The substratostat - an automated near-infrared spectroscopy-based variable-feed system for the continuous alcoholic fed-batch fermentation of high gravity grape must at static sugar concentrations

Abstract

Aim: High sugar concentrations in musts cause a hyperosmotic stress response in Saccharomyces cerevisiae increasing the risk of sluggish and stuck alcoholic fermentations and/or causing high acetic acid levels. Applying a fed-batch technique where sugar levels are kept at a constant, low rate throughout fermentations reduces this stress but requires in-situ quantification of sugars and process automation for practicability. The aim of this work was to develop and validate a near-infrared (NIR) spectroscopy method allowing for the continuous in-situ quantification of total fermentable sugars in fully turbid alcoholic fermentations of grape musts. Calibration models for glucose, fructose and the fermentation product ethanol were also established.Methods and results: A research Fourier-transform NIR spectrophotometer equipped with a transflectance probe was used to acquire spectra from 240 natural and semisynthetic standards from fermentations conducted using varying concentrations of yeast and yeast nutrients. Using chemometric software, calibration models for total sugars, glucose, fructose and ethanol demonstrated R2 values >0.93 and prediction error (RMSEP) values of 11.6 g l-1, 12.3 g l-1, 10.2 g l-1, and 0.328 % v/v, respectively. The method was integrated with modern process automation technology and was able to maintain sugar concentrations within 5 g l-1 of the 45 g l-1 setpoint adjusted during alcoholic fermentations.Conclusions: The NIR calibration models generated allow prediction of total sugar levels accurately enough to conduct fully automated fed-batch grape must fermentations at constant substrate concentrations. Application of a transflectance probe measuring a high proportion of back-scattered radiation proved useful and necessary considering the high degree of turbidity during fermentations. Placement of the measurement probe in a recirculation loop decreased interference from biomass sedimentation and adherence of CO2 bubbles.Significance and impact of the study: This study presents a fully automated system to carry out fed-batch fermentations which allow circumventing the hyperosmotic stress response of S. cerevisiae during alcoholic fermentations. Calibrated for other substrates, the system may be used in other food and non-food fermentations, too.