Refractive index detector

Samples for metabolites and OD were regularly withdrawn from the reactor. For biomass determination, 5 mL culture was washed with distilled water and dried on Gelman filters (ø 47 mm Supor-450, 0.45 μm) in a microwave oven (350 W) for 8 minutes. The biomass concentration was correlated with OD₆₂₀ by dry weight measurements. Glucose, ethanol, HMF, glycerol and acetic acid were analysed by high performance liquid chromatography (HPLC; Waters Corporation, MA, USA) on an Aminex HPX-87H column (Bio-Rad, CA, USA) at 65°C. The mobile phase was 5 mM sulphuric acid with a flow of 0.6 mL/min. All compounds were detected with a refractive index detector (Shimadzu, Tokyo, Japan).
The specific HMF uptake rates were calculated according to the formula: ${\mathrm{q}}_{\mathrm{hmf}}=\mathrm{\mu }.\mathrm{m}$
where:
μ = maximum specific growth rate (1/h)
m: slope obtained after plotting HMF concentration (gHMF/L) in the Yaxis vs. cdw (g cell/L) in the X-axis at each time point of the exponential phase.

Digestion of β-1,3:1,4-glucan with enzyme was performed using a reaction mixture (total volume, 1 mL) consisting of the enzyme, 0.3% (w/v) β-1,3:1,4-glucan, and 50 mM 3-morpholinopropanesulfonic acid-NaOH buffer (pH 6.5). The apparent molecular weight (M_r) of the β-glucan was estimated by high-performance liquid chromatography (HPLC) with a Shimadzu LC-10 A (Shimadzu, Kyoto, Japan) fitted with a refractive index detector (Shimadzu) and tandem columns (each 7.8 × 300 mm) of TSKgel G3000PW_XL and G2500PW_XL (Tosoh, Tokyo, Japan), equilibrated and eluted with 0.2 M potassium phosphate buffer (pH 6.9) at a flow rate of 0.8 mL/min and at 40 °C. The void volume (V₀) and inner volume (V_i) were determined with pullulan markers (Shodex Standard P-82; Showa Denko, Tokyo, Japan) and Glc.

Sugar analysis was performed by HPLC as described previously [43 (link)]. Samples were prepared from 100 µl of the hydrolysis supernatants. The HPLC system comprised a Micro-Guard De-Ash pre-column (Bio-Rad, USA), an SPO810 column (Shodex) coupled to a refractive index detector (Shimadzu). Deionised water delivered at a flow rate of 0.7 mL min⁻¹ was used to elute the columns at 60 °C.
Phenolic analysis was based on the Folin–Ciocalteau reaction following the micro-scale protocol by Waterhouse [44 ]. In short, a hydrolysate sample of 20 μl was reacted with commercial Folin–Ciocalteau reagent (Merck) and NaCO₃ in a plastic cuvette in a total volume of 2 mL. The phenol concentration was determined spectrophotometrically at 765 nm against a gallic acid standard as mg mL⁻¹ gallic acid equivalent (GAE).

The hydrolysis tests for Avicel (Sigma-Aldrich), holocellulose, and PASC were conducted using the enzyme and each lignin (3.3%, 5.4 mg protein g^-1 lignin) at 45°C for 48 h in a rotary shaker (QB-228). The substrate for loading was double lignin. In the supernatant, glucose and xylose were determined using HPLC (Shimadzu, Kyoto, Japan) with a refractive index detector (Shimadzu) on an Aminex HPX-87P column (Bio-Rad, Hercules, CA, USA) at a flow rate of 0.5 mL/min at 78°C, with water as the eluent. Samples lacking lignin or enzyme served as control.

Controlled volumes of samples were taken regularly for analysis. Biomass was followed by OD620 measurements during the length of the fermentations and determination of cell dry weight measurements were also done at time zero (just after inoculation) and at times 42 h and 92 h. For biomass determination, the cell pellet from 5 mL culture was washed with distilled water and dried on Gelman filters (ø 47 mm Supor-450, 0.45 μm) in a microwave oven (350 W) for 8 minutes. Ethanol, glycerol, acetic acid, HMF and furfural were analysed by high performance liquid chromatography (HPLC; Waters Corporation, MA, USA) using an Aminex HPX-87H column (Bio-Rad, CA, USA) at 65°C. The mobile phase was 5 mM sulphuric acid with a flow rate of 0.6 mL.min⁻¹. Analysis of glucose, mannose, xylose, galactose and xylitol was performed on a Shodex™ SP-0810 sugar column (Showa Denko K.K, Japan) at 85°C with water as mobile phase and 0.6 mL.min⁻¹ flow rate. All compounds were detected with a refractive index detector (Shimadzu, Tokyo, Japan, and Waters 2414, MA, USA respectively). Yields were calculated based on HPLC measurements.

The MOS were identified and quantified using a CarboSep CHO 411 column (Concise Separations, San Jose, USA) connected to a Shimadzu HPLC system with a refractive index detector (Shimadzu Corp, Kyoto, Japan) as described in our previous work.^{9 (link)}