Biotransformation and Degradation of the Insensitive Munitions

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Biotransformation and Degradation of the Insensitive Munitions Compound, 3-nitro-1,2,4-triazol-5-one (NTO), by Soil Bacterial Communities Mark James Krzmarzick, Raju Khatiwada, Christopher I. Olivares, Leif Abrell, Reyes Sierra-Alvarez, Jon Chorover, and James A. Field Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b00511 • Publication Date (Web): 03 Apr 2015 Downloaded from http://pubs.acs.org on April 5, 2015

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Environmental Science & Technology

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Biotransformation and Degradation of the Insensitive Munitions Compound,

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3-nitro-1,2,4-triazol-5-one (NTO), by Soil Bacterial Communities

3 4

Mark J. Krzmarzick†,§; Raju Khatiwada‡; Christopher I. Olivares†, Leif Abrell‡, Reyes Sierra-

5

Alvarez†, Jon Chorover‡ and James A. Field*,†

6 7

†

8

Arizona, United States

Department of Chemical and Environmental Engineering, University of Arizona, Tucson,

9 10

‡

11

United States

Department of Soil, Water & Environmental Science, University of Arizona, Tucson, Arizona,

12 13 14 15 16 17 18 19 20 21

§

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MJ Krzmarzick; School of Civil and Environmental Engineering, Oklahoma State University, 207

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Engineering South, Stillwater, OK 74078

Present Address:

ACS Paragon Plus Environment


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ABSTRACT: Insensitive munitions (IM) are a new class of explosives that are increasingly

25

being adopted by the military. The ability of soil microbial communities to degrade IMs is

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relatively unknown. In this study, microbial communities from a wide range of soils were tested

27

in microcosms for their ability to degrade the IM, 3-nitro-1,2,4-triazol-5-one (NTO). All seven

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soil inocula tested were able to readily reduce NTO to 3-amino-1,2,4-triazol-5-one (ATO) via 3-

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hydroxylamino-1,2,4-triazol-5-one (HTO), under anaerobic conditions with H2 as an electron

30

donor. Numerous other electron donors were shown to be suitable for NTO-reducing bacteria.

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The addition of a small amount of yeast extract (10 mg/L), was critical to diminish lag times and

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increased the biotransformation rate of NTO in nearly all cases indicating yeast extract provided

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important nutrients for NTO-reducing bacteria. The main biotransformation product, ATO, was

34

degradable only in aerobic conditions, as evidenced by a rise in the inorganic nitrogen species

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nitrite and nitrate, indicative of nitrogen-mineralization. NTO was non-biodegradable in aerobic

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microcosms with all soil inocula.

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TOC/Abstract Art

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41

INTRODUCTION

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Conventional munition compounds, such as 2,4,6-trinitrotoluene (TNT), hexahydro-1,3,5-

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trinitro-1,3,5-triazacyclohexane (RDX),

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(HMX) are often associated with problematic contamination issues and often degrade slowly, if

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at all, in environmental systems [1]. The toxicity and mutagenicity of these compounds is widely

46

known [2-4], and in soils with a long-term exposure to TNT, RDX or HMX, a significant loss in

47

bacterial activity and fungal populations has been observed [5]. The heterocyclic compounds

48

RDX and HMX have a greater propensity towards mineralization [6], while TNT is likely to

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incorporate into bound residues [7, 8]. Recently, insensitive munitions (IM)s are being proposed

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and tested as alternatives to TNT, RDX, and HMX due to their relative safety with regards to

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accidental explosions [9-11]. However little is known concerning their fate and biodegradation

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potential in the environment. One of the IM compounds, 3-nitro-1,2,4-triazol-5-one (NTO), has

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been shown to have lower toxicity and mutagenicity properties and may be less harmful to

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human health than traditional explosives [12-15]. However, NTO may be problematic in the

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environment due to its high solubility [16, 17] and thus increased mobility into groundwater and

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through water systems.

and

octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine

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The biodegradation of NTO has been observed in preliminary studies [16, 18]. Mammalian

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cytochrome P450 enzymes biotransformed NTO to both urazole or 3-amino-1,2,4-triazol-5-one

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(ATO) under aerobic conditions, while anaerobically NTO is primarily reduced to ATO with

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only a minor yield of urazole [18]. A Bacillus licheniformis was isolated from NTO production

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wastewater and was shown to biotransform NTO to ATO with sucrose at pH 6. ATO was then

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ring cleaved at a higher pH of 8 [16, 18]. This bacterial process was reported to be oxygen

63

insensitive and was carried out with high quantities of NTO, cell mass, and glucose.


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Although there is initial evidence of NTO biotransformation and biodegradation, studies are

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needed to evaluate the biodegradability of NTO in soils where residues of unexploded ordnance

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may end up as contamination in military firing ranges. We report the biodegradation of NTO and

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its main metabolite ATO by microbial communities in diverse soils under aerobic and anaerobic

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conditions. Several different electron donors and the presence and absence of a nutritional

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amount of yeast extract (YE) were also tested. This constitutes the first study to determine if soil

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microbial communities can mineralize NTO nitrogen.

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MATERIAL AND METHODS

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Materials.

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NTO was purchased from Interchem (San Pedro, CA). The synthesis of ATO from NTO was

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adapted from Le Campion and Ouazzani [19]. During the same procedure, 3-hydroxyamino-

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1,2,4-triazol-5-one (HTO) was also synthesized. Details of ATO and HTO synthesis are

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described in Supporting Information (SI). The end product, ATO, was a white, or slightly

78

yellow, powder. The structures of ATO and HTO were verified with high resolution quadrupole-

79

time of flight mass spectrometry (see qTOF-MS below).

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All soils were collected within 20 cm from the surface. Roger Road soil was collected from

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the University of Arizona Campus Agricultural Center in Tucson, AZ. Catlin agricultural soil

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was from Illinois and was described in [20]. Maricopa soil was collected from the University of

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Arizona Maricopa County Agricultural Station in Maricopa County, AZ. Camp Navajo (AZ),

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Camp Butner (NC), Camp Ripley (MN), and Florence (AZ) soils, were collected by CH2M

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HILL at U.S. National Guard bases. All soils, with the exception of Catlin and Roger Road soils,

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were immediately placed on ice and shipped overnight to the laboratory. Roger Road (AZ) soil



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was immediately transferred to the laboratory. All soils with the exception of Catlin were sealed

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to maintain original moisture, while Catlin soil was air dried. All soils were sieved (2 mm) and

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stored at 4oC until used in experiments.

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Solid Phase Characterization of the Soils. The seven soils used in this study were

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characterized for various soil properties (Table 1) including pH, total organic carbon (TOC),

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particle size distribution, and Brunauer-Emmett-Teller (BET) specific surface area.

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mineralogy was also characterized (Supplementary Information (SI), Tables S1-S2). The pH was

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measured on a 1:5 (w/w) mixture of soil and water using a VWR Symphony pH Electrode (VWR

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International, Randor, PA). External specific surface area was determined using the BET

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dinitrogen gas adsorption method (Beckman Coulter SA-3100). TOC was calculated from the

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difference between total carbon and total inorganic carbon. Total carbon was determined by

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combustion at 900ºC and total inorganic carbon was determined by phosphoric acid addition

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followed by combustion at 200ºC using a Shimadzu 5000A-SSM TOC Analyzer (Columbia,

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MD). For particle size analysis, samples were pretreated for organic matter removal and

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analyzed with a fully automated Beckman Coulter LS 13 320 Laser Diffraction Particle Size

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Analyzer (Fullerton, CA). Mineralogical analysis was conducted on both the bulk soil (powder)

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and oriented clays using a PANalytical X’Pert Pro MPD X-ray Diffractometer (XRD) with Cu-

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Kα radiation source for qualitative and quantitative identification of the soil minerals. Samples

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were pretreated for the organic matter removal by oxidation with sodium hypochlorite. For

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oriented clay slides, the soil was dispersed using sodium hexametaphosphate and allowed to

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settle overnight. The clay suspension was used for preparing the oriented aggregate for clay

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mineral identification. Minerals were identified based on the expansion and contraction of d-

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spacing of clay minerals with various treatments. The treatments include saturation with MgCl2,


The

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ethylene glycol, KCl at 25ºC and subsequent heating at 300ºC and 550ºC. Quantitative phase

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analysis was performed using the Rietveld module in X’Pert High Score Plus software following

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the methodology described previously [21].

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Degradation Assays. Anaerobic Assays. Anaerobic microcosms were incubated at 30ºC

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in 160 mL serum bottles with butyl-rubber septa. Assays composed of 100 mL contained basal

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mineral medium with trace elements as described previously [22] with the only exception that

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resazurin was not included here. The headspace of the microcosms (60 mL) was flushed with

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He/CO2 (80/20%). In microcosms with H2, 1 bar of H2/CO2 (80/20%) over pressure was added.

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The microcosms were inoculated with 5 g L-1 soil dry weight (or autoclaved soil for killed

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controls). For killed controls, soil was autoclaved for 60 min for three consecutive days.

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Microcosms were autoclaved prior to addition of soil and NTO or ATO was added via filtering a

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20 mM stock solution through a 0.22 µm syringe filter. Samples were taken with sterile 1 mL

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sterile syringes through the septum. Samples (0.5 mL) were immediately spiked with 1 mL of

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ascorbic acid (300 mg L-1). Samples were stored at -20oC until analysis and centrifuged (10 min,

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13,000×g) prior to analytical analysis.

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Aerobic Assays. Aerobic microcosms were incubated at 30oC on a shaker table (180 rpm) in

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200 mL flasks topped with cotton. Assays composed of 100 mL of basal mineral medium

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prepared as above with the exception of 1.1 g L-1 K2HPO4 and 1.7 g L-1 KH2PO4 in lieu of

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NaHCO3. 5 g L-1 of dry weight soil (or autoclaved soil for killed controls) was added to

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microcosms and NTO or ATO was added via a 20 mM stock solution filtered through a 0.22 µm

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syringe filter. NH4Cl and/or YE were excluded in microcosms to further study ATO degradation

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(see below). Samples were taken from sterile pipettes, and flasks with initial medium were

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weighed to correct the concentrations of analytes due to evaporation. All microcosm conditions



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were run in duplicates. Samples (0.5 mL) were immediately spiked with 1 mL of ascorbic acid

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(300 mg L-1). Samples were stored at -20oC and centrifuged (10 min, 13,000×g) prior to

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analytical analysis.

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Initial degradation experiments for NTO were conducted under anaerobic and aerobic

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conditions for all seven soils at pH 7.2. H2 was added in anaerobic microcosms. These

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experiments included killed controls for all soils (aerobic and anaerobic), basal-medium only

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controls (aerobic and anaerobic), and for selected anaerobic soils (Camp Ripley Camp Butner,

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Camp Navajo, and Florence) endogenous controls (no external electron donor).

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Additional anaerobic experiments were conducted with various electron donors (20 mM),

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with and without YE, with Camp Butner soil and included the following: acetate, lactate,

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ethanol, methanol, glucose, pyruvate, citrate, lactose, butyrate, propionate, and formate. H2-

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amended (0.8 bar) microcosms and endogenous controls were also run in parallel.

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Lastly, we tested the variation of pH and glucose amendment on NTO and ATO degradation.

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In these experiments, ATO degradation was tested using both aerobic and anaerobic microcosms

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while NTO degradation was tested only with aerobic microcosms. Assays were run with Camp

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Butner and Camp Navajo soils for each set (ATO, aerobic; ATO, anaerobic; and NTO, aerobic).

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Because previous research shows that ATO can be ring cleaved at mildly alkaline pH [16],

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microcosms were operated at either pH 7.2 or 8.4 and with or without glucose (20 mM) for a

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total of four different treatments for each soil. NH4Cl was excluded from the basal medium to

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quantify inorganic nitrogen resulting from degradation. An additional aliquot of glucose (20

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mM) was added at day 14 to microcosms with glucose. The pH was measured and, if needed,

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adjusted every 7 days. Killed controls were operated for both soils as well as basal-medium only


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controls (at pH 7.2, no glucose). For ATO-aerobic assays, microcosms without ATO were run

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for both soils to characterize soil endogenous release of inorganic nitrogen.

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Analytical Methods. HPLC-DAD. NTO, HTO, and ATO were analyzed using an Agilent

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1200 series (Santa Clara, CA) HPLC-DAD. Samples were diluted (1:3) into 0.1% trifluoroacetic

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acid (TFA) buffer prior to analysis. Injections (5 µL) were separated with a Hypercarb column

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[23] (150 mm × 4.6 mm, 5 µm pore size) at a temperature of 30 oC. The mobile phase (1 mL

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min-1) was operated under the following v/v ratios of 0.1% TFA aqueous buffer and acetonitrile:

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0-3 min 100/0; 11 min 85/15; 15 min 50/50; 17 min 50/50; 19 min 100/0; 20 min 100/0. NTO

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was detected at 15 min/ 340 nm, HTO at 13 min/ 360 nm, and ATO at 8.9 min/ 216.5 nm. Stock

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solutions of NTO and ATO were prepared from dry powder and standards were prepared from

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dilutions of this stock solution in 0.1% TFA to final concentrations of 1.56, 3.13, 6.25, 12.5, 25,

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and 50 mg L-1. HTO stock solution was prepared from a mixed solution of NTO, HTO, and ATO

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(from the synthesis above). This stock solution was diluted in 0.1% TFA buffer and the

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concentration of HTO was determined via a molar balance of the synthesis experiment: the HTO

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was equal to the amount of the initial NTO in the reaction microcosm minus the amount of NTO

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and ATO in the final mixture. Standards of HTO were prepared from this stock solution and

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were at a final concentration of 0.1, 1.5, 2.0, 4.0, 8.0, and 16.0 mg L-1. All standards were linear

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with respect to peak area.

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QToF-MS. Quadrupole time-of-flight mass spectrometry (QToF-MS) analysis was

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performed with aqueous solutions by direct infusion on a TripleTOF 5600 QTOF-MS (AB Sciex,

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Framingham, MA). QToF-MS analysis was used to confirm the MW of ATO and HTO. ATO

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([M+H]+ = 101.0447 detected, 1.1 ppm from expected) was verified via direct infusion of a 1 mg

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L-1 solution in positive mode. HTO ([M+H]+= 117.0390 observed, 1.7 ppm from expected) was



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verified as well. QToF-MS analyses were also used to screen for metabolites in selected

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microcosms. Microcosm contents were centrifuged, as above, and diluted (1:10-1:20 final

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dilution in water v/v) before infused directly into the QToF-MS. Spectra were obtained in both

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positive and negative mode and a mass range of 35-600 m/z was acquired. Analyst TF 15.1 and

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Formula Finder 2.02.0 were used to process data.

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IC. Ammonium (NH4+), nitrite (NO2-), and nitrate (NO3-) were measured with ion

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chromatography (IC) for assays investigating the aerobic degradation of ATO. The IC analyses

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was performed on an ICS-3000 system (Dionex, Sunnyvale, CA) with a split flow for

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simultaneous anion and cation analysis on an AG18 RFIC column (4 × 50 mm, Dionex) and

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IonPac CG16 RFIC column (3 × 50 mm, Dionex), respectively. The eluent flow rate for anion

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analysis was 1 mL min-1 and for cation analysis 0.5 mL min-1. Standards were 3.125, 6.25, 12.5,

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25, 50, and 100 mg L-1 for NH4+ and NO2- and 1.5625, 3.125, 6.25, 12.5, 25 and 50 mg L-1 for

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NO3-.

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Data Analysis. Lag time, degradation rate, initial ATO yield, and fraction of NTO

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converted to ATO were calculated for the anaerobic microcosms. Spearman’s correlation tests

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were performed with Stata 10.1 software.

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RESULTS

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Soil Characteristics. Soils collected and used in this study covered a broad range of

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characteristics (Table 1; and Tables S1-S2 and Figure S1). Five unique textural classes were

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found for the seven soils (Table 1). The clay content ranged from 8% (Camp Ripley) to 42%

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(Roger Road) and the sand content ranged from 14% (Catlin) to 78% (Camp Ripley) The

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amount of secondary minerals also covered a broad range, from a low of 0.62% (Camp Ripley)


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to a high of 44.0% (Catlin). The BET specific surface area of Camp Ripley soil was lowest (1.72

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m2 g-1) and Maricopa was highest (34.58 m2g-1). Maricopa and Roger Road soils had pH in the

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alkaline range (7.75) while Florence soil had near neutral pH, and other soils were on the slightly

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acidic range (6-6.5). TOC content of the soils ranged from 4.16 to 52.36 g kg-1, with Camp

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Navajo soil having the lowest and Flagstaff soil having the highest TOC concentration. Total

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nitrogen, like TOC, was highest for the Flagstaff soil (3.65 g kg-1). Maricopa and Camp Navajo

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had the lowest (0.8 g kg-1) values.

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Anaerobic Reduction of NTO to ATO. In anaerobic microcosms with H2 added as an

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electron donor, NTO was fully biotransformed (Figure 1, Table S3). No biotransformation was

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observed in killed controls or non-inoculated basal media. Endogenous controls (no external

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electron donor) displayed NTO bioconversions rates of 0.03 mM d-1 and 0.006 mM d-1 in Camp

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Navajo soil and in Florence soil microcosms, respectively. When H2 was amended as an electron

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donor, the lag phase prior to NTO biotransformation was less than 5 days in all soils except for

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Roger Road and Maricopa, who had lag phases of ~12 days. This lag phase is putatively due to

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the growth of nitro-group reducing bacteria. Amendments with H2 as electron donor increased

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biotransformation rates, ranging from 0.23 (Roger Road) to 1.25 mM d-1 (Camp Navajo). No

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clear connection between biotransformation rates and TOC or total nitrogen of the soil was

218

observed. There was a negative correlation with soils with alkaline pH that corresponded to

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slower biotransformation rates (Spearman’s ρ= -0.85, P= 0.016).

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ATO was the dominant product from NTO biotransformation under anaerobic conditions.

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ATO formation was concomitant to the removal of NTO, and the yield of ATO production as a

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fraction of NTO removed was stoichiometric, averaging 95.3 ± 9.4% in the H2 amended

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microcosms. Consequently, the final molar concentration of ATO in H2-amended microcosms



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was also nearly equivalent with the initial amount of NTO added to the microcosms (98.1 ± 4.7%

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amongst the 7 soils) since NTO was totally consumed. Thus any loss of NTO to sorption or other

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reactions was minimal. NTO conversion to ATO was dependent on the presence of an external

227

electron donor; the conversion NTO to ATO was very low in endogenous microcosms although

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the yield per unit of NTO removed was similar to that observed in H2-amended microcosms.

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The ability of Camp Butner soil microbial communities to use various electron donors to

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degrade NTO was tested, both with and without a nutritional amount of YE (Figures 2 and S2).

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The electron donors stimulated biotransformation of NTO compared to endogenous controls

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(there was only one exception, methanol without the addition of YE). In almost all cases a

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nutritional quantity of YE (10 mg L-1) decreased the lag phase for NTO reduction. YE did not

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improve the rates of biotransformation if the electron donor could readily serve as carbon source,

235

such as the cases with the citrate or pyruvate amended microcosms (Figure S2 in SI).

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Conversely, YE was responsible for a major improvement in the rate for the electron donors that

237

did not include carbon such as with the H2-amended microcosms, or with the microcosms with

238

electron donors that readily yield H2 upon anaerobic fermentation (e.g. lactate).

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With many of the electron donors, minor amounts of the intermediate HTO were observed

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(Figure 2, Table S4). HTO did not accumulate, probably because it was quickly reduced to ATO.

241

Aerobic Degradation Microcosms. NTO was not degraded under fully aerobic

242

conditions with any of the soils (Table S4). In these microcosms, pH was maintained at 7.2 for at

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least 62 days (and up to 112 days). Two soils, Camp Butner and Camp Navajo, were also tested

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for their ability to aerobically degrade NTO with glucose addition, increased pH, and under

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nitrogen-limiting conditions (no exogenous nitrogen source). With these treatments, NTO was

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not degraded with either soil.


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Degradation of ATO. The degradation of ATO was tested under various conditions with

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Camp Butner and Camp Navajo soils under aerobic or anaerobic conditions, with and without

249

glucose addition, and with neutral pH (7.2) or increased pH (8.5) (Figure 3). ATO did not

250

degrade under any anaerobic condition with either soil. Under aerobic conditions, ATO was

251

found to degrade relatively slowly with both soils as evidenced by loss of parent compound and

252

detection of inorganic N species. With the Camp Butner soil, ATO was degraded in all aerobic

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microcosms tested and the fastest degradation occurred at neutral pH without glucose addition.

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With the Camp Navajo soil, the fastest degradation occurred with pH 8.5 without glucose

255

addition. At pH 7.2 and with glucose addition, the duplicate microcosms were dissimilar, with

256

only one replicate degrading the ATO. Generally, nitrite was found to initially rise, followed by a

257

permanent rise of nitrate, far above the background amount of inorganic nitrogen (0.5 mM). In

258

controls without ATO amendment and with killed controls and non-inoculated media-only

259

controls, inorganic nitrogen species remained very low and did not increase (Figure S3). Thus,

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this increase in inorganic nitrogen observed in live-soil inoculated treatments after the decrease

261

in ATO concentration is indicative of the mineralization of the ATO in these microcosms.

262

Intermediate(s) between ATO and inorganic nitrogen species must exist as is seen from a lack of

263

a molar balance between the amount of degraded ATO and the production of inorganic nitrogen

264

products. Samples from these microcosms were analyzed with QToF-MS but no organic

265

intermediates were detected.

266 267

DISCUSSION

268

Lack of NTO Biodegradation Under Aerobic Conditions. Nitro groups are electron

269

withdrawing making direct oxidation of the molecule difficult. However, nitroaromatic

270

compounds with two or less nitro-groups have been degraded by several oxidative pathways



271

[24]. In other cases, aerobic bacteria will reduce nitro-aromatics to aromatics with

272

hydroxylamino- groups [24, 25] or to amino- groups [26, 27], which are then further

273

metabolized. Less is known concerning the aerobic biodegradation of nitro- heterocyclic

274

compounds that may also require a reduction of the nitro group, but may involve different

275

mechanisms. The reduction of NTO to ATO in this study was only achieved under anaerobic

276

conditions, suggesting the need of an anaerobic step for bioremediation. On the other hand, Le

277

Campion et al. [16] and Richard and Weidhaas [28] observed reduction of NTO in the presence

278

of oxygen using high amounts of cells and rich organic broths as medium (e.g. glucose). High

279

levels of organic substrates in those experiments may have unknowingly produced O2-deficient

280

conditions as the organic substrates were consumed.

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Nitro-group containing organic compounds are likely difficult to biodegrade aerobically in

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soils. Similar to our findings, the degradation of the heterocyclic compound RDX was not

283

degraded by soil microbial communities under aerobic conditions [29]. TNT is also not readily

284

degraded aerobically except when co-amended with other substrates which cause nitro-group

285

reduction to occur [30]. In our study, however, the addition of glucose as a cosubstrate did not

286

aid in the biodegradation of NTO under aerobic conditions.

287

Anaerobic Biotransformation. The anaerobic reduction of NTO to ATO occurred in all

288

seven soils tested indicating that this pathway should be ubiquitous in soil environments if

289

subjected to anaerobic conditions. The facile anaerobic reduction of nitro-groups is common [31-

290

33]. The complete reduction of the nitro- to the amino- group in NTO occurred readily with

291

nearly stoichiometric amounts of ATO produced in nearly all cases. Stoichiometric reduction of

292

NTO to ATO was observed in previous literature with Bacillus licheniformis and mammalian


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liver microsomes [18] and has been observed in environmental soils for RDX [29]. The results

294

here indicate this reduction process is very ubiquitous.

295

Nitroreductases. Specific nitroreductase enzymes have been implicated in some aerobic

296

nitroaromatic degrading bacteria [24, 32]. Less specific oxygen-insensitive nitroreductases of

297

enteric bacteria have been shown to reduce TNT and RDX [32, 34, 35]. Hydrogenases and

298

carbon monoxide dehydrogenase pyruvate:ferrodoxin oxidoreductase of Clostridia have been

299

shown to reduce TNT [32, 33]. Bulk reducing agents generated in anaerobic environments such

300

as sulfide together with redox mediating natural organic matter and ferrous iron adsorbed to iron

301

oxide minerals are also known to reduce nitro-groups abiotically [36, 37]. Though such a

302

mechanism is theoretically possible in our experiments, the relatively low concentrations of

303

sulfate (0.4 mM) amended in the mineral medium would need to be cycled many fold in order to

304

provide the reducing electrons needed to transform the amount of NTO added (3.8 mM). The IM

305

compound, 2,4-dintroanisole (DNAN) was transiently converted to both nitroso- and

306

hydroxylamine intermediates as it was being reduced to 2-amino-4-nitroanisole by an aerobic

307

soil bacteria, Bacillus sp. strain 13G [38].

308

Physiology. An external electron donor was required for rapid and complete

309

biotransformation. In previous studies, the reduction of nitro-organic explosive compounds also

310

needed electron donors, and similar to this study, diverse sources of electron donors were found

311

suitable [33, 39-41]. H2 or substrates that generate H2 during their conversion were the best

312

electron donorsin agreement with the findings in this work on NTO. YE provided a critical

313

nutritional requirement to the soil microflora that enabled them to reduce NTO with a decreased

314

lag phase with nearly every electron donor tested and an increased degradation rate with many of

315

the electron donors. YE provided the greatest improvement of NTO degradation rates when H2



316

was used as an electron donor, indicating that YE may have also served as a heterotrophic carbon

317

source. . This has been observed previously for chemolithotrophic microbial reactions where

318

either pyruvate or YE was needed to stimulate the reduction of perchlorate with elemental sulfur

319

[42]. YE has also been shown to increase the nitro-group reduction of TNT by a Pseudomonas

320

strain [43].

Page 16 of 32

321

Degradation Products. The reduction of nitro- groups occurs via three steps of two

322

electron reductions of nitro- to nitroso-, nitroso- to hydroxylamino-, and hydroxylamino- to

323

amino groups [32]. In this study, NTO was reduced to ATO with small amounts of the

324

hydroxylamino intermediate (HTO) being observed temporally in some assays. The HTO as an

325

intermediate of NTO biotransformation has not been reported previously. TNT is often observed

326

to degrade to mixed amounts of hydroxylamino- and amino- derivatives by bacteria [44-46] and

327

RDX is most commonly associated with a reduction to nitroso-interemediates [38, 47, 48].

328

Hydroxylamino- intermediates are known to be reactive [3, 49]. In studies with DNAN, the

329

mixture of hydroxylamino- and nitroso-intermediates may lead to dimerization products when

330

degraded by bacteria [22, 38]. A similar high level of reactivity was not observed in this study,

331

since there was not a major loss in the stoichiometric yield of ATO in biodegradation

332

experiments even under conditions with the highest HTO intermediate concentration or

333

conditions with the most prolonged exposure to HTO. Additionally, coupling products were

334

sought using MS-QToF yet these products were not found.

335

ATO was never degraded under any of the anaerobic conditions or soils used in this study but

336

degradation was observed under aerobic conditions. In the aerobic microcosms, the nitrogen

337

balance was incomplete after the degradation of ATO and prior to the production of inorganic

338

nitrogen products indicating that intermediates not measured nor detected in this study were


Page 17 of 32


339

produced. Heterocyclic explosives such as RDX and HMX become unstable after initial

340

reduction of the nitro- groups due to the weak energy of carbon-nitrogen bond leading to abiotic

341

hydrolysis [6, 50, 51]. In contrast, ATO was stable in anaerobic conditions and degradation in

342

aerobic conditions occurred slowly. Previous research shows that ATO can be ring cleaved at

343

mildly alkaline pH [16]. A higher pH did accompany a faster degradation rate in the Camp

344

Navajo soil without glucose addition, but otherwise an increase in pH was not accompanied with

345

significantly better degradation of ATO. Le Campion et al. [16, 18] found ring cleavage products

346

such as CO2 and urea. Likewise they putatively identified hydroxyurea. Urea and hydroxyurea

347

could potentially represent N-compounds that were missing in the N-balance of this study.

348

Additionally, denitrification may have occurred if there were anaerobic niches in the aerobic

349

assays, causing nitrogen to be released as nitrogen gas.

350

A recent study [28] reported aerobic conversion of NTO using an enrichment culture exposed

351

to the munitions formula IMX-101 as an N source and organic co-substrates. They indicated an

352

NTO degradation product lacking the nitro group, however the LC-MS evidence provided did

353

not support the proposed structure.

354

Environmental Implications. In soils, the degradation of NTO may be stimulated by

355

promoting an initial anaerobic phase to form ATO followed by an aerobic phase for ATO

356

biodegradation. In aerobic conditions, our research suggests that NTO will be non-biodegradable

357

in soils. In anaerobic environments, it will most likely be readily converted to ATO but ATO,

358

will then persist in anaerobic conditions. In aerobic conditions, ATO may be susceptible to

359

mineralization depending on the soil microbial community and pH conditions. The addition of

360

cosubstrates (e.g. glucose) was not observed in our study to necessarily enhance ATO

361

degradation.



362 363

ASSOCIATED CONTENT

364

Supporting Information

365

ATO and HTO synthesis.; soil characteristics; mineralogical content of the oriented clays;

366

concentrations of the intermediate 5-hydroxylamino-1,2,4-triazol-3-one; lag times, degradation

367

rates, and ATO yields for anaerobic soil microcosms. Summary of experiments were degradation

368

of ATO or NTO was not observed. This material is available free of charge via the Internet at

369

http://pubs.acs.org/.

370 371

AUTHOR INFORMATION

372 373

Corresponding Author:

374

* Phone: 1-520-621-2591 Fax: 1-520-621-6048 E-mail: [email protected]

375

Notes

376

The authors declare no competing financial interest.

377 378

AKNOWLEDGEMENTS

379

This study was supported by the Strategic Environmental Research and Development Program

380

(SERDP) project ER-2221. We thank Katerina Donstova for the Catlin soil and Stefan Walston

381

for the Maricopa soil. CIO was funded in part by the Mexican National Council for Science and

382

Technology (CONACyT).

383 384 385

REFERENCES


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Preuss, A.; Fimpel, J.; Diekert, G., Anaerobic transformation of 2,4,6-trinitrotoluene

McCormick, N. G.; Cornell, J. H.; Kaplan, A. M., Biodegradation of hexahydro-1,3,5trinitro-1,3,5-triazine. Appl. Environ. Microb. 1981, 42, (5), 817-823.

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50.

Bhushan, B.; Trott, S.; Spain, J. C.; Halasz, A.; Paquet, L.; Hawari, J., Biotransformation of

520

hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: insight

521

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522

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523

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Halasz, A.; Hawari, J., Degradation routes of RDX in various redox systems. In Aquatic

524

Redox Chemistry, Tratnyek, P. G.; Grundl, T. J.; Haderlein, S. B., Eds. 2011; Vol. 1071, pp

525

441-462.

526



527

Page 26 of 32

Tables

528 529

Table 1. Selected properties of the soils used in this study. Soil Texture Soils

pH

BET SA† m2 g-1

TN§

TC¶

TOC ‡

Sand

Silt

Clay

-----------------------g kg-1 ---------------------

-----------------%-------------

530

531 Textural Class 532

Camp Ripley (MN)

5.96 ± 0.06

1.72 ± 0.03

1.27 ± 0.20

12.55 ± 1.50

12.5 ± 1.50

78.20

13.99

7.82

Loamy Sand

533

Camp Butner (NC)

6.36 ± 0.02

4.85 ± 0.07

1.33 ± 0.05

20.69 ± 1.20

20.69 ± 1.20

68.68

19.83

11.50

Sandy Loam

534

Florence (AZ)

6.96 ± 0.11

32.45 ± 1.73

0.81± 0.02

4.16 ± 0.20

4.16 ± 0.20

44.20

28.50

27.30

Clay Loam

535

Camp Navajo (AZ)

6.32 ± 0.01

21.5 ± 0.56

3.65 ± 0.21

52.36 ± 3.70

52.36 ± 3.70

21.48

38.10

40.43

Clay

536

Maricopa (AZ)

7.75 ± 0.07

34.58 ± 1.70

0.80 ± 0.05

7.07 ± 0.40

4.65 ± 0.40

37.48

21.98

40.55

Clay

537

Roger Road (AZ)

7.75 ± 0.01

27.69 ± 0.80

1.54 ± 0.03

18.25 ± 0.10

7.07 ± 0.40

23.33

35.10

41.58

Clay

538

Catlin Soil (IL)

6.42 ± 0.06

5.05 ± 0.44

2.81 ± 0.18

45.44 ± 1.10

44.08 ± 1.10

13.50

54.98

31.53

539 Silty Clay Loam 540

541

†Brunauer, Emmett and Teller (BET) Surface area; § TN = Total nitrogen; ¶ TC = Total Carbon ‡ TOC = Total Organic Carbon.


Page 27 of 32


542

Figure 1. The anaerobic degradation of NTO to ATO in microcosms inoculated with (A) Camp

543

Navajo (AZ) soil, and (B) Florence (AZ) soil in H2 amended microcosms (squares) endogenous

544

controls (triangles), and killed controls (circles). NTO concentrations are shown with solid

545

symbols and solid lines and ATO concentrations are shown with open symbols, dotted lines).

546

Error bars indicate standard deviation of duplicate microcosms.

547 548

Figure 2. The degradation of NTO to ATO in microcosms with H2 as an electron acceptor

549

without YE (A) and with 10 mg L-1 YE (B), or with 20 mM of citrate without YE (C) and with

550

10 mg L-1 YE (D), or with 20 mM pyruvate without YE (E) and with 10 mg L-1 YE (F). The

551

concentration of NTO (black squares) and ATO (triangles) are shown on the primary axis; the

552

concentration of the hydroxyl-amino intermediate (circles) is shown on the secondary axis. Error

553

bars indicate standard deviations of duplicate microcosms.

554 555

Figure 3. The degradation of ATO under aerobic conditions and release of nitrogen species: (A)

556

Camp Butner Soil, pH 7.2; (B) Camp Navajo soil, pH 7.2; (C) Camp Butner, pH 8.5; (D) Camp

557

Navajo soil, pH 8.5; (E) Camp Butner soil, pH 7.2, with glucose; (F) Camp Navajo soil, pH 7.2,

558

with glucose; (G) Camp Butner soil, pH 8.5, with glucose; (H) Camp Navajo soil, pH 8.5, with

559

glucose. Solid triangles show ATO concentrations (as per mol of N), squares correspond to

560

ammonia concentration, circles correspond to nitrite concentration, diamonds correspond to

561

nitrate concentration, and dashed line represents the sum of nitrogen species (both inorganic and

562

ATO). Mineral medium used in these experiments initially contained 0.5 mM of inorganic

563

nitrogen (as ammonia). Error bars represent standard deviations of duplicate microcosms.

564

Autoclaved controls and non-inoculated controls are available in the Supplementary Information

565

(Figure S3 in SI).



566 567

Figure 4. The degradation pathway of NTO found in this study. The dotted arrows signify minor

568

reactions.

569


Page 28 of 32

Page 29 of 32

570


Figures

571

572 573 574 575 576 577

Figure 1

578



579

Page 30 of 32

.

580 581 582 583 584

Figure 2

585 586


Page 31 of 32


587 588 589 590

Figure 3

591



Page 32 of 32

592 593 594 595

Figure 4


Biotransformation and Degradation of the Insensitive Munitions

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