Microculture and fermentative phenotypic characterization
The microculture assay was performed in liquid media as previously
described (Nespolo et al. , 2020). Briefly, isolates were
pre-cultivated in 200 μl 0.67% YNB medium supplemented with glucose 2%
for 48 h at 25°C. Each pre-inoculum (optical density (OD) of 0.03–0.1)
from N. atacamensis isolates was inoculated in 200 μL of media
with the following carbon sources: glucose 2%, fructose 2% and sucrose
2% for 64 h incubated without agitation using a Tecan Sunrise
absorbance microplate reader. Additionally, we included environmental
stressors such as ethanol 4%, 6% and 8%, and glucose 20% during 64
h. The OD was measured every 30 minutes using a 630 nm filter. Each
experiment was carried out in triplicate. Maximum growth rate, lag time,
and OD max parameters were obtained for each isolate using the
GrowthRates software as previously described (Villarreal et al. ,
2022). All statistical analyses were performed using biological
replicates. One-way ANOVAs (Analysis of Variance) were performed using
GraphPad Prism 8.01 for Windows (GraphPad Software, La Jolla, CA, USA,www.graphpad.com).
Fermentations were carried out as previously described (Villarrealet al. , 2022). Briefly, for each experiment, yeast cells were
initially grown under constant agitation in 10 mL of SWM for 16 hours at
25°C. Next, 1x106 cells/mL were inoculated into 50 mL
SWM (in 250 mL flasks) and incubated at 25°C with constant agitation for
7 days. As wine fermentation control, we used the strain S.
cerevisiae EC1118 strain. The experiments were carried out in
triplicates. Micro-fermentations were weighed every day to calculate the
CO2 output. Sugar consumption and metabolite production
were estimated using HPLC and a Bio-Rad HPX-87H column. In this way, we
estimate the consumption of glucose and fructose, together with the
production of glycerol and ethanol.