Supporting Information for: Iron and electron shuttle mediated (bio)degradation of the military next

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1 Supporting Information for: Iron and electron shuttle mediated (bio)degradation of the military next generation insensitive munition 2,4-dinitroanisole (DNAN) Jolanta B. Niedźwiecka #, Scott R. Drew Ψ, Mark A. Schlautman #, Kayleigh A. Millerick #&, Erin Grubbs #, Nishanth Tharayil, and Kevin T. Finneran *# # Environmental Engineering and Earth Sciences, Clemson University, 168 Rich Laboratory, Anderson, SC, 29625, Ψ Geosyntec Consultants, Ewing, NJ 08628, & Department of Civil and Environmental Engineering, University of Illinois at Urbana-Champaign, Urbana, IL, 61801, School of Agriculture, Forestry, and Environmental Sciences, Clemson University, 218 Biosystems Research Complex, Clemson, SC *Corresponding author footnote: Phone: , ktf@clemson.edu Pages: 9 (including this cover sheet) Equations: 4 Tables: 1 Figures: 4 S1

2 Reactions equations + Equation S1. Iron reduction Equation S2. Reduction of anthraquinone-2,6-disulfonate (AQDS) to anthrahydroquinone-2,6- disulfonate (AH2QDS) / +2 Equation S3. Reduction of 2,4-dinitroanisole (DNAN) to 2-methoxy-5-nitroaniline (MENA) or 4-methoxy-3-nitroaniline (imena) / Equation S4. Reduction of MENA or imena to 2,4-diaminoanisole (DAAN). S2

3 Table S1. DNAN degradation rates. Treatment Degradation rate (hr -1, unless specified otherwise) Chemical reactions ph 7 Fe(II) unmixed Fe(II) mixed ph 8 Fe(II) unmixed Fe(II) mixed min -1 ph 9 Fe(II) unmixed Fe(II) mixed um/hr* AH2QDS at ph um /hr* Mixed biological and chemical reactions DNAN+GS DNAN+GS-15+Ac DNAN+GS-15+Ac+AQDS DNAN+GS-15+Ac+FeGel DNAN+GS-15+Ac+ AQDS+FeGel DNAN+GS-15+Ac+FeCit *Zero order reaction rates. All other rates are first order. S3

4 Figure S1. DNAN control (no cells added) in the cell suspension experiment with Geobacter metallireducens, strain GS-15. Experimental conditions: 100 µm DNAN, buffered with 30 mm bicarbonate at ph 7. The error bars represent the standard deviation with n=3. S4

5 Objective: The objective was to determine DNAN degradation product based on the stoichiometry of Fe(II) and anthrahydroquinone-2,6-disulfonate (AH 2 QDS) required to reduce 100 µm DNAN. Methods: Different concentrations of Fe(II) (100 µm, 200 µm, 600 µm, 1200 µm) and AH 2 QDS (50 µm, 100 µm, 150 µm, 300 µm, 600 µm) were added to anoxic bottles buffered with 30 mm 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) at ph 8 and 9. Positive control was run with 300 µm AH 2 QDS. DNAN was periodically analyzed as described in the main text. Results and Discussion: DNAN concentrations recovered after 24 hrs in bottles with specified initial Fe(II) amount, suggested a reduction of a nitro functional group to yield MENA or imena. For example, 600 µm Fe(II) would be required to reduce 100 µm DNAN to 100 µm MENA or imena. At both ph values, 90 % of DNAN was degraded, which supported the predicted degradation pathway. Also, when initial 200 µm Fe(II) was used, 60 % DNAN was recovered, which is close to expected 1/3 of DNAN degradation based on the stoichiometry of the reaction (Equation S1, S3). DNAN degradation with AH 2 QDS yielded less accurate results, but some treatments confirm the stoichiometry for proposed degradation pathway. Based on calculations, 100 µm AH 2 QDS should degrade 1/3 initial DNAN (200 µm) and data for ph 8 show that approximately that DNAN amount was removed. Treatment with 150 µm AH 2 QDS should degrade 50 % DNAN and the final concentration at ph 8 reflected that loss. S5

6 Figure S2. Influence of Fe(II) concentration on DNAN degradation at ph 8 (A) and 9 (B). Experimental conditions: 100 µm DNAN, buffered with 30 mm HEPES, Fe(II) concentrations were added as specified in the legend. The error bars represent the standard deviation with n=3. S6

7 Figure S3. Influence of AH 2 QDS concentration on DNAN degradation at ph 8 (A) and 9 (B). Experimental conditions: 100 µm DNAN, buffered with 30 mm HEPES, AH 2 QDS concentrations were added as specified in the legend. Controls were run with 300 µm AQDS. The error bars represent the standard deviation with n=3. S7

8 Objective: The objective was to test the sub stoichiometric DNAN reduction and to determine degradation products, based on the amount of amended Fe(II). Methods: Solutions of 100 µm DNAN were buffered at ph 8 with 30 mm HEPES and at ph 9 with 2-(cyclohexylamino)ethanesulfonic acid (CHES). The reactions were initiated with 100 µm Fe(II), and then the batches were amended step-wise with 11 injections of 100 µm Fe(II) to reach the final concentration of 1200 µm Fe(II). DNAN samples taken before and after each amendment were analyzed as described in the main text. Results and Discussion: Results at ph 8 indicate that approximately 500 µm Fe(II) had to be present to initiate DNAN degradation. It may be due to formation of Fe(III) solids that originate from amended Fe(II) oxidation. Once Fe(III) precipitated, it could serve as a solid surface for Fe(II) adsorption. Adsorbed Fe(II) species are more reactive than dissolved Fe(II) so they could reduce DNAN. There was no lag phase at ph 9. DNAN reduction started from the first Fe(II) amendment and 90 % DNAN was reduced using 600 µm Fe(II), suggesting that the main degradation product is MENA or imena. S8

9 Figure S4. The stepwise amendment of Fe(II) to DNAN at ph 8 (A) and 9 (B). Experimental conditions: 100 µm DNAN, buffered with 30 mm HEPES at ph 8 and 30 mm CHES at ph 9. The controls showed no DNAN degradation and are represented in plot A. The error bars represent the standard deviation with n=3. S9