Ethanol, lactate, formate, acetate, pyruvate, fumarate, pyroglutamate, isobutanol, meso-2,3-butanediol, and RR/SS-2,3-butanediol and xylitol were quantified by HPLC (Waters, Milford, MA) with refractive index (RI) and UV detection using an Aminex HPX-87H column (Bio-Rad, Hercules, CA) with a 5-mM sulfuric acid solution eluent. Malate was quantified by colorimetric assay (BioVision, Milpitas, CA).
Since measurements of CO2 are frequently unreliable due to the accumulation of CO2 in the liquid fraction, for the purposes of carbon balances CO2 production in moles was estimated by stoichiometry using the following formulas:
Formula1 is used when looking at “commonly reported” fermentation products. Formula 2 is a more comprehensive version of formula 1 .
To account for solubilized, non-utilized oligosaccharides present at the end of the fermentation, the sugar content of the supernatant was determined by acid hydrolysis and measured by HPLC.
The higher alcohols 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, isobutanol and the 2,3-butanediols were identified by gas chromatography with flame ionization detector (GC/FID)(Agilent 7890A, RESTEK-FAMEWAX, 30 m column, helium carrier) and by matching retention times to standards on two different liquid chromatography columns: H-column (Agilent 1100, Aminex HPX-87H column, Bio-Rad, Hercules, CA, 5 mM H2SO4, 0.7 mL/min) and P-column (Thermo Spectra, Aminex HPX-87P column, Bio-Rad, Hercules, CA, distilled water, 0.65 mL/min). Refractive index (RI) was used to detect compounds during HPLC analysis. LC-MS was used to identify fumaric acid and pyroglutamate (L-5-oxo-proline) (Thermo LTQ, Aminex HPX-87H column with 0.1% formic acid eluent, 0.7 mL/min). The chiral isomers SS/RR 2,3-butanediol could not be resolved on either the GC or LC.
Since measurements of CO2 are frequently unreliable due to the accumulation of CO2 in the liquid fraction, for the purposes of carbon balances CO2 production in moles was estimated by stoichiometry using the following formulas:
Formula
To account for solubilized, non-utilized oligosaccharides present at the end of the fermentation, the sugar content of the supernatant was determined by acid hydrolysis and measured by HPLC.
The higher alcohols 1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, isobutanol and the 2,3-butanediols were identified by gas chromatography with flame ionization detector (GC/FID)(Agilent 7890A, RESTEK-FAMEWAX, 30 m column, helium carrier) and by matching retention times to standards on two different liquid chromatography columns: H-column (Agilent 1100, Aminex HPX-87H column, Bio-Rad, Hercules, CA, 5 mM H2SO4, 0.7 mL/min) and P-column (Thermo Spectra, Aminex HPX-87P column, Bio-Rad, Hercules, CA, distilled water, 0.65 mL/min). Refractive index (RI) was used to detect compounds during HPLC analysis. LC-MS was used to identify fumaric acid and pyroglutamate (L-5-oxo-proline) (Thermo LTQ, Aminex HPX-87H column with 0.1% formic acid eluent, 0.7 mL/min). The chiral isomers SS/RR 2,3-butanediol could not be resolved on either the GC or LC.
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