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 (www.interscience.wiley.com).

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

Materials
Ethanol as a renewable fuel can be produced domesti-
cally,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 develop-
ment of cost-effective cellulosic biomass pretreatment and
Correspondence concerning this article should be addressed to N. S.
Mosier at mosiern@purdue.edu.
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 enzy-
matic hydrolysis and ethanol fermentation efficiencies.11 To
tackle this important issue and provide insight into the devel-
opment of an advanced biomass deconstruction platform, the
Biomass Refining Consortium for Applied Fundamentals and
Innovation (CAFI) was formed in 2000 and initiated collabo-
rative research on this subject. The initial studies by the
CAFI groups focused on corn stover as a biomass feedstock,

C 2009 American Institute of Chemical Engineers

V 349
350
and results, conclusions, and recommendations were dis-
cussed 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 Renew-
able Energy Laboratory (NREL) LAPs], and a consistent
approach to data interpretation employed by all the partici-
pating groups.11 Such an approach allows valuable compari-
sons among each of the optimized pretreatment technologies,
and sheds lights on the direction for improvement/develop-
ment 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 pre-
treatment 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 fermenta-
tion 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 hy-
drolysis.12–18 Therefore, it is critically important to evaluate
pretreatment by examining the glucose/xylose fermentation
performance simultaneously during the course of pretreat-
ment optimization.
This study is the evaluation of ethanol fermentation per-
formance of mixed sugar streams from pretreatment and en-
zymatic 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 exam-
ined with respect to their impacts on the glucose/xylose
cofermentation in YEPDX medium. After that, poplar bio-
mass processed by the various pretreatments under their re-
spective 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. Understand-
ing to the key influential inhibitory factors to fermentation
suggests on strategies to optimize upstream processes, i.e.
pretreatment and enzymatic hydrolysis, depending on com-
positional characteristics of the biomass.
Materials and Methods
Materials
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 1=4 in. screen. Milled poplar was stored at

20 C until used. All chemicals were obtained from Sigma-
Aldrich (St. Louis, MO) unless otherwise specified.
Biotechnol. Prog., 2009, Vol. 25, No. 2
S. cerevisiae 424A (LNH-ST) used in this study was devel-
oped and provided by Dr. Nancy Ho. The genetically engi-
neered S. cerevisiae 424A (LNH-ST) was constructed by
integrating multiple copies of XD, XR, and XK into the chro-
mosomes of S. cerevisiae ATCC 4124 according to the tech-
nology 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 incu-
bator 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.
Methods
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 dis-
cussed 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 mate-
rial was submitted to the following conditions: 150 C reac-
tion 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 Uni-
versity)
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 temper-
ature 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)
Biotechnol. Prog., 2009, Vol. 25, No. 2
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 þ 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. Depend-
ing 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 resid-
uals and blown-out liquid separately. The pH of the liquid
was adjusted to 4.8 by addition of Ca(OH)2. The solid pre-
treatment 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 pretreat-
ment 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-cat-
alyzed steam-explosion pretreated poplar was directly hydro-
lyzed 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 load-
ing. 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), supple-
mented with an equivalent amount (mass of protein) of mul-
tifect xylanase. The enzymatic hydrolysis (with a total liquid
volume of 100 mL) was held in an Innova 2000 shaker incu-
bator set at 50 C, 190 rpm for a 7-day saccharification reac-
tion. 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 cofer-
mentation was carried out at 28.5 C for 48 h using the glu-
cose 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 over-
night, 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 hydrol-
ysate 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 incu-
bated at 28.5 C. At regular intervals 1 mL samples of the
fermentation mixture were removed for HPLC analysis. The
351
sampling was accomplished by removing the plastic wrap-
ping and foam plug from the top of the flasks and removing
an aliquot with a pipette. Samples were obtained more fre-
quently early in the fermentation (every 3–6 h) and less fre-
quently (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 experi-
ments 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 sug-
ars were added to autoclaved YEP to avoid thermal degrada-
tion 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 col-
umn (Bio-Rad Laboratories, Hercules, CA) in a HPLC sys-
tem consisting of a Milton Roy minipump (Milton Roy,
Ivyland, PA), Waters 717 plus autosampler, Waters R401
differential refractometer (Waters, Milford, MA), and a per-
sonal computer with Empower software for HPLC opera-
tion control. The mobile phase was 5 mM sulfuric acid in
distilled, deionized water filtered through 0.2 lm nylon fil-
ters. Operating conditions for the HPLC column were 60 C
with a mobile phase flow rate of 0.6 mL/min. For sample
analysis, 50 lL of sample was injected and complete sam-
ple elution could be accomplished within 55 min per injec-
tion. 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 regres-
sion for the curves between the elution peak area and con-
centration was computed to give [99.9% R-square values
for all compounds. Other sugars and organic acids (fruc-
tose, 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 pro-
ductivity yields were calculated to evaluate and compare the
fermentation performance of the enzymatic poplar hydroly-
sates from the different pretreatments examined. The meta-
bolic 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.
352
Figure 1. Fermentation of glucose, and xylose to ethanol by S.
cerevisiae 424A (LNH-ST) in the presence of varying
initial concentrations of furfural (l – 0 g/L furfural;
!– 5.0 g/L furfural; n –10 g/L furfural; h –15 g/L
furfural).
Metabolic Yield ¼ ½ethanol=½0:51  ðconsumed glucose
þ consumed xyloseÞ ð1Þ
Productive Yield ¼ ½ethanol=½0:51  ðinitial glucose
þ initial xyloseÞ ð2Þ
Results and Discussions
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 sub-
figure 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 glu-
cose and xylose averaged 85% of theoretical. These results
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 L
1 hr
1) 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 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 approxi-
mately the same ([2.0–2.8 g L
1 hr
1), and furfural concen-
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
furfural, glucose consumption is 30% of the control case.
Furfural is metabolized concurrently with glucose consump-
tion. 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 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
extend beyond the effects of the presence of furfural in the
fermentation media. Membrane adsorbed furfural, intracellu-
lar 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 inhib-
itory 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 fermenta-
tion 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
Biotechnol. Prog., 2009, Vol. 25, No. 2
Figure 2. The interaction effect of pH and acetic acid concen-
tration on xylose consumption rates during the glu-
cose/xylose cofermentation by S. cerevisiae 424A
(LNH-ST).
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 concen-
tration, 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 cel-
lular metabolism. However, the charged, dissociated form of
acetic acid cannot diffuse across the cytoplasmic mem-
brane.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.
Acetic acid is ubiquitously present in all types of cellu-
losic 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),
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-
lar 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 materi-
als, 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 elimi-
nate the confounding effect of potential differences in enzy-
matic hydrolysis rates and difficulty in computing metabolic
yield of ethanol production due to the difficulty in accurately
measuring total sugar release in SSF.
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 sum-
marized in Figure 3. Since all the CAFI pretreatments have
been optimized to reduce furfural/HMF accumulation, mini-
mal amounts of furfural/HMF were detected in the enzy-
matic 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.
354 Biotechnol. Prog., 2009, Vol. 25, No. 2

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 detec-
Figure 3. Glucose/xylose fermentation profiles of poplar hydro-
lysates processed by various pretreatments under
optimal conditions (l – Glucose, * – Xylose, ! –
Xylitol, ~ – Glycerol, n – Acetic acid, h – Ethanol).
tible xylitol formation. The metabolic ethanol yield reached
86.8%. No acetic acid was detectible in any of these sam-
ples. During the fermentation of dilute sulfuric acid pre-
treated 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 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
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

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 L
1 h
1) Productivity (g L
1 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 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 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.

Figure 4. Inhibitors effects on xylose fermentation (1) ARP
pretreated poplar liquor (mainly phenolic com-
pounds 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 (l – Glucose, * – Xylose, ! – Xylitol, ~
– Glycerol, n – Acetic acid, h – Ethanol).
the fermentations of hydrolysates from optimized pretreat-
ments yielded ethanol productivities comparable to the con-
trol 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 fer-
mented quite poorly, as shown in Figure 4. The ARP pre-
Conclusions
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 fur-
fural, acetic acid and pH on the ethanol fermentation per-
formance by a recombinant S. cerevisiae strain (LNH-ST
424A) in YEPDX medium were investigated. With this par-
ticular yeast strain, initial furfural concentrations below 5 g/
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
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 fur-
ther demonstrated by the fermentation results of poplar hy-
drolysates processed by the various CAFI pretreatments.
Examples of low-efficiency fermentations were presented to
illustrate the inhibitory effects of high acetic acid concentra-
tion, dissolved phenolic compounds, and low pH conditions.
Therefore, a pretreatment process that is capable of
356
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 pre-
treatment and hydrolysis steps should be optimized to
improve fermentation performance in an integrated cellulosic
ethanol production process.
Acknowledgments
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 opti-
mally 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 fermen-
tations, and Youngmi Kim and Eduardo Ximenes for internal
review of the manuscript.
Literature Cited
1. Lin Y, Tanaka S. Ethanol fermentation from biomass resources:
current state and prospects. Appl Microbiol Biotechnol.
2006;69:627–642.
2. Wang M. The debate on energy and greenhouse gas emissions
impacts of fuel ethanol, www.transportation.anl.gov/pdfs/TA/
347.pdf. 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 sys-
tems utilized for producing biofuels: bioethanol and biodiesel.
Biomass Bioenergy. 2005;29:426–439.
4. Farrell AE, Plevin RJ, Turner BT, Jones AD, O’Hare M, Kam-
men DM. Ethanol can contribute to energy and environmental
goals. Science. 2006;311:506–508.
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;311:484–
489.
6. Somerville C. The billion-ton biofuels vision. Science. 2006;
312:1277–1277.
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;315:804–
807.
8. Mosier N, Wyman C, Dale B, Elander R, Lee YY, Holtzapple
M, Ladisch M. Features of promising technologies for pretreat-
ment of lignocellulosic biomass. Biores Technol. 2005;96:673–
686.
9. Stephanopoulos G. Challenges in engineering microbes for bio-
fuels production. Science. 2007;315:801–804.
10. Lynd LR, Wyman CE, Gerngross TU. Biocommodity engineer-
ing. Biotechnol Prog. 1999;15:777–793.
11. Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR,
Lee YY. Coordinated development of leading biomass pretreat-
ment technologies. Biores Technol. 2005;96:1959–1966.
Biotechnol. Prog., 2009, Vol. 25, No. 2
12. Palmqvist E, Hahn-Hagerdal B. Fermentation of lignocellulosic
hydrolysates. II: inhibitors and mechanisms of inhibition. Biores
Technol. 2000;74:25–33.
13. Palmqvist E, Grage H, Meinander NQ, Hahn-Hagerdal B. Main
and interaction effects of acetic acid, furfural, and p-hydroxy-
benzoic acid on growth and ethanol productivity of yeasts. Bio-
technol Bioeng. 1999;63:46–55.
14. Luo CD, Brink DL, Blanch HW. Identification of potential fer-
mentation inhibitors in conversion of hybrid poplar hydrolyzate
to ethanol. Biomass Bioenergy. 2002;22:125–138.
15. Lawford HG, Rousseau JD, Mohagheghi A, McMillan JD. Fer-
mentation performance characteristics of a prehydrolyzate-
adapted xylose-fermenting recombinant Zymomonas in batch
and continuous fermentations. Appl Biochem Biotechnol. 1999;
77–79:191–204.
16. Larsson S, Palmqvist E, Hahn-Hagerdal B, Tengborg C, Sten-
berg K, Zacchi G, Nilvebrant NO. The generation of fermenta-
tion inhibitors during dilute acid hydrolysis of softwood.
Enzyme Microb Technol. 1999;24:151–159.
17. Oliva JM, Negro MJ, Saez F, Ballesteros I, Manzanares P, Gon-
zalez A, Ballesteros M. Effects of acetic acid, furfural and cate-
chol combinations on ethanol fermentation of Kluyveromyces
marxianus. Process Biochem. 2006;41:1223–1228.
18. Klinke HB, Thomsen AB, Ahring BK. Inhibition of ethanol-pro-
ducing yeast and bacteria by degradation products produced
during pre-treatment of biomass. Appl Microbiol Biotechnol.
2004;66:10–26.
19. Ho NWY, Chang SF. Cloning of yeast xylulokinase gene by
complementation of Escherichia coli and yeast mutations.
Enzyme Microb Technol. 1989;11:417–421.
20. Ho NWY, Chen ZD, Brainard AP. Genetically engineered Sacc-
charomyces yeast capable of effective cofermentation of glucose
and xylose. Appl Environ Microbiol. 1998;64:1852–1859.
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 cellu-
losic biomass to fuel ethanol. In: Tsao GT, 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 glycoly-
sis by furfural in Saccharomyces cerevisiae. Eur J Appl Micro-
biol Biotechnol. 1981;11:226–228.
25. Axe DD, Bailey JE. Transport of lactate and acetate through the
energized cytoplasmic membrane of Escherichia coli. Biotech-
nol Bioeng. 1995;47:8–19.
26. Taherzadeh MJ, Niklasson C, Liden G. Acetic acid—friend or
foe in anaerobic batch conversion of glucose to ethanol by Sac-
charomyces cerevisiae? Chem Eng Sci 1997;52:2653–2659.
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;96:2026–2032.
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;21:671–684.
29. Sedlak M, Ho NWY. Production of ethanol from cellulosic bio-
mass hydrolysates using genetically engineered Saccharomyces
yeast capable of cofermenting glucose and xylose. Appl Bio-
chem Biotechnol. 2004;113–116:403–416.
Manuscript received July 29, 2008, and revision received Oct. 13, 2008.