Soluble and Solid Iron Reduction Assays with Desulfitobacterium hafniense

Materials and Reagents

1. 1.7 ml Eppendorf centrifuge tubes (Corning, Axygen^®, catalog number: MCT-175-C )
2. Syringes
   1 ml (CODAN, catalog number: 621640 )
   5 ml (CODAN, catalog number: 625607 )
   20 ml (CODAN, catalog number: 627602 )
3. Needle for syringes (Henke-Sass, Wolf, catalog number: 4710005016 )
4. 1,000, 500, 100 and 50 ml serum bottle for anaerobic bacterial culture (DWK Life Sciences, Wheaton, catalog number: W012467A [100 ml])
5. 100 ml serum bottle with large bottleneck (Merck, catalog number: STBMRFA12 )
6. Rubber stoppers for serum bottles (VWR, special request)
7. Metal caps for serum bottle (Thermo Fisher Scientific, catalog number: C4020-3A )
8. Serum bottle seal crimper (DWK Life Sciences, Wheaton, catalog number: 224322 )
9. 0.2 μm sterile filter (SARSTEDT, catalog number: 83.1826.001 )
10. 96-well polypropylene microplate (SARSTEDT, catalog number: 82.1581 )
11. 96-well microcentrifuge tube flipper rack with Lid (Fisher Scientific, catalog number: 11710344 )
12. Desulfitobacterium hafniense strain TCE1 (Gerritse et al., 1999)
13. Desulfitobacterium hafniense strain LBE (Comensoli et al., 2017)
14. Ethanol (Thommen Furler, catalog number: 180-VL54K )
15. Corroded iron coupons (steel coupons presenting a natural corrosion layer produced after outdoor exposure in the city of Zurich, Switzerland)
16. Adhesive Carbon Tape 12 mm x 20 m (Agar Scientific, catalog number: AGG3939A )
17. N₂ gas cylinder (Carbagas, catalog number: I4001 )
18. NH₄HCO₃ (Sigma-Aldrich, catalog number: A6141 )
19. NaHCO₃ (Sigma-Aldrich, catalog number: S5761 )
20. K₂HPO₄•3H₂O (Sigma-Aldrich, catalog number: P5504 )
21. NaH₂PO₄•2H₂O (Sigma-Aldrich, catalog number: 71505 )
22. Peptone (BD, catalog number: 211677 )
23. Resazurin sodium salt (Sigma-Aldrich, catalog number: R7017 )
24. Cyanocobalamin (Acros Organics, catalog number: 405920010 )
25. Riboflavin (Sigma-Aldrich, catalog number: R4500 )
26. Thiamine-hydrochloride (AppliChem, catalog number: A0955 )
27. Biotin (Thermo Fisher Scientific, Alfa Aesar, catalog number: A14207 )
28. P-aminobenzoate (sodium salt) (Sigma-Aldrich, catalog number: A9878 )
29. Pantothenate (sodium salt) (Sigma-Aldrich, catalog number: P3161 )
30. Folic acid•2H₂O (Sigma-Aldrich, catalog number: F7876 )
31. Lipoic acid (Sigma-Aldrich, Fluka, catalog number: 62320 )
32. Pyridoxine hydrochloride (Acros Organics, catalog number: 150770500 )
33. Nicotinic acid (Sigma-Aldrich, catalog number: N4126 )
34. EDTA disodium salt•2H₂O (Sigma-Aldrich, catalog number: E1644 )
35. FeCl₂•4H₂O (Sigma-Aldrich, catalog number: 44939 )
36. MnCl₂•4H₂O (Sigma-Aldrich, catalog number: M3634 )
37. CoCl₂•6H₂O (Sigma-Aldrich, catalog number: C8661 )
38. ZnCl₂ (Sigma-Aldrich, catalog number: 793523 )
39. CuCl₂•2H₂O (Sigma-Aldrich, catalog number: C3279 )
40. AlCl₃ (Sigma-Aldrich, catalog number: 237051 )
41. H₃BO₃ (Sigma-Aldrich, catalog number: B6768 )
42. Na₂MoO₄•2H₂O (Sigma-Aldrich, catalog number: 331058 )
43. NiCl₂•6H₂O (Sigma-Aldrich, catalog number: N6136 )
44. CaCl₂•2H₂O (Sigma-Aldrich, catalog number: 223506 )
45. MgCl₂•6H₂O (Sigma-Aldrich, catalog number: M2393 )
46. Na₂S•9H₂O (Sigma-Aldrich, catalog number: 208043 )
47. Sodium DL-lactate 60% solution (Sigma-Aldrich, catalog number: L1375 )
48. Disodium fumarate (Sigma-Aldrich, catalog number: F1506 )
49. HCl 37% (S-20) (Honeywell International, catalog number: 30721-1L-GL )
50. MilliQ water
51. Fe(II)-ammonium sulfate (Honeywell International, Fluka, catalog number: 09720 )
52. Fe(III)-citrate (Sigma-Aldrich, Fluka, catalog number: 44941-250G )
53. 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl) piperazine-N′-(2-ethanesulfonic acid) (HEPES) (Sigma-Aldrich, catalog number: H3375-250G )
54. NaOH (Sigma-Aldrich, catalog number: 71690 )
55. Goethite: α-FeO(OH) (Sigma-Aldrich, catalog number: 71063-100G ) (alternative source of solid Fe(III)-phase to akaganeite)
56. Fe₂O₃ (Sigma-Aldrich, catalog number: 529311-5G ) (alternative source of solid Fe(III)-phase to akaganeite)
57. Growth medium for D. hafniense (see Recipes)
    
    1. N₂-degassed H₂O 
    2. Sterile serum bottles
    3. Solution of sodium DL-lactate 40% (v/v)
    4. Solution of disodium fumarate 16% (v/v)
    5. Reducing agent solution 1 M
    6. Resazurin solution 0.5 g/L
    7. Vitamin solution 1
    8. Vitamin solution 2
    9. Vitamin solution 3
    10. Vitamin solution 4
    11. Trace elements solution
    12. Carbonate solution
    13. Solution A (basal medium)
    14. Solution B (vitamin solution)
    15. Solution C (buffering/reducing solution)
    16. Solution D
58. Soluble Fe(III)-citrate (35 g/L) – 100 ml (see Recipes)
    
    1. HCl solutions to adjust pH
    2. NaOH solutions to adjust pH
    3. Fe(III) solution
59. Solid Fe(III) suspension (see Recipes)
    
    1. Solid Fe(III) source
    2. Preparation of the suspension of solid Fe(III)-phase (akaganeite or goethite)
60. Ferrozine^® reagents (see Recipes)
    
    1. HCl solution 5 M
    2. Stock solution of Fe(II) 1 M for calibration curve
    3. Ferrozine^® reagent

Equipment

1. 1 L graduated flasks (SciLabware, catalog number: 1132/26 )
2. Magnetic bars (Sigma-Aldrich, BRAND, catalog numbers: Z328774 , Z328812 )
3. Stainless steel spatula (Sigma-Aldrich, catalog number: HS15909 )
4. Balance (Mettler-Toledo International, catalog number: PG5002 )
5. P20 pipetman (Gilson, catalog number: F123600 )
6. P200 pipetman (Gilson, catalog number: F123601 )
7. P1000 pipetman (Gilson, catalog number: F123602 )
8. pH meter
   
9. Bunsen burner (FIREBOY Plus) (Integra Biosciences, catalog number: 144000 )
10. Autoclave (Fedegari Autoklav FOB5/TS) (VITARIS, catalog number: 260000-FED , serial number: NBD801AV)
11. Orbital shaker (Kühner, model: SMX1200 )
12. Hotplate and magnetic Stirrer (Heidolph Instruments, catalog number: MR2002 )
13. Spinbar^® Magnetic Stir bar (Sigma-Aldrich, SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: Z126942-1EA )
14. Spectrophotometer cuvettes (Sigma-Aldrich, catalog number: C5291-100EA )
15. Spectrophotometer UV-visible (GENESYS^TM 10S) (Thermo Fisher Scientific, catalog number: 840-208100 )
16. Microplate reader (Biochrom, Asys Hitech, catalog number: UVM 340 )
17. pH meter (Benchtop Meter AE150) (Fisher Scientific, catalog number: 15524693 )
18. Biosafety cabinet equipped with UV lamp at 254 nm (Azbil Telstar, catalog number: Bio II Advance )
19. Chemical fume hood
20. Desiccator (BRAND, catalog number: 65815 )
21. Scanning electron microscope (SEM) (Philips ESEM XL30 FEG environmental scanning electron microscope equipped with an energy-dispersive X-ray analyzer (Philips)
22. Raman Microscope (HORIBA, JOBIN YVON, LabRAM Aramis microscope equipped with a Nd:YAG laser of 532 nm and controlled by LabSpec NGS spectral software. HORIBA, JOBIN YVON, catalog number: LabRAM Aramis, 3 lasers and xyz stage)
23. Vortex
24. Vacuum pump
25. Fridge
    

Procedure

1. Biomass production
   
   1. An estimation of the volume of the medium and the number of serum bottles is needed according to the number of bacterial strains to investigate. Include always an abiotic control. To test the ability of the strains TCE1 and LBE of D. hafniense, prepare 3 different bottles of medium (abiotic control, strain TCE1 and strain LBE). For the composition, see recipe point B. Growth medium for D. hafniense.
   2. Prepare the inoculum using normal growth medium for D. hafniense and incubate the bacterial strains in standard conditions (30 °C under agitation at 100 rpm) for approximatively 3 days. 
   3. Final OD₆₀₀ of the inoculum should be in the range of 0.1-0.15. To verify the OD₆₀₀, take a 1 ml sample from the culture using a sterile syringe and transfer it to a spectrophotometer cuvette. Measure OD₆₀₀. 
   4. Using a sterile syringe, add aseptically 40 ml of inoculum (5%) to 800 ml of growth medium. For this volume, a 1 L serum bottle is required (Figure 2).
      
      
      Figure 2. Scheme showing the inoculation procedure for the production of the bacterial biomass
      
      
   5. Incubate the cultures at 30 °C under agitation at 100 rpm until reaching an OD₆₀₀ between 0.12 and 0.18 for both the strains.
      
      
      
2. Incubation with iron sources
   
   1. Prepare the required number of empty 50 ml sterile serum bottles in N₂ atmosphere (see Recipe 2). Include triplicates for each iron source, strain used, and abiotic controls. Autoclave.
   2. To seal standard serum bottles, insert rubber stoppers and cover them with metal caps with hollow opening. When sealed, the hollow opening allows sampling with syringes during the experiment. Finally, use the seal crimper to tie and fix the metal caps to the serum bottles. 
   3. For the experiments with strains LBE and TCE1, prepare 18 empty 50 ml sterile serum bottles in N₂ atmosphere.
   4. In 9 of the empty bottles add aseptically 1.5 ml of Fe(III)-citrate solution (see Recipes). In the other 9 bottles add aseptically 1.5 ml of akageneite suspension (see Recipes).
   5. To test bacterial reduction on corroded coupons, sterilize 9 corroded coupons by spraying a solution of ethanol 70% (v/v), followed by exposure to UV radiation (20 min each side at 254 nm) under sterile conditions. Perform the sterilization procedure inside a biosafety cabinet.
   6. Add the 9 coupons in serum bottles with large bottlenecks, seal the bottles, vacuum the headspace and replace the atmosphere using N₂ Autoclave.
   7. Once all the bottles are autoclaved (Steps B1 and B6), add aseptically 20 ml of either, the sterile medium (abiotic control), or the biomass prepared in Procedure A. The procedure is done for the different iron sources (Fe(III)-citrate, akageneite, iron coupons). These proportions are calculated to obtain a culture with a starting concentration of 10 mM of soluble and solid Fe(III) phases. In the culture amended with the corroded coupons, iron concentration is unknown.
   8. To keep bacteria and the iron sources well mixed, incubate the serum bottles at 30 °C under agitation at 100 rpm. Incubate the bottles until a black precipitate is formed (7 days of incubation with D. hafniense). During incubation, the medium changes color from orange/green to black in the cultures amended with soluble Fe(III)-citrate and akaganeite suspension. The formation of black precipitates is an indication of iron reduction. The same phenomenon can be observed in the cultures amended with iron coupons, as the surface of the coupons and medium turn black.
   9. During the 7 days of incubation, collect daily 0.5 ml supernatant sample from each treatment and replicate. Make sure to take a representative sample. When the black precipitates form, insert the needle of the syringe in the rubber stopper of the serum bottle. Overturn the serum bottle. Shake gently and collect 0.5 ml of sample. 
   10. Transfer the sample into a 1.7 ml Eppendorf tube. 
   11. To all the tubes, add 50 μl of 5 M HCl.
   12. Mix by vortexing for 5 sec and incubate for 15 min at room temperature. This step will allow the dissolution of iron by the acid (HCl) and prevent the oxidation of Fe(II) ions.
       
   13. Freeze all the samples at -20 °C.
       
       
       
3. Validation of iron reduction
   
   1. Quantification of Fe(II) by spectrophotometry
      The Ferrozine^® reagent will become violet upon reaction with Fe(II). Color intensity is proportional to the concentration of Fe(II). Performing a calibration curve will then allow to quantify Fe(II) content in the samples.
      
      1. Switch on the microplate reader and set the wavelength at 562 nm.
      2. Take the standard solutions for the calibration curve from the fridge (see Recipes). 
      3. Thaw the frozen samples (Steps B9-B12) taken from the cultures before Fe(II) quantification at room temperature.
      4. Mix by vortexing for 5 sec.
      5. Transfer 10 μl of the reaction mixtures (standard solution or samples) to a 96-well microplate.
         Note: when taking the samples make sure to mix them well with a vortex before pipetting.
      6. Add 90 μl of Ferrozine^® reagent to the microplate wells. 
      7. Do not forget to perform blank samples using the standard growth medium of D. hafniense. 
      8. Measure absorbance at 562 nm in the following 2 min using the microplate reader. If the absorbance values are higher than the value of the more concentrated sample in the calibration curve (in our case 0.239 nm for the 1000 μM standard), prepare a dilution of the corresponding sample starting from the original sample and repeat the measure from Step C1d. 
      9. Measure each replicate and perform 2-3 measures for each sample.
         
         
         
   2. Characterization of Fe(II)-biogenic minerals
      
      1. When the cultures become black and a precipitate is observed, remove the coupons from the culture and sterilize them by spraying a solution of ethanol 70% (v/v), followed by exposure to UV radiation (20 min on each side). With the bacterium D. hafniense, coupons can be removed from serum bottles after 7 days.
      2. Store the treated coupons under vacuum in a desiccator to avoid changes in the oxidation state of the biogenic minerals produced during the experiment.
      3. To study the morphology, the distribution and the elemental composition of the newly formed biogenic minerals, analyze coupons with SEM by simply positioning them inside the microscope chamber. To fix coupons to the sample holder use the carbon tape as illustrated in Figure 3. Observe samples in secondary electrons mode at an acceleration potential of 10-25 keV.
         
         
         Figure 3. Sample holder with coupons and carbon tape, prepared for the SEM analysis
         
         
      4. To study the molecular composition of the newly formed Fe(II)-biogenic minerals, perform a Raman spectroscopy analysis directly on the surface of the coupons. To do so, simply position the coupons under the lowest objective and focus on the area to be analyzed. Change the objective and make the focus again until reaching the 400x magnification. Use the following set-up to obtain good quality spectra and to avoid burning of the surface of the sample: laser at 532 nm at power lower than 1 mW (600 g/mm), spectral interval between 100 and 1,600 cm^-1 and 1,000 μm hole, 100 μm slit and 5 accumulations of 100 sec.

Data analysis

1. Calculation of Fe(II) content in mM
   Convert the absorbance values in Fe(II) concentrations using the calibration curve with μM as unit, as illustrated in Figure 4. Consider only the absorbance values that are in the range of the calibration curve (in our case between 0.011 and 0.239). If the measured absorbance is lower than that, consider Fe(II) content as 0 μM. If values are higher, dilute the original sample and repeat the measurement until absorbance is within the linear range of the calibration curve.
   
   
   Figure 4. Procedure for the quantification of Fe(II) with the Ferrozine^® reactive. A. Example of the calibration curve. On the left a table with the numeric data is presented; on the right the corresponding graph showing the equation and correlation coefficient of the calibration curve is shown. Data represent average values of duplicates. The increase in absorbance was linear between 50 and 1,000 μM and the correlation coefficients were 0.9999. B. Example of data processing. On the left, the equation extrapolated from the calibration curve is shown, and on the right, an example of data processing is presented. Data are from abiotic controls amended with Fe(II)-citrate sampled at day 0.
   
   When all the absorbance values are converted to Fe(II) content, data can be presented in histograms as shown in Figure 5.
   
   
   Figure 5. Quantification of Fe(II) content with the Ferrozine^® assay. Graph represents Fe(II) content in the abiotic control as well as in cultures amended with soluble Fe(III)-citrate (left) and solid Fe(III) (right) as calculated from the calibration curve.
   
   
2. Characterization of biogenic minerals
   Morphology of the biogenic crystals can be studied directly by observing the SEM micrographs. In order to identify the newly formed biogenic minerals, compare the obtained spectra (recorded on the surface of the treated coupons) with reference spectra found in the software library as well as with Raman shifts presented in literature (Frost et al., 2002; Monnier et al., 2011; Rémazeilles et al., 2013). Figure 6 shows an example of the results obtained for the corroded coupons treated with D. hafniense strains TCE1 and LBE.
   
   
   Figure 6. Iron reduction tests on corroded iron coupons treated with cultures of D. hafniense strains TCE1 and LBE. A. Morphology of the newly formed biogenic minerals. Left column: appearance of the coupons; right column: corresponding SEM images taken in the area indicated by the black square on the pictures of the coupons. B. Molecular composition of the surface of the coupons after incubation. Left column: area analyzed by Raman spectroscopy (black squares); and right column: corresponding Raman spectra. Minerals are identified as: 1: Lepidocrocite (Le), 2: Mixture of poorly crystallized mackinawite (M) and elemental sulfur (S), 3: Vivianite (Vi), and 4: Mixture of vivianite (Vi) and lepidocrocite (Le).

Notes

1. Reproducibility
   The collection of culture samples is a delicate step. In fact, due to the formation of iron precipitates during incubation, it is difficult to collect representative samples. Therefore, before sampling the cultures, makes sure to mix them well, otherwise the amount of iron in the sample will not be representative.
2. Abiotic control
   Media for growing anaerobic bacteria are often complex and contain reducing agents such as Na₂S. Therefore, in order to exclude abiotic reduction of Fe(III), it is essential to perform abiotic controls with all the iron sources tested.
3. Ferrozine^® reagent
   The intensity of the mixture containing the Ferrozine^® reagent and the samples changes over time. Therefore, it is important to measure the absorbance precisely after 2 min of incubation, and to use the same procedure for all the samples.
4. Storage of treated coupons
   The stability of the newly-formed biogenic minerals is unknown. So, until identification with Raman spectroscopy, store the iron coupons in a desiccator to avoid changes in the oxidation state of the biogenic minerals produced during the treatment.
   

Recipes

1. Growth medium for D. hafniense
   
   1. N₂-degassed H₂O
      
      1. Boil, 500 ml of MilliQ water, with a hotplate stirrer
      2. Cool down under N₂, distribute 80 ml to serum bottle, gas exchange for N₂, autoclave (120 °C, 20 min)
      3. Store at room temperature up to 12 months
   2. Sterile serum bottles
      Gas exchange for N₂ and autoclave
      Store at room temperature up to 12 months
   3. Solution of sodium DL-lactate 40% (v/v)
      
      1. Dilute the 60% stock solution to 40% with MilliQ water
      2. Distribute 100 ml to serum bottle, gas exchange for N₂, autoclave
      3. Store at 4 °C up to 12 months
   4. Solution of disodium fumarate 16% (v/v)
      
      1. Add 80 g of disodium fumarate to 500 ml of MilliQ water
      2. Distribute 100 ml to serum bottle, gas exchange for N₂, autoclave
      3. Store at room temperature up to 12 months
   5. Reducing agent solution 1 M
      
      1. Wash crystals of Na₂S•9H₂O with N₂-degassed H₂O to remove the already oxidized part of the crystals
      2. Dry crystals with a tissue paper
      3. Weight 24.02 g of this compound (dry weight)
      4. Dissolve it in 100 ml of degassed-MilliQ water
      5. Filter sterilize into serum bottles with a 0.2 μm filter
      6. Gas exchange for N₂
      7. Store at 4 °C up to 12 months
   6. Resazurin solution 0.5 g/L
      Add 0.1 g of resazurin sodium salt to 200 ml of MilliQ water
      Store at 4 °C up to 12 months
   7. Vitamin solution 1
      
      1. Add 250 mg of cyanocobalamin to 1 L of MilliQ water
      2. Filter sterilize into a sterile serum bottle, gas exchange for N₂
      3. Store at 4 °C up to 12 months
   8. Vitamin solution 2
      
      1. Add 50 mg of riboflavin to 1 L of MilliQ water
      2. Filter sterilize into a sterile serum bottle, gas exchange for N₂
      3. Store at 4 °C up to 12 months
   9. Vitamin solution 3
      
      1. Add 100 mg of thiamine-hydrochloride to 1 L of MilliQ water
      2. Filter sterilize into a sterile serum bottle, gas exchange for N₂
      3. Store at 4 °C up to 12 months
   10. Vitamins solution 4
       
       1. Add all of the components to 1 L of MilliQ water
          50 mg of biotin
          250 mg of p-aminobenzoate (sodium salt)
          50 mg of pantothenate (sodium salt)
          20 mg of folic acid•2H₂O
          50 mg of lipoic acid
          100 mg of pyridoxine-hydrochloride
          550 mg of nicotinic acid
       2. Filter sterilize into sterile serum bottle with a 0.2 μm filter, gas exchange for N₂
       3. Store at 4 °C up to 12 months
   11. Trace elements solution
       
       1. Dissolve 500 mg of EDTA in 900 ml of MilliQ water, adjust the pH to 7.0 with HCl, then add the following compounds:
          2 mg of FeCl₂•4H₂O
          100 mg of MnCl₂•4H₂O
          190 mg of CoCl₂•6H₂O
          70 mg of ZnCl₂
          2.55 mg of CuCl₂•2H₂O
          5.52 mg of AlCl₃
          6 mg of H₃BO₃
          41.4 mg of Na₂MoO₄•2H₂O
          24 mg of NiCl₂•6H₂O
       2. Add MilliQ water to 1 L
       3. Store at 4 °C up to 12 months
   12. Carbonate solution
       
       1. Add 9.01 g of NH₄HCO₃ and 76.11 g of NaHCO₃ to 1 L of MilliQ water
       2. Boil, cool down under N₂/CO₂ (4:1), distribute 49 ml to each serum bottle, gas exchange for N₂/CO₂ (4:1), autoclave
       3. Store at RT up to 12 months
   13. Solution A (basal medium)
       
       1. Add all of the components to 1 L of MilliQ water:
          0.958 g of K₂HPO₄•3H₂O
          0.218 g of NaH₂PO₄•2H₂O
          0.1 g of Peptone
          1 ml of Resazurin solution 0.5 g/L
       2. Boil, cool down under N₂/CO₂ (4:1), distribute to serum bottles, gas exchange for N₂/CO₂ (4:1), autoclave
       3. Store at room temperature up to 12 months
   14. Solution B (vitamin solution)
       To 20 ml of anaerobic sterile MilliQ water, add aseptically the following solutions with syringes:
       1 ml Trace elements solution
       1 ml Vitamins solution 1
       1 ml Vitamins solution 2
       1 ml Vitamins solution 3
       1 ml Vitamins solution 4
       Store at 4 °C up to 3 months
   15. Solution C (buffering/reducing solution)
       To 49 ml of carbonate solution, add 1 ml of reducing agent solution
       Store at 4 °C up to 3 months
   16. Solution D
       
       1. Add 4.40 g of CaCl₂•2H₂O and 4.06 g of MgCl₂•6H₂O to 1 L of MilliQ water
       2. Distribute 200 ml in serum bottle, gas exchange for N₂ and autoclave
       3. Store at 4 °C up to 12 months
          
          
   Growth medium completion
   To 45 ml of solution A, add the following components aseptically by syringe:
   1.25 ml of solution B
   2 ml of solution C
   1.25 ml of solution D
   1 ml of lactate solution
   1 ml of fumarate solution
   The pH should be between 7.0 and 7.6
   To verify the pH, collect a 1 ml sample with a syringe and measure on a pH meter
   
   
2. Soluble Fe(III)-citrate (35 g/L) – 100 ml
   
   1. HCl solutions to adjust pH
      
      1. Fill a part of a 100-ml graduated flask with MilliQ water
      2. Put 41.5 ml of HCl 37%
      3. Fill up to 100 ml with MilliQ water
      4. Perform dilutions in order to obtain a range of concentrations from 5 M (starting solution) to 0.01 M
      5. Store at room temperature up to 6 months
   2. NaOH solutions to adjust pH
      
      1. Dissolve 20 g of NaOH pellets in 100 ml of MilliQ water
      2. Perform dilutions in order to obtain a range of concentrations from 5 M (starting solution) to 0.01 M
      3. Store at room temperature up to 6 months
   3. Fe(III) solution
      
      1. Dissolve 3.5 g of Fe(III)-citrate in 100 ml of milliQ water. To facilitate dissolution, add a magnetic bar and mix the solution with a magnetic stirrer at 80 °C. This step can take 1-2 h. When the powder is completely dissolved no residual particle should be visible, the color of the solution becomes yellow and the pH is extremely acidic (pH 1-2)
      2. Adjust the pH of the solution to 7 by adding drops of NaOH. After this procedure, the solution becomes brown-orange in color
      3. To remove oxygen mark with an indelible pen the level of the solution in the flask, add extra MilliQ water to the solution, and let the solution boil until all the added water is evaporated (help yourself with the pen mark to detect the original volume of the solution). Then cool down the solution by flushing N₂/CO₂, and seal the serum bottle with rubber stoppers and metal caps using the serum bottle seal crimper. Sterilize the solution by autoclaving (120 °C, 20 min).
      4. Store at 4 °C up to 6 months
         
         
         
3. Solid Fe(III) suspension
   
   1. Solid Fe(III) source
      
      1. In the original experiment, akaganeite (FeO_0.833(OH)_1.167Cl_0.167) was used. Akaganeite was provided by the Swiss National Museum. This compound was synthesized following the protocols by Schwertmann and Cornell (2008). However, for this test any kind of insoluble Fe(III)-oxides or Fe(III)-oxyhydroxides can be used to prepare the suspension (i.e., Fe₂O₃ or α-FeO(OH), Sigma-Aldrich)
      2. Store at 4 °C up to 6 months
   2. Preparation of the suspension of solid Fe(III)-phase (akaganeite or goethite)
      
      1. For akaganeite suspension (10 g/L – 100 ml):
         Add 1.0 g of akaganeite to 100 ml of MilliQ water
      2. For goethite suspension (13 g/L – 100 ml):
         Add 1.3 g of goethite to 100 ml of MilliQ water
      3. Control the pH of the suspension and adjust it with drops of HCl or NaOH solutions to pH 7
      4. Repeat all the steps already described for the preparation of the soluble-Fe(III) solution in order to remove oxygen and sterilize the solution
      5. Store at 4 °C up to 6 months
         
         
         
4. Ferrozine^® reagents
   
   1. HCl solution 5 M
      Same procedure previously described (B1. HCl solutions to adjust pH)
   2. Stock solution of Fe(II) 1 M for calibration curve
      
      1. Clean a graduated flask of 1 L with HCl (5 M)
      2. Wash with MilliQ water
      3. Fill with a part of MilliQ water
      4. Add 41.55 ml of HCl 37% (S-20)
      5. Add 392.14 mg of Fe(II)-ammonium sulfate
      6. Fill up to 1 L with MilliQ water
      7. Dilute the stock solution to obtain the following concentrations: 50, 100, 250, 500, 750 and 1,000 μM.
      8. Aliquot and store at 4 °C up to 6 months (protect from light)
   3. Ferrozine^® reagent
      
      1. Clean a graduated flask of 1 L with HCl (5 M)
      2. Wash with MilliQ water
      3. Fill with a part of MilliQ water
      4. Add 11.9 ml of HEPES buffer (final concentration 50 mM)
      5. Clean the flask wall with MilliQ water
      6. Add 1 g of Ferrozine^®
      7. Fill up to 1 L with MilliQ water
      8. Adjust the pH with drops of NaOH solution to 7
      9. Store at 4 °C up to 2 months

Acknowledgments

The authors are grateful to the Swiss National Science Foundation for the Ambizione grant (PZ00P2_142514, 2013-2016, Pi: Edith Joseph). The authors also want to acknowledge the research conservation laboratory of the Swiss National Museum for the help in conducting Raman investigations (Dr. Marie Woerle and Dr. Tiziana Lombardo) and providing the iron coupons used in the experiments.
This protocol is a modified version of the method described by Comensoli et al. (2017). In addition, the composition of the culture media was adapted from Gerritse et al. (1999), while the Ferrozine^® assay employed to quantify Fe(II) ions in liquid solutions was adapted from Stookey (1970).

Competing interests

The authors have no conflicts of interest or competing interests to declare.

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