EINEMANN

Modeling simultaneous
sac~hari~cation and fermentation of
lignocellulose to ethanol in batch and
continuous reactors
C. R. South, D, A. L. Hogsett and L. R. Lynd

Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire

An adsorption-based kinetic model was sought that accurately predicts simultaneous saccharifcation and fer-
mentation (SSF) of insoluble lignoceihdosic substrates in batch and continuous reactors. With a common set of
three heuristic parameters, a hydrolysis rate equation of the form r = (k X (I - x)” + cl x (ES)/(C,) in
conjunction with Langmuir a~orption is capable of accurately representing batch SSF data from we&mixed
bioreactors for a variety of feed substrate concentrations and cellulase loadings (root mean squared (RMS)
difierence in predicted and measured conversion = 5.2%). Using a particle population model in conjunction
with the batch kinetics, conversion in a CSTR as a function of residence time is also well predicted (4.6% RMS
difference). In&ding both batch and continuous data, the model is successful over a four-fold range of enzyme
loadings and a 12 .&fold range of substrate concentrations. Commentary is offered on useful features of kinetic
models for processes including cellulose hydrolysis

Keywords: Enzymatic hydrolysis; celiulase kinetics; SSF: ethanol

Introduction Trichoderma reesei cellulase and Saccharomyces cerevisiae

The bioreactor in which enzymatic hydrolysis and fermen-
tation occurs is a logical focal point in pursuing cost reduc-
tions for ethanol production from ceiluIosic biomass. ‘+2A
validated kinetic model capable of predicting simultaneous
sacch~~cation and fc~entation (SSF) performance over a
range of substrate concentrations, cellulase loadings, and
reactor configurations would be a useful tool in this en-
deavor, but is not available at present. The absence of such
a model is attributable primarily to the complexity and in-
complete state of understanding of the action of cellulase
enzyme systems acting on nonideal particulate lignocellu-
losic substrates. By contrast, kinetic models have been pre-
sented in the literature for other processes involved in SSF
(see below) such as the action of &glucosidase and asso-
ciated inhibition by soluble sugars, and the fermentation of
soluble substrates and associated inhibition by ethanol.
We recently reported experimental results for SSF using
Address reprint requests to Dr. L. R. Lynd, Thayer Schoof of Enginee~ng,
Dartmouth College, Hanover, NH 037%
Received 26 August 1994
fermentation in both batch and continuous well-mixed sys-
tems.3 The purpose of this study was to develop a kinetic
model and modeling approach for ethanol production from
cellulosic biomass as exemplified by SSF, and to test the
adequacy of the proposed model for correlating our previ-
ous ex~~mental results. Continuous systems are a partic-
ular focus, as these have both significant applied potential
and interesting fundamental features. The specific continu-
ous system considered here is well mixed and at steady state
with continuous substrate and enzyme addition, and with
enzyme and residual substrate continuously existing in the
reactor effluent.
Analytic framework
To be an effective tool for reactor design, a kinetic model
for SSF should predict substrate conversion, x [X = 1 -
(Remaining cellulose)/(Initial cellulose)], with reasonable
accuracy over a significant range with respect to input sub-
strate concentration, cellulase(s) loading, reaction time, and
substrate conversion. We sought to develop such a model.
The approach taken here was to use the minimum level
of complexity required to obtain a useful model. Specifi-

Enzyme and Microbial Technology 17:797-803, 1995

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Papers

tally, we considered a single measure of cellulase activity
(FPU), and used a Langmuir adsorption model as presented
by Ooshima et al. 4 in combination with a conversion-
dependent rate constant. The Langmuir affinity constants
for cellulose and lignin, respectively, were defined as:
(1)
(2)
ticulate biomass in a CSTR can be considered equivalent to
segregated micromixing with respect to the substrate and
complete micromixing with respect to the aqueous phase.
For such conditions, mean conversion can be calculated
from the relationship between conversion and time, x(t), for
a given particle and the exit age distribution E(t,r), where t
is time and T = (Reactor volume)/(Volumetric flow rate) is
the mean residence time. Overall conversion as a function
of mean residence time under these conditions is given by a
particle population model as represented by Equation (8):

where C, and C1 represent the capacity of substrate and

lignin, respectively, to bind enzyme, and may also be in-
X(T) =
I cc
0
x(t) x E(t,T) dt (8)

terpreted as the ratio of E to S or L in each enzyme com-
plex. The Langmuir constants reported by Ooshima et al.
were for a similar, though not identical, substrate to that
modeled here.
Conservation equations for substrate, lignin, and enzyme
respectively are:
(3)
(4)
L$ = Er + ES + EL (5)
Enzyme adsorbed to cellulose and lignin is calculated from
E,, S, and L, with the adsorption parameters of Ooshima et
~1.~We also used the assumption by Ooshima et al. that the
capacity constants for both the lignin and cellulose do not
change during the course of hydrolysis. Equations (1)
through (5) can readily be solved simultaneously to give ES
for values of initial substrate (cellulose and lignin), E,, K,,
Kr, Cs, and Cr.
All studies known to us indicate a notable decline in
(r)/(ES) over the course of the reaction. Working with T.
reesei cellulase and a pretreated hardwood substrate similar
to that used here, Nutor and Converse’ found (r)/(ES) at
high conversion to be one to two orders of magnitude less
than at low conversion. Other investigators?” also re-
ported decreasing reactivity as reaction proceeds. Declining
reactivity with increasing conversion in combination with
an adsorption model suggests a rate equation of the general
form
where E(t,r), the reactor exit age distribution for an ideally
mixed CSTR, is given by Equation (9)“:
E(t,T) = i X exp - i (9)
( 1
Equation (8) is used to account for the conversion and res-
idence time-dependent behavior of enzyme and substrate
over the course of the reaction of a population of individual
particles in a CSTR, and represents a useful framework
within which to analyze continuous nonhomogeneous par-
ticulate reactors. Thus, to successfully model particulate
substrate reactions that incorporate factors such as changing
adsorptive capacity or accessibility of substrate with con-
version, 12.13 declinin reactivity of the enzyme bound to
P
cellulose over time,7, or declining substrate reactivity as in
this analysis, it is necessary to account for the conversion
and residence time-dependent behavior of enzyme and sub-
strate over the course of the reaction of a population of
individual particles.
SSF simulation
Rate equations used to simulate SSF are presented below,
where r is the rate of formation of the component of interest.
Equations (10) and (11) account for the enzymatic hydro-
lysis of cellulose and cellobiose, respectively; Equations
(12) through (14) account for cell production, substrate up-
take, and solvent production by the biocatalyst:
ES
rs = - {k x (1 - x)” + c} x cs x

ES x [(Elhk:rE:&] (10)
r = k(x) X -
cs
Consistent with the trend exhibited by the data of Nutor and rc = 1.056 X rs -
Converse, we chose to consider k(x) of the form i(K- xk[:+‘k$ + C)]
k(x) = k x (1 - x)” + c (7)
(11)
Modeling reaction of particulate substrates in (Xc x vmaxx G) Eth

( --I
rx =
(12)
continuous reactors G+ko ’ ‘- kxiEth
The simplest type of continuous reactor for hydrolysis of
cellulose is the continuous stirred tank reactor (CSTR). Us- ro=(-l.O56~rs-rc)~ 1.053-rX (13)
ing the nomenclature of Danckwerts,” the reaction of par- YX/G

798 Enzyme Microb. Technol., 1995, vol. 17, September

 Modeling simultaneous saccbarification and fermentation of ~ignocelluiose to ethanol: C. R. South et al.

YEtWG
r&h = rx x -
Y%G
The form and associated constants for terms in the above
equations are from Gustakov et ~1.‘~ for the rate of cello-
biose formation, Phillippidis et ~1.‘~ for inhibition by eth-
anol on cellulose hydrolysis, cellobiose inhibition of cellu-
lose hydrolysis, and inhibition of P-glucosidase by glucose,
and van Uden” for the effect of ethanol on biocatalyst
growth. The Monod model is used for biocatalyst growth
with kinetic constants from Ghose and Tyagii6 and van
Uden. The constants 1.053 and 1.056 arise from the addi-
tion of water during hydrolysis.
For continuous SSF at steady state the cont~butions of
the range of particle reaction times comprising the overall
hydraulic residence time distribution E(t,r) is represented
by Equation (9), with x(t) evaluated in the presence of a
common aqueous environment. Cellobiose concentration is
calculated from the CSTR mass balance Equation (15),
which balances the rate of cehobiose pr~uction and con-
sumption with loss due to liquid outflow:
c batch SSF conve~ion .
rc = -
7
enzyme between lignin and cellulose fractions of the bio-
(14) mass, then summing the enzyme forms in the reactor. E,
was varied iteratively until the enzyme inventory agreed
with the specified value of E, to within 0.1%. The conver-
sion of each particle residing for a time t in the reactor in the
presence of the free enzyme concentration calculated as
above was then determined. Cellulose solubilization, and
hence cellobiose formation from the particulate substrate in
the CSTR, was computed from the discrete form of Equa-
tion (8), given by Equation (18).
x
X(T) = c q(t) x Wi(t) (18)
Cellobiose pr~uction Equation (IO), and consumption,
Equations ( 11) and (15), are balanced by iteratively adjust-
ing the aqueous cellobiose level. A dilution curve is ob-
tained by repeating this procedure at different hydraulic
residence times. In this manner conversion in a CSTR as a
function of hydraulic residence time can be predicted with
no new parameter estimation from that used to calculate the

The CSTR glucose concentration can be determined explic- Results

itly from he Monod equation and the steady-state material ~ar~~~ter fitting for Dutch data
balance for a well-mixed reactor by:
A single set of the parameters k, n, and c for the conversion-
ki dependent rate constant (7) were fit to the batch SSF data at
G= (16)
tT x Pmax - 1) all enzyme loadings, with the sum of squared residuals
(simuIated minus ex~~mental time to achieve the experi-
mental conversion) minimized by the use of a Hooke-
Methods Jeeves optimization procedure. Optimization using time-
The SSF data and associated methods have been previously re- based residuals weights the high conversion fit more heav-
ported.? Batch SSF performance was predicted by simultaneously ily; this is appropriate, as high-substrate conversion is a
solving Equations (10) through (14) via a fourth-order Runge- likely to be a requirement for financial viability. Figure 1
Kutta algorithm. At each time step, E, was varied iteratively, presents experimental data for batch SSF run together with
allowing calculation of ES and EL using ~uat~ons (1) and (2).
until convergence to Equation (5) (within a 0.1% tolerance) was
found. Enzyme p~itioning could be solved directly for the batch
case; however, an iterative approach was necessary for the simu-
lation of the continuous SSF case, and a common algorithm was
used.
In accordance with Equation (g) the composition of the partic-
ulate substrate was calculated as the weighted sum of particles that
spent varying times in the reactor as defined by the residence time
distribution E(t.7). The weight, W, allocated to the conversion of
a particle that resided in the reactor between t, and time ti + At
was given by evaluating the integral of the exit age distribution
E(t,r) over this time interval as per Equation (17),

Wi(t) = :;+‘t E(t,r) dt (17)

f
To ensure an adequate range of particle residence times, the
procedure was repeated until the time integral of the exit age 0 0.5 I 1.5 2 2.5 3 3.5 4 4.5 5
distribution, Zi[E(ti) X At], reached 0.999 (as Jb”E(t,r) dt Residencetime (days)
= 1).
In a steady-state CSTR, enzyme in the aqueous phase is Figure 1 Predicted and experimental conversion for batch SSF
with pretreated wood at 35 g I-’ cellulose, and cellulase load-
modeled as spatially homogeneous. The total enzyme in the ings as shown, using the parameter values given in Table 1.
reactor is calculated by solving the sets of coupled differ- Cellulase loadings were: U, 5 U g-‘; 0, 10 U g-‘; 0, 15 U g-l;
ential and Langmuir equilib~um equations that partition the andA,ZOUg-’

Enzyme Microb. Technol., 1995, vol. 17, September 799

 Papers
the predicted time-course conversion based on integration
of Equations (10) through (14) and the parameter values
indicated in Table I. Figure 2 compares the residuals of
Figure 2 with those obtained using a best-fit model with
constant k. We were unable to accurately represent the
batch data at varying cellulase loadings using a single pa-
rameter model for the rate coefficient k. The root mean
squared error in the time to reach a given conversion of the
constant rate model is five times higher than the declining
rate model (0.45 days versus 0.09 days). Further, the re-
siduals for the constant rate model are generally positive for
low conversion and negative for high conversion, whereas
the declining rate model residuals are distributed about zero
-1.5 ~,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,~,~
for all conversions. Figure 3 shows the (r)/(Eadsorbed) data of
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Nutor and Converse’ (specific rate based on total adsorbed
Conversion
enzyme), along with the rate per total adsorbed enzyme
independently calculated using the SSF adsorption model Figure 2 Normalized residuals for batch SSF predictions using
and the best-fit values for k, c, and n. The fit to Nutor and declining (r)/(EC) (open symbols), and constant (r)/(EC) (closed
Converse’s data is surprisingly good considering that the symbols). Cellulase loading symbols as per Figure 1
substrates used by Nutor and Converse and Ooshima et al.
were different from those used here, and that the data of
4.2%, respectively, indicating that the proposed CSTR
Nutor and Converse are for cell-free enzymatic hydrolysis,
framework performs well, particularly when the inherent
whereas our data are for SSF.
variability in the steady-state results is considered (standard
deviation conversion = 3.5%).
Prediction of conversion in a CSTR The particle population model can be compared to a
CSTR model, where the substrate is immediately diluted to
Table 2 shows the application of the kinetic model and its exit reactivity, as is the case for a soluble substrate. In
CSTR prediction algorithm to continuous data. To obtain this case the substrate reactivity corresponds to that of the
these predictions no further parameters were fit beyond material leaving the reactor, and conversion is given by:
those previously used to predict batch conversion. Equa-
tions (8) and (9) were used together with the rate equations ES
x(7) = k[x(T)] x -
and adsorption parameters used in the batch simulations as S, x Cs x 7
well as the CSTR solution framework described in Meth-
ods. The average absolute error (predicted - actual conver- Figure 4 presents the ratio of reaction times required to
sion) in the batch and continuous models were 3.8 and achieve a given conversion in a CSTR relative to a batch
reactor as a function of conversion. Whereas this ratio is
approximately 2.5 for prediction according to the soluble
Table 1 Parameters used in the generation of the models in substrate model, it is significantly lower (approximately 1.4
Figures 2 through 4 with some increase at high conversions) for prediction ac-

Symbol Value Source

C 0.18125 h-’ This work 45 d 0

k 2.8625 h -’ This work
k, 0.020 g/(U h - ’) Gusakov and Sinitsyn14
0.05 g I-’ Ghose and Tyagi”
? 1.49 I u-1 Ooshima et a/.4
K: 0.66 I W’ Ooshima et a/.4
K, 10.56 g I-’ Phillippidis et a/l3
kC/G 0.62 g I-’ Phillippidis et aLI
ks/c 5.85 g 1-1 Phillipidis eta/.13
kS/Eth 50.35 g I -’ Phillipidis eta/.13
kWEth van Uden”
z?-’ This work
5s 98.3 U g-i Ooshima et aL4 L
I 1z.i pi- Ooshima et a/.4
bmax Ghose and Tyagi16 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Y x/S 0.69 Ghose and Tyagi” Conversion
Y Eth/S 0.47 Ghose and Tyagi”
Figure 3 Hydrolysis rate per cellulase adsorbed to cellulose, in
The data of Ooshima et aL4 for b, K,, C., and C, are reported in relation to conversion (data for similar substrate concentration
terms of adsorbed protein measurements. Activity was deter- and cellulase loading from Nutor and Converse5), shown with
mined from filter paper activity of the same enzyme solution declining MEadsorbed ) curve calculated from the batch SSF sim-
(1.22 U mg-‘1 measured by Girard and Conversez3 ulations and best-fit (r)/(EC) parameters

800 Enzyme Microb. Technol., 1995, vol. 17, September

 Modeling simultaneous saccharification and fermentation of lignocellulose to ethanol: C. R. South et al.
TABLE 2 Summary of continuous SSF data and model predic- Although the hydrolysis rate equation used here does not
tions incorporate mechanistic detail the success of this rate equa-
tion as well as the general modeling approach is not without
fundamental implications, and may provide some guidance
g I-’
Hydraulic Cellulase in understanding biomass hydrolysis and subsequent formu-
residence loading Experimental Predicted lation of mechanistically based models. In particular, our
time (days) In out (U g-11 conversion conversion
results support the utility and validity of the following fea-
tures in the context of modeling processes that involve cel-
0.93 39.9 17.5 12.9 0.54 0.58
lulose hydrolysis:
1.25 60.5 25.8 14.6 0.62 0.67
1.39 40.4 15.6 14.6 0.65 0.69 An adsorption model that allows for either enzyme or
2.03 60.8 12.5 11.6 0.79 0.73
substrate to be in relative excess;
3.26 33.9 6.1 15.3 0.86 0.83
1.50 42.1 10.2 24.0 0.76 0.77 A conversion-dependent rate equation that reflects the
0.58 4.7 3.3 16.0 0.31 0.38 declining ratio of (r)/(ES) as the reaction proceeds;
For CSTRs, and we believe continuous reactors in gen-
Experimental data is from South et al.3 eral, a particle-population model that accounts for vari-
ation in rate over the time individual particles spend in
the reactor.
cording to the particle population model. Experimental data
agree much better with the particle population model, sug- These features are discussed successively in the paragraphs
gesting that such an approach is necessary to accurately that follow.
represent continuous conversion of particulate substrates. The incorporation of an adsorption model into the hy-
drolysis rate equation allows for the prediction of rate sat-
uration with enzyme, a phenomenon that has frequently
Discussion been reported for biomass hydrolysis’7-‘0 and limits the
utility of the Michaelis-Menten model to biomass hydroly-
The proposed SSF model satisfactorily fit batch data. Data sis. Langmuir models have been shown to adequately
obtained in continuous culture are predicted well by the describe adsorption of T. reesei cellulase onto bio-
same model with the application of a particle population- mass, 4.‘o,2@22 although the matter of adsorption reversibil-
based analytic framework. The average error for fitted batch ity appears to be an open question.“.*’
data and predicted continuous data was comparable in mag- In light of the success of the model reported here, and the
nitude to that of the analytic techniques used. We thus con- similar pattern for measured and fitted data for (r)/(ES), we
clude that the model works well for reproducing, and at believe it highly likely that a sharp decline in (r)/(ES) over
least for a CSTR predicting, experimental SSF data over the the course of hydrolysis actually occurs and should be in-
range of conditions to which it has been applied. As this corporated into kinetic models for biomass hydrolysis. Al-
range includes conditions encompassing enzyme loadings though we use Ooshima et ~1,‘s [4] assumptions with re-
that differ by a factor of 4 and feed substrate concentrations spect to lignin availability we have found that the trend of
that differ by a factor of 12, and encompass both batch and declining (r)/(ES) is insensitive to lignin availability as-
continuous systems, the model would appear to be rather sumptions, including full availability over the course of
robust. reaction to availability proportional to conversion (data not
shown). Loss of cellulase activity over time has been re-
ported by investigators.7s However, the high recovery of
free-enzyme activity (up to 50% relative to initial activity),
and the conservation of specific enzyme activity in the study
of Ooshima et al., exclude loss of activity as the major
factor underlying the observed decline in (r)/(ES). More
widespread investigation of this reactivity decline and ex-
planation at the level of interactions between individual en-
zyme components and their binding sites are important ar-
eas for future research.
Based on the results presented here, and Figure 4 in
particular, it is believed that variation of substrate reactivity
over the time individual substrate elements spend in a re-
actor is a fundamental difference between insoluble and
soluble substrates. Whereas soluble substrates are brought
0.5 0.55 0.6 0.65 0.7 0.75 0.6 0.65 0.9 by dilution to their exit reactivity upon introduction into the
Conversion reactor, this does not occur with an insoluble substrate.
During the time a biomass particle spends in a CSTR, for
Figure 4 Ratio of time achieve conversion in CSTR versus that example, the fractional conversion of an individual particle
in batch for population-based model, and a soluble substrate
CSTR model. Simulations are using 50 g cellulose I-’ pretreated
progresses from zero upon entry to some value < 1 upon
wood, with cellulase loading of 15 U g-’ cellulose, and the pa- exit. Simultaneously, the reactivity of the particle declines
rameter values given in Table 1. Experimental data, A radically, with a maximum at x = 0 and declining there-

Enzyme Microb. Technol., 1995, vol. 17, September 801

Papers
after. The reactivity of an element of a particulate substrate Cs Specific ca acity of cellulosic component for cellu-
over time in a CSTR is thus much like that of a soluble lase, U g- P
substrate in a batch or plug flow reactor, but is decidedly C, Specific capacity of lignaceous component for cellu-
different from that of a soluble substrate in a CSTR. lase, U gP
p+,,,,Maximum cell growth rate, hh ’
7 Average CSTR hydraulic residence time, h
Symbols
B B-glucosidase concentration in solution, U 1-l
Acknowledgments
C Conversion-independent component in rate
function, h- ’ The authors are grateful to A. 0. Converse for useful dis-
C Cellobiose concentration, g l- ’ cussions, and for the support provided by NREL subcon-
Eth Ethanol concentration, g 1- ’ tract RE-2-13005-1, as well as the John Merck Fund and
Er Cellulase enzyme in solution, U 1- ’ NSF Grant BCS 9058392.
E, Total cellulase enzyme added, U 1-l
EL Concentration of lignin-cellulase complex, References
u 1-l
ES Concentration of cellulose~ellulase complex, I Chem Systems. Technical and Economic Evaluation, Wood IO Erh-
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u1-’
ergy, Washington, DC, 1992.
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G Glucose concentration, g 1- ’ 1991. Fuel ethanol from cellulosic biomass. Science 251, 1318-
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f(
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