The Influence of Pretreatment and Enzyme Loading on the Effectiveness of Batch

and Fed-Batch Hydrolysis of Corn Stover

Richard P. Chandra, Kathy Au-Yeung, Carolina Chanis, Alexandra A. Roos, Warren Mabee, Pablo A.
Chung, Sonia Ghatora, and Jack N. Saddler
Forest Products Biotechnology Group, Dept. of Wood Science, University of British Columbia, Vancouver, BC, Canada V6T 1Z4

DOI 10.1002/btpr.508
Published online in Wiley Online Library (wileyonlinelibrary.com).

To try to improve hydrolysis yields at elevated solids loadings, a comparison was made
between batch and fed-batch addition of fresh substrate at the initial and later phases of hy-
drolysis. Both ethanol (EPCS) and steam-pretreated corn stover (SPCS) substrates were
tested at low (5 FPU) and high (60 FPU) loadings of cellulase per gram of cellulose. The
fed-batch addition of fresh substrate resulted in a slight decrease in hydrolysis yields when
compared with the corresponding batch reactions. A 72-h hydrolysis of the SPCS substrate
resulted in a hydrolysis yield of 66% compared with 51% for the EPCS substrate. When the
enzyme adsorption and substrate characteristics were assessed during batch and fed-batch
hydrolysis, it appeared that the irreversible binding of cellulases to the more recalcitrant
original substrate limited their access to the freshly added substrate. After 72-h hydrolysis
of the SPCS substrate at low enzyme loadings,
40–50% of the added cellulases were de-
sorbed into solution, whereas only 20% of the added enzyme was released from the EPCS
substrate. Both simultaneous and sequential treatments with xylanases and cellulases
resulted in an up to a 20% increase in hydrolysis yields for both substrates at low enzyme
loading. Simons’ stain measurements indicated that xylanase treatment increased cellulose
access, thus facilitating cellulose hydrolysis. V
C 2010 American Institute of Chemical Engi-

neers Biotechnol. Prog., 000: 000–000, 2010

Keywords: cellulases, bioconversion, hydrolysis, lignocellulosic, cellulose, xylan, corn stover

Introduction ques can be reduced by
33%.4–6 One possible method for

The world is becoming increasingly aware of climate
change issues and the finite nature of our fossil fuel resour-
ces. This has prompted a considerable amount of research
aimed at reducing environmental pollution and securing sus-
tainable fuel sources. The bioconversion of cellulosic bio-
mass to ethanol is one technology currently being assessed
for its potential to supplement the use of current fossil fuel-
derived gasoline/petrol.1 Based on availability, it has been
suggested that a likely initial biomass source for any poten-
tial bioconversion processes would be an already available
agricultural residue such as corn stover (CS).2 The main
steps in the enzymatically mediated bioconversion of bio-
mass to ethanol process include pretreatment to reduce the
recalcitrance of the substrate, subsequent hydrolysis by car-
bohydrate degrading enzymes, fermentation of the resulting
sugars to ethanol, and ethanol recovery.
In previous biomass-to-ethanol work, it has been noted
that there is a need to minimize water requirements,3 which
has the benefit of increasing the concentration of the sugar
stream for fermentation and decreasing the energy required
for ethanol recovery during subsequent distillation. It has
been estimated that, by doubling the ethanol concentration
from 2.5 to 5%, the energy required to distill a fermentation
broth to 93.5% ethanol using conventional distillation techni-
Correspondence concerning this article should be addressed to R. P.
Chandra at richard.chandra@ubc.ca.
increasing the concentration of sugar streams used in the
subsequent fermentation step is to perform the hydrolysis
reactions at a higher solids concentration. However, medium
(10–20% w/v) and high (20–30% w/v) solids loadings bring
about their own set of obstacles. For example, at higher sol-
ids loadings, the reaction viscosity increases, therefore
increasing the energy required for mixing,7 in addition to
possible shear inactivation of cellulases,8 end product inhibi-
tion, and inhibition by lignin either through nonproductive
binding or chemical/physical interference.9,10
Because of the challenges encountered at higher solids
loadings, there have been several studies that have explored
a fed-batch approach of gradually adding the substrate to the
hydrolysis reaction.11 A potential advantage of using a fed-
batch approach is the maintenance of a lower solids loading
during the course of the hydrolysis reaction.12 Fed-batch
experiments have been shown to improve ethanol yields
when a simultaneous saccharification and fermentation (SSF)
approach is used.12,13 However, other work has suggested
that the fed-batch approach is no better than the traditional
batch approach.11 The reported differences in the various
fed-batch hydrolysis results are likely due to that fact that,
when studying fed-batch hydrolysis, the time when the fresh
substrate is added to the actively hydrolyzing reaction is crit-
ical as is the nature of the substrate that is used. It is also
likely that the efficiency of hydrolysis is substrate and
enzyme dependent, with substrates more amenable to enzy-
matic hydrolysis and/or the addition of higher cellulase

C 2010 American Institute of Chemical Engineers

V 1
2 Biotechnol. Prog., 2010, Vol. 00, No. 0
Table 1. Chemical Composition of Ethanol-Pretreated and Steam-Pretreated Corn Stover Water-Insoluble Fractions
Substrate Abbreviation Ara
Steam-pretreated corn stover SPCS 0.6
Ethanol-pretreated corn stover EPCS 0.3
Ara, arabinose; Gal, galactose; Glu, glucose; Xyl, xylose; Man, mannose; AIL, acid-insoluble lignin; ASL, acid-soluble lignin.
enzyme dosages resulting in a greater overall cellulose
conversion.
A typical cellulose hydrolysis initially progresses at a rela-
tively rapid rate, followed by a slowing of the rate of hydro-
lysis and then often ending with the incomplete hydrolysis
of the substrate unless excessive dosages of enzyme are
used.14–16 There have been varying mechanisms proposed to
explain the observed decrease in the hydrolysis rate during
the later stages of lignocellulosic hydrolysis including the
loss of stability or activity of cellulases,17–25 end product in-
hibition by liberated glucose,26–29 a potential increase in sub-
strate recalcitrance over the hydrolysis time period, or
competition between the various cellulase components for re-
active sites on the substrate.30,31
It is possible that during fed-batch substrate addition, if
fresh substrate was added to the hydrolysis reaction at the
‘‘plateau phase,’’ where the potential exists for enzymes to
be either inactive or unable to transition to the freshly added
substrate,31 this could possibly result in the fed-batch reac-
tion being less effective in comparison to substrate addition
at the initial, rapid phase of hydrolysis. The fed-batch sub-
strate addition during hydrolysis is likely an interplay
between the ability of cellulases to desorb and transition to
freshly added substrate and find reactive sites on the freshly
added substrate. Thus, it could be challenging for enzymes
to desorb from recalcitrant, less accessible pretreated
substrates.
In the work described here, we hoped to determine if the
fed-batch hydrolysis of pretreated CS substrates was affected
when the substrate was added at either the initial or plateau
phases of hydrolysis. We also wanted to see if the type of
pretreatment and the nature of the resulting substrate had an
influence on enzyme action and desorption and its possible
influence on overall substrate accessibility.
Materials and Methods
Materials
The cellulase preparation used in this work was Spezyme
CP (Spezyme-CP, Genencor-Danisco, Palo Alto, CA), which
had an activity of 59 FPU/mL and a protein content of 123
mg/mL. The b-glucosidase preparation used was Novozym
188 (Novozymes, Bagsværd, Denmark; 350 IU/mL and 120
mg protein/mL). Multifect xylanase (Genencor-Danisco)
with a protein content of 43.7 mg/mL was also used in this
work. The CS was grown in 2004 and was kindly provided
by the National Renewable Energy Laboratory (Colorado).
Before pretreatment, the CS was milled and sieved to a size
between 2 and 10 mm.
Ethanol pretreatment
In brief, 200 g of CS was pretreated in aqueous ethanol
with sulfuric acid as a catalyst using a four-vessel (2 L each)
rotating digester (Aurora Products, Savona, BC, Canada).
Two hundred grams of CS (200 g oven dry weight) was
Gal Glu Xyl Man AIL ASL Ash
0.2 57.8 8.5 0.6 23.2 2.0 5.4
0.1 67.8 12.3 0.6 14.0 1.1 5.6
treated in each vessel at 200 C for 50 min at a 65% ethanol
concentration and 1.1% (w/w on wood) H2SO4 according to
Pan et al.32 At the conclusion of the pretreatment run, the
vessels were cooled to room temperature in a water bath.
The substrate (solid fraction) and spent liquor were then sep-
arated using a nylon bag. The substrate was washed three
times (300 mL each) with 60 C aqueous ethanol, which was
at the same concentration of ethanol as the original pretreat-
ment liquor. The washes were combined with the spent liq-
uor. The substrate was then washed three times with water at
60 C. The water washes were discarded. The washed sub-
strate was homogenized in a standard British disintegrator
for 5 min and filtered. The yield of solid (substrate) was
49%. All substrates were subsequently stored at 4 C for
analysis and hydrolysis. Only the solid fractions of pretreated
materials were used for this study and are referred to as
ethanol-pretreated CS (EPCS).
Steam pretreatment
The CS was impregnated with 3% SO2 (w/w, based on the
dry weight of the material). The impregnated CS was pre-
treated at 190 C for 5 min33 in 50 g batches in a 2-L steam
gun (Stake Tech II batch reactor, SunOpta (formerly Stake
Technologies) of Norval, ON, Canada). The material was
then collected for analysis. The CS was washed with 10 L of
deionized water, and the solid fraction was used in the hy-
drolysis experiments. The steam-pretreated CS solid fraction
was referred to as SPCS.
Substrate analysis
The composition of the pretreated substrates is detailed in
Table 1. The carbohydrate and the lignin contents were
determined according to the analytical procedure recom-
mended by NREL.34 Fiber quality analysis (FQA), which
measures the length of the fibers, was performed on an Opt-
est Hi-Resolution benchtop fiber quality analyzer.35 Water
retention value (WRV) was performed and calculated
according to TAPPI Useful Method-256.36 Carboxylic acid
groups on substrates were measured using the conductomet-
ric method according to Katz et al.37 Simons’ staining (SS)
was performed according to the modified method by Chandra
et al.38 For hydrolysis reactions that were stopped during hy-
drolysis for subsequent analysis by the above methods, the
reactions were transferred into a KapakTM thermal bag and
submerged into boiling water for 30 min. The samples were
subsequently filtered and stored at 4 C for subsequent
analysis.
Enzymatic hydrolysis
Batch enzymatic hydrolysis experiments were conducted
in 30 mL total volume in 125-mL screw-top Erlenmeyer
flasks at 10% substrate consistency [(g dry substrate)/(g dry
substrate þ g water)] in 50 mM acetate buffer at pH 4.8.
Flasks and substrates were preincubated at 50 C for 20 min
Biotechnol. Prog., 2010, Vol. 00, No. 0
before the addition of enzymes. Reactions were performed at
50 C at a shaking speed of 150 rpm. Reactions were started
by the addition of cellulase and b-glucosidase at either 5 or
60 FPU and CBU per gram of cellulose, respectively. Ali-
quots of 0.15 mL were taken at various times and were
boiled for 20 min at 100 C, followed by centrifugation at
13,000g for 10 min. When specified, enzymatic hydrolysis
reactions were supplemented simultaneously with xylanases
at a 1:7.5 cellulase:xylanase protein ratio.39 Sequential treat-
ment with xylanases involved reacting 3.0 g substrate at
10% substrate consistency with xylanases for 72 h after
which a sample of the liquid was taken for sugar analysis
and the sample was then transferred into a KapakTM thermal
bag and submerged into boiling water for 30 min. Samples
were then washed through a Buchner funnel containing a
Whatman no.1 filter paper and recycling of the filtrate
through the filter cake. The filter cake was then subsequently
washed with an additional 4 L of water. The remaining sub-
strate was subsequently treated with cellulases at 5 FPU/g
cellulose, as described above. Aliquots of 0.15 mL were
taken out before the addition of the substrates for sugar anal-
ysis. For all hydrolysis experiments, samples were removed
from the reaction at specified time intervals and centrifuged
to remove the insoluble materials. The reducing sugar con-
tent was measured by high-performance liquid chromatogra-
phy as described previously.40 The hydrolysis yield of the
substrate was calculated from the measured reducing sugar
content as a percentage of the theoretical reducing sugar
available in each substrate.
Fed-batch substrate additions
Fed-batch hydrolysis experiments were conducted under
the same conditions as batch hydrolysis with the exception
that four portions of 0.75 g of either the SPCS or EPCS sub-
strates were added to the hydrolysis reaction at various
specified times (see below) as opposed to adding the entire
3.0 g of the substrate at the beginning of the reaction. Fed-
batch reactions performed at 5 FPU/g cellulose, involved
adding the substrates to the reaction at 3 h (4  3 h) and 24
h (4  24 h), whereas for reactions at 60 FPU/g cellulase,
substrates were added at 1 h (4  1 h) and 24 h (4  24 h).
The amounts of enzymes required for the whole 3.0 g sub-
strate were added at the beginning of the reaction. For exam-
ple, in the case of the ‘‘4  1 h’’ hydrolysis reaction at 60
FPU/g, all the required enzymes for the 3.0 g of substrate
were added at the beginning of the reaction along with 0.75
g of substrate after which an additional 0.75 g of substrate
was added every hour for 3 h for a total of 3.0 g.
Protein content
A modified ninhydrin method was used to measure the
protein content in the hydrolysates.41 Bovine serum albumin
was used as a standard, ranging from 0.5 to 4.0 mg/mL. In
brief, all samples and standards were diluted accordingly to
fit in the range mentioned above. An aliquot of 40 lL of the
each diluted samples and standards was hydrolyzed over-
night in 100 lL of 1 N HCl in microcentrifuge tubes at
105 C. The next day, 200 lL of 0.2% ninhydrin reagent
(Sigma) was added to the protein hydrolysates. The protein
hydrolysates were boiled in a water bath at 100 C for 20
min. Upon addition of 50% (v/v) ethanol to the cooled sam-
ples, the absorbance was subsequently measured at 570 nm.
3
Control samples were prepared using the same reaction mix-
ture and conditions as the hydrolysis reactions without the
addition of enzyme proteins. The values of protein measure-
ment obtained from the control experiments were subtracted
from the respective experiments where enzyme was added.
Error analysis
A coefficient of variance (COV) value was obtained for
the hydrolysis yield measurements by using the standard
deviation of the hydrolysis yield measurement of the SPCS
substrate hydrolyzed with 5 FPU/g cellulase and 5 CBU/g b-
glucosidase at 10% consistency, 50 C, and pH of 4.8
repeated five times which resulted in a COV of 2.3%.
Results and Discussion
We wanted to initially determine the effects of differential
addition of substrates during the fed-batch hydrolysis of
lignocellulosic substrates at both relatively low (5 FPU/g cel-
lulose) and high (60 FPU/g cellulose) enzyme dosages. The
5 FPU/g enzyme dosage was used to assess whether a fed-
batch approach would improve hydrolysis efficiency at the
lower enzyme loadings as it is apparent that, in an effort to
reduce the cost of the enzymatic hydrolysis process, we need
to maximize enzymatic hydrolysis at minimal enzyme load-
ings.42 The 60 FPU/g cellulose dosage was used, both to
compare the variations in performance and enzyme adsorp-
tion behavior between the low and high enzyme loadings
and to obtain as near complete hydrolysis as possible, so we
could determine if there were any differences in nonproduc-
tive enzyme binding during the hydrolysis of the two sub-
strates. Before the hydrolysis of each of the SPCS and EPCS
substrates, their chemical composition was determined
(Table 1).
Previous work has shown that both the steam- and etha-
nol-pretreatment processes are promising candidates for use
in the bioconversion of lignocellulosics to ethanol.33 Typi-
cally, we have not applied ethanol pretreatment to the, gen-
erally, more easily hydrolyzed substrates such as CS (when
compared with more recalcitrant softwoods) as the primary
mechanism involves the delignification of the substrate. This
delignification mechanism has been shown to increase the
free phenolic groups on the substrate10,43 that have been
shown to influence cellulase inhibition.9,10 With the lower
lignin content of most agricultural substrates, we have found
that steam pretreatment, because of its lower cost and sim-
plicity, is generally preferred. Although SPCS could be
effectively hydrolyzed at enzyme loadings of 15 FPU/g,44
this level of protein loading is still estimated to be higher
than can be justified in a potential commercial bioconversion
process.42 To try to reduce the protein loading, we assessed
the hydrolysis of the SPCS substrate at an enzyme loading
of 5 FPU/g cellulose combined with a fed-batch approach of
substrate addition.
The higher lignin content of the SPCS substrate (23.2%)
compared with the EPCS substrate (14.0%) was due to the
delignification that occurred during the ethanol pretreatment
(Table 1). The EPCS substrate contained a greater amount of
glucan and xylan than did the SPCS substrate. Although pre-
vious work had shown that the ethanol pretreatment of pop-
lar resulted in a significant loss of hemicellulose,33 EPCS
showed significant lignin removal while retaining glucan and
xylan in the substrate (Table 1).
4 Biotechnol. Prog., 2010, Vol. 00, No. 0

When a fed-batch approach is used, where the fresh sub-
strate is added periodically to a hydrolysis reaction that is al-
ready in progress, the point in time at which the fresh
substrate is added to the active hydrolysis reaction may have
a significant effect on the resulting hydrolysis yields. For
example, with batch and fed-batch hydrolysis of substrates
Figure 1. Batch and fed-batch hydrolysis of steam (SPCS)-
and ethanol (EPCS)-pretreated corn stover at an
enzyme loading of (a) 5 FPU/g cellulose and (b) 60
FPU/g cellulose.
The arrows indicate the times where the subsequent addition of
substrate was performed during fed-batch hydrolysis experi-
ments: (a) 3 and 24 h and (b) 1 and 24 h after the start of the
hydrolysis reactions. Reactions performed at 10% solids (%
solids ¼ grams solid/grams solid þ grams liquid) at a constant
reaction volume of 30 mL, pH 4.8, in 50 mM sodium acetate
buffer, 50 C, and a shaker speed of 150 rpm with a 1:1 ratio
of b-glucosidase to cellulase activity (IU:FPU).
containing a higher proportion of lignin, this may influence
nonproductive binding of cellulases, potentially hindering
their ability to transition to allow them to hydrolyze the
freshly added substrate. Alternatively, if more substrate is
added during the initial, rapid phase of hydrolysis, there
should be a ready supply of more easily hydrolyzed cellulose
available to the enzymes.
The fed-batch hydrolysis experiments described in Figure 1
involved adding four batches of 0.75 g of substrate per addi-
tion. Initially, 0.75 g of substrate was hydrolyzed (at
4%
solids) to mimic the first addition of fed-batch hydrolysis to
determine the addition point for subsequent substrate addi-
tions. The initial, rapid phase of hydrolysis at the 5 FPU/g
loading for both substrates occurred during the first 10 h
while the reaction slowed and reached a plateau after 24 h
(Figure 1a). For both the EPCS and SPCS substrates, at the
5 FPU/g enzyme loading, the addition points that were used
for subsequent fed-batch reactions were every 3 and 24 h af-
ter the start of the reaction. The higher amount of enzymes
available to hydrolyze the cellulose at the 60 FPU/g enzyme
loading was the likely reason that both substrates quickly
reached a plateau and were effectively hydrolyzed (Figure
1b). As a result of this efficient hydrolysis at 60 FPU/g, in
subsequent fed-batch experiments additional substrate was
added every 1 and 24 h.
When the one-step batch reaction (3.0 g at 10% solids
loading) was compared to the fed-batch reaction (substrate
added at four distinct times during the hydrolysis reaction) at
both the initial and plateau phases for both the SPCS and
EPCS substrates, it was evident that the batch reaction at 5
FPU/g cellulose had the highest overall yield after 72 h of
hydrolysis (Figures 2a,c). At both enzyme loadings, the addi-
tion of substrates at the later stages of hydrolysis (24-h addi-
tions) during fed-batch reactions resulted in a lower overall
hydrolysis yield (Figures 2a,b). These results suggested that

Figure 2. Hydrolysis yields of SPCS (a and b) and EPCS (c and d) at enzyme loadings of 5 FPU/g cellulose and 60 FPU/g cellulose.
For fed-batch experiments, substrate was added at (a) 3, 6, 9 h and 24, 48, 72 h and (b) 1, 2, 3 h and 24, 48, 72 h after the start of the hydrolysis
reactions. Reactions performed at 10% solids (% solids ¼ grams solid/grams solid þ grams liquid) in a constant reaction volume of 30 mL and a
1:1 ratio of b-glucosidase to cellulase activity (IU:FPU). at pH 4.8 in 50 mM sodium acetate buffer, 50 C, and a shaker speed of 150 rpm.
Biotechnol. Prog., 2010, Vol. 00, No. 0 5

Figure 3. Protein adsorption during hydrolysis of SPCS (a and b) and EPCS (c and d) with an enzyme loading of 5 FPU/g cellulose
and 60 FPU/g cellulose.
For fed-batch experiments, substrate was added at (a) 3, 6, 9 h and 24, 48, 72 h and (b) 1, 2, 3 h and 24, 48, 72 h after the start of the hydrolysis
reactions. Reactions performed at 10% solids (% solids ¼ grams solid/grams solid þ grams liquid) in a constant reaction volume of 30 mL and a
1:1 ratio of b-glucosidase to cellulase activity (IU:FPU). at pH 4.8 in 50 mM sodium acetate buffer, 50 C, and a shaker speed of 150 rpm.

the enzymes were unable to transition from the initial par-
tially hydrolyzed substrate to the freshly added substrate dur-
ing the fed-batch reaction, perhaps because of the
irreversible binding of the enzymes to the substrate and/or
the inability of the remaining unabsorbed enzymes to find
accessible cellulose on the freshly added substrate. The
poorer performance of the fed-batch reactions was unex-
pected, but was in general agreement with previous fed-batch
studies which used barley straw as the substrate.11 It is pos-
sible that glucose may have accumulated more quickly in
the fed-batch reactions because of more efficient mixing at
the initial low solids loading used during the fed-batch hy-
drolysis (
2.5%), resulting in end product inhibition, espe-
cially when lower enzyme loadings were used.
To ensure that an accumulation of cellobiose was not the
cause of inhibition, additional experiments with the EPCS
and SPCS substrates were performed using a cellulase load-
ing of 5 FPU/g cellulose and b-glucosidase loading of 20
CBU/g cellulose against controls using only b-glucosidase at
20 CBU/g cellulose (data not shown). These experiments
showed that the extra b-glucosidase was ineffective in pro-
viding an increase in hydrolysis yields. Therefore, some
other factor such as nonproductive binding or substrate recal-
citrance was responsible for the decrease in performance of
the fed-batch reactions. To try to assess if there was any
nonproductive binding of the added enzymes to the substrate,
we next measured protein adsorption during the batch/fed-
batch reactions to determine whether the added cellulase
enzyme proteins were adsorbed or free in solution.
For both the batch and fed-batch hydrolysis of the SPCS
substrate at the lower enzyme loading, a 66% hydrolysis
yield was obtained after 72 h (Figure 2a), whereas the free
protein decreased in proportion to the amount of substrate
added during each of the hydrolysis reactions. Thus, it
appeared that the 0.75 g (4  3 h) substrate fraction was
only able to absorb 70% of the added enzymes, whereas the
full 3.0 g of substrate (batch reaction) absorbed 90% of the
added enzyme. The maximum protein absorption of the 4 
24-h fed-batch reaction was also 70% (Figure 3a). At the 5
FPU/g cellulase dosage, the EPCS batch reaction reached
close to 0% free protein at the initial stages of the reaction
and the 4  3 h fed-batch reaction behaved quite similarly
(Figure 3c). This was likely due to the slower reaction rate
at the low enzyme loading, which did not show the differen-
ces between the fed-batch and the batch reaction scheme
(Figure 3a). As was observed with the SPCS substrate, the
percentage of free enzymes reached the same level for the
EPCS substrate regardless of whether the reaction was a
batch or fed-batch addition of substrate at both the low and
high enzyme loading. It is also interesting to note that
although the substrate was added every 24 h up to 72 h, the
amount of free enzymes in solution stabilized after the first
48 h even though the hydrolysis yield continued to slowly
increase (Figure 2).
It seems that for both the SPCS and the EPCS substrate,
the addition of fresh substrates at the shorter time periods
(3 h for 5 FPU/g and 1 h for 60 FPU/g) resulted in a slightly
higher adsorption of protein compared with the batch reac-
tion (Figure 3). This was likely due to the residual free
enzymes finding reactive sites on the next batch of substrate
added shortly after the first hydrolysis reaction. The fed-
batch hydrolysis, where substrate was added every 3 h,
resulted in a rapid hydrolysis rate, which resembled a batch
hydrolysis, as the initial enzyme loading was as high as 240
FPU/g cellulose. Likely because there were longer time peri-
ods between the fresh substrate additions, the 4  24-h fed-
6 Biotechnol. Prog., 2010, Vol. 00, No. 0
Table 2. Characteristics and Hydrolysis Yields of Ethanol-Pretreated (EPCS) and Steam-Pretreated Corn Stover (SPCS) Substrates Measured
Using Water Retention Value, Fiber Quality Analysis, Simons’ Staining, and at 0, 7, and 48 h Timepoints During the Hydrolysis Reaction*
Fiber Quality Analysis
Technique Water Retention Average
Sample Value % Particle Size (mm)
SPCS 0 h 305 0.38
SPCS 7 h 425 0.34
SPCS 48 h 174 0.22
EPCS 0 h 381 0.44
EPCS 7 h 609 0.34
EPCS 48 h 293 0.25
* Each timepoint reaction performed separately and boiled for 30 min before filtering and measurement using the above methods. Reactions performed
at 10% solids, a constant reaction volume of 15 mL, and a 1:1 ratio of b-glucosidase to cellulase activity (IU:FPU) at pH 4.8 in 50 mM sodium acetate
buffer, 50 C, and a shaker speed of 150 rpm.
batch hydrolysis reactions resulted in lower yields. This was
also probably because of the nonproductive binding of the
substrate that occurred after the first substrate addition and a
lack of available enzymes to quickly hydrolyze the subse-
quently added fresh substrates. The lower hydrolysis yields
observed for the fed-batch reactions could also be due to the
accumulation of noncellulosic components such as xylan and
lignin at the later stages of the reaction, which could result
in the nonproductive binding of the cellulases to these sub-
strate components.
In addition to differences in protein adsorption at the 5
FPU/g enzyme loading, which may be explained by the
higher amount of cellulose in the EPCS substrate, the hydro-
lysis of the two substrates also resulted in slightly different
hydrolysis yields (
66% for SPCS vs.
50% for EPCS),
suggesting differences between the interaction of the
enzymes with the two substrates. Of the various substrate
factors thought to influence the hydrolysis of cellulosic sub-
strates, the specific surface area of the cellulose available to
the cellulases has been shown to play a pivotal role in deter-
mining the ease of hydrolysis of a given substrate.38 Accessi-
bility of the substrate to the enzyme is governed both by the
physical features of the substrates such as pores and ‘‘swell-
ability’’ and by the chemical features of the substrate, as
both the lignin and hemicellulose components can present a
barrier to the cellulases.45 Therefore, by using the SS and
water retention methods, we next tried to determine which
substrate characteristics, at the initial and plateau phases of
hydrolysis, might influence the ability of the enzymes to cat-
alyze hydrolysis and transition to the freshly added substrate
during fed-batch hydrolysis at low enzyme loadings.
Previous work that had assessed changes in the physio-
chemical properties of dilute acid-pretreated substrates dur-
ing hydrolysis showed a rapid accumulation of lignin and
hemicellulose because of hydrolysis of the cellulose.46 We
therefore assessed changes in the SPCS and EPCS substrates
at 0, 7, and 48 h after hydrolysis at low (5 FPU/g) enzyme
loadings. When we used the SS technique to estimate the
changes in substrate accessibility, the EPCS substrate was
shown to have a higher dye adsorption, most likely because
of its higher cellulose content (Table 2). In addition, the
EPCS substrate also exhibited a higher DO:DB dye ratio,
indicating that the EPCS substrate contained a greater
amount of accessible area to accommodate the DO dye.47
Although the higher cellulose content of the EPCS substrate
may have accounted for an increase in the absorption of the
SS dye, it is possible that some other factor such as nonpro-
ductive binding of the enzymes may play a role in the
increased recalcitrance of the EPCS to enzymatic hydrolysis.
Simons’ Staining Conductometric Titration
Total Dye Carboxylic Cellulose
Absorption Orange:Blue Acid Groups Hydrolysis
(mg/g) Dye Ratio (mmol/kg) Yield (%)
162 2.1 52 0
143 2.2 23
104 1.4 58
217 2.5 34 0
199 2.5 20
142 1.6 41
For example, after 72 h of hydrolysis at the 5 FPU/g cellu-
lose enzyme loading, it was apparent that although the hy-
drolysis reaction had slowed, only about 20% of the added
enzyme protein desorbed from the EPCS substrate. This
effectively slowed the hydrolysis reaction after 72 h com-
pared with the 50% added protein that desorbed from the
SPCS substrate where the hydrolysis continued to increase to
nearly 72% after 96 h (Figure 3). It was also apparent that at
lower solids (
2.5%) the EPCS and SPCS hydrolysis pro-
files were the same when the
20 FPU/g cellulase loading
was used to simulate the first addition of fed-batch hydroly-
sis (Figure 1), while the batch hydrolysis reactions at 5 FPU/
g and 10% solids differentiated the two substrates.
The WRV measurement estimates the amount of water
retained by the substrate after centrifugation, estimating the
degree of substrate swelling,48 which has also been shown to
be an indicator of substrate accessibility to cellulases.49 Meas-
urements on the initial SPCS and EPCS substrates (t ¼ 0)
indicated that, similar to the SS test, the EPCS had a
greater ability to retain water and thus a greater swelling
potential compared with the SPCS substrate (Table 2). This
may be due to the lower lignin content and higher hemicellu-
lose (xylan) content of the EPCS substrate (Table 1) as lignin
has been shown to restrict swelling and hemicellulose has
been shown to promote water uptake in fibers.50 Similarly to
the SS results, there was also a significant increase in the
WRV at 7 h for both substrates suggesting the creation of a
more swollen substrate via the action of the enzymes. It was
apparent that the substrates retained a level of accessibility to
the SS dye and an increase in swelling even while the
substrate cellulose was hydrolyzed. There have been several
examples where cellulases, especially endoglucanases,
have been implicated in the swelling of the substrate during
cellulolytic hydrolysis.51–53
Another measurement associated with lignocellulosic fiber
swelling is the conductometric measurement of carboxylic
acid group functionality.54 An increase in the bulk anionic
charge of a lignocellulosic substrate may also potentially
result in changes in enzymatic binding, as the major compo-
nents of cellulases possess a predominantly negative charge
at a pH of 4.8, which is typically used during hydrolysis
using commercial cellulases.55 It is possible that this could
result in a repelling of cellulase enzymes from the substrate.
It is also possible that the higher number of carboxylic acid
groups may contribute to a greater substrate hydrophilicity,
especially of the lignin component,56 thus potentially
decreasing the likelihood of nonproductive binding of cellu-
lases to the substrate.9,10 The SPCS substrate had a higher
carboxylic group content than the EPCS substrate, which
Biotechnol. Prog., 2010, Vol. 00, No. 0
Figure 4. The simultaneous and sequential additions of xyla-
nases to the reactions of cellulases with (a) steam-
pretreated corn stover (SPCS) and (b) ethanol-pre-
treated corn stover (EPCS).
C, cellulase; X, xylanase. A ‘‘þ’’ indicates a simultaneous treat-
ment and a ‘‘,’’ indicates a sequential treatment. Reactions per-
formed at 10% solids (% solids ¼ grams solid/grams solid þ
grams liquid) in a constant reaction volume of 30 mL and a
1:1 ratio of b-glucosidase to cellulase activity (IU:FPU) at pH
4.8 in 50 mM sodium acetate buffer, 50 C, and a shaker speed
of 150 rpm.
may have contributed to the increase in nonproductive bind-
ing observed with the EPCS substrate compared with the
SPCS substrate at the 5 FPU/g enzyme loading.
In addition to swelling and accessible surface area, the
particle sizes of the substrates were measured during the
course of hydrolysis as particle size has been shown to have
a significant influence on the ease of hydrolysis of lignocel-
lulosic substrates.57 FQA showed that the particle lengths for
both the SPCS and EPCS substrates were quite similar (Ta-
ble 2). The initial fiber size data were unexpected as the
ethanol organosolv pretreatment results in delignification of
the biomass, which presumably facilitates fiber separation,
whereas SO2-catalyzed steam pretreatment does not usually
result in significant delignification. Therefore, the EPCS sub-
strate was expected to be composed of longer fibers. The
fiber length decreased as the hydrolysis reactions progressed,
likely because of the hydrolysis of the cellulose component.
Overall, with the exception of the chemical composition and
carboxylic acid group content (Table 1), the SS and WRVs
were quite similar for the two substrates. Therefore, it was
possible that the differences in hydrolysis yields and the
reduction in performance for both substrates during fed-batch
hydrolysis were due to a lack of accessibility because of the
residual xylan and/or lignin.
There have been numerous studies showing that xylanases
improve cellulolytic hydrolysis when added to cellulase
7
enzyme cocktails58,59 likely through the removal of xylan
increasing accessibility to the cellulose33,60,61 In this study,
the EPCS substrate that contained 12.5% xylan reached a
lower hydrolysis yield at 10% solids compared with the
SPCS substrate, which had a xylan content of 8.5% (Table 1).
It is possible that the recalcitrance of the substrate may have
increased during the course of hydrolysis because of the
increase in the proportion of lignin and xylan as the cellulose
was hydrolyzed, thus limiting further hydrolysis by the cellu-
lases. Therefore, xylanases were added to determine if their
addition could improve hydrolysis yields to assess whether
the accumulated hemicellulose limited accessibility to
cellulases.
It was apparent that the addition of xylanases enhanced
the hydrolysis yields by 14 and 20%, respectively, for the
SPCS and EPCS substrates (Figures 4a,b). To further assess
the beneficial effect of xylanase addition to the hydrolysis
reaction, substrates were treated sequentially with xylanases,
followed by cellulase addition. This prehydrolysis of the
EPCS and SPCS substrates by xylanases with subsequent
cellulase addition (X,C) showed a similar trend to the results
shown for the simultaneous addition of xylanases (CþX)
(Figures 4a,b).
To assess whether the xylanase treatment created a substrate
more accessible to the subsequent hydrolysis by the cellulases,
the SS of the substrate after xylanase treatment was per-
formed. The SS results indicated that the xylanase treatment
of the pretreated SPCS and EPCS substrates increased the total
dye accessibility of the substrates from 162 to 224 mg/g sub-
strate and from 217 to 350 mg/g substrate in the case of the
SPCS and EPCS substrates, respectively. These results indi-
cate that the xylanase treatment increased the overall accessi-
bility of the substrates to cellulases, thereby improving the
overall hydrolysis yields. It was apparent that the xylan played
a limiting role as the yield of the EPCS substrate increased
close to the level of the SPCS substrate upon the sequential
xylanase treatment. It is also likely that, during the fed-batch
reactions using the SPCS and EPCS substrates, the ability for
enzymes to effectively transition to the freshly added substrate
to find a viable reactive site to hydrolyze cellulose was
adversely affected by the xylan.
Conclusions
The fed-batch hydrolysis of both the EPCS and SPCS sub-
strates at a 10% solids loading did not result in increases in hy-
drolysis yields compared with the corresponding batch
reactions. At lower enzyme loadings (5 FPU/g cellulose), the
enzymes appeared to be irreversibly bound during the fed-
batch substrate addition. It seemed that accessibility was a key
factor governing the ability of the cellulases to catalyze hydro-
lysis, as the addition of xylanase resulted in significant
improvements in both hydrolysis yields and accessibility
measurements. To facilitate efficient hydrolysis at lower
enzyme loadings and elevated solids levels, it is likely that ei-
ther the addition of ancillary enzymes and/or pretreatments
that create more accessible substrates with a lower tendency to
nonproductively bind cellulolytic enzymes will be required.
Acknowledgments
The support of the Natural Sciences and Engineering
Research Council of Canada (NSERC), Natural Resources
Canada (NRCan), and BIOCAP Canada is gratefully
8 Biotechnol. Prog., 2010, Vol. 00, No. 0
acknowledged. The authors also thank Genencor-Danisco (Palo
Alto, CA) and Novozymes (Bagsværd, Denmark) for providing
the enzymes used in this study and NREL for providing the
feedstock. They also thank Marc Irawan for his support during
preparation of the protein assays.
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Manuscript received July 29, 2010.