Mix ingredients except KP buffer, vitamin and Na₂CO₃. Dispense the medium into suitable culture vessels under a N₂/CO₂ (80/20) and seal with butyl rubber stoppers. Separately autoclave KP buffer under a N₂. Sterilize vitamin and 10% Na₂CO₃ solutions by filtration. Prior to inoculation, aseptically and anaerobically add KP buffer, vitamin and Na₂CO₃ solutions. Add O₂ at 5% concentration in the headspace, and pressurize it to 150 kPa with N₂/CO₂ (80/20). Final pH is 6.0-6.5.

*KP buffer

  Autoclave under N₂.

**Trace element solution

First dissolve NTA and adjust pH to 6.5 with NaOH, and then add minerals. Final pH is 7.0.

***Vitamin solution

           Adjust pH to 7.0 with NaOH.