Prediction of Weak Acid Toxicity in Saccharomyces cerevisiae Using Genome-Scale Metabolic Models Patrick B Hyland, 1 Serene Lock-Sow Mun, 1,2 and Radhakrishnan Mahadevan 1 1 Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada 2 CO 2 Management Mission Oriented Research, Universiti Teknologi PETRONAS, Tronoh, Malaysia Abstract The use of lignocellulosic biomass is critical for the economic production of transportation fuels and chemicals in renewable bioprocesses. While biomass is an abundant resource, neces- sary pretreatment to yield fermentable monosaccharides pro- duces toxic compounds that dramatically affect fermentation performance. Weak acids such as acetic acid play an important role in the toxicity of lignocellulosic hydrolysate to Sacchar- omyces cerevisiae, a commonly used industrial organism. In order to explore the ramifications of weak acid inhibition on cellular metabolism, we adapted a genome-scale metabolic model of S. cerevisiae to describe toxicity of acetic acid by a decoupling mechanism. We evaluated the performance of the model in predicting growth rates and ethanol production characteristics under aerobic and anaerobic cultivations. We found that the model was able to capture the decreased growth during aerobic cultivations in the presence of acetic acid, but was unable to capture the increase in ethanol yield observed. The model was able to predict anaerobic growth rates and ethanol yields; however, at conditions of higher toxicity levels, discrepancies arose. We expect that a model such as this may find application in the optimization of lignocellulose-based bioprocesses in which there exists a critical economic trade-off between neutralization costs and product yields. Introduction I n recent years, renewable means of producing transpor- tation fuel and chemicals have been heavily investigated in order to reduce reliance on natural oil resources. It has been previously noted that as a result of increasing global rate of oil consumption and decreasing rate at which new fossil fuel resources are discovered, alternative sources of transpor- tation fuels and chemicals are highly sought after. 1 Bioprocesses offer an attractive alternative to petroleum-based processes by potentially converting renewable biomass feedstock to drop-in replacements for petroleum-derived chemicals. In particular, bioprocesses that use lignocellulosic biomass, an abundant and renewable resource, as feedstock are among the current areas of focus in the field of biotechnology. Lignocellulose is composed of cellulose, hemicellulose, and lignin. Cellulose and hemicellulose are long-chain polymers of saccharides that must undergo enzymatic treatment to release fermentable monomers. This process is complicated by the presence of lignin—a complex aromatic polymer—that makes it necessary for the biomass to undergo a pretreatment such as weak acid hydrolysis prior to enzymatic treatment. 2 A signifi- cant limitation of pretreatment processes is that inhibitory compounds are produced that significantly impact fermentation characteristics. Inhibitory compounds can be classified as phe- nolics, furan derivatives, or weak acids, and mechanisms of these inhibitor classes have been reviewed extensively. 2–4 Weak acid toxicity is a particularly interesting physical phe- nomenon arising from the interaction of internal and external pH and cellular response mechanisms. The toxicity of weak acids has largely been attributed to the acidification of the cytosol by decoupling and anion accumulation. 3,5 The decoupling mecha- nism of weak acid toxicity is a result of the ability of non-polar undissociated weak acid species to diffuse across the cellular membrane to the near-neutral pH cytosol. Upon entering the cytosol, the acid dissociates, yielding a proton and the conjugate base. To maintain neutral cytosolic pH, the cell uses the reverse action of adenosine triphosphate (ATP) synthase, exporting protons at the expense of ATP. In addition, the anion accumu- lation theory proposed by Russell states that the anionic form of the acid species may accumulate in the cytosol and drive the diffusion of undissociated acid across the cell membrane to- wards equilibrium. 3,5 A significant weak acid inhibitor is acetic acid, which is formed largely from the degradation of hemicellulose and is present in hydrolysates in concentrations up to 10 g/L, depend- ing on the biomass source. 6 While many studies have focused on strain engineering for tolerance to furan derivatives and phe- nolics, there are significantly fewer that focus on improving tolerance to weak acids. 6–8 Notably, Hasunuma et al. demon- strated that a recombinant xylose-utilizing strain of Saccharo- myces cerevisiae over-expressing the TAL1 gene had improved ethanol yield in the presence of acetic acid. 6 Recently, an acetic- acid-tolerant strain of S. cerevisiae was generated using a genome shuffling method. 9 However, hypothesis discovery remains a bottleneck in rational strain engineering for tolerance to weak acids. Mathematical modeling is a powerful tool for examining the metabolism of microorganisms. Previous studies have modeled DOI: 10.1089/ind.2013.0004 ª MARY ANN LIEBERT, INC.  VOL. 9 NO. 4  AUGUST 2013 INDUSTRIAL BIOTECHNOLOGY 229