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Original data for Figure 1-6 & 8-9
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Microbial Cultivation and Batch Experiment Protocol The Fe(III)-rich nontronite NAu-2 (source: Clay Minerals Society) was Na-saturated, sonicated, and fractionated to 0.02-0.5 µm particles. Shewanella oneidensisMR-1 was cultured in Luria-Bertani medium, while Desulfovibrio vulgariswas grown in a specialized anoxic medium containing CaSO₄, NH₄Cl, KH₂PO₄, MgSO₄·7H₂O, yeast extract, sodium lactate, and resazurin. For bioreduction experiments, batch incubations were set up under strictly anoxic conditions (95% N₂, 5% H₂ glove box) using 5 mg/mL NAu-2 as the sole electron acceptor and 20 mM lactate as the electron donor. Experimental variables included the initial MR-1:SRB cell ratio (1:0, 0:1, 0.5:0.5, 1:1), Na₂SO₄ concentration (0, 5, 10 mM), and the presence/absence of the electron shuttle anthraquinone-2,6-disulfonate (AQDS). All treatments, including abiotic controls and a chemical reduction control with 5 mM sodium sulfide, were incubated at 25°C for 30 days, with triplicate sampling at 15 time points. Chemical Analysis Methods for Figures 1-6 The production of Fe(II) (Figures 1-4) was quantified using the 1,10-phenanthroline assay. Sulfate consumption (Figure 5) was determined by BaCl₂ titration, and sulfide production (Figure 6) was measured spectrophotometrically according to the method of Cline (1969). The initial iron reduction rate was calculated mathematically from the Fe(II) production data. Spectroscopic Analysis and Instrumentation for Figures 8-9 Fourier-transform infrared (FTIR) spectra (Figures 8-9) were acquired using a Midac M2000 FTIR spectrometer. Self-supporting sample films were prepared by mixing ~2 mg of dried clay pellets with 200 mg of KBr and pressing into discs under anoxic conditions. Spectra were recorded from 4000-400 cm⁻¹ at a resolution of 4 cm⁻¹ (50 scans). The raw absorbance data for the regions 900-700 cm⁻¹ (M-O-H deformation; Figure 8) and 3800-3200 cm⁻¹ (O-H stretching; Figure 9) were mathematically processed into their second derivatives using the GRAMS/32 software for detailed analysis of structural changes. Key Reagents and Software Key reagents included nontronite NAu-2, sodium lactate, Na₂SO₄, AQDS, and sodium sulfide. Data processing and mineral phase identification for the associated XRD analyses (not in the specified figures but part of the overall methodology) utilized the JADE 7 software package.
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- Henan University