Development of a phenotypic assay for characterisation of ethanologenic yeast strain sensitivity to inhibitors released from lignocellulosic feedstocks

Abstract

Inhibitors released by the breakdown of plant cell walls prevent efficient conversion of sugar into ethanol. The aim of this study was to develop a fast and reliable inhibitor sensitivity assay for ethanologenic yeast strains. The assay comprised bespoke 96-well plates containing inhibitors in isolation or combination in a format that was compatible with the Phenotypic Microarray Omnilog reader (Biolog, hayward, CA, USA). A redox reporter within the assay permits analysis of inhibitor sensitivity in aerobic and/or anaerobic conditions. Results from the assay were verified using growth on spot plates and tolerance assays in which maintenance of viability was assessed. The assay allows for individual and synergistic effects of inhibitors to be determined. It was observed that the presence of both acetic and formic acid significantly inhibited the yeast strains assessed, although this impact could be partially mitigated by buffering to neutral pH. Scheffersomyces stipitis, Candida spp., and Pichia guilliermondii demonstrated increased sensitivity to short chain weak acids at concentrations typically present in lignocellulosic hydrolysates. S. cerevisiae exhibited robustness to short chain weak acids at these concentrations. However, S. stipitis, Candida spp., and P. guilliermondii displayed increased tolerance to HMF when compared to that observed for S. cerevisiae. The results demonstrate that the phenotypic microarray assay developed in the current study is a valuable tool that can be used to identify yeast strains with desirable resistance to inhibitory compounds found in lignocellulosic hydrolysates.

Acetic Acid Causes Endoplasmic Reticulum Stress and Induces the Unfolded Protein Response in Saccharomyces cerevisiae

“…According to the types of biomass and pretreatment methods, levels of acetic acid often reach very high in lignocellulosic hydrolysates (Klinke et al, 2004; Nakata et al, 2006; Almeida et al, 2007). Additionally, contamination with lactic acid bacteria and acetic acid bacteria increases the concentration of acetic acid in open fermenters (Beckner et al, 2011).…”

“…Since hemicellulose and lignin are highly acetylated, acetic acid is released during the pretreatment of the lignocellulosic biomass prior to bioethanol production and remains in hydrolysates (Palmqvist and Hahn-Hägerdal, 2000; van Maris et al, 2006; Vilela-Moura et al, 2011; Koppram et al, 2014). Although the concentration of acetic acid in the lignocellulosic hydrolysates varies widely according to the types of treatment methods and biomass used, a wide range of acetic acid concentrations (0 – 178 mM) was reported in previous studies (Klinke et al, 2004; Nakata et al, 2006; Almeida et al, 2007). Additionally, acetic acid is easily produced in open fermenters due to contamination by lactic acid bacteria and acetic acid bacteria.…”

Acetylcholine Protects against Candida albicans Infection by Inhibiting Biofilm Formation and Promoting Hemocyte Function in a Galleria mellonella Infection Model

“…Biofilm biomass of C. albicans cultured in RPMI for 24 h was quantified using the crystal violet assay, as previously described (18). Real-time cellular metabolic activity (respiration) was evaluated using phenotypic microarray (PM) analysis as previously described (19). Briefly, a suspension of C. albicans was adjusted to a transmittance of 62% (ϳ5 ϫ 10 6 cells · ml Ϫ1 ) using a turbidimeter.…”

Evaluation of different lignocellulosic biomass pretreatments by phenotypic microarray-based metabolic analysis of fermenting yeast

“…Furfural and HMF have been shown to be derived from degradation of sugar molecules when under acid catalysed or high temperature hydrolysis (Taherzadeh and Karimi, 2008). Previous studies have demonstrated that 20 mM furfural inhibits S. cerevisiae (Park et al, 2011;Greetham et al, 2014). This would suggest that the concentrations detected in these hydrolysates would not be significantly problematic for S. cerevisiae.…”