Cellulosic ethanol might serve as a sustainable biofuel that could replace gasoline use as a transportation fuel [1, 2], and it can be generated from a variety of cellulosic biomass types, such as wood . One challenge that is particularly acute with woody biomass, such as pine, is that the pretreatment process releases a number of compounds that are inhibitory to the growth and/or metabolic activity of the fermenting organism . These chemicals act through a variety of mechanisms to reduce ethanol production efficiency, including inhibition of cell growth, reduction of cell metabolic activity, or inhibition of enzymatic activity. Thus, it is important to use a fermenting organism that is able to tolerate these compounds, especially at the high solids loadings required for industrial fermentations to produce the ethanol concentrations necessary for cost-effective distillation.
Inhibitors found in biomass fermentations are determined by conditions used during pretreatment (temperature, pH, time, and any chemicals used), and act in various ways to inhibit efficient fermentation of sugars to ethanol [5–10]. Inhibitors can be divided into three general categories: aromatic compounds, furan derivatives, and weak aliphatic acids. Aromatic compounds, such as vanillin and 4-hydroxybenzaldehyde, are generated when the lignin in the wood is degraded . Furan derivatives are generated from sugar portions of the feedstock during pretreatment: with furfural (FF) from degradation of pentose sugars, and 5-hydroxymethylfurfural (HMF) from hexose sugars . HMF can be further degraded during pretreatment to produce the weak acids levulinic acid and formic acid. Acetic acid, another weak acid, is formed by hydrolysis of hemicellulose. HMF and FF can decrease ethanol yield and productivity, and slow the organism's growth . FF and HMF act synergistically to decrease ethanol production . The most concentrated weak acids present in pine-wood fermentations are acetic, levulinic, and formic acids, acting to inhibit cellular activity by mechanisms of uncoupling and intracellular anion accumulation . Uncoupling results in a dissipation of the cell's proton gradient; thus hindering its ability to generate ATP . During intracellular anion accumulation, the undissociated form of the acid will diffuse across the plasma membrane, and then dissociate inside the cell, thus decreasing the cytosolic pH . The cell must then correct this pH imbalance. Mechanisms by which aromatics inhibit are not completely elucidated, presumably due to the complex structure of lignin. Proposed mechanisms include a loss of integrity in the cell membrane, and destruction of the electrochemical gradient by transporting protons back into the mitochondria similar to the weak acids [9, 13]. Furthermore, it has been shown that FF and aromatic compounds can lead to reactive oxygen species that can randomly oxidize proteins, lipids, and other structures in Saccharomyces cerevisiae, and if the damage is too great, the cells will not survive [6, 14].
Inhibitory compounds may be removed before fermentation, resulting in increased ethanol production [4, 15, 16]. Although effective, ameliorating these compounds from fermentations increases overall production costs. The ethanologenic yeast, S. cerevisiae, displays relatively robust growth in the presence of inhibitory compounds , although the response of individual strains varies widely . Some Saccharomyces strains convert HMF to the less toxic 2,5-bis-hydroxymethylfuran , and the ADH6 gene product (alcohol dehydrogenase 6) has been shown to increase the rate at which cells metabolize HMF . S. cerevisiae is also able to partially metabolize some of the phenolic compounds, probably via phenylacrylic acid decarboxylase conversion of cinnamic, p-coumaric, and ferulic acids to their less toxic vinyl derivatives [21, 22]. Furan reductase or laccase have been expressed in yeast [23, 24], and these increased fermentation rates. Other efforts to reduce the detrimental effects of inhibitors include optimizing process configurations, such as using fed-batch pulse feeding of hydrolysate instead of immersing the yeast in hydrolysate all at once. Saccharomyces strains are able to adapt to some degree if precultured on hydrolysate or via cell recycling [25–27], although the exact mechanisms that result in increased performance are still unknown for many strains.
Previous efforts have described approaches to improve fermentation performance of S. cerevisiae strains with respect to inhibitor tolerance. When an industrial strain of S. cerevisiae was cultured in increasing concentrations of FF, the time spent in lag phase by the adapted strain was significantly reduced compared with the parental strain . In a later study, this reduction in lag phase was attributed to increased oxireductase activity in the evolved strain . Other researchers have increased xylose utilization in engineered strains through a process called chemostat evolution . In this process, the strain was kept under constant xylose limitation in a chemostat, and the resulting pressure selected for strains that are best able to use xylose as a carbon source. Because of the large natural biodiversity in S. cerevisiae, other approaches have focused on the isolation from distilleries of natural strains with the desired phenotypes .
In this paper, we describe the directed evolution and adaptation of an industrial Saccharomyces yeast strain, XR122N, currently used in corn-ethanol fermentations for the production of ethanol from pretreated lignocellulose. We selected sulfur dioxide-pretreated pine wood as the substrate, because of the high level of inhibitory compounds found in this feedstock. In order to generate a strain with improved tolerance of inhibitory compounds found in pretreated pine, XR122N was evolved using SO2-pretreated pine directly, without separating the liquid from the solids and without ameliorating the toxic compounds, rather than using a single inhibitory compound such as FF for directed evolution. The strain was also subjected to additional evolutionary adaptation at high solids loadings in order to increase ethanol concentrations in the fermentation. Growth and ethanol production of the evolved strain in various combinations of 13 inhibitory compounds found in pretreated pine was also investigated. The final evolved strain, AJP50, possesses greater fermentation capability than its parent in both rich liquid media supplemented with various combinations of inhibitory compounds, and in pretreated pine fermentations at high solids loadings.