Eliodorio, K. P. | Search bioRxiv

bioRxiv2023

A Fully Defined Synthetic Medium Mimicking Sugar Cane Molasses

BackgroundYeast research in the context of food/beverage production and industrial biotechnology faces a dilemma: to use real industrial media or to use fully defined laboratory media? While the former option might lead to experiments closer to industrial conditions, the latter has the advantage of allowing for reproducibility and comparability of results among different laboratories, as well as being suitable for the investigation of how different individual components affect microbial or process performance. It is undoubtable that the development of a synthetic must a few decades ago led to important advances in wine yeast research. ResultsWe developed a fully defined medium that mimics sugarcane molasses, a frequently used medium in different industrial processes where yeast is cultivated. The medium, named 2SMol, builds upon a previously published semi-defined formulation and is conveniently prepared from some stock solutions: C-source, organic N, inorganic N, organic acids, trace elements, vitamins, Mg+K, and Ca. We validated the 2SMol recipe in a scaled-down sugarcane biorefinery model, comparing the performance of different yeast strains in different real molasses-based media. We also showcase the flexibility of the medium by investigating the effect of nitrogen availability on the ethanol yield during fermentation. ConclusionsHere we present in detail the development of a fully defined synthetic molasses medium, and we hope the 2SMol formulation will be valuable to researchers both in academia and industry to obtain new insights and developments in industrial yeast biotechnology.

bioengineering↗

bioRxiv2021

Blocking mitophagy does not improve fuel ethanol production in Saccharomyces cerevisiae

Ethanol fermentation is frequently performed under conditions of low nitrogen. In Saccharomyces cerevisiae, nitrogen limitation induces macroautophagy, including the selective removal of mitochondria, also called mitophagy. Shiroma and co-workers (2014) showed that blocking mitophagy by deletion of the mitophagy specific gene ATG32 increased the fermentation performance during the brewing of Ginjo sake. In this study, we tested if a similar strategy could enhance alcoholic fermentation in the context of fuel ethanol production from sugarcane in Brazilian biorefineries. Conditions that mimic the industrial fermentation process indeed induce Atg32-dependent mitophagy in cells of S. cerevisiae PE-2, a strain frequently used in the industry. However, after blocking mitophagy, no differences in CO2 production, final ethanol titres or cell viability were observed after five rounds of ethanol fermentation, cell recycling and acid treatment, as commonly performed in sugarcane biorefineries. To test if S. cerevisiaes strain background influences this outcome, cultivations were carried out in a synthetic medium with strains PE-2, Ethanol Red (industrial) and BY (laboratory), with and without a functional ATG32 gene, under oxic and oxygen restricted conditions. Despite the clear differences in sugar consumption, cell viability and ethanol titres, among the three strains, we could not observe any improvement in fermentation performance related to the blocking of mitophagy. We conclude with caution that results obtained with Ginjo sake yeast is an exception and cannot be extrapolated to other yeast strains and that more research is needed to ascertain the role of autophagic processes during fermentation. ImportanceBioethanol is the largest (per volume) ever biobased bulk chemical produced globally. The fermentation process is very well established, and industries regularly attain nearly 85% of maximum theoretical yields. However, because of the volume of fuel produced, even a small improvement will have huge economic benefits. To this end, besides already implemented process improvements, various free energy conservation strategies have been successfully exploited at least in laboratory strains to increase ethanol yields and decrease by-product formation. Cellular housekeeping processes have been an almost unexplored territory in strain improvement. Shiroma and co-workers previously reported that blocking mitophagy by deletion of the mitophagy receptor gene ATG32 in Saccharomyces cerevisiae led to a 2.12% increase in final ethanol titres during Japanese sake fermentation. We found in two commercially used bioethanol strains (PE-2 and Ethanol Red) that ATG32 deficiency does not lead to an improvement in cell viability or ethanol levels during fermentation with molasses or in a synthetic complete medium. More research is required to ascertain the role of autophagic processes during fermentation conditions.

microbiology↗