| Summary: | The effect of temperature (32–50 °C) on bacterial dynamics and taxonomic structure was evaluated during spontaneous whey fermentation for lactic acid production. Bacterial plate count in fresh whey (5 log CFU/mL) increased in two orders of magnitude after 60 h of fermentation (7 log CFU/mL), followed by one log reduction after 120 h (6 log CFU/mL) at 37 and 42 °C. <i>Streptococcus</i> and <i>Lactobacillus</i> counts ranged between 5–9 and 5–8 log CFU/mL, respectively. High-throughput sequencing of the 16S rRNA gene (V3-V4 region) used as a taxonomic marker revealed thirteen different bacterial phyla. <i>Actinobacteria</i>, <i>Bacteroidetes</i>, <i>Firmicutes</i>, and <i>Proteobacteria</i> were detected in all fermentation treatments (32–50 °C, 0–120 h), where <i>Firmicutes</i> was the predominant phylum. Bacterial diversity included more than 150 bacterial genera with predominant lactic acid bacteria (belonging to <i>Firmicutes</i>) such as <i>Lactobacillus</i>, <i>Lactococcus</i>, <i>Streptococcus</i>, and <i>Tetragenococcus</i>. At the species level, fresh whey presented 61 predominant species (relative abundance > 0.05%); however, only 57.4% of these resisted the fermentation conditions (most of them belonging to lactic acid bacteria genera). <i>Tetragenococcus halophilus</i>, <i>Lactococcus lactis</i>, and <i>Enterococcus casseliflavus</i> were the predominant bacteria found in all treatments. Temperatures between 37–42 °C were more favorable for lactic acid production and could be considered appropriate conditions for fermented whey production and for the standardization of some artisanal cheese-making processes requiring acid whey addition for milk coagulation. The diversity of native beneficial bacteria found in fresh whey offers attractive technological characteristics, and their fermentative capacity would represent a biotechnological option to add value to cheese whey.
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