Cell growth was monitored by measuring OD600 utilizing a spectrophotometer (Shimazu, Japan). Glucose and acetate were quantitatively analyzed by high-performance liquid chromatography (HPLC) (Shimazu, Japan) which equipped with a refractive index detector (RID-10A) and an Ion Exclusion column (Bio-Rad, HPX-87H). The samples were first centrifuged at 12,000 rpm for 10 min, and then the supernatant was filtrated with a 0.22 μm filter membrane. 5 mM sulfuric acid was utilized as the mobile phase of HPLC with the flow rate of 0.6 mL/min and the utilized column temperature was 65 °C.
Rid 10a
Manufactured by Bio-Rad
Sourced in Japan, United States
The RID-10A is a lab equipment product manufactured by Bio-Rad. It is a radial immunodiffusion (RID) system used for the quantitative determination of specific proteins in biological samples.
Automatically generated - may contain errors
6 protocols using rid 10a
1
Cell Growth Monitoring and Metabolite Analysis
2
HPLC Quantification of Glucose and Xylose
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Glucose and xylose in culture supernatant were quantified using a HPLC (LC-20AB, Shimadzu) equipped with a Fast Acid Analysis Column (Biorad, Hercules) and a differential refractive detector (RID-10A). 5 mM H2SO4 served as the mobile phase at a flow rate of 0.6 mL min−1 at 65 °C for 15 min.
3
Sugar Quantification in Fiber Samples
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The supernatatant was used to determine glucose, xylose and arabinose by high-performance liquid chromatography (HPLC) (Detector: Shimazu RID-10 A; Column: BioRad (Hercules, CA, USA) Aminex HPX-87H (300 × 7.8)) at 65 °C. The eluent was 5 mmol/L sulphuric acid, and the flow rate was 0.5 mL/min.
The recoverable sugars measurement was carried out in triplicate. The glucan, xylan and arabinan contents of the fibre samples were determined, taking into account the depolymerisation factor of the monosaccharides [8 ]. The glucan includes all polysaccharides consisting of glucose monomers.
The recoverable sugars measurement was carried out in triplicate. The glucan, xylan and arabinan contents of the fibre samples were determined, taking into account the depolymerisation factor of the monosaccharides [8 ]. The glucan includes all polysaccharides consisting of glucose monomers.
4
Quantifying Residual Sugars in Fermentation
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The residual sugars in the fermented residue were extracted by ultrapure water and were analysed using high performance liquid chromatography (HPLC) (Shimadzu LC-20A) equipped with a strong acid cation-exchange resin column (Bio-Rad Aminex HPX-87H) operating at 55°C and a differential refractive index detector (RID-10A) operating at 40°C. The mobile phase was dilute H2SO4 solution of 0.005 mol l−1 at the flow rate of 0.6 ml min−1.
5
Quantifying Biomass and Metabolites
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Biomass concentration was measured through optical density at 600 nm (OD600) in 1 cm cuvettes using Spectrophotometer (Biospectrometer, Eppendrof, USA). To determine cell dry weight 3–5 ml of a culture was withdrawn, centrifuged at 5,000 rpm at 4 °C for 5 mins to collect cell pellet. The pellet was washed once with deionized water, added into a pre-weighed tube and dried in an 80 °C oven for 12–24 hrs followed by putting in desiccator for 24 hrs before being subsequently weighed. The following correlation: cell dry weight (g/l) = 0.359 × OD600 (R2 = 0.96) was used for estimation of cell dry weight from OD600 measurement. Sugar and fermentative byproduct concentration were measured using an HPLC system equipped with a cation exchange column (HPX-87H, Biorad Labs, Hercules, CA) and a refractive index detector (RID-10A). Samples from the culture supernatant were collected, filtered using 0.2 µm sterile filter and stored at −20 °C before analysis. The column was run in an isocratic mode using 5 mM H2SO4 mobile phase at 60 °C and a flow rate of 0.6 ml/min for 30 min. A standard curve correlating area to concentration of metabolites was used to determine concentration in the sample.
6
Biomass and Metabolite Quantification Protocol
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Biomass was determined indirectly by spectrophotometry at 600 nm using ThermoScientific Evolution 201, and the dry weight was calculated using the calibration curve. Substrate glycerol and the products PDO, butyric acid, acetic acid, lactic acid, and butanol were quantified using ultra-fast liquid chromatography (UFLC) with a refractive index detector (Shimadzu RID 10A) at 60°C and AMINEX HPX— 87H column (Biorad) at 63°C, a solution of 3 mM of sulfuric acid as the phase mobile, and 0.5 mL/min flow during 45 minutes of run time. The samples had a volume of 750 μL and were located in a Shimadzu Prominence LC-20AD autosampler, which ultimately injected 20 μL to UFLC. Lab Solutions software V. 1.25 (Shimadzu) was used for calculating retention time, the slope of the straight line, and linear ratio coefficients (R2) to determine the concentrations of the compounds evaluated.
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