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SI text
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X-ray Absorption Near Edge Structure (XANES) spectroscopy
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Samples were prepared for the XANES measurements under strictly anoxic conditions in a
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glovebox under an N2:H2; 95:5 vol:vol) atmosphere. Two silicon nitride Si3N4 membranes
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(Silson Ltd, UK) of 100nm thickness were used to seal the sample and avoid oxygen
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contamination as previously described (Thieme et al., 2006). Samples were filtered using a GE
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Polycarbonate black filter (0.2uM, 25mm) (GE Waters & process Technologies) and then placed
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into a glass vial sealed with thick butyl-rubber stoppers and aluminum cap. Samples were kept at
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4 ºC prior to analysis. The prepared filters were placed between two silicon nitride membranes
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(Si3N4) of 100nm thickness and sealed with glue. This “sandwich” is impenetrable to oxygen for
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more than 1020 seconds and allows easy transfer of the samples into the X-ray spectrometer at
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Beamline X15 in the National Synchrotron Light Source, NSLS, at Brookhaven National
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Laboratory. The beamline X15B is located at a bending magnet and operates in an energy range
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of 1.2 keV< E <8 keV. Si(111) with an energy resolution of 2 × 10−4. XANES spectra were
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obtained at the K-absorption edge of sulfur around E=2472 eV. The beam size on the sample is
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1mm × 1mm with a photon flux of around 1012 photons/sec at a ring current of 250mA. The
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sample can be rotated around the vertical axis to optimize the position for X-ray fluorescence
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detection. The instrument is equipped with an ion chamber for transmission and a single-element
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Germanium fluorescence detector for fluorescence measurements. For measurements around the
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sulfur K-edge, the sample chamber is purged with Helium. A detailed description of X15B can
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be found in MacDowell et al., 1989.
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Taking the complexity of the sample under investigation and of XANES spectra in general into
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account, constraining assumptions had to be made with respect to the fitting approach in order to
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achieve consistent results.
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All data were analyzed using standardized techniques. Raw spectra were baseline-corrected,
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normalized to the edge jump, and smoothed with the Golay–Savitzky algorithm, using an
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interval length of 5 points in both directions from the central point and a fifth grade polynom.
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Major components were fitted into the spectrum, using a combination of Gaussian and
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Lorentzian analysis for each component. The ratio of Gaussian to Lorentzian was optimized at
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0.6 based on iterative determinations. Absorption spectroscopy lines are asymmetric and can be
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described in theory using Lorentzian analysis. However, instrumental influences broaden the
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line. These influences are symmetrical and are best described using Gaussian analysis. A
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convolution of both, Lorentzian and Gaussian provides the optimum approach for fitting a
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spectrum. Arctangent functions were used to describe edge jumps. As each sulfur species can be
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presumed to be independent from others, an arctangent function was assigned to every peak. The
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width of the arctangent function was set to 1 eV, the height relative to the peak height was set to
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approximately 0.15. All analytical operations were performed using the program Specfit (Gleber
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et al., 2003). A diluted sulfate solution was used for calibration, for which the spectrum showed
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the peak position of the sulfate S at E = 2483.5 eV. Using the well accepted value of E = 2482.5
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eV from literature (Huffmann et al. 1991, Prietzel et al. 2007), all measured spectra were
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corrected accordingly. After that correction, the various sulfur species in the samples were
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identified by comparing the maximum energies of the fitted peaks to well-known white-line
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energies of different standard compounds, taken from literature. The absorption cross section of
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the S white line peak is proportional to the number of 3p orbital vacancies in a single one-
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electron model, and thus increases as the oxidation state of the S atom increases (Xia et al.,
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1998). Therefore, the contribution of S species with different electronic oxidation states of the S
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atoms to total S can be determined by correcting the measured peak areas with the respective
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absorption cross section.,
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Gleber, G., J. Thieme, J. Niemeyer, and M. Feser. 2003. Interaction of organic substances with
iron studied by O1s spectroscopy - Development of an analysis program. J Phys IV 104: 31533168.
Huffman, G., S. Mitra, F. E. Huggins, N. Shah, S. Vaidya, and F. Lu. 1991. Quantitative
Analysis of All Major Forms of Sulfur in Coal by X-ray Absorption Fine Structure
Spectroscopy. Energy & Fuels 5:574-581.
MacDowell, A. A., T. Hashizume, and P. H. Citrin. 1989. A soft/hard x-ray beamline for surface
EXAFS studies in the energy range 0.8-15 keV. Review of Scientific Instruments 60:1901-1904.
Prietzel, J., J. Thieme, M. Salome, and H. Knicker. 2007. Sulfur K-edge XANES spectroscopy
reveals differences in sulfur speciation of bulk soils, humic acid, fulvic acid, and particle size
separates. Soil Biology & Biochemistry 39:877-890.
Thieme, J., J. Prietzel, N. Tyufekchieva, D. Paterson, and I. McNulty. 2006. Speciation of sulfur
in oxic and anoxic soils using x-ray spectromicroscopy. IPAP Conference Series 7:318-320.
Xia K., F. Weesner, W. F. Bleam, P. R. Bloom, U. L. Skyllberg, and P. Helmke. 1998. XANES
Studies of Oxidation States of Sulfur in Aquatic and Soil Hunlic Substances. Soil Sci. Soc. Am. J.
(1998) 62, pp. 1240-1246
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Isotopic fractionation
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Isotopic fractionation analysis was performed looking at the ratios of
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elemental sulfur. Dissolved sulfide samples were precipitated by adding AgNO3. The Ag2S
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formed was purified and washed with NH4OH and deionized water. Elemental sulfur samples
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were rinsed with ethanol and deionized water. All samples were dried prior to analysis. Isotope
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ratios were measured using a Eurovector model 3028 elemental analyzer in helium continuous
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S to
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S in sulfide and
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flow mode interfaced with a GV Isoprime isotope ratio mass spectrometer. Isotope ratios are
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reported in delta notation: δ34S (‰) = 1000([34S/32S]sample /[34S/32S]standard -1).
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Transmission Electron Microscopy imaging
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Bacteria, from a 0.53 OD600nm growth suspension, were added undiluted to 100-400 mesh
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carbon-coated and glow-discharged grids for imaging. The solution was blotted off, and a dilute
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trehalose solution was applied for 30s. The solution was then blotted again and stored at room
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temperature until imaging. Grids were imaged in a JEOL JEM-1200 electron microscope
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operated at 120 kV at 5000X nominal magnification. Images were recorded with a Gatan
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1024x1024-pixel CCD camera (Gatan, Inc.).
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a
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0.3
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Lactate
Chlorate
OD 600nm
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0.2
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95
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Time (hr)
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SI Figure 1a. Growth coupled to lactate oxidation and chlorate reduction by a reinoculated
culture of A. suillum in unfiltered spent broth after the initial sulfide oxidation was complete.
The results depicted are the average of triplicate cultures.
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0.4
14
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0.3
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Lactate
Chlorate
OD 600nm
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4
0.2
OD 600nm
Chlorate and Lactate consumption
(mM)
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b
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OD 600nm
Chlorate and Lactate consumption
(mM)
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Time (hr)
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SI Figure 1b. Growth coupled to lactate oxidation and chlorate reduction by a reinoculated
culture of A. suillum in 0.22m filtered spent broth after the initial sulfide oxidation was
complete. The results depicted are the average of triplicate cultures.
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0.3
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Lactate
Chlorate
OD 600nm
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0.2
OD 600nm
Chlorate and Lactate consumption
(mM)
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0
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Time (hr)
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SI Figure 2. Growth coupled to lactate oxidation and chlorate reduction by the static H2Soxidizing culture of A. suillum when it is used to inoculate fresh BM amended with lactate
(10mM) and chlorate (10mM)
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Gregoire et al – SI Figure 2
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XANES analysis T0
XANES analysis T24
SI Figure 3. XANES spectroscopy of washed culture retentate of A. suillum initially and after
24 hours incubation in the presence of H2S and chlorate.
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SI Figure 4. EDX spectra confirming three sulfur inclusions in A. sullium on the carbon film
support. Chemical analyses of the whole mount bacterial cell TEM samples were carried out in a
JEOL 2100-F 200 kV Field-Emission Analytical Transmission Electron Microscope (TEM)
equipped with Oxford INCA Energy Dispersive Spectroscopy (EDX) X-ray detection system at
the Molecular Foundry at LBL. EDX spectra were acquired for 60 live seconds with a 0.4 μm
probe at 200 kV.
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