Mix ingredients except for bicarbonate, yeast extract, fructose, vitamin solution, cysteine-HCl and Na2S·9H2O, sparge medium with N2/CO2 (80/20) gas, adjust pH to 5.0 and autoclave at 121°C for 15 min in Hungate-type tubes of serum vials. Concentrated  yeast extract, fructose, cysteine-HCl and sodium sulfide solutions are sterilized separately under a N2 atmosphere in tightly closed vessels. Bicarbonate solution are also sterilized under an N2/CO2 (80/20) gas atmosphere in tightly closed vesseles. Prior to inoculation, add bicarbonate, yeast extract, fructose, filter-sterile vitamin solution, cysteine-HCl and sodium sulfide solutions. Adjust the pH of the complete medium to 5.9.


*Modified Wolin's mineral solution 

1. First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, then add minerals. Adjust final to pH 7.0 with KOH.


**Wolin's vitamin solution (10x)