Hpx 87h ion exclusion column

The reducing sugar yield in the supernatant was analyzed by 2,4-dinitrosalicyclic acid (DNS) assay [25 (link)], while the monosaccharide composition was determined by comparing the retention time against standards using HPLC (Agilent), where an HPX-87H ion exclusion column (BioRad Aminex) was used at 35 °C with 5 mM H₂SO₄ as the mobile phase. A flow rate of 0.6 mL/min and an injection volume of 20 μL were used with a 30 min analysis time. HPLC was also used to analyze the glucose consumption and ethanol yield using the following equation [30 (link)]: $$\text{Ethanol yield}\left(\%\right)=\frac{\text{Final ethanol concentration}\left({\text{g L}}^{-1}\right)}{\text{Initial glucose and xylose}\text{concentration}\times 0.511\left({\text{g L}}^{-1}\right)}\times 100\%.$$
The total nitrogen and carbon contents in pretreated, enzymatic hydrolyzed, and fermented solutions were analyzed by an spectrophotometry and auto analyzer (AA3, Bran + Luebbe, Norderstedt, Germany) [31 (link)].
All experimental data are presented as the mean of samples performed in triplicate, where error bars indicate standard deviation.

SCFA concentrations were measured according to the method of Wielen et al. (2020) (link). Frozen ileal digesta (about 0.5 g) was thawed at 4°C and diluted fourfold with double-distilled water. After thawing, samples were centrifuged (10 min at 12,000 rpm), and 12.5 ml of a xylitol solution (0.586 M in 1.5 M HCl; internal standard) was added. Then, samples were centrifuged again (10 min at 12,000 rpm) before analysis using HPLC. Samples (20 μl) were injected into the HPLC with a Spark Holland autosampler (Emmen, the Netherlands). The HPLC was equipped with a Waters 2,996 Photodiode Array Detector and an organic acid column HPX-87H ion exclusion column (300 mm × 7.8 mm, Bio-Rad Laboratories Inc., Hercules, CA, United States). The column was operated at 40°C, with 5 mM NH₂SO₄ at 0.6 ml/min as the eluent.

Glucose and ethanol were calculated using high-performance liquid chromatography (HPLC; Agilent 1200), according to the description by You et al. [50 (link)]. Briefly, an HPX-87H ion exclusion column (Bio-Rad Aminex) was used and 5 mM H₂SO₄ worked as the mobile phase. The chromatographic system was run at a flow rate of 0.6 mL/min and 35 °C. The injection volume was set to 20 μL.
Cells were harvested for assaying intracellular NAD using a NAD/NADH Assay Kit (ab65348, Abcam Inc.). Briefly, one milliliter of fresh cells was harvested by centrifuging at 4000 rpm and 4 °C for 10 min. After washing with cold phosphate buffer solution (PBS), cells were extracted using NADH/NAD extraction buffer via two freeze/thaw cycles of 20 min at − 80 °C followed by 10 min at room temperature (RT). After a short vortex, supernatants were collected by centrifuging at 4 °C for 5 min. For measuring total NAD, 50 μL of extracted aliquots and 100 μL of reaction mix were added in each well of a 96-well microplate, incubated at RT for 5 min. 10 μL of NADH developer were added in each well and mixed, and OD_450nm values were then read using a microplate reader (Spectro MAX190, Molecular Devices, USA). For measuring NADH, extracted aliquots were heated to decompose NAD⁺ before blending with reaction mix.

Bioreactor batch cultures were performed in a Sartorius Biostat B plus bioreactor in 1 liter of M9 medium with 90 mMeqC of glucose and/or xylose mixes. The temperature was set at 37°C and pH at 7. Nonlimiting aeration conditions were obtained with an airflow set at 0.35 liter · min⁻¹ and adaptation of stirring to maintain pO₂ of >20%. Growth was assessed by OD₆₀₀ measurement at 30-min intervals with a LibraS4 spectrophotometer (Biochrom, UK). Glucose, xylose, and acetate concentrations were quantified by high-performance liquid chromatography (HPLC) (Agilent, USA) with an HPX 87H ion exclusion column (Bio-Rad) as described in reference 66 (link).

For L. casei Zhang fermented with LSF-MRS medium, samples (2.0 mL) were centrifuged at 5000× g for 10 min to obtain cell-free cultured supernatants. The supernatants were diluted 10 times and then filtered through disposable syringe filters (Millipore, 0.22 μm). For L. casei Zhang fermented with SSF-SW medium, samples (10.0 g) were added into 90.0 mL of 5 mM H₂SO₄ solution and then mixed in shaker at 180 rpm for 20 min. The mixture was centrifuged at 5000× g for 10 min to obtain cell-free cultured supernatants. The supernatants were also diluted 10 times and then filtered through disposable syringe filters (Millipore, 0.22 μm). Finally, 20 μL of the obtained solutions were analyzed by HPLC (LC-20A, Shimadzu, Japan) on a Bio-Rad HPX-87H ion-exclusion column (300 × 7.8 mm). Organic acids (lactate and acetate) were detected by a differential refraction detector and mobile phase was 5 mM H₂SO₄ that was pumped through the column at a flow rate of 0.6 mL/min at column temperature 40 °C. The peak area was used to calculate the concentrations of lactate and acetate according to corresponding standard curves.

From the supernatant samples, the concentration of various organic acids was measured by HPLC (Agilent, 1260 Infinity), using a standard analytical system (Shimadzu, Kyoto, Japan) equipped with an Organic Acid Analysis column (Bio-Rad, HPX-87H ion exclusion column) at 35°C. The eluent was 5 mm sulfuric acid, used at a flow rate of 0.6 mL min^-1 and compounds were detected by refractive index. A five-point calibration curve based on peak area was generated and used to calculate concentrations in the unknown samples. For determination of ammonia concentrations, we employed assay kits (Sigma, AA0100) according to the manufacturer’s protocols.