Abstract Description

Acidity is key for wine freshness, organoleptic balance, and microbial stability. However, global warming has led to a decline in grape acidity at harvest. While acid additions are commonly used, they are costly, can cause stability issues, and face increasing consumer and regulatory rejection. The yeast Lachancea thermotolerans offers a promising biological alternative through its production of lactic acid during fermentation. However, production varies significantly between strains and is strongly influenced by environmental conditions, limiting its reliable use in industry. Increasing evidence suggests that oxygen availability is a critical, yet understudied, factor influencing this variability. To investigate this, three genetically distinct L. thermotolerans strains with differing lactic acid production abilities were inoculated in synthetic grape must under defined oxygenation regimes (fully anaerobic, timed oxygen pulses, and fully aerobic) to assess the impact of oxygen on fermentation performance and primary metabolites, with a focus on lactic acid. Oxygen availability strongly influenced fermentation kinetics and yeast growth, with consistent trends across all strains. Fully aerobic conditions enabled complete fermentation and resulted in ~20-fold higher biomass production compared to fully anaerobic, which fermented only ~30 % of sugars. Timed oxygen pulses had intermediate effects, with the strongest response observed for a pulse at 25 % maximum growth, followed by 20 h of fermentation, while at 40 g/L CO₂ loss had no effect. Oxygen availability also clearly affected carbon metabolism, particularly lactic acid yield, which was highest under fully anaerobic conditions and nearly absent under fully aerobic, consistent with previous reports. Other metabolites showed mixed trends compared to those typically observed in the main wine yeast Saccharomyces cerevisiae. The main differences were increased acetic acid under anaerobic conditions and stable glycerol. These show different carbon flux regulation in L. thermotolerans in response to oxygen availability. Timed oxygen pulses caused no significant changes in lactic acid, ethanol, or glycerol; however, pulses at 20 h of fermentation and at 25 % maximum growth significantly increased biomass and succinic acid while reducing acetic acid, whereas a pulse at 40 g/L CO₂ loss had no effect. The minimal impact on lactic acid suggests that earlier or longer oxygen exposure may be required, as lactic acid is primarily produced during the early growth phase. This work highlights oxygen as a key driver of variability in lactic acid production by L. thermotolerans and its broader impact on fermentation and carbon metabolism. These findings provide a basis for developing strategies to optimise acidity in wines affected by climate change, with future work needed to refine oxygenation regimes in natural grape must and elucidating the genetic mechanisms underlying the regulation of lactic acid production by oxygen.