Enhanced ethanol fermentation by thermotolerant Pichia kudriavzevii LC671435 in the presence of major lignocellulosic inhibitors.

Lignocellulosic biomass is a sustainable feedstock for bioethanol production, but its industrial utilization is hindered by fermentation inhibitors generated during biomass pretreatment, including furfural, acetic acid, and formic acid. Developing robust thermotolerant yeasts capable of maintaining high ethanol productivity under these inhibitory conditions is essential for economically viable bioprocesses. This study evaluated the growth, viability, and fermentative performance of the thermotolerant yeast Pichia kudriavzevii LC671435 in the presence of major lignocellulosic inhibitors at 42 °C. The strain exhibited remarkable tolerance, remaining viable at concentrations of up to 35 mM furfural, 100 mM acetic acid, and 50 mM formic acid. Under these conditions, ethanol production reached 40.4 g/L (79.2% theoretical yield) in the presence of 35 mM furfural, 41.2 g/L (80.8% theoretical yield) with 80 mM acetic acid, and 39.3 g/L (77.1% theoretical yield) with 50 mM formic acid. Ethanol productivity remained above 2.0 g/L/h during the first 16 h of fermentation across all inhibitor treatments. Although combined inhibitor cocktails reduced fermentation efficiency, the strain consistently maintained ethanol production and retained fermentative activity over four consecutive repeated-batch fermentations although ethanol production progressively declined. These findings demonstrate that P. kudriavzevii LC671435 combines thermotolerance with broad inhibitor tolerance, enabling efficient ethanol production under conditions relevant to lignocellulosic biorefineries. This unique multi-stress-tolerant feature of P. kudriavzevii LC671435 makes it a potential strain for second-generation bioethanol production as well as positions it as a suitable candidate for investigating the molecular basis underlying the robust stress tolerance.