Inhibitor tolerance and flocculation of a yeast strain suitable for second generation bioethanol production

Abstract

Background: Robust second generation bioethanol processes require microorganisms able to ferment inhibitory lignocellullosic hydrolysates. In this study, the inhibitor tolerance and flocculation characteristics of Saccharomyces cerevisiae CCUG53310 were evaluated in comparison with S. cerevisiae CBS8066. Results: The flocculating strain CCUG53310 could rapidly ferment all hexoses in dilute acid spruce hydrolysate, while CBS8066 was strongly inhibited in this medium. In synthetic inhibitory media, CCUG53310 was more tolerant to carboxylic acids and furan aldehydes, but more sensitive than CBS8066 to phenolic compounds. Despite the higher tolerance, the increase in expression of the YAP1, ATR1 and FLR1 genes, known to confer resistance to lignocellulose-derived inhibitors, was generally smaller in CCUG53310 than in CBS8066 in inhibitory media. The flocculation of CCUG53310 was linked to the expression of FLO8, FLO10 and one or more of FLO1, FLO5 or FLO9. Flocculation depended on cell wall proteins and Ca 2+ ions, but was almost unaffected by other compounds and pH values typical for lignocellulosic media. Conclusions: S. cerevisiae CCUG53310 can be characterised as being very robust, with great potential for industrial fermentation of lignocellulosic hydrolysates relatively low in phenolic inhibitors.

Encapsulation-Induced Stress Helps Saccharomyces cerevisiae Resist Convertible Lignocellulose Derived Inhibitors

“…We have previously shown that S. cerevisiae CBS8066 was strongly inhibited by both furan aldehydes and carboxylic acids at the same concentrations in the medium as used in the current study, as well as by a dilute acid spruce hydrolysate [14]. The rate of consumption of the first 12 g/L glucose in the media containing carboxylic acid or furan aldehydes was roughly 40% of the rate obtained in the non-inhibitory medium.…”

Flocculation Causes Inhibitor Tolerance in Saccharomyces cerevisiae for Second-Generation Bioethanol Production

“…Flocculation trials. The flocculation trials were performed with stationary-phase yeast cells harvested 48 h after inoculation in YPD medium according to a previously reported protocol (26), with slight modifications. In short, EDTA-deflocculated cells were heat killed and mixed at a concentration of ϳ10 8 cells/ml in citrate buffer (50 mM, pH 4.5) containing 4 mM CaCl 2 and various concentrations of the sugars tested in a total volume of 2 ml placed in a 12-ml round-bottom tube.…”

“…The hydrophobicity of cells was tested by the MATH assay according to the method of Westman et al (26). The hydrophobicity is reported as the relative difference between the absorbance of the aqueous phase before and after vortexing with octane and was calculated as follows: hydrophobicity ϭ [1 Ϫ (OD 600 after vortexing/OD 600 before vortexing)] ϫ 100.…”

“…Batch cultivations with inhibitors. Due to the inherent difficulties of cultivating a flocculating yeast strain reproducibly (growth cannot be monitored by OD 600 measurements or withdrawal of samples for dry weight determination), a previously developed cultivation method was used (26) in order to avoid deflocculation of the cells, e.g., with EDTA. Hence, the entire precultivations were used as inocula for the batch cultivations in order to get reproducible data.…”

“…The latter is the case for the recently sequenced S. cerevisiae CEN.PK 113-7D, where the major flocculation genes FLO1, FLO5, and FLO9 are missing (23). However, naturally flocculating strains have been shown to be good at fermenting inhibitory hydrolysates (24)(25)(26), and flocculation has also been shown to increase the ethanol tolerance of S. cerevisiae (27,28). Therefore, it is plausible that flocculation can indeed be a beneficial trait in second-generation bioethanol production.…”