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RES E AR C H Open Access
A comparative study of ethanol production using
dilute acid, ionic liquid and AFEX™ pretreated
corn stover
Nirmal Uppugundla
1
, Leonardo da Costa Sousa
1
, Shishir PS Chundawat
1,2
, Xiurong Yu
3
, Blake Simmons
4,5
,
Seema Singh
4,5
, Xiadi Gao
6,7,8
, Rajeev Kumar
6,8
, Charles E Wyman
6,7,8
, Bruce E Dale
1
and Venkatesh Balan
1*
Abstract
Background: In a biorefinery producing cellulosic biofuels, biomass pretreatment will significantly influence the
efficacy of enzymatic hydrolysis and microbial fermentation. Comparison of different biomass pretreatment
techniques by studying the impact of pretreatment on downstream operations at industrially relevant conditions
and performing comprehensive mass balances will help focus attention on necessary process improvements, and
thereby help reduce the cost of biofuel production.
Results: An on-going collaboration between the three US Department of Energy (DOE) funded bioenergy research
centers (Great Lakes Bioenergy Research Center (GLBRC), Joint BioEnergy Institute (JBEI) and BioEnergy Science
Center (BESC)) has given us a unique opportunity to compare the performance of three pretreatment processes,
notably dilute acid (DA), ionic liquid (IL) and ammonia fiber expansion (AFEX
TM
), using the same source of corn
stover. Separate hydrolysis and fermentation (SHF) was carried out using various combinations of commercially
available enzymes and engineered yeast (Saccharomyces cerevisiae 424A) strain. The optimal commercial enzyme
combination (Ctec2: Htec2: Multifect Pectinase, percentage total protein loading basis) was evaluated for each
pretreatment with a microplate-based assay using milled pretreated solids at 0.2% glucan loading and 15 mg total
protein loading/g of glucan. The best enzyme combinations were 67:33:0 for DA, 39:33:28 for IL and 67:17:17 for
AFEX. The amounts of sugar (kg) (glucose: xylose: total gluco- and xylo-oligomers) per 100 kg of untreated corn
stover produced after 72 hours of 6% glucan loading enzymatic hydrolysis were: DA (25:2:2), IL (31:15:2) and AFEX
(26:13:7). Additionally, the amounts of ethanol (kg) produced per 100 kg of untreated corn stover and the respective
ethanol metabolic yield (%) achieved with exogenous nutrient supplemented fermentations were: DA (14.0, 92.0%),
IL (21.2, 93.0%) and AFEX (20.5, 95.0%), respectively. The reason for lower ethanol yield for DA is because most of
the xylose produced during the pretreatme nt was removed and not converted to ethanol during fermentation.
Conclusions: Compositional analysis of the pretreated biomass solids showed no significant change in composition
for AFEX treated corn stover, while about 85% of hemicellulose was solubilized after DA pretreatment, and about
90% of lignin was removed after IL pretreatment. As expected, the optimal commercial enzyme combination was
different for the solids prepared by different pretreatment technologies. Due to loss of nutrients during the
pretreatment and washing steps, DA and IL pretreated hydrolysates required exogenous nutrient supplementation
to ferment glucose and xylose efficiently, while AFEX pretreated hydrolysate did not require nutrient supplementation.
Keywords: AFEX, Dilute acid, Ionic liquid, Pretreatment, Enzymatic hydrolysis, Cellulosic ethanol
* Correspondence: balan@msu.edu
1
Department of Chemical Engineering and Materials Science, Department of
Energy (DOE) Great Lakes Bioenergy Research Center (GLBRC), Michigan
State University, East Lansing, MI 48824, USA
Full list of author information is available at the end of the article
© 2014 Uppugundla et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
article, unless otherwise stated.
Uppugundla et al. Biotechnology for Biofuels 2014, 7:72
http://www.biotechnologyforbiofuels.com/content/7/1/72
Background
Crude oil is the primary feedstock source for producing
transportation fuels , industrial chemicals and polymers.
Rising crude oil prices, political/social tensions in major
oil-producing nations, and greenhouse gas emissions driv-
ing climate change have triggered worldwide research to-
wards the development of alternative, sustainable sources
of energy [1]. Lignocellulosic biofuels are recognized as a
potential alternative to fossil fuels. Considerable research
has been conducted in recent years to develop efficient
technologies to convert plant-derived polysaccharides to
ethanol [2]. Unlike corn grain-based ethanol, where the
starch can be readily hydrolyzed to fermentable sugars
using enzymes, the production of lignocellulosic ethanol is
limited by biomass recalcitrance. Biomass recalcitrance is
thought to arise largely due to the complex intertwined
network of cellulose, hemicellulose and lignin that re-
stricts enzyme accessibility [3,4]. Numerous pretreatment
processes have been developed to overcome biomass re-
calcitrance, such as: steam explosion, hot water, dilute acid
(DA), lime, phosphoric acid, ammonia (for example,
ammonia fiber expansion ( AFE X™), soaking in aqueous
ammonia (SAA) and ammonia recycled percolation (ARP))
and ionic liquid (IL)-based pretreatments [5,6]. However,
most pretreatments pose various challenges in terms of
costs incurred by use of excess water, expensive chemicals
and chemical recovery, feedstock handling, energy require-
ments and downstream processing [7].
DA, IL and AFEX pretreatments are part of the core
research programs studied at the three US Department of
Energy (DOE) funded bioenergy research centers, namely:
Great Lakes Bioenergy Research Center (GLBRC), Joint
Bioenergy Institute (JBEI) and the BioEnergy S cience
Center (BE SC). DA (acidic) and IL (acidic or alkaline)
are dry to wet product pretreatment processes , whe reas
AFEX (alkaline) is a dry biomass to dry product process.
Considerable research has been done to streamline these
processes in order to make them cost effective. Though
these pretreatments still face various challenges, they can
efficiently pretreat lignocellulosic biomass to aid higher
sugar release during enzyme hydrolysis.
In our previous research, developed under the Consor-
tium for Applied Fundamentals and Innovation (CAFI) I,
II and III programs [8,9], we compared the performance
of several pretreatment technologies. In these studies, the
performance of AFEX and DA pretreatments was com-
pared for different types of feedstock (corn stover (CS),
poplar and switchgrass) [6,9,10]. However, IL pretreat-
ment was not a part of such comparative studies. In this
work, we take into consideration the fact that, after being
exposed to different pretreatment methods, the resulting
biomass materials do not necessarily have the same en-
zyme requirements (for example, greater hemicellula se
requirement for AFEX versus DA treated corn stover) to
achieve maximum yields. Similarl y, enzymatic hydr oly-
sates derived from the various pretreatment methods
have different nutrient requirements to maximize ethanol
yields. Thus, by studying downstream processing condi-
tions that maximize product yields, we can better compare
the potential of each pretreatment method on a level play-
ing field (for example, enzyme loading, glucan loading,
residence time, and so on). We carried out separate hy-
drolysis and fermentation (SHF) and compared the per-
formance of corn stover solids prepared by DA, IL and
AFEX pretreatments. To achieve this, we first optimized
the commercial enzyme cocktails for each pretreated bio-
mass prior to subsequent high-solids loading saccharifica-
tion and fermentation. The fermentability of pretreated
corn stover hydrolysates was evaluated using a recombin-
ant yeast strain of Saccharomyces cerevisiae 424-A (LNH-
ST) (with and without external nutrient supplementation)
[11]. Material balances around pretreatment, hydrolysis
and fermentation were developed to determine the fates
of key biomass components (cellulose, hemicellulose and
lignin) and highlight the differences in sugar and ethanol
yields for the three pretreatment methodologies at indus-
trially relevant saccharification/fermentation conditions.
Results and discussion
Composition of AFEX, DA and IL pretreated biomass
Conditions and parameters for DA, IL and AFEX pre-
treatments are summarized in Table 1. As expected, all
three pretreatments exhibit distinct chemistries, as dem-
onstrated by differences in cell wall composition among
pretreated materials (see Table 2). For example, the alkaline
AFEX process cleaves most of the ester linkages present in
the plant cell wall [12], as confirmed by the absence of
acetyl content in the AFEX pretreated corn stover (Table 2).
Almost all the acetyl groups are converted to acetic acids
and acetamide due to the corresponding hydrolysis or
ammonolysis reactions during AFEX pretreatment. In this
process, lignin and hemicellulose are partially solubilized
and relocated to the biomass surface during pretreatment,
leaving behind a highly porous cell wall that helps to im-
prove enzyme access to the embedded cellulose and hemi-
cellulose [13]. AFEX pretreatment is a dry to dry process
(material enters the process dry and also leaves the process
in the dry state) during which minimal carbohydrate deg-
radation takes place [12] and negligible modifications in
total polysaccharide composition are seen compared to un-
treated biomass (see Table 2). The reduced acid insoluble
or Klason lignin (approximately 30%) levels observed after
AFEX pretreatment may result from currently unknown
chemical modifications at the lignin level that improve lig-
nin solubility during sample preparation (that is , extrac-
tion with hot water and ethanol to remove interfering
extractives prior to sulfuric acid hydrolysis) prior to com-
position analysis based on current National Renewable
Uppugundla et al. Biotechnology for Biofuels 2014, 7:72 Page 2 of 14
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Energy Laboratory (NREL) laboratory analytical procedure
(LAP).
On the other hand, DA pretreatment cleaves not only
ester linkages, but also some ether linkages present in
lignin. Use of dilute sulfuric acid favors the chemical con-
version of xylan to xylose, which can be further dehydrated
to furfural under certain conditions [14]. These hydrolyzed
sugars and their respective decomposition products are
soluble in the acidic pretreatment liquor, which are hence
separated from the solids after pretreatment. Subsequent
washing steps (mostly to remove r esidual acid) aid in
further removal of these soluble components from the
pretreated residual solids. Therefore, these solids have a
reduced xy lan content compared to untreated corn sto-
ver. As a result, the percentage of glucan a nd lignin in
the pretreated solids increased by 26% and 5%, respect-
ively. The ace tyl content decrease d from 2.1% to 0.6%,
possibly due to incomplete hydrolysis of ester linkages
under these pretreatment conditions.
ILs, such as 1-ethyl-3-methylimidazolium acetate ([C2
mim][OAc]), can solubilize plant cell wall components,
which can then be selectively precipitated from solution
by adding an anti-solvent (for example, water and etha-
nol), leaving most of the lignin behind in solution, as
carried out in this study. The composition of the solids
after precipitation and washing (Table 2) clearly shows
an enriched fraction of glucan (46.9%) and xylan (29.8%),
with only 2.7% lignin content. The residual acetyl content
of IL treated corn stover is higher than that of DA and
AFEX treated corn stover. This suggests that IL pretreat-
ment may not effectively cleave acetyl residues. Since
acetyl ester residues remain intact after IL treatment, it is
likely that most of the ferulate ester crosslinks that are re-
sponsible for the lignin-carbohydrate complexes (LCCs)
are intact after IL pretreatment. Previous work has sug-
gested that [C
2
mim][OAc]-based pretreatment deacety-
lated the xylan backbone, while simultaneously acetylating
the lignin from Eucalyptus globulus, and partially cleaving
the β-ether linkages from lignin [15]. Bas ed on this ob-
ser vation, it may be that the acetyl content of the IL
pretreated solids might have derived from the residual
acetylated lignin. Detailed work is required to further
characterize the acetylated components of IL pretreated
cell walls and the role of [C2mim][OAc] in acetylation
reactions during IL pretreatment of corn stover.
Enzyme optimization
Commercial enzyme cocktail mixtures were optimized for
DA, IL and AFEX treated corn stover to maximize sacchar-
ification yields for each pretreated substrate. It is reasonable
to assume that corn stover subjected to different pretreat-
ment methodologies will require a unique cocktail of en-
zymes to achieve optimum conversion. For example, DA
pretreatment removes most of the xylan from corn stover
and should therefore require lower xylanase activity when
compared to AFEX and IL treated corn stover. To account
for these differences in cell wall composition, enzyme cock-
tails were optimized for each pretreat ed substrate.
The design of experiments was carried out using Minitab®
software (Coventry, UK), utilizing 31 unique enzyme com-
binations, composed of various ratios of three commercial
Table 1 Pretreatment conditions used to pretreat biomass using different methods
Method
Pretreatment Post-wash
Temperature (°C) Residence
time (minutes)
Catalyst type Catalyst
loading (kg)
a
Catalyst
recyclable?
Water
loading (kg)
b
Post-wash
water use (kg)
b
Water
temperature (°C)
DA 160 20 Sulfuric acid 4.5 No 895.5 1,000 Room
temperature
IL 140 180 IL [C2mim][OAc] 900 Yes N/R 10,000 Room
temperature
AFEX 140 15 Anhydrous
ammonia
100 Yes 60 N/R -
a
Catalyst loading: kg/100 kg dry biomass;
b
water use: L/100 kg dry biomass. AFEX, ammonia fiber expansion; [C2mim][OAc], 1-ethyl-3-methylimidazolium acetate;
DA, dilute acid; IL, ionic liquid; N/R, not required.
Table 2 Compositional analysis (% wt/wt, dry weight basis)
for untreated, AFEX, DA and IL treated MSU corn stover
Composition Untreated AFEX DA IL
Glucan 33.4 ± 3.2 33.5 ± 0.5 59.1 ± 3.0 46.9 ± 1.9
Xylan 24.9 ± 2.0 24.8 ± 0.9 6.5 ± 0.1 29.8 ± 0.5
Arabinan 3.7 ± 0.5 3.3 ± 0.4 3.6 ± 0.1 0.3 ± 0.0
Acetyl 2.1 ± 0.2 0.0 ± 0.0 0.6 ± 0.6 1.5 ± 0.1
Acid insoluble lignin
a
17.2 ± 0.6 12.2 ± 0.2 22.2 ± 0.2 2.7 ± 0.5
Ash 3.6 ± 0.0 4.4 ± 0.3 2.5 ± 0.0 1.3 ± 0.26
Extractives 10.4 ± 0.4 24.8 ± 0.8 15.4 ± 0.8 13.1 ± 2.0
All experiments were carried out in triplicates with averages and standard
deviations reported here.
a
The acid insoluble or Klason lignin analysis method was modified to use
47 mm, 0.22 μm pore size mixed cellulose ester filter disks (Millipore
Corporation, Bedford, MA, USA) during the filtration process instead of the
fritted crucibles. The filtered lignin residues were dried overnight in a
desiccator prior to weighing. AFEX does not remove any particular
components during pretreatment. Ammonia is evaporated and all the
ammonia soluble biomass components condense on the surface of the
biomass [13]. However, some reactions do occur to lignin during AFEX,
transforming Klason lignin (acid insoluble) to acid soluble lignin, which was
not quantified in this study. AFEX, ammonia fiber expansion; DA, dilute acid;
IL, ionic liquid; MSU, Michigan State University.
Uppugundla et al. Biotechnology for Biofuels 2014, 7:72 Page 3 of 14
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enzymes: CTec2 (cellulase), HTec2 (hemicellulase) and
Multifect Pectinase (accessory) (Figure 1). The enzymatic
hydrolysis wa s performed in a 96-well microplate at
0.2 wt% glucan loading and total enzyme protein loading of
15 mg/g glucan with the relative protein ratios varying from
0 to 100% of the total added protein. The performance
of different enzyme combinations with respect to glucan
and xylan conversions for each pretreatment is shown
in Figure 2. The results demonstrate that for maximum
polysaccharide conversion, DA-CS requires 67% Ctec2
and 33% Htec2 enzymes, IL-CS requires all three enzymes
in similar proportions, whereas AFEX-CS requires 66.7%
Ctec2, 16.7% Htec2 and 16.7% Multifect Pectinase.
It is not surprising that DA-CS requires no additional
accessory enzymes and only requires 33% hemicellulase
(Htec2) due to the lower levels of xylan (6.5%) and arabinan
(3.6%). Similarly, 67% cellulase (Ctec2) in the optimized
cocktail for DA-CS correlates with the higher glucan con-
tent, which represents approximately 85% of the total car-
bohydrates in the DA pretreated solids.
On the other hand, AFEX and IL pretreated corn sto-
ver contains more hemicelluloses, and thus required
16.7% and 33%, respectively, of each accessory enzyme.
Even though the ratio of glucan to xylan is higher in IL-
CS as compared to AFEX-CS, it requires a lower ratio of
cellulase to hemicellulase. This observation correlates with
the fact that AFEX-CS has higher lignin content (approxi-
mately 4.5 fold) than IL-CS. Lignin content in biomass is
known to inhibit cellulases more readily than hemicellu-
lases [16]. Moreover, IL-CS is known to decrystallize cellu-
lose and partially convert native crystalline cellulose I to
highly amorphous cellulose II [17]. Decrystallized cellulose
is more amenable to enzymatic hydrolysis than the native
cellulose, thereby requiring relatively less cellulase in the
enzyme mixture. Thus, from our results, optimal enzyme
mixtures correlate well with the content of cellulose,
hemicellulose and lignin, as well as the crystalline state of
cellulose. Other factors influencing these results may in-
clude the relative influence of non-native decomposition
products formed during pretreatment on enzyme activity
[18,19].
Enzymatic conversion of biomass to sugars at high
solid loading
The pretreated substrates were enzymatically hydrolyzed
at high solid loading (6% glucan loading) in a fermenter,
using the respective optimized enzyme mixtures, operating
at 50°C with pH controlled at 4.8. As mentioned previ-
ously, these optimized enzyme mixtures were determined
by 0.2% glucan loading enzymatic hydrolysis, performed in
a high-throughput microplate format.Itispossiblethatthe
optimum enzyme mixtures may vary with increased solid
loadings and saccharification conditions, since some of
these enzymes may be more susceptible to shear stress and
substrate/end product inhibition [20]. These factors may
promote deactivation/inhibition of enzymes and thereby
change the relative ratios of enzymes for an optimum mix-
ture. Thus, the extrapolation of optimum enzyme mixtures
to higher solid loadings represents a limitation of this
study. However, this factor should not significantly impact
the final conclusions of this paper and is a previously ac-
knowledged limitation in peer-reviewed literature [21].
Throughout the course of high solid loading hydrolysis of
the pretreated substrates, liquid samples were taken every
24 hours to estimate monomeric sugar concentrations.
Time course data for the monomeric sugar concentrations
Figure 1 Three commercial enzyme combinations (based on total protein loading) used for optimizing enzyme cocktail for DA, IL and
AFEX biomass. AFEX, ammonia fiber expansion; DA, dilute acid; IL, ionic liquid.
Uppugundla et al. Biotechnology for Biofuels 2014, 7:72 Page 4 of 14
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are given in Figure 3. The first 24 hours were critical for en-
zymatic hydrolysis, since over 50 g/L of glucose was
achieved for all three pretreated substrates (Figure 3A). On
the other hand, only 4 to 6 g/L of additional glucose was
produced in the following 48 hours. Moreover, the decrease
in the rate of enzymatic hydrolysis observed between 24 to
72hoursisnotsignificantlydifferentbetweenthethreepre-
treatments/substrates, and is approximately 4% to 5% of
the rates of enzymatic hydrolysis achieved in the first
24 hours.
During high solid loading enzymatic hydrolysis, the li-
quid volume does not represent the total reaction volume.
As the solids concentration increases (from 1% glucan
loading to 6% as used in this study), the assumption that
the solution volume is all liquid is increasingly incorrect
as a larger fraction of the volume is occupied by solids.
Therefore, it is not possible to calculate conversion at high
solid loading without performing solid–liquid separations,
measuring liquid volume and concentration of sugars
in the liquid fraction. For this reason a thorough mass
balance was performed on the hydrol ysis products as
described pre viously by Garlock et al. [6] to determine
the final glucan and xylan conversion to mono- and
oligosaccharides.
IL-CS gave higher glucan conversion after 24 hours,
yielding 66 g/L of glucose and therefore achieving the
highest final concentration of glucose of all three sub-
strates after 72 hours (72 g/L, 100% glucan conversion).
DA-CS produced 59 g/L of glucose in the first 24 hours
and reached a maximum of 65 g/L after 72 hours (88%
Figure 2 Optimum commercial enzyme mixture ratio-based Minitab model predicted contour plots for ternary combinations of CTec2,
HTec2 and Multifect Pectinase. (I) DA, (II) IL and (III) AFEX pretreated corn stover at 0.2% glucan loading. (A) Glucan conversions are shown on
the left and (B) xylan conversions are shown on the right. The total protein loading was 15 mg protein/g glucan. AFEX, ammonia fiber expansion;
DA, dilute acid; IL, ionic liquid.
Uppugundla et al. Biotechnology for Biofuels 2014, 7:72 Page 5 of 14
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glucan conversion). Similarly, AFEX produced 56 g/L of
glucose in 24 hours; however, since the rate of glucose
release is slightly lower than that of DA-CS during the
remaining 48 hours, the final concentration of glucose
peaked at 60 g/L (79% glucan conversion).
These differences in glucose release rate and fin al glu-
cose concentrations are a consequence of the various
physicochemical properties of the differently pretreated
substrates. For example, lignin content is low in IL-C S.
Lignin is widely known to affect enzymatic hydrolysis by
inhibiting cellulases and hemicellulases [16,22-25]. More-
over, IL pretreatment modifies cellulose structure by con-
verting native cellulose I
β
to cellulose II with a significantly
reduced crystallinity index (CrI) [17,26-28]. Cellulose II
prepared by mercerization with 25% NaOH had enhanced
the performance of cellulases by 1.6 fold when compared
to native cellulose I
β
[29]. However, it is likely that the
amorphous cellulose that is also produced during forma-
tion of cellulose II is the primary cause for improvement
in net hydrolysis rate. The other pretreatments in this
study do not modify the native crystal structure of cellu-
lose present in corn stover. DA pretreatment is known to
increase cellulose CrI due to a selective decomposition
of the amorphous portions of cellulose, which are more
susceptible to acid degradation as suggested by Kumar
et al. [27,30]. With respect to AFEX pretreatment, earlier
reports did not observe any major effect on cellulose CrI
under current AFEX conditions [26]. Even though previ-
ous observations suggest a decrease of cellulose digestibil-
ity with increasing CrI of cellulose (on pure substrate), it is
not possible to address differences in enzymatic hydrolysis
performance based on CrI measurements with these sub-
strates. This is because the pretreated biomass materials
are composed of different fractions of amorphous -like
materials that impact not only enzymatic digestibility
(for example, lignin and hemicellulose) but also affect
the accuracy of CrI estimation using X-ray diffraction.
High concentrations of xylo-oligomers inhibit cellu-
lases and could also contribute to the lower glucan con-
version seen for AFEX-CS compared to DA-CS [30,31].
This hypothesis is supported by the accumulation of
xylo-oligomers during enzymatic hydrolysis of AFEX-CS,
which reaches 19 g/L after 72 hours of incubation.
In Figure 3B, xylose concentrations of hydrolysates are
presented as a function of enzymatic hydrolysis time for
the various pretreated feedstocks. Since DA-CS contains
only 6.5% of xylan, lower xylose concentrations (4 g/L)
can be expected in the hydrolysate. This xylose concen-
tration corresponds to 48% xylan conversion, which is
relatively low when compared to the other pretreated
substrates (79% for IL-CS and 52% for AFEX-CS). It is
possible that the xylan present in DA-CS is more recalci-
trant to enzymatic hydrolysis since it could not be hy-
drolyzed to soluble oligomeric and monomeric xylose
during acid pretreatment.
Similar to glucan hydrolysis, most of the hydrolyzed
xylan was released in the first 24 hours in pretreated
corn stover with all three pretreatment processes. IL-CS
produced the highest concentration of xylose among all
pretreated feedstocks (35 g/L) after 72 hours, which rep-
resents 79% of the total xylan conversion. The low lignin
composition of IL-CS could be an important factor con-
tributing to high xylan conversion and reduced enzyme
inhibition during enzymatic hydrolysis [16]. AFEX-CS
released 29 g/L of xylose in 72 hours of enzymatic hydroly-
sis, representing about 52% xylan conversion. However,
78% of AFEX-CS xylan was solubilized to monomeric and
oligomeric sugars. The xylo-oligomers content is much
higher in AFEX (33%) than IL (11%) and DA (23%) hydro-
lysates. The total xylan conversions for IL and DA (th at
is, xylose and xylo-oligomers) reached 88% and 62%, re-
spectively, a fter 72 hours. Although the yeast used in
this study is able to ferment only xylose to ethanol, it is
not capa ble of utilizing xylo-oligomers, in common with
most industrial e thanologens. Therefore, ethanol yields
will depend on the conversion of xylan to monomeric
xylose and n ot the total xylan conversion, that is, xylose
and x ylo-oligomers.
Ethanol fermentation
Fermentation experiments were performed using the re-
combinant xylose-fermenting yeast strain S. cerevisiae
Figure 3 Time course profile of glucose and xylose concentrations during high solid loading hydrolysis of pretreated corn stover.
(A) Glucose and (B) xylose. AFEX, ammonia fiber expansion.
Uppugundla et al. Biotechnology for Biofuels 2014, 7:72 Page 6 of 14
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424A (LNH-ST), kindly provi ded by Professor Nancy Ho
of Purdue University (West Lafayette, IN, USA) [32].
Fermentation product profiles for IL, AFEX and DA corn
stover hydrolysates are shown in Figure 4, where glucose,
xylose, ethanol and cell density (OD
600
)weremonitored
during 120 hours of fermentation. Since the three pre-
treatment methodologies considered in this work exhibit
distinct mechanisms of action, the relative chemical com-
position of the resulting hydrolysates varies significantly
[12,26,30]. In this context, since nutrient availability is one
of the major factors that influences fermentation perform-
ance, we compared the fermentability of these hydroly-
sates both in the absence (Figure 4A) and in the presence
(Figure 4B) of adequate external nutrient supplementation
(yeast extract peptone and urea) required for an efficient
microbial sugar metabolism [33]. The hydrolysates contain
different initial concentrations of glucose and xylose,
which will result in different concentrations of ethanol.
Figure 4 Fermentation product and cell growth profiles for three different pretreated corn stover hydrolysates. (I) DA, (II) IL and (III) AFEX.
(A) Without external nutrients and (B) with external nutrient supplementation. AFEX, ammonia fiber expansion; DA, dilute acid; IL, ionic liquid.
Uppugundla et al. Biotechnology for Biofuels 2014, 7:72 Page 7 of 14
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Since final ethanol concentration is not an adequate metric
by itself for comparing fermentati on performances in this
study, the data are analyzed and discussed with respect to
ethanol metabolic yield, fermentation rates and percentage
sugar consumption. Ethanol metabolic yield was calculated
from consumed glucose and xylose during fermentation,
based on the theoretical ethanol yield (0.51 g ethanol per
g glucose/xylose).
In the absence of nutrient supplementation, glucose
metabolism was fastest for AFEX-CS hydrolysate, with a
maximum rate of 5.72 g.L
−1
.h
−1
(Table 3). In agreement
with previous reports, xylose fermentation occurred only
after glucose was totally consumed by S. cere visiae 424A
(LNH-ST). Diauxic lag is typical of this microbial strain,
which can be observed in nutrient-rich environments such
as synthetic media composed of Yeast Extract Peptone
(YEP), glucose and xylose [32]. The AFEX-CS hydrolysate
also provided the highest xylose consumption rate (0.47 g.
L
−1
.h
−1
) without additional nutrients. However, even in
these optimal conditions, S. cerevisiae 424A (LNH-ST)
was unable to completely consume xylose in all the hydro-
lysates within the first 120 hours of fermentation. DA-CS
hydrolysate showed the highest percentage xylose con-
sumption among the three substrates (91%), which relates
to low initial concentration of x ylose (4 g/L) in DA-CS
hydrolysate, whereas AFEX-CS and IL-CS hydrolysates
contained 29 g/L and 35 g/L of xylose, respectively.
The superior fermentation performance of the AFEX
hydrolysate without nutrient supplementation when com-
pared to IL-CS and DA-CS hydrolysates is likely due to
the fact that the AFEX process requires no washing steps
thereby preserving essential biomass nutrients for fermen-
tation. Additionally, unlike IL and DA, AFEX pretreat-
ment does not produce a highly inhibitory pretreatment
mixture [12], thus avoiding the detoxification and exten-
sive washing steps that remove nutrients from the biomass
[34,35]. This hypothesis is supported by the lower cell
growth rates observed in both IL-CS and DA-CS hydroly-
sates, when compared to AFEX-CS hydrolysate (Figure 4A),
suggesting that both IL-CS and DA-CS hydrolysates
were nutrient-limited for yeast cell growth. The superior
fermentability of AFEX-CS hydrolysates in the absence
of nutrient supplementation was also refle cted by a
higher metabolic yield of 98%, compared to 93% and
90% for DA-CS and IL-CS hydrolysates , respe ctively
(Table 3).
When the hydrolysates were supplemented with nutri-
ents, an increase in cell growth (Figure 4B) and sugar
consumption rate were observed for all three hydroly-
sates. The highest final cell density (18.5 at OD
600
)was
observed for the IL-CS hydrolysate as it contains a
higher concentration of fermentable sugars (glucose and
xylose) compared to the other two pretreated corn stover
hydrolysates. This observation also supports the earlier
hypothesis that IL-CS and DA-CS hydrolysates were
nutrient-limited for S. cerevisiae 424A (LNH-ST) fermen-
tation (Figure 4A). Nutrient supplementation helped the
yeast to ferment most glucose in 8 hours and xylose in
48 hours (Figure 4B and Table 3). From these observations
we conclude that xylose fermentation performance by S.
cerevisiae 424A (LNH-ST) depends on nutrient availability
in the media, as previously obser ved by Lau et al. [34].
Comparing the xylose fermentation rates between the hy-
drolysates, IL-CS hydrolysate leads with the highest rate
(1.18 g.L
−1
.h
−1
), followed by AFEX-CS (0.87 g.L
−1
.h
−1
)and
DA-CS hydrolysates (0.16 g.L
−1
.h
−1
). The lower xylose
consumption rate observed in DA-CS hydrolysate can be
attributed to its low xylos e concentration (4 g/L), while
the superior xylose fermentation performance of IL-CS
hydrolysate demonstrates that it benefited the most
from nutrient supplementation since only 70% of the xy-
lose was fermented at a maximum rate of 0.24 g.L
−1
.h
−1
when no nutrients were supplemented. This hypothesis is
further supported by the marginal increase in metabolic
Table 3 Ethanol fermentation performances of different pretreated biomass hydrolysates
Ethanol fermentation Parameter DA IL AFEX
Without nutrient supplementation Metabolic yield (%) 93 90 98
Glucose consumption (%) 100 100 99
Xylose consumption (%) 91 70 84
Maximum glucose consumption rate (g.L
−1
.h
−1
) 4.95 5.36 5.72
Maximum xylose consumption rate (g.L
−1
.h
−1
) 0.07 0.24 0.47
With nutrient supplementation Metabolic yield (%) 90 93 97
Glucose consumption (%) 100 100 99
Xylose consumption (%) 100 97 94
Maximum glucose consumption rate (g.L
−1
.h
−1
) 8.09 8.80 7.30
Maximum xylose consumption rate (g.L
−1
.h
−1
) 0.16 1.18 0.87
Metabolic yield calculated based on the theoretical ethanol yield from consumed glucose and xylose, 0.51 g ethanol/g sugar. AFEX, ammonia fiber expansion; DA,
dilute acid; IL, ionic liquid.
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yield from 90% to 93% after supplementing the IL-CS hy-
drolysate with nutrients, while the opposite trend was ob-
served for the other two hydrolysates. The medium that
benefited the least from nutrient supplementation was
DA-CS hydrolysate, since the metabolic yield decreased
by 3% (Table 3) and the final ethanol concentration did
not vary significantly. This result was predictable consider-
ing that nutrient supplementation tends to benefit xylose
fermentation to a greater extent than glucose fermentation
and DA-CS hydrolysate contains low levels of xylose.
Process mass balances
The results obtained from pretreatment, enzymatic hy-
drolysis and fermentation were used to develop a process
mass balance for each pretreatment technology (Figure 5).
AFEX pretreatment utilized 100 kg of ammonia and 60 kg
of water per 100 kg of dry untreated corn stover, generat-
ing a solid stream (pretreated corn stover) and a gaseous
stream (mostly composed of ammonia and water vapor).
The ammonia released after AFEX pretreatment can be
recycled and reused in an industrial system; however, the
mass balance for ammonia was not performed in this
study. From previous studies, it is known that about 3 kg
of ammonia left behind per 100 kg of dry untreated
corn stover, mostly due to the ammonolysis and hydrolysis
reactions mentioned previously [12,13]. Corn stover was
essentially completely recovered after AFEX pretreatment
and only minor changes were observed in its composition.
DA pretreatment utilized 4.5 kg of sulfuric acid and
895.5 kg of water per 100 kg of corn stover, followed by
washing steps utilizing additional water at room tempera-
ture. In an industrial setup, after the pretreatment process,
sulfuric acid is carried with the liquid stream and neutral-
ized with an alkali (for example, CaO, CaCO
3
or NH
4
OH).
The liquid stream is rich in xylose that can be fermented
to ethanol by xylose-fermenting microbes when properly
conditioned and detoxified [36,37]; however, in this work
this stream was not evaluated for ethanol conversion. The
recovered solid stream represents about 49.3% of the un-
treated corn stover, primarily because of the xylan and
other extractives removal during pretreatment.
In the case of IL pretreatment, 900 kg of [C
2
mim]
[OAc] was used per 100 kg of corn stover. The residual
IL was removed from the solid biomass after pretreat-
ment with a ser ies of water and ethanol washes. In an
industrial setup, the liquid stream can be processed to
recycle and reuse the IL in subsequent pretreatment steps
[38,39]; however, the mass balance over the recycling
process was not performed in this study. IL pretreatment
was able to remove most of the lignin from corn stover,
generating a solid fraction that is enriched in glucan
(46.9%) and xylan (29.8%). This washed solid fraction rep-
resents 64% of the total inlet biomass and was the stream
subjected to the enzymatic hydrolysis step.
Enzymatic hydrolysis of the pretreated corn stover was
performed at 6% glucan loading for 72 hours using the
optimal combination of commercial enzyme cocktails
determined by the previously discussed combinatorial
experiments. For AFE X pretreated biomass, 78.6% of the
glucan wa s converted to monomeric glucose during en-
zymatic hydrolysis, while 52% of the xylan was converted
to monomeric xylose. However, the total soluble sugars
present in the hydrolysate (monomeric and oligomeric)
represent 88.3% and 77.7% of the initial glucan and xy-
lan, respectively. The residual solids stream is composed
of 20.5% (wt/wt) of unhydrolyzed carbohydrates and
26% (wt/wt) of lignin [40,41].
During enzymatic hydrolysis of DA-CS, it was possible
to convert 87.6% and 48% of initial glucan and xylan to
monomeric sugars, respectively. However, the mono-
meric xylose that was converted during enzymatic hy-
drolysis represents only 1.5 kg per 100 kg of the initial
corn stover that was fed into the pretreatment operation.
This is a consequence of the initial xylan solubilization
during DA pretreatment. The total soluble glucan and
xylan produced during enzymatic hydrolysis of DA corn
stover represents 92.2% and 62.3% of the total glucan and
xylan fed into the hydrolysis vessel, respectively. Similar to
the AFEX pretreatment, the solid fracti on leaving after
enzymatic hydrolysis represents 23% of the raw biomass
input a nd can be used to generate electrical power to
supply for biorefinery operations [41].
Enzymatic hydro lysis of IL-CS was able to produce the
highest glucan and xylan conversions to monomeric sugars
among the three processes considered in this study. The
monomeric glucose and xylose conversions during enzym-
atic hydrolysis were 100% and 78.9%, respectively. When
compared to the other pretreate d feedstocks, IL corn
stover did not generate a significant amount of g luco-
oligomers during enzymatic hydrolysis. Howe ver, we ob-
ser ved 1.7 kg of xylo-oligomers for 100 kg of corn stover
fed. This corresponds to 9.4% of the xylan that was carried
through enzymatic hydrolysis. More importantly, the fer-
mentable sugar recovery (based on the initial corn stover
input) for the IL-based process was the highest among the
three cases considered here. The IL-based process was
able to convert 79% of the i nitial sugars present in the
untreated corn stover to fermentable sugars, while AFEX -
and DA-based processes converted 67% and 46%, respect-
ively. Potentially, the DA-based process can utilize the
xylose-rich residue derived from pretreatment wash stream
during fermentation. In this scenario, the sugar recovery
for a DA-based process will improve about 30%, yielding
76% of total fermentable sugar recovery. However, the fer-
mentability of this pretreatment stream was not considered
in this work.
The final ethanol yields of the pro cesses evaluated in
this work are a direct consequence of the nature of the
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Dilute Acid
Pretreatment
Corn Stover
5 kg Sulfuric Acid
895.5 kg Water
100 kg dry weight
33.4 kg glucan
24.9 kg xylan
3.7 kg arabinan
3.6 kg ash
17.2 kg lignin
17.0 kg others
160
o
C
20 mins
46.7 kg dry
weight
4.0 kg glucan
21.0 kg xylan
3.0 kg arabinan
7.0 kg lignin
12.0 kg others
Liquid
Solids
49.3 kg dry
weight
29.0 kg glucan
3.2 kg xylan
1.7 kg arabinan
11.0 kg lignin
4.0 kg others
Enzymatic
Hydrolysis
Hydrolysis
50
o
C and 72 h
Biomass: 29 kg glucan
Ctec 2: 583 g
Htec 2: 287 g
23 kg dry
weight
2.3 kg glucan
1.2 kg xylan
11.0 kg lignin
8.5 kg others
Solids
Liquid
Fermentation
Hydrolysate
28.2 kg glucose
1.3 kg glc olig
1.7 kg xylose
0.4 kg xyl olig
14 kg Ethanol
1.3 kg glc olig
0.7 kg xylose
0.4 kg xyl olig
0.28 g dry-cell-wt./L
Yeast - 424A
A
Ionic Liquid
Pretreatment
Corn Stover
900 kg [C2mim][OAc]
100 kg dry weight
33.4 kg glucan
24.9 kg xylan
3.7 kg arabinan
3.6 kg ash
17.2 kg lignin
17.0 kg others
140
o
C
ATM Pressure
3h
36 kg dry weight
1.9 kg glucan
5.0 kg xylan
3.0 kg arabinan
10.0 kg lignin
Other
Streams
Solids
64 kg dry weight
31.7 kg glucan
19.0 kg xylan
0.2 kg arabinan
6.0 kg lignin
6.0 kg others
Enzymatic
Hydrolysis
Hydrolysis
50
o
C and 72 h
Biomass: 31.7 kg glucan
Ctec 2: 371 g
Htec 2: 314 g
Multifect Pectinase: 266 g
13.5 kg dry
weight
2.3 kg xylan
6.0 kg lignin
5.5 kg others
Solids
Liquid
Fermentation
Hydrolysate
34.4 kg glucose
17.0 kg xylose
1.7 kg xyl olig
21.2 kg Ethanol
6.8 kg xylose
1.7 kg xyl olig
0.28 g dry-cell-wt./L
Yeast - 424A
B
AFEX
Pretreatment
Corn Stover
100 kg Ammonia
60 kg Water
100 kg dry weight
33.4 kg glucan
24.9 kg xylan
3.7 kg arabinan
3.6 kg ash
17.2 kg lignin
17.0 kg others
140
o
C
300 psi
15 mins
Ammonia
for
recovery
Solids
Enzymatic
Hydrolysis
Hydrolysis
50
o
C and 72 h
Biomass: 33.5 kg glucan
Ctec 2: 670 g
Htec 2: 167.5 g
Multifect Pectinase: 167.5 g
47. 3 kg dry
weight
3.9 kg glucan
5.5 kg xylan
0.3 kg arabinan
12.2 kg lignin
Solids
Liquid
Fermentation
Hydrolysate
29.2 kg glucose
3.2 kg glc olig
14.5 kg xylose
3.5 kg xyl olig
20.5 kg Ethanol
0.3 kg glucose
3.2 kg glc olig
2.9 kg xylose
3.5 kg xyl olig
0.28 g dry-cell-wt./L
Yeast - 424A
100 kg dry weight
33.5 kg glucan
24.8 kg xylan
3.3 kg arabinan
4.3 kg ash
17.2 kg lignin
18.0 kg others
C
Figure 5 Material balances during pretreatment, hydrolysis and fermentation for three different processes. (A) DA-, (B) IL- and (C)
AFEX-based pretreatments. AFEX, ammonia fiber expansion; DA, dilute acid; IL, ionic liquid.
Uppugundla et al. Biotechnology for Biofuels 2014, 7:72 Page 10 of 14
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different pretreatments. The AFEX-based process was
able to produce 20.5 kg of ethanol per 100 kg of bio-
mass, while DA- and IL-based biorefineries were able to
produce 14 kg and 21.2 kg, respectively. The lower etha-
nol value for the DA process is a consequence of the ex-
tensive xylan removal during pretreatment. Although the
AFEX process presented the lowest monomeric glucose
and xylose conversions during enzymatic hydrolysis, it
preserved most of the sugars throughout the ethanol pro-
duction process and capitalized on high ethanol metabolic
yields during fermentation (Table 3). AFEX has another
property that was not explored in this study. Since bio-
mass is left dry following AFEX, it can be fed in very high
concentrations, thereby achieving potentially higher etha-
nol concentrations, in contrast with DA and IL pretreat-
ments. In this study, pretreated DA and IL corn stover
was freeze-dried, thereby enabling the 6% glucan loadings
used here. In the case of the IL process, the high mono-
meric glucose and xylose conversions coupled with the
preservation of carbohydrates up to the fermentation stage
contributed to the highest ethanol yield among the three
processes. The viability of these three processes is strongly
dependent on the ethanol yield. However, it should be
noted that the cost of ethanol production (for example,
US$/gallon of ethanol) is the most important metric to
evaluate the commercial potential of these three processes.
Even though this work does not present full techno-
economic evaluations for the various pretreatments in a
process context, it provides useful insights on different as-
pects of the ethanol production platforms and how they
depend on the choice of pretreatment.
Conclusions
In this work, AFEX, DA and IL pretreatments were eval-
uated from a biorefinery processing perspective, using
industrially relevant conditions for converting corn sto-
ver to ethanol. The physicochemical differences between
the pretreated substrates were acknowledged in this
work by optimizing the commercial enzyme cocktails to
maximize sugar yields for each individual substrate. The
optimum enzyme combinations were correlated to the
composition of the pretreated biomass. IL pretreated
corn stover was the most readily digestible among the
substrates considered in this work given the nature and
composition of the substrate, and it also gave improved
fermentability when supplemented with nutrients. Similar
to IL-CS, DA-CS was amenable to enzymatic hydrolysis
and fermentation when supplemented with adequate nu-
trients. In this work, the soluble sugars generated during
DA pretreatment were not considered for fermentation. If
we consider these soluble sugars, the DA pretreatment
process could potentially recover 76% of the total sugars
present in untreated corn stover. Finally, AFEX pretreat-
ment was able to produce highly digestible substrates,
conserving most of the carbohydrates during the pretreat-
ment step. AFEX was able to produce high fermentation
metabolic yield (98%) even without external nutrient sup-
plementation. The ethanol yields calculated for DA, IL
and AFEX pretreated residual solids were 14, 21.2 and
20.5 kg of ethanol per 100 kg of corn stover, respectively.
Materials and methods
Biomass
Corn stover har vested in September 2008, was o btained
from Michigan State University (MSU) Farms (Ea st
Lansing, MI, USA ). The corn hybrid used w as NK 49-E 3
(Syngenta, Basel, S wit zerland) which is a typic al c orn
stover hybrid used in the Great Lakes region. We refer
to it as MSU corn stover. The biomass wa s milled to a
40 mesh size and stored at 4°C until further use.
Enzymes
Cellic® CTec2 (138 mg protein/mL, batch number VCNI
0001), a complex blend of cellulase, β-glucosidase and
hemicellulase, and Cellic® HTec2 (157 mg protein/mL,
batch number VHN00001) were generously provided by
Novozymes (Franklinton, NC, USA). Multifect Pectinase®
(72 mg protein/mL, batch number 4861295753) was a gift
from DuPont Industrial Biosciences (Palo Alto, CA, USA).
The protein concentrations of the enzymes were deter-
mined by estimating the protein (and subtracting the non-
protein nitrogen contribution) using the Kjeldahl nitrogen
analysis method (AOAC Method 2 001.11, Dairy One
Cooperative Inc., Ithac a , NY, USA).
Biomass pretreatment
DA pretreatment was performed at BESC (University of
California, Riverside, CA, USA) at 160°C for 20 minutes
with 10% w/w solid loading and 0.5% w/w sulfuric acid
using a 1 L Parr reactor with two stacked pitched blade
impellers (Model 4525, Parr Instruments Company, Moline,
IL, USA). It took 2 minutes for the reactor to reach 160°C
and another 2 minutes to bring the biomass temperature
down to ambient conditions after pretreatment completion.
The heating system was a 4 kW model SBL-2D fluidized
sand bath (Techne, Princeton, NJ, USA). After the pretreat-
ment, the residual solids were washed with water to remove
acid and other degradation compounds produced during
the process.
IL pretreatment was performed at JBEI (Berkeley, CA,
USA) using 1-ethyl-3-methylimidazolium acetate, abbre-
viated as [C2mim ][OAc], at 140°C for 3 hours using 15%
(wt/wt) loading of biomass to IL in a controlled tem-
perature oil bath using a sealed stirred vessel. It took
30 minutes for the reactor to reach 140°C and 20 minutes
to cool down to 60°C. The residual IL was removed and
pretreated biomass material was recovered with a series of
water and ethanol washes.
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AFEX pretreatment was performed at the GLBRC (Bio-
mass Conversion Research Laboratory, MSU, Lansing, MI,
USA). The conditions were 140°C for 15 minutes at 60%
(wt/wt) moisture with 1:1 anhydrous ammonia to biomass
loading in a bench-top stainless steel batch reactor (Parr
Instruments Company) [33]. It took 30 minutes for the re-
actor to reach 140°C and the ammonia was rapidly re-
leased, which immediately brought the biomass to room
temperature. After the treatment, ammonia was removed
by evaporation, leaving an essentially dry material. Hence
AFEX is a dry to dry process, while IL and DA are dry to
wet processes, as noted above.
Compositional analysis
Extractive-based compositional analyses of the samples
were performed according to the NREL LAPs: Prepar-
ation of Samples for Compositional Analysis (NREL/
TP-510-42620) [42] and Determination of Structural
Carbohydrates and Lignin in Biomass (NREL/TP-510-
42618) [43]. The biomass was extracted with water and
ethanol prior to the acid hydrolysis step. The sugar
concentrations of the extracts were included in t he
composition.
Microplate-based saccharification of pre treated biomass
Ternary enzyme mixture optimization assays were per-
formed on AFEX, freeze-dried DA and freeze-dried IL
pretreated corn stov er samples at 0.2% glucan loading in
a 96-well microplate as described by Gao et al. [44]. The
assay was carried out at 15 mg protein/g glucan loading
using three commercial enzyme mixtures (CTec2, HTec2
and Multifect Pectinase) at different ratios. The pH was
maintained at 4.8 with 50 mM citrate buffer. The micro-
plates were incubated at 50°C for 24 hours at 250 rpm.
After hydrolysis, monomeric glucose and xylose concen-
trations of the liquid samples were determined colorimet-
rically using enzymatic assay kits, as described previously
by Gao et al. [44].
High solid loading hydrolysis
The washed (when used) solid streams from the three dif-
ferent pretreatments were hydrolyzed at 6% glucan loading
in a fermenter equipped with a pitched blade impeller. The
previously determined optimum enzyme mixtures were
used for hydrolysis of the respective pretreated feedstocks.
Hydrolysis was performed over a period of 3 days with
30 mg protein/g glucan enzyme loading at 50°C and
1,000 rpm. Samples were taken every 24 hours and the
sugar concentrations were measured by HPLC. After 3 days
of hydrolysis, the overall mass balances for the pretreated
solids were determined as described previously by Garlock
et al. [6].
Fermentation
The sterile filtered hydrolysates resulting from DA, IL and
AFEX treated corn stover at 6% glucan loading were fer-
mented using a recombinant S. cerevisiae 424A (LNH-ST)
strain capable of metabolizing both glucose and xylose to
ethanol. Fermentations were carried out in 125 mL baffled
shake flasks at 50 mL reaction volume. The experiments
were initiated with an initial cell optical density (OD) of 2
measured at 600 nm, with and without nutrient supple-
mentation (yeast extract (5 g/L) and tryptone (10 g/L)).
Samples were taken at 4, 8, 12, 18, 24, 48, 72, 96 and
120 hours and their glucose, xylose and ethanol concen-
trations were determined by HPLC.
Analytical methods
Monomeric sugars were quantified using a Shimadzu
HPLC system equipped with an Aminex HPX-87P carbo-
hydrate analysis column maintained at 60°C (Bio-Rad,
Hercules, CA, USA) and Shimadzu refractive index de-
tector (RID). Degassed HPLC grade water was used as a
mobile phase at 0.6 mL/min. Injection volume was 10 μL
with a run length of 20 minutes. Fermentation samples
were analyzed for ethanol and residual sugars with the
above me ntioned HPLC system equipped with an Ami-
nex HPX-87H column maintained at 50°C. Sulfuric acid
(5 mM) wa s used as an eluent at 0.6 mL/min. Inje ction
volume and run length was similar to HPX-87P column.
Oligosaccharide analysis
Oligomeric sugar analysis was conducted on the hydrolys-
ate liquid streams using an autoclave-based acid hydrolysis
method at a 2 mL scale. Hydrolysate samples were mixed
with 69.7 μL of 72% sulfuric acid in 10 mL screw-cap cul-
ture tubes and incubated in a 121°C bench-top hot plate
for 1 hour, cooled on ice and filtered into HPLC vials. The
concentration of o ligomeric sugar wa s determined by
subtracting the monomeric sugar concentration of the
non-hydrolyzed samples from the total sugar concentra-
tion of the acid hydrolyzed samples. Sugar degradation was
accounted for by running the appropriate sugar recovery
standards along with the samples during acid hydrolysis.
Abbreviations
[C2mim][OAc]: 1-Ethyl-3-methylimidazolium acetate; AFEX: Ammonia fiber
expansion; ARP: Ammonia recycled percolation; BESC: BioEnergy Science
Center; CAFI: Consortium for Applie d Fundamentals and Innovation;
CrI: Crystallinity index; CS: Corn stover; DA: Dilute acid; DOE: Department of
Energy; GLBRC: Great Lakes Bioenergy Research Center; HPLC: High
performance liquid chromatography; IL: Ionic liquid; JBEI: Joint BioEnergy
Institute; LAP: Laboratory analytical procedure; LCC: Lignin-carbohydrate
complex; MSU: Michigan State University; NREL: National Renewable Energy
Laboratory; OD: Optical density; RID: Refractive index detector; SAA: Soaking
in aqueous ammonia; SHF: Separate hydrolysis and fermentation; YEP: Yeast
Extract Peptone.
Competing interests
The authors declare no competing interests.
Uppugundla et al. Biotechnology for Biofuels 2014, 7:72 Page 12 of 14
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Authors’ contributions
NU designed and executed all experiments. LDS and SPSC guided the
design of experiments and data analysis. XY undertook preliminary
composition analysis. BD initiated the collaboration and reviewed the
manuscript. BS and SS prepared IL pretreated biomass and provided IL
pretreatment-related data. XG, CW and RK prepared DA pretreated biom ass
and provided all DA pretreatment-related data. VB led and coordinated the
overall project. NU, VB and LDS wrote the manuscript. All authors read,
edited and approved the final manuscript.
Acknowledgements
We gratefully acknowledge funding support for this research by the Office of
Biological and Environmental Research in the Department of Energy (DOE)
Office of Science through the BESC at Oak Ridge National Laboratory
(contract DE-PS02-06ER64304), GLBRC (grant DE‐FC02 ‐07ER64494), and JBEI
(grant DE-AC02-05CH11231). We would like to thank Professor Nancy Ho at
Purdue University (West Lafayette, IN, USA) for providing the 424A yeast
strain. We thank Novozymes and DuPont Industrial Biosciences for supplying
commercial enzymes for this collaborative work. Also, we thank Charles
Donald, Margaret Magyar, Christa Gunawan, Rebecca Garlock, Mingjie Jin and
James Humpula (Biomass Conversion Research Laboratory, MSU, Lansing, MI,
USA) for additional laboratory assistance. MBI International (Lansing, MI, USA)
kindly provided access to their 5 gallon batch reactor for AFEX pretreatment.
AFEX
TM
is a trademark of MBI International.
Author details
1
Department of Chemical Engineering and Materials Science, Department of
Energy (DOE) Great Lakes Bioenergy Research Center (GLBRC), Michigan
State University, East Lansing, MI 48824, USA.
2
Department of Biochemistry,
Department of Energy (DOE) Great Lakes Bioenergy Research Center (GLBRC),
University of Wisconsin, Madison, WI 53706, USA.
3
Jilin TuoPai Agriculture
Products Development Ltd, Jilin, China.
4
Deconstruction Division, Joint
BioEnergy Institute (JBEI), Emeryville, CA 94608, USA.
5
Biological and Material
Science Center, Sandia National Laboratories, Livermore, CA 94550, USA.
6
BioEnergy Science Center (BESC), Oak Ridge National Laboratory, Oak Ridge,
TN 37831, USA.
7
Department of Chemical and Environmental Engineering,
Bourns College of Engineering, University of California Riverside, Riverside, CA
92507, USA.
8
Center for Environmental Research and Technology (CE-CERT),
Bourns College of Engineering, University of California Riverside, 1084
Columbia Avenue, Riverside, CA 92507, USA.
Received: 11 October 2013 Accepted: 19 February 2014
Published: 13 May 2014
References
1. Huber GW, Dale BE: Grassoline at the pump. Sci Am 2009, 301(1):52–59.
2. Chundawat SPS, Beckham GT, Himmel ME, Dale BE: Deconstruction of
lignocellulosic biomass to fuels and chemicals. Annu Rev Chem Biomol
Eng 2011, 2(1):121–145.
3. Weber C1, Farwick A, Benisch F, Brat D, Dietz H, Subtil T, Boles E: Trends
and challenges in the microbial production of lignocellulosic bioalcohol
fuels. Appl Microbiol Biotechnol 2010, 87(4):1303–1315.
4. Cosgrove DJ: Growth of the plant cell wall. Nat Rev Mol Cell Biol 2005,
6(11):850–861.
5. da Costa SL, Chundawat SP, Balan V, Dale BE: Cradle-to-grave’ assessment
of existing lignocellulose pretreatment technologies. Curr Opin Biotechnol
2009, 20(3):339–347.
6. Garlock RJ, Balan V, Dale BE, Pallapolu VR, Lee YY, Kim Y, Mosier NS,
Ladisch MR, Holtzapple MT, Falls M, Sierra-Ramirez R, Shi J, Ebrik MA,
Redmond T, Yang B, Wyman CE, Donohoe BS, Vinzant TB, Elander RT,
Hames B, Thomas S, Warner RE: Comparative material balances around
pretreatment technologies for the conversion of switchgrass to soluble
sugars. Bioresour Technol 2011, 102(24):11063–11071.
7. Brodeur G, Yau E, Badal K, Collier J, Ramachandran KB, Ramakrishnan S:
Chemical and physicochemical pretreatment of lignocellulosic biomass: a
review. Enzyme Res 2011. doi:10.4061/2011/787532.
8. Tao L, Aden A, Elander RT, Pallapolu VR, Lee YY, Garlock RJ, Balan V, Dale BE,
Kim Y, Mosier NS, Ladisch MR, Falls M, Holtzapple MT, Sierra R, Shi J, Ebrik MA,
Redmond T, Yang B, Wyman CE, Hames B, Thomas S, Warner RE: Process and
technoeconomic analysis of leading pretreatment technologies for
lignocellulosic ethanol production using switchgrass. Bioresour Technol 2011,
102(24):11105–11114.
9. Elander RT, Dale BE, Holtzapple M, Ladisch MR, Lee YY, Mitchinson C,
Saddler JN, Wyman CE: Summary of findings from the biomass refining
consortium for applied fundamentals and innovation (CAFI): corn stover
pretreatment. Cellulose 2009, 16(4):649–659.
10. Wyman CE, Dale BE, Elander RT, Holtzapple M, Ladisch MR, Lee YY, Mitchinson
C, Saddler JN: Comparative sugar recovery and fermentation data following
pretreatment of poplar wood by leading technologies. Biotechnol Prog 2009,
25(2):333–339.
11. Lau M, Gunawan C, Dale B: The impacts of pretreatment on the
fermentability of pretreated lignocellulosic biomass: a comparative
evaluation between ammonia fiber expansion and dilute acid
pretreatment. Biotechnol Biofuels 2009,
2(1):30.
12. ChundawatSPS,VismehR,SharmaLN,HumpulaJF,daCostaSL,ChamblissCK,
Jones AD, Balan V, Dale BE: Multifaceted characterization of cell wall
decomposition products formed during ammonia fiber expansion (AFEX) and
dilute acid based pretreatments. Bioresour Technol 2010, 101(21 ):8429–8438.
13. Chundawat SPS, Donohoe BS, da Costa SL, Elder T, Agarwal UP, Lu F, Ralph J,
Himmel ME, Balan V, Dale BE: Multi-scale visualization and characterization
of lignocellulosic plant cell wall deconstruction during thermochemical
pretreatment. Energy Environ Sci 2011, 4(3):973–984.
14. Larsson S, Palmqvist E, Hahn-Hägerdal B, Tengborg C, Stenberg K, Zacchi G,
Nilvebrant NO: The generation of fermentation inhibitors during dilute
acid hydrolysis of softwood. Enzyme Microb Technol 1999, 24(3–4):151–159.
15. Çetinkol ÖP, Dibble DC, Cheng G, Kent MS, Knierim B, Auer M, Wemmer DE,
Pelton JG, Melnichenko YB, Ralph J, Simmons BA, Holmes BM: Understanding
the impact of ionic liquid pret reatment on eucalyp tus. Biofuels 2010,
1(1):33–46.
16. Berlin A, Balakshin M, Gilkes N, Kadla J, Maximenko V, Kubo S, Saddler J:
Inhibition of cellulase, xylanase and beta-glucosidase activities by
softwood lignin preparations. J Biotechnol 2006, 125(2):198–209.
17. Cheng G, Varanasi P, Li C, Liu H, Melnichenko YB, Simmons BA, Kent MS,
Singh S: Transition of cellulose crystalline structure and surface
morphology of biomass as a function of ionic liquid pretreatment and
its relation to enzymatic hydrolysis. Biomacromolecules 2011,
12(4):933–941.
18. García-Aparicio MP, Ballesteros I, González A, Oliva JM, Ballesteros M,
Negro MJ: Effect of inhibitors released during steam-explosion pretreatment
of barley straw on enzymatic hydrolysis. Appl Biochem Biotechnol 2006,
129(1):278–288.
19. Kothari U, Lee Y: Inhibition effects of dilute-acid prehydrolysate of corn
stover on enzymatic hydrolysis of solka floc. Appl Biochem Biotechnol
2011, 165(5):1391–1405.
20. Gunjikar TP, Sawant SB, Joshi JB: Shear deactivation of cellulase,
exoglucanase, endoglucanase, and β-glucosidase in a mechanically
agitated reactor. Biotechnol Prog 2001, 17(6):1166–1168.
21. Banerjee G, Car S, Scott-Craig JS, Hodge DB, Walton JD: Alkaline peroxide
pretreatment of corn stover: effects of biomass, peroxide, and enzyme
loading and composition on yields of glucose and xylose.
Biotechnol
Biofuels 2011, 4(1):16.
22. Tejirian A, Xu F: Inhibition of enzymatic cellulolysis by phenolic
compounds. Enzym Microb Technol 2010, 48(3):239–247.
23. Ximenes E, Kim Y, Mosier N, Dien B, Ladisch M: Inhibition of cellulases by
phenols. Enzyme Microb Technol 2010, 46:170–176.
24. Li X, Ximenes E, Kim Y, Slininger M, Meilan R, Ladisch M, Chapple C: Lignin
monomer composition affects Arabidopsis cell-wall degradability after
liquid hot water pretreatment. Biotechnol Biofuels 2010, 3(1):27.
25. Lee SH, Doherty TV, Linhardt RJ, Dordick JS: Ionic liquid-mediated selective
extraction of lignin from wood leading to enhanced enzymatic cellulose
hydrolysis. Biotechnol Bioeng 2009, 102(5):1368–1376.
26. Li C, Cheng G, Balan V, Kent MS, Ong M, Chundawat SP, Sousa L, Melnichenko
YB, Dale BE, Simmons BA, Singh S: Influence of physico-chemical changes on
enzymatic digestibility of ionic liquid and AFEX pretreated corn stover.
Bioresour Technol 2011, 102(13):6928–6936.
27. Li C, Knierim B, Manisseri C, Arora R, Scheller HV, Auer M, Vogel KP,
Simmons BA, Singh S: Comparison of dilute acid and ionic liquid
pretreatment of switchgrass: biomass recalcitrance, delignification and
enzymatic saccharification. Bioresour Technol 2010, 101(13):4900–4906.
28. O'Sullivan A: Cellulose: the structure slowly unravels. Cellulose 1997,
4(3):173–207.
Uppugundla et al. Biotechnology for Biofuels 2014, 7:72 Page 13 of 14
http://www.biotechnologyforbiofuels.com/content/7/1/72
29. Chundawat SP, Bellesia G, Uppugundla N, da Costa SL, Gao D, Cheh AM,
Agarwal UP, Bianchetti CM, Phillips GN Jr, Langan P, Balan V, Gnanakaran S,
Dale BE: Restructuring the cr ystalline cellulose hydrogen bond
network enhances its depolymerization rate. JAmChemSoc2011,
133(29):11163–11174.
30. Kumar R, Mago G, Balan V, Wyman CE: Physical and chemical
characterizations of corn stover and poplar solids resulting from leading
pretreatment technologies. Bioresour Technol 2009, 100(17):3948–3962.
31. Qing Q, Wyman C: Supplementation with xylanase and beta-xylosidase
to reduce xylo-oligomer and xylan inhibition of enzymatic hydrolysis of
cellulose and pretreated corn stover. Biotechnol Biofuels 2011, 4(1):18.
32. Sedlak M, Ho N: Production of ethanol from cellulosic biomass
hydrolysates using genetically engineered Saccharomyces yeast capable
of cofermenting glucose and xylose. Appl Biochem Biotechnol 2004,
114(1):403–416.
33. Balan V, Bals B, Chundawat SP, Marshall D, Dale BE: Lignocellulosic biomass
pretreatment using AFEX. Biofuels. Methods Mol Biol 2009, 581:61–77.
34. Lau MW, Dale BE: Cellulosic ethanol production from AFEX-treated corn
stover using Saccharomyces cerevisiae 424A(LNH-ST). Proc Natl Acad
Sci U S A 2009, 106(5):1368–1373.
35. Lau M, Dale B, Balan V: Ethanolic fermentation of hydrolysates from
ammonia fiber expansion (AFEX) treated corn stover and distillers grain
without detoxification and external nutrient supplementation. Biotechnol
Bioeng 2008, 99:529–539.
36. Carter B, Squillace P, Gilcrease PC, Menkhaus TJ: Detoxification of a
lignocellulosic biomass slurry by soluble polyelectrolyte adsorption for
improved fermentation efficiency. Biotechnol Bioeng 2011,
108(9):2053–2060.
37. Cheng KK, Ge JP, Zhang JA, Ling HZ, Zhou YJ, Yang MD, Xu JM:
Fermentation of pretreated sugarcane bagasse hemicellulose
hydrolysate to ethanol by Pachysolen tannophilus. Biotechnol Lett 2007,
29(7):1051–1055.
38. Haerens K, Van Deuren S, Matthijs E, Van der Bruggen B: Challenges for
recycling ionic liquids by using pressure driven membrane processes.
Green Chem 2010, 12(12):2182–
2188.
39. Dibble DC, Li C, Sun L, George A, Cheng A, Cetinkol OP, Benke P, Holmes
BM, Singh S, Simmons BA: A facile method for the recovery of ionic liquid
and lignin from biomass pretreatment. Green Chem 2011, 13(11):3255–3264.
40. Sendich EN, Laser M, Kim S, Alizadeh H, Laureano-Perez L, Dale B, Lynd L:
Recent process improvements for the ammonia fiber expansion (AFEX)
process and resulting reductions in minimum ethanol selling price.
Bioresour Technol 2008, 99(17):8429–8435.
41. Humbird D, Davis R, Tao L, Kinchin C, Hsu D, Aden A, Schoen P, Lukas J,
Olthof B, Worley M, Sexton D, Dudgeon D: Process Design and Economics for
Biochemical Conversion of Lignocellulosic Biomass to Ethanol. Technical Report
NREL/TP-5100-47764. Golden, CO: National Renewable Energy
Laboratory; 2011.
42. Hames B, Ruiz R, Scarlata C, Sluiter A, Sluiter J, Templeton D: Preparation of
Samples for Compositional Analysis. Technical Report NREL/TP-510-42620.
National Renewable Energy Laboratory: Golden, CO; 2008.
43. Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D:
Determination of Structural Carbohydrates and Lignin in Biomass. Technical
Report NREL/TP-510-42618. National Renewable Energy Laboratory: Golden,
CO; 2012.
44. Gao D, Chundawat SP, Krishnan C, Balan V, Dale BE: Mixture optimization
of six core glycosyl hydrolases for maximizing saccharification of
ammonia fiber expansion (AFEX) pretreated corn stover. Bioresour Technol
2010, 101(8):2770–2781.
doi:10.1186/1754-6834-7-72
Cite this article as: Uppugundla et al.: A comparative study of ethanol
production using dilute acid, ionic liquid and AFEX™ pretreated corn
stover. Biotechnology for Biofuels 2014 7:72.
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