Adaptation of Scheffersomyces stipitis to hardwood spent sulfite liquor by evolutionary engineering


Background: Hardwood spent sulfite liquor (HSSL) is a by-product of acid sulfite pulping process that is rich in xylose, a monosaccharide that can be fermented to ethanol by Scheffersomyces stipitis. However, HSSL also contains acetic acid and lignosulfonates that are inhibitory compounds of yeast growth. The main objective of this study was the use of an evolutionary engineering strategy to obtain variants of S. stipitis with increased tolerance to HSSL inhibitors while maintaining the ability to ferment xylose to ethanol. Results: A continuous reactor with gradually increasing HSSL concentrations, from 20% to 60% (v/v), was operated for 382 generations. From the final obtained population (POP), a stable clone (C-4) was isolated and characterized in 60% undetoxified HSSL. C-4 isolate was then compared with both the parental strain (PAR) and POP. Both POP and C-4 were able to grow in 60% undetoxified HSSL, with a higher capability to withstand HSSL inhibitors than PAR. Higher substrate uptake rates, 7% higher ethanol efficiency and improved ethanol yield were obtained using C-4. Conclusion: S. stipitis was successfully adapted to 60% (v/v) undetoxified eucalyptus HSSL. A stable isolate, C-4, with an improved performance in undetoxified HSSL compared to PAR was successfully obtained from POP. Owing to its improved tolerance to inhibitors, C-4 may represent a major advantage for the production of bioethanol using HSSL as substrate.



subject category

Biotechnology & Applied Microbiology; Energy & Fuels


Pereira, SR; Nogue, VSI; Frazao, CJR; Serafim, LS; Gorwa-Grauslund, MF; Xavier, AMRB

our authors


This work was partly developed within the scope of the project CICECO-Aveiro Institute of Materials (Ref. FCT UID/CTM/50011/2013), financed by national funds through the FCT/MEC and when appropriate co-financed by FEDER under the PT2020 Partnership Agreement. VSN was partly funded by the Swedish National Energy Administration (Energimyndigheten). Authors acknowledge Prof. Dmitry V. Evtyugin for helpful consultations regarding the HSSL and Eng. A. Prates from CAIMA - ALTRI Group, Industria de Celulose S.A., Constancia, Portugal for providing the HSSL. Authors also thank FCT for the PhD grant of S. R. Pereira (SFRH/BD/64552/2009).

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