Mutants of the pentose‐fermenting yeast Pichia stipitis with improved tolerance to inhibitors in hardwood spent sulfite liquor

Abstract

Mutants of Pichia stipitis NRRL Y-7124 able to tolerate and produce ethanol from hardwood spent sulfite liquor (HW SSL) were obtained by UV mutagenesis. P. stipitis cells were subjected to three successive rounds of UV mutagenesis, each followed by screening first on HW SSL gradient plates and then in diluted liquid HW SSL. Six third generation mutants with greater tolerance to HW SSL as compared to the wild type (WT) were isolated. The WT strain could not grow in HW SSL unless it was diluted to 65% (v/v). In contrast, the third generation mutants were able to grow in HW SSL diluted to 75% (v/v). Mutants PS301 and PS302 survived even in 80% (v/v) HW SSL, although there was no increase in cell number. All the third generation mutants exhibited higher growth rates but significantly lower growth yields on xylose or glucose compared to the WT. The mutants fermented 4% (w/v) glucose as efficiently as the WT and fermented 4% (w/v) xylose more efficiently with a higher ethanol yield than the WT. In a medium containing 4% (w/v) each of xylose and glucose, all the third generation mutants utilized glucose as efficiently and xylose more efficiently than the WT. This resulted in higher ethanol yield by the mutants. The mutants retained the ability to utilize galactose and mannose and ferment them to ethanol. Arabinose was consumed slowly by both the mutants and WT with no ethanol production. In 60% (v/v) HW SSL, the mutants utilized and fermented glucose, mannose, galactose and xylose while the WT could not ferment any of these sugars.

Repression of xylose‐specific enzymes by ethanol inScheffersomyces(Pichia)stipitisand utility of repitching xylose‐grown populations to eliminate diauxic lag

“…For major xylose‐fermenting strains, such as S. stipitis and P. tannophilus , future research is needed to address the management of a competent cell population through many cycles of hydrolyzate fermentation. Optimization of nutrient and culture environment (Slininger et al, 2006, 2009), as well as selective culture enrichment approaches to improve yeast survival on inhibitory hydrolyzates (Bajwa et al, 2009), have shown promise and warrant further investigation. Preservation of positive xylose utilization properties during a strain selection process involving recycle in hydrolyzate would need to be evaluated in progeny to ensure that progress is made toward more competent hydrolyzate fermenting strains that perform well under more economical conditions—such as warmer fermentation temperatures in the 30–37°C range and no or low oxygen delivery.…”

Saccharomyces cerevisiae Genome Shuffling through Recursive Population Mating Leads to Improved Tolerance to Spent Sulfite Liquor

“…For the creation of agar plates with a growth inhibition gradient, undiluted HWSSL agar (2%, wt/vol) was overlaid with inhibitor-free minimal medium to establish a gradient from higher to lower HWSSL concentrations (6). The HWSSL used for all experiments was kindly supplied by Tembec Inc. and was adjusted to pH 5.5 with 10 M NaOH before use.…”

Evaluation of a kinetic model for computer simulation of growth and fermentation by Scheffersomyces (Pichia) stipitis fed D‐xylose

“…However, there is still strong interest in applying S. stipitis to whole hydrolyzates of lignocellulosic biomass since it has been considered the native pentose‐fermenting yeast with most promise for commercial application (Agbogbo et al, , 2008; Agbogbo and Wenger, ; Lin et al, ). Advancements are being made toward application of S. stipitis to lignocellulosic hydrolyzate fermentation, including strain improvement (Bajwa et al, , ; Caspeta and Nielsen, ; Hughes et al, ; Jeffries et al, ; Liu et al, , , ; Slininger et al, ), new chemical and enzyme hydrolysis technologies (Balan et al, ; Chundawat et al, ), and new understandings of nutritional requirements for hydrolyzate fermentation (Slininger et al, ; Wang et al, ). For the future, the model represents a good basic framework for addition of mathematical expressions to accommodate the prediction of fermentation of mixed sugars, particularly glucose and xylose, and the impacts of limiting or inhibitory factors in a hydrolyzate environment.…”