698 Biotechnol. Prog.

2004, 20, 698−705

Development and Validation of a Kinetic Model for Enzymatic

Saccharification of Lignocellulosic Biomass

Kiran L. Kadam,*,† Eric C. Rydholm,‡ and James D. McMillan†

National Renewable Energy Laboratory, National Bioenergy Center, Biotechnology Division for Fuels and
Chemicals, 1617 Cole Blvd., Golden, Colorado 80401, and Department of Chemical Engineering,
University of Colorado, Boulder, Colorado 80309

A multireaction kinetic model was developed for closed-system enzymatic hydrolysis

of lignocellulosic biomass such as corn stover. Three hydrolysis reactions were modeled,
two heterogeneous reactions for cellulose breakdown to cellobiose and glucose and
one homogeneous reaction for hydrolyzing cellobiose to glucose. Cellulase adsorption
onto pretreated lignocellulose was modeled via a Langmuir-type isotherm. The sugar
products of cellulose hydrolysis, cellobiose and glucose, as well as xylose, the dominant
sugar prevalent in most hemicellulose hydrolyzates, were assumed to competitively
inhibit the enzymatic hydrolysis reactions. Model parameters were estimated from
experimental data generated using dilute acid pretreated corn stover as the substrate.
The model performed well in predicting cellulose hydrolysis trends at experimental
conditions both inside and outside the design space used for parameter estimation
and can be used for in silico process optimization.

Introduction challenges faced in efficiently hydrolyzing biomass feed-

Abundantly available lignocellulosic biomass poten-
tially can be converted to ethanol; this approach, besides
yielding a renewable liquid fuel, offers concomitant
environmental benefits (1). Corn stover is an example
biomass feedstock that can generate more than three
billion gallons per year of ethanol in the United States
(2). Sugar production from cellulose is a major step in
the biomass-to-ethanol process. The enzymatic hydrolysis
of cellulose offers advantages over other chemical conver-
sion routes of higher yields, minimal byproduct forma-
tion, low energy requirements, and mild operating con-
ditions (3-6). Although the enzymatic route has the
highest current costs, it has long-term potential for cost
reductions compared to other more established routes
such as concentrated acid and two-stage dilute acid
hydrolysis (7). Simultaneous saccharification and fer-
mentation (SSF) is one of several process configurations
by which ethanol can be produced from biomass; organ-
isms and configurations have been reviewed by Wyman
(8). In simultaneous saccharification and cofermentation
(SSCF), a variant of SSF, sugars from lignocellulosic
biomass are produced and converted in situ to ethanol;
hemicellulosic sugars already present in the hydrolyzate
are also fermented to ethanol (9).
Irrespective of processing schemes, enzymatic saccha-
rification of lignocellulose is a key cost center in the
overall bioconversion process. Gregg et al. (10) and
Esteghlalian et al. (11) provide an excellent overview of
* To whom correspondence should be addressed. Fax: 303 384
6877. Email: kiran_kada@nrel.gov. Corresponding author’s cur-
rent address: Quintessence Technologies LLC, 16696 W. Bayaud
Dr., Golden, CO 80401. Fax: 303 986 2530. E-mail: kkadam1@
yahoo.com.
† National Bioenergy Center.
‡ University of Colorado.
10.1021/bp034316x CCC: $27.50 © 2004 American Chemical Society and American Institute of Chemical Engineers
Published on Web 04/23/2004
stocks. A kinetic model based on observable, macroscopic
properties of the overall system can be helpful in the
design and economic evaluation of processes for sugar
and ethanol production. Experimental data generation
is labor-intensive and analytically challenging; a kinetic
model is a useful forecasting tool in this context.
Previous Modeling Efforts. Cellulose hydrolysis is
effected via a synergistic action of endo-β-1,4-glucanase
(EG), exo-β-1,4-cellobiohydrolase (CBH), exo-β-1,4-glucan
glucohydrolase, and β-glucosidase (12-15). The first
three enzymes produce cellobiose and glucose; cellobiose
is hydrolyzed to glucose by the action of β-glucosidase.
Both sugars cause end-product inhibition; however, cel-
lobiose is a much stronger inhibitor than glucose.
Previously reported saccharification models depict
enzymatic hydrolysis of lignocellulose in a variety of ways
and can be broadly characterized as being theoretical
(16-20), empirical (21-24), or based on SSF (25-27).
The most relevant previous modeling efforts are sum-
marized below.
The kinetic model of enzymatic hydrolysis of cellulose
by Wald et al. (19) incorporates enzyme adsorption,
product inhibition, and a multiple enzyme system. The
simultaneous adsorption of cellulase components, which
have different adsorption characteristics, is responsible
for hydrolysis; enzyme adsorption had not been ad-
equately addressed before this effort. The Michaelis-
Menten model used in earlier modeling attempts is
applicable to low substrate concentrations when the
substrate is not close to maximum loading. In the Wald
et al. (19) representation, enzyme adsorption is a function
of available sorption sites and, thus, of accessible surface
area via a Langmuir-type isotherm relationship. This
model does a good job of recognizing and representing
surface phenomenon and is a first attempt to realistically
portray enzymatic hydrolysis. Although the model does
Biotechnol. Prog., 2004, Vol. 20, No. 3
not consider glucose end-product inhibition, it was ca-
pable of simulating saccharification of rice straw ligno-
cellulose at high substrate (up to 333 g/L) and high
enzyme (up to 9.2 FPU/mL) concentrations.
Gusakov et al. (28, 29) mathematically describe the
kinetics of glucose and cellobiose formation from cellulose
for batch and plug-flow processes for enzymatic cellulose
hydrolysis and take into account the composition of the
cellulase complex, the structural complexity of cellulose,
inhibition by reaction products, and enzyme inactivation
during enzymatic hydrolysis. Inactivation of enzymes as
modeled is limited to that due to unproductive adsorption
to substrate. Thus, while these models are realistic and
sophisticated, thermal inactivation is not modeled and
the representation of cellulase adsorption is rudimen-
tary: [enzyme] + [substrate] S [enzyme-substrate]ads.
Nonetheless, the reported models satisfactorily predicted
batch glucose and cellobiose accumulation over the
substrate concentration range of 5-100 g/L and the
enzyme concentration range of 5-60 g/L.
The field was further advanced by South et al. (26) and
Philippidis and Hatzis (27), who developed SSF-based
models to describe lignocellulose conversion to ethanol.
The model by South et al. (26), which applies to batch
and continuous operations, incorporates a hydrolysis rate
equation (for cellulose to cellobiose conversion) and uses
the following equation for the adsorption of cellulase to
cellulose: [bound enzyme] ) constant × [free enzyme]
× [substrate]. Adsorption of cellulase to the lignin portion
of the biomass is similarly modeled. The authors report
that the model accurately describes batch SSF data from
well-mixed bioreactors over a wide range of substrate and
enzyme concentrations. In the SSF model reported by
Philippidis and Hatzis (27), cellulose hydrolysis rate
equations include cellulose-to-cellobiose conversion as
well as direct cellulose-to-glucose conversion. This model
does not include enzyme-substrate adsorption equilibria,
and cellobiose-to-glucose conversion is assumed to be
independent of enzyme concentration. Moreover, enzyme
inactivation is modeled as an exponential function of
cellulose conversion rather than time, making the model
less general. This diminution in generality stems from
the fact that enzyme inactivation is known to be related
to the duration of exposure to the temperature and is
usually modeled as a first-order kinetics, which leads to
a mechanism of exponential decay with respect to time
(16, 30). However, the model derived by the above
authors was able to predict SSF performance well.
Together, these previous efforts incorporate many of the
important phenomena that a kinetic model of enzymatic
cellulose hydrolysis needs to describe.
Study Objective. Kinetic modeling of enzymatic hy-
drolysis is complicated by the heterogeneous nature of
the substrate and multiple enzyme activities. Rigorous
models must consider the phenomena of enzyme adsorp-
tion, sugar inhibition, temperature effects, and substrate
reactivity. Our objective was to formulate and validate
a kinetic model that incorporates these features. This
paper describes the development and validation of a
kinetic model for enzymatic cellulose hydrolysis that is
capable of predicting performance over a range of operat-
ing conditions encompassing various background sugar
concentrations, temperatures, and mixing regimes.
Model Development
The general modeling philosophy followed in this work
is that a kinetic model should be sophisticated enough
to describe the complexities of enzymatic hydrolysis of
699
lignocellulosic biomass, but the parameters should be
based on observable phenomena to the extent possible.
The model should be grounded in the biochemistry of
enzymatic hydrolysis and should include adsorption of
cellulase components onto and desorption off of the
lignocellulosic substrate, thermal effects, and end-product
inhibition. However, not all system complexities must be
incorporated to develop an effective model; the general
preference is to minimize the number of model param-
eters.
Substrate Reactivity. There are several factors
besides end-product inhibition that reduce hydrolysis
rates as saccharification progresses including lower
substrate reactivity (variations in crystal structure,
degree of polymerization, substrate accessibility, etc.),
enzyme inactivation, and loss of enzyme due to adsorp-
tion on lignin. Cellulose contains both amorphous and
crystalline regions. In studies with pure celluloses,
amorphous cellulose is degraded 5-10 times more rapidly
when compared to highly crystalline celluloses by both
fungal enzymes and ruminal bacteria (12, 31-33). It has
been suggested that the high initial rates are due to
preferential hydrolysis of the amorphous region and that
the rate decreases later as the enzyme encounters the
more recalcitrant crystalline region (34). However, as
mentioned above several researchers have observed no
substantial change in crystallinity as the saccharification
progresses (35-37) or obtained equivocal results. Also,
Wald et al. (19) and Desai and Converse (38) argue that
the two-substrate hypothesis suffers from making an
assumption, i.e., the easier to digest substrate is not
shrouded or otherwise blocked by the recalcitrant one,
which is phenomenologically not sound. Thus, changing
intrinsic substrate reactivity may be a more practical way
to model the biphasic behavior than resorting to an
assumed dichotomy between amorphous and crystalline
regions. Hence, although structure in cellulose can be
introduced by dividing total cellulose into amorphous and
crystalline fractions, we chose to represent cellulose as
a uniform substrate. This approach simplifies cellulase
adsorption modeling and obviates the need to indepen-
dently measure distinct enzyme adsorption equilibria for
the putative amorphous and crystalline cellulose.
Assuming that the normalized initial saccharification
rates during secondary hydrolysis of residual substrates
(see Materials and Methods for details) are indicative of
substrate reactivity, the following correlation is then
proposed:
S
RS ) R (1)
S0
where RS is a substrate reactivity parameter (dimension-
less), R is a constant (dimensionless), S is the substrate
concentration at a given time (g/kg), S0 is the initial
substrate concentration (g/kg), and S/S0 is the dimen-
sionless substrate concentration at a given time.
Enzyme Adsorption. Both Langmuir and Freundlich-
Langmuir adsorption models have been used to describe
enzyme adsorption onto solid lignocellulosic substrates
(39-41). Although the Langmuir model has been used
for describing enzyme adsorption in this heterogeneous
system, it should be noted that the underlying assump-
tions for the Langmuir model, uniform binding sites and
no interaction between the adsorbing molecules, are not
valid for cellulase adsorption onto cellulose. Nevertheless,
the Langmuir formulation remains useful for mathemati-
cally describing the phenomenon of enzyme adsorption,
and we have used it in this study.
700 Biotechnol. Prog., 2004, Vol. 20, No. 3

temperature on saccharification is captured via the

Arrhenius rate relationship indicated in eq 10; this
equation is only valid at temperatures below the enzyme’s
temperature maximum. Although we are currently using
a mesophilic enzyme preparation with an optimal tem-
perature of 50 °C, being able to describe the temperature
dependence of enzymatic hydrolysis over a broad tem-
perature range will be very important for modeling and
optimizing processes based on the use of more thermo-
stable enzymes currently being developed.
Enzyme Adsorption

Eimax KiadEiFS
Langmuir isotherm EiB ) (2)
1 + KiadEiF

Figure 1. Reaction scheme for modeling cellulose hydrolysis. Cellulose-to-Cellobiose Reaction with Competi-
Enzymes involved in r1: endo-β-1,4-glucanase (EC 3.2.1.4) and tive Glucose, Cellobiose and Xylose Inhibition
exo-β-1,4-cellobiohydrolase (CBH) (EC 3.2.1.91). Enzymes in-
volved in r2: exo-β-1,4-cellobiohydrolase (CBH) (EC 3.2.1.91) and k1rE1BRSS
exo-β-1,4-glucan glycohydrolase (EC 3.2.1.74). Enzymes involved r1 ) (3)
in r3: β-bludosidase or cellobiases (EC 3.2.1.21). G2 G X
1+ + +
K1IG2 K1IG K1IX
Proposed Kinetic Model. Figure 1 depicts the sim-
plified cellulose hydrolysis reaction sequence that forms Cellulose- to-Glucose Reaction with Competitive
the basis for the model. Each enzymatic reaction is Glucose, Cellobiose and Xylose Inhibition
potentially inhibited by the sugar it generates or by the
six sugars already present in the system, i.e., glucose, k2r(E1B + E2B)RSS
cellobiose, galactose, mannose, xylose, and arabinose. To r2 ) (4)
G2 G X
simplify model development, the sugar system was 1+ + +
consolidated to three sugars: cellobiose, glucose, and K2IG2 K2IG K2IX
xylose. In terms of inhibition capacity, xylose was used
as the surrogate for both pentoses (xylose and arabinose) Cellobiose- to-Glucose Reaction with Competi-
and glucose was used as the surrogate for all hexoses tive Glucose and Xylose Inhibition
(glucose, galactose, and mannose).
The potential for complicated sugar inhibition phe- k3rE2FG2
r3 )
( )
nomena coupled with the presence of both heterogeneous (5)
G X
and homogeneous reaction types necessitates a sophis- K3M 1+ + + G2
K3IG K3IX
ticated model to effectively simulate the system. The
Langmuir adsorption model is shown in eq 2. Rate
Mass Balances
equations based on a competitive mode (42) of sugar
inhibition are shown below. Equation 3 describes the dS
conversion of cellulose to cellobiose, eq 4 the conversion cellulose: ) - r1 - r2 (6)
dt
of cellulose to glucose, and eq 5 the conversion of
cellobiose to glucose. The competitive mode of sugar dG2
inhibition assumes that inhibitor sugars are substrate cellobiose: ) 1.056r1 - r3 (7)
analogues and bind competitively to the active site, dt
thereby retarding the formation of enzyme-substrate
complex. In the noncompetitive mode of inhibition, dG
glucose: ) 1.111r2 + 1.053r3 (8)
inhibitors do not bind to the active site but bind rather dt
at a remote positions causing the maximum specific
reaction rate to diminish. Unlike competitive inhibition, enzyme: ETi ) EFi + EBi (9)
noncompetitive inhibition is irreversible (42). We chose
to model competitive inhibition as it is mechanistically Temperature Dependence
more realistic.
These rate equations assume that (1) enzyme adsorp- Arrhenius eq: kir(T2) ) kir(T1) e- Eai/R{1/T1-1/T2};
tion follows a Langmuir-type isotherm with the first- 30 °C e T e 55 °C (10)
order reactions (r1 and r2) occurring on the cellulose
surface; (2) the cellulose matrix is uniform in terms of The proposed model distinguishes between the β-glu-
its susceptibility to enzymatic attack (i.e., no provision cosidase and CBH/EG enzymes, representing their ad-
was made to include separately more reactive amorphous sorption via a Langmuir-type isotherm. This more struc-
and more recalcitrant crystalline cellulose fractions); (3) tured formulation permits the model to better describe
enzyme activity remains constant; and (4) conversion of the kinetics of cellulase preparations having different
cellobiose to glucose occurs in solution and follows proportions of these components. Beyond this, the model
classical Michaelis-Menton kinetics. incorporates potential inhibition by xylose, a prominent
The mass balances for cellulose, cellobiose, glucose, and sugar in hydrolyzates of dilute acid pretreated biomass;
cellulase enzymes are listed in eqs 6-9, respectively, and inhibition of cellulases by xylose has not been considered
are based on fundamental principles of mass conserva- previously.
tion; these equations will not change if modifications are Computational Methodology. The kinetic model
made in the kinetic rate expressions. The effect of was coded and solved using Matlab programming envi-
Biotechnol. Prog., 2004, Vol. 20, No. 3 701

ronment (The MathWorks, Natick, MA). The Matlab Table 1. Estimated Model Parameters
optimization function “lsqnonlin” was used to simulta- parameter value
neously estimate the various model parameters. Param-
Independently Established Parameters
eters characterizing the Langmuir, Arrhenius, and sub- K1ad (g protein/g substrate) 0.4
strate reactivity correlations were determined indepen- K2ad (g protein/g substrate) 0.1
dently and served as inputs. E1max (g protein/g substrate) 0.06
E2max (g protein/g substrate) 0.01
Materials and Methods Ea (cal/mole) -5540
Rs RS/S0, R ) 1
The substrate used in the enzymatic hydrolysis studies Parameters Obtained by
was pretreated corn stover (composed of approximately Regression of Saccharification Data
60% cellulose) that was water-washed to remove en- k1r (g/mg‚h) 22.3
trained soluble sugars. Corn stover was pretreated with K1IG2 (g/kg) 0.015
dilute sulfuric acid in an engineering-scale countercur- K1IG (g/kg) 0.1
rent pretreatment reactor system, a 200 kg dry biomass/ K1IX (g/kg) 0.1
day capacity Sunds hydrolyzer (Metso Paper USA Inc., k2r (g/mg.h) 7.18
K2IG2 (g/kg) 132.0
Norcross, GA) using 1.4% acid concentration, 8-min K2IG (g/kg) 0.04
residence time, and 190 °C temperature. The details of K2IX (g/kg) 0.2
the hydrolyzer setup and operation are described by k3r (h-1) 285.5
Nguyen et al. (43) and Tucker et al. (44). The pretreated K3M (g/kg) 24.3
corn stover slurry was separated into liquor and solid K3IG (g/kg) 3.9
fractions, and the solid fraction was washed (45) and used K3IX (g/kg) 201.0
in the experiments.
For shake flasks the standard operating conditions Results and Discussion
were as follows: 100 g total charge in 250-mL baffled The basic approach followed was to obtain experimen-
Erlenmeyer flasks; 10% corn stover solids by weight (dry tal data, estimate model parameters, and conduct simu-
basis); enzyme loading of 45 mg protein/g cellulose (CPN lations using these parameters. Model predictions were
commercial cellulase, Iogen Corp., Ottawa, Ontario, compared with experimental data to validate the model
Canada), equivalent to approximately 15 FPU/g cellulose and assess its utility in predicting hydrolytic performance
(no β-glucosidase supplementation); 45 °C temperature beyond the range of experimental conditions employed
of incubation; shaker (Innova 4000, New Brunswick in parameter estimation. In the ensuing figures, experi-
Scientific, Edison, NJ) speed of 130 rpm; initial pH of mental data are presented on a weight/weight basis
4.8 (0.05 M citrate buffer added, pH uncontrolled). For because of the heterogeneous, two-phase nature of the
stirred-tank reactors (BioFlo III, New Brunswick Scien- system. Doing so also readily allows one to calculate
tific, Edison, NJ) the standard operating conditions were conversion yields. Sugar and substrate concentrations are
same as above, except as follows: 700 g total charge and expressed as g/kg, i.e., grams of entity per kilogram of
impeller speed of 250 rpm. total system mass.
Liquid samples were analyzed for glucose, xylose, and Parameter Estimation. There are two types of model
cellobiose using HPLC (Agilent Technologies, Palo Alto, parameters: independently established parameters that
CA; Bio-Rad Laboratories, Hercules, CA). For character- are derived through separate experimentation and ki-
izing Langmuir adsorption behavior, free enzyme was netic parameters that are derived via Matlab regression
assumed to be the protein concentration in the superna- of hydrolysis data; both are listed in Table 1.
tant and was measured using the Bradford assay using Adsorption. Langmuir parameters estimated from
Coomassie blue dye (Pierce Biotechnology, Rockford, IL). enzyme adsorption studies are as follows (in g protein/g
Bound enzyme was calculated by subtracting the free substrate): for EG/CBH E1max ) 0.06 and K1ad ) 0.4, and
enzyme concentration from the initial protein concentra- for β-glucosidase E2max ) 0.01 and K2ad ) 0.1.
tion charged to each flask. Adsorption of one cellulase Substrate Reactivity. The initial hydrolysis rates
preparation (CPN cellulase) and one β-glucosidase prepa- during secondary enzymatic hydrolysis were plotted as
ration (Novo 188 from Novozymes Biotech, Davis, CA) a function of S/S0 (Figure 2) to arrive at substrate
were tested. reactivity. Given that the best-fit value for R is close to
The issue of substrate reactivity was studied using unity (0.97 with R2 ) 0.97, data not shown), the substrate
washed pretreated corn stover solids and CPN cellulase reactivity correlation of eq 1 simplifies to
preparation. During hydrolysis, two flasks were har-
vested at each time point, and the residual substrate was S
RS ) (11)
then subjected to secondary enzymatic hydrolysis for an S0
additional 4 h using fresh enzyme added at a constant
enzyme/substrate ratio. The residual cellulose was esti- The experimental 0-4 h rate data can be described
mated from net glucose and cellobiose produced. Washing well by this simplified substrate reactivity correlation
the substrate removes end-product inhibition, and adding (Figure 2).
fresh enzyme eliminates enzyme inactivation as a factor. Temperature Dependence. To predict the effect of
Hence, this assay attempts to isolate intrinsic substrate temperature on cellulose saccharification, a single Ar-
reactivity. It should be noted, however, that the washing rhenius relationship was assumed for all reactions. It
step does not necessarily dislodge bound enzyme. should be noted that the Arrhenius relationship would
The standard conditions using the shake-flask system be different for each enzyme component because of their
described above were used for parameter estimation. This varying degrees of thermostability, with CBH being the
defines the standard design space. Experiments for model most thermostable. Hence, the above assumption is a
validation were conducted using conditions outside the simplification of reality. This equation was used in model
design space with different background sugar concentra- validation experiments performed at temperatures be-
tions, temperatures, and mixing regimes. sides 45 °C, the standard operating temperature used.
702
Figure 2. Relative overall glucose production rates during
secondary hydrolysis using constant enzyme/substrate ratio.
The best-fit activation energy, Ea, based on initial rate
data was estimated to be -5540 cal/mol for the CPN
enzyme (Figure 3). We expect thermostable enzymes to
be eventually employed, and the activation energy for
these enzymes will be different.
Kinetic Parameters. The independently established
parameters were used as input to the model, and the
kinetic parameters were subsequently regressed; Table
1 lists best-fit estimates of these parameters. A compari-
son was attempted between the parameters listed in
Table 1 and those reported by Oh et al. (46), who
determined the competitive glucose inhibition parameter
for r3 to be 0.33 g/L. Their method was to determine the
initial rate of cellobiose consumption during SSF and
back calculate the cellobiose-to-glucose conversion rates.
This approach makes it more difficult to pinpoint the true
effect of glucose on β-glucosidase activity. Oh et al. (46)
also determined K1IG2 ) 6.1 g/kg and K1IG ) 12.4 g/kg for
competitive inhibition. The lower inhibition they found
for reaction r1 may be balanced by the higher inhibition
they observed for the cellobiose-to-glucose reaction (r3).
Oh et al. (46) did not model the direct conversion of
cellulose to glucose and report that, at a glucose concen-
tration of 20 g/L, β-glucosidase was practically inactive.
Our results using CPN cellulase enzyme show a less
dramatic reduction of β-glucosidase activity above 20 g/L
glucose. When 30 g/kg glucose (our concentrations are
on a w/w basis) is added initially, the cellobiose conver-
sion is slower compared to the case with no added
glucose, but cellobiose conversion still occurs. These
findings illustrate how different reaction schemes and
model structures can result in different parameter values
and make direct comparisons of different models difficult
(see also ref 47). Such comparisons are further con-
founded by different researchers using different enzyme
mixtures, different enzyme and substrate loading ranges,
different hydrolysis temperatures, etc.
Model Validation. As shown in Figure 4, which is
generated using standard conditions, the proposed model
predicts hydrolysis behavior with reasonable accuracy.
However, the kinetic model for cellulose saccharification
needs to be validated using datasets not used to estimate
model parameters. Simulations were run using the
parameters listed in Table 1 to predict cellulose hydroly-
sis performance at background sugar concentrations,
temperatures, and mixing regimes different from those
in the standard design space.
The data used for parameter estimation were gener-
ated using 10% w/w insoluble solids in a shake-flask
system. Enzymatic cellulose hydrolysis was also con-
Biotechnol. Prog., 2004, Vol. 20, No. 3
Figure 3. Arrhenius plot based on initial glucose production
rates.
Figure 4. Enzymatic cellulose hydrolysis was conducted in the
shake-flask system and a stirred-tank reactor using 10% w/w
corn stover solids with no added background sugars.
ducted in stirred-tank reactors using 10% w/w corn stover
solids. Results showed that the model does a fairly good
job of predicting glucose production in a shake flask or a
stirred-tank reactor (Figure 4). Hence, the parameters
estimated on the basis of shake-flask data appear ap-
plicable in predicting hydrolysis in a stirred-tank reactor
exhibiting different hydrodynamics.
Validation tests also show that the model does a sound
job of predicting hydrolysis behavior at high initial
glucose, xylose, and cellobiose concentrations (Figures
5-7, respectively). Thus, inhibition by these three sugars
is well captured by the model. Xylose inhibition as
predicted by the model is authentic in the sense that the
24-h hydrolysis efficiencies at 0 and 50 g/kg background
xylose are 53% vs 46%, respectively, suggesting signifi-
cant xylose inhibition. The high cellobiose concentration
used is only for model validation given that cellobiose
concentrations typically peak at much lower levels (10-
12 g/L); cellobiose concentrations are mainly dependent
on the β-glucosidase content of a given enzyme prepara-
tion.
Model Predictions of Sugar Inhibition. Model
fidelity can be further assessed by examining if it
correctly describes sugar inhibition. Inhibition by each
sugar is inversely proportional to its respective inhibition
constant. On the basis of this fact, inhibition of reaction
r1 by cellobiose is more potent than that by glucose, which
is expected. Conversely, inhibition of reaction r2 by
glucose is much higher than that by cellobiose because
glucose is the reaction product obtained directly from
Biotechnol. Prog., 2004, Vol. 20, No. 3
Figure 5. Kinetic model validation. Enzymatic cellulose hy-
drolysis was conducted in the shake-flask system using 10% w/w
corn stover solids with initial background glucose of 30 or 50
g/kg.
Figure 6. Kinetic model validation. Enzymatic cellulose hy-
drolysis was conducted in the shake-flask system using 10% w/w
corn stover solids with initial background xylose of 40 g/kg.
Figure 7. Kinetic model validation. Enzymatic cellulose hy-
drolysis was conducted in the shake-flask system using 10% w/w
corn stover solids with initial background cellobiose of 30 g/kg.
cellulose. Xylose inhibition of reaction r1 is of the same
order of magnitude as that by glucose. Inhibition of
reaction r3 is dominated by glucose, with inhibition by
xylose being two orders of magnitude less than for
glucose. Hence, the model is consistent with expected
inhibition by glucose and cellobiose, and the predicted
role for xylose seems reasonable as well. To further
illustrate sugar inhibition patterns, inhibition by each
sugar, which is the ratio of sugar concentration and its
703
Figure 8. Sugar inhibition patterns for cellulose-to-cellobiose
reaction (r1). Enzymatic cellulose hydrolysis was conducted in
the shake-flask system using 10% w/w corn stover solids with
initial background xylose of 30 g/kg. The overall hydrolysis
profile was similar to that shown in Figure 5.
Figure 9. Kinetic model validation. Enzymatic cellulose hy-
drolysis was conducted in the shake-flask system using 10% w/w
corn stover solids at temperatures of 40, 50, and 55 °C.
respective inhibition constant, was calculated for reaction
1. The relative contribution of each sugar to the total to
inhibition was then calculated. Figure 8 depicts the
resulting sugar inhibition patterns along with predicted
reaction rates normalized to the time-zero reaction rate.
As expected, normalized reaction rates decrease with
time. The model predicts that inhibition due to cellobiose
and xylose plays a major role during the early phase.
However, as glucose concentration builds up, its contri-
bution to total inhibition also rises. The overall inhibition
pattern mechanistically makes sense.
Model Predictions of Temperature Effects. The
model was also used to predict hydrolytic performance
at different temperatures using eq 10. The model predicts
hydrolysis behavior reasonably well for 40° and 50 °C
for the first 96 h but falters after 24 h for 55 °C (Figure
9). This is explained by the fact that the temperature
optimum for the CPN enzyme is near 50 °C, and the
enzyme is slowly inactivated at higher temperatures.
Therefore, this feature of the model needs more fine-
tuning. Obviously, more thermostable second-generation
enzymes should be available before putting an extensive
effort into modeling enzyme inactivation.
Conclusion
We have developed and validated a kinetic model for
batch enzymatic saccharification of dilute acid pretreated
704
corn stover that incorporates structure in how the cel-
lulase enzyme system is represented and considers
feedback inhibition by biomass-derived sugars. The model
is capable of predicting hydrolysis performance beyond
the range of experimental conditions used to estimate the
model parameters. The model differs from previous
models in that it considers inhibition by xylose, a major
sugar in hemicellulose-derived hydrolyzates. Moreover,
the expected benefit of β-glucosidase supplementation is
captured by adding structure, thereby rendering the
model potentially useful for predicting the hydrolysis
performance of cellulase preparations having different
levels of β-glucosidase. A key limitation identified is that
the effect of temperature is not sufficiently well repre-
sented in the model. Nevertheless, the model provides a
solid foundation for future model refinements and has
the potential to be used for in silico optimization of the
hydrolysis portion of a bioethanol process.
Acknowledgment
This work was funded by the Office of the Biomass
Program of the U.S. Department of Energy. We thank
Jeffrey S. Knutsen of the University of Colorado, Boulder,
CO, for his considerable help with enzyme adsorption
experiments.
Notation
Ea activation energy (cal/mol)
ET total enzyme concentration (g/kg)
EB bound enzyme concentration (g/kg)
EF free enzyme concentration (g/kg)
E1B bound concentration of CBH and EG (g/kg)
E2B bound concentration of β-glucosidase (g/kg)
E2F concentration of β-glucosidase in solution (g/kg)
Emax maximum mass of enzyme that can adsorb onto
a unit mass of substrate (g protein/g cellulose)
G glucose concentration (g/kg)
G2 cellobiose concentration (g/kg)
Kad dissociation constant for the enzyme adsorption/
desorption reaction (g protein/g cellulose)
kir reaction rate constants (kg/g.h)
KiIG inhibition constants for glucose (g/kg)
KiIG2 inhibition constants for cellobiose (g/kg)
KiIX inhibition constants for xylose (g/kg)
K3M substrate (cellobiose) saturation constants (g/kg)
ri reaction rate (g/kg‚h)
R universal gas constant (cal/mol‚K)
Rs substrate reactivity
S substrate concentration (g/kg)
T temperature (K)
X xylose concentration (g/kg)
R constant relating substrate reactivity with de-
gree of hydrolysis
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Accepted for publication January 28, 2004.
BP034316X