Biocatalysis and Biotransformation, JanuaryFebruary 2009; 27(1): 2735

ORIGINAL ARTICLE

Modelling ethanol production from cellulose: separate hydrolysis and

fermentation versus simultaneous saccharification and fermentation

R. E. T. DRISSEN1, R. H. W. MAAS2, J. TRAMPER1, & H. H. BEEFTINK1

Wageningen University and Research Center, 1Food and Bioprocess Engineering Group, PO Box 8129, 6700 EV Wageningen
and 2Agrotechnology and Food Sciences Group, PO Box 17, 6700 AA Wageningen, The Netherlands
(Received 19 August 2007; accepted 25 February 2008)

Abstract
In ethanol production from cellulose, enzymatic hydrolysis, and fermentative conversion may be performed sequentially
(separate hydrolysis and fermentation, SHF) or in a single reaction vessel (simultaneous saccharification and fermentation,
SSF). Opting for either is essentially a trade-off between optimal temperatures and inhibitory glucose concentrations on the
one hand (SHF) vs. sub-optimal temperatures and ethanol-inhibited cellulolysis on the other (SSF). Although the impact of
ethanol on cellobiose hydrolysis was found to be negligible, formation of glucose and cellobiose from cellulose were found to
be significantly inhibited by ethanol. A previous model for the kinetics of enzymatic cellulose hydrolysis was, therefore,
extended with enzyme inhibition by ethanol, thus allowing a rational evaluation of SSF and SHF. The model predicted SSF
processing to be superior. The superiority of SSF over SHF (separate hydrolysis and fermentation) was confirmed
experimentally, both with respect to ethanol yield on glucose (0.41 g g
1 for SSF vs. 0.35 g g
1 for SHF) and ethanol
production rate, being 30% higher for an SSF type process. High conversion rates were found to be difficult to achieve since
at a conversion rate of 52% in a SSF process the reaction rate dropped to 5% of its initial value. The model, extended with
the impact of ethanol on the cellulase complex proved to predict reaction progress accurately.

Keywords: SSF, SHF, ethanol inhibition, glucose inhibition, model

Introduction waste materials, such as grass or wheat straw, which

can act as an abundant and cheap source of fermen-
With decreasing oil availability and the negative
table sugars (Wyman 1994). In addition to a neces-
effects of traditional fossil fuels on the environment,
initiatives have started to look for alternative sources sary pretreatment of the cellulosic material, this will
of liquid fuel. One of these promising sources is involve two consecutive catalytic steps, an enzymatic
ethanol, which can be produced through fermenta- conversion of the cellulose to fermentable sugars and
tion of a sugar source, such as sugar cane or glucose a fermentative conversion of these sugars to ethanol
syrup from starch. However, for introduction of (Sheehan &d Himmel 1999). These hydrolysis and
ethanol for fuel purposes to be successful, the price fermentation steps may be operated sequentially
of ethanol should be competitive with the price of (separate hydrolysis and fermentation, SHF) or
petrol. Until the recent oil price rises, ethanol from concurrently (simultaneous saccharification and fer-
traditional sugar sources, such as starch has been mentation, SSF; Wingren et al. 2003). Diluted
more expensive than petrol because of the relatively ethanol is finally concentrated by, for instance,
high sugar cost. Furthermore, as demand for ethanol distillation or pervaporation (O’Brien et al. 2000).
rises, a dramatic increase in sugar price is expected When opting for either SSF or SHF, one faces a
to occur (Lynd et al. 1999) with consequences for trade-off in terms of process variables (Takagi et al.
the price of food. 1977). With a sequential SHF process, both the
Therefore, research over the past years has focused hydrolysis and the fermentation may be performed
on making available glucose from (ligno)cellulosic at their optimum temperature: 378C for yeast

Correspondence: R.E.T. Drissen, Food and Bioprocess Engineering Group, PO Box 8129, 6700 EV Wageningen, The Netherlands.
E-mail: [email protected]

ISSN 1024-2422 print/ISSN 1029-2446 online # 2009 Informa UK Ltd

DOI: 10.1080/10242420802564358
28 R. E. T. Drissen et al.

fermentation and 508C for a cellulase complex b-Glucosidase activity in 1.0 mL of enzyme
(Stenberg et al. 2000). The downside, however, is preparation was determined by addition of 1.0
accumulation of the hydrolysis product glucose in mL of a 15 mM solution of cellobiose in 0.05 M
the enzyme reactor. As a result, the cellulase com- citrate buffer (pH 4.8), and incubation for 30 min
plex would, at increasing glucose concentration, at 508C. The enzyme was then inactivated (5 min
greatly suffer from product inhibition. Such product at 1008C) after which glucose was measured. The
inhibition may be avoided by continuous sugar b-glucosidase activity was found to be 204 units per
removal by a yeast, i.e. in an SSF type of process. mL of Cellubrix (1 unit forming 1 mmol glucose per
In the case of SSF, however, both the hydrolysis and min, i.e. a maximum turnover rate of 0.0108 g
the fermentation are performed at some sub-optimal glucose unit
1 h
1).
temperature (the pH optimum is approximately 5 for
both reactions). In addition, possible effects of Substrates
ethanol on the performance of the cellulase have to
be considered. As model cellulose, Avicel was used. This was
Previous work has often limited itself to proof of supplied by Merck (order nr. 1.02330.0500).
principle without actually weighing the two pro- The additional nutrients used for fermentation,
were: 5 g L
1 (NH4)2SO4, 3 g L
1 KH2PO4, 0.5 g
cesses on a quantitative basis (Mohagheghi et al.
L
1 MgSO4 ×/7H2O, 1 mL L
1 trace elements
1992; Alkasrawi et al. 2002). Some comparisons
solution, 1 mL L
1 vitamin solution, 1.25 mL
were based on rather limited mathematical models of
L
1 ergosterol solution, 0.5 mL L
1 anti-foam.
both processes and for comparison. Previous math-
ematical models of an SSF-type process (Philippidis
et al. 1992; Philippidis & Wyman 1992; Wright et al. HPLC analysis
1987; Wyman et al. 1986) have often taken only Samples were centrifuged (1 min, 13,000 rpm); the
parts of the complete trade-off into consideration, supernatant was inactivated by diluting 1:1 v/v
which should include the impact of various sugar with 1 M sulphuric acid. and filtered (0.2 mm) for
inhibitions, as well as ethanol inhibition, an impact removal of any residual proteins or yeast. Samples
of reaction temperature and a changing substrate were then analysed on a Waters (USA) HPLC with a
reactivity (South et al. 1993, 1995; Desai & Con- refractive index detector and a IOA-1000 column
verse 1997; Yang et al. 2006). (Alltech, The Netherlands) at 908C and 0.4 mL
In this article both the enzymatic hydrolysis and min
1 of 3.0 mM as mobile phase.
fermentation to ethanol will be topic of investigation.
A previously developed model, adequately describ-
Ethanol inhibition
ing hydrolysis and glucose production of both a
pretreated lignocellulosic substrate and Avicel, a Six shake flasks with 40 g L
1 of Avicel were
model substrate, will be expanded to describe incubated in 0.1 M citrate buffer (pH 4.8) with
ethanol production from cellulosic material. There- Cellubrix (20 FPU g
1 cellulose) at a temperature
fore, effects of ethanol on cellulase are presented. of 378C. Ethanol was added to each of the shake
Secondly, SSF and SHF are weighed against one flasks to create a series of increasing ethanol con-
another, and a model presented to simulate both centrations of 0, 20, 40, 60, 80 and 100 g L
1
processes. This might be used for an economical (density ethanol determined to be 0.790 kg L
1).
assessment and process optimization of the produc- Samples were taken at t 1, 3, 7, 15 and 30 min, and
tion process of ethanol from cellulosic sources. at t2, 3, 4, 20 and 28 h. Sample analysis was done
by HPLC as described above. Initial glucose pro-
duction rates were calculated as the slope of a linear
Materials and methods fit to the data points of the first 30 min. During this
Enzyme and enzyme assay period, glucose was seen to accumulate linearly and,
therefore, enzymatic activity was not yet influenced
Cellubrix (Novozymes Corp. Denmark) was used as by inhibitory effects and inactivation.
cellulase preparation for experiments presented in
this article. Its cellulolytic activity was 56 filter paper
units (FPU) per mL of solution as determined SSF in shake flasks
according to Ghose (1987). The concentration of Two 200 mL bottles were filled with 100 mL of
protein per millilitres of Cellubrix was determined suspension of 40 g L
1 Avicel (being 95% pure
according to Bradford (1976) and found to be 32.3 cellulose) with pH 4.8 (citrate buffer) and incubated
mg/mL. All experiments were conducted at pH 4.8 in a 378C water bath with 7.4 and 74 g L
1 yeast
(citrate buffer). (equalling 2 g L
1 and 20 g L
1 based on dry mass,
 SHF vs. SSF 29

respectively). The micro-organism was a commer- various ethanol concentrations. Values for R2 were
cially available Saccharomyces cerevisiae (DSM Delft, 0.95 and higher.
The Netherlands). Samples were taken every hour Initial glucose production rates from Avicel were
for HPLC analysis on glucose and ethanol concen- found to be inhibited by ethanol (Figure 1). Regres-
trations. The cellulose concentration was chosen to sion with a general type of product inhibition
mimic the maximum workable dry-mass substrate equation (eqn 1) yielded an inhibition constant
concentration found in previous research (Drissen et Ki 95 g L
1.
al. 2007), i.e. 100 g L
1 of wheat straw with an
approximately 40% cellulose concentration. Limita- vv(0) × [Ki;EtOH =(Ki;EtOH CEtOH )] (1)
tions in handling were due to high viscosity.
The model fit using eqn 1 and an inhibition constant
of 95 g L
1 is also plotted in Figure 1. This ethanol
SSF and SHF in fermentors inhibition is somewhat less severe than reported
elsewhere: Phillipidis and Hatzis (1997) found a 1st-
For these experiments two 3-L fermentors with a order inhibition constant of approximately 50 g L
1,
working volume of 2 L (Applikon BV, The Nether- whereas Wu and Lee (1997) found almost complete
lands, type ADI 1020) were used. For both experi- inhibition of the reaction at approximately 65 g L
1
ments, the initial suspension contained 40 g L
1 ethanol. Apparently, the current Cellubrix prepara-
Avicel and additional nutrients in 0.1 M citrate tion is less susceptible to ethanol inhibition.
buffer (pH 4.8) and Cellubrix (12 FPU g
1 Previously (Drissen et al. 2007), it was shown that
cellulose). cellobiose activity is in excess in Cellubrix. Potential
The SHF-process was operated at 508C for the effects of ethanol were further investigated by study-
first 42 h to allow for the build-up of fermentable ing the accumulation of the intermediate cellobiose
glucose. After 42 h, 2 g L
1 (based on dry mass) of over time for various reaction mixtures with Cellu-
yeast was added and the process was continued at brix, Avicel and a specific ethanol concentration
378C. For the SHF-process all concentrations were (Figure 2). In all cases, the cellobiose concentration
monitored by measuring of samples by HPLC. was found to rise quickly due to initial formation of
The SSF-process was continuously operated at the dimeric cellobiose from polymeric cellulose by
378C and 2 g L
1 yeast (based on dry mass) was the cellulase activity of Cellubrix. Due to the
added at the start of the experiment. For the SSF- increasing cellobiose concentration, however, cello-
type fermentation, cumulative carbon dioxide pro- biose degradation then started to increase and the
duction was measured with an online gas analyser net formation of cellobiose decreased,, until, finally,
(1440, Servomex), using 1 L min
1 N2 as carrier production and degradation of cellobiose were
gas. A condenser (set at 28C) was used to avoid balanced and a plateau value was reached.
ethanol stripping from the broth. Glucose and
ethanol concentrations were also monitored by 40
HPLC analysis of samples.
glucose production rate (mg.l-1.h-1)

Numerical procedure 30
For modelling purposes and solution of differential
equations, a first-order Euler forward algorithm was
used (Press 2007).
20

Results and discussion

Ethanol inhibition of cellulase activity
10
When contemplating a process for bio-ethanol pro-
duction where both the enzymatic conversion of
cellulose to glucose and the parallel fermentation of
glucose to ethanol are combined, one must take into 0
consideration possible effects of ethanol on the 0 20 40 60 80 100
performance of the cellulase complex, as has been ethanol concentration (g/l)
suggested previously (Wu & Lee 1997). In the present Figure 1. Effect of ethanol concentration on the production rate
research, initial glucose production rates were mea- of glucose by Cellubrix (20 FPU g
1 cellulose) from Avicel (40 g
sured for incubations of Avicel and Cellubrix at L
1), experimental data (j) and model fit (**) at 378C.
30 R. E. T. Drissen et al.
v1
1.0 cellulose 0 cellobiose
v2
cellobiose 0 glucose
v3
0.8 cellulose 0 glucose (2)
cellobiose concentration (g.l-1)

It will be used as a starting point for this paper.

Combined with the effects of ethanol on cellulase
0.6 (eqn 1), the hydrolysis model is:
kmax;1 × CE e
Ea =(R×T)
KD (T)×t
v1 (t; T) × ×e ×CC (t)
0.4 KL  CE e
Ea =(R×313)
 
CC (t)

Krec × 1

CC (0)
Ki;G Ki;EtOH

×e × (3)
0.2 Ki;G  CG (t) Ki;EtOH  CEtOH

e
Ea =(RT)
0.0 v2 (t; T)kmax;2 eg etotal × ×e
KD (T)t
e
Ea =(R×313)
0 200 400 1000 1200 1400 1600
time (min)
CCb (t)

(4)
Km (1  CG (t)=Ki;2 )  CCb
Figure 2. Accumulation of cellobiose from Avicel (40 g L
1) after
incubation with Cellubrix (20 FPU g
1 cellulose) and various
concentrations of ethanol (", 0 g L
1, 2, 20 g L
1, ', 40 g kmax;3 × CE e
Ea =(RT)
KD (T)×t
L
1, ^, 60 g L
1, %, 80 g L
1, \, 100 g L
1). v3 (t; T) × ×e
KL  CE e
Ea =(R×313)
 
CC (t)
Lower plateau values for cellobiose were observed
Krec × 1

CC (0)
at higher ethanol concentrations. Given the fact that Ki;G

CC (t)× ×e
the plateau values are below 1.92 g L
1, the Km of Ki;G  CG (t)
the cellobiose (Dekker 1986), the conversion of Ki;EtOH
cellobiose to glucose can be considered to be first
(5)
Ki;EtOH  CEtOH
order in cellobiose. A plateau value means the
production rate of glucose is equal to consumption For reactions with cellulose as a substrate (reaction 1
rate. and 3), the active amount of enzyme was assumed to
The ratio between glucose production rate and be determined by enzyme adsorption onto the
maximum glucose consumption rate by cellobiose cellulose substrate (adsorption constant KL). For
equals Cg(t)/[Cg(t)Km]. At increasing ethanol con- reactions with glucose as a reaction product (reac-
centrations, this ratio becomes smaller; therefore, tions 2 and 3), glucose product inhibition was
the ratio between glucose production rate and assumed (inhibition constants Ki,G and Ki,2). For
maximum glucose consumption rate becomes smal- all three reactions, the zero-order rate constant was
ler, leading to the conclusion that the cellulase given as a function of temperature (activation energy
activity is more strongly inhibited by ethanol than Ea); in addition, all enzyme activity was assumed to
the cellobiose activity. The impact of ethanol on the be subject to thermal inactivation (kD). The nature
conversion reaction of cellobiose to glucose can of the cellulose substrate was assumed to be conver-
therefore be neglected. sion-dependent; to this end, a recalcitrance constant
Krec was used. Finally, ethanol inhibition was
assumed to affect the rates of reactions 1 and 3
Model development
(inhibition constant Ki,EtOH).
A generic model for cellulose hydrolysis and glucose For the thermal inactivation constant kD(T), the
production from various cellulose sources by a following temperature dependency was assumed:
commercial cellulase complex has been studied
previously (Drissen et al. 2007). During this model kD AD ×e
DH=RT (6)
development, the impact of various enzyme concen- Values for the relevant kinetic parameters for
trations, various temperatures, and the effects and Cellubrix on Avicel were obtained from the literature
kinetics of using various cellulose sources (both (Dekker 1986; Drissen et al. 2007): kmax,1 0.081
Avicel as model substrate and pretreated wheat h
1; KL 18.2 FPU L
1; Ea 29.8 kJ mol
1;
straw) were examined. The model assumes three AD 3.64 ×/1018 h
1; DH 148 kJ mol
1; Ki 6.3
enzyme-catalysed reactions (reaction rates v1, v2 and g L
1; Krec 2.8 (
); kmax,2 0.0108 g U
1 h
1;
v3) for cellulose hydrolysis: eg 6320 U g
1; Km 1.92 g L
; Ki,2 0.54 g L
;
 SHF vs. SSF 31

kmax,3 0.058 h
1. Ce and etotal depend on the requirement for an effective SSF-type of process is
specific enzyme dosage for a specific experiment. that the production rate of glucose is the rate limiting
Yeast (Saccharomyces cerevisiae) metabolism has al- step in the process, i.e. the catalytic activity of the
ready been topic of extensive research. For modelling yeast is in excess in comparison with the catalytic
glucose consumption and biomass formation, stan- activity of the cellulase. In this way, the concentra-
dard Monod kinetics (Groot et al. 1993; Oliveira et al. tion of the inhibiting sugars will remain negligibly
1999) were assumed, expanded to include ethanol low, thus avoiding product inhibition of the en-
inhibition (Philippidis & Hatzis 1997) on yeast. zymes. The experiments have been executed with
This yields the following overall model for the yeast various yeast concentrations, both an excessive (i.e.
growth rate (v4) and substrate consumption rate (v5): 20 g L
1) concentration and a concentration that
mmax × Cg (t) would, according to initial model predictions, be just
v4 (t) ×Cx (t) sufficient to avoid glucose accumulation (i.e. 2 g
Kg  Cg (t)
L
1 dry yeast). Figure 3 shows the production of
Kiy;EtOH ethanol over time and model predictions.

(biomass) (7)
Kiy;EtOH  CEtOH (t) Final analysis of the reaction mixture showed that
v4 (t) possible other components (i.e. sugars, acetic acid)
v5 (t)

Cx (t)×ms (substrate) (8) were negligible (i.e. concentrations below 0.25 g
Yxs
L
1).
The following values for kinetic constants were As can be seen in Figure 3, the model for
adopted from the literature (Groot et al. 1993; Hatzis production of ethanol from cellulose, with a com-
et al. 1996; Oliveira et al. 1999): Kiy,EtOH 50 g L
; mercial cellulase complex and S. cerevisiae accurately
Kg 0.0252 g L
; ms 0.02 g g
1 h
1; Yxg 0.11 g predicts ethanol formation. Final glucose accumula-
g
1; mmax 0.250 h
1. tion has been determined, being 0.5 g L
1 and 0.8 g
Overall, changes in concentrations of cellulose, L
1 for the experiments with 20 g L
1 yeast and 2 g
cellobiose, and glucose in a batch reactor are now L
1 yeast, respectively. The production of ethanol
described by: with time using 2 g L
1 of yeast was almost identical
dCC =dt
[v1 (t)v3 (t)] (9) to that using 20 g L
1 of yeast. Apparently, only a
small amount of yeast is sufficient to conduct an
dCCb =dt 1; 053×v1 (t)
v2 (t) (10)
SSF-type process, therefore adding to the ease of
dCG =dt1; 053×v2 (t)1; 111×v3 (t)v5 (t) (11) implementing this type of process. The production
dCx =dt v4 (t) (12) of glucose itself can accurately be described by the
The formation of ethanol and CO2 are calculated proposed model for cellulose degradation to glucose,
by closing the overall carbon mass balance (Hatzis et
al. 1996). It is assumed that all carbon, not used for 12
biomass formation, is used for energy production
according to:
10
C6 H12 O6 0 2 CO2 2 C2 H5 OH:
ethanol concentration (g.l-1)

Taking into consideration the slightly higher molar 8

mass of ethanol (46 g mol
1) vs. that of carbon
dioxide (44 g mol
1), on a weight base 0.511 g of
ethanol and 0.489 g of CO2 will be produced from 6
every gram of glucose.
The overall mass balance describing ethanol con-
centration with time as a function of yeast and 4
cellulase activity becomes:
Cethanol 
0:511×½1:111×(Cc (t)
Cc (0)) 2
1:053×(Ccb (t)
Ccb (0))
(Cg (t)
Cg (0))(Cx (t)
Cx (0)) (13)
0
0 5 10 15 20
time (h)
Model testing
Figure 3. Ethanol concentration in time for an SSF process using
Experiments have been conducted to validate the Avicel (40 g L
1) at 378C, experimental data (20 g L
1 yeast 
model for a simultaneous saccharification and fer-
mentation (SSF) as presented in this paper. A
and 2 g L
1 yeast k) and model prediction (20 g L
1
L
1 ). *
and 2 g *
32 R. E. T. Drissen et al.

expanded with the effect of ethanol on cellulase

16
activity. Therefore, the yeast has no negative impact
on cellulase activity through, e.g. cellulase protein 14
consumption for biomass formation.
12

concentration (g.l-1)
Comparing SSF and SHF 10
The model as described by eqns 214 allows a
comparison between an SSF and an SHF process. 8
Input parameters for model calculations are listed in
6
Table I.
In order to compare both configurations, SSF and 4
SHF experiments were conducted in a 2-L fermen-
tor, and were compared with model predictions. 2
Figure 4 shows the measured glucose and ethanol
concentrations (dots) as a function in time for an 0
SHF process and the corresponding model predic- 0 10 20 30 40 50
tions. After 42 h, i.e. shortly after the yeast was time (h)
added, the glucose concentration dropped very Figure 4. Experimental glucose (m) and ethanol (k) concentra-
rapidly and the ethanol concentration rose rapidly.
At t 44 h, all accumulated glucose had been
tions in time, and model predictions ( ). *
converted; from then on, the process continued as the SHF process, i.e. with respect to ethanol yield
an SSF-type process. Cellulase activity continued to from a cellulose source, an SSF-type process seems
produce new glucose, which was directly converted the preferable set-up. The negative effects of product
to ethanol by the yeast. This can be seen by the inhibition of sugars on cellulase in an SHF process
glucose concentration remaining low (BB1 g L
1) therefore outweigh the negative effects of ethanol
and a slow further increase in ethanol concentration, inhibition and a sub-optimal saccharification tem-
compared with the rapid initial increase. Calculated perature in an SSF-type process, especially given the
ethanol yield was 0.35 g ethanol g
1 glucose. fact that all other factors, such as variance in
Figure 5 shows the result for an SSF process in a substrate crystallinity were identical for both pro-
2-L fermentor. Both the ethanol concentration, cesses.
measured through sampling and HPLC analysis, Secondly, the ethanol yield in an SHF process is
and the ethanol concentration, measured indirectly lower than in an SSF process. This is in line with
through cumulative CO2 production, are shown and conclusions from previous research, although the
compared with a model prediction. The glucose benefits of an SSF process are more pronounced
concentration (not shown in Figure 5) was mon- than found by, e.g. Wingren et al. (2003), but less
itored by HPLC analysis of samples taken every 3 h than found by Stenberg et al. (2000).
and remained negligibly low (i.e. below 0.3 g L
1). In an SHF process, shortly after yeast addition,
After completion of the experiment a complete the excess glucose allows for biomass formation. In
carbon analysis and carbon balance were made. an SSF process, glucose concentration is continu-
The final ethanol yield on glucose was 0.41 g g
1. ously low and will mainly be used for cell main-
Comparing Figure 4 and 5 reveals that after 48 h, tenance, i.e. energy and, therefore, ethanol
the SSF process produced 30% more ethanol than production. For this, it will, however, be imperative

Table I. Input parameters for model comparison of SSF and SHF.

Parameter SSF SHF

Total process time modelled 48 h 48 h

Moment of yeast addition t 0 h t 40 h
Temperature 378C 508C (saccharification first 40 h)
378C (fermentation, last 8 h)
Yeast concentration 2 g L
1 2 g L
1
Avicel concentration 38 g L
1 38 g L
1
Enzyme loading
k1 and k3 12 FPU g
cellulose 12 FPU g
1 cellulose
0.032 h
1 and 0.023 h
1 0.032 h
1 and 0.023 h
1
 SHF vs. SSF 33
100
14

12 80
ethanol concentration (g.l-1)

relative reaction rate (%)

10
60
8

6
40

4
20
2

0
0
0 10 20 30 40 50 0 10 20 30 40 50
time (h) time (h)
Figure 5. Ethanol concentration as a function of time for an SSF Figure 6. Relative CO2 production rate (%) for an SSF process,
process, experimental results (k) and model prediction (black); indirectly indicating overall ethanol production rate.
direct EtOH measurement (dots) and calculated through CO2-
measurement (grey line).

Conclusions
during an SSF process that the catalytic activity of A model to describe the hydrolysis of cellulose by a
the yeast at all times is larger than the catalytic commercial cellulase was successfully expanded to
activity of the cellulase complex used, in order to describe the conversion of cellulose to ethanol
avoid sugar accumulation and therefore product through the process of simultaneous saccharification
inhibition. Figure 3 demonstrates yeast’s potential and fermentation. Ethanol has been demonstrated to
for an SSF process in this respect. This has further cause first-order inhibition of cellulase with a Ki of
been affirmed by the results presented in Figure 4 95 g L
1. No impact of ethanol on conversion of
and 5. cellobiose to glucose could be found. It was shown
The online measurement of CO2-production in that with a relatively small inoculum of yeast,
the SSF configuration enabled a constant determi- glucose accumulation in a SSF-type process could
nation of the reaction rate during the course of the be avoided. Furthermore, it was shown that yeast
process (Figure 6). had no negative impact on the activity of cellulase.
Clearly, the reaction proceeded at only approxi- The SSF type process proved to be superior to the
mately 5% of the original reaction rate after 48 h. Due SHF type process, both with respect to ethanol yield
to this rapid decline in conversion rate, it was per gram of glucose produced (0.41 for SSF vs. 0.35
considered impractical to progress further with the for SHF) and ethanol production rate, being 30%
reaction. As Figure 5 shows, this correlates with higher for an SSF type process. However, for the
approximately 11 g L
1 ethanol, i.e. a conversion of cellulose source used it was difficult to obtain a high
conversion rate and yield. Due to the increasing
53%. This drop in reaction rate is attributed to a
recalcitrance of the substrate the conversion rate
change in the nature of the substrate. Since the yeast
dropped to 5% of its initial value at a conversion of
can consume all of the sugar and ethanol concentra-
approximately 53%.
tions are still non-inhibitory (Figure 1), product
inhibition does not explain the drop in the reaction
rate. Enzyme inactivation was excluded as an ex- Notation
planation since addition of fresh enzyme showed no
A Arrhenius constant for (h
1)
increase in reaction rate. However, the addition of increase of initial rate at
fresh cellulose substrate showed an increase in reac- increasing temperatures
tion rate, leading to the conclusion that the enzyme AD Arrhenius constant for (h
1)
still had catalytic activity and the presence of less thermal enzyme deactivation
recalcitrant cellulose could restart glucose produc- CC Cellulose concentration (g L
1)
tion. CCb Cellobiose concentration (g L
1)
34 R. E. T. Drissen et al.
Acknowledgements
CE Enzyme concentration (FPU L
1)
CEtOH Ethanol concentration (g L
1) The project was financially supported through a
CG Glucose concentration (g L
1) grant from the Programme Economy, Ecology and
CX Yeast concentration (dry (g L
1) Technology (EET) by the Netherlands’ Department
weight) of Economic Affairs, the Department of Public
eg b-glucosidase activity per g of (units g
1) Housing, Spatial Planning and Environmental Pro-
protein in the enzyme tection, and the Department of Education, Cultural
preparation Affairs and Sciences. The authors thank Arianne
etotal Protein (enzyme) (g L
1
) Huijnen, Iris Camp and Steven Rothuizen for help-
concentration per L reaction ing to determine the relevant kinetic parameters.
volume
Ea Activation energy (J mol
1) Declaration of interest: The authors report no
h Planck’s constant (J s) conflicts of interest. The authors alone are respon-
(6.626 ×/10
34) sible for the content and writing of the paper.
ki Reaction rate constant (h
1)
(i 13) References
k2 Reaction rate constant of g U
1 ×/h
1
Alkasrawi M, Galbe M, Zacchi G. 2002. Recirculation of process
b-glucosidase

1 streams in fuel ethanol production from softwood based on
kmax,i Maximum specific rate (h ) simultaneous saccharification and fermentation. Appl Biochem
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This paper was first published online on iFirst on 9 December

2008.