Fong:MJ

Description

MJ chemically defined minimal medium is described by Johnson et al., 1981 to support the growth of Clostridium thermocellum. If using cellobiose or another soluble sugar, a 20x solution of the sugar should be either sterile filtered or autoclaved separately, depending on whether a precise initial concentration of the carbon source is needed.

MJ Minimal Medium for C. thermocellum

Salt solution	Final Amt. (1L)	10x stock (g/L)
Calcium chloride dehydrate - CaCl2 * 2H2O	0.150g	1.50g
Magnesium Chloride hexahydrate - MgCl2 * 6H2O	1.0g	10.0g
Ferrous sulfate heptahydrate - FeSO4 * 7H2O	1.17mg	11.7mg
Vitamin solution	Final Amt. (1L)	100x stock (g/L)
Pyradoxamine HCl	2.0mg	0.2g
Biotin	0.2mg	20mg
p-Aminobenzoic acid	0.4mg	40mg
Vitamin B12	0.2mg	20mg
MJ Solution A	Final Conc. (g/L)
Potassium phosphate monobasic - KH2PO4	1.5
Potassium phosphate - K2HPO4	2.9
Urea	2.1
Cystein-HCl (or Cystein-HCl monohydrate)	1.0 (1.12)
MOPS	10.0
Sodium citrate tribasic dihydrate - Na3C6H5O7 * 2H2O	3.0
Carbon source*	5.0

'* If using a soluble carbon source, such as cellobiose, make a separate 20x stock in serum bottles to be autoclaved separately and added with other solutions. Otherwise, add insoluble carbon source to individual serum bottles to which MJ Solution A will be added.

Procedure

1. Prepare 10x stock salt solution, distribute in serum bottles, purge, and autoclave.
2. Prepare a 100x solution of each vitamin, distribute in serum bottles and purge, leaving ≥10psi positive pressure in each bottle. In addition, purge one empty serum bottle for each vitamin bottle, and autoclave empty bottles. Using syringe filters and sterile technique, filter vitamin solutions into purged, autoclaved, empty bottles.
3. In a 1L beaker containing ~600ml ddH2O, add the above ingredients for MJ Solution A in the order listed, omitting carbon source if applicable. Add 0.1 ml of 1.0% stock solution of Resazurin dye.
4. Adjust pH to 7.05 - 7.10. Using calibrated pH meter, submerge probe in media and allow pH to stabilize. Use disposable transfer pipette to add 2.0 M KOH dropwise until pH is steady.
   • NB: this step is important because a low pH will inhibit C. therm growth, while a higher pH, even around 7.4, results in significant salt precipitation when all medium components are combined.
5. Adjust total volume to 860mL (810mL if carbon source is omitted). Dispense among serum bottles. Always leave about 50% of total volume of serum bottles as empty headspace, e.g., in a 100ml serum bottle use final volume of 50ml. This allows gas production by C. thermocellum to occur without too much pressure accumulating. Stopper with blue or black butyl stoppers and seal with aluminum crimp seals. Purge bottles with ultra high purity N₂ gas.
6. Label bottles with “MJ, Carbon source/conc., date.” Autoclave bottles, including separate 20x carbon source, if applicable, for 15 min at 121°C (liquid cycle). Anaerobic sterile technique should be used for all procedures after this step.
7. Use anaerobic sterile technique to transfer appropriate volumes (depending on serum bottle size) of salt solution and each vitamin solution. If carbon source is separate, transfer appropriate volume of carbon source to each serum bottle of MJ medium.