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Medium No.
Methanobacterium alcaliphilum medium
 Bacto Yeast Extract (Difco)2g
 Carbonate mixture*210ml
 Tris-HCl buffer**10ml
 Vitamin solution***10ml
 Trace elements solution****10ml
 Distilled water760ml
  Mix ingredients except for carbonate mixture, Tris-HCl buffer, vitamin solution, cysteine-HCl and Na2S·9H2O, and autoclave under a H2 atmosphere. Carbonate mixture, Tris-HCl buffer, cysteine-HCl (3% solution) and Na2S·9H2O (3% solution) are separately autoclaved under a N2 atmosphere. Prior to inoculation, add filter-sterile vitamin solution, carbon mixture, Tris-HCl buffer, cysteine-HCl and Na2S·9H2O. Pressurize the inoculated vessels to 150 kPa with H2.
  *Carbonate mixture
   Distilled water210ml
  Mix ingredients and autoclave under a N2 atmosphere.
  **Tris-HCl buffer
  Dissolve 2 M Tris, adjust pH to 8.4 with HCl, and autoclave under a N2 atmosphere.
  ***Vitamin solution
   Folic acid2mg
   Nicotinic acid5mg
   p-Aminobenzoic acid1mg
   Vitamin B120.01mg
   Distilled water1L
  ****Trace elements solution
   Distilled water1L
  Dissolve the 2Na-EDTA in 500 ml of distilled water, and after adding the salts, make up to 1 L.