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EXPERIMENTAL PROCEDURES
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Clay minerals: smectite. Smectite was extracted from bentonite (Georgia, Europe) from the
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mine in Ozurgeti. A suspension of 20 g of bentonite in 700 mL of distilled water, containing
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40 mL of Amberlite IR 1200 Na resin (Rohm and Haas) to facilitate dispersion, was shaken
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for 16 hrs. The mixture was filtered and poured into 30 cm measuring cylinders, mixed for 1
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minute and decanted (between 14 and 16 hrs). After decantation, the supernatant (the upper
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20 cm according to Stokes’ law) corresponding to the clay fraction (particles of 2 m in
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diameter) was recovered, transferred in small bottles and centrifuged (4,000 g, 1 h). The
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pellets were dried at 75°C for 24 hrs.
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TEM micrographs, X-ray and infrared spectroscopic analyses were performed and detected no
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iron oxides at the surface of the purified clay, contrarily to quartz and carbonates which were
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present before purification of the smectites. However, infrared spectroscopy showed the
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possible presence of residual silica, colloidal or crystalline (Devineau et al., 2004).
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To measure the ξ potential of the clay sample, a suspension of the smectitic material used in
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the present study (1 g/L) was prepared in 0.01M NaCl electrolyte at pH 3.1 in an individual
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HDPE tube, and the tube was gently shaken at 298K. At the end of the shaking period (3hs),
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the final pH value of the sample was taken and was found to be equal to 3.9. The
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electrophoretic mobility (EM) of the particles dispersed in the suspension was measured with
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a Malvern NanoZS apparatus. The EM value was converted into ξ (surface) potential value by
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using the Smolukowski equation. The ξ potential of the suspended particles was found to be
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equal to - 41mV. The ξ measurement provided strong evidence for existence in the suspension
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of only one type of particles, which show a negative value of surface potential at acidic pH.
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This result is in good accordance with previously published data for smectites, which have
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been widely reported to exhibit an overall negative charge at acidic pH (Delgado et al., 1986;
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Horikawa et al., 1988; Zarzycki et al., 2007; Zadaka et al., 2010), unlike what is usually
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observed for particles of (oxihydr)oxides of aluminum or iron (Del Nero et al, 2010). The ξ
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potential result obtained here for the smectite rules out the existence of Fe-(oxihydr)oxides in
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our smectite sample, either as coatings at the clay surface and/or as individual phases of Fe-
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(oxyhydr)oxides of very small size (colloids).
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The CEC of the smectite was determined using a hexamminecobalt trichloride solution
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(Ciesielski and Sterckeman, 1997). The smectite preparation was sterilized by a combination
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of heat and chemical treatments chosen to preserve the layer structures: the samples were
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subjected to tyndallization treatment, with three cycles of heating for 2 hrs per day at 90°C,
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followed by 15 days of incubation in chloroform (Hasnaoui et al., 2001). The smectite was
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then rehydrated to a concentration of 20 g L-1 in distilled deionized water.
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Bacterial strains and growth conditions. We used the wild-type P. aeruginosa ATCC 15692
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strain and three siderophore mutants: PAO6297 (a pyochelin-deficient strain, (Serino et al.,
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1995)), PAO6382 (a pyoverdine-deficient strain, (Braud et al., 2009)) and PAO6383 (a
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pyoverdine- and pyochelin-deficient strain, (Michel et al., 2007)). Bacteria were grown at
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30°C overnight in LB broth medium at 200 rpm. For iron-depleted culture, cells were
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harvested from a LB pre-culture (24 hrs incubation), washed twice in an iron-deficient
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succinate medium (composition in g L-1: K2HPO4, 6.0; KH2PO4, 3.0; (NH4)2SO4, 1.0;
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MgSO4.7H2O, 0.2; sodium succinate, 4.0; pH adjusted to 7.0 by addition of NaOH) (Meyer
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and Abdallah, 1978) and used to inoculate fresh medium. The cultures were incubated for 48
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hrs under shaking (200 rpm) and then centrifuged (10 min at 8,600 g) and washed twice with
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succinate medium. Bacterial cell density was determined by measuring the optical density
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(OD) at 600 nm.
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Growth in the presence of smectite was performed in polycarbonate erlenmeyer flasks
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(125 mL) in 20 mL of succinate medium supplemented with one of a series of concentrations
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of smectite (0, 0.2, 1 and 10 g L-1). Each Erlenmeyer flask was inoculated with bacterial cells
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to a final OD600 of 0.05 per mL. Controls without bacteria or without clay were included.
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Erlenmeyer flasks were incubated at 30°C and shaken at 200 rpm for 28 hrs. Each condition
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was replicated three times. One mL aliquots were removed after 4, 8, 24, and 28 hrs of
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incubation and bacteria enumerated: samples were serially diluted and plated on LB agar
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plates incubated at 30°C for 24 hrs. Results are expressed as “colony forming units” (CFU)
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per mL. For each growth condition, the pH and the redox potential (Eh) were measured at the
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beginning and at the end of the experiment. For the initial measurements of pH and Eh, a
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flask from each experiment was sacrified to avoid adventitious bacteria that might be
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associated with the electrodes. The redox potential was measured with an Ag/AgCl electrode.
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Monitoring siderophore production in batch experiments. For each time point of bacterial
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growth, 1 mL of culture was centrifuged for 10 min at 8,600 g and the amount of pyoverdine
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in solution in the supernatant was estimated by monitoring absorbance at 400 nm
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(Folschweiller et al., 2002). To measure pyoverdine trapped in the biofilm matrix, pyoverdine
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was extracted using sodo-calcic glass beads in 10 mL of 200 mM Tris-HCl pH 7.0 buffer and
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the absorbance monitored at 400 nm ( = 19 000 M-1 cm-1 for pyoverdine at pH 7).
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Fluorescence microscopy was used to check the destruction of the biofilm.
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The bacteria produce less pyochelin than pyoverdine and pyochelin cannot be detected
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directly in culture supernatants without prior extraction. Due to the limited volumes of culture
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supernatant available, this study focused only on pyoverdine.
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Metal detection in bacterial batch growth experiments. P. aeruginosa cells were incubated
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in the presence of smectite in succinate media. After 28 hrs of growth, the cultures were
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centrifuged for 15 min at 4,500 g, and filtered through a 0.2 m pore-size membrane. The
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filtrate was acidified to pH 1.0 with 70 % HNO3, and Al, Fe and Si were assayed by
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Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES, Liberty II, Varian,
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Inc, Palo Alto, California). A certified solution (EU-H-2, SCP Science, Courtaboeuf, France)
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was included in the analyses as a control for the determination step. Detection and
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quantification limits were calculated for each batch of analysis according to Neuilly (1998)
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(Neuilly, 1998). Quantification limits ranged from 0.67 to 1.00 µM for Al, 0.09 to 0.21 µM
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for Fe and 7.12 to 12.46 M for Si.
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Biofilm quantification. Biofilm formation in the presence or absence of smectite was tested
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in 6-well plates (Greiner, TC-plate CELLSTAR), filled with 3 mL of succinate medium
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supplemented with a series of concentrations of smectite (0, 0.2, 1 and 10 g L-1). Each well
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was inoculated with bacterial cells to a final OD600 of 0.05 per ml and incubated at 30 °C for
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28 hrs on a shaker (50 rpm). Controls without bacteria were included. Each condition was
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repeated three times. For violet crystal treatment, culture supernatants were carefully removed
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and the biofilm washed twice with sterile distilled water. One mL of 0.5 % crystal violet was
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added and left in contact for 15 min at room temperature with gentle agitation. The solution
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was removed, the biofilm was washed twice in sterile distilled water, and then the crystal
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violet was eluted with 2 mL of ethanol. Biofilm was then quantified by measuring the
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absorbance at 570 nm of the eluted crystal violet.
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Microscopy. Smectite incubated for 28 hrs with or without bacteria in the succinate medium
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was examined under light and epifluorescence microscope (Nikon Eclipse 50i, objective: CFI
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Achroplan 100 x A ON 1.25 DT 0.18) connected to a numerix 12 bits DS-Fi1 camera.
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Biofilm, stained naturally by the pyoverdine produced, was detected with a Bv2A Filter set
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(excitation 400-440 nm, emission 470 nm, dichroic filter 455 long pass). Images were
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captured using NIS imaging software elements.
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Pyoverdine sorption on smectite. One g L-1 of smectite was incubated in the presence of 250
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µM of pyoverdine in 10 mL of 2 mM MES, 725 µM Ca(NO3)2 buffer in 50 mL Falcon tubes
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and shaken at 220 rpm. Controls without siderophore or without smectite were run in parallel.
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Purification of pyoverdine accumulated in the culture supernatant of iron-starved growth of P.
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aeruginosa was done by the XAD procedure, as described previously (Meyer et al, 1997).
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Briefly, supernatant was acidified with HCl 6M (pH 6) and passed through an XAD-4
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Amberlite column which retained the pyoverdine. After washing with 500 ml distilled water,
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the pyoverdine was eluted with 250 ml 50% ethanol, concentrated using vacuum using
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rotavapor and lyophilized (Meyer et al, 1997). Each treatment was replicated three times. At
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various times (0, 1, 8, 24, 48 hrs), samples were centrifuged (8.000 g, 20 min), and the
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supernatants were filtered (0.22 µm pore-size filters), acidified (to pH 1.0 with 70% HNO3)
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and assayed for Fe and Al contents by ICP-AES. One mL aliquots of the supernatant
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collected before acidification were used to measure siderophore concentrations by visible
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light absorption (OD400 nm).
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Statistical analysis. Data were analyzed using analysis of variance (ANOVA) and differences
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among treatments were separated using Tukey’s test (Turkey, 1951), under R environment (R
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Core Team (2012) R: A language and environment for statistical computing. R Foundation
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for Statistical Computing, Vienna, Austria. ISBN 3-900051-07-0).
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Figure 1s : (A) XRD, (B) FTIR profile and (C) TEM micrograph of smectite.
A, XRD profile of smectite, d-values are given in A
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B. FTIR profile of smectite (JASCO, 4100 type A, 2 cm-1 resolution)
C. TEM micrograph of smectite (JEOL 2100F Cs 200KV 0.1nm resolution)
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