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Comparison of Glucose/Xylose Cofermentation of Poplar Hydrolysates Processed

by Different Pretreatment Technologies

Yulin Lu
Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907
Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN 47907

Ryan Warner
Genencor, a Danisco Division, Palo Alto, CA

Miroslav Sedlak
Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907
Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN 47907
Nancy Ho
Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN 47907
School of Chemical Engineering, Purdue University, West Lafayette, IN 47907
Nathan S. Mosier
Department of Agricultural and Biological Engineering, Purdue University, West Lafayette, IN 47907
Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, IN 47907

DOI 10.1021/bp.158
Published online March 24, 2009 in Wiley InterScience (

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 inhibi-
tion 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 sig-
nificant with xylose consumption rates especially affected. Interactive inhibition between ace-
tic 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 explo-
sion, and controlled-pH) under respective optimal conditions was enzymatically hydrolyzed,
and the mixed sugar streams in the hydrolysates were fermented. The 5-hydroxymethyl furfu-
ral (HMF) and furfural concentrations were low in all hydrolysates and did not pose nega-
tive 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. V C 2009

American Institute of Chemical Engineers Biotechnol. Prog., 25: 349–356, 2009

Keywords: recombinant Saccharomyces cerevisiae LNH-ST 424A, xylose fermentation,
ethanol, pretreatment, acetic acid, furfural, fermentation inhibitors

Introduction hydrolysis processes,7,8 and engineering of robust industrial

microbes that are capable of fermenting mixed streams of
Materials hexose and pentose derived from lignocellulosic biomass.9,10
Ethanol as a renewable fuel can be produced domesti- Among the major technical hurdles for cellulosic ethanol
cally,1 can displace greenhouse gas emissions from the use production, pretreatment is one of the most costly processing
of petroleum-based transportation fuels,2–4 and provide a steps, and has significant influences on the following enzy-
renewable energy source to combat the diminishing global matic hydrolysis and ethanol fermentation efficiencies.11 To
energy supply.5,6 Realizing these advantages will require tackle this important issue and provide insight into the devel-
several technological advancements, which include develop- opment of an advanced biomass deconstruction platform, the
ment of cost-effective cellulosic biomass pretreatment and Biomass Refining Consortium for Applied Fundamentals and
Innovation (CAFI) was formed in 2000 and initiated collabo-
Correspondence concerning this article should be addressed to N. S. rative research on this subject. The initial studies by the
Mosier at CAFI groups focused on corn stover as a biomass feedstock,

C 2009 American Institute of Chemical Engineers

V 349
350 Biotechnol. Prog., 2009, Vol. 25, No. 2

and results, conclusions, and recommendations were dis- S. cerevisiae 424A (LNH-ST) used in this study was devel-
cussed in a special volume of ‘‘Bioresource Technology’’.11 oped and provided by Dr. Nancy Ho. The genetically engi-
The unique strength of the CAFI research approach lies in neered S. cerevisiae 424A (LNH-ST) was constructed by
results comparability, through the use of a single feedstock, integrating multiple copies of XD, XR, and XK into the chro-
identical analytical methods [adapted from National Renew- mosomes of S. cerevisiae ATCC 4124 according to the tech-
able Energy Laboratory (NREL) LAPs], and a consistent nology reported by Ho et al.18–21 The 424A (LNH-ST) strain
approach to data interpretation employed by all the partici- was maintained in liquid YEPX medium, which includes 20 g/
pating groups.11 Such an approach allows valuable compari- L of Difco peptone (Becton Dickinson, St. Louis, MO), 10 g/L
sons among each of the optimized pretreatment technologies, of Difco yeast extract (Becton Dickinson), and 20 g/L of
and sheds lights on the direction for improvement/develop- xylose. Fresh seed cultures were prepared by inoculating the
ment of advanced biomass depolymerization platform at respective seed cultures in 100 mL of YEPX in 300-mL
reduced cost. baffled Erlenmeyer flask equipped with a sidearm (BELLCO).
In the second phase of the CAFI project, a woody biomass The cultures were incubated in a controlled environment incu-
feedstock, poplar, was the feedstock of study. Compared to bator shaker (New Brunswick Scientific, Edison, NJ) at 30 C
corn stover (11.0% mass in lignin), polar has a significantly and 200 rpm and grown aerobically overnight. The following
higher percentage of lignin content (29.1% mass in lignin), morning, when the cultures reached OD 450–500 Klett-unit
which causes additional structural barriers to effective pre- (KU) (OD 400 KU corresponds to a cell mass concentration of
treatment and enzymatic hydrolysis. This additional hurdle is about 5 g dry wt/L), the flasks containing the seed cultures
discussed in the other articles of this special issue as the were stored in a refrigerator at 4 C until used.
CAFI teams optimized the pretreatment technologies (dilute
sulfuric acid, ammonia recycle percolation, lime, ammonia
fiber/freeze expansion, SO2-catalyzed steam explosion, and Methods
controlled-pH liquid hot water). Although pretreatment is Pretreated Solids Preparation. The detailed description
required to improve reactivity of this recalcitrant substrate to and modes of action of the various pretreatment processes
enzymatic hydrolysis,8 an effective pretreatment can also can be found in previously published articles8,11 and will not
create compounds that are inhibitory to microbial fermenta- be reiterated here. The optimization methodologies for these
tion through degradation of monomeric sugars (especially pretreatments with poplar as the biomass substrate are dis-
furfural from pentoses and 5-hydroxymethyl furfural (HMF) cussed in other articles of this issue. A brief summary on
from hexoses), through solubilization of lignin compounds, how the poplar materials were pretreated for use in this
and through release of acetic acid during hemicellulose hy- study is given below:
drolysis.12–18 Therefore, it is critically important to evaluate • Dilute sulfuric acid pretreatment (National Renewable
pretreatment by examining the glucose/xylose fermentation
Energy Laboratory)
performance simultaneously during the course of pretreat-
A 900 kg/day pilot-scale reactor at NREL was used to
ment optimization.
carry out the dilute sulfuric acid pretreatment of poplar. The
This study is the evaluation of ethanol fermentation per- reaction condition was set at 190 C, 0.048 g sulfuric acid
formance of mixed sugar streams from pretreatment and en- per g biomass (dry); the residence time was about 1.5 min,
zymatic hydrolysis by a single robust microorganism. The at a solid-loading of 250 g/L biomass loading.
availability of recombinant Saccharomyces cerevisiae (LNH- • Oxidative lime pretreatment (Texas A&M University)
ST 424A)19–21 that ferments both glucose and xylose made it During the oxidative lime pretreatment, the poplar mate-
possible for a side-by-side comparison of the individually rial was submitted to the following conditions: 150 C reac-
optimized pretreatment processes. The two main potential tion temperature, oxygen was supplied at 200 psi, lime
fermentation inhibitors, furfural and acetic acid, were exam- loading was controlled in excess of 0.4 g lime/g-dry-poplar,
ined with respect to their impacts on the glucose/xylose and the pretreatment lasted for 6 h. Once the pretreatment
cofermentation in YEPDX medium. After that, poplar bio- time elapsed, the pretreatment liquor was separated from the
mass processed by the various pretreatments under their re- solids. The excess of lime in the solids was neutralized using
spective optimal conditions was subjected to a 7-day water and 5N HCl, and was extensively washed afterwards.
enzymatic hydrolysis, and the hydrolysates were fermented • Ammonia recycle percolation (Auburn University)
for 48 h. The relative inhibitory effects of furfural, acetic The ARP reactor was operated in a flow-through mode,
acid and pH of the hydrolysates are discussed. Understand- with a reaction temperature of 185 C, a residence time of
ing to the key influential inhibitory factors to fermentation 27.5 min, and a NH3 to biomass ratio of 3.667:1. The solids
suggests on strategies to optimize upstream processes, i.e. loading in the reactor was 260 g/L poplar (dry).
pretreatment and enzymatic hydrolysis, depending on com- • Ammonia fiber/freeze expansion (Michigan State Uni-
positional characteristics of the biomass. versity)
During the AFEX pretreatment, the poplar biomass was
presoaked in water for 24 h, NH3-to-poplar ratio was set at
Materials and Methods 1:1, and the residence time was 30 min at a reaction temper-
ature of 180 C.
Materials • SO2-catalyzed steam explosion (University of British
The poplar used in this study was supplied by USDA and Columbia)
distributed by the NREL to the CAFI project participants. The SO2-catalyzed steam explosion pretreatment was
NREL debarked, chipped and milled the poplar materials to carried out at 200 C, for 5 min of reaction, with a 3%
pass through a 1=4 in. screen. Milled poplar was stored at SO2-gas load.
20 C until used. All chemicals were obtained from Sigma- • Controlled-pH, Liquid hot water pretreatment (Purdue
Aldrich (St. Louis, MO) unless otherwise specified. University)
Biotechnol. Prog., 2009, Vol. 25, No. 2 351

The pretreatments were carried out in 1 in. stainless steel sampling was accomplished by removing the plastic wrap-
tubes (45 mL total volume). The reaction temperature was ping and foam plug from the top of the flasks and removing
controlled at 200 C and the pretreatment lasted for 15 min an aliquot with a pipette. Samples were obtained more fre-
(include 5-min heat-up time þ 10-min reaction time). A hot- quently early in the fermentation (every 3–6 h) and less fre-
washing strategy was used to remove inhibitors to enzymatic quently (every 12–24 h) although the xylose was fermented
hydrolysis. Each batch of pretreated solids was washed with alone in the later stages. The times of sampling are shown in
500 mL 80–90 C DI H2O. the graphs in Figures 1, 3, and 4. The fermentation experi-
Enzymatic Hydrolysis of the Pretreated Poplar. Depend- ments were run in duplicate at each condition.
ing on the physical nature of the received pretreated poplar, For the furfural inhibition experiments, 0, 5, 10, and 15 g/
the pretreated poplar materials were prepared as follows L furfural was added to the YEPDX medium. YEPDX media
before enzymatic hydrolysis: The dilute acid pretreated was prepared by dissolving 10 g of yeast extract and 20 g of
NREL sample was received as wet solid pretreatment resid- peptone per liter of distilled water. After autoclaving the
uals and blown-out liquid separately. The pH of the liquid YEP media, dry dextrose (glucose) and dry xylose were
was adjusted to 4.8 by addition of Ca(OH)2. The solid pre- added to achieve initial concentrations of 35 g/L. Dry sug-
treatment residuals were then added into the pH 4.8 liquid at ars were added to autoclaved YEP to avoid thermal degrada-
200 g/L dry solid-loading. The AFEX, Lime (O2) and ARP tion of the sugars during autoclaving. Acetic acid inhibition
pretreated samples were received as solids without pretreat- experiments used a 4  3 full factorial experimental design,
ment liquid/liquor. Therefore 50 mM citrate buffer (pH 4.8) with initial acetic acid concentrations of 0, 5, 10, and 15 g/
was added to prepare a 200 g/L dry solid-loading solution. L, and pH of 5.0, 5.5, and 6.0. Each experimental condition
No overliming was used for the pH adjustment. The SO2-cat- was repeated twice.
alyzed steam-explosion pretreated poplar was directly hydro- HPLC Analysis. The enzymatic hydrolysate and ethanol
lyzed in 50 mM citrate buffer (pH 4.8) by our colleagues at fermentation samples were analyzed by HPLC. The HPLC
the University of British Columbia and filtered. The liquid system consists of a Bio-Rad HPX-87H organic acid col-
hydrolysate was shipped frozen and stored at 20 C until umn (Bio-Rad Laboratories, Hercules, CA) in a HPLC sys-
used. The controlled-pH, liquid hot water pretreated poplar tem consisting of a Milton Roy minipump (Milton Roy,
was hot-washed and hydrolyzed at 150 g/L dry solids load- Ivyland, PA), Waters 717 plus autosampler, Waters R401
ing. See Kim et al. for details in this issue. differential refractometer (Waters, Milford, MA), and a per-
Enzymes used in this study, spezyme CP (Lot no. 301- sonal computer with Empower software for HPLC opera-
04075-054) and multifect xylanase (Lot no. 301-04021-015), tion control. The mobile phase was 5 mM sulfuric acid in
were supplied by Genencor, a Danisco Division (Rochester, distilled, deionized water filtered through 0.2 lm nylon fil-
NY). The spezyme CP had a cellulase activity of 59 FPU/ ters. Operating conditions for the HPLC column were 60 C
mL, with a total protein content of 123 mg/mL; the multifect with a mobile phase flow rate of 0.6 mL/min. For sample
xylanase had a xylanase activity (Oat spelt xylan) of 25,203 analysis, 50 lL of sample was injected and complete sam-
IU/mL, and a total protein content of 42 mg/mL. For each ple elution could be accomplished within 55 min per injec-
hydrolysis, 60 FPU/g-total-glucan spezyme CP was added to tion. Each sample was analyzed by HPLC in triplicate. The
the 200 g/L dry solid-loading solution (pH 4.8), supple- average coefficient of variation (standard deviation divided
mented with an equivalent amount (mass of protein) of mul- by mean) for the replication injections was less than 3% for
tifect xylanase. The enzymatic hydrolysis (with a total liquid all compounds reported. A standard solution was prepared
volume of 100 mL) was held in an Innova 2000 shaker incu- by dissolving pure ([99% purity) compounds (glucose,
bator set at 50 C, 190 rpm for a 7-day saccharification reac- xylose, glycerol, xylitol, furfural, acetic acid, and ethanol)
tion. The resulting hydrolysate liquid was separated from the in the HPLC mobile phase. Fractional dilutions of the
solid by filtration, and the liquid was adjusted to pH 5.5–6.0 standard solution ranging from 0.5–4 g/L were prepared to
before fermentation by addition of Ca(OH)2 or NaOH. provide standards for HPLC calibration. The linear regres-
Glucose/Xylose Cofermentation. Glucose/xylose cofer- sion for the curves between the elution peak area and con-
mentation was carried out at 28.5 C for 48 h using the glu- centration was computed to give [99.9% R-square values
cose and xylose-fermenting recombinant yeast 424A (LNH- for all compounds. Other sugars and organic acids (fruc-
ST). Eight milliliter of seed culture was used to inoculate tose, lactic acid, formic acid, 4-O-methyl glucuronic acid)
100 mL YEPD (YEP plus 2% glucose) in a 300 mL baffled are resolved by this HPLC method, but were either not
sidearm Erlenmeyer flask. Cultures were incubated in a detected or detected as minor peaks in the samples and thus
shaker at 28.5 C and 200 rpm and grown aerobically over- not quantified. Xylose and galactose coelute from the HPX-
night, after which a cell optical density (OD) between 450– 87H column operated under these conditions. However,
500 KU was obtained. Cultured yeast cells were harvested complete compositional analyses of the poplar substrate
by centrifugation (J-21 Beckman) at 5000 rpm for 5 min at performed at the National Renewable Energy Laboratory
room temperature. The supernatant was discarded and the found that the galactan content of the raw poplar was 1%
cells were transferred into a 300 mL baffled Erlenmeyer of dry mass, compared to 14.9% of dry matter that was
flask containing 100 mL of either YEPDX medium (for the xylan, and thus not quantified for this study.
furfural/acetic acid effects experiments) or the poplar hydrol- Calculation of Yields. Both metabolic and overall pro-
ysate from enzymatic hydrolysis. Concentration of initial cell ductivity yields were calculated to evaluate and compare the
mass before fermentation in each experiment was about 5 g fermentation performance of the enzymatic poplar hydroly-
dry weight/L. The flasks were then sealed with plastic wrap sates from the different pretreatments examined. The meta-
to allow fermentation to be carried out under microaerobic bolic yield determines the efficiency of the fermentative
conditions. The cultures were placed in the shaker and incu- metabolism, while the productive yield evaluated the ethanol
bated at 28.5 C. At regular intervals 1 mL samples of the yield compared to the theoretical yield based upon the sugars
fermentation mixture were removed for HPLC analysis. The initially present.
352 Biotechnol. Prog., 2009, Vol. 25, No. 2

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 Furfural (Initial)
Production Rates
(g L1 hr1) 0 g/L 5 g/L 10 g/L 15 g/L
Glucose 9.5 8.1 6.6 2.8
Xylose 2.5 2.0 0.5 0.3
Furfural n/a [2.0 2.8 2.9
Ethanol 4.8 3.8 3.0 1.2

are consistent with previously reported glucose/xylose cofer-

mentation results for this yeast strain.22,23
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 furfu-
ral concentrations. Above 5 g/L initial furfural, more signifi-
cant 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 L1 hr1, 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 L1 hr1, 80% lower than the control (2.5 g L1
hr1). Furthermore, the fermentation of xylose largely occurs
Figure 1. Fermentation of glucose, and xylose to ethanol by S. after the furfural concentration falls below the detectable
cerevisiae 424A (LNH-ST) in the presence of varying limit. In all cases the rate of furfural metabolism is approxi-
initial concentrations of furfural (l – 0 g/L furfural;
!– 5.0 g/L furfural; n –10 g/L furfural; h –15 g/L mately the same ([2.0–2.8 g L1 hr1), and furfural concen-
furfural). trations in the media fall below the detectable limit before
all of the sugars are consumed.
This trend continues to 15 g/L initial furfural concentra-
tion, which corresponds to the tolerance limit for this yeast
strain at this cell concentration. In the case of 15 g/L initial
Metabolic Yield ¼ ½ethanol=½0:51  ðconsumed glucose furfural, glucose consumption is 30% of the control case.
þ consumed xyloseÞ ð1Þ Furfural is metabolized concurrently with glucose consump-
tion. The furfural concentration fell below detectable limit
after 12 h and glucose was completely consumed within
Productive Yield ¼ ½ethanol=½0:51  ðinitial glucose 20 h. However the consumption of xylose, which begins at
þ initial xyloseÞ ð2Þ after 12 h, was significantly affected (10% of the consump-
tion rate of the control). No significant amount of xylose
was consumed after 30 h. These results suggest that the in-
hibitory effects of furfural with regard to xylose fermentation
Results and Discussions
extend beyond the effects of the presence of furfural in the
The inhibitory effects of furfural/acetic acid on fermentation media. Membrane adsorbed furfural, intracellu-
glucose-xylose cofermentation lar furfural, the metabolic product of furfural conversion
The effects of furfural on glucose/xylose cofermentation (furfuryl alcohol and furoic acid), or byproducts of furfural
in YEPDX medium are summarized in Figure 1. Each sub- metabolism (redox imbalance and/or oxidative damage) may
figure is a side-by-side comparison of glucose, xylose, or continue to affect the metabolism of the yeast long after the
ethanol concentrations over the time course of fermentation. furfural in the bulk media is removed.
Under the control conditions (no furfural added), complete At higher than 15 g/L initial furfural, little metabolism of
fermentation of glucose (30 g/L initial concentration) either sugar was observed. The results suggest that the inhib-
occurred in less than 6 h and complete xylose (35–40 g/L itory effects of furfural is due to more than one mechanism,
initial concentration) fermentation was accomplished in less such as inhibition of glycolytic enzymes,24 redox imbalance,
than 48 h. The average maximum glucose consumption rate, or inactivation of the cells over time due to toxicity from the
computed by averaging the slopes of the tangent line to the accumulation of intracellular acetaldehyde as the furfural is
greatest rate of consumption, was 9.5 g L1 hr1 (Table 1). metabolized.12 The reason for increased sensitivity of xylose
By contrast, the average maximum xylose consumption rate fermentation to furfural is not yet clear.
was approximately one quarter that of glucose (2.5 g L1 Previous research on inhibition of Zymomonas fermenta-
hr1). The maximum average ethanol production rate, which tion of pretreated poplar showed that acetic acid/acetate was
occurs during glucose consumption, was calculated to be 4.8 the predominant inhibitor.15 Thus impact of acetic acid on
g L1 hr1. The metabolic yield of ethanol from both glu- xylose consumption rates in this strain of S. cerevisiae was
cose and xylose averaged 85% of theoretical. These results examined (Figure 2). Not only acetic acid concentration, but
Biotechnol. Prog., 2009, Vol. 25, No. 2 353

Table 2. Composition of Enzyme Hydrolysates from Poplar

Pretreated by Various Pretreatment Technologies (60 FPU/g-total
Glucan Spezyme CP 1 Equivalent Mass of Protein of Multifect
Xylanase for 168 h at 508C)
Glucose Xylose Acetic
Pretreatment* (g/L; Yield) (g/L; yield) Acid (g/L)
Dilute acid 41.4 41.6% 22.3 67.3% 5.1
SO2 explosion 33.2 33.4% 25.8 77.9% 6.2
Controlled pH† 56.1 54.0% 12 27.0% 2.3
Lime (O2) 52.8 53.0% 16 48.3% n/d
ARP 32.7 32.8% 8 24.2% n/d
AFEX 62.3 62.6% 16.2 48.9% 3.5
* HMF/furfural was not detected in any hydrolysate, which suggested
that sugar degradation reaction was minimized for the optimal pretreat-
ments. † Hydrolysis continued for 5 days, Enzyme loading: 15 FPU/g-
total glucan Spezyme CP þ 40 U/ g-total glucan Novo188, No Multifect
Xylanase was used. n/d ¼ not detected by HPLC.

which may lead to 7.2–11.2 g/L acetic acid in the hydroly-

sate 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 ace-
tic acid, a careful conditioning of hydrolysate is necessary to
mitigate its inhibitory effects on glucose/xylose cofermenta-
tion. 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 pop-
Figure 2. The interaction effect of pH and acetic acid concen-
tration on xylose consumption rates during the glu- lar hydrolysates from the CAFI pretreatments as follows.
cose/xylose cofermentation by S. cerevisiae 424A
Glucose/xylose cofermentation of poplar hydrolysates
The composition pretreated poplar hydrolysates are listed
also the interactive effect of pH and acetate concentration in Table 2. Because of the fact that the enzyme mixture used
was evaluated with respect to xylose consumption rate. In in this study was not optimal, but rather dosed to provide a
the range of acetic acid concentrations from 5–15 g/L and consistent biocatalyst loading to all pretreated poplar materi-
pH from 5–6, the xylose consumption rates varied from als, the sugar yields from the 7 day saccharification were
0.135 (4.8% of control) to 2.8 g L1 hr1 (control). When lower than expected: glucose yields ranged from 33 to 63%,
there was no acetic acid present, varying the fermentation while xylose yields were in the range of 24–78% (Table 2).
pH in the range of 5–6 did not affect the xylose consumption Compared to yields obtained with corn stover as the biomass
rate; however, the pH-dependant inhibition became evident feedstock ([85%) under optimal conditions,27 the combined
when 5–20 g/L acetic acid was added to the fermentation. sugar yields from pretreatment and enzymatic hydrolysis of
When 5 g/L acetic acid was present, the xylose consumption poplar seemed to be significantly less efficient. The higher
rates were between 0.86 (34.4% of control)–1.55 g L1 hr1 lignin content of the poplar (29% compared to 17% in corn
(59.6% of control) with correspondent pH values between 5 stover) may account for this. In addition, inhibition of the
and 6. An increasing inhibitory trend was observed as the enzymes by high concentrations of glucose and xylose likely
acetic acid concentration was raised to 10–15 g/L (Figure 2). limited the extent of conversion. Conducting a simultaneous
The pH effect became profound at high acetic acid concen- saccharifaction and fermentation (SSF) would likely alleviate
tration, e.g. at 15 g/L acetic acid concentration, the xylose this inhibition. However, for these experiments we chose to
consumption rate at pH 5 was 0.135 g L1 hr1, only of examine separate saccharifaction and fermentation to elimi-
20.1% of that in pH 6 medium (0.67 g L1 hr1). This can nate the confounding effect of potential differences in enzy-
be explained by the membrane transportability of acetic acid matic hydrolysis rates and difficulty in computing metabolic
at the two different pHs. The undissociated form of acetic yield of ethanol production due to the difficulty in accurately
acid can freely diffuse across the cytoplasmic membrane, measuring total sugar release in SSF.
dissociate, lower the intracellular pH, and disturb normal cel- After the 7 day enzymatic hydrolysis of poplar pretreated
lular metabolism. However, the charged, dissociated form of by each CAFI pretreatment was completed, the hydrolysate
acetic acid cannot diffuse across the cytoplasmic mem- liquid was filtered from the residual pretreated poplar solids,
brane.25,26 Since the pKa of acetic acid is 4.75, at pH 5 the adjusted to pH 5.5–6.0, and subjected to a 48 h glucose/
undissociated [HA]-to-dissociated [A] ratio is 0.56:1, while xylose cofermentation. The fermentation profiles are sum-
at pH 6 this value changes to 0.056:1 (a magnitude lower). marized in Figure 3. Since all the CAFI pretreatments have
Therefore, there is 10 times more undissociated acetic acid been optimized to reduce furfural/HMF accumulation, mini-
at pH 5 than that at pH 6. mal amounts of furfural/HMF were detected in the enzy-
Acetic acid is ubiquitously present in all types of cellu- matic hydrolysate. However, acetic acid concentrations
losic biomass materials and its release is inevitable during varied from 0 to 6.2 g/L in these hydrolysates (Table 2). A
hemicellulose hydrolysis (5.6% acetyl by mass in CAFI 2 summary of the fermentation products composition at the
corn stover, and 3.6% acetyl by mass in CAFI 2 poplar), conclusion of the 48 h fermentation is also shown in Table 3.
354 Biotechnol. Prog., 2009, Vol. 25, No. 2

For the fermentation of poplar hydrolysate prepared from tible xylitol formation. The metabolic ethanol yield reached
controlled-pH pretreatment, glucose was readily fermentable 86.8%. No acetic acid was detectible in any of these sam-
within 6 h, however only of 38.0% of the initial xylose was ples. During the fermentation of dilute sulfuric acid pre-
fermented in that time. A total of 80.0% of available xylose treated poplar hydrolysates, the glucose was completely
was fermented by the end of the fermentation, with no detec- 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 through-
out 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 detecti-
ble acetic acid. After 6 h of fermentation, 81.2% of the glu-
cose 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 fermen-
tation 90.4% of xylose was consumed, and the metabolic
ethanol yield was near quantitative (Table 4). The ARP proc-
essed 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 pre-
treatment, 96.5% of glucose and 12.2% of xylose were con-
sumed 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 perform-
ance of poplar hydrolysate prepared from the SO2-catalyzed
steam explosion pretreatment was similar to the ARP fer-
mentation 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).
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, pro-
ductive yield, and average and maximum ethanol productiv-
ity had to be calculated to differentiate the fermentation
performances. Based on calculated metabolic, productive
yields and average ethanol productivity, hydrolysates proc-
essed by the lime (O2) pretreatment fermented most effec-
tively. 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
Figure 3. Glucose/xylose fermentation profiles of poplar hydro- there were differences between the hydrolysates in the initial
lysates processed by various pretreatments under rates of fermentation. Therefore, the 6 h maximum ethanol
optimal conditions (l – Glucose, * – Xylose, ! –
Xylitol, ~ – Glycerol, n – Acetic acid, h – Ethanol).
productivities were computed and compared (Table 4). All

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)
Dilute acid 0 2.7 0.6 3.9 5.0 26.4
SO2 Explosion 0 2.4 0.8 3.8 6.4 25.9
Controlled pH 0.4 2.4 0.0 2.7 2.5 29.4
Lime (O2) 0.3 1.5 0.0 6.5 0.6 39.9
ARP 0 0 0.0 3.5 0.0 20.5
AFEX 0.2 3.5 0.5 5.5 3.9 35.5
Biotechnol. Prog., 2009, Vol. 25, No. 2 355

Table 4. Summary of Yields and Ethanol Productivities in the Cofermentation of Poplar Hydrolysates
From Different Pretreatment Technologies
Metabolic Yield Productive Yield Xylose Consumption Average Ethanol 6-h Maximum Ethanol
Pretreatment (% of Theoretical)* (% of Theoretical)† in 48 h (%) Productivity (g L1 h1) Productivity (g L1 h 1)
Dilute acid 85.0% 81.4% 87.8% 0.55 4.7
SO2 explosion 89.9% 86.2% 90.8% 0.54 4.7
Controlled pH 86.8% 82.7% 80.0% 0.60 4.7
Lime (O2) 100% 100% 90.4% 0.83 4.7
ARP 98.6% 98.6% 100% 0.43 4.6
AFEX 93.0% 88.6% 78.7% 0.74 4.8
* Metabolic Yield ¼ [ethanol]/[0.51 * (consumed glucose þ consumed xylose)]. † Productive Yield ¼ [ethanol]/[0.51 * (initial glucose þ initial xylose)].

treated 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 con-
sumption rate was unaffected, and complete glucose fermen-
tation was reached within 6 h. However, xylose consumption
was substantially affected: only 64.3% of xylose was con-
sumed in the 48 h fermentation period (Figure 4), with a
maximum xylose consumption rate of only 0.83 g L1 h1.
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 fer-
mentation (Figure 4). Poplar hydrolysate pretreated by the
SO2-catalyzed steam-explosion was also subjected to fermen-
tation, 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 undisso-
ciated acetic acid.

Inhibitors resulting from biomass pretreatment processes
can pose serious hurdles to subsequent ethanol fermentation.
Poor performance of ethanol fermentation in S. cerevisiae
Figure 4. Inhibitors effects on xylose fermentation (1) ARP
and other microorganisms has been linked to the presence of
pretreated poplar liquor (mainly phenolic com- furfural, HMF, and their synergistic action with acetic acid
pounds from lignin) supplemented with chemical- in the hydrolysate. In this study, the inhibitory effects of fur-
grade glucose/xylose (2) Diluted acid pretreated corn fural, acetic acid and pH on the ethanol fermentation per-
stover hydrolysate (with 13.3 g/L acetic acid, 2.1 g/L formance by a recombinant S. cerevisiae strain (LNH-ST
furfural, and 2.7 g/L HMF) (3) Uncatalyzed steam-
explosion pretreated poplar with pH 5 fermentation 424A) in YEPDX medium were investigated. With this par-
condition (l – Glucose, * – Xylose, ! – Xylitol, ~ ticular yeast strain, initial furfural concentrations below 5 g/
– Glycerol, n – Acetic acid, h – Ethanol). L did not inhibit glucose/xylose consumption in batch fer-
mentations with high inoculum (4.5–6.0 g/L). At higher ini-
tial furfural concentrations (10 g/L or more), the xylose
consumption rate is more significantly affected than glucose
the fermentations of hydrolysates from optimized pretreat- consumption rate. Initial acetic acid concentrations higher
ments yielded ethanol productivities comparable to the con- than 10 g/L slows the xylose fermentation rate, and coupled
trol experiments values (Table 1). The values suggest that with low pH the inhibitory effect is especially severe. Based
even though the initial acetic acid concentrations ranged on these principles, optimal fermentation can be obtained by
from 0 to 6.2 g/L in the poplar hydrolysates, the acetic acid controlling the acetic acid level and the pH, which was fur-
at these levels did not pose measurable inhibition to the final ther demonstrated by the fermentation results of poplar hy-
ethanol productivity, mainly because the pH of fermentation drolysates processed by the various CAFI pretreatments.
was maintained at 5.5–6.0. Examples of low-efficiency fermentations were presented to
In contrast, hydrolysates with low pH, high concentrations illustrate the inhibitory effects of high acetic acid concentra-
of phenolic compounds, or high acetic acid hydrolysates fer- tion, dissolved phenolic compounds, and low pH conditions.
mented quite poorly, as shown in Figure 4. The ARP pre- Therefore, a pretreatment process that is capable of
356 Biotechnol. Prog., 2009, Vol. 25, No. 2

preferentially removing inhibitory compounds from the final 12. Palmqvist E, Hahn-Hagerdal B. Fermentation of lignocellulosic
hydrolysate liquid, fractionates the acetate from the sugars, hydrolysates. II: inhibitors and mechanisms of inhibition. Biores
or improvements to microbial strains to resist or metabolize Technol. 2000;74:25–33.
13. Palmqvist E, Grage H, Meinander NQ, Hahn-Hagerdal B. Main
these inhibitors may improve fermentation performance. and interaction effects of acetic acid, furfural, and p-hydroxy-
Control of fermentation pH conditions is a remedial benzoic acid on growth and ethanol productivity of yeasts. Bio-
approach to minimize acetic acid inhibition. The results from technol Bioeng. 1999;63:46–55.
this study provide feedback on the direction of how the pre- 14. Luo CD, Brink DL, Blanch HW. Identification of potential fer-
treatment and hydrolysis steps should be optimized to mentation inhibitors in conversion of hybrid poplar hydrolyzate
improve fermentation performance in an integrated cellulosic to ethanol. Biomass Bioenergy. 2002;22:125–138.
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Acknowledgments and continuous fermentations. Appl Biochem Biotechnol. 1999;
Material in this work is supported by US Department of 16. Larsson S, Palmqvist E, Hahn-Hagerdal B, Tengborg C, Sten-
Energy Office of the Biomass Program, Contract DE-FG36- berg K, Zacchi G, Nilvebrant NO. The generation of fermenta-
04GO14017 as part of the Biomass Refining Consortium for tion inhibitors during dilute acid hydrolysis of softwood.
Enzyme Microb Technol. 1999;24:151–159.
Applied Fundamentals and Innovation (CAFI). The authors 17. Oliva JM, Negro MJ, Saez F, Ballesteros I, Manzanares P, Gon-
thank all the CAFI team collaborators for providing the opti- zalez A, Ballesteros M. Effects of acetic acid, furfural and cate-
mally pretreated poplar samples and for comments on this chol combinations on ethanol fermentation of Kluyveromyces
work. They thank Genencor, a Danisco Division, for gifts of the marxianus. Process Biochem. 2006;41:1223–1228.
enzymes used in this work. They also thank Chia-Ling Wu and 18. Klinke HB, Thomsen AB, Ahring BK. Inhibition of ethanol-pro-
Shanying Tao in LORRE for technical support with the fermen- ducing yeast and bacteria by degradation products produced
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