1
Prepare the medium under an atmosphere of H2/CO2 (80:20) without adding the vitamins and NaHCO3 in serum bottles and seal the serum tubes with butyl rubber stoppers. Steam medium for 3 hours on each of 3 successive days. To the sterile medium add, from filter sterilised stock solutions, the NaHCO3 and vitamin solution.
2
Increase the 80% H2 + 20% CO2 gas phase pressure to 300 kPa. The final pH is 7.0.
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