A strain of Saccharomyces cerevisiaeevolved for fermentation of lignocellulosic biomass displays improved growth and fermentative ability in high solids concentrations and in the presence of inhibitory compounds
© Hawkins and Doran-Peterson; licensee BioMed Central Ltd. 2011
Received: 23 December 2010
Accepted: 10 November 2011
Published: 10 November 2011
Softwoods are the dominant source of lignocellulosic biomass in the northern hemisphere, and have been investigated worldwide as a renewable substrate for cellulosic ethanol production. One challenge to using softwoods, which is particularly acute with pine, is that the pretreatment process produces inhibitory compounds detrimental to the growth and metabolic activity of fermenting organisms. To overcome the challenge of bioconversion in the presence of inhibitory compounds, especially at high solids loading, a strain of Saccharomyces cerevisiae was subjected to evolutionary engineering and adaptation for fermentation of pretreated pine wood (Pinus taeda).
An industrial strain of Saccharomyces, XR122N, was evolved using pretreated pine; the resulting daughter strain, AJP50, produced ethanol much more rapidly than its parent in fermentations of pretreated pine. Adaptation, by preculturing of the industrial yeast XR122N and the evolved strains in 7% dry weight per volume (w/v) pretreated pine solids prior to inoculation into higher solids concentrations, improved fermentation performance of all strains compared with direct inoculation into high solids. Growth comparisons between XR122N and AJP50 in model hydrolysate media containing inhibitory compounds found in pretreated biomass showed that AJP50 exited lag phase faster under all conditions tested. This was due, in part, to the ability of AJP50 to rapidly convert furfural and hydroxymethylfurfural to their less toxic alcohol derivatives, and to recover from reactive oxygen species damage more quickly than XR122N. Under industrially relevant conditions of 17.5% w/v pretreated pine solids loading, additional evolutionary engineering was required to decrease the pronounced lag phase. Using a combination of adaptation by inoculation first into a solids loading of 7% w/v for 24 hours, followed by a 10% v/v inoculum (approximately equivalent to 1 g/L dry cell weight) into 17.5% w/v solids, the final strain (AJP50) produced ethanol at more than 80% of the maximum theoretical yield after 72 hours of fermentation, and reached more than 90% of the maximum theoretical yield after 120 hours of fermentation.
Our results show that fermentation of pretreated pine containing liquid and solids, including any inhibitory compounds generated during pretreatment, is possible at higher solids loadings than those previously reported in the literature. Using our evolved strain, efficient fermentation with reduced inoculum sizes and shortened process times was possible, thereby improving the overall economic viability of a woody biomass-to-ethanol conversion process.
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.
Results and Discussion
Pine fermentations with the industrial yeast strain XR122N
Compositional analysis of pine subjected to sulfur dioxide steam explosiona
Untreated Pine d
3.3% SO2 213°Ce
Concentrations (g/L) of each inhibitory compound studied, divided into classes
The effect of inoculum size on pretreated pine fermentations at a 12% w/v solids loading is presented in Figure 1B. Initial attempts at inoculation of pretreated pine solids at or above 5% w/v using a low inoculum level equal to 0.2 g/L dcw resulted in cell death of XR122N (absence of growth on solid or liquid medium) and no ethanol was detected in these cultures. An inoculum size of 0.5 g/L produced ethanol in pretreated pine fermentations at a 10% w/v solids concentration (data not shown), but at a 12% w/v solids concentration no ethanol production was detected (Figure 1B). Increasing the inoculum level to 1 g/L dcw in 12% w/v solids fermentations resulted in ethanol production, albeit with a pronounced lag phase of 72 hours. An inoculum of 2 g/L dcw produced almost as much ethanol as 4 g/L, and was selected as the inoculum size for further studies.
Comparison of simultaneous saccharification and fermentation methods using SO2 pretreatment of softwoods with Saccharomyces cerevisiae strains
Solids, % dry weight/volume
Cellulase, FPU/gram dry weight
Max EtOH, g/L
Time to maximum EtOH production, hours
Reaction temp, °C
40 FPU/g cellulase, 20 CU/g cellobiase
Ewanick et al.  (note: 6 hours enzyme preincubation)
40 mL, WSF
No enzymes added
Keating et al. 
32 FPU/g cellulase, 28 IU/g cellobiase
Alkasrawi et al. 
15 FPU/g cellulose, 23 CU/g cellobiase
Söderström et al. 
15 FPU/g cellulose, 23 CU/g cellobiase
Hoyer et al. 
15 FPU/g cellulose, 60 CU cellobiase
15 FPU/g cellulose, 60 CU cellobiase
Evolution of XR122N for fermentation at high solids loading
To reach the ethanol concentrations necessary for cost-efficient distillation, the solids loading must be 15 to 20% w/v . However, as the biomass content increases in the fermentation, the concentration of inhibitory compounds also increases. Previous studies with Saccharomyces spp. illustrated that some strains are able to adapt to varying degrees by preculturing on hydrolysate or via cell recycle [25–27]; the exact mechanisms for increased performance are still unknown for many of these strains. Using FF alone for adaptation experiments results in different phenotypes, depending upon the method used for selection. In previous work, increased rates of FF reduction were seen in selection regimens in which FF was added during logarithmic growth . By contrast, challenging cells at a low inoculum size to relatively high concentrations of FF did not change the FF reduction rates, but significantly reduced the lag phases and allowed growth in glucose minimal medium containing 40% v/v of spruce acid hydrolysate sample, a medium that killed the parental strain .
The resulting strain exhibiting the phenotype of increased ethanol production and decreased lag time in high solids fermentations was designated AJP50, and used for subsequent studies. Inoculation of 17.5% w/v solids fermentations with AJP50 taken directly from revived freezer stocks (0.2 g/L dcw) did not produce ethanol levels of above 10 g/L. However, inoculating AJP50 (0.2 g/L dcw) into a fermentation with reduced (7% w/v) solids loading for a short adaptation period (24 hours), followed by removal of a 10% v/v inoculum (approximately 1 g/L dcw) into 17.5% w/v solids fermentations, improved ethanol production significantly (Figure 3). With this short adaptation period, the evolved strain, AJP50, had a reduced lag time and produced over 80% of the maximum theoretical yield in 72 hours of fermentation and over 90% of the maximum theoretical yield of ethanol in 120 hours.
Growth and ethanol production in the presence of inhibitors
The inhibitory factors present in the largest concentrations in biomass fermentations are HMF, FF, and acetic acid, thus both strains were also grown in the presence of a mixture of these; the parental strain was strongly inhibited while the evolved strain showed an increase in lag phase. (Figure 4C). Growth of both strains was strongly inhibited by the mixture of the furan compounds HMF, FF, and furoic acid (Figure 4D). With this combination, no growth of XR122N was seen over 30 hours. Growth of AJP50 had a longer lag phase than in the other conditions; however, the furan-inhibited AJP50 cultures did eventually reach the same final optical density (OD) as the uninhibited cultures.
The effects of FF and HMF on certain strains of S. cerevisiae have been described previously by a number of groups [12, 19, 41]. FF completely inhibited the growth of yeast strains at a concentration of 5.76 mg/ml. and partially inhibited growth at a concentration of 2.88 mg/ml during an incubation period of 125 hours. HMF completely inhibited one strain, and partially inhibited another at 7.6 mg/ml; various degrees of partial inhibition were seen at a concentration of 3.8 mg/ml. These concentrations are higher than those previously reported for pine-wood biomass fermentations, however, the amounts of inhibitory compounds might increase with increased severity of the pretreatment, and with increased concentrations of biomass at high solids loadings.
Conversion of furfural and hydroxymethylfurfural to alcohol derivatives
Stability of the AJP50 inhibitor-resistant phenotype on rich media
Optical densities at OD580 of AJP50 cultures in inhibitory media after growth on rich media
0.0 to < 0.3
0.3 to <0.6
0.6 to < 0.9
0.9 to < 1.2
1.2 to < 1.5
Analysis of isolated clones and verification of the inhibitor-resistant phenotype
Reactive oxygen species in AJP50 and XR122N cultures incubated with inhibitory compounds
Pretreatment of pine-wood biomass
Loblolly pine from Georgia, USA, was debarked and chipped to a particle size of 10 mm or smaller. The chips were pretreated with gaseous sulfur dioxide [42, 43], and subjected to steam explosion in the Process Development Unit (PDU) at the Chemical Engineering Department, Lund University, Sweden, or in a similar PDU located at the Georgia Institute of Technology under the direction of C2Biofuels (Atlanta, GA). A known weight of chips was pretreated with 3.3% SO2 (w/w moisture content of chips) and held at 215°C for 5 minutes in the PDU in a single-step process. The resulting material consisted of a mixture of liquids and solids. These phases were not separated, pressed, drained, or washed to remove potentially inhibitory compounds. Materials were stored at 4°C until use.
Pretreated pine fermentations with XR122N
Fermentations were performed in small-scale bioreactors with a working volume of 200 mL using pretreated pine-wood biomass as feedstock essentially as described previously . The percentage moisture was determined using the IR-35 moisture analyzer as before and samples containing 5, 10, and 12% w/v dry solids were weighed, added to a 500 ml flask, and autoclaved at 121°C for 20 minutes to ensure sterility (although this could be considered an additional pretreatment). Upon cooling, double-strength tryptic soy broth (TSB, containing 15 g pancreatic digest of casein, 5 g papaic digest of soybean meal, and 5 g NaCl per liter; Difco, Detroit, MI, USA), and sterile water were added, and the pH adjusted to 5.0 with 2 mol/L KOH. The S. cerevisiae strain XR122N (North American Bioproducts Corporation, Duluth, GA, USA) was inoculated in a freeze-dried state at an initial concentration of 4 g/L dcw similar to its use in corn-ethanol fermentations. Cellulases and cellobiase (Novozymes Inc., Franklinton, NC, USA) were added simultaneously with the inoculum at concentrations of 15 FPU and 60 CU per gdw of pretreated pine, respectively. Fermentations were maintained at 35°C and pH 5.0, sampled every 24 hours, and ethanol concentration estimated using gas chromatography as previously described . All fermentations were performed in triplicate, and error bars represent one standard deviation from the mean. Inoculation of pretreated pine at 10, and 12% w/v solids loading was performed using 0.2, 0.5, 1, 2, and 4 g/L dcw.
Evolutionary adaptation of XR122N
A 2 g/L dcw inoculum of XR122N was added to pretreated pine fermentations at a 17.5% w/v solids loading for simultaneous saccharification and fermentation at 37°C and pH 5.0. The fermentation was allowed to proceed for 168 hours, and aliquots equal to 10% v/v were transferred to fresh fermentations containing 17.5% w/v solids, enzymes, and TSB, as described previously. Measurements of cell biomass using optical-density readings or dcw were not possible, because of the particulate matter present from the pretreated biomass, therefore cultures were monitored for ethanol production every 24 hours. Cultures were plated during transfer to the fresh 17.5% w/v solids fermentation, and were approximately equivalent to 1 g/L dcw. After no ethanol was detected at 96 hours, an additional 2 g/L dcw of XR122N cells were added to the fermentation vessels. Ethanol production was measured every 24 hours, and ethanol concentrations in one of the fermentation vessels continued to increase for an additional total of 72 hours. A 10% v/v inoculum was removed from the fermentation vessel in which ethanol production was detected, and used to inoculate a third fermentation vessel containing 17.5% w/v pretreated pine and enzymes. Ethanol production was measured every 24 hours; no additional ethanol was produced after 96 hours of fermentation. Again, another 2 g/L dcw of XR122N was added to the fermentation at this point. This process of inoculating a 17.5% w/v solids fermentation with a 10% v/v inoculum from a previous fermentation, monitoring ethanol production for 96 hours without observing an increase in ethanol content, and adding 2 g/L dcw of XR122N was repeated for a total of six full cycles. During the seventh cycle, measurement at 24 hours showed the ethanol production had increased, and it continued to increase up to 48 hours. At 48 hours of fermentation, a 10% v/v inoculum was transferred to a fresh 17.5% w/v solids fermentation, and the ethanol production monitored. Samples from this fermentation were frozen in glycerol at -80°C, and designated AJP40. A similar set of fermentations using 20% w/v solids failed to produce high concentrations of ethanol, even after the addition of 2 g/L dcw of XR122N.
Glycerol stocks of AJP40 were subjected to additional transfers. First, AJP40 (approximately 0.2 g/L dcw) was inoculated into fermentations containing 17.5% w/v solids loading of pretreated pine; these produced little ethanol. Inoculation of the same amount of AJP40 into a 7% w/v solids fermentation resulted in maximum ethanol production after 24 hours of fermentation, and a 10% v/v aliquot of this fermentation was used to inoculate a 17.5% w/v solids fermentation. Ethanol production was seen at 48 hours, and upon transfer of a 10% v/v inoculum into another 17.5% w/v solids fermentation, ethanol was detected after 24 hours. Additional transfers into 17.5% w/v solids were made by removing a 10% v/v inoculum from a 17.5% solids fermentation that was producing ethanol after 48 hours, and placing it into a new flask containing 17.5% w/v solids and enzymes for saccharification. Transfers were made every 48 hours for a total of 50 transfers. Aliquots from the final (50th) fermentation were frozen in glycerol stocks and designated AJP50.
Growth in combinations of inhibitory compounds
Stock solutions of each inhibitor were prepared fresh on the day they were to be used. Typical compounds found in pretreated pine wood were grouped by inhibitor class, and were examined in various mixtures. These inhibitory compounds comprised weak acids (acetic, formic, levulinic, lactic, and succinic acids), aromatics (3,4-dihydroxybenzaldehyde, 4-hydroxybenzaldehyde, vanillic acid, vanillin, and benzoic acid), and furans (FF, HMF, and 2-furoic acid). The effects of all 13 compounds were also examined simultaneously, and a mixture of HMF, FF, and acetic acid was also evaluated. The concentrations of each compound were similar to those seen in pretreated pine-wood fermentations (Table 2).
Freezer stocks were created from 7% w/v pretreated pine-wood fermentations for both AJP50 and XR122N. Freezer stocks were revived briefly (<10 minutes) in 9 ml TSB, and microscopic cell counts performed with a hemocytometer were used to standardize the initial inoculum concentration to 4.0 × 105 cells/ml in each well, which contained 20 g/L glucose and TSB. The starting OD for XR122N appeared to be higher than that of AJP50 because the presence of more particulate matter in the original inoculum from the freezer stocks, thus a larger volume of material was required to obtain an initial cell concentration of 4.0 × 105 cells/ml for XR122N. The initial pH of each well was 5.0, and temperature was maintained at 37°C in a growth curves machine (Bioscreen C; Oy Growth Curves Ab Ltd. Helsinki, Finland) without shaking. OD of the wells was recorded every 30 minutes at 580nm. Each well was replicated on the plate five times, and used to calculate the mean and standard deviation.
Ethanol production in model media containing various combinations of inhibitory compounds and glucose as the carbon source
Ethanol production was measured by inoculating wells of a plate with the inhibitor stock to be studied and the culture of interest as described above. Ethanol samples were taken in triplicate at each time point. Ethanol was sampled every 6 hours by removing the plate and removing full 300 μl volume of the appropriate wells by pipette into separate 0.22 μm centrifuge filtration tubes. These were then separated by centrifugation at 10,956 × g for 1 minute at room temperature before being frozen at -20°C until further analysis. Ethanol concentration in the samples was determined using gas chromatography as previously described .
Conversion of furfural and hydroxymethylfurfural to alcohol derivatives
These samples were also evaluated for the conversion of FF and HMF at 6-hour intervals in fermentations described above. FF, HMF, FF alcohol, and HMF alcohol concentrations were determined using HPLC as described previously .
Examination of the inhibitor-resistant phenotype of AJP50
AJP50 was cultured overnight on YPD agar at 37°C and a single colony was used to inoculate a 50-ml flask of YPD broth. The inoculated flask was incubated overnight at 37°C with shaking. The overnight YPD broth culture was examined (Bioscreen) in the presence of all 13 inhibitors as before. All 100 wells of the plate were identical in media composition and initial inoculum level; the OD of the wells was determined at 24, 30, and 48 hours after inoculation to determine how well AJP50 retained its resistance to the inhibitors after culture on rich media lacking any inhibitory compounds.
Analysis of isolated clones and verification of inhibitor-resistant phenotype
AJP50 glycerol stocks from the directed evolution were used to inoculate 7% w/v pretreated pine solids fermentations, and incubated at 37°C for 24 hours with shaking. Samples from the 7% w/v solids fermentation were removed, and frozen as 40% w/v glycerol stock cultures. Aliquots of these glycerol stocks were revived in YPD-BI, and incubated for 24 hours at 37°C with shaking. Isolated colonies were obtained by plating onto YPD-AI, and incubated at 37°C. Colonies took an average of 7 days to develop on the YPD-AI plates. Individual colonies from these plates were subcultured onto a second YPD-AI plate, and incubated at 37°C for approximately 7 days. Isolated colonies from this second plate were used to inoculate YPD-BI, and incubated for 24 hours at 37°C with shaking. Aliquots from this broth were used to inoculate wells in plates used for growth curve experiments in the Bioscreen apparatus as described previously, to ascertain if the inhibitor-resistant phenotype was being maintained during isolation and culturing.
A second round of experiments involved selection of isolated colonies from the second YPD-AI plate described above, and subculture onto a third YPD-AI plate. Isolated colonies from the third plate were then inoculated into YPD-BI, and used for growth-curve experiments.
A third set of experiments involved selection of isolated colonies from the third YPD-AI plate, and subculture for isolated colonies onto a fourth YPD agar plate. Isolated colonies were inoculated into YPD-BI, and screened for growth as described previously.
Comparison of the effect of reactive oxygen species on XR122N & AJP50
The effect of ROS on XR122N and AJP50 was measured using 2' 7'-dichlorofluorescein diacetate (DCF; Sigma-Aldrich Corp., St. Louis, MO, USA), which fluoresces in the presence of ROS, as described previously [14, 47]. XR122N and AJP50 were inoculated at 4.0 × 105 cells/ml from freezer stocks into 50 ml YPD media containing either: all 13 inhibitors; HMF, FF, and acetic acid; 5 mM H2O2; or no inhibitors. Cultures were maintained at 37°C with shaking, and samples taken at the indicated time points. Samples were examined for fluorescence using a reflected fluorescence microscope (BX61; Olympus Corp., Tokyo, Japan) with a fluorescein isothiocyanate filter. For each time point, at least 100 cells were examined, and the percentage of cells exhibiting fluorescence determined; this reflects the portion of the cell population experiencing ROS damage.
A strain of Saccharomyces cerevisiae (XR122N) was evolved by continuous exposure to pretreated pine-wood biomass to develop the daughter strain AJP50. Adding a preculture or short adaptation phase of 24 hours in 7% w/v pretreated pine enhanced the performance of the all strains, including AJP50. AJP50 more rapidly fermented pretreated pine-wood biomass at a high solids loading than its parent, or other Saccharomyces strains reported in the literature. Growth comparisons between XR122N and AJP50 in a model hydrolysate medium containing inhibitory compounds found in pretreated biomass showed that AJP50 exited lag phase faster under all conditions tested. This ability is due, in part, to AJP50 rapidly converting FF and HMF to their less toxic alcohol derivatives and recovering from ROS damage more quickly than XR122N. Under industrially relevant conditions of 17.5% w/v pretreated pine solids loading, additional evolutionary engineering was required to decrease the pronounced lag phase. Using a combination of adaptation by inoculation first into a fermentation with a solids loading of 7% w/v for 24 hours, followed by a 10% v/v inoculum (approximately equivalent to 1 g/L cell dry weight) into 17.5% w/v solids, the final strain (AJP50) produced ethanol at more than 80% of the maximum theoretical yield after 72 hours of fermentation and reached more than 90% of the maximum theoretical yield after 120 hours of fermentation.
Our results show that that fermentations of pretreated pine containing liquid and solids, including any inhibitory compounds generated during pretreatment, are possible at higher solids loadings than previously reported in the literature. These fermentations used reduced inoculum sizes and had shortened process times, thereby improving the overall economic viability of a pine-to-ethanol conversion process. Results from future studies characterizing the stability of the strain and analyzing the performance under conditions used with industrial processes (for example, after lyophilization) will be important for optimizing use of AJP50 in industrial applications.
List of abbreviations
cell dry weight
filter paper units
gram dry weight
high-performance liquid chromatography
simultaneous saccharification and fermentation
tryptone soy broth
YPD agar with inhibitors
YPD broth with inhibitors
We thank the following staff for their assistance: our technician Amruta Jangid for adaptation of XR122N and pine-wood fermentations; our technician Neffy Burgess and undergraduate student researcher, Divya Bansal for additional pine-wood fermentations; our undergraduate student researcher Debashis Ghose for Bioscreen comparisons and stability studies; and our UGA Research Experience for Undergraduates (REU) student Lydia Howes for Bioscreen optimization. The pretreated pine substrate and partial funding were provided by C2 biofuels LLC (Atlanta, GA, USA). Professor John Muzzy (Georgia Institute of Technology) assisted with the pretreatment studies of pine in the PDU. XR122N was provided by North American Bioproducts Corp. (Duluth, GA, USA). Additional support provided by the Department of Energy (DOE-EE000410) and the UGA Bioenergy Systems Research Institute. GMH was supported by a Graduate School Assistantship Award and the Microbiology Department at the University of Georgia.
- Vertes AA, Inui M, Yukawa H: Implementing biofuels on a global scale. Nat Biotechnol. 2006, 24 (7): 761-764. 10.1038/nbt0706-761.View ArticleGoogle Scholar
- Lin Y, Tanaka S: Ethanol fermentation from biomass resources: current state and prospects. Appl Microbiol Biotechnol. 2006, 69 (6): 627-642. 10.1007/s00253-005-0229-x.View ArticleGoogle Scholar
- Hahn-Hägerdal B, Galbe M, Gorwa-Grauslund MF, Lidén G, Zacchi G: Bio-ethanol-the fuel of tomorrow from the residues of today. Trends Biotechnol. 2006, 24 (12): 549-556. 10.1016/j.tibtech.2006.10.004.View ArticleGoogle Scholar
- Palmqvist E, Hahn-Hägerdal B: Fermentation of lignocellulosic hydrolysates. I: inhibition and detoxification. Bioresource Technology. 2000, 74: 17-24. 10.1016/S0960-8524(99)00160-1.View ArticleGoogle Scholar
- Larsson S, Palmqvist E, Hahn-Hägerdal B, Tengborg C, Stenberg K, Zacchi G, Nilvebrant N: The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme and Microbial Technology. 1999, 24: 151-159. 10.1016/S0141-0229(98)00101-X.View ArticleGoogle Scholar
- Almeida J, Modig T, Petersson A, Hahn-Hägerdal B, Lidén G, Gorwa-Grauslund MF: Increased tolerance and conversion of inhibitors in lignocellulosic hydrolysates by Saccharomyces cerevisiae. J Chem Technol Biotechnol. 2007, 82: 340-349. 10.1002/jctb.1676.View ArticleGoogle Scholar
- Taherzadeh MJ, Gustafsson L, Niklasson C, Lidén G: Physiological effects of 5-hydroxymethylfurfural on Saccharomyces cerevisiae. Appl Microbiol Biotechnol. 2000, 53 (6): 701-708. 10.1007/s002530000328.View ArticleGoogle Scholar
- Russell JB: Another explanation for the toxicity of fermentation acids at low pH: anion accumulation versus uncoupling. Journal of Applied Bacteriology. 1992, 73: 363-370. 10.1111/j.1365-2672.1992.tb04990.x.View ArticleGoogle Scholar
- Terada H: Uncouplers of oxidative phosphorylation. Environ Health Perspect. 1990, 87: 213-218.View ArticleGoogle Scholar
- Pampulha ME, Loureiro-Dias MC: Combined effect of acetic acid, pH and ethanol on intracellular pH of fermenting yeast. Appl Microbiol Biotechnol. 1989, 31: 547-550. 10.1007/BF00270792.View ArticleGoogle Scholar
- Ando H, Arai I, Kiyoto K, Hanai S: Identification of aromatic monomers in steam-exploded poplar and their influences on ethanol fermentation by Saccharomyces cerevisiae. J Ferment Technol. 1986, 64 (6): 567-570. 10.1016/0385-6380(86)90084-1.View ArticleGoogle Scholar
- Sanchez B, Bautista J: Effects of furfural and 5-hydroxymethylfurfural on the fermentation of Saccharomyces cerevisiae and biomass production from Candida guilliermondii. Enzyme Microb Technol. 1988, 10: 315-318. 10.1016/0141-0229(88)90135-4.View ArticleGoogle Scholar
- Heipieper HJ, Weber FJ, Sikkema J, Keweloh H, de Bont JAM: Mechanisms of resistance of whole cells to toxic organic solvents. TIBTECH. 1994, 12: 409-415.View ArticleGoogle Scholar
- Allen SA, Clark W, McCaffery JM, Cai Z, Lanctot A, Slininger PJ, Liu ZL, Gorsich SW: Furfural induces reactive oxygen species accumulation and cellular damage in Saccharomyces cerevisiae. Biotechnol Biofuels. 2010, 3: 2-10.1186/1754-6834-3-2.View ArticleGoogle Scholar
- Palmqvist E, Hahn-Hägerdal B: Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Bioresource Technology. 2000, 74: 25-33. 10.1016/S0960-8524(99)00161-3.View ArticleGoogle Scholar
- Olsson L, Hahn-Hägerdal B, Zacchi G: Kinetics of ethanol production by recombinant Escherichia coli KO11. Biotechnol Bioeng. 1995, 45 (4): 356-365. 10.1002/bit.260450410.View ArticleGoogle Scholar
- Olsson L, Hahn-Hägerdal B: Fermentative performance of bacteria and yeasts in lignocellulose hydrolysates. Process Biochemistry. 1993, 28: 249-257. 10.1016/0032-9592(93)80041-E.View ArticleGoogle Scholar
- Modig T, Almeida JR, Gorwa-Grauslund MF, Lidén G: Variability of the response of Saccharomyces cerevisiae strains to lignocellulose hydrolysate. Biotechnol Bioeng. 2008, 100 (3): 423-429. 10.1002/bit.21789.View ArticleGoogle Scholar
- Liu ZL, Slininger PJ, Dien BS, Berhow MA, Kurtzman CP, Gorsich SW: Adaptive response of yeasts to furfural and 5-hydroxymethylfurfural and new chemical evidence for HMF conversion to 2,5-bis-hydroxymethylfuran. J Ind Microbiol Biotechnol. 2004, 31 (8): 345-352.View ArticleGoogle Scholar
- Petersson A, Almeida JR, Modig T, Karhumaa K, Hahn-Hägerdal B, Gorwa-Grauslund MF, Lidén G: A 5-hydroxymethyl furfural reducing enzyme encoded by the Saccharomyces cerevisiae ADH6 gene conveys HMF tolerance. Yeast. 2006, 23 (6): 455-464. 10.1002/yea.1370.View ArticleGoogle Scholar
- Larsson S, Quintana-Sainz A, Reimann A, Nilvebrant NO, Jonsson LJ: Influence of lignocellulose-derived aromatic compounds on oxygen-limited growth and ethanolic fermentation by Saccharomyces cerevisiae. Appl Biochem Biotechnol. 2000, 84-86: 617-632. 10.1385/ABAB:84-86:1-9:617.View ArticleGoogle Scholar
- Clausen M, Lamb CJ, Megnet R, Doerner PW: PAD1 encodes phenylacrylic acid decarboxylase which confers resistance to cinnamic acid in Saccharomyces cerevisiae. Gene. 1994, 142: 107-112. 10.1016/0378-1119(94)90363-8.View ArticleGoogle Scholar
- Larsson S, Nilvebrant NO, Jonsson LJ: Effect of overexpression of Saccharomyces cerevisiae Pad1p on the resistance to phenylacrylic acids and lignocellulose hydrolysates under aerobic and oxygen-limited conditions. Appl Microbiol Biotechnol. 2001, 57 (1-2): 167-174.View ArticleGoogle Scholar
- Nilsson A, Gorwa-Grauslund MF, Hahn-Hägerdal B, Lidén G: Cofactor dependence in furan reduction by Saccharomyces cerevisiae in fermentation of acid-hydrolyzed lignocellulose. Appl Environ Microbiol. 2005, 71 (12): 7866-7871. 10.1128/AEM.71.12.7866-7871.2005.View ArticleGoogle Scholar
- Alkasrawi M, Rudolf A, Lidén G, Zacchi G: Influence of strain and cultivation procedure on the performance of simultaneous saccharification and fermentation of steam pretreated spruce. Enzyme Microb Tech. 2006, 38: 279-286. 10.1016/j.enzmictec.2005.08.024.View ArticleGoogle Scholar
- Keller FA, Bates D, Ruiz R, Nguyen Q: Yeast adaptation on softwood prehydrolysate. Appl Biochem Biotechnol. 1998, 70-72: 137-148. 10.1007/BF02920131.View ArticleGoogle Scholar
- Martin C, Marcet M, Almazan O, Jonsson LJ: Adaptation of a recombinant xylose-utilizing Saccharomyces cerevisiae strain to a sugarcane bagasse hydrolysate with high content of fermentation inhibitors. Bioresour Technol. 2007, 98 (9): 1767-1773. 10.1016/j.biortech.2006.07.021.View ArticleGoogle Scholar
- Heer D, Sauer U: Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain. Microb Biotechnol. 2008, 1 (6): 497-506. 10.1111/j.1751-7915.2008.00050.x.View ArticleGoogle Scholar
- Heer D, Heine D, Sauer U: Resistance of Saccharomyces cerevisiae to high concentrations of furfural is based on NADPH-dependent reduction by at least two oxireductases. Appl Environ Microbiol. 2009, 75 (24): 7631-7638. 10.1128/AEM.01649-09.View ArticleGoogle Scholar
- Kuyper M, Toirkens MJ, Diderich JA, Winkler AA, van Dijken JP, Pronk JT: Evolutionary engineering of mixed-sugar utilization by a xylose-fermenting Saccharomyces cerevisiae strain. FEMS Yeast Res. 2005, 5 (10): 925-934. 10.1016/j.femsyr.2005.04.004.View ArticleGoogle Scholar
- Basso LC, de Amorim HV, de Oliveira AJ, Lopes ML: Yeast selection for fuel ethanol production in Brazil. Fems Yeast Research. 2008, 8 (7): 1155-1163. 10.1111/j.1567-1364.2008.00428.x.View ArticleGoogle Scholar
- Gauss WF, Suzuki S, Takagi M: Manufacture of alcohol from cellulosic materials using plural ferments. US Patent No. 3,990,944. 1976Google Scholar
- Takagi M, Abe S, Suzuki S, Emert GH, Yata N: A method for production of alcohol directly from cellulose using cellulase and yeast. Proceedings of Bioconversion of cellulosic substances into energy, chemicals and microbial protein. Edited by: Ghose TK. 1977, New Delhi: I.I.T, 551-571.Google Scholar
- Wingren A, Soderstrom J, Galbe M, Zacchi G: Process considerations and economic evaluation of two-step steam pretreatment for production of fuel ethanol from softwood. Biotechnol Prog. 2004, 20 (5): 1421-1429. 10.1021/bp049931v.View ArticleGoogle Scholar
- Soderstrom J, Galbe M, Zacchi G: Effect of washing on yield in one- and two-step steam pretreatment of softwood for production of ethanol. Biotechnol Prog. 2004, 20 (3): 744-749. 10.1021/bp034353o.View ArticleGoogle Scholar
- Ewanick SM, Bura R, Saddler JN: Acid-catalyzed steam pretreatment of lodgepole pine and subsequent enzymatic hydrolysis and fermentation to ethanol. Biotechnol Bioeng. 2007, 98 (4): 737-746. 10.1002/bit.21436.View ArticleGoogle Scholar
- Keating JD, Robinson J, Cotta MA, Saddler JN, Mansfield SD: An ethanologenic yeast exhibiting unusual metabolism in the fermentation of lignocellulosic hexose sugars. J Ind Microbiol Biotechnol. 2004, 31 (5): 235-244.View ArticleGoogle Scholar
- Hoyer K, Galbe M, Zacchi G: Production of fuel ethanol from softwood by simultaneous saccharification and fermentation at high dry matter content. J Chem Technol Biotechnol. 2009, 84: 570-577. 10.1002/jctb.2082.View ArticleGoogle Scholar
- Wingren A, Galbe M, Zacchi G: Techno-economic evaluation of producing ethanol from softwood: comparison of SSF and SHF and identification of bottlenecks. Biotechnol Prog. 2003, 19 (4): 1109-1117.View ArticleGoogle Scholar
- Liu ZL, Slininger PJ, Gorsich SW: Enhanced biotransformation of furfural and hydroxymethylfurfural by newly developed ethanologenic yeast strains. Appl Biochem Biotechnol. 2005, 121-124: 451-460.View ArticleGoogle Scholar
- Modig T, Lidén G, Taherzadeh MJ: Inhibition effects of furfural on alcohol dehydrogenase, aldehyde dehydrogenase and pyruvate dehydrogenase. Biochem J. 2002, 363 (Pt 3): 769-776.View ArticleGoogle Scholar
- Brownell HH, Yu EK, Saddler JN: Steam-explosion pretreatment of wood: Effect of chip size, acid, moisture content and pressure drop. Biotechnol Bioeng. 1986, 28 (6): 792-801. 10.1002/bit.260280604.View ArticleGoogle Scholar
- Boussaid AL, Esteghlalian AR, Gregg DJ, Lee KH, Saddler JN: Steam pretreatment of Douglas-fir wood chips. Can conditions for optimum hemicellulose recovery still provide adequate access for efficient enzymatic hydrolysis?. Appl Biochem Biotechnol. 2000, 84-86: 693-705. 10.1385/ABAB:84-86:1-9:693.View ArticleGoogle Scholar
- NREL: Standard Biomass Analytical Procedures. [http://www.nrel.gov/biomass/analytical_procedures.html]
- Doran-Peterson J, Jangid A, Brandon SK, DeCrescenzo-Henriksen E, Dien B, Ingram LO: Simultaneous saccharification and fermentation and partial saccharification and co-fermentation of lignocellulosic biomass for ethanol production. Methods Mol Biol. 2009, 581: 263-280. 10.1007/978-1-60761-214-8_17.View ArticleGoogle Scholar
- Brandon SK, Eiteman MA, Patel K, Richbourg MM, Miller DJ, Anderson WF, Peterson JD: Hydrolysis of Tifton 85 bermudagrass in a pressurized batch hot water reactor. J Chem Technol Biotechnol. 2008, 83: 505-512. 10.1002/jctb.1824.View ArticleGoogle Scholar
- Ohba M, Shibanuma M, Kuroki T, Nose K: Production of hydrogen peroxide by transforming growth factor-beta 1 and its involvement in induction of egr-1 in mouse osteoblastic cells. J Cell Biol. 1994, 126 (4): 1079-1088. 10.1083/jcb.126.4.1079.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.