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Medium Detail Information

Medium No.
Medium for haloalkaliphilic methanogen
  Mineral base 1* 500 ml
  Mineral base 2** 500 ml
  MgCl2·6H2O 0.2 g
  Bacto Yeast Extract (Difco) 0.02 g
  Coenzyme M 0.014 g
  Vitamin solution*** 5 ml
  Trace element solution**** 10 ml
  Methanol 2 ml
  Sodium formate 3.4 g
  Sodium acetate 0.16 g
  FeS slurry solution***** 2 ml
  Na2S·9H2O 0.12 g
    Mix mineral base 1 with mineral base 2 in 1:1 proportion in tightly closed vessels under a N2 atmosphere. Separately autoclave concentrated Bacto Yeast Extract, coenzyme M, sodium acetate, Na2S·9H2O and FeS solution under a N2 atmosphere in tightly closed vessels. Add the autoclaved concentrated solutions, and filter-sterile concentrated MgCl2·6H2O, vitamin solution, trace element solution, methanol and sodium formate solution. After re-exchanging with N2, add 1 drop of filter-sterile 10% sodium dithionaite solution in 1 M NaHCO3 by syringe. Final pH of the complete medium should be 9.
    *Mineral base 1
      Na2CO3 185 g
      NaHCO3 35 g
      NaCl 16 g
      K2HPO4 1 g
      Distilled water 1 L
    Mix ingredients and autoclave under a N2 atmosphere. Because some precipitate appears on the bottom for 1 day after autoclaving, use only supernatant for making the medium. Final pH should be 10.
    **Mineral base 2
      NaCl 240 g
      NH4Cl 0.5 g
      K2HPO4 2.5 g
      KCl 5 g
      Distilled water 1 L
    Mix ingredients and autoclave under a N2 atmosphere. Final pH should be 7.3.
    ***Vitamin solution
      Biotin 2 mg
      Folic acid 2 mg
      Pyridoxine-HCl 10 mg
      Thiamine-HCl 5 mg
      Riboflavin 5 mg
      Nicotinic acid 5 mg
      Ca-pantothenate 5 mg
      p-Aminobenzoic acid 1 mg
      Vitamin B12 0.01 mg
      Distilled water 1 L
    ****Trace elements solution
      Nitrilotriacetic acid (NTA) 12.8 g
      FeCl3·6H2O 1.35 g
      MnCl2·4H2O 0.1 g
      CoCl2·6H2O 0.024 g
      CaCl2·2H2O 0.1 g
      ZnCl2 0.1 g
      CuCl2·2H2O 0.025 g
      H3BO3 0.01 g
      Na2MoO4·2H2O 0.024 g
      NaCl 1 g
      NiCl2·6H2O 0.12 g
      Na2SeO4 0.004 g
      Na2WO4 0.004 g
      Distilled water 1 L
    First dissolve nitrilotriacetic acid and adjust pH to 6.5 with NaOH, and then add minerals. Final pH 7.0.
    *****FeS slurry solution
    Mix 0.5 M FeSO4·7H2O and 0.5 M Na2S·9H2O solutions, wash with pure water several times, finally resuspend the FeS precipitate as 20% solution and autoclave (110°C, 30 min) under a N2 atmosphere.