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Medium No.	1356
Medium	Medium for haloalkaliphilic methanogen
Composition
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.
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