Bioresource Technology 130 (2013) 757762

Contents lists available at SciVerse ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

A mathematical model for the inhibitory effects of lignin

in enzymatic hydrolysis of lignocellulosics
Roger H. Newman , Alankar A. Vaidya, Sylke H. Campion
Scion, Private Bag 3020, Rotorua Mail Centre, Rotorua 3046, New Zealand

h i g h l i g h t s
" Cellulose conversion was calculated at cessation of cellulase activity.
" Steam-exploded pine feedstock provided data for a worked example.
" Conversion at relatively low enzyme loading characterized enzyme deactivation.
" Conversion at relatively high enzyme loading characterized cellulose occlusion.
" Enzyme requirements doubled when the temperature was raised from 30 C to 50 C.

a r t i c l e

i n f o

Article history:
Received 20 August 2012
Received in revised form 9 November 2012
Accepted 16 December 2012
Available online 23 December 2012
Keywords:
Softwood
Steam explosion
Enzymatic hydrolysis
Enzyme deactivation

a b s t r a c t
A new model for enzymatic hydrolysis of lignocellulosic biomass distinguishes causal inuences from
enzyme deactivation and restrictions on the accessibility of cellulose. It focuses on calculating the
amount of unreacted cellulose at cessation of enzyme activity, unlike existing models that were constructed for calculating the time dependence of conversion. There are three adjustable parameters: (1)
occluded cellulose is dened as cellulose that cannot be hydrolysed regardless of enzyme loading or
incubation time, (2) a characteristic enzyme loading is sufcient to hydrolyse half of the non-occluded
cellulose, (3) a mechanism index measures deviations from rst-order kinetics. This model was used to
predict that the optimal incubation temperature is lower for lignocellulosics than for pure cellulose. For
steam-exploded pine wood after 96 h incubation, occluded cellulose was 24% and 26% at 30 C and 50 C,
and the characteristic enzyme loadings were 10 and 18 FPU/g substrate, respectively.
2013 Elsevier Ltd. All rights reserved.

1. Introduction
The lignin in lignocellulosic biomass inhibits enzymatic hydrolysis, and the unique chemistry of softwood lignin makes softwoods particularly challenging for bioconversion (Mabee et al.,
2006). Manseld et al. (1999) suggested that two distinct mechanisms are involved: lignin binds cellulases in non-productive complexes, while also blocking cellulose from being accessible to
cellulases. This paper describes a mathematical model developed
to assist in distinguishing between the two mechanisms, and uses
softwood biomass to illustrate use of the model.
Lignin and other phenolic substances can inhibit enzymatic
hydrolysis through non-productive binding (Pan, 2008; Ximenes
et al., 2010) or permanent deactivation (Ximenes et al., 2011).
The distinction between the two mechanisms is important. Nonproductive binding to the substrate or hydrolysis products slows

Corresponding author. Tel.: +64 7 343 5899.

E-mail address: Roger.Newman@scionresearch.com (R.H. Newman).
0960-8524/$ - see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.12.122

the hydrolysis of cellulose to glucose, but the enzymes are eventually released to continue the hydrolysis process. On the other hand,
permanent deactivation, e.g. through denaturation or chemical
deactivation reactions with the substrate, can lead to cessation of
hydrolysis before all of the cellulose has been converted to glucose.
Ximenes et al. (2011) studied the effects of phenolic substances
formed by degradation of lignin, e.g. vanillin, cinnamic acid and
4-hydroxycinnamic acid, and reported considerable enzyme deactivation for substances that showed only traces of non-productive
binding. Sinitsyn et al. (1982) washed steam-exploded hardwood
and found that the wash water contained cellulase inhibitors. They
also found that adding the wash water to the washed wood resulted in markedly inferior glucose yields, with hydrolysis halted
after incubation for 84 h. The latter observation indicated enzyme
deactivation by water-soluble components of pretreated wood.
Sewalt et al. (1996) suggested that deactivation of cellulase by
lignin involves chemical reactions with quinone methide
intermediates.
Permanent deactivation can also occur in the absence of lignin,
e.g. as a result of shear forces generated by agitation (Taneda et al.,

758

R.H. Newman et al. / Bioresource Technology 130 (2013) 757762

2012), particularly in the presence of airliquid interfaces (Basu

and Pal, 1956; Kim et al., 1982). Thermal deactivation can also be
important. Studies of enzymatic hydrolysis of cellulose usually report incubation temperatures between 45 C and 50 C, maximizing the initial rate of conversion of cellulose to glucose (Baker
et al., 1992; Tengborg et al., 2001). If the goal is to maximize the
nal degree of conversion, rather than the initial rate of conversion,
then a lower incubation temperature might be more appropriate
(Eklund et al., 1990; Tengborg et al., 2001). It is important that
any mathematical model for deactivation incorporates the inuences of shear-force and thermal deactivation, as well as deactivation by chemical reactions.
Delignication is effective, but expensive (Cullis and Manseld,
2004). Cellulose microbrils can be made more accessible by thermochemical treatment, during which lignin is depolymerized, relocated and repolymerized as droplets (Hansen et al., 2011).
Accessibility is readily measured by adsorption of dyes or uorescently-labeled enzymes (Esteghlalian et al., 2001), and sometimes
changes during conversion (Kumar and Wyman, 2009). In this paper, the cellulose that never becomes accessible, even after an innitely long incubation time, is referred to as occluded cellulose.
The distinction between inaccessible cellulose and occluded cellulose is important. Cellulose that is initially inaccessible might become accessible through the action of enzymes, digesting layers
of hemicelluloses that obstruct the movement of cellulase into
the pores of the substrate. Occluded cellulose never becomes
accessible, because it is surrounded by substances that are not digested by any component of the enzyme mixture.
Numerous mathematical models have described the kinetics of
enzymatic hydrolysis of cellulose over periods of hours or days
(Zhang and Lynd, 2004; Zhou et al., 2009; Wang and Feng, 2010).
Most of the models were based on an implicit assumption that
all of the available cellulose would eventually be consumed after
long incubation times. Some models have showed that deactivation can slow the release of glucose as conversion proceeds (Gusakov et al., 1992; Shen and Agblevor, 2008; Zhang et al., 2010; Ye
and Berson, 2011). A different approach was taken in the mathematical modeling reported here. Instead of describing conversion
as a function of time, the goal of the present work was to derive
a mathematical relationship between enzyme loading and the extent of conversion attained when enzymatic hydrolysis halts. This
was achieved by considering the ratio of rates of hydrolysis and
deactivation, so that incubation time was eliminated from the
equations.
2. Theory
The model was kept at a semi-mechanistic level for simplicity.
In other words, the details of reaction mechanisms were not incorporated. As an example of a simplifying assumption, a single symbol E was used to represent cellulase activity in a broad sense.
Commercial products contain mixtures of enzymes including
endoglucanases, exoglucanases and b-glucosidases (Zhang et al.,
2006). In this work, only enzyme activity associated with consumption of cellulose was considered relevant to the symbol E. It
was assumed that cellobiose and other intermediate products were
eventually hydrolyzed to glucose by enzymes other than those included in the activity represented by E.
The process was assumed to start with enzymes added to a slurry of lignocellulosic substrate, bringing the enzyme concentration
to E = E0, expressed in FPU (lter paper units) per liter, and the cellulose concentration to C = C0, expressed in grams per liter. The
process was assumed to end when the enzyme was fully deactivated, i.e., E1 = 0, leaving a concentration C1 of cellulose unreacted. The percent conversion of cellulose was dened as:

Conversion % AC 0  C 1 C 1
0

Here A is the percentage of cellulose that can be hydrolyzed at relatively high enzyme loadings and long incubation times, i.e., 100A
is the percentage of occluded cellulose. The rate of deactivation of
the enzyme was assumed proportional to a function f(E):

dE=dt kd f E

Here kd is a rate constant for deactivation, assumed to involve a

mixture of contributing processes including shear deactivation,
thermal denaturation and chemical reactions with lignin. If deactivation is rst order in E, as suggested by Gusakov et al. (1992), then
f(E) = E. If a portion of the enzyme is temporarily involved in nonproductive binding, f(E) might represent the non-bound portion.
The form of f(E) is not relevant here, as will be discussed further below. The rate of deactivation by lignin will, of course, depend on the
shear forces applied during agitation, the temperature of incubation, and the concentration of lignin, so it was assumed that all
these factors remain constant through the period of incubation.
The function f(E) includes deactivation by substrate components
other than lignin, and deactivation mechanisms that do not involve
the substrate, such as thermal denaturation or deactivation at air
liquid interfaces as discussed in the Section 1.
The rate of change of C was likewise assumed proportional to
f(E):

dC=dt kh C m1 f E

Here kh is a rate constant for enzymatic hydrolysis. The exponent of

m + 1 in Eq. (3) allows for a decline in reaction rate as conversion
proceeds: the larger the value of the mechanism index m, the steeper the decline in reaction rate. If m = 0, conversion follows a kinetic
equation that is rst-order in cellulose. Any decline in the reaction
rate can then be attributed to enzyme deactivation. If m > 0, at least
a portion of the decline can be attributed to decreasing accessibility
of cellulose.Dividing Eq. (3) by Eq. (2) eliminates incubation time
and f(E) from a differential equation:
1

dE=dC kd kh C m1

The elimination of f(E) from Eq. (4) means that it is not necessary to
consider details of the conversion processes that are time dependent, e.g., the rate of exchange of enzymes between non-bound
and non-productively bound states.
A general relationship between enzyme loading and nal conversion can be obtained by integrating Eq. (4) from the initial condition to the nal condition. In the present work, just two values of
m are considered: m = 0 or 1 in Models 1 and 2, respectively.
In Model 1, any decline in reaction rate is attributed to deactivation of the enzyme rather than changes in accessibility of cellulose. Integration of Eq. (4) with m = 0 gives:
1

E0 kd kh ln C 0  ln C 1

Rearranging Eq. (5) and insertion into Eq. (1) gives:

1

Conversion % A1  expkd kh E0

The initial enzyme loading EL was expressed as FPU per gram of

substrate, and dened as EL = E0X/C0, where X is the mass fraction
of cellulose in the substrate. A characteristic enzyme loading e
was dened as the value of EL that is sufcient to achieve conversion of half of the cellulose, other than occluded cellulose, before
enzyme activity ceases. Eq. (6) then becomes:

Conversion % A1  expce1 EL

Here c = ln(2) = 0.693, and:

1
e cXC 1
0 kd kh

R.H. Newman et al. / Bioresource Technology 130 (2013) 757762

In Model 2, the rate of enzymatic hydrolysis declines more steeply than in Model 1. Integrating Eq. (4) with m = 1 and inserting the
result in Eq. (1) gives:

Conversion % AEL EL e1

Here e remains dened as the value of EL required to achieve conversion of half of the cellulose, other than occluded cellulose, and
in Model 2 the value of e is:
1
e XC 2
0 kd kh

10

Eq. (9) is identical to an empirical relationship proposed by Sattler

et al. (1989) in their study of enzymatic hydrolysis of steam-pretreated willow wood.
In both special cases of the general model, e involves a ratio of
rate constants for deactivation and enzyme hydrolysis. Both rate
constants are expected to increase with temperature, but the rate
constant for a non-catalyzed reaction is expected to rise more steeply than that for a catalyzed reaction, so the value of e is expected
to be a function of incubation temperature. This prediction was
tested by measuring cellulose conversion for two different incubation temperatures and several different enzyme loadings.
3. Methods
3.1. Substrate
Steam-exploded softwood was used as the substrate, because of
the high lignin contents reported for similar substrates. Lignin contents between 39% and 46% have been reported for steam-exploded
and steam-pretreated woods from three different softwood species
(Tengborg et al., 2001; Lu et al., 2002; Kumar et al., 2010).
Pinus radiata wood chips were obtained from a local sawmill
and used to produce steam-exploded wood (SEW). The steam
explosion apparatus was built around a 75 mm diameter Type
316 stainless tube with a ball valve mounted at each end, designed
to be heated by steam injected directly into the tube. The lower
ball valve discharged into a cyclone. A portion of 0.75 kg chips,
with a moisture content of 60%, was impregnated with SO2
(3% w/w) and heated with steam at 215 C for 3 min, and the lower
ball valve was opened. The pulp was washed four times with water
to obtain a 54% yield of water-insoluble matter on an oven-dried
basis. It was freeze-dried, Wiley milled at 80 mesh and sieved
through a 250 lm screen to reject coarse particles. Milling and
sieving ensured uniform sub-samples for small-scale enzymatic
hydrolysis experiments. Preliminary enzymatic hydrolysis experiments on SEW that was neither freeze-dried nor milled showed
poor reproducibility, attributed to the sample size being too small
for successful assembly of a collection of fragments to represent
the mixture in the bulk supply.
Extractives were determined using a FOSS Soxtec System 1043
extraction unit with dichloromethane as the solvent. Lignin was
determined using methods based on TAPPI Standard Method T
222 om-88 and TAPPI Useful Method UM 250. Fucose was added
to the hydrolysate from lignin analysis, as an internal standard,
and the carbohydrates were analyzed by ion chromatograph using
a Dionex ICS 3000 instrument (Pettersen and Schwandt, 1991). The
composition of the freeze-dried SEW was: 53.6% glucosyl residues,
36.2% Klason lignin, 5.5% extractives, 0.4% acid-soluble lignin, 0.2%
mannosyl residues and 0.2% xylosyl residues. The composition was
similar to published compositions for steam-exploded and steampretreated woods from other softwood species (Tengborg et al.,
2001; Lu et al., 2002; Kumar et al., 2010). In particular, hemicellulose contents are typically <3%, most of the hemicelluloses having
been hydrolyzed during pretreatment.

759

3.2. Enzymatic hydrolysis

Hydrolysis was performed on a 5 ml scale at 30 C or 50 C, pH
4.8, in 0.05 M sodium citrate buffer containing 0.01% w/v sodium
azide, in 20-ml screw-capped glass tubes agitated at 180 rpm.
Rotational motion was used, rather than shaking which caused turbulence and might have increased the rate of deactivation of enzymes (Basu and Pal, 1956). Experiments were carried out in
duplicates or triplicates using a substrate concentration of 1.5%
dry matter (DM). The SEW absorbed moisture from room air, and
this was allowed for in weighing out each portion of 75 mg substrate on a dry basis. The low substrate concentration was used
to avoid mass transfer problems encountered when agitating higher consistency samples.
Celluclast 1.5 l was supplemented with Novozym 188 to provide additional cellobiase activity. The cellobiohydrolase in Celluclast 1.5 l converts cellulose to cellobiose, but is inhibited by that
cellobiose. Novozym 188 converts cellobiose to glucose and is, in
turn, inhibited by that glucose (Hong et al., 1981). The enzymes
were mixed in an activity ratio of 1 FPU1.25 IU, in order to ensure
adequate conversion of cellobiose to glucose. In published experiments enzymatic hydrolysis of pretreated woods, ratios have usually been chosen within the range 1:0.51:2.0 (Sinitsyn et al.,
1982; Sattler et al., 1989; Gusakov et al., 1992; Esteghlalian
et al., 2001; Lu et al., 2002; Cullis and Manseld, 2004; Mabee
et al., 2006; Brjesson et al., 2007; Pan, 2008; Kumar and Wyman,
2009; Kumar et al., 2010), although lower and higher ratios have
also been tested (Eklund et al., 1990; Tengborg et al., 2001; Inoue
et al., 2008). Enzyme activities were measured by standard IUPAC
methods (Ghose, 1987). Activity values were 67 FPU/ml for Celluclast 1.5 l and 498 IU/ml for Novozym 188. These activity values
were similar to those published in other studies (Brjesson et al.,
2007; Tengborg et al., 2001). Protein was analyzed by the method
of Bradford (1976), using bovine serum albumin as the standard.
The mixture of the two enzymes showed an activity-to-protein ratio of 1.7 FPU/mg.
The samples for sugar analysis were taken at different time
points up to 28 days. For each sample, enzymatic hydrolysis was
stopped by plunging the tube into boiling water for 5 min and then
cooling it to room temperature in water. The mixture was then
centrifuged at 4000 rpm for 10 min at 25 C and the supernatant
was collected for glucose analysis by a YSI 2700 SELECT Biochemistry Analyzer (YSI Life Sciences) congured in single analyte
mode. The manufacturers specications for precision indicated
that the coefcient of variation should be <2% for 10 measurements of glucose concentration. Results were corrected for small
amounts of glucose introduced along with the enzyme solution.
The standard deviation was used to calculate the 95% condence
interval for each mean value from duplicate or triplicate hydrolysis
experiments.
4. Results and discussion
4.1. Cessation of enzymatic hydrolysis
For an enzyme loading of 5 FPU/g substrate, conversion of cellulose to glucose reached a plateau at about 12% conversion after 24 h
of incubation at 50 C (Fig. 1). Hydrolysis was not limited by occlusion of the cellulose, since treatment with larger enzyme loadings
resulted in conversion of 74% of the cellulose as discussed below.
4.2. Occlusion of cellulose
Final conversion from cellulose to glucose was plotted against
enzyme loading, i.e. EL, for incubation temperatures of 30 C and

760

R.H. Newman et al. / Bioresource Technology 130 (2013) 757762

Table 1
Best-t parameters for enzymatic hydrolysis of pine SEW.

e (FPU/g substrate)

RMSDa (%)

Model 1, Eq. (7)

30 C
24
50 C
26

10
18

1.4
1.1

Model 2, Eq. (8)

30 C
13
50 C
18

12
19

2.3
2.2

Model

100A (%)

a
Root-mean-square deviation of the best-t curve from experimental data
points.

Fig. 1. Enzymatic hydrolysis at 50 C and an initial enzyme loading of 5 FPU/g

substrate. Error bars show 95% condence intervals based on replication of data.

50 C (Fig. 2). For both incubation temperatures, Eq. (7) (Model 1,

m = 0) gave a better t than Eq. (9) (Model 2, m = 1). This comparison was reected in the root-mean-square-deviations (Table 1).
Model 1 led to a correct prediction that the curves would reach a
plateau at relatively high enzyme loadings, i.e., EL > 50 FPU/g substrate at 30 C or EL > 100 FPU/g cellulose at 50 C (Fig. 2). Model
2 led to a prediction that the curves would continue to rise steadily
on the right-hand side of Fig. 2. That was not observed. The differences between the qualities of the ts were, however, too small to
warrant investigation of intermediate values of the mechanism index, i.e., 0 < m < 1.
Experiments carried out at longer incubation times pointed to a
limitation in the model, in that the parameter A showed a small
dependence on incubation time (Fig. 3). The best-t value for
occlusion dropped from 100A = 26% at 96 h to 20% at 168 h and
13% at 336 h of incubation. The slow increase in the apparent value
of A was attributed to slow diffusion of enzymes into poorly-accessible domains, rather than fully occluded domains. The indicator
for deactivation showed little change, with best-t values of
e = 18, 18 and 20 FPU/g substrate after incubation times of 96,
168 and 336 h, respectively.

Fig. 2. Enzymatic hydrolysis by incubation for 96 h at 30 C and 50 C. Error bars

show 95% condence intervals based on replication of data. Best-t curves were
based on Model 1 (m = 0, solid line) and Model 2 (m = 1, broken line).

Fig. 3. Enzymatic hydrolysis by incubation at 50 C. The incubation time (h) is

shown beside each curve. Error bars show 95% condence intervals based on
replication of data. Best-t curves were based on Model 1 (m = 0).

Sattler et al. (1989) studied enzymatic hydrolysis of steam-pretreated willow wood and reported good ts to Eq. (9), i.e., Model 2.
Their procedure for assessing the quality of the t was reassessed.
Sattler et al. (1989) plotted the reciprocal of conversion against the
reciprocal of enzyme loading and reported linear plots, consistent
with the inverted form of Eq. (9):
1

Conversion

1
eA1 E1
L A

11

Close inspection of their plots revealed slight deviations from linearity. Results from the present work (Fig. 4) likewise showed a deviation from linearity for high enzyme loadings, i.e., at the left-hand
side of the plot. A broken line in Fig. 4 indicates extrapolation from
the linear portion of the plot, as carried out by Sattler et al. (1989) in
order to determine the intercept 1/A in Eq. (11). In Fig. 4, the intercept of the broken line on the vertical axis corresponds to A > 100%,
yet the experimental data points curve away from broken line towards the intercept indicated by the solid curve. That intercept corresponds to A = 76% as determined by direct least-squares tting to
Model 1 (Table 1). Clearly it is important to use direct least-squares
tting rather than the procedure suggested by Sattler et al. (1989).
It is also important to use enzyme loadings much greater than the
loadings compatible with commercial processing, i.e., it is important to place multiple data points on the right-hand side of Fig. 2
or left-hand side of Fig. 4.
Sattler et al. (1989) and Lu et al. (2002) used wet substrates obtained by pretreating willow and Douglas r, respectively, and reported that cellulose conversion rose to >90% at relatively high
enzyme loadings. Luo and Zhu (2011) studied pretreated lodgepole
pine and showed that air drying led to decreased enzyme digestibility, attributed to hornication and consequent loss of enzyme

R.H. Newman et al. / Bioresource Technology 130 (2013) 757762

Fig. 4. The inverse of cellulose conversion plotted against the inverse of initial
enzyme loading, for incubation at 30 C. Error bars show 95% condence intervals
based on replication of data. The solid curve represents the best t to Model 1
(m = 0), and the broken line indicates extrapolation from the linear portion of the
solid curve as in Eq. (11) for Model 2 (m = 1).

accessibility. Inoue et al. (2008) freeze dried pretreated eucalypt

wood, rewet it for incubation at 45 C for 72 h and plotted conversion to glucose against enzyme loading. Their plots showed plateaus at conversion values between 75% and 80%, depending on
the nature of pretreatment, corresponding to between 20% and
25% occlusion. The best-t values of the occlusion parameter for
pine SEW (Table 1) were similar to those reported by Inoue et al.
(2008). The amounts of occluded cellulose reported by Inoue
et al. (2008), along with those reported here, were attributed to
hornication processes during freeze drying.
4.3. Capacity for deactivation
When the steam-exploded wood was incubated at 30 C for
96 h with an enzyme loading of 2.0 FPU/g substrate, 13% of the cellulose was converted to glucose (Fig. 2). Enzyme loadings of just a
few FPU/g substrate have rarely been investigated in studies of
pretreated softwoods. The closest comparison available was a
study by Kumar et al. (2010), in which six pretreated softwood
samples were incubated for 72 h of incubation at 50 C at enzyme
loadings between 2.5 and 2.9 FPU/g substrate. Between 18% and
26% of the cellulose was converted to glucose. The results reported
in the present work are consistent with those reported by Kumar
et al. (2010) after allowance for the difference in enzyme loadings.
The relevance of lignin was demonstrated by Kumar et al. (2010)
when they delignied one of their substrates with sodium chlorite
and incubated it under the same conditions, to achieve 94% conversion of cellulose to glucose.
The best-t values of e in Eq. (7) were 10 and 18 FPU/g substrate
for incubation at 30 C and 50 C, respectively, after 96 h incubation (Table 1). The quality of the ts to Eq. (7) were consistent with
the mechanism of Model 1, i.e., m = 0 corresponding to constant
accessibility of cellulose throughout conversion, and a declining
reaction rate because of deactivation of enzymes through chemical
reactions with lignin.
Pan (2008) tested the hypothesis that lignin is responsible for
deactivation of cellulase. When microcrystalline cellulose was
incubated with enzymes at a loading of 15 FPU/g cellulose, the enzymes remained active after 72 h at 45 C. When softwood ethanolysis lignin was added at a loading of 0.2 g/g cellulose, conversion
of cellulose to glucose leveled off at approximately 50% after 72 h.

761

This result indicated a deactivation capacity of e = 75 FPU/g lignin.

Pan (2008) tested four other types of lignins, and found them less
effective in deactivating enzymes. In the present work, pine SEW
contained 36.2% lignin, so the best-t values of e (Table 1), expressed in terms of the weight of lignin, were 28 and 50 FPU/g lignin for incubation temperatures of 30 C and 50 C, respectively.
The capacity for deactivation in the present work was similar to
that reported by Pan (2008), and therefore consistent with deactivation by lignin.
Other authors have reported lower deactivation capacities, expressed in terms of the weight of lignin. Lu et al. (2002) hydrolyzed
softwood SEW for 72 h at 45 C, and reported that hydrolysis
stopped at approximately 50% conversion when the enzyme loading was 10 FPU/g substrate. Their substrate contained 46% lignin,
so their observation indicated e = 22 FPU/g lignin. Sattler et al.
(1989) steam-pretreated hardwood at 200 C and hydrolyzed it
for 72 h at 50 C. Hydrolysis stopped at approximately 50% conversion when the enzyme loading was 5 FPU/g substrate. They did not
report the lignin content of their substrate, but a value in the typical range 3350% would correspond to a value of e in the range
1015 FPU/g lignin. According to Eq. (8), the value of e decreases
when the rate of deactivation decreases, or when the rate of enzymatic hydrolysis increases. The low capacity for enzyme deactivation observed by Sattler et al. (1989) might reect greater
accessibility of the hardwood substrate in the initial stages of enzymatic hydrolysis, so that a larger amount of cellulose was converted before enzyme deactivation caused hydrolysis to halt.
Other studies of pretreated wood have drawn attention to the
advantages of incubation at temperatures below 50 C, expressing
those advantages in terms of improved yields at xed enzyme
loadings rather than in terms of enzyme consumption. Eklund
et al. (1990) and Brjesson et al. (2007) studied enzymatic hydrolysis of steam-pretreated woods and found improvements in glucose yield when the incubation temperature was lowered from
50 C to 40 C. Tengborg et al. (2001) studied enzymatic hydrolysis
of steam-pretreated spruce at 38, 45 and 52 C, and found that the
lowest of those incubation temperatures gave the highest levels of
conversion from cellulose to glucose. They also found that the sensitivity to temperature increased when the incubation time was increased from 72 h to 144 h, indicating that maximizing nal
conversion might require an incubation temperature below 38 C.
The temperature dependence of e reported here is therefore consistent with reported results.
5. Conclusion
A mathematical model containing just three adjustable parameters proved adequate to describe the relationship between enzyme loading and the amount of unreacted cellulose when
enzyme activity ceased, at least for pine SEW. The model accounted for declining reaction rates as conversion proceeded.
Best-t values of the parameters were consistent with cellulose
accessibility remaining constant during conversion, so the declining reaction rates were attributed to enzyme deactivation, e.g.
through chemical reactions with lignin. The capacity for enzyme
deactivation was almost doubled by raising the incubation temperature from 30 C to 50 C.
Acknowledgements
The authors thank Daniel van de Pas and Peter Dare for steam
explosion processing, Katrina Martin for analysis of the substrate,
and Sara Carey and Keryn Tutt for assistance with enzymatic
hydrolysis. Financial support from the Ministry for Science and
Innovation under contract CO4X0802 is gratefully acknowledged.

762

R.H. Newman et al. / Bioresource Technology 130 (2013) 757762

References
Baker, J.O., Tatsumoto, K., Grohmann, K., Woodward, J., Wichert, J.M., Shoemaker,
S.P., Himmel, M.E., 1992. Thermal denaturation of Trichoderma reesei cellulases
studied by differential scanning calorimetry and tryptophan uorescence. Appl.
Biochem. Biotechnol. 34 (35), 217231.
Basu, S.N., Pal, P.N., 1956. An unfavourable effect of shaking on fungal cellulases.
Nature 178, 312313.
Brjesson, J., Peterson, R., Tjerneld, F., 2007. Enhanced enzymatic conversion of
softwood lignocellulose by poly(ethylene glycol) addition. Enzyme Microb.
Technol. 40, 754762.
Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem. 72, 248254.
Cullis, I.F., Manseld, S.D., 2004. Optimized delignication of wood-derived
lignocellulosics for improving enzymatic hydrolysis. Biotechnol. Bioeng. 106,
884893.
Eklund, R., Galbe, M., Zacchi, G., 1990. Optimization of temperature and enzyme
concentration in the enzymatic saccharication of steam-pretreated willow.
Enzyme Microb. Technol. 12, 225228.
Esteghlalian, A., Bilodeau, M., Manseld, S.D., Saddler, J.N., 2001. Do enzymatic
hydrolyzability and Simons stain reect the changes in the accessibility of
lignocellulosic substrates to cellulase enzymes? Biotechnol. Prog. 17, 1047
1054.
Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257
268.
Gusakov, A.V., Sinitsyn, A.P., Manakova, J.A., Protas, O.V., 1992. Enzymatic
saccharication of industrial and agricultural lignocellulosic wastes. Appl.
Biochem. Biotechnol. 34 (35), 625637.
Hansen, M.A.T., Kristensen, J.B., Felby, C., Jorgensen, H., 2011. Pretreatment and
enzymatic hydrolysis of wheat straw (Triticum aetivum L.) the impact of lignin
relocation and plant tissues on enzymatic accessibility. Bioresour. Technol. 102,
28042811.
Hong, J., Ladisch, M.R., Gong, C.S., Wankat, P.C., Tsao, G.T., 1981. Combined product
and substrate inhibition equation for cellobiase. Biotechnol. Bioeng. 23, 2779
2788.
Inoue, H., Yano, S., Endo, T., Sakaki, T., Sawayama, S., 2008. Combining hotcompresed water and ball milling pretreatments to improve the efciency of
the enzymatic hydrolysis of eucalyptus. Biotechnol. Biofuels 1, 2.
Kim, M.H., Lee, S.B., Ryu, D.D.Y., Reese, E.T., 1982. Surface deactivation of cellulase
and its prevention. Enzyme Microb. Technol. 4, 99103.
Kumar, R., Wyman, C., 2009. Does change in accessibility with conversion depend
on both the substrate and pretreatment technology? Bioresour. Technol. 100,
41934202.
Kumar, L., Chandra, S., Chung, P.A., Saddler, J., 2010. Can the same steam
pretreatment conditions be used for most softwoods to achieve good,
enzymatic hydrolysis and sugar yields? Bioresour. Technol. 101, 78277833.

Lu, Y., Yang, B., Gregg, D., Saddler, J.N., Manseld, S.D., 2002. Cellulase adsorption
and an evaluation of enzyme recycle during hydrolysis of steam-exploded
softwood residues. Appl. Biochem. Biotechnol. 98200, 641654.
Luo, X., Zhu, J.Y., 2011. Effects of drying-induced ber hornication on enzymatic
saccharication of lignocelluloses. Enzyme Microb. Technol. 48, 9299.
Mabee, W., Gregg, D., Arato, C., Berlin, A., Bura, R., Gilkes, N., Mirochnik, O., Pan, X.,
Pye, E., Saddler, J., 2006. Updates on softwood-to-ethanol process development.
Appl. Biochem. Biotechnol. 129, 5570.
Manseld, S.D., Mooney, C., Saddler, J.N., 1999. Substrate and enzyme
characteristics that limit cellulose hydrolysis. Biotechnol. Prog. 15, 804816.
Pan, X., 2008. Role of functional groups in lignin inhibition of enzymatic hydrolysis
of cellulose to glucose. J. Biobased Mat. Bioeng. 2, 2532.
Pettersen, R.C., Schwandt, V.H., 1991. Wood sugar analysis by anion
chromatography. J. Wood Chem. Technol. 11, 495501.
Sattler, W., Esterbauer, H., Glatter, O., Steiner, W., 1989. The effect of enzyme
concentration on the rate of the hydrolysis of cellulose. Biotechnol. Bioeng. 33,
12211234.
Sewalt, V.J.H., Glasser, W.G., Fontenot, J.P., Allen, V.G., 1996. Lignin impact on bre
degradation: 1 Quinone methide intermediates formed from lignin during
in vivo fermentation of corn stover. J. Sci. Food Agric. 71, 195203.
Shen, J., Agblevor, F.A., 2008. Kinetics of enzymatic hydrolysis of steam-exploded
cotton gin waste. Chem. Eng. Commun. 195, 11071121.
Sinitsyn, A.P., Clesceri, L.S., Bungay, H.R., 1982. Inhibition of cellulases by impurities
in steam-exploded wood. Appl. Biochem. Biotechnol. 7, 455458.
Taneda, D., Ueno, Y., Ikeo, M., Okino, S., 2012. Characteristics of enzyme hydrolysis
of cellulose under static condition. Bioresour. Technol. 121, 154160.
Tengborg, C., Galbe, M., Zacchi, G., 2001. Inuence of enzyme loading and physical
parameters on the enzymatic hydrolysis of steam-pretreated softwood.
Biotechnol. Prog. 17, 110117.
Wang, Z., Feng, H., 2010. Fractal kinetic analysis of the enzymatic saccharication of
cellulose under different conditions. Bioresour. Technol. 101, 79958000.
Ximenes, E., Kim, Y., Mosier, N., Dien, B., Ladisch, M., 2010. Inhibition of cellulases by
phenols. Enzyme Microb. Technol. 46, 170176.
Ximenes, E., Kim, Y., Mosier, N., Dien, B., Ladisch, M., 2011. Deactivation of cellulases
by phenols. Enzyme Microb. Technol. 48, 5460.
Ye, Z., Berson, R.E., 2011. Kinetic modeling of cellulose hydrolysis with rst order
inactivation of adsorbed cellulase. Bioresour. Technol. 102, 1119411199.
Zhang, Y.-H.P., Lynd, L.R., 2004. Toward an aggregated understanding of enzymatic
hydrolysis of cellulose noncomplexed cellulase systems. Biotechnol. Bioeng. 88,
797824.
Zhang, Y.-H.P., Himmel, M.E., Mielenz, J.R., 2006. Outlook for cellulase
improvement: screening and selection strategies. Biotechnol. Adv. 24, 452481.
Zhang, Y., Xu, J.-L., Xu, H.-J., Yuan, Z.-H., Guo, Y., 2010. Cellulase deactivation based
kinetic modeling of enzymatic hydrolysis of steam-exploded wheat straw.
Bioresour. Technol. 101, 82618266.
} ttler, H.-B., Hao, Z., Xu, Y., 2009. Cellulose hydrolysis in evolving
Zhou, W., Schu
substrate morphologies I: a general modeling formalism. Biotechnol. Bioeng.
104, 261274.