Mutants of the pentose-fermenting yeast Pachysolen tannophilus tolerant to hardwood spent sulfite liquor and acetic acid

Abstract

A strain development program was initiated to improve the tolerance of the pentose-fermenting yeast Pachysolen tannophilus to inhibitors in lignocellulosic hydrolysates. Several rounds of UV mutagenesis followed by screening were used to select for mutants of P. tannophilus NRRL Y2460 with improved tolerance to hardwood spent sulfite liquor (HW SSL) and acetic acid in separate selection lines. The wild type (WT) strain grew in 50 % (v/v) HW SSL while third round HW SSL mutants (designated UHW301, UHW302 and UHW303) grew in 60 % (v/v) HW SSL, with two of these isolates (UHW302 and UHW303) being viable and growing, respectively, in 70 % (v/v) HW SSL. In defined liquid media containing acetic acid, the WT strain grew in 0.70 % (w/v) acetic acid, while third round acetic acid mutants (designated UAA301, UAA302 and UAA303) grew in 0.80 % (w/v) acetic acid, with one isolate (UAA302) growing in 0.90 % (w/v) acetic acid. Cross-tolerance of HW SSL-tolerant mutants to acetic acid and vice versa was observed with UHW303 able to grow in 0.90 % (w/v) acetic acid and UAA302 growing in 60 % (v/v) HW SSL. The UV-induced mutants retained the ability to ferment glucose and xylose to ethanol in defined media. These mutants of P. tannophilus are of considerable interest for bioconversion of the sugars in lignocellulosic hydrolysates to ethanol.

Adaptation of Saccharomyces cerevisiae in a concentrated spent sulphite liquor waste stream for increased inhibitor resistance

“…As adaptation requires a relationship or link between growth and the desired phenotype, under selective pressure, superior producers are advantaged (Winkler and Kao 2014). An improved growth phenotype, however, may not necessarily result in an improved fermentation phenotype, which makes it crucial that adapted strains maintain competitive fermentation ability and sugar utilisation phenotypes (Harner et al 2014). Therefore, adaptation experiments often reveal phenotypic trade-offs, as seen by the evolutionary engineering of S. cerevisiae to high-temperature tolerance (Mans et al 2018).…”

“…The SSL derived from softwood feedstocks is rich in hexoses such as glucose, whereas SSL derived from hardwood feedstocks is rich in pentoses such as xylose (Pereira et al 2013(Pereira et al , 2015. SSL is of particular interest as a potential feedstock for bioethanol production given that it (i) is low priced, (ii) has a global annual production capacity of 90 billion litres (Pereira et al 2013) and (iii) contains fermentable monomeric sugars that eliminate the need for extensive pre-treatment (Harner et al 2014;Branco et al 2019).…”

“…Currently, SSL is primarily used for chemical recovery as well as energy generation (Abdulkhani et al 2019;Humpert et al 2019), which is recycled back for use in the mill (Pereira et al 2015;Branco et al 2019). Alternatively, these waste streams are (i) sold for lignosulphonate recovery (Petersen et al 2014), (ii) disposed of (restricted as high oxygen demands limit surface water discharge (Harner et al 2014;Branco et al 2019;Xing et al 2019)) and/or (iii) fermented before initiating the chemical recovery step (Sinner et al 2021). Hardwood-derived SSL is particularly underutilised as a potential fermentation feedstock.…”

“…SSL obtained from hardwoods pulping composes of a significant portion of hemicellulose derived pentose sugars such as xylose (Branco et al 2019;Villain-Gambier et al 2020), which cannot naturally be fermented by S. cerevisiae (Harner et al 2014;Cunha et al 2019a). Additionally, microbial inhibitory compounds derived from lignin and sugar degradation such as phenolics, furans, and weak acids are released during the chemical pulping process (Pereira et al 2015;He and Chen 2020), which severely impacts the yeasts biocatalyst ability for economical bioconversion of sugars to ethanol (Bhatia et al 2020;Vees et al 2020).…”

“…Microbial inhibitors have also been shown to negatively impact heterologous xylose metabolism in S. cerevisiae (Deparis et al 2017), with different effects depending on the pathway(s) being expressed (Cunha et al 2019b) and the xylose isomerase (XI) pathway being particularly vulnerable (Xie et al 2020). In addition, the concentrated thick liquor has high viscosity, osmolality, biological oxygen demand and low pH (Pereira et al 2013;Harner et al 2014). SSL also contains microbial inhibitors beyond the typical furans, weak acids and phenolics, such as lignosulphonates, extractives, SO 2 and MgO (He and Chen 2020), that are also concentrated during the evaporation-concentration process.…”