Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain

Abstract

The production of fuel ethanol from low-cost lignocellulosic biomass currently suffers from several limitations. One of them is the presence of inhibitors in lignocellulosic hydrolysates that are released during pre-treatment. These compounds inhibit growth and hamper the production of ethanol, thereby affecting process economics. To delineate the effects of such complex mixtures, we conducted a chemical analysis of four different real-world lignocellulosic hydrolysates and determined their toxicological effect on yeast. By correlating the potential inhibitor abundance to the growth-inhibiting properties of the corresponding hydrolysates, we identified furfural as an important contributor to hydrolysate toxicity for yeast. Subsequently, we conducted a targeted evolution experiment to improve growth behaviour of the half industrial Saccharomyces cerevisiae strain TMB3400 in the hydrolysates. After about 300 generations, representative clones from these evolved populations exhibited significantly reduced lag phases in medium containing the single inhibitor furfural, but also in hydrolysate-supplemented medium. Furthermore, these strains were able to grow at concentrations of hydrolysates that effectively killed the parental strain and exhibited significantly improved bioconversion characteristics under industrially relevant conditions. The improved resistance of our evolved strains was based on their capacity to remain viable in a toxic environment during the prolonged, furfural induced lag phase.

Figure 1

GC‐TOF‐MS identified compounds in the four hydrolysates. *Compounds only found in wheat and barley straw hydrolysate.

Figure 2

Inhibitory effect of 5‐hydroxy‐methylfurfural (▴), vanillin (♦) and furfural (●) on the growth rate of TMB3400 in aerobic glucose minimal medium.

Figure 3

Correlation between EC₅₀ and single inhibitor concentration in different lignocellulosic hydrolysates. HMF, 5‐hydroxy‐methylfurfural.

Figure 4

Growth of evolved populations in aerobic minimal medium containing 17 mM furfural.

Figure 5

Growth of TMB3400 (●) and the evolved clone TMB3400‐FT30‐3 (○) in anaerobic glucose minimal medium containing 40% (v/v) spruce hydrolysate.

Figure 6

Time‐courses of cell growth (A), furfural (B) and furfuryl alcohol concentrations in cultures of TMB3400 (●), TMB3400‐FT30‐3 (○) and without the addition of cells (▪) (evaporation) in aerobic minimal medium containing 17 mM furfural.

Figure 7

Colony‐forming unit counts (triangles) and furfural concentration (bullets) of TMB3400 (closed symbols) and TMB3400‐FT30‐3 (open symbols) in aerobic glucose minimal medium with 17 mM furfural.

Figure 8

Time‐courses of glucose (bullets) and ethanol (triangles) concentrations during an anaerobic bioconversion in 80% v/v barley straw hydrolysate with TMB3400 (closed symbol) and TMB3400‐FT30‐3 (open symbols) with 5 g_cdw l⁻¹ initial biomass.