswere incubated in the absence (A) or presence (B) of poorly crystalline Fe(III). Results are the meansof triplicate analyses and bars indicate one standard deviation.

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194 M. J. Kwon and K. T. Finneran

(Table 1). A. dehalogenans directly reduced RDX slowly relative to electron shuttle amendedcells; more than 70% of initial RDX concentration still remained when extracellular electronshuttles were not present. A. dehalogenans did not degrade RDX effectively with poorlycrystalline Fe(III) hydroxide regardless of the presence of extracellular electron shuttles(Figure 1B). MNX, the one nitroso metabolite of RDX, was initially detected but quicklyfell below detection limit in cells plus HS incubations and cells only incubations. Nitrosometabolites did not accumulate in AQDS and/or Fe(III) amended incubations (Table 2).

RDX Degradation by Shewanella oneidensis Strain MR1

RDX was completely reduced within 3 h in S. oneidensis suspensions containing AQDS(Figure 2A). HS-amended RDX reduction rates were relatively slow compared to the othercultures. S. oneidensis also used RDX directly as an electron acceptor. RDX degradationrates were similar regardless of the presence or absence of lactate (Figure 2A and 2B).RDX in poorly crystalline Fe(III) amended incubations was reduced to 30 µM in 65 h;however, AQDS and HS increased RDX reduction when Fe(III) was present (Figure 2B).All incubations except the AQDS-amended suspensions accumulated nitroso metabolitesat concentrations higher than other cultures (Table 2).

RDX Degradation by Desulfitobacterium chlororespirans Strain Co23

D. chlororespirans completely reduced RDX within 4 h and 45 h in suspensions that con-tained AQDS and HS, respectively (Figure 3A). RDX was not completely reduced in anyother treatment. Cells alone (with lactate as the electron donor) reduced RDX to 20 µMin 43 h, but further reduction was limited. D. chlororespirans did not reduce RDX in theabsence of electron donor (lactate). RDX was reduced in the presence of Fe(III); however,this rate was increased by adding AQDS or HS to Fe(III) incubations (Figure 3B). Ni-troso metabolites accumulated in the presence of HS and/or Fe(III). Nitroso metabolitesdid not accumulate in AQDS amended incubations without Fe(III) or within the cells onlyincubations (Table 2).

HMX Degradation by G. metallireducen

The initial concentration of HMX in the resting cell suspensions of G. metallireducenswas ca. 7 µM, due to the low solubility of HMX in water. Extracellular electron shuttlesstimulated HMX reduction; 7 µM HMX was reduced to approximately 0.5 µM in 35 hin AQDS- or HS-amended incubations (data not shown). Cells-only incubations reducedHMX to 4 µM in the same time period. Resting cell suspensions with only poorly crystallineFe(III) oxide reduced HMX to 4.5 µM in 35 h, while adding AQDS to Fe(III) increasedHMX reduction rates (Table 1). Nitroso metabolites of HMX accumulated in the presenceof HS and/or Fe(III), while their accumulation in AQDS-only amended incubations andcells only incubations was insignificant (Table 2).

Discussion

These results demonstrate that four genera of Fe(III)-reducing bacteria transform RDX(and one at least HMX) and that extracellular electron shuttles increase the rate and extentof RDX and HMX biodegradation catalyzed by Fe(III)-reducing microorganisms. Cyclicnitramine biodegradation (directly and via extracellular electron transfer) is ubiquitous

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196

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Fe(III)-Reduction Mediated RDX Biodegradation 197

Figure 2. RDX reduction in resting cell suspensions of Shewanella oneidensis strain MR1 with orwithout extracellular electron shuttling compounds. Lactate was the sole electron donor. Cells wereincubated in the absence (A) or presence (B) of poorly crystalline Fe(III). Results are the means oftriplicate analyses and bars indicate one standard deviation.

among the “model” Fe(III)-reducing Bacteria (i.e. Fe(III)-reducing microorganisms thatdominate anoxic environments both in cell density and phylogenetic distribution). Thissuggests that cyclic nitramines may not persist in subsurface environments dominated byFe(III) reduction, particularly when extracellular electron shuttling compounds are present.Although it is possible to stimulate the reduction of explosives with the addition of organiccarbon alone, the reported degradation rates of cyclic nitramines under anaerobic conditionshave varied (Hawari et al., 2000; Zhao et al., 2002; Wani and Davis, 2003; Wani andDavis, 2006). Electron shuttling can promote nitramine reduction by accepting electrons

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198 M. J. Kwon and K. T. Finneran

Figure 3. RDX reduction in resting cell suspensions of Desulfitobacterium chlororespirans strainCo23 with or without extracellular electron shuttling compounds. Lactate was the sole electron donor.Cells were incubated in the absence (A) or presence (B) of poorly crystalline Fe(III). Results are themeans of triplicate analyses and bars indicate one standard deviation.

in microbial respiration and abiotically transferring the electrons to RDX or HMX at ratessignificantly faster than those previously reported. The electron shuttles are re-oxidizedand available again for microbial respiration; in this manner the shuttles are catalytic andonly a small concentration would be needed to promote these reactions in bioremediationapplications.

RDX reduction rates were slightly different between two previously tested strains(Kwon and Finneran, 2006); G. sulfurreducens degraded RDX 2 or 6 times faster than G.metallireducens when amended with AQDS or HS, respectively. HS had a more marked

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Fe(III)-Reduction Mediated RDX Biodegradation 199

effect on G. sulfurreducens; however, this may be due to the fact that cells alone of G.metallireducens reduced RDX more efficiently. Reduction rates were also different amongall of the species tested above.

Several of the genera reduced RDX efficiently without an added electron donor. Thisis likely due to a phenomenon called endogenous respiration, in which the cells utilizedecayed cellular biomass as a substrate for respiratory processes (which is common for cellsuspensions) (Fredrickson et al., 2000). The organisms tested do not form storage moleculesunder the conditions tested. RDX reduction was also not attributed to non-specific reductionvia reduced membrane cytochromes, as air-oxidized cells did not behave differently whenincubated in the cell suspension. D. chlororespirans was the exception, and it required anelectron donor to promote these reactions. Further investigation is necessary to identifythe reason that the sole gram-positive member of the experiments did not follow the sameendogenous respiration trend.

Degradation of RDX/HMX by Fe(III)-Reducing Microorganisms

Anaeromyxobacter species are a newly defined group of facultative Fe(III) and electronshuttle reducing bacteria. The full extent of their capacity to reduce extracellular quinonesand HS has not been characterized, but those tested respire and grow via AQDS reduction(data not shown). Cells directly reduced RDX (Table 1). Although AQDS and HS increasedthe rate and extent of RDX reduction, the degradation rates of RDX by Anaeromyxobacterspecies were relatively slow compared to other species tested in this study. Given thatthey are facultative anaerobes, they may also be relevant in subsurface environments wheredominant electron accepting processes are shifting, such as the boundary between aerobicand anaerobic zones.

Shewanella represent one of the model Fe(III) and electron shuttle reducing genera. Inaddition, several reports suggest RDX reducing enrichments from marine sediment weredominated by Shewanella species, and an isolate from the enrichment was within the genusShewanella (Zhao et al., 2004; Zhao et al., 2005). Shewanella are reported to producesoluble electron transfer molecules which can reduce electron acceptors outside of thecell membrane (Hernandez and Newman, 2001). Perhaps this metabolic capacity makesShewanella particularly adept with cyclic nitramines, which are reduced quickly by extra-cellular electron shuttling compounds. Also, like Anaeromyxobacter, the Shewanella arefacultative anaerobes, which suggests that they may dominate RDX (or other nitramines)reduction at the fringe between aerobic and anaerobic conditions.

D. chlororespirans is a gram positive, low G+C content, spore-forming bacterium(Sanford et al., 1996). It is reported to reduce HS but to date has not been reported toreduce Fe(III). Other members of the genus including D. metallireducens and D. hafniensecan reduce Fe(III) as well as HS (Beaudet et al., 1998; Niggemyer et al., 2001; Finneranet al., 2002; Rhee et al., 2003; Shelobolina et al., 2003). D. chlororespirans can fermentpyruvate, and it grows via respiration with several electron acceptors (Sanford et al., 1996).Although D. chlororespirans is the only gram positive species among organisms tested, italso directly reduced RDX to some extent suggesting cell membrane/cell wall differencesdid not inhibit direct RDX reduction. AQDS- and HS-mediated RDX reduction was veryfast (Table 1). AQDS and HS also promoted RDX reduction when Fe(III) oxide was present.Given that D. chlororespirans cannot directly reduce Fe(III) oxide electron transfer fromthe reduced electron shuttles was probably split between Fe(III) and RDX, which accountsfor the differences in the rate and extent of RDX degradation when Fe(III) is present.

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200 M. J. Kwon and K. T. Finneran

G. metallireducens stimulated HMX reduction with extracellular electron shuttles(Table 1). HMX was reduced much more slowly than RDX, which is likely due to thepresence of an additional nitro group and possibly differences in standard redox potential.However, the trends of HMX reduction were similar to those of RDX reduction with respectto electron shuttles and Fe(III). These data suggest that the direct or indirect electron trans-fer via extracellular electron shuttles can increase the reduction rates of other explosivescontaining nitro groups in their structure.

Nitroso metabolites (MNX, DNX, and TNX) of RDX accumulated to different ex-tents among the five microorganisms tested and among different amendments (Table 2).In the resting cell suspensions of G. metallireducens, nitroso metabolites of HMX alsoaccumulated to different extents among different treatments (Table 2). The accumulationof nitroso metabolites of RDX (or HMX) in AQDS amended incubations was not sig-nificant or was not detected regardless of different microbial species. Nitroso metabo-lites accumulated to a greater extent in HS and/or poorly crystalline Fe(III) amendedincubations. Other than the AQDS/donor incubations, the decay rates of RDX in twofacultative microorganisms (Anaeromyxobacter and Shewanella) were similar. However,more nitroso metabolites accumulated in Shewanella suspensions than Anaeromyxobactersuspensions.

Competition for Electrons Between RDX and Fe(III)

Fe(III) oxides influenced RDX reduction in the presence or absence of electron shuttles.Reduced shuttles transfer electrons quickly to Fe(III) (Kwon and Finneran, 2006); however,these and previous data indicate that electron transfer to RDX is also very fast (Kwon andFinneran, 2006). The data suggest that there are two possible pathways for RDX reduc-tion: 1) reduced electron shuttles transfer electrons to Fe(III) and RDX is subsequentlyreduced by surface bound Fe(II) (which has been reported by others (Gregory et al., 2004;Williams et al., 2005), or 2) reduced electron shuttles are simultaneously transferring elec-trons to both Fe(III) and RDX. Although there may be some combination of both, thesecond pathway is likely correct based on reaction kinetics and metabolite accumulationpatterns.

The nitroso metabolites MNX, DNX, and TNX also accumulated to higher concentra-tions in systems where Fe(II) was the primary reductant (Gregory et al., 2004; Williamset al., 2005; Kwon and Finneran, 2006). The sequential reduction of HMX to the nitrosometabolites (1-NO-HMX, octahydro-1,3-dinitroso-5,7-dinitro-1,3,5,7-tetrazocine (2-NO-HMX), occasionally octahydro-1,3,5-trinitroso-7-nitro-1,3,5,7-tetrazocine (3-NO-HMX),and octahydro-1,3,5,7-tetranitroso-1,3,5,7-tetrazocine (4-NO-HMX)) by zero-valent ironunder anoxic conditions has also been reported (Monteil-Rivera et al., 2005). Nitrosometabolite accumulation was low or undetectable with AQDS or HS, which suggests fasterreduction through the nitroso series to ring-cleavage products.

This dynamic between Fe(III) and RDX has implications for the in situ strategy; how-ever, the results will likely be similar despite the electron transfer pathway. Both Fe(II)and reduced electron shuttles reduce RDX. The kinetic differences do not preclude us-ing this strategy in sediments with high bioavailable Fe(III). The data clearly indicatethat adding electron shuttles increases RDX reduction when Fe(III) is present. Althoughthe number of possible reactions increases in heterogeneous environments, the prepon-derance of data demonstrates that stimulating Fe(III) reduction increases RDX and HMXtransformation.

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Fe(III)-Reduction Mediated RDX Biodegradation 201

Environmental Implications

The results demonstrate that cyclic nitramine transformation is ubiquitous among theFe(III)-reducing microbial genera most often cited as “environmentally relevant.” Thedegradation patterns amongst all species tested were consistent when electron shuttles wereadded, and electron shuttle amendment resulted in rapid degradation of RDX/HMX. Pastreports indicate that extracellular electron shuttles are catalytic and therefore in situ appli-cations may only require a low concentration of these compounds to stimulate nitraminereduction. Given that that Fe(III)/electron shuttle-reducing microorganisms in general, andin particular the genera tested above, are ubiquitous throughout all environmental media,bioremediation strategies predicated on their physiology and catalytic effects of electronshuttles may be a good approach in situ for both Fe(III)-rich and Fe(III)-poor environments.

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