Miroorganism and culture maintenance
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.
Measurement of total sugar and reducing sugar concentration
Total sugar concentration was measured by phenol–sulfuric acid method. Reducing sugar was measured by DNS method, as d-glucose equivalents.
Alcohol concentration
Alcohol concentration was measured by a gas chromatograph GC-2010plus (Shimadzu, Kyoto, Japan) with a capillary column Rt-Q-BOND (30 m, inner diameter. 0.32 mm; RESTEK, PA, USA). The oven temperature was 250 °C and the column temperature was 150 °C. Nitrogen was the carrier gas and set at a flow rate of 1.21 mL/min.
Determination of cell growth
Cell growth was measured by Lumitester PD-30, LuciPac Pen and ATP eliminating enzyme (Kikkoman Biochemifa, Tokyo, Japan). It is known that integrated intracellular ATP concentration correlates with cell growth (Miyake et al. 2016). Cell growth was estimated by measuring ATP concentration of 0.1 mL of cell culture according to the manufacturer’s instruction.
Preparation of substrates from mandarin
Mandarin oranges purchased at a grocery store was used. Flavedo and albedo, hereafter called removed peel, were removed before squeezing (Fig. 1a). Whole mandarin oranges except removed peel were squeezed by a squeezing device (Fig. 1b). 10 vials of a medium containing removed peel were prepared. Mandarin oranges were squeezed by SJC-75-W (Irisohyama, Miyagi, Japan).
Statistics
The data were analyzed for statistical significances using Welch’s t test. Difference was assessed with two-side test with an α level of 0.05.