Quantification of Fermentation Metabolites

Concentrations of cellobiose, succinate, formate and acetate in culture samples were analyzed with HPLC. Samples taken at different time points were stored at −20 °C. Before HPLC analysis, the samples were thawed, filtered through a 0.2-μm nylon filter and acidified with 2 M sulfuric acid. Twenty microliters each of the acidified samples were then applied to an Aminex HPX-87H, 300 × 7.8 mm column (Bio-Rad, Hercules, CA) on a Hitachi LaChrom Elite System (Hitachi High Technologies America, Inc., San Jose, CA) or a Waters Breeze system (Waters Corp., Milford, MA). Analysis was performed at a flow rate of 0.5 ml/min in 5 mM H₂SO₄ for 35 min as previously described^{39 (link)}. Soluble fermentation products were identified by comparison with retention times and peak areas of corresponding standards.