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Cultivate Clostridium sp. DSM 754 for 5 to 8 days at 37°C in the medium 1022, but omit 4-hydroxybenzoic acid, replace the C. sporogenes supernatant with distilled water and add after autoclaving 0.30 g/l Na2S x 9 H2O from a sterile anoxic stock solution prepared under 100% N2 gas. Adjust pH of the complete medium to 7.0.
Disrupt cells of the grown culture by autoclaving at 121°C for 20 min . Centrifuge autoclaved culture at 18000 x g for 20 min. Discard cell pellet and store the supernatant in screw capped bottles at -20°C .
Before use sterilize the supernatant by autoclaving under 100% N2 gas atmosphere in vials suitable for anaerobic cultivation.
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