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emi412080-sup-0001-si.tif985K

Fig. S1. Representative chromatograms from HPLC of (A) a mixture of 7.5 nmol each of cysteine, coenzyme M and sulfide from commercial sources. Numbers represent the retention times in minutes. (B) and (C) show chromatograms from acid extracts (0.5–0.75 mg cell protein) of methanol and acetate HDCS cells, respectively, with 0 (black) or 0.63 mM CdCl2 (red). It is noted that a significant fraction of sulfide is lost in the acid extracts; therefore, sulfide was quantified by the methylene blue method (see Appendix S1).

emi412080-sup-0002-si.tif1029K

Fig. S2. Methane production of CdPA methanol-grown cells cultured in 0 (squares), 0.054 (226 pM free, circles), 0.63 (12.9 μM free up triangles), 1.4 (78.4 μM free; down triangles), 1.94 (339 μM free; diamonds) or 2.5 mM CdCl2 (835 μM free; cross). Values are the mean ± SD of three different batches. *P > 0.005 versus control without cadmium.

emi412080-sup-0003-si.tif7214K

Fig. S3. Cell aggregates formation induced by cadmium in cultures of M. acetivorans with methanol. Representative images of cultures with different concentrations of CdCl2. It is noted that control cultures without cadmium did not show any cell aggregates, whereas cultures of Cd2+-preadapted cells with 1.4 and 1.94 mM CdCl2 showed a metabolically active aggregate.

emi412080-sup-0004-si.tif1672K

Fig. S4. Effect of DNase I on the turbidity of 1 mg protein extracted from control cells (blue) and biofilm (black). For comparison, homogenized biofilm without DNase I is shown (red).

emi412080-sup-0005-si.tif5687K

Fig. S5. Intracellular and extracellular cadmium clusters in M. acetivorans cultures. Atomic-resolution HAADF-STEM mages of Cd2+-preadapted cells cultured in the presence of 1.4 mM CdCl2 for 18 days revealed white spots localized inside (A) and outside along the biofilm (C). Regions enclosed in the white circle were analysed, and cadmium-sulphur grains were detected (B, D). Y-axis units are given in counts from 0 up to 1800. X-axis units are given in keV from 0 up to 20.48. The Cu2+ detected derived from the grid in which samples were fixed.

emi412080-sup-0006-si.tif7418K

Fig. S6. Atomic resolution HAADF-STEM images from cells cultured in the absence of cadmium for 18 days revealed no dense clusters (A) of cadmium-sulphur. Well-defined dense grains, acidocalcisomes were detected (white region enclosed in a square) (B) and in preadapted cells further exposed to 1.4 mM CdCl2 acidocalcisomes shown larger (C).

emi412080-sup-0007-si.doc59K

Table S1. Content of malate, citrate and total phosphate in M. acetivorans in the absence or presence of Cd2+ in the culture medium.

Table S2. Dry weight and Cd2+ removal in biofilms.

emi412080-sup-0008-si.docx21K

Appendix S1. Supplementary materials.

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