These other stressors may also be why cells incubated in Cauldron were small (Table 3) and unbudded when observed by
microscopy (Supplemental Fig. 1C) relative to cells incubated in
YPD-pH3 medium (Supplemental Fig. 1G). The low-oxygen, lownutrient, high-EtOH, acidic, complex chemical makeup of Cauldron
cannot be completely duplicated in the lab to determine the relative effects of individual stressors on the yeast. Thankfully, simply
mixing Cauldron with rich medium was enough to both maintain
active metabolism by the yeast and “prime” them for bottle conditioning under the harsher 100% Cauldron conditions (Fig. 3C,D).
We hypothesize that diluting the Cauldron stressors by half in
YPD-Cauldron medium, as well as growing the cells with aeration
and reintroducing nutrients, is sufficient to generate a population
of healthy cells that have adapted to tolerate the bioactive chemicals in Cauldron. Similarly, adapting the cells to a significant
Cauldron stressor (e.g., lactic acid) by growing them in YPD-pH3
medium also yielded cells capable of bottle conditioning the beer.
These cells were not completely adapted to the Cauldron milieu as
they took twice as long to bottle condition the beer as cells grown
in YPD-Cauldron medium, but our hypothesis remains the same.
Thus, pre-adapting yeast in a mixture of rich medium and beer may
be a general method to ensure proper bottle conditioning of any
beer.