Metabolite Analysis Protocol using Enzymatic Assay

Clarified supernatant (20 800 rcf, 2 min) was used for metabolite analysis. Residual glucose and fructose were measured enzymatically (Boehringer-Mannheim 1989 ) with volumes adjusted to 100 μl for microtiter plate analysis (Walker et al., 2014 (link)). A liquid handling robot (Tecan EVO 150) collected samples for absorbance readings using an Infinite® 200 PRO microplate reader (Tecan Group Ltd). Samples were diluted 1 in 10, and 1 in 100 for analysis (Walker et al. 2014 (link)).
Other analyses included nitrogen by spectrophotometry (Dukes and Butzke 1998 ) using the Primary amino acid nitrogen (PAN) kit (K- PANOPA; Megazyme), and major metabolites (organic acids, glycerol and ethanol) by HPLC (Lin et al. 2020 (link)).
Data was organized, analyzed and graphed using GraphPad Prism software (versions 8.0.0 and 9.0.0 for Windows; GraphPad Software, San Diego, www.graphpad.com). Area Under the Curve (AUC) calculations and statistical analysis using One-way Analysis of Variance Analysis (ANOVA) and multiple comparisons testing was also with GraphPad Prism.