• recombinant Saccharomyces cerevisiae LNH-ST 424A;
  • xylose fermentation;
  • ethanol;
  • pretreatment;
  • acetic acid;
  • furfural;
  • fermentation inhibitors


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgements
  8. Literature Cited

The inhibitory effects of furfural and acetic acid on the fermentation of xylose and glucose to ethanol in YEPDX medium by a recombinant Saccharomyces cerevisiae strain (LNH-ST 424A) were investigated. Initial furfural concentrations below 5 g/L caused negligible inhibition to glucose and xylose consumption rates in batch fermentations with high inoculum (4.5–6.0 g/L). At higher initial furfural concentrations (10–15 g/L) the inhibition became significant with xylose consumption rates especially affected. Interactive inhibition between acetic acid and pH were observed and quantified, and the results suggested the importance of conditioning the pH of hydrolysates for optimal fermentation performance. Poplar biomass pretreated by various CAFI processes (dilute acid, AFEX, ARP, SO2-catalyzed steam explosion, and controlled-pH) under respective optimal conditions was enzymatically hydrolyzed, and the mixed sugar streams in the hydrolysates were fermented. The 5-hydroxymethyl furfural (HMF) and furfural concentrations were low in all hydrolysates and did not pose negative effects on fermentation. Maximum ethanol productivity showed that 0–6.2 g/L initial acetic acid does not substantially affect the ethanol fermentation with proper pH adjustment, confirming the results from rich media fermentations with reagent grade sugars. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgements
  8. Literature Cited


Ethanol as a renewable fuel can be produced domestically,1 can displace greenhouse gas emissions from the use of petroleum-based transportation fuels,2–4 and provide a renewable energy source to combat the diminishing global energy supply.5, 6 Realizing these advantages will require several technological advancements, which include development of cost-effective cellulosic biomass pretreatment and hydrolysis processes,7, 8 and engineering of robust industrial microbes that are capable of fermenting mixed streams of hexose and pentose derived from lignocellulosic biomass.9, 10

Among the major technical hurdles for cellulosic ethanol production, pretreatment is one of the most costly processing steps, and has significant influences on the following enzymatic hydrolysis and ethanol fermentation efficiencies.11 To tackle this important issue and provide insight into the development of an advanced biomass deconstruction platform, the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI) was formed in 2000 and initiated collaborative research on this subject. The initial studies by the CAFI groups focused on corn stover as a biomass feedstock, and results, conclusions, and recommendations were discussed in a special volume of “Bioresource Technology”.11 The unique strength of the CAFI research approach lies in results comparability, through the use of a single feedstock, identical analytical methods [adapted from National Renewable Energy Laboratory (NREL) LAPs], and a consistent approach to data interpretation employed by all the participating groups.11 Such an approach allows valuable comparisons among each of the optimized pretreatment technologies, and sheds lights on the direction for improvement/development of advanced biomass depolymerization platform at reduced cost.

In the second phase of the CAFI project, a woody biomass feedstock, poplar, was the feedstock of study. Compared to corn stover (11.0% mass in lignin), polar has a significantly higher percentage of lignin content (29.1% mass in lignin), which causes additional structural barriers to effective pretreatment and enzymatic hydrolysis. This additional hurdle is discussed in the other articles of this special issue as the CAFI teams optimized the pretreatment technologies (dilute sulfuric acid, ammonia recycle percolation, lime, ammonia fiber/freeze expansion, SO2-catalyzed steam explosion, and controlled-pH liquid hot water). Although pretreatment is required to improve reactivity of this recalcitrant substrate to enzymatic hydrolysis,8 an effective pretreatment can also create compounds that are inhibitory to microbial fermentation through degradation of monomeric sugars (especially furfural from pentoses and 5-hydroxymethyl furfural (HMF) from hexoses), through solubilization of lignin compounds, and through release of acetic acid during hemicellulose hydrolysis.12–18 Therefore, it is critically important to evaluate pretreatment by examining the glucose/xylose fermentation performance simultaneously during the course of pretreatment optimization.

This study is the evaluation of ethanol fermentation performance of mixed sugar streams from pretreatment and enzymatic hydrolysis by a single robust microorganism. The availability of recombinant Saccharomyces cerevisiae (LNH-ST 424A)19–21 that ferments both glucose and xylose made it possible for a side-by-side comparison of the individually optimized pretreatment processes. The two main potential fermentation inhibitors, furfural and acetic acid, were examined with respect to their impacts on the glucose/xylose cofermentation in YEPDX medium. After that, poplar biomass processed by the various pretreatments under their respective optimal conditions was subjected to a 7-day enzymatic hydrolysis, and the hydrolysates were fermented for 48 h. The relative inhibitory effects of furfural, acetic acid and pH of the hydrolysates are discussed. Understanding to the key influential inhibitory factors to fermentation suggests on strategies to optimize upstream processes, i.e. pretreatment and enzymatic hydrolysis, depending on compositional characteristics of the biomass.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgements
  8. Literature Cited


The poplar used in this study was supplied by USDA and distributed by the NREL to the CAFI project participants. NREL debarked, chipped and milled the poplar materials to pass through a ¼ in. screen. Milled poplar was stored at −20°C until used. All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

S. cerevisiae 424A (LNH-ST) used in this study was developed and provided by Dr. Nancy Ho. The genetically engineered S. cerevisiae 424A (LNH-ST) was constructed by integrating multiple copies of XD, XR, and XK into the chromosomes of S. cerevisiae ATCC 4124 according to the technology reported by Ho et al.18–21 The 424A (LNH-ST) strain was maintained in liquid YEPX medium, which includes 20 g/L of Difco peptone (Becton Dickinson, St. Louis, MO), 10 g/L of Difco yeast extract (Becton Dickinson), and 20 g/L of xylose. Fresh seed cultures were prepared by inoculating the respective seed cultures in 100 mL of YEPX in 300-mL baffled Erlenmeyer flask equipped with a sidearm (BELLCO). The cultures were incubated in a controlled environment incubator shaker (New Brunswick Scientific, Edison, NJ) at 30°C and 200 rpm and grown aerobically overnight. The following morning, when the cultures reached OD 450–500 Klett-unit (KU) (OD 400 KU corresponds to a cell mass concentration of about 5 g dry wt/L), the flasks containing the seed cultures were stored in a refrigerator at 4°C until used.


Pretreated Solids Preparation

The detailed description and modes of action of the various pretreatment processes can be found in previously published articles8, 11 and will not be reiterated here. The optimization methodologies for these pretreatments with poplar as the biomass substrate are discussed in other articles of this issue. A brief summary on how the poplar materials were pretreated for use in this study is given below:

  • Dilute sulfuric acid pretreatment (National Renewable Energy Laboratory)

    A 900 kg/day pilot-scale reactor at NREL was used to carry out the dilute sulfuric acid pretreatment of poplar. The reaction condition was set at 190°C, 0.048 g sulfuric acid per g biomass (dry); the residence time was about 1.5 min, at a solid-loading of 250 g/L biomass loading.

  • Oxidative lime pretreatment (Texas A&M University)

    During the oxidative lime pretreatment, the poplar material was submitted to the following conditions: 150°C reaction temperature, oxygen was supplied at 200 psi, lime loading was controlled in excess of 0.4 g lime/g-dry-poplar, and the pretreatment lasted for 6 h. Once the pretreatment time elapsed, the pretreatment liquor was separated from the solids. The excess of lime in the solids was neutralized using water and 5N HCl, and was extensively washed afterwards.

  • Ammonia recycle percolation (Auburn University)

    The ARP reactor was operated in a flow-through mode, with a reaction temperature of 185°C, a residence time of 27.5 min, and a NH3 to biomass ratio of 3.667:1. The solids loading in the reactor was 260 g/L poplar (dry).

  • Ammonia fiber/freeze expansion (Michigan State University)

    During the AFEX pretreatment, the poplar biomass was presoaked in water for 24 h, NH3-to-poplar ratio was set at 1:1, and the residence time was 30 min at a reaction temperature of 180°C.

  • SO2-catalyzed steam explosion (University of British Columbia)

    The SO2-catalyzed steam explosion pretreatment was carried out at 200°C, for 5 min of reaction, with a 3% SO2-gas load.

  • Controlled-pH, Liquid hot water pretreatment (Purdue University)

    The pretreatments were carried out in 1 in. stainless steel tubes (45 mL total volume). The reaction temperature was controlled at 200°C and the pretreatment lasted for 15 min (include 5-min heat-up time + 10-min reaction time). A hot-washing strategy was used to remove inhibitors to enzymatic hydrolysis. Each batch of pretreated solids was washed with 500 mL 80–90°C DI H2O.

Enzymatic Hydrolysis of the Pretreated Poplar

Depending on the physical nature of the received pretreated poplar, the pretreated poplar materials were prepared as follows before enzymatic hydrolysis: The dilute acid pretreated NREL sample was received as wet solid pretreatment residuals and blown-out liquid separately. The pH of the liquid was adjusted to 4.8 by addition of Ca(OH)2. The solid pretreatment residuals were then added into the pH 4.8 liquid at 200 g/L dry solid-loading. The AFEX, Lime (O2) and ARP pretreated samples were received as solids without pretreatment liquid/liquor. Therefore 50 mM citrate buffer (pH 4.8) was added to prepare a 200 g/L dry solid-loading solution. No overliming was used for the pH adjustment. The SO2-catalyzed steam-explosion pretreated poplar was directly hydrolyzed in 50 mM citrate buffer (pH 4.8) by our colleagues at the University of British Columbia and filtered. The liquid hydrolysate was shipped frozen and stored at −20°C until used. The controlled-pH, liquid hot water pretreated poplar was hot-washed and hydrolyzed at 150 g/L dry solids loading. See Kim et al. for details in this issue.

Enzymes used in this study, spezyme CP (Lot no. 301-04075-054) and multifect xylanase (Lot no. 301-04021-015), were supplied by Genencor, a Danisco Division (Rochester, NY). The spezyme CP had a cellulase activity of 59 FPU/mL, with a total protein content of 123 mg/mL; the multifect xylanase had a xylanase activity (Oat spelt xylan) of 25,203 IU/mL, and a total protein content of 42 mg/mL. For each hydrolysis, 60 FPU/g-total-glucan spezyme CP was added to the 200 g/L dry solid-loading solution (pH 4.8), supplemented with an equivalent amount (mass of protein) of multifect xylanase. The enzymatic hydrolysis (with a total liquid volume of 100 mL) was held in an Innova 2000 shaker incubator set at 50°C, 190 rpm for a 7-day saccharification reaction. The resulting hydrolysate liquid was separated from the solid by filtration, and the liquid was adjusted to pH 5.5–6.0 before fermentation by addition of Ca(OH)2 or NaOH.

Glucose/Xylose Cofermentation

Glucose/xylose cofermentation was carried out at 28.5°C for 48 h using the glucose and xylose-fermenting recombinant yeast 424A (LNH-ST). Eight milliliter of seed culture was used to inoculate 100 mL YEPD (YEP plus 2% glucose) in a 300 mL baffled sidearm Erlenmeyer flask. Cultures were incubated in a shaker at 28.5°C and 200 rpm and grown aerobically overnight, after which a cell optical density (OD) between 450–500 KU was obtained. Cultured yeast cells were harvested by centrifugation (J-21 Beckman) at 5000 rpm for 5 min at room temperature. The supernatant was discarded and the cells were transferred into a 300 mL baffled Erlenmeyer flask containing 100 mL of either YEPDX medium (for the furfural/acetic acid effects experiments) or the poplar hydrolysate from enzymatic hydrolysis. Concentration of initial cell mass before fermentation in each experiment was about 5 g dry weight/L. The flasks were then sealed with plastic wrap to allow fermentation to be carried out under microaerobic conditions. The cultures were placed in the shaker and incubated at 28.5°C. At regular intervals 1 mL samples of the fermentation mixture were removed for HPLC analysis. The sampling was accomplished by removing the plastic wrapping and foam plug from the top of the flasks and removing an aliquot with a pipette. Samples were obtained more frequently early in the fermentation (every 3–6 h) and less frequently (every 12–24 h) although the xylose was fermented alone in the later stages. The times of sampling are shown in the graphs in Figures 1, 3, and 4. The fermentation experiments were run in duplicate at each condition.

For the furfural inhibition experiments, 0, 5, 10, and 15 g/L furfural was added to the YEPDX medium. YEPDX media was prepared by dissolving 10 g of yeast extract and 20 g of peptone per liter of distilled water. After autoclaving the YEP media, dry dextrose (glucose) and dry xylose were added to achieve initial concentrations of ∼35 g/L. Dry sugars were added to autoclaved YEP to avoid thermal degradation of the sugars during autoclaving. Acetic acid inhibition experiments used a 4 × 3 full factorial experimental design, with initial acetic acid concentrations of 0, 5, 10, and 15 g/L, and pH of 5.0, 5.5, and 6.0. Each experimental condition was repeated twice.

HPLC Analysis

The enzymatic hydrolysate and ethanol fermentation samples were analyzed by HPLC. The HPLC system consists of a Bio-Rad HPX-87H organic acid column (Bio-Rad Laboratories, Hercules, CA) in a HPLC system consisting of a Milton Roy minipump (Milton Roy, Ivyland, PA), Waters 717 plus autosampler, Waters R401 differential refractometer (Waters, Milford, MA), and a personal computer with Empower software for HPLC operation control. The mobile phase was 5 mM sulfuric acid in distilled, deionized water filtered through 0.2 μm nylon filters. Operating conditions for the HPLC column were 60°C with a mobile phase flow rate of 0.6 mL/min. For sample analysis, 50 μL of sample was injected and complete sample elution could be accomplished within 55 min per injection. Each sample was analyzed by HPLC in triplicate. The average coefficient of variation (standard deviation divided by mean) for the replication injections was less than 3% for all compounds reported. A standard solution was prepared by dissolving pure (>99% purity) compounds (glucose, xylose, glycerol, xylitol, furfural, acetic acid, and ethanol) in the HPLC mobile phase. Fractional dilutions of the standard solution ranging from 0.5–4 g/L were prepared to provide standards for HPLC calibration. The linear regression for the curves between the elution peak area and concentration was computed to give >99.9% R-square values for all compounds. Other sugars and organic acids (fructose, lactic acid, formic acid, 4-O-methyl glucuronic acid) are resolved by this HPLC method, but were either not detected or detected as minor peaks in the samples and thus not quantified. Xylose and galactose coelute from the HPX-87H column operated under these conditions. However, complete compositional analyses of the poplar substrate performed at the National Renewable Energy Laboratory found that the galactan content of the raw poplar was 1% of dry mass, compared to 14.9% of dry matter that was xylan, and thus not quantified for this study.

Calculation of Yields

Both metabolic and overall productivity yields were calculated to evaluate and compare the fermentation performance of the enzymatic poplar hydrolysates from the different pretreatments examined. The metabolic yield determines the efficiency of the fermentative metabolism, while the productive yield evaluated the ethanol yield compared to the theoretical yield based upon the sugars initially present.

  • equation image(1)
  • equation image(2)

Results and Discussions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgements
  8. Literature Cited

The inhibitory effects of furfural/acetic acid on glucose-xylose cofermentation

The effects of furfural on glucose/xylose cofermentation in YEPDX medium are summarized in Figure 1. Each subfigure is a side-by-side comparison of glucose, xylose, or ethanol concentrations over the time course of fermentation. Under the control conditions (no furfural added), complete fermentation of glucose (30 g/L initial concentration) occurred in less than 6 h and complete xylose (35–40 g/L initial concentration) fermentation was accomplished in less than 48 h. The average maximum glucose consumption rate, computed by averaging the slopes of the tangent line to the greatest rate of consumption, was 9.5 g L−1 hr−1 (Table 1). By contrast, the average maximum xylose consumption rate was approximately one quarter that of glucose (2.5 g L−1 hr−1). The maximum average ethanol production rate, which occurs during glucose consumption, was calculated to be 4.8 g L−1 hr−1. The metabolic yield of ethanol from both glucose and xylose averaged 85% of theoretical. These results are consistent with previously reported glucose/xylose cofermentation results for this yeast strain.22, 23

thumbnail image

Figure 1. Fermentation of glucose, and xylose to ethanol by S. cerevisiae 424A (LNH-ST) in the presence of varying initial concentrations of furfural (• – 0 g/L furfural; ▾– 5.0 g/L furfural; ▪ –10 g/L furfural; □ –15 g/L furfural).

Download figure to PowerPoint

Table 1. Maximum Glucose and Xylose Consumption Rates, Maximum Ethanol Production Rates, and Maximum Furfural Metabolism Rates for Varying Initial Concentrations of Furfural/Acetic Acid
Consumption or Production Rates (g L−1 hr−1)Furfural (Initial)
0 g/L5 g/L10 g/L15 g/L

For initial furfural concentrations below 5 g/L, the glucose and xylose consumption rates are not substantially slower than the control (Table 1). The complete fermentation of both glucose and xylose occurred within 48 h at these furfural concentrations. Above 5 g/L initial furfural, more significant impacts on sugar consumption were observed, although the degree of inhibition differs between glucose and xylose. The maximum rate of glucose consumption in the presence of 10 g/L furfural was ∼6.6 g L−1 hr−1, which was nearly 31% lower than the control rate. However, the maximum rate of xylose consumption in the presence of 10 g/L furfural was ∼0.5 g L−1 hr−1, 80% lower than the control (2.5 g L−1 hr−1). Furthermore, the fermentation of xylose largely occurs after the furfural concentration falls below the detectable limit. In all cases the rate of furfural metabolism is approximately the same (>2.0–2.8 g L−1 hr−1), and furfural concentrations in the media fall below the detectable limit before all of the sugars are consumed.

This trend continues to 15 g/L initial furfural concentration, which corresponds to the tolerance limit for this yeast strain at this cell concentration. In the case of 15 g/L initial furfural, glucose consumption is ∼30% of the control case. Furfural is metabolized concurrently with glucose consumption. The furfural concentration fell below detectable limit after 12 h and glucose was completely consumed within 20 h. However the consumption of xylose, which begins at after 12 h, was significantly affected (10% of the consumption rate of the control). No significant amount of xylose was consumed after 30 h. These results suggest that the inhibitory effects of furfural with regard to xylose fermentation extend beyond the effects of the presence of furfural in the fermentation media. Membrane adsorbed furfural, intracellular furfural, the metabolic product of furfural conversion (furfuryl alcohol and furoic acid), or byproducts of furfural metabolism (redox imbalance and/or oxidative damage) may continue to affect the metabolism of the yeast long after the furfural in the bulk media is removed.

At higher than 15 g/L initial furfural, little metabolism of either sugar was observed. The results suggest that the inhibitory effects of furfural is due to more than one mechanism, such as inhibition of glycolytic enzymes,24 redox imbalance, or inactivation of the cells over time due to toxicity from the accumulation of intracellular acetaldehyde as the furfural is metabolized.12 The reason for increased sensitivity of xylose fermentation to furfural is not yet clear.

Previous research on inhibition of Zymomonas fermentation of pretreated poplar showed that acetic acid/acetate was the predominant inhibitor.15 Thus impact of acetic acid on xylose consumption rates in this strain of S. cerevisiae was examined (Figure 2). Not only acetic acid concentration, but also the interactive effect of pH and acetate concentration was evaluated with respect to xylose consumption rate. In the range of acetic acid concentrations from 5–15 g/L and pH from 5–6, the xylose consumption rates varied from 0.135 (4.8% of control) to 2.8 g L−1 hr−1 (control). When there was no acetic acid present, varying the fermentation pH in the range of 5–6 did not affect the xylose consumption rate; however, the pH-dependant inhibition became evident when 5–20 g/L acetic acid was added to the fermentation. When 5 g/L acetic acid was present, the xylose consumption rates were between 0.86 (34.4% of control)–1.55 g L−1 hr−1 (59.6% of control) with correspondent pH values between 5 and 6. An increasing inhibitory trend was observed as the acetic acid concentration was raised to 10–15 g/L (Figure 2). The pH effect became profound at high acetic acid concentration, e.g. at 15 g/L acetic acid concentration, the xylose consumption rate at pH 5 was 0.135 g L−1 hr−1, only of 20.1% of that in pH 6 medium (0.67 g L−1 hr−1). This can be explained by the membrane transportability of acetic acid at the two different pHs. The undissociated form of acetic acid can freely diffuse across the cytoplasmic membrane, dissociate, lower the intracellular pH, and disturb normal cellular metabolism. However, the charged, dissociated form of acetic acid cannot diffuse across the cytoplasmic membrane.25, 26 Since the pKa of acetic acid is 4.75, at pH 5 the undissociated [HA]-to-dissociated [A] ratio is 0.56:1, while at pH 6 this value changes to 0.056:1 (a magnitude lower). Therefore, there is 10 times more undissociated acetic acid at pH 5 than that at pH 6.

thumbnail image

Figure 2. The interaction effect of pH and acetic acid concentration on xylose consumption rates during the glucose/xylose cofermentation by S. cerevisiae 424A (LNH-ST).

Download figure to PowerPoint

Acetic acid is ubiquitously present in all types of cellulosic biomass materials and its release is inevitable during hemicellulose hydrolysis (5.6% acetyl by mass in CAFI 2 corn stover, and 3.6% acetyl by mass in CAFI 2 poplar), which may lead to 7.2–11.2 g/L acetic acid in the hydrolysate after a typical 200 g/L dry-solid-loading pretreatment (without considering stream recycles that would increase the steady-state concentration). To address the toxicity from acetic acid, a careful conditioning of hydrolysate is necessary to mitigate its inhibitory effects on glucose/xylose cofermentation. The set of experiment described above demonstrated that controlling of the fermentation pH to above 5.5 would be beneficial for minimizing the inhibition of acetic acid. This inhibition control strategy was examined with the poplar hydrolysates from the CAFI pretreatments as follows.

Glucose/xylose cofermentation of poplar hydrolysates

The composition pretreated poplar hydrolysates are listed in Table 2. Because of the fact that the enzyme mixture used in this study was not optimal, but rather dosed to provide a consistent biocatalyst loading to all pretreated poplar materials, the sugar yields from the 7 day saccharification were lower than expected: glucose yields ranged from 33 to 63%, while xylose yields were in the range of 24–78% (Table 2). Compared to yields obtained with corn stover as the biomass feedstock (>85%) under optimal conditions,27 the combined sugar yields from pretreatment and enzymatic hydrolysis of poplar seemed to be significantly less efficient. The higher lignin content of the poplar (29% compared to 17% in corn stover) may account for this. In addition, inhibition of the enzymes by high concentrations of glucose and xylose likely limited the extent of conversion. Conducting a simultaneous saccharifaction and fermentation (SSF) would likely alleviate this inhibition. However, for these experiments we chose to examine separate saccharifaction and fermentation to eliminate the confounding effect of potential differences in enzymatic hydrolysis rates and difficulty in computing metabolic yield of ethanol production due to the difficulty in accurately measuring total sugar release in SSF.

Table 2. Composition of Enzyme Hydrolysates from Poplar Pretreated by Various Pretreatment Technologies (60 FPU/g-total Glucan Spezyme CP + Equivalent Mass of Protein of Multifect Xylanase for 168 h at 50°C)
Pretreatment*Glucose (g/L; Yield)Xylose (g/L; yield)Acetic Acid (g/L)
  • *

    HMF/furfural was not detected in any hydrolysate, which suggested that sugar degradation reaction was minimized for the optimal pretreatments.

  • Hydrolysis continued for 5 days, Enzyme loading: 15 FPU/g-total glucan Spezyme CP + 40 U/ g-total glucan Novo188, No Multifect Xylanase was used.

  • n/d = not detected by HPLC.

Dilute acid41.441.6%22.367.3%5.1
SO2 explosion33.233.4%25.877.9%6.2
Controlled pH56.154.0%1227.0%2.3
Lime (O2)52.853.0%1648.3%n/d

After the 7 day enzymatic hydrolysis of poplar pretreated by each CAFI pretreatment was completed, the hydrolysate liquid was filtered from the residual pretreated poplar solids, adjusted to pH 5.5–6.0, and subjected to a 48 h glucose/xylose cofermentation. The fermentation profiles are summarized in Figure 3. Since all the CAFI pretreatments have been optimized to reduce furfural/HMF accumulation, minimal amounts of furfural/HMF were detected in the enzymatic hydrolysate. However, acetic acid concentrations varied from 0 to 6.2 g/L in these hydrolysates (Table 2). A summary of the fermentation products composition at the conclusion of the 48 h fermentation is also shown in Table 3. For the fermentation of poplar hydrolysate prepared from controlled-pH pretreatment, glucose was readily fermentable within 6 h, however only of 38.0% of the initial xylose was fermented in that time. A total of 80.0% of available xylose was fermented by the end of the fermentation, with no detectible xylitol formation. The metabolic ethanol yield reached 86.8%. No acetic acid was detectible in any of these samples. During the fermentation of dilute sulfuric acid pretreated poplar hydrolysates, the glucose was completely fermented within 6.5 h, whereas only 24.8% of the total xylose was consumed. At the end of the 48-h fermentation, 87.8% of xylose was fermented (Table 4). The metabolic ethanol yield reached 85.0% of the theoretical yield. Among the consumed xylose, only 2.9% was converted to xylitol (0.57 g/L). The acetic acid level remained 5.1 g/L throughout the fermentation period. The poplar hydrolysate prepared from the lime (supplemented with O2 supply) pretreatment contained 52.8 g/L glucose, 16.0 g/L xylose, and no detectible acetic acid. After 6 h of fermentation, 81.2% of the glucose was consumed while only 2.3% xylose was simultaneously fermented. The rate of xylose fermentation significantly increased after the glucose was consumed. This is likely because of competition between glucose and xylose for HTX transport proteins.29 At the end of the 48 h fermentation 90.4% of xylose was consumed, and the metabolic ethanol yield was near quantitative (Table 4). The ARP processed hydrolysates fermentation seemed to be more rapid than the other fermentations: 98.0% of glucose and 28.4% of xylose were completely consumed after 3 h of fermentation. At the completion of the 48 h fermentation, all of the xylose was consumed and the metabolic ethanol yield reached 98.6% of theoretical yield. For the fermentation of poplar hydrolysate prepared from the ammonia fiber/explosion pretreatment, 96.5% of glucose and 12.2% of xylose were consumed within 6.5 h. After 48 h 78.7% of total xylose (of which 3.7% went to xylitol) was consumed, with a metabolic ethanol yield at 93.0% (Table 4). The fermentation performance of poplar hydrolysate prepared from the SO2-catalyzed steam explosion pretreatment was similar to the ARP fermentation results, 98.8% of glucose and 18.6% of xylose were consumed within 3 h of fermentation. After 48 h, 90.8% of xylose was fermented (3.3% of the xylose was converted to xylitol), and the metabolic ethanol yield was 89.9% of theoretical (Table 4).

thumbnail image

Figure 3. Glucose/xylose fermentation profiles of poplar hydrolysates processed by various pretreatments under optimal conditions (• – Glucose, ○ – Xylose, ▾ – Xylitol, ▵ – Glycerol, ▪ – Acetic acid, □ – Ethanol).

Download figure to PowerPoint

Table 3. Summary of Major Fermentation Substrates/Products at the Conclusion of 48 h Glucose/Xylose cofermentation
PretreatmentGlucose (g/L)Xylose (g/L)Xylitol (g/L)Glycerol (g/L)Acetic Acid (g/L)Ethanol (g/L)
Dilute acid02.
SO2 Explosion02.
Controlled pH0.
Lime (O2)
Table 4. Summary of Yields and Ethanol Productivities in the Cofermentation of Poplar Hydrolysates From Different Pretreatment Technologies
PretreatmentMetabolic Yield (% of Theoretical)*Productive Yield (% of Theoretical)Xylose Consumption in 48 h (%)Average Ethanol Productivity (g L−1 h−1)6-h Maximum Ethanol Productivity (g L−1 h −1)
  • *

    Metabolic Yield = [ethanol]/[0.51 * (consumed glucose + consumed xylose)].

  • Productive Yield = [ethanol]/[0.51 * (initial glucose + initial xylose)].

Dilute acid85.0%81.4%87.8%0.554.7
SO2 explosion89.9%86.2%90.8%0.544.7
Controlled pH86.8%82.7%80.0%0.604.7
Lime (O2)100%100%90.4%0.834.7

It should be noted that due to the different sugar yields from the enzymatic hydrolysis, the starting glucose/xylose ratio and concentrations in the different hydrolysates were not statistically equivalent. Therefore, metabolic yield, productive yield, and average and maximum ethanol productivity had to be calculated to differentiate the fermentation performances. Based on calculated metabolic, productive yields and average ethanol productivity, hydrolysates processed by the lime (O2) pretreatment fermented most effectively. The average ethanol productivity values shown in Table 4 were calculated over the total fermentation time (48 h). This illustrates the final ethanol productivity; however ethanol productivity does not provide insights into whether there were differences between the hydrolysates in the initial rates of fermentation. Therefore, the 6 h maximum ethanol productivities were computed and compared (Table 4). All the fermentations of hydrolysates from optimized pretreatments yielded ethanol productivities comparable to the control experiments values (Table 1). The values suggest that even though the initial acetic acid concentrations ranged from 0 to 6.2 g/L in the poplar hydrolysates, the acetic acid at these levels did not pose measurable inhibition to the final ethanol productivity, mainly because the pH of fermentation was maintained at 5.5–6.0.

In contrast, hydrolysates with low pH, high concentrations of phenolic compounds, or high acetic acid hydrolysates fermented quite poorly, as shown in Figure 4. The ARP pretreated liquor consists of a representative stream of lignin and its degradation phenolic compounds, which is believed to be potential inhibitors to fermentation.16 The fermentation was carried out by supplementing the liquor with chemical-grade glucose and xylose, resulting in 45 g/L glucose and 28.8 g/L xylose. The pH was adjusted to 6. Glucose consumption rate was unaffected, and complete glucose fermentation was reached within 6 h. However, xylose consumption was substantially affected: only 64.3% of xylose was consumed in the 48 h fermentation period (Figure 4), with a maximum xylose consumption rate of only 0.83 g L−1 h−1. In another experiment, corn stover pretreated by diluted acid pretreatment (supplied by NREL) generated a pretreatment liquor containing 13.3 g/L initial acetic acid, which led to only 60.3% xylose consumption at the end of the 48-h fermentation (Figure 4). Poplar hydrolysate pretreated by the SO2-catalyzed steam-explosion was also subjected to fermentation, while the pH was not adjusted to six but instead was around five. There was 7.3 g/L initial acetic acid present, and the xylose barely fermented (only 7.5% was consumed as shown in Figure 4). The poor fermentation performance could be associated with the high concentrations of undissociated acetic acid.

thumbnail image

Figure 4. Inhibitors effects on xylose fermentation (1) ARP pretreated poplar liquor (mainly phenolic compounds from lignin) supplemented with chemical-grade glucose/xylose (2) Diluted acid pretreated corn stover hydrolysate (with 13.3 g/L acetic acid, 2.1 g/L furfural, and 2.7 g/L HMF) (3) Uncatalyzed steam-explosion pretreated poplar with pH 5 fermentation condition (• – Glucose, ○ – Xylose, ▾ – Xylitol, ▵ – Glycerol, ▪ – Acetic acid, □ – Ethanol).

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgements
  8. Literature Cited

Inhibitors resulting from biomass pretreatment processes can pose serious hurdles to subsequent ethanol fermentation. Poor performance of ethanol fermentation in S. cerevisiae and other microorganisms has been linked to the presence of furfural, HMF, and their synergistic action with acetic acid in the hydrolysate. In this study, the inhibitory effects of furfural, acetic acid and pH on the ethanol fermentation performance by a recombinant S. cerevisiae strain (LNH-ST 424A) in YEPDX medium were investigated. With this particular yeast strain, initial furfural concentrations below 5 g/L did not inhibit glucose/xylose consumption in batch fermentations with high inoculum (4.5–6.0 g/L). At higher initial furfural concentrations (10 g/L or more), the xylose consumption rate is more significantly affected than glucose consumption rate. Initial acetic acid concentrations higher than 10 g/L slows the xylose fermentation rate, and coupled with low pH the inhibitory effect is especially severe. Based on these principles, optimal fermentation can be obtained by controlling the acetic acid level and the pH, which was further demonstrated by the fermentation results of poplar hydrolysates processed by the various CAFI pretreatments. Examples of low-efficiency fermentations were presented to illustrate the inhibitory effects of high acetic acid concentration, dissolved phenolic compounds, and low pH conditions. Therefore, a pretreatment process that is capable of preferentially removing inhibitory compounds from the final hydrolysate liquid, fractionates the acetate from the sugars, or improvements to microbial strains to resist or metabolize these inhibitors may improve fermentation performance. Control of fermentation pH conditions is a remedial approach to minimize acetic acid inhibition. The results from this study provide feedback on the direction of how the pretreatment and hydrolysis steps should be optimized to improve fermentation performance in an integrated cellulosic ethanol production process.


  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgements
  8. Literature Cited

Material in this work is supported by US Department of Energy Office of the Biomass Program, Contract DE-FG36-04GO14017 as part of the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI). The authors thank all the CAFI team collaborators for providing the optimally pretreated poplar samples and for comments on this work. They thank Genencor, a Danisco Division, for gifts of the enzymes used in this work. They also thank Chia-Ling Wu and Shanying Tao in LORRE for technical support with the fermentations, and Youngmi Kim and Eduardo Ximenes for internal review of the manuscript.

Literature Cited

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussions
  6. Conclusions
  7. Acknowledgements
  8. Literature Cited
  • 1
    Lin Y, Tanaka S. Ethanol fermentation from biomass resources: current state and prospects. Appl Microbiol Biotechnol. 2006; 69: 627642.
  • 2
    Wang M. The debate on energy and greenhouse gas emissions impacts of fuel ethanol, Presented at the Energy Systems Division Seminar, Argonne National Laboratory, Chicago, IL, Aug. 3, 2005.
  • 3
    Kim S, Dale BE. Life cycle assessment of various cropping systems utilized for producing biofuels: bioethanol and biodiesel. Biomass Bioenergy. 2005; 29: 426439.
  • 4
    Farrell AE, Plevin RJ, Turner BT, Jones AD, O'Hare M, Kammen DM. Ethanol can contribute to energy and environmental goals. Science. 2006; 311: 506508.
  • 5
    Ragauskas AJ, Williams CK, Davison BH, Britovsek G, Cairney J, Eckert CA, Frederick WJ, Hallett JP, Leak DJ, Liotta CL, Mielenz JR, Murphy R, Templer R, Tschaplinski T. The path forward for biofuels and biomaterials. Science. 2006; 311: 484489.
  • 6
    Somerville C. The billion-ton biofuels vision. Science. 2006; 312: 12771277.
  • 7
    Himmel ME, Ding SY, Johnson DK, Adney WS, Nimlos MR, Brady JW, Foust TD. Biomass recalcitrance: engineering plants and enzymes for biofuels production. Science. 2007; 315: 804807.
  • 8
    Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple M, Ladisch M. Features of promising technologies for pretreatment of lignocellulosic biomass. Biores Technol. 2005; 96: 673686.
  • 9
    Stephanopoulos G. Challenges in engineering microbes for biofuels production. Science. 2007; 315: 801804.
  • 10
    Lynd LR, Wyman CE, Gerngross TU. Biocommodity engineering. Biotechnol Prog. 1999; 15: 777793.
  • 11
    Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY. Coordinated development of leading biomass pretreatment technologies. Biores Technol. 2005; 96: 19591966.
  • 12
    Palmqvist E, Hahn-Hagerdal B. Fermentation of lignocellulosic hydrolysates. II: inhibitors and mechanisms of inhibition. Biores Technol. 2000; 74: 2533.
  • 13
    Palmqvist E, Grage H, Meinander NQ, Hahn-Hagerdal B. Main and interaction effects of acetic acid, furfural, and p-hydroxybenzoic acid on growth and ethanol productivity of yeasts. Biotechnol Bioeng. 1999; 63: 4655.
  • 14
    Luo CD, Brink DL, Blanch HW. Identification of potential fermentation inhibitors in conversion of hybrid poplar hydrolyzate to ethanol. Biomass Bioenergy. 2002; 22: 125138.
  • 15
    Lawford HG, Rousseau JD, Mohagheghi A, McMillan JD. Fermentation performance characteristics of a prehydrolyzate-adapted xylose-fermenting recombinant Zymomonas in batch and continuous fermentations. Appl Biochem Biotechnol. 1999; 77–79: 191204.
  • 16
    Larsson S, Palmqvist E, Hahn-Hagerdal B, Tengborg C, Stenberg K, Zacchi G, Nilvebrant NO. The generation of fermentation inhibitors during dilute acid hydrolysis of softwood. Enzyme Microb Technol. 1999; 24: 151159.
  • 17
    Oliva JM, Negro MJ, Saez F, Ballesteros I, Manzanares P, Gonzalez A, Ballesteros M. Effects of acetic acid, furfural and catechol combinations on ethanol fermentation of Kluyveromyces marxianus. Process Biochem. 2006; 41: 12231228.
  • 18
    Klinke HB, Thomsen AB, Ahring BK. Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass. Appl Microbiol Biotechnol. 2004; 66: 1026.
  • 19
    Ho NWY, Chang SF. Cloning of yeast xylulokinase gene by complementation of Escherichia coli and yeast mutations. Enzyme Microb Technol. 1989; 11: 417421.
  • 20
    Ho NWY, Chen ZD, Brainard AP. Genetically engineered Sacccharomyces yeast capable of effective cofermentation of glucose and xylose. Appl Environ Microbiol. 1998; 64: 18521859.
  • 21
    Ho N, Chen ZD. Stable recombinant yeasts capable of effective fermentation of both glucose and xylose. WO 97/42307, 1997.
  • 22
    Ho N, Chen ZD, Brainard AP, Sedlak M. Successful design and development of genetically engineered Saccharomyces yeasts for effective cofermentation of glucose and xylose from cellulosic biomass to fuel ethanol. In: TsaoGT, editor. Advances in Biochemical Engineering/Biotechnology. Berlin, Heidelberg: Springer-Verlag; 1999.
  • 23
    Ho N, Tsao GT. Recombinant yeasts for effective fermentation of glucose and xylose, US Patent 5,789,210, 1998.
  • 24
    Banerjee N, Bhatnagar R, Viswanathan L. Inhibition of glycolysis by furfural in Saccharomyces cerevisiae. Eur J Appl Microbiol Biotechnol. 1981; 11: 226228.
  • 25
    Axe DD, Bailey JE. Transport of lactate and acetate through the energized cytoplasmic membrane of Escherichia coli. Biotechnol Bioeng. 1995; 47: 819.
  • 26
    Taherzadeh MJ, Niklasson C, Liden G. Acetic acid—friend or foe in anaerobic batch conversion of glucose to ethanol by Saccharomyces cerevisiae? Chem Eng Sci 1997; 52: 26532659.
  • 27
    Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY. Comparative sugar recovery data from laboratory scale application of leading pretreatment technologies to corn stover. Biores Technol. 2005; 96: 20262032.
  • 28
    Sedlak M, Ho NWY. Characterization of the effectiveness of hexose transporters for transporting xylose during glucose and xylose co-fermentation by a recombinant Saccharomyces yeast. Yeast. 2004; 21: 671684.
  • 29
    Sedlak M, Ho NWY. Production of ethanol from cellulosic biomass hydrolysates using genetically engineered Saccharomyces yeast capable of cofermenting glucose and xylose. Appl Biochem Biotechnol. 2004; 113–116: 403416.