Bacterial community dynamics during distilled spirit fermentation: influence of mash recipes and fermentation processes

The popularity and production of whiskey have grown dramatically in recent years. During whiskey fermentation, lactic acid bacteria (LAB) are a major concern since they can outcompete yeast and spoil the fermentation. However, some bacteria present in the fermentation could potentially counter this effect and promote fermentation efficiency. To better understand the possible roles bacteria play in yeast-based whiskey fermentations, we examined bacterial community dynamics across fermentation stages and investigated how variation in the mash recipe affects bacterial community composition and fermentation efficiency. To this end, we collected 193 samples from three distilleries at the beginning (Cook/set), middle (Fermentation), and end (Drop) of whiskey fermentation, with six mash recipes sampled from one distillery. We used high-performance liquid chromatography (HPLC) to quantify the contents of sugars, organic acids, and ethanol, which revealed distinct differences between distilleries and mash recipes. High-throughput Illumina Miseq sequencing of the 16S rRNA gene V4 region revealed that bacterial communities shifted toward Firmicutes during the fermentative conversion of sugar to ethanol, especially Lactobacillales. Mash recipes also influenced sugar composition, fermentation efficiency, and microbial dynamics. Recipe-specific operational taxonomic unit (OTU) biomarkers in Drop samples included Leuconostoc for corn, Lactococcus for wheat, and Lactobacillaceae_unclassified for rye, while Escherichia/Shigella was associated with sorghum, suggesting potential suppression of LAB.IMPORTANCEProduction of ethanol from sugars and yeast is an ancient, ostensibly simple process. The source of sugars varies depending on the desired product and can include fruits, vegetables, molasses, honey, or grains, among other things. The source of yeast can be natural in the case of spontaneous ferments, but dry yeast addition is typical for large-scale fermentations. While the polymicrobial nature of some alcoholic fermentations is appreciated (e.g., for wine), most grain-based ethanol producers view microbes, apart from the added yeast, as "contaminants" meant to be controlled in order to maximize efficiency of ethanol production per unit of sugar. Nonetheless, despite rigorous cleaning-in-place measures and cooking the mash, bacteria are routinely cultured from these fermentations. We now know that bacteria can contribute to fermentation efficiency on an industrial scale, yet nothing is known about the makeup and stability of microbial communities in distilled spirit fermentations. The work here establishes the roles of mash recipes and distillery practices in microbial community assembly and dynamics over the course of fermentation. This represents an important first step in appreciating the myriad roles of bacteria in the production of distilled spirits. Production of ethanol from sugars and yeast is an ancient, ostensibly simple process. The source of sugars varies depending on the desired product and can include fruits, vegetables, molasses, honey, or grains, among other things. The source of yeast can be natural in the case of spontaneous ferments, but dry yeast addition is typical for large-scale fermentations. While the polymicrobial nature of some alcoholic fermentations is appreciated (e.g., for wine), most grain-based ethanol producers view microbes, apart from the added yeast, as "contaminants" meant to be controlled in order to maximize efficiency of ethanol production per unit of sugar. Nonetheless, despite rigorous cleaning-in-place measures and cooking the mash, bacteria are routinely cultured from these fermentations. We now know that bacteria can contribute to fermentation efficiency on an industrial scale, yet nothing is known about the makeup and stability of microbial communities in distilled spirit fermentations. The work here establishes the roles of mash recipes and distillery practices in microbial community assembly and dynamics over the course of fermentation. This represents an important first step in appreciating the myriad roles of bacteria in the production of distilled spirits.