Isolation and Characterization of Resilient Thermotolerant Yeasts from Animal Manure for 2G Bioethanol Production from Sugarcane Bagasse Hydrolysate

The economic viability of second-generation (2G) bioethanol production depends on the availability of robust, multistress-tolerant yeast strains capable of withstanding harsh industrial conditions. This study investigates animal manure as a novel ecological niche for discovering such strains, as microbes in these environments naturally adapt to high organic loading and fluctuating temperatures. From eighty-six initial isolates, twenty-nine demonstrated superior xylose fermentation at 37 °C. Eight high-performing isolates (C2-1, B1-2, B1-6, B2-6, B2-8, G1-4, G1-5, and G2-4) exhibited exceptional tolerance to ethanol, high temperatures, and lignocellulosic-derived inhibitors (acetic acid, formic acid, furfural, and vanillic acid). Molecular identification classified isolate C2-1 as Pichia kudriavzevii and the remaining seven as Candida tropicalis. In synthetic media, C. tropicalis B2-8 produced up to 16.33 g/L of ethanol using xylose (60 g/L) as the sole carbon source. While the undetoxified, highly acidic sugarcane bagasse hydrolysate completely inhibited yeast growth, the industrial potential of these strains was successfully validated using the concentrated, undetoxified enzymatic hydrolysate derived from the acid-pretreated sugarcane bagasse solids, which contained 30.15 g/L glucose and 25.58 g/L xylose. P. kudriavzevii C2-1 achieved ethanol titers of 6.02 g/L and 5.71 g/L at 37 °C and 40 °C, respectively. The C. tropicalis strains outperformed P. kudriavzevii, yielding 6.12–6.35 g/L at 37 °C and maintaining 5.75–6.19 g/L at 40 °C. These findings underscore the potential of manure-derived yeasts as resilient biocatalysts. Although their fermentation yields remain relatively low and require further metabolic optimization, their ability to survive and ferment in this concentrated, undetoxified enzymatic hydrolysate at elevated temperatures makes them promising candidates for further development in high-temperature ethanol fermentation (HTEF), offering a potential pathway toward reducing cooling costs associated with 2G biorefineries.