Clostridium cellulovorans 743B (ATCC35296) was grown anaerobically as described previously (Han et al. 2004), with the exception of carbon source in the media. As carbon sources, 2 % (w/v) glucose (Nacalai tesque, Kyoto, Japan), 2 % (w/v) cellobiose (Sigma, MO, USA), 2 % (w/v) microcrystalline cellulose (Merck, Darmstadt, Germany), 1 % (w/v) xylan (Sigma), 1 % (w/v) pectin (Sigma), 1 % (w/v) locust bean gum (LBG; galactomannan, Sigma), or 0.3 % (w/v) phosphoric acid swollen cellulose (PASC) was used. PASC can be easily degraded compared to microcrystalline cellulose, because the cellulose in PASC is digested in smaller particles by the acid. PASC was prepared from microcrystalline cellulose as described previously (Zhang et al. 2006).
Estimation of cell growth
Growth was measured by quantitation of intracellular ATP concentration by luciferase-based luminescence with a Lumitester PD-30 and LuciPac Pen (Kikkoman biochemifa, Tokyo, Japan) according to the manufacturer’s instruction. It is known that integrated intracellular ATP concentration correlates with cell growth (Miyake et al. 2016). Cell culture (100 μL) was incubated with 10 μL of ATP eliminating enzyme (Kikkoman) for 30 min at room temperature to remove extracellular ATP. Subsequently, cell growth was estimated by measuring ATP concentration of 100 μL of cell culture.
Measurement of saccharide concentration in supernatant
Glucose and cellobiose concentrations in culture supernatants were measured by HPLC (Prominence; Shimadzu, Kyoto, Japan) equipped with an electrochemical detector (Coulochem III; thermo scientific, MA, USA). Supernatants were separated using a Sugar-D column (250 mm long, 4.6 mm inner diameter; Nacalai tesque) and the mobile phase was 80 % acetonitrile at a flow rate of 500 μL/min. The sample injection volume was 1 μL.
Measurement of residual carbon source in the media
Residual carbon source in the culture supernatant and precipitation were measured. Cell culture (50 μL) were incubated with 930 μL of 50 mM citrate buffer (pH 5.0) and 20 μL of cellulase SS (Nagase Chemtex, Osaka, Japan) for 24 h at 50 °C. After degradation, reacting solution was centrifuged for 10 min at 13,000×g. Polysaccharide concentration was measured by GC–MS.
Degraded residual carbon sources (20 μL) and 60 μL of 2 mg/mL ribitol, as an internal standard were stored in a deep freezer (−80 °C) until frozen (30 min), and were subsequently lyophilized. Lyophilized sample was incubated with 20 mg/mL pyridine methoxyamine (Sigma) for 90 min at 30 °C. Silylation was performed using MSTFA (N-methyl-N-TMS-trifluoro-acetamide) (GL Sciences, Tokyo, Japan) for 30 min at 37 °C. We used a gas chromatograph GCMS-QP2010 ultra (Shimadzu) equipped with a capillary column (CP-SIL 8CB; 30 m, inner diameter. 0.25 mm; Agilent Technologies, CA, USA). The oven temperature was 80 °C at 0–2 min, increased to 200 °C at a rate of 100 °C per minute, then at a rate of 50 °C per minute to 330 °C, and was finally maintained at 330 °C for 5 min. The interface temperature was 250 °C, and the source temperature was 200 °C. Helium was the carrier gas and was set at a flow rate of 1.12 mL/min.