Precipitating growth medium - (Jul/25/2012 )

I have been given the task of growing sulphate reducing bacteria for the production of magnetic iron sulphide. In a paper by Marius et al. (2005, J. Physics, 17, pp65-69), the following recipe is given:

Per litre of water:

FeSO4.7H2O 3.22g
Fe2(SO4)3.2H2O 0.58g
(NH4)2SO4 0.07g
KH2PO4 0.5g
Na2SO4 4.5g
CaCl2.2H2O 0.06g
MgSO4.7H2O 0.06g
Sodium lactate 5ml @ 70% w/v
Adjust to pH 6.7 using NaOH

I have tried adding these compounds to deionised water in the order shown. When I add the KH2PO4, the liquid become slightly cloudy. If I continue to add all the ingredients it continues to be cloudy. I have left this for a day and the precipitate settles.
In addition, when I adjust the pH of the medium, a greenish (more khaki) precipitate forms, which falls out of suspension very quickly.

What I am wondering is if there is any way of combining these ingredients without precipitation. I find it difficult to believe that the authors of the paper could have used the medium in this state because I believe the precipitate to be an iron compound, is one of the most important elements for this particular culture setup.

On a side note, I have found other media for growing sulphate reducers, with a high concentration of iron sulphates as above, which do not precipitate. However their pHs tend to be low (~4) and it seems that it is important that the medium pH is close to neutral.

David

-David Ian Walker-

Hi David,

I find it difficult to believe that the authors of the paper could have used the medium in this state because I believe the precipitate to be an iron compound, is one of the most important elements for this particular culture setup.

I've worked with sediments before on a totally different subject but decided to give the experiment a try since I've prepared a considerable amount of microelement (ME) stock. I'm certain the sedimented solution (swirled to suspend prior to adding the required amount) worked because I didn't observe growth in another batch minus the ME.

Caveat against self: I understand that I'm working on a totally different bacteria (and physiology)

I have since switched to a colleague's recipe with slightly different elements dissolved in 0.1M HCl instead of dH2O (which I used). The stock stays clear since the day it was prepared but when it's added it into a pH7 medium, I'd still get a precipitate. Apparently, those elements prefer to "stay" in an acidic milieu.

On a side note, I have found other media for growing sulphate reducers, with a high concentration of iron sulphates as above, which do not precipitate. However their pHs tend to be low (~4) and it seems that it is important that the medium pH is close to neutral.

You could try adjusting the pH of these "other media" above 4 (if it's important for SRB activity) to a point before they precipitate, if they do precipitate.

Just curious, what's present/absent in the other media compared to this one?

I found this paper by the same group and I noticed that they worked with unfiltered sediments and it's likely that the precipitating medium is part of the setup.

Also, pH fluctuates throughout the cultivation period:

The periods of highest pH (>7) for the SBC-bioreactor coincide with the batch phase of the culture period. This correlation is probably as a result of the bacteria altering the internal conditions of the reactor as a result of their metabolic processes.

Perhaps they could grow at the range of 4 < pH < 6.7, albeit slower, and raise the pH accordingly -- just a guess

-Julio-Claudian-

Having so much iron without precipitate at that pH is impossible unless you use a chelator such EDTA.

In the paper, they say:

containing a modified Freke and Tate <7>/Postgate C growth medium <2>

The Postgate C medium, I found it in a handbook of media but it is not even close in Fe concentration (0.0004g/L ferrous sulfate). Recipe if you want it:

Medium C for Sulfate Reducers
(Postgate’s Medium C for Sulfate Reducers)
Composition per liter:
Sodium lactate............................................................................... 6.0g
Na2SO4.......................................................................................... 4.5g
NH4Cl ........................................................................................... 1.0g
Yeast extract.................................................................................. 1.0g
KH2PO4......................................................................................... 0.5g
Sodium citrate·2H2O..................................................................... 0.3g
CaCl2·6H2O................................................................................. 0.06g
MgSO4·7H2O.............................................................................. 0.06g
FeSO4·7H2O.............................................................................. 0.004g
pH 7.5 ± 0.2 at 25°C
Preparation of Medium: Add components to distilled/deionized water and bring volume to 1.0L. For marine bacteria, NaCl may be added or sea water used in place of distilled/deionized water. Mix thoroughly. Adjust pH to 7.5. Distribute into tubes or flasks. Autoclave for 15 min at 15 psi pressure–121°C.
Use: For detection, culturing, and storage of Desulfovibrio species and many Desulfotomaculum species. This medium should be used when a clear culture medium is desired such as for chemostat culture. This medium may be cloudy after sterilization but usually clears on cooling. It turns black as a result of H2S production due to bacterial growth

And the Freke and Tate medium is not defined in their paper but they state:

Some of the iron entering the culture vessel was in suspension, as the operating pH was consistent with the formation of some ferric hydroxide and ferrous hydroxide. The latter, when employing only ferrous iron solutions, in turn caused the formation of a small quantity of ferric hydroxide by oxidation, since although the influent reservoir was scoured with nitrogen no exacting precautions were taken to remove the last traces of dissolved oxygen present. However, the system converted iron present, whether in solution or in suspension, in the ferrous or ferric state, with apparent impartiality. Static cultures containing hydrated ferric oxides, ferrous hydroxide and mixtures thereof were converted to iron sulphide which was, however, non-magnetic.

-El Crazy Xabi-