Anaerobic Preculture and Culture Media

Saccharomyces cerevisiae BY4741 was used and precultured anaerobically in YPD media with 2.0% glucose (w/v) (Wako, Osaka, Japan) at 30 °C for 72 h without shaking. YPD media was used for one litter of medium: 10 g of yeast extract (Bacto, MD, USA), 20 g of Pepton (Bacto), 20 g of glucose, and adjusted to pH 6.
The preculture and culture medium for C. cellulovorans 743B (ATCC 35296) and C. beijerinckii NCIMB8052 (ATCC 51743) was partially modified and used [5]. For one litter of medium, it was prepared with 4 g of yeast extract, 1 mg of Resazurin salt, 1 g of l-cysteine HCl, 5 g of NaHCO₃, 0.45 g of K₂HPO₄, 0.45 g of KH₂PO₄, 0.3675 g of NH₄Cl, 0.9 g of NaCl, 0.1575 g of MgCl₂∙6H₂O, 0.12 g of CaCl₂∙2H₂O, 0.85 mg of MnCl₂∙4H₂O, 0.942 mg of CoCl₂∙6H₂O, 5.2 mg of Na₂EDTA, 1.5 mg of FeCl₂∙4H₂O, 0.07 mg of ZnCl₂, 0.1 mg of H₂BO₃, 0.017 mg of CuCl₂∙2H₂O, 0.024 mg of NiCl₂∙6H₂O, 0.036 mg of Na₂MoO₄∙2H₂O, 6.6 mg of FeSO₄∙7H₂O, 0.1 g of p-aminobenzoic acid), and was adjusted to pH 7 for C. cellulovorans and to pH 5 for C. beijerinckii, respectively. C. cellulovorans and C. beijerinckii were anaerobically precultured in the above medium with 0.5% (w/v) cellobiose (Sigma, MO, USA) and with 2.0% (w/v) glucose, respectively, at 37 °C for 23 h without shaking.