Formation of Sphalerite (ZnS) Deposits in Natural Biofilms of Sulfate-Reducing Bacteria Matthias Labrenz, Gregory K. Druschel, Tamara Thomsen-Ebert, Benjamin Gilbert, Susan A. Welch, Kenneth M. Kemner, Graham A. Logan, Roger E. Summons, Gelsomina De Stasio, Philip L. Bond, Barry Lai, Shelly D. Kelly, and Jillian F. Banfield

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Supplemental Figure 2. Fairly typical SEM image of ZnS biofilm showing elongate cells and spherical ZnS aggregates. Thick ropy structures are completely mineralized cells. Arrows indicate dehydrated filaments of cells (most filaments run NNW - SSE).

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Supplemental Figure 3. Another typical field of view of the biofilm obtained using high-resolution scanning electron microscopy. Rounded objects are ZnS aggregates (themselves composed of ~ 3 nm particles, as shown below). Polymer material (cells) is evident in some regions (esp. lower right side of this image). Cells are dehydrated in the SEM vacuum.

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Supplemental Figure 4. TEM cross section image of a ZnS aggregate. Sub-horizontal fractures are due to sample preparation by ultramicrotomy.

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Supplemental Figure 5. HRTEM image of region from above microtome-sectioned ZnS aggregate showing that it consists of small particles ~ 3 nm in diameter (regions where lattice fringes are visible and continuous represent single particles). The scale bar is 2. 5 nm.

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Supplemental Figure 6. Epifluorescent microscope image of a fragment of the biofilm. Left side is stained with with a DNA-binding stain (DAPI), the right side with a probe specific for the Desulfobacteriaceae (see methods section, below). Note that in the right hand image, the brightest cells occur at the ends of the chains, indicating a higher RNA content (more active cells).

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Supplemental Figure 7. Model (see Fig. 6) for precipitation from water as the solution becomes reduced (moving left to right across the diagram). This calculation (with 1 ppm Pb) shows that PbS (galena) precipitates at an Eh somewhat below that where ZnS precipitates. This suggests that the system could switch from ZnS to PbS+ZnS precipitation as the solution becomes slightly more reduced (due to higher activity of sulfate reducing microbes or by the existence of a microbial population optimized to live at under more reducing conditions). This phenomenon may have direct relevance to formation of a variety of low-temperature Pb-Zn deposits.

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Supplemental Figure 8. SEM image of cultured cells (diagonal) and bright contrast aggregates of ZnS

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Supplemental Figure 9. The energy-dispersive x-ray spectra from the aggregates shows that they are essentially Fe-free, despite abundant Fe in the culture media.

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METHODS AND NOTES

Scanning electron microscopy

Field-emission scanning electron micrographs were collected using a Leo 1530 low voltage high-resolution scanning electron microscope operated at 3 kV and ~ 4 mm working distance using an in lens secondary electron detector. Samples were coated with Pt before examination to prevent charging. TEM images were collected using a Philips CM200 UT high-resolution instrument operated at 200 kV. Energy dispersive x-ray analyses were collected using an ultra thin window Ge detector.

X-ray fluorescence (XRF) microprobe analyses

X-ray fluorescence (XRF) microprobe analyses (0.20 X 0.35 mm2 beam size) experiments were performed at the SRICAT high energy x-ray microprobe (24) at the Advanced Photon Source. Incident x-ray energies greater and less than the absorption LIII edge of Pb (13.055 and 13.000 keV) were used to distinguish between Pb La and As Ka fluorescence signals. XRF measurements were made at both energies on thin film glass standards (NIST thin glass film on polycarbonate standard reference material #1832 and #1833) and the elemental area densities of the precipitates were converted to elemental percentage concentrations (relative to ZnS mass) by normalization to precipitate thickness and a ZnS density of 4.102 g/cm3.

Synchrotron-based x-ray photoelectron emission microscopy (PEEM)

X-ray PEEM images and spectra (not shown) were collected with the MEPHISTO instrument at the University of Wisconsin Synchrotron Radiation Center (25) using the PGM undulator beamline (2 ( 1012 photons/second, 60 meV resolution). The sample voltage was -15 kV, the aperture diameters in the back-focal plane of the objective lens were 50 or 20 mm. Micro-x-ray absorption near-edge structure spectra, acquired while scanning the photon energy across the S2p core level edge (130-160 eV), were taken from individual cell-associated mineral aggregates. These spectra exhibit the same line shape observed in ZnS reference standards. Local areas of sulfate (identified by comparison to CaSO4 reference standards) were detected over much of the sample.

Sample collection from the field site

Samples were collected in sterile 50 ml Falcon tubes. Following DNA extraction (26), microbial community 16S ribosomal RNA genes were amplified by polymerase chain reaction with bacterial, universal and archaeal primers. 16S rRNA genes were cloned for analysis using pGEM(r)-T and pGEM(r)-T Easy Vector Systems (Promega). 122 clones were analyzed by restriction fragment length polymorphism and 46 clones with distinctive patterns were totally or partially sequenced (27) (650-1400 bases). Sequences of representative phylogenetic relatives of the SRB clones from the Ribosomal Database Project (28) and GenBank databases (29) were aligned using the ARB software package (30). Sequences were reduced to comparable positions and evolutionary distance dendograms were constructed using the Jukes-Cantor correction and neighbour joining in PAUP (31).

Lipid analyses

Freeze dried biomass was solvent extracted by sequential sonication steps using gradational mixtures of methanol and dichloromethane. Fatty acid methyl esters were prepared using BF3/methanol. They were quantified and identified using GC-MS and relative retention times (14, 32).

Fluorescence in situ hybridization (FISH)

Samples for FISH were fixed in 4 % paraformaldehyde soon after collection. 5 ml of homogenized fixed samples were spotted onto gelatin coated multi-welled slides. FISH staining was carried out with 50 ng of oligonucleotide probes SRB385 and SRB385Db (33) under optimal conditions (35% formamide) determined as described previously (33) and finally stained with DAPI in conditions described previously (34). SRB385 and SRB385Db were used to identify sulfate-reducing bacteria of the families Desulfovibrionaceae and Desulfobacteriaceae, respectively. Desulfobacter postgatei DSM 2034 was used as positive control for hybridizations with SRB385Db and Desulfovibrio desulfuricans DSM 642 as the positive control for hybridizations with SRB385. Bacterial cultures used as negative hybridization controls included Thiobacillus caldus (isolate from Katrina J. Edwards), Sulfolobus thermosulfidooxidans DSM 9293, Acidiphilium strain SJH, and Leptospirillum ferrooxidans DSM 2705.

REFERENCES FOR THE METHODS SECTION

24. Z. Cai et al., in: X-RAY MICROSCOPY, W. Meyer-Ilse, T. Warwick, D. Attwood, Eds. (American Institute of Physics, Melville, N. Y., 1999). pp. 472-477.

25. G. De Stasio et al., Rev. Sci. Instrum. 70, 1740 (1999) and the references therein.

26. S. M. Barns, R. E. Fundyga, M. W. Jeffries, N. R. Pace, Proc. Natl. Acad. Sci. U.S.A. 91, 1609 (1994).

27. P. L. Bond, S. P. Smriga, J. F. Banfield, Appl. Environ. Microbiol., in press (2000).

28. B. L. Maidak et al., Nucleic Acids Res. 25, 109 (1997).

29. D. A. Benson, M. S. Boguski, D. J. Lipman, J. Ostell, B. F. Ouellette, Nucleic Acids Res. 26, 1 (1998).

30. O. Strunk, W. Ludwig, ARB. Computer program distributed by the Technical University Munich, Munich, Germany (1998).

31. D. L. Swofford, PAUP*. Phylogenetic Analysis using Parsimony (* and other Methods). (Sinauer Associates, Sunderland, Massachusetts, 1998), vol. 4.

32. M. Rhomer, P. Bouvier-Nave, G. Ourisson, J. Gen. Microbiol. 130, 1137 (1984).

33. R. Rabus, M. Fukui, H. Wilkes, F. Widdel, Appl. Environ. Microbiol. 62, 3605 (1996).

34. P. L. Bond, J. F. Banfield, Microbial Ecology, in press (2000)