Identification of the major fermentation inhibitors of recombinant 2G yeasts in diverse lignocellulose hydrolysates

Abstract

Background Presence of inhibitory chemicals in lignocellulose hydrolysates is a major hurdle for production of second-generation bioethanol. Especially cheaper pre-treatment methods that ensure an economical viable production process generate high levels of these inhibitory chemicals. The effect of several of these inhibitors has been extensively studied with non-xylose-fermenting laboratory strains, in synthetic media, and usually as single inhibitors, or with inhibitor concentrations much higher than those found in lignocellulose hydrolysates. However, the relevance of individual inhibitors in inhibitor-rich lignocellulose hydrolysates has remained unclear. Results The relative importance for inhibition of ethanol fermentation by two industrial second-generation yeast strains in five lignocellulose hydrolysates, from bagasse, corn cobs and spruce, has now been investigated by spiking higher concentrations of each compound in a concentration range relevant for industrial hydrolysates. The strongest inhibition was observed with industrially relevant concentrations of furfural causing partial inhibition of both D-glucose and D-xylose consumption. Addition of 3 or 6 g/L furfural strongly reduced the ethanol titer obtained with strain MD4 in all hydrolysates evaluated, in a range of 34 to 51% and of 77 to 86%, respectively. This was followed by 5-hydroxymethylfurfural, acetic acid and formic acid, for which in general, industrially relevant concentrations caused partial inhibition of D-xylose fermentation. On the other hand, spiking with levulinic acid, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid or vanillin caused little inhibition compared to unspiked hydrolysate. The further evolved MD4 strain generally showed superior performance compared to the previously developed strain GSE16-T18. Conclusion The results highlight the importance of individual inhibitor evaluation in a medium containing a genuine mix of inhibitors as well as the ethanol that is produced by the fermentation. They also highlight the potential of increasing yeast inhibitor tolerance for improving industrial process economics.

Saccharomyces cerevisiae Cells Lacking the Zinc Vacuolar Transporter Zrt3 Display Improved Ethanol Productivity in Lignocellulosic Hydrolysates

“…It is well established that AA originates from acetyl-groups of hemicellulose during the pre-treatment that lignocellulosic biomass undergoes to release fermentable sugars [17,62]. Depending on the biomass and hydrolytic process used, AA can be found in concentrations between 8 and 192 mM (0.5 and 11.5 g/L) [63]. Here, the zrt3∆ mutant stood out when compared with the wild-type regarding both cell growth and fermentative performance under 60 mM (3.6 g/L) AA stress, with a 2.6-fold improvement in ethanol productivity.…”

Cell Immobilization Using Alginate-Based Beads as a Protective Technique against Stressful Conditions of Hydrolysates for 2G Ethanol Production

“…Acetic acid (HAc) is a well-known inhibitor of S. cerevisiae, and its production during biomass pretreatment for 2G ethanol production is inevitable due to deacetylation of the hemicellulose fraction [12]. It strongly affects T18 free cell metabolism, reducing the xylose fermentation rate by 10-fold in the presence of 8 g/L of acetic acid [15].…”

“…To overcome this challenge, it is necessary to develop strains capable of fermenting xylose efficiently, which will increase ethanol production from biomass without increasing the cultivation area [9]. In this sense, with advances in molecular biology and metabolic engineering, recombinant strains with superior C5 sugar assimilation have been developed [10][11][12]. This is an essential requirement to circumvent the underutilization of the hemicellulose biomass fraction and a prerequisite for feasible bioeconomic production, so that bioethanol generation can also become based on xylose.…”

Utilization of Bioetanol Fermentation Waste Pineapple and Coconut Water as Disinfectants With Bacteria Saccharomyces Cerevisiae

“…Presence of inhibitory chemicals in lignocellulose hydrolysates is a major hurdle for production of second-generation bioethanol. Especially cheaper pre-treatment methods that ensure an economical viable production process generate high levels of these inhibitory chemicals (Vanmarcke, Demeke, Foulquié-Moreno, & Thevelein, 2021).…”