Sampling for extracellular metabolites was performed every 30 min after culture inoculation. Glucose and acetate were quantified by the use of an H+ high-performance liquid chromatography (HPLC) system (Agilent Technologies 1200 Series HPLC and Aminex HPX-87h column for separation of acids and sugar). The analysis was carried out at 48°C using H2SO4 5 mM as an eluent.
1200 series hplc
Manufactured by Aminex
The 1200 Series HPLC is a high-performance liquid chromatography system designed for separation, identification, and quantification of chemical compounds. It features precision pumps, an autosampler, and a variety of detectors to facilitate diverse analytical applications.
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6 protocols using 1200 series hplc
1
Extracellular Metabolite Sampling and Analysis
2
HPLC Analysis of Carbon Metabolites
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HPLC analysis of select carbon metabolites including glucose and organic acids was performed with an Agilent 1200 series HPLC equipped with a refractive index detector (RID) and an Aminex HPX-87H ion exclusion column, 300 mm × 7.8 mm. A mobile phase of 5 mM H2SO4 was run at a flow rate of 0.6 mL/min for 25 min/injection. A volume of 200 µL of sample was added to an HPLC vial with 200 µL of an internal standard of 1 g/L fucose dissolved in 10 mM H2SO4. Each sample was injected twice for a total of two technical replicates for each of the three biological replicates for each time point. HPLC analysis of culture supernatants were compared to NMR analysis (Chenomx library and an internal standard of DSS) as a verification of metabolite identities and to ensure no major metabolites were being missed34 (link). Supplementary material S3 includes a comparison of HPLC- and NMR-based analyses of select metabolites during batch growth.
3
Metabolite Profiling in Microbial Cultures
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The extracellular metabolites were identified and quantified by H+ high-performance liquid chromatography (HPLC) (Agilent Technologies 1200 Series HPLC and an Aminex HPX-87h column to separate acid and sugar). The analysis was carried out at 48°C with 5 mM H2SO4 as the eluent. Intracellular metabolites were sampled at four different times during culture by the differential method (45 (link)). The metabolic intracellular content was assessed by LC-MS/MS as previously described (16 (link)). The AEC was quantified as previously described (35 (link)). Specific growth rates and consumption and production rates were determined as previously described (46 (link)).
4
Multi-Analytical Approach for Electrochemical CO2 Reduction
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X-ray diffraction (XRD) was done using
a Rigaku Miniflex instrument with a Cu Kα (1.5406 Å) radiation.
The X-ray photoelectron spectroscopy (XPS) measurement was done with
a Thermo Scientific instrument, and the same was corrected for carbon
by 284.6 eV. The surface morphologies of the thin film were investigated
using a Hitachi model field-emission scanning electron microscope
(FE-SEM). The products dissolved in the electrolyte, viz., HCOOH and
acetic acid, were analyzed using ion chromatography (Metrohm 881)
in 1 mM HClO4 with acetone (5%) and 10 mM LiCl as the suppressor.
In our work, only the liquid was analyzed with the importance given
to understand the formation of formic acid, and all of the dilution
factors were taken for analyses and calculations. Further, the liquid
products were investigated using the high-performance liquid chromatography
(HPLC; Agilent HPLC-1200 series) technique with a UV detector, with
the column being Aminex HPX-87H, 300 mm × 7.8 mm, and the mobile
phase used for the study being 5 mM H2SO4.
a Rigaku Miniflex instrument with a Cu Kα (1.5406 Å) radiation.
The X-ray photoelectron spectroscopy (XPS) measurement was done with
a Thermo Scientific instrument, and the same was corrected for carbon
by 284.6 eV. The surface morphologies of the thin film were investigated
using a Hitachi model field-emission scanning electron microscope
(FE-SEM). The products dissolved in the electrolyte, viz., HCOOH and
acetic acid, were analyzed using ion chromatography (Metrohm 881)
in 1 mM HClO4 with acetone (5%) and 10 mM LiCl as the suppressor.
In our work, only the liquid was analyzed with the importance given
to understand the formation of formic acid, and all of the dilution
factors were taken for analyses and calculations. Further, the liquid
products were investigated using the high-performance liquid chromatography
(HPLC; Agilent HPLC-1200 series) technique with a UV detector, with
the column being Aminex HPX-87H, 300 mm × 7.8 mm, and the mobile
phase used for the study being 5 mM H2SO4.
5
Biomass Compositional Analysis Protocol
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The chemical composition of the WIS fraction was analyzed by using the standard methods for determination of structural carbohydrates and lignin in biomass (LAP-002, LAP- 003, and LAP-019) of the National Renewable Energy Laboratory (NREL). The full description for these methods can be found in the following link [https://www.nrel.gov/bioenergy/biomass-compositional-analysis.html ].
Glucose, xylose, xylitol and ethanol were determined and quantified by high-performance liquid chromatography (HPLC) using an Agilent HPLC 1200 Series equipped with a refractive index detector and an Aminex HPX-87H Ion Exclusion column operating at 50 °C with 5 mM H2SO4 (0.6 mL/min) as elution buffer.
Statistics were performed to estimate the mean and standard deviation during fermentation and SSF assays. Analysis of variance (ANOVA) was used for comparison between assays using the software Statgraphics Centurion XVIII. The level of significance was set at P < 0.05, P < 0.01, and P < 0.001.
Glucose, xylose, xylitol and ethanol were determined and quantified by high-performance liquid chromatography (HPLC) using an Agilent HPLC 1200 Series equipped with a refractive index detector and an Aminex HPX-87H Ion Exclusion column operating at 50 °C with 5 mM H2SO4 (0.6 mL/min) as elution buffer.
Statistics were performed to estimate the mean and standard deviation during fermentation and SSF assays. Analysis of variance (ANOVA) was used for comparison between assays using the software Statgraphics Centurion XVIII. The level of significance was set at P < 0.05, P < 0.01, and P < 0.001.
6
Analytical Quantification of Biomass Fermentation
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Glucose, xylose, acetic acid, furfural, 5-hydroxymethylfurfural (HMF), and ethanol were analysed on an Agilent HPLC 1200 Series using an Aminex HPX-87H column, as described elsewhere.35 (link) All samples were centrifuged (5810R, Eppendorf, Inc.) and filtered through 0.45 μm filters and then properly diluted before analysis. The theoretical ethanol yield was calculated taking into consideration that the S. cerevisiae BY4741 consumes only C6 sugars as reported previously.36 (link) The theoretical ethanol from the PCTR-pretreated biomass was calculated based on their glucan content as % of the theoretical, following eqn (3) : where TYE = theoretical ethanol yield (g L−1), CG = glucan concentration (%), and glucan to glucose unit ratio. The yield of ethanol (%) was calculated according to the ethanol produced during SSF and the theoretical ethanol yield, according to the following equation: where YE = ethanol yield (%).
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