Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-basedSaccharomyces cerevisiaestrain

Abstract

Acetic acid, an inhibitor released during hydrolysis of lignocellulosic feedstocks, has previously been shown to negatively affect the kinetics and stoichiometry of sugar fermentation by (engineered) Saccharomyces cerevisiae strains. This study investigates the effects of acetic acid on S. cerevisiae RWB 218, an engineered xylose-fermenting strain based on the Piromyces XylA (xylose isomerase) gene. Anaerobic batch cultures on synthetic medium supplemented with glucose-xylose mixtures were grown at pH 5 and 3.5, with and without addition of 3 g L(-1) acetic acid. In these cultures, consumption of the sugar mixtures followed a diauxic pattern. At pH 5, acetic acid addition caused increased glucose consumption rates, whereas specific xylose consumption rates were not significantly affected. In contrast, at pH 3.5 acetic acid had a strong and specific negative impact on xylose consumption rates, which, after glucose depletion, slowed down dramatically, leaving 50% of the xylose unused after 48 h of fermentation. Xylitol production was absent (<0.10 g L(-1)) in all cultures. Xylose fermentation in acetic -acid-stressed cultures at pH 3.5 could be restored by applying a continuous, limiting glucose feed, consistent with a key role of ATP regeneration in acetic acid tolerance.

Elimination of Glycerol Production in Anaerobic Cultures of a Saccharomyces cerevisiae Strain Engineered To Use Acetic Acid as an Electron Acceptor

“…1 and Table 2). This effect has been attributed to the higher rate of glucose dissimilation for intracellular pH homeostasis due to diffusion of acetic acid into the cell, which in turn results in a lower biomass yield on glucose (7). Under the same conditions, an isogenic gpd1⌬ gpd2⌬ strain, in which an absence of NAD ϩ -dependent glycerol-3-phosphate dehydrogenase activity was confirmed in cell extracts (Table 2), was completely unable to grow anaerobically (data not shown), consistent with the notion that glycerol production via Gpd1 and Gpd2 is essential for NADH reoxidation in anaerobic cultures of S. cerevisiae (8).…”

“…In addition to reducing the organic carbon content of spent media and increasing the ethanol yield, the reduction of acetic acid to ethanol may at least partially alleviate acetate inhibition of yeast growth and metabolism, which is especially problematic at low pH and during the consumption of pentose sugars by engineered yeast strains (7). However, before industrial implementation can be contemplated, several issues remain to be addressed.…”

“…Significant amounts of acetic acid are released upon hydrolysis of lignocellulosic biomass, and, in fact, acetic acid is studied as an inhibitor of yeast metabolism in lignocellulosic hydrolysates (5,7,26). The S. cerevisiae genome already contains genes encoding acetyl coenzyme A (acetyl-CoA) synthetase (32) and NAD ϩ -dependent alcohol dehydrogenases (ADH1-5 [12]).…”

Enhanced biofuel production through coupled acetic acid and xylose consumption by engineered yeast

“…Further, hemicellulose and lignin in the plant cell wall are acetylated 8 , yielding acetic acid as an unavoidable component in cellulosic hydrolyzates with acetic acid concentrations ranging from 1 to 15 g l À 1 (refs 8,9). Acetic acid is toxic to fermenting microorganisms and negatively influences sugar fermentation and biofuel yields 8,[10][11][12] . Xylose conversion and acetic acid detoxification are two major problems that must be solved to make cellulosic biofuels economically viable.…”

Can Microbially Derived Advanced Biofuels Ever Compete with Conventional Bioethanol? A Critical Review

“…Construction of a genetically engineered strain to consume multiple sugars has been shown to compromise product tolerance and yield as well as the ability to tolerate inhibitors (Bellissimi et al 2009;Clomburg and Gonzalez 2010;Vickers et al 2012). Wisselink and coworkers (2009) attempted to evolve the engineered S. cerevisiae on the pentose sugars without losing their performance on hexose sugars.…”