Rezex roa

Ethanol quantification analysis was conducted by using ROA (organic acid H +) determination method by HPLC system 1200 series (Agilent Technologies, Germany), which was equipped with an auto sampler and refractive index detector. The column used was Rezex ROA (Phenomenex, USA) with 300 × 7.8 mm in size. Sulfuric acid in water, at 0.005 N (normality), was used as mobile phase with a flow rate of 0.6 mL/min. The chromatography method was performed at a temperature of 60 °C with injection volume of 20 µL. Five ethanol standard solutions were injected at different concentrations in the range of 0.5–4 mg/mL to produce the standard curve with linear regression. P. niruri extracts were dissolved in dimethyl sulfoxide (DMSO) in the range concentration of 1 to 5 mg/mL and ethanol chromatographic peak in the extracts was identified through comparison with the retention time of ethanol standard reference.

Rumen fluid samples (2.0 mL) were thawed at room temperature, centrifuged at 12,000 x g for 10 min. Then, cell-free supernatants were treated as described by Siegfried et al. [22 ]. Volatile fatty acid concentrations were quantified using a Dionex Ultimate 3000 Dual detector HPLC (Dionex Corporation, Sunnyvale, CA, USA) coupled to a refractive index (RI) Shodex RI-101 maintained at 40°C. The ion exchange column (300 x 7.8) used was a Phenomenex Rezex ROA (Phenomenex Inc. Torrance, CA, USA), which was maintained at 45°C. Mobile phase was prepared with 5 mmol/L H₂SO₄, and the flow was set as 0.7 mL/min. The following volatile fatty acids were used to calibrate the standard curve: acetic, succinic, formic, lactic, propionic, valeric, isovaleric, isobutyric and butyric acid. The volatile fatty acids were prepared with a final concentration of 10 mmol/L, except isovaleric acid (5 mmol/L) and acetic acid (20 mmol/L).

Fungal cell wall polysaccharides were extracted from 10 mg dry-frozen biomass as described previously (54 (link)). One milliliter of extracted samples was concentrated 10× by lyophilization, and sugars were subsequently analyzed by high-performance liquid chromatography (HPLC) using a Young Lin YL9100 series system (Young Lin, Anyang, South Korea) equipped with a YL9170 series refractive index (RI) detector at 40°C. Samples were loaded in a Rezex ROA (Phenomenex, USA) column (300 by 7.8 mm) at 85°C and eluted with 0.05 M sulfuric acid at a flow rate of 1.5 ml/min.

The organic acid quantification was based on Rawi et al. (2021) (link). The fermentation sample from each sampling period was pipetted into a 2 mL microcentrifuge tube for centrifugation (Centrifuge-5804, Eppendorf) at 13000 rpm for 10 min to obtain a clear supernatant. The supernatant was filtered through a 0.22 μm syringe filter unit (Millipore) into an HPLC vial (Agilent Technologies, Cheshire, United Kingdom). Prominence Series Liquid Chromatography (Shimadzu Corp., Japan) using a reverse phase ion-exclusion C12 column (Rezex ROA, Phenomenex) was used for SCFA analysis. Analytes were detected using a UV detector at a wavelength of 210 nm. The isocratic mobile phase used was 0.25 mM sulphuric acid (H₂SO₄). A 15 μL sample was injected into the heated column (40°C) programmed to run in isocratic elution at a flow rate of 0.5 mL/min for 40 min. The peaks and response factors within the sample were calibrated and calculated using the LC Solutions software (Shimadzu). The standard solution contained of 12.5, 25, 50, 75, 100, 125, 150, 175, and 200 mM acetate, butyrate, propionate, and lactate.

The two fractions (fiber rich and vessel rich) were hydrolyzed following the laboratory analytical procedure for the determination of structural carbohydrates and lignin in biomass, from the National Renewable Energy Laboratory (NREL)²⁶ . Concisely, 300 mg of sample was successively submitted to a 72% (m/m) and 3%(m/m) sulfuric acid hydrolysis during 1 h at ambient temperature and 1 h at 121 °C, respectively. Neutral sugars, organic acids, and carbohydrate byproducts (furfural and hydroxymethylfurfural, HMF) in the hydrolysates were analyzed by using the high-performance liquid chromatography (HPLC) system. In this case a Rezex ROA (Phenomenex^®) organic acid column was used, using a 0.005N sulfuric acid ultra-pure water, as eluent. The column was maintained at 60 °C, and the flow was 400 µL/min. Cellobiose, glucose, xylose, mannose, galactose, and arabinose were used as standards. Although this column elutes xylose, mannose, and galactose at the same retention time, not enabling their separation, it has the advantage of analyzing the samples without post-treatment procedures.