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May 2017

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Soluble and Solid Iron Reduction Assays with Desulfitobacterium hafniense
哈夫尼脱亚硫酸杆菌用于可溶和固体铁的还原试验   

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Abstract

There is a pressing need to develop sustainable and efficient methods to protect and stabilize iron objects. To develop a conservation-restoration method for corroded iron objects, this bio-protocol presents the steps to investigate reductive dissolution of ferric iron and biogenic production of stabilizing ferrous iron minerals in the strict anaerobe Desulfitobacterium hafniense (strains TCE1 and LBE). We investigated iron reduction using three different Fe(III) sources: Fe(III)-citrate (a soluble phase), akaganeite (solid iron phase), and corroded coupons. This protocol describes a method that combines spectrophotometric quantification of the complex Fe(II)-Ferrozine® with mineral characterization by scanning electron microscopy and Raman spectroscopy. These three methods allow assessing reductive dissolution of ferric iron and biogenic mineral production as a promising alternative for the development of an innovative sustainable method for the stabilization of corroded iron.

Keywords: Reductive dissolution of ferric iron (铁离子的还原性溶解作用), Fe(II) quantification (铁离子定量), Biogenic minerals (生物矿物), Desulfitobacterium hafniense (哈夫尼脱亚硫酸杆菌), Iron passivation (铁钝化), Heritage conservation (文物保护)

Background

Since the Iron Age, iron has been used to produce everyday utensils. Therefore, archaeological iron findings are an extremely important testimony of the past and should be preserved. However, due to its reactivity, iron can be easily corroded and archaeological iron objects risk to be completely damaged. When buried, iron artifacts develop a complex corrosion layer according to the environmental conditions of the burial site. After excavation, conditions change and the corrosion layer becomes unstable. To avoid complete destruction, archaeological iron objects require a rapid stabilization treatment. Currently, available stabilization treatments do not provide long-term protection and have substantial drawbacks, such as toxicity, low efficiency, and production of large amount of waste (Scott and Eggert, 2009; Rimmer et al., 2012). Consequently, it is necessary to develop new technologies to stabilize archaeological iron artifacts.

Exploiting a microbial metabolism is increasingly considered for the development of more efficient, sustainable and eco-friendly treatments in conservation-restoration (Ranalli et al., 2005; Cappitelli et al., 2006 and 2007; Jonkers, 2011; Joseph et al., 2011, 2012 and 2013; Bosch-Roig and Ranalli, 2014). Our research team is developing a treatment based on the reductive dissolution of ferric iron under anaerobic conditions (Kooli et al., 2018; Comensoli et al., 2017). The unstable corrosion products are converted into more stable biogenic minerals (i.e., magnetite and vivianite), as a byproduct of bacterial iron reduction. This conversion would stabilize the corrosion layer of the object.

In order to study the suitability of the chosen bacteria, iron reduction has to be carefully monitored. Several methods are available to quantify iron. Inductive coupled plasma mass spectrometry (ICP-MS) is useful to measure trace elements with concentrations of less than 1 ppm (Meissner et al., 2004). However, it requires expensive equipment and does not provide information on the oxidation state of iron if not combined with chromatographic separation devices such as high-performance liquid chromatography (HPLC), ion chromatography (IC), gas chromatography (GC), and capillary electrophoresis (CE) (Thomas, 2013). A spectrophotometric method to measure Fe(II) uses the metal-ligand ortho-phenanthroline (Fortune and Mellon, 1938). This compound is now considered carcinogenic (Whittaker et al., 2001). Therefore, for this protocol we selected the spectrophotometric quantification of Fe(II) with the Ferrozine® assay. This simple and reliable method requires standard lab equipment and can be used to analyze many samples. In addition, the characterization of biogenic minerals was made based on their appearance, morphology and molecular composition. For these analyses, we used scanning electron microscopy and Raman spectroscopy.

This Bio-protocol consists of three main steps (Figure 1): A. Biomass production; B. Incubation with iron sources; C. Validation of iron reduction.


Figure 1. Graphical summary of the overall structure of this bio-protocol

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 H2
    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 (GENESYSTM 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 (FeO0.833(OH)1.167Cl0.167) 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.

References

  1. Bosch-Roig, P. and Ranalli, G. (2014). The safety of biocleaning technologies for cultural heritage. Front Microbiol 5: 155.
  2. Cappitelli, F., Toniolo, L., Sansonetti, A., Gulotta, D., Ranalli, G., Zanardini, E. and Sorlini, C. (2007). Advantages of using microbial technology over traditional chemical technology in removal of black crusts from stone surfaces of historical monuments. Appl Environ Microbiol 73(17): 5671-5675.
  3. Cappitelli, F., Zanardini, E., Ranalli, G., Mello, E., Daffonchio, D. and Sorlini, C. (2006). Improved methodology for bioremoval of black crusts on historical stone artworks by use of sulfate-reducing bacteria. Appl Environ Microbiol 72(5): 3733-3737.
  4. Comensoli, L., Maillard, J., Albini, M., Sandoz, F., Junier, P. and Joseph, E. (2017). Use of bacteria to stabilize archaeological iron. Appl Environ Microbiol 83(9).
  5. Fortune, W. B. and Mellon, M. G. (1938). Determination of iron with o-phenanthroline: a spectrophotometric study. Ind Eng Chem 10(2): 60-64.
  6. Frost, R. L., W. Martens, P. Williams. and J. T. Kloprogge. (2002). Raman and infrared spectroscopic study of the vivianite-group phosphates vivianite, baricite and bobierrite. Mineral Mag 66(6): 1063-1073.
  7. Gerritse, J., Drzyzga, O., Kloetstra, G., Keijmel, M., Wiersum, L. P., Hutson, R., Collins, M. D. and Gottschal, J. C. (1999). Influence of different electron donors and acceptors on dehalorespiration of tetrachloroethene by Desulfitobacterium frappieri TCE1. Appl Environ Microbiol 65(12): 5212-5221.
  8. Jonkers, H. M. (2011). Bacteria-based self-healing concrete. Heron 56(1/2)
  9. Joseph, E., Cario, S., Simon, A., Worle, M., Mazzeo, R., Junier, P. and Job, D. (2011). Protection of metal artifacts with the formation of metal-oxalates complexes by Beauveria bassiana. Front Microbiol 2: 270.
  10. Joseph, E., Letardi, P., Comensoli, L., Simon, A., Junier P., Job, D. and Wörle, M. (2013). Assessment of a biological approach for the protection of copper alloys artefacts. In: Hyslpop, E., Gonzalez, V., Troalen, L. and Wilson, L. (Eds.). Conference Proceedings of Metal 2013, Interim Meeting of the ICOM-CC Metal WG. Historic Scotland, Edinburgh, 203-208.
  11. Joseph, E., Simon, A., Mazzeo. R., Job. Daniel and Wörle, M. (2012). Spectroscopic characterization of an innovative biological treatment for corroded metal artefacts. Raman Spectroscopy in Art and Archaeology 43(11): 1612-1616.
  12. Kooli, W. M., Comensoli, L., Maillard, J., Albini, M., Gelb, A., Junier, P. and Joseph, E. (2018). Bacterial iron reduction and biogenic mineral formation for the stabilisation of corroded iron objects. Sci Rep 8(1): 764.
  13. Meissner, K., T. Lippert, A. Wokaun and D. Guenther (2004). Analysis of trace metals in comparison of laser-induced breakdown spectroscopy with LA-ICP-MS. Thin Solid Films 453: 316-322.
  14. Monnier, J., L. Bellot-Gurlet, D. Baron, D. Neff, I. Guillot and P. Dillmann. (2011). A methodology for Raman structural quantification imaging and its application to iron indoor atmospheric corrosion products. J Raman Spectrosc 42(4): 773-781.
  15. Ranalli, G., Alfano, G., Belli, C., Lustrato, G., Colombini, M. P., Bonaduce, I., Zanardini, E., Abbruscato, P., Cappitelli, F. and Sorlini, C. (2005). Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes. J Appl Microbiol 98(1): 73-83.
  16. Rémazeilles, C., K. Tran, E. Guilminot, E. Conforto and P. Refait. (2013). Study of Fe (II) sulphides in waterlogged archaeological wood. Stud Conserv 58(4): 297-307.
  17. Rimmer, M., Watkinson, D. and Wang, Q. (2012). The efficiency of chloride extraction from archaeological iron objects using deoxygenated alkaline solutions. Stud Conserv 57(1): 29-41
  18. Schwertmann, U. and Cornell, R. M. (2008). Iron oxides in the laboratory: preparation and characterization. John Wiley & Sons.
  19. Scott, D. A. and Eggert, G. (2009). Iron and steel in art: corrosion, colorants, conservation. Archetype Publications.
  20. Stookey, L. L. (1970). Ferrozine - a new spectrophotometric reagent for iron. Anal Chem 42(7): 779-781.
  21. Thomas, R. (2013). Practical guide to ICP-MS: a tutorial for beginners. CRC Press.
  22. Whittaker, P., Seifried, H. E., San, R. H., Clarke, J. J. and Dunkel, V. C. (2001). Genotoxicity of iron chelators in L5178Y mouse lymphoma cells. Environ Mol Mutagen 38(4): 347-356.

简介

迫切需要开发可持续和有效的方法来保护和稳定铁制物体。为了开发腐蚀铁物体的保护 - 恢复方法,该生物方案提出了研究严格厌氧菌[Desulfitobacterium hafniense (菌株TCE1)中三价铁的还原溶解和稳定亚铁矿物质的生物产生的步骤。和LBE)。我们使用三种不同的Fe(III)来源研究了铁还原:Fe(III) - 柠檬酸盐(可溶相),akaganeite(固体铁相)和腐蚀的试样。该协议描述了一种方法,该方法结合了复杂的Fe(II)-Ferrozine ®的分光光度定量,通过扫描电子显微镜和拉曼光谱进行矿物表征。这三种方法可以评估三价铁的还原溶解和生物矿物质生产,作为开发一种创新的可持续方法来稳定腐蚀铁的有希望的替代方法。

【背景】自铁器时代以来,铁已被用于生产日常用具。因此,考古学上的铁试验是过去极其重要的证据,应予以保留。然而,由于其反应性,铁容易被腐蚀并且考古铁物体可能被完全损坏。埋藏时,铁制品会根据埋葬地点的环境条件形成复杂的腐蚀层。挖掘后,条件发生变化,腐蚀层变得不稳定。为避免完全破坏,考古铁制物需要快速稳定处理。目前,可用的稳定化处理不能提供长期保护并且具有实质性缺点,例如毒性,低效率和大量废物的产生(Scott和Eggert,2009; Rimmer 等人, 2012)。因此,有必要开发新技术来稳定考古铁器。

越来越多地考虑利用微生物代谢来开发更有效,可持续和环保的保护 - 恢复治疗(Ranalli et al。,2005; Cappitelli et al。 ,2006和2007; Jonkers,2011; Joseph et al。,2011,2012和2013; Bosch-Roig和Ranalli,2014)。我们的研究团队正在开发一种基于厌氧条件下三价铁还原溶解的处理方法(Kooli et al。,2018; Comensoli et al。,2017)。不稳定的腐蚀产物转化为更稳定的生物矿物质(即,磁铁矿和vivianite),作为细菌铁还原的副产品。这种转换将稳定物体的腐蚀层。

为了研究所选细菌的适用性,必须仔细监测铁的还原。有几种方法可用于量化铁。电感耦合等离子体质谱(ICP-MS)可用于测量浓度小于1 ppm的微量元素(Meissner et al。,2004)。然而,如果不与色谱分离装置如高效液相色谱(HPLC),离子色谱(IC),气相色谱(GC)和毛细管电泳相结合,它需要昂贵的设备并且不提供有关铁的氧化态的信息。 (CE)(Thomas,2013)。用于测量Fe(II)的分光光度法使用金属配体邻菲啰啉(Fortune和Mellon,1938)。该化合物现在被认为是致癌物(Whittaker et al。,2001)。因此,对于该方案,我们选择用Ferrozine ®测定法对Fe(II)进行分光光度定量。这种简单可靠的方法需要标准的实验室设备,可用于分析许多样品。此外,生物矿物的表征是基于它们的外观,形态和分子组成。对于这些分析,我们使用扫描电子显微镜和拉曼光谱。

该生物方案包括三个主要步骤(图1):A。生物量生产; B.与铁源孵育; C.验证铁还原。


图1.该生物方案总体结构的图形摘要

关键字:铁离子的还原性溶解作用, 铁离子定量, 生物矿物, 哈夫尼脱亚硫酸杆菌, 铁钝化, 文物保护

材料和试剂

  1. 1.7毫升Eppendorf离心管(Corning,Axygen ®,目录号:MCT-175-C)
  2. 注射器
    1毫升(CODAN,目录号:621640)
    5毫升(CODAN,目录号:625607)
    20毫升(CODAN,目录号:627602)
  3. 注射器针头(Henke-Sass,Wolf,目录号:4710005016)
  4. 用于厌氧细菌培养的1,000,500,100和50 ml血清瓶(DWK Life Sciences,Wheaton,目录号:W012467A [100 ml])
  5. 100毫升含大瓶颈的血清瓶(默克,目录号:STBMRFA12)
  6. 血清瓶用橡胶塞(VWR,特殊要求)
  7. 用于血清瓶的金属盖(Thermo Fisher Scientific,目录号:C4020-3A)
  8. 血清瓶密封压接器(DWK Life Sciences,Wheaton,目录号:224322)
  9. 0.2μm无菌过滤器(SARSTEDT,目录号:83.1826.001)
  10. 96孔聚丙烯微孔板(SARSTEDT,目录号:82.1581)
  11. 带盖的96孔微量离心管鳍架(Fisher Scientific,目录号:11710344)
  12. Desulfitobacterium hafniense 菌株TCE1(Gerritse et al。,1999)
  13. Desulfitobacterium hafniense 菌株LBE(Comensoli et al。,2017)
  14. 乙醇(Thommen Furler,目录号:180-VL54K)
  15. 腐蚀铁试样(在瑞士苏黎世市户外暴露后产生天然腐蚀层的钢试样)
  16. 粘合碳带12 mm x 20 m(Agar Scientific,目录号:AGG3939A)
  17. N 2 气瓶(Carbagas,目录号:I4001)
  18. NH 4 HCO 3 (Sigma-Aldrich,目录号:A6141)
  19. NaHCO 3 (Sigma-Aldrich,目录号:S5761)
  20. K 2 HPO 4 •3H 2 O(Sigma-Aldrich,目录号:P5504)
  21. NaH 2 PO 4 •2H 2 O(Sigma-Aldrich,目录号:71505)
  22. 蛋白胨(BD,目录号:211677)
  23. 刃天青钠盐(Sigma-Aldrich,目录号:R7017)
  24. Cyanocobalamin(Acros Organics,目录号:405920010)
  25. 核黄素(Sigma-Aldrich,目录号:R4500)
  26. 盐酸硫胺素(AppliChem,目录号:A0955)
  27. 生物素(Thermo Fisher Scientific,Alfa Aesar,目录号:A14207)
  28. 对氨基苯甲酸盐(钠盐)(Sigma-Aldrich,目录号:A9878)
  29. 泛酸(钠盐)(西格玛奥德里奇,目录号:P3161)
  30. 叶酸•2H 2 O(Sigma-Aldrich,目录号:F7876)
  31. 硫辛酸(Sigma-Aldrich,Fluka,目录号:62320)
  32. 盐酸吡哆醇(Acros Organics,目录号:150770500)
  33. 烟酸(Sigma-Aldrich,目录号:N4126)
  34. EDTA二钠盐•2H 2 O(Sigma-Aldrich,目录号:E1644)
  35. FeCl 2 •4H 2 O(Sigma-Aldrich,目录号:44939)
  36. MnCl 2 •4H 2 O(Sigma-Aldrich,目录号:M3634)
  37. CoCl 2 •6H 2 O(Sigma-Aldrich,目录号:C8661)
  38. ZnCl 2 (Sigma-Aldrich,目录号:793523)
  39. CuCl 2 •2H 2 O(Sigma-Aldrich,目录号:C3279)
  40. AlCl 3 (Sigma-Aldrich,目录号:237051)
  41. H 3 BO 3 (Sigma-Aldrich,目录号:B6768)
  42. Na 2 MoO 4 •2H 2 O(Sigma-Aldrich,目录号:331058)
  43. NiCl 2 •6H 2 O(Sigma-Aldrich,目录号:N6136)
  44. CaCl 2 •2H 2 O(Sigma-Aldrich,目录号:223506)
  45. MgCl 2 •6H 2 O(Sigma-Aldrich,目录号:M2393)
  46. Na 2 S•9H 2 O(Sigma-Aldrich,目录号:208043)
  47. DL-乳酸钠60%溶液(Sigma-Aldrich,目录号:L1375)
  48. 富马酸二钠(Sigma-Aldrich,目录号:F1506)
  49. HCl 37%(S-20)(Honeywell International,目录号:30721-1L-GL)
  50. MilliQ水
  51. Fe(II) - 硫酸铵(Honeywell International,Fluka,目录号:09720)
  52. Fe(III) - 柠檬酸盐(Sigma-Aldrich,Fluka,目录号:44941-250G)
  53. 4-(2-羟乙基)哌嗪-1-乙磺酸,N-(2-羟乙基)哌嗪-N' - (2-乙磺酸)(HEPES)(Sigma-Aldrich,目录号:H3375-250G)
  54. NaOH(Sigma-Aldrich,目录号:71690)
  55. 针铁矿:α-FeO(OH)(西格玛奥德里奇,目录号:71063-100G)(固体Fe(III) - 相对于akaganeite的替代来源)
  56. Fe 2 O 3 (Sigma-Aldrich,目录号:529311-5G)(固体Fe(III) - 相到akaganeite的替代来源)
  57. D的生长培养基。 hafniense (见食谱)
    1. N 2 - 脱气H 2 O 
    2. 无菌血清瓶
    3. DL-乳酸钠溶液40%(v / v)
    4. 富马酸二钠溶液16%(v / v)
    5. 还原剂溶液1 M.
    6. 刃天青溶液0.5克/升
    7. 维生素溶液1
    8. 维生素溶液2
    9. 维生素溶液3
    10. 维生素溶液4
    11. 微量元素解决方案
    12. 碳酸盐溶液
    13. 溶液A(基础培养基)
    14. 溶液B(维生素溶液)
    15. 解决方案C(缓冲/还原溶液)
    16. 解决方案D.
  58. 可溶性柠檬酸铁(III)(35克/升) - 100毫升(见食谱)
    1. HCl溶液调节pH值
    2. NaOH溶液调节pH值
    3. Fe(III)溶液
  59. 固体Fe(III)悬浮液(见食谱)
    1. 固体Fe(III)源
    2. 制备固体Fe(III) - 相(akaganeite或针铁矿)的悬浮液
  60. Ferrozine ®试剂(见食谱)
    1. HCl溶液5M
    2. Fe(II)1M的储备溶液用于校准曲线
    3. Ferrozine ®试剂

设备

  1. 1升刻度烧瓶(SciLabware,目录号:1132/26)
  2. 磁棒(Sigma-Aldrich,BRAND,目录号:Z328774,Z328812)
  3. 不锈钢刮刀(Sigma-Aldrich,目录号:HS15909)
  4. Balance(Mettler-Toledo International,目录号:PG5002)
  5. P20移液器(Gilson,产品目录号:F123600)
  6. P200移液器(Gilson,目录号:F123601)
  7. P1000移液器(Gilson,产品目录号:F123602)
  8. pH计
  9. 本生灯(FIREBOY Plus)(Integra Biosciences,目录号:144000)
  10. 高压灭菌器(Fedegari Autoklav FOB5 / TS)(VITARIS,目录号:260000-FED,序列号:NBD801AV)
  11. 轨道振动筛(Kühner,型号:SMX1200)
  12. 电炉和磁力搅拌器(Heidolph Instruments,目录号:MR2002)
  13. Spinbar ®磁力搅拌棒(Sigma-Aldrich,SP Scienceware - Bel-Art Products - H-B Instrument,目录号:Z126942-1EA)
  14. 分光光度计比色皿(Sigma-Aldrich,目录号:C5291-100EA)
  15. 紫外可见分光光度计(GENESYS TM 10S)(Thermo Fisher Scientific,目录号:840-208100)
  16. 酶标仪(Biochrom,Asys Hitech,目录号:UVM 340)
  17. pH计(台式仪表AE150)(Fisher Scientific,目录号:15524693)
  18. 生物安全柜配备254 nm紫外灯(Azbil Telstar,目录号:Bio II Advance)
  19. 化学通风橱
  20. 干燥器(BRAND,目录号:65815)
  21. 扫描电子显微镜(SEM)(飞利浦ESEM XL30 FEG环境扫描电子显微镜配备能量色散X射线分析仪(飞利浦)
  22. 拉曼显微镜(HORIBA,JOBIN YVON,LabRAM Aramis显微镜,配备Nd:YAG激光,波长为532 nm,由LabSpec NGS光谱软件控制.HORIBA,JOBIN YVON,目录号:LabRAM Aramis,3个激光器和xyz阶段)
  23. 涡流
  24. 真空泵
  25. 冰箱

程序

  1. 生物质生产
    1. 根据要研究的细菌菌株的数量,需要估计培养基的体积和血清瓶的数量。始终包括非生物控制。测试 D菌株TCE1和LBE的能力。 hafniense ,准备3瓶不同的培养基(非生物对照,菌株TCE1和菌株LBE)。对于组合物,参见配方点B. D. hafniense的生长培养基。
    2. 使用正常生长培养基为 D制备接种物。 hafniense 并在标准条件下(30°C,100rpm搅拌下)孵育细菌菌株约3天。 
    3. 接种物的最终OD 600 应在0.1-0.15的范围内。为了验证OD 600 ,使用无菌注射器从培养物中取出1ml样品,并将其转移到分光光度计比色皿中。测量OD 600 。 
    4. 使用无菌注射器,将无菌40ml接种物(5%)加入800ml生长培养基中。对于该体积,需要1L血清瓶(图2)。


      图2.显示生产细菌生物质的接种程序的方案

    5. 将培养物在30℃下在100rpm的搅拌下孵育直至在两种菌株的OD 600 0.12和0.18之间孵育。

  2. 与铁源孵化
    1. 在N 2 气氛中准备所需数量的空的50ml无菌血清瓶(参见配方2)。包括每个铁源,使用的菌株和非生物控制的一式三份。高压灭菌。
    2. 要密封标准血清瓶,请插入橡胶塞并用带有空心开口的金属盖盖住。当密封时,中空开口允许在实验期间用注射器取样。最后,使用密封压接器将金属帽系在一起并固定在血清瓶上。 
    3. 对于菌株LBE和TCE1的实验,在N 2 气氛中制备18个空的50ml无菌血清瓶。
    4. 在9个空瓶中,无菌加入1.5ml柠檬酸铁(III)溶液(见食谱)。在其他9个瓶中加入无菌1.5毫升的akageneite悬浮液(见食谱)。
    5. 为了测试腐蚀的试样上的细菌减少,通过喷洒70%乙醇溶液(v / v)对9个腐蚀的试样进行灭菌,然后在无菌条件下暴露于UV辐射(每侧20分钟,254nm)。在生物安全柜内执行灭菌程序。
    6. 在具有大瓶颈的血清瓶中添加9个试样,密封瓶子,抽空顶部空间并使用N 2 替换大气。高压灭菌。
    7. 一旦所有瓶子都经过高压灭菌(步骤B1和B6),无菌添加20毫升无菌培养基(非生物对照)或程序A中制备的生物质。对不同的铁源(Fe(III)进行操作 - 柠檬酸盐,akageneite,铁优惠券)。计算这些比例以获得起始浓度为10mM的可溶性和固态Fe(III)相的培养物。在使用腐蚀的优惠券修改的文化中,铁浓度未知。
    8. 为了使细菌和铁源充分混合,将血清瓶在30℃下在100rpm的搅拌下孵育。孵育瓶子直至形成黑色沉淀(与 D.hafniense 孵育7天)。在培养期间,培养基在用可溶性柠檬酸铁(III)和碱金属盐悬浮液修饰的培养物中从橙色/绿色变为黑色。黑色沉淀物的形成是铁还原的指示。在用铁试样修正的培养物中可以观察到相同的现象,因为试样和介质的表面变黑。
    9. 在孵育7天期间,每天从每次处理中收集0.5ml上清液样品并复制。确保采取有代表性的样本。当形成黑色沉淀物时,将注射器的针头插入血清瓶的橡胶塞中。翻倒血清瓶。轻轻摇动,收集0.5毫升样品。 
    10. 将样品转移到1.7毫升Eppendorf管中。 
    11. 向所有管中加入50μl5MHCl。
    12. 通过涡旋混合5秒并在室温下孵育15分钟。该步骤将允许通过酸(HCl)溶解铁并防止Fe(II)离子的氧化。
    13. 在-20°C冷冻所有样品。

  3. 确认铁减少
    1. 用分光光度法定量测定Fe(II)
      Ferrozine ®试剂在与Fe(II)反应时会变成紫色。颜色强度与Fe(II)的浓度成比例。然后,执行校准曲线可以量化样品中的Fe(II)含量。
      1. 打开酶标仪,将波长设置为562 nm。
      2. 从冰箱中取出校准曲线的标准溶液(参见食谱)。 
      3. 在室温下定量Fe(II)之前解冻从培养物中取出的冷冻样品(步骤B9-B12)。
      4. 通过涡旋混合5秒。
      5. 将10μl反应混合物(标准溶液或样品)转移至96孔微量培养板中。
        注意:取样时,确保在移液前将它们与涡旋混合均匀。
      6. 在微孔板孔中加入90μl的Ferrozine ®试剂。 
      7. 不要忘记使用 D的标准生长培养基进行空白样品。 。hafniense  
      8. 使用酶标仪在接下来的2分钟内测量562 nm处的吸光度。如果吸光度值高于校准曲线中浓度较高的样品的值(在我们的情况下,对于1000μM标准品为0.239 nm),则从原始样品开始准备相应样品的稀释液,并重复步骤C1d中的测量值 
      9. 测量每个重复,并为每个样本执行2-3个测量。

    2. Fe(II) - 生物矿物的表征
      1. 当培养物变黑并观察到沉淀时,从培养物中取出试样并通过喷洒70%乙醇溶液(v / v)对其进行灭菌,然后暴露于UV辐射(每侧20分钟)。用细菌 D。 hafniense ,7天后可以从血清瓶中取出优惠券。
      2. 将处理过的试样在真空下储存在干燥器中,以避免在实验过程中产生的生物矿物的氧化态发生变化。
      3. 为了研究新形成的生物矿物的形态,分布和元素组成,通过简单地将它们放置在显微镜室内来用SEM分析试样。要将试样固定到样品架,请使用如图3所示的碳带。以10-25 keV的加速电位观察二次电子模式下的样品。


        图3.带有试样和碳带的样品架,为SEM分析准备

      4. 为了研究新形成的Fe(II) - 生物矿物的分子组成,直接在试样表面上进行拉曼光谱分析。为此,只需将优惠券放在最低物镜下,并专注于要分析的区域。更改目标并再次聚焦,直到达到400倍放大率。使用以下设置获得高质量光谱并避免样品表面燃烧:激光在532 nm,功率低于1 mW(600 g / mm),光谱间隔在100和1,600 cm 之间 - 1 和1,000μm孔,100μm狭缝和5次累积100秒。

数据分析

  1. 以Mn计算Fe(II)含量
    使用以μM为单位的校准曲线转换Fe(II)浓度的吸光度值,如图4所示。仅考虑校准曲线范围内的吸光度值(在我们的情况下介于0.011和0.239之间)。如果测得的吸光度低于此值,则将Fe(II)含量视为0μM。如果值较高,请稀释原始样品并重复测量,直到吸光度在校准曲线的线性范围内。


    图4.使用Ferrozine ®反应物定量Fe(II)的程序。 A.校准曲线的示例。在左边是一个带有数字数据的表格;在右边,显示了显示校准曲线的方程和相关系数的相应图表。数据代表重复的平均值。吸光度的增加在50和1,000μM之间是线性的,相关系数是0.9999。 B.数据处理的例子。在左侧,显示了从校准曲线外推的等式,在右侧,给出了数据处理的示例。数据来自非生物对照,用在第0天取样的柠檬酸铁(II)修正。

    当所有吸光度值转换为Fe(II)含量时,数据可以直方图显示,如图5所示。


    图5.使用Ferrozine ®测定法定量Fe(II)含量。图表表示非生物对照中的Fe(II)含量以及用可溶性Fe修饰的培养物中的Fe(II)含量(III) - 由校准曲线计算的 - 柠檬酸盐(左)和固体Fe(III)(右)。

  2. 生物矿物的表征
    可以通过观察SEM显微照片直接研究生物晶体的形态。为了识别新形成的生物矿物,将获得的光谱(记录在处理过的试样表面上)与软件库中的参考光谱以及文献中提供的拉曼位移进行比较(Frost et al。,2002; Monnier et al。,2011;Rémazeilles et al。,2013)。图6显示了用 D处理的腐蚀试样获得的结果的实例。 hafniense 菌株TCE1和LBE。


    图6.用 D培养物处理的腐蚀铁试样的铁还原试验。 hafniense 菌株TCE1和LBE。 A.新形成的生物矿物的形态学。左栏:优惠券的外观;右栏:在优惠券图片上由黑色方块表示的区域中拍摄的相应SEM图像。 B.孵育后试样表面的分子组成。左栏:通过拉曼光谱分析的区域(黑色方块);和右列:相应的拉曼光谱。矿物质被鉴定为:1:纤铁矿(Le),2:结晶不良的mackinawite(M)和元素硫(S)的混合物,3:Vivianite(Vi),和4:vivianite(Vi)和纤铁矿(Le)的混合物。

笔记

  1. 再现性
    收集培养样品是一个微妙的步骤。事实上,由于在培养期间形成铁沉淀物,因此难以收集代表性样品。因此,在对培养物进行取样之前,确保将它们混合均匀,否则样品中的铁含量将无法代表。
  2. 非生物控制
    用于培养厌氧细菌的培养基通常很复杂并且含有还原剂,例如Na 2 S.因此,为了排除Fe(III)的非生物减少,必须用所有测试的铁源进行非生物控制。
  3. Ferrozine ®试剂
    含有Ferrozine ®试剂和样品的混合物的强度随时间而变化。因此,重要的是在孵育2分钟后精确测量吸光度,并对所有样品使用相同的程序。
  4. 储存经过处理的优惠券
    新形成的生物矿物的稳定性尚不清楚。因此,在用拉曼光谱法进行鉴定之前,将铁试样存放在干燥器中,以避免在处理过程中产生的生物矿物的氧化状态发生变化。

食谱

  1. D的生长培养基。 hafniense
    1. N 2 - 脱气H 2 O
      1. 煮沸,500毫升MilliQ水,加热板搅拌器
      2. 在N 2 下冷却,将80 ml分配到血清瓶中,气体交换为N 2 ,高压灭菌器(120°C,20分钟)
      3. 在室温下储存长达12个月
    2. 无菌血清瓶
      N 2 和高压釜的气体交换
      在室温下储存长达12个月
    3. DL-乳酸钠溶液40%(v / v)
      1. 用MilliQ水将60%的储备溶液稀释至40%
      2. 将100ml分配至血清瓶,气体交换N 2 ,高压灭菌
      3. 储存在4°C至12个月
    4. 富马酸二钠溶液16%(v / v)
      1. 将80g富马酸二钠加入500ml MilliQ水中
      2. 将100ml分配至血清瓶,气体交换N 2 ,高压灭菌
      3. 在室温下储存长达12个月
    5. 还原剂溶液1 M
      1. 用N 2 -degassed H 2 O洗涤Na 2 S•9H 2 O的晶体以除去已经氧化部分的晶体
      2. 用薄纸干燥晶体
      3. 重量24.02克该化合物(干重)
      4. 将其溶于100毫升脱气的MilliQ水中
      5. 使用0.2μm过滤器过滤灭菌到血清瓶中
      6. N 2 的气体交换
      7. 储存在4°C至12个月
    6. 刃天青溶液0.5 g / L
      将0.1g刃天青钠盐加入200ml MilliQ水中 储存在4°C至12个月
    7. 维生素溶液1
      1. 在1升MilliQ水中加入250毫克氰钴胺素
      2. 过滤灭菌到无菌血清瓶中,气体交换N 2
      3. 储存在4°C至12个月
    8. 维生素溶液2
      1. 在1升MilliQ水中加入50毫克核黄素
      2. 过滤灭菌到无菌血清瓶中,气体交换N 2
      3. 储存在4°C至12个月
    9. 维生素溶液3
      1. 向1升MilliQ水中加入100毫克硫胺素 - 盐酸盐
      2. 过滤灭菌到无菌血清瓶中,气体交换N 2
      3. 储存在4°C至12个月
    10. 维生素溶液4
      1. 将所有组分添加到1L MilliQ水中
        50毫克生物素
        250毫克对氨基苯甲酸盐(钠盐)
        50毫克泛酸(钠盐)
        20毫克叶酸•2H 2 O
        50毫克硫辛酸
        100毫克吡哆醇盐酸盐
        550毫克烟酸
      2. 用0.2μm过滤器过滤灭菌到无菌血清瓶中,对N 2 进行气体交换
      3. 储存在4°C至12个月
    11. 微量元素解决方案
      1. 将500毫克EDTA溶于900毫升MilliQ水中,用HCl调节pH至7.0,然后加入以下化合物:
        2毫克FeCl 2 •4H 2 O
        100mg MnCl 2 •4H 2 O
        190mg CoCl 2 •6H 2 O
        70毫克ZnCl 2
        2.55mg CuCl 2 •2H 2 O
        5.52毫克AlCl 3
        6mg H 3 BO 3
        41.4毫克Na 2 MoO 4 •2H 2 O
        24毫克NiCl 2 •6H 2 O.
      2. 将MilliQ水加入1升
      3. 储存在4°C至12个月
    12. 碳酸盐溶液
      1. 将9.01克NH 4 HCO 3 和76.11克NaHCO 3 加入1升MilliQ水中
      2. 煮沸,在N 2 / CO 2 (4:1)下冷却,向每个血清瓶分配49 ml,气体交换为N 2 / CO 2 (4:1),高压灭菌器
      3. 在室温下储存长达12个月
    13. 溶液A(基础培养基)
      1. 将所有组分添加到1L MilliQ水中:
        0.958克K 2 HPO 4 •3H 2 O
        0.218克NaH2PO 4 •2H 2 O
        0.1克蛋白胨
        1ml刃天青溶液0.5g / L.
      2. 煮沸,在N 2 / CO 2 (4:1)下冷却,分配到血清瓶中,气体交换为N 2 / CO 2 (4:1),高压灭菌器
      3. 在室温下储存长达12个月
    14. 溶液B(维生素溶液)
      向20毫升厌氧无菌MilliQ水中,用注射器无菌添加以下溶液:
      1毫升微量元素溶液
      1毫升维生素溶液1
      1毫升维生素溶液2
      1毫升维生素溶液3
      1毫升维生素溶液4
      储存在4°C至3个月
    15. 解决方案C(缓冲/还原溶液)
      向49毫升碳酸盐溶液中加入1毫升还原剂溶液 储存在4°C至3个月
    16. 解决方案D
      1. 加入4.40克CaCl 2 •2H 2 O和4.06克MgCl 2 •6H 2 O至1 L MilliQ水
      2. 在血清瓶中分配200ml,用于N 2 的气体交换和高压灭菌
      3. 储存在4°C至12个月

    生长培养基完成
    向45 ml溶液A中,用注射器无菌添加以下成分:
    1.25毫升溶液B
    2毫升溶液C
    1.25毫升溶液D
    1毫升乳酸盐溶液
    1毫升富马酸盐溶液
    pH值应介于7.0和7.6之间 为了验证pH值,用注射器收集1 ml样品并在pH计上测量

  2. 可溶性柠檬酸Fe(III)(35 g / L) - 100 ml
    1. HCl溶液调节pH值
      1. 用MilliQ水填充100ml带刻度量瓶的一部分
      2. 将41.5毫升HCl 37%
      3. 用MilliQ水填充至100毫升
      4. 进行稀释以获得从5 M(起始溶液)到0.01 M的浓度范围
      5. 在室温下储存长达6个月
    2. NaOH溶液调节pH值
      1. 将20g NaOH颗粒溶解在100ml MilliQ水中
      2. 进行稀释以获得从5 M(起始溶液)到0.01 M的浓度范围
      3. 在室温下储存长达6个月
    3. Fe(III)溶液
      1. 将3.5g柠檬酸铁(III)溶于100ml milliQ水中。为了促进溶解,添加磁棒并在80℃下用磁力搅拌器混合溶液。此步骤可能需要1-2小时。当粉末完全溶解时,不应看到残留的颗粒,溶液的颜色变为黄色,pH值极差(pH 1-2)
      2. 通过添加NaOH滴将溶液的pH调节至7。在此过程之后,溶液变成棕橙色
      3. 用不可磨灭的笔除去氧标记,烧瓶中的溶液水平,向溶液中加入额外的MilliQ水,让溶液沸腾,直到所有添加的水都蒸发掉(帮助自己用笔痕检测原始体积解决方案)。然后通过冲洗N 2 / CO 2 冷却溶液,并使用血清瓶密封压接器用橡胶塞和金属盖密封血清瓶。通过高压灭菌(120℃,20分钟)对溶液灭菌。
      4. 储存在4°C至6个月

  3. 固体Fe(III)悬浮液
    1. 固体Fe(III)源
      1. 在最初的实验中,使用了akaganeite(FeO 0.833 (OH) 1.167 Cl 0.167 )。 Akaganeite由瑞士国家博物馆提供。按照Schwertmann和Cornell(2008)的方案合成该化合物。然而,对于该测试,可以使用任何种类的不溶性Fe(III) - 氧化物或Fe(III) - 羟基氧化物来制备悬浮液(即,Fe 2 O 3 或α-FeO(OH),Sigma-Aldrich)
      2. 储存在4°C至6个月
    2. 制备固体Fe(III) - 相(akaganeite或针铁矿)的悬浮液
      1. 对于akaganeite悬浮液(10 g / L - 100 ml):
        将1.0g的akaganeite加入100ml MilliQ水中
      2. 对于针铁矿悬浮液(13 g / L - 100 ml):
        在100毫升MilliQ水中加入1.3克针铁矿
      3. 控制悬浮液的pH并用HCl或NaOH溶液滴加至pH7
      4. 重复已经描述的制备可溶性Fe(III)溶液的所有步骤,以除去氧气并对溶液进行灭菌
      5. 储存在4°C至6个月

  4. Ferrozine ®试剂
    1. HCl溶液5 M
      先前描述的相同程序(B1。用于调节pH的HCl溶液)
    2. Fe(II)1M的储备溶液用于校准曲线
      1. 用HCl(5 M)清洗1升的刻度瓶
      2. 用MilliQ水清洗
      3. 填充部分MilliQ水
      4. 加入41.55毫升37%的HCl(S-20)
      5. 加入392.14mg硫酸铁铵(II)
      6. 用MilliQ水填充高达1升
      7. 稀释储备溶液以获得以下浓度:50,100,250,500,750和1,000μM。
      8. 分装并储存在4°C至6个月(避光)
    3. Ferrozine ®试剂
      1. 用HCl(5 M)清洗1升的刻度瓶
      2. 用MilliQ水清洗
      3. 填充部分MilliQ水
      4. 加入11.9毫升HEPES缓冲液(终浓度50 mM)
      5. 用MilliQ水清洗烧瓶壁
      6. 加入1克Ferrozine ®
      7. 用MilliQ水填充高达1升
      8. 用NaOH溶液调节pH至7
      9. 储存在4°C至2个月

致谢

作者感谢瑞士国家科学基金会的Ambizione资助(PZ00P2_142514,2013-2016,Pi:Edith Joseph)。作者还要感谢瑞士国家博物馆的研究保护实验室,以帮助进行拉曼调查(Marie Woerle博士和Tiziana Lombardo博士),并提供实验中使用的铁券。
该协议是Comensoli 等人描述的方法的修改版本(2017)。此外,培养基的组成改编自Gerritse et al。(1999),而Ferrozine ®测定用于量化液体溶液中的Fe(II)离子改编自Stookey(1970)。

利益争夺

作者没有利益冲突或竞争利益申报。

参考

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引用:Comensoli, L., Maillard, J., Kooli, W. M., Junier, P. and Joseph, E. (2018). Soluble and Solid Iron Reduction Assays with Desulfitobacterium hafniense. Bio-protocol 8(17): e3002. DOI: 10.21769/BioProtoc.3002.
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