231. CARBOXYDOCELLA SPOROPRODUCENS MEDIUM

KCl 0.33 g

MgCl₂ × 6 H₂O 0.52 g

CaCl₂ × 2 H₂O 0.29 g

NH₄Cl 0.33 g

KH₂PO₄ 0.33 g

NaHCO₃ 1.00 g

Trace element solution SL-4 (see below) 10.00 ml

Resazurin 0.50 mg

Vitamin solution (see below) 10.00 ml

Pyruvate 2.5 g

Yeast extract 0.05 g

Na₂S × 9 H₂O 0.30 g

Distilled water 1000.00 ml

Trace element solution SL-4

EDTA 0.5 g

FeSO₄ × 7H₂O 0.2 g

Trace element solution SL-6 (see below) 100.0 ml

Distilled water 900.0 ml

Vitamin solution:

Biotin 2.0 mg

Folic acid 2.0 mg

Pyridoxine 0.1 mg

Riboflavin 5.0 mg

Pantotenoic acid 5.0 mg

p-Aminobenzoic acid 5.0 mg

Thiamine-HCl 5.0 mg

Nicotinic acid 5.0 mg

Vitamin B₁₂ 0.1 mg

Lipoic (thioctic) acid 5.0 mg

Distilled water 1000.0 ml

Trace element solution SL-6:

ZnSO₄ × 7 H₂O 0.1 g

MnCl₂ × 4 H₂O 0.03 g

H₃BO₃ 0.3 g

CoCl₂ × 6 H₂O 0.2 g

CuCl₂ × 2 H₂O 0.01 g

NiCl₂ × 6 H₂O 0.02 g

Na₂MoO₄ 0.03 g

Distilled water 1000.0 ml

Dissolve ingredients except bicarbonate, yeast extract, vitamins, pyruvate and sulfide, boil medium for 1 min, then cool to room temperature under N₂ gas atmosphere. Dispense medium under same gas atmosphere in tubes or serum bottle and autoclave at 121°C for 15 min. Add vitamins (sterilized by filtration), yeast extract, pyruvate and sulfide from sterile anoxic stock solutions prepared under N₂ gas atmosphere and bicarbonate from a sterile anoxic solution prepared under 80% N₂ and 20% CO₂ gas mixture (all solutions sterilize separately at 121°C 15 min). Adjust pH to 7.0 with sterile anoxic solution of 10% (w/v) NaHCO₃. Inoculated vessels are pressurized with carbon monoxide gas to 2 bar overpressure.