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Keywords:

  • ethanolic fermentation;
  • inhibitors;
  • interaction effects;
  • experimental design;
  • yeast

Abstract

The influence of the factors acetic acid, furfural, and p-hydroxybenzoic acid on the ethanol yield (YEtOH) of Saccharomyces cerevisiae, bakers' yeast, S. cerevisiae ATCC 96581, and Candida shehatae NJ 23 was investigated using a 23-full factorial design with 3 centrepoints. The results indicated that acetic acid inhibited the fermentation by C. shehatae NJ 23 markedly more than by bakers' yeast, whereas no significant difference in tolerance towards the compounds was detected between the S. cerevisiae strains. Furfural (2 g L−1) and the lignin derived compound p-hydroxybenzoic acid (2 g L−1) did not affect any of the yeasts at the cell mass concentration used. The results indicated that the linear model was not adequate to describe the experimental data (the p-values of curvatures were 0.048 for NJ 23 and 0.091 for bakers' yeast). Based on the results from the 23-full factorial experiment, an extended experiment was designed based on a central composite design to investigate the influence of the factors on the specific growth rate (μ), biomass yield (Yx), volumetric ethanol productivity (QEtOH), and YEtOH. Bakers' yeast was chosen in the extended experiment due to its better tolerance towards acetic acid, which makes it a more interesting organism for use in industrial fermentations of lignocellulosic hydrolysates. The inoculum size was reduced in the extended experiment to reduce any increase in inhibitor tolerance that might be due to a large cell inoculum. By dividing the experiment in blocks containing fermentations performed with the same inoculum preparation on the same day, much of the anticipated systematic variation between the experiments was separated from the experimental error. The results of the fitted model can be summarised as follows: μ was decreased by furfural (0–3 g L−1). Furfural and acetic acid (0–10 g L−1) also interacted negatively on μ. Furfural concentrations up to 2 g L−1 stimulated Yx in the absence of acetic acid whereas higher concentrations decreased Yx. The two compounds interacted negatively on Yx and YEtOH. Acetic acid concentrations up to 9 g L−1 stimulated QEtOH, whereas furfural (0–3 g L−1) decreased QEtOH. Acetic acid in concentrations up to 10 g L−1 stimulated YEtOH in the absence of furfural, and furfural (0–2 g L−1) slightly increased YEtOH in the absence of acetic acid whereas higher concentrations caused inhibition. Acetic acid and furfural interacted negatively on YEtOH. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 46–55, 1999.