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Techniques - How to make Fe(III)-reducing media

It is not difficult to grow anaerobic organisms with Fe(III) as a terminal electron acceptor. You will need an anaerobic gas set-up to flush oxygen out of the medium and buffer the pH, and you will need containers with a gas tight seal, such as thick butyl rubber stoppers.

Suggested Electron donors and acceptors (choose one each):

electron donors
suggested acceptors
acetate (20 mM)
FeOOH (amorph solid)
Hydrogen
Ferric Citrate (50mM)
ethanol
Fe(III)-NTA* (10mM)
*Nitrilotriacetic acid

Note that other VFA's may be substituted for acetate, but fermentable substrates may not result in respiratory Fe(III) reduction.

Bicarbonate-buffered medium for freshwater bacteria includes the following salts (per L medium) to provide major nutrients for growth:

NaHCO3 2.5 g
NH4Cl 0.5 g
NaH2PO4.6H2O 0.6 g
KCl 0.1 g

To buffer the medium, flush the medium with N2/CO2 (80:20). Final pH=6.8

In addition, a source of trace nutrients is needed (add 10mL of mineral stock solution), and vitamins (10mL of vitamin stock solution) may be required.

Trace minerals:

STOCK SOLUTION

(in g/L)

NTA (free acid form) 1.5
MgSO4-7H2O 3.0
MnSO4-H2O 0.5
NaCl 1.0
FeSO4-7H2O 0.1
CaCl2-2H2O 0.1
CoCl2-6H2O 0.1
ZnCl 0.13
CuSO4-5H2O 0.01
AlK(SO4)2-12H2O 0.01
H3BO3 0.01
Na2MoO4 0.025
NiCl2-6H2O 0.024
Na2WO4-2H2O 0.025

Vitamins:

(mg/L)

biotin 2
folic acid 2
pyridoxine-H2O 10
riboflavin 5
thiamine 5
nicotinic acid 5
pantothenic acid 5
cyanocobalamin 0.1
p-aminobenzoic acid 5
thioctic acid 5

Note that it may be possible to substitute yeast extract for trace minerals and vitamins.

For amorphous ferric oxyhydroxide (FeOOH):

  1. Carefully add NaOH to a 0.2M solution of FeCl3, adjusting the pH to 7 (but never over 7).
  2. Wash the resulting suspension by centrifuging, decanting, and adding deionized water.
  3. Repeat the washing step until the Cl- is <1mM.
  4. Store refrigerated, as a suspension.
  5. Add (1:9) to medium.
  6. Autoclave.

For Ferric Citrate Medium

  1. Dissolve 50mM ferric citrate in water by heating.
  2. Add NaOH to increase pH to 6.
  3. Add the other media components and flush with N2/CO2 (80:20).
  4. Seal and autoclave.
  5. Final pH should be about 6.8.

For 100mM Fe(III) NTA stock:

  1. Dissolve 1.64 g sodium bicarbonate in 100 mL
  2. Add 2.56 g Nitrilotiacetic acid (trisodium salt)
  3. Add 2.70 g FeCl3
  4. Gas out the mixture with N2/CO2 (80:20)
  5. Filter sterilize into a sterile, sealed, anaerobic 100mL bottle. (Final pH is ~7)
  6. Add from sterile stock into sterilized media (1:9).

It is possible to make a 1M stock for use with larger volumes of media. (But difficult to filter). Some organisms will not tolerate more than 10mM Fe(III)-NTA.

Anaerobic System Set-up


anaerobic set-up