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
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/L||5 g/L||10 g/L||15 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.
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)|
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).
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
|Pretreatment||Glucose (g/L)||Xylose (g/L)||Xylitol (g/L)||Glycerol (g/L)||Acetic Acid (g/L)||Ethanol (g/L)|
Table 4. Summary of Yields and Ethanol Productivities in the Cofermentation of Poplar Hydrolysates From Different Pretreatment Technologies
|Pretreatment||Metabolic 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)|
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.
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