Process Biochemistry 42 (2007) 834–839

www.elsevier.com/locate/procbio

A comparison between simultaneous saccharification and fermentation and

separate hydrolysis and fermentation using steam-pretreated corn stover
Karin Öhgren a, Renata Bura b, Gary Lesnicki c, Jack Saddler b, Guido Zacchi a,*
a
Department of Chemical Engineering, Lund University, P.O. Box 124, SE-22100 Lund, Sweden
b
Forest Products Biotechnology, Department of Wood Science, 4th Floor, Forest Science Centre, 4042-2424 Main Mall,
University of British Columbia, Vancouver, BC, Canada
c
Michael Smith Laboratories, Fermentation Pilot Plant Facility, 2185 East Mall, University of British Columbia, Vancouver, BC, Canada
Received 10 July 2006; received in revised form 19 January 2007; accepted 9 February 2007

Abstract
Two different process configurations, simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF),
were compared, at 8% water-insoluble solids (WIS), regarding ethanol production from steam-pretreated corn stover. The enzymatic loading in
these experiments was 10 FPU/g WIS and the yeast concentration in SSF was 1 g/L (dry weight) of a Saccharomyces cerevisiae strain. When the
whole slurry from the pretreatment stage was used as it was, diluted to 8% WIS with water and pH adjusted, SSF gave a 13% higher overall ethanol
yield than SHF (72.4% versus 59.1% of the theoretical). The impact of the inhibitory compounds in the liquid fraction of the pretreated slurry was
shown to affect SSF and SHF in different ways. The overall ethanol yield (based on the untreated raw material) decreased when SSF was run in
absence on inhibitors compared to SSF with inhibitors present. On the contrary, the presence of inhibitors decreased the overall ethanol yield in the
case of SHF. However, the SHF yield achieves in the absence of inhibitors was still lower than the SSF yield achieves with inhibitors present.
# 2007 Elsevier Ltd. All rights reserved.

Keywords: Steam pretreatment; SSF; SHF; Ethanol production; SO2; Corn stover; Ethanol concentration

1. Introduction Bio-ethanol can be produced from any biomass, thus access to

Ethanol, produced from sugar, starch and lignocellulosic
biomass, is a liquid bio-fuel with the potential to replace some
of the liquid fossil fuels used in transportation. Bio-ethanol,
produced from corn grain (starch) and sugar cane (sucrose) is
currently the most common renewable fuel [1] and the
introduction of ethanol on to the fuel market has been
facilitated by the positive effects of low-blend ethanol–petrol
mixtures [2]. However, it is clear that the large-scale use of bio-
ethanol will require lignocellulosic biomass to be used as raw
material [3,1]. Furthermore, current data suggest that only
lignocellulosic ethanol (ethanol made from lignocellulosic
biomass) offers large reductions in greenhouse gas emissions
compared with fossil fuels [3]. Bio-fuels also provide the
opportunity for non-oil-producing countries to be self-sufficient
in fuel.
* Corresponding author. Tel.: +46 46 222 82 97; fax: +46 46 222 45 26.
E-mail address: guido.zacchi@chemeng.lth.se (G. Zacchi).
1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2007.02.003
raw material is virtually unlimited. For example, agricultural by-
products (straw, sugar cane bagasse, stover) provide a readily
available, vast source of cheap biomass [4]. However, the
production of ethanol from lignocellulosic raw materials is more
difficult than from sugar or starch. Lignocellulosic materials
consist primarily of three components, namely cellulose,
hemicellulose and lignin, of which the first two can be
hydrolysed to monomeric sugars [5], which can then be
fermented to ethanol using a hexose- and pentose-fermenting
organism. The hydrolysis of lignocellulose to monomeric sugars
can be achieved in many different ways. One thoroughly
investigated method is to first treat the material using steam
pretreatment [6], with or without a catalyst. Several studies have
been carried out on steam explosion as a method of pretreating
corn stover, the raw material used in this study, using dilute
sulphuric acid or SO2 as a catalyst, with the aim of solubilizing
hemicellulosic sugars, and rendering the remaining cellulose
accessible to subsequent enzymatic hydrolysis [7–11].
After pretreatment, enzymatic hydrolysis is used to convert
the residual cellulose and hemicellulose into monomeric sugars.
 K. Öhgren et al. / Process Biochemistry 42 (2007) 834–839
The sugars are then fermented to ethanol using yeast. When
enzymatic hydrolysis and fermentation are performed sequen-
tially, it is referred to as separate hydrolysis and fermentation
(SHF). However, the two process steps can be performed
simultaneously, i.e. simultaneous saccharification and fermenta-
tion (SSF). This was first done by Takagi et al. in 1977 [12]. SSF
was shown to be superior to separate hydrolysis and fermentation
when the whole slurry from steam pretreatment of softwood was
used [13]. Combining the two process steps also results in a lower
capital cost, and the fact that the ethanol concentration is higher
during SSF than SHF reduces the risk of contamination [14].
However, mixing the lignin residues with the yeast (as in SSF)
makes yeast recirculation very difficult. In addition, the
temperature optima for the yeast and the enzymes used differ,
which means that the conditions used in SSF cannot be optimal
for both the enzymes and the yeast.
Pretreatment hydrolysate has an inhibitory effect on
cellulose conversion in the enzymatic step [15,16] but this
can be overcome by fermentation of the pretreatment
hydrolysate prior to enzymatic hydrolysis [17]. SSF may thus
exhibit lower inhibition of the enzymes due to the concomitant
fermentation. The pretreatment hydrolysate also has an
inhibitory effect on the yeast, however, at low concentrations
some of the inhibitors can actually have a positive effect on the
ethanol productivity and the ethanol yield by stressing the yeast
[18].
In this study, bench-scale (5 L) SSF and SHF at 8% water
insoluble solids (WIS) were compared using steam-pretreated
corn stover. A WIS content of 8% is high enough to obtain
reasonable high ethanol concentrations, but low enough to
ensure that complete cell death is avoided. Both the whole
slurry (with all the inhibitors present) and washed slurry were
used in order to distinguish between inhibition due to by-
products formed in the steam pretreatment stage and sugar-
inhibition of the enzymes. The enzymes used were commercial
enzyme mixtures from Novozymes A/S, Denmark, and the
yeast was a Saccharomyces cerevisiae strain cultivated in the
steam-pretreatment hydrolysate and thus adapted to the harsh
environment of SSF.
2. Materials and methods
2.1. Raw material
All the investigations were performed using corn stover from North
America, kindly supplied by NREL, Golden, Colorado, USA. After collection,
the corn stover was chopped, air-dried and then stored at room temperature. The
sugar and lignin contents of the raw material were determined according to
NREL [19].
2.2. Experimental set-up
After impregnation with SO2, the stover was steam-pretreated at 190 8C for
5 min. These conditions have previously been determined to be optimal for
SO2-impregnated, steam pretreatment of corn stover [11]. The resulting slurry
was then analysed and used in SSF and enzymatic hydrolysis (EH). The WIS
content was adjusted with water, after which enzymes or enzymes and yeast
were added. The initial WIS concentration in all the experiments was 8%.
An overview of the experimental set-up is shown schematically in Fig. 1.
835
Fig. 1. The experimental procedure.
2.3. Steam pretreatment
The water content in the corn stover was determined and the corn stover was
impregnated with 3% SO2 (w/w, based on the dry weight). Steam pretreatment
was carried out in 50 g batches in a 2-L steam gun (Stake Tech II batch reactor,
Stake Tech-Norvall, Ontario, Canada). Several batches were pretreated, after
which the material was collected for analysis and stored at ÿ20 8C before use.
Solid fractions, generated by pretreatment, were analysed in the same way as the
raw material, and the liquid fraction was analysed with respect to monomeric
and oligomeric sugars, acetic acid, 5-hydroxy methyl furfural (HMF) and
furfural.
2.4. SSF and EH
The SSF and EH experiments were performed in a 15-L bioreactor with a
working weight of 5 kg (Applikon, Schiedam, The Netherlands). The slurry
from the pretreatment stage was either used as it was or was thoroughly washed
with tap water. The pH was then adjusted to 5.0 with concentrated NaOH and
the WIS concentration was adjusted by the addition of tap water. In one series of
experiments, additional glucose and xylose were added to the washed and
conditioned slurry to adjust the sugar concentrations in the liquid fraction to the
same level as in the experiment with the whole slurry. Sugar addition was based
on the sugar content in the liquid fraction of the pretreated slurry.
A commercial cellulase mixture, cellulase NS 50013 (69.5 Filter Paper Unit
(FPU)/mL), supplemented with the b-glucosidase preparation, beta-glucosidase
NS 50010, both from Novozymes A/S, Bagsværd, Denmark, was used. The
enzymatic activity in the experiments was 10 FPU/g WIS and the b-glucosidase
supplementation constituted 25% of the volume of cellulase added. The
commercial xylanase mixture, Multifect xylanases, (Genencor International,
Rochester, NY, USA), was also added in one series of experiments. The dosage
of xylanases was based on the protein content in the xylanase mixture (43 g/mL)
and equalled 0.006 g xylanase protein/g WIS, which was equivalent to 20% of
the protein content in the cellulase added.
A spent sulphite liquor-adapted strain of S. cerevisiae, (Tembec I) provided
by Tembec Ltd. (Témiscaming, QU, Canada), was used at a concentration of
1 g/L (dry yeast). This yeast ferments glucose but not xylose. The yeast was
purified and then cultivated on the liquid obtained after pretreatment of corn
stover to adapt it to the conditions used in SSF (see Section 2.5 below).
Nutrients were added in the SSF experiments so that the concentrations in
the fermentor were 0.5 g/L (NH4)2HPO4, 0.025 g/L MgSO4
7H2O and 1.0 g/L
yeast extract. The temperature in the fermenters was kept at 35 8C during SSF
and 45 8C during EH, and all experiments were run for 120 h with the pH being
maintained at 5.0 by manual addition of a 50% NaOH. Samples were withdrawn
after 0, 2, 4, 8, 24, 28, 32, 48, 72, 96 and 120 h, and analysed regarding ethanol,
sugars, acetic acid, lactic acid and sugar degradation products.
The dry matter of the liquid, DMliquid, and of the whole slurry, DMslurry,
were measured by drying a sample of the liquid fraction and of the slurry,
836 K. Öhgren et al. / Process Biochemistry 42 (2007) 834–839
respectively. The WIS content in the pretreated slurry was calculated accord-
ing to [20]:
WIS ¼ ðDMslurry ÿ ð1 ÿ DMslurry Þ  DMliquid Þ
2.5. Yeast cultivation
2.5.1. Inoculum culture
Pure yeast culture (50 mg) was added to a 300-mL Erlenmeyer flask, which
contained 100 mL sterile medium at a pH of 5.0. The medium composition was
as follows: 16.5 g/L glucose, 7.5 g/L (NH4)2SO4, 3.5 g/L KH2PO4, 0.75 g/L
MgSO4, 10 mL/L trace metal solution [21] and 5 g/L yeast extract. The culture
was incubated at 30 8C for 24 h.
2.5.2. Aerobic batch cultivation
In order to produce cell mass before the fed-batch phase, batch cultivation
on glucose, with a volume of 0.750 L, was carried out at 30 8C under sterile
conditions. A 2.5-L bioreactor (Applikon, Schiedam, The Netherlands) was
used for the batch and fed-batch cultivation stages. The medium contained the
following components: 30 g/L glucose, 23 g/L (NH4)2SO4, 11 g/L KH2PO4,
2.6 g/L MgSO4, 34 mL/L trace metal solution and 5 g/L yeast extract. The pH
was maintained at 5.0 by automatic addition of 10% NaOH. Cultivation was
started by adding 60 mL inoculum culture. The dissolved oxygen tension (DOT)
was measured with a DOT electrode and was not allowed to fall below 40%.
This was ensured by regulating the stirrer speed and air and oxygen gas flow.
2.5.3. Aerobic fed-batch cultivation
After 24 h of batch cultivation, feeding of the pretreatment liquid started.
The pretreatment liquid was enriched with 60 g glucose/L, as it contained a very
low concentration of hexoses, and pH-adjusted to 5.0 with 50% NaOH.
Glucose-enriched pretreatment liquid (1 L) was added over a period of 19 h
at a feed rate of 0.88 mL/min, and the pH was maintained at 5.0 by automatic
addition of 10% NaOH. The DOT was, again, not allowed to fall below 40%.
2.5.4. Cell harvest
When feeding had ceased, the cultivation liquid containing the yeast was
transferred from the fermenter to centrifugation vessels and centrifuged at
3500 rpm for 5 min (Multifuge 3 L-R (HWS 6027), Heraeus, Hanau, Germany).
The supernatant was discarded and the amount of pellet corresponding to 1 g/L
dry yeast was transferred to a beaker. Tap water was added to 200 mL,
whereupon the cell suspension was added to the SSF. The time lapse between
the end of the fed-batch phase and the addition of the harvested cells to the SSF
fermentation phase was less than 1 h.
2.6. Analysis
The sugar and the lignin contents in the raw materials were determined
using concentrated acid hydrolysis at room temperature, followed by dilute acid
hydrolysis at 121 8C to hydrolyse the cellulose and hemicellulose, according to
the analytical procedure recommended by NREL [19]. The sugars were then
analysed with HPLC using a Dionex DX-2500 with an ion-exchange (PA1,
Dionex) column and a pulsed amperometric detector with a gold electrode. The
remaining acid-insoluble lignin was measured by weighing, and the amount of
acid-soluble lignin was determined from the adsorption at 205 nm against a 4%
H2SO4 blank.
The amounts of monosaccharides and inhibitors in the liquid fraction after
pretreatment were determined by HPLC. Glucose, xylose, galactose and
mannose, lactic acid, glycerol, acetic acid, ethanol, HMF and furfural were
separated on an Aminex HPX-87-H column at 65 8C using 5 mmol/L H2SO4 as
eluent at a flow rate of 0.6 mL/min. All samples were filtered through a 0.20-mm
filter and diluted properly analysis. To measure the total amount of sugars
(monomers and oligomers) in the liquid fraction after pretreatment, the samples
were hydrolysed with 4% H2SO4 at 121 8C for 1 h. They were then analysed
with HPLC as described above.
The FPU activity of the cellulase mixture used was determined according to
Adney and Baker [22] and the protein content of the xylanase mixture was
determined using a BCS Protein Assay (Pierce, Rockford, IL, USA).
3. Results and discussion
3.1. Raw material and steam pretreatment
The composition of the raw material is presented in Table 1. It
was found to be within the normal range [23–27], although,
the normal range is quite wide since the content of corn stover
tends to differ depending on region, year, crop maturity, etc.
(Thomas S. Golden, Colorado, USA: National Bioenergy
Center, National Renewable Energy Laboratory; 2003, personal
communication).
Table 2 summarizes the composition of the pretreated slurry.
The sugar concentration in the liquid fraction includes both
oligomeric and monomeric sugars.
The recovery of sugars in the pretreatment stage (in both the
solid and liquid fractions) was 92.6% for glucose and 73.0% for
xylose.
3.2. Simultaneous saccharification and fermentation
When SSF was performed using the whole slurry the ethanol
productivity during the first 24 h was 0.55 g/L
h. After 120 h,
20.5 g/L ethanol had been produced, which corresponds to an
overall ethanol yield of 72.4% based on the theoretical ethanol
production from glucose (Fig. 2(a)). In order to evaluate the
effect of the inhibitors present in the liquid fraction of the slurry,
the pretreated material was washed thoroughly with tap water,
after which the WIS was adjusted with water and additional
sugars were added in order to compensate for the sugars in the
liquid fraction that had been lost during washing. Washing the
material and then adding sugars is not a feasible process
alternative, but was done in this study to evaluate the inhibitory
effect in SSF.
The concentration of acetic acid was much lower in SSF
with washed material (Fig. 2(b)) since the acetic acid liberated
when hemicellulose is hydrolysed during pretreatment is
removed during washing. A minor amount of hemicellulose
was still present in the solid fraction after pretreatment
(Table 2). When this hemicellulose is hydrolysed in the
enzymatic hydrolysis, the concentration of acetic acid increases
slightly. The difference in sugar concentration between washed
and non-washed slurry at 0 h is due to uncertainties in the
analysis procedure as the high viscosity which makes it difficult
to take a representative sample. After 4 h, the viscosity had
decreased significantly and the samples taken then and
thereafter were considered to be representative. The ethanol
Table 1
Composition of corn stover expressed as % of dry matter
Glucan 36.1
Xylan 21.4
Arabinan 3.5
Galactan 2.5
Mannan 1.8
Lignina 17.2
Ash 7.1
Acetyl 3.2
a
Acid-soluble lignin and lignin ash included.
 K. Öhgren et al. / Process Biochemistry 42 (2007) 834–839
Table 2
The concentration of sugars, degradation products, DM and WIS in pretreated
material (solid and liquid)
DMslurry (%)
WIS in slurry (%)
Content in WIS (% of dry weight)
Glucan
Xylan
Lignin
Concentration in the liquid fraction (g/L)
Glucose
Xylose
Acetic acid
HMF
Furfural
a
Includes both acid-soluble and acid-insoluble lignin and lignin ash.
productivity during the first 24 h of SSF with washed slurry was
0.62 g/L
h, and the ethanol concentration after 120 h reached
18.2 g/L, corresponding to an overall ethanol yield of 64.1%
(Fig. 2(b)). The higher overall ethanol yield when non-washed
slurry is presumably due to the fact that inhibitors present in the
pretreatment liquid can increase the ethanol production if they
are not present at too high concentrations. At low concentra-
tions, up to 100 mmol/L, acetic acid, for example, has been
shown to increase the ethanol yield [18]. Removing the
inhibitors can thus decrease the ethanol yield. The slight
increase in ethanol productivity during the first 24 h with
washed solids can be explained by the lower concentration of
fermentable inhibitors such as furfural and HMF. These
inhibitors are metabolized by the yeast, leading to a lag phase in
ethanol production [18]. The glucose concentration decreased
to zero much faster when the washed solids was used in SSF
than when the whole slurry was used (Fig. 2(a) and (b)) since
the yeast does not need to metabolize any inhibitors in the
washed slurry.
3.3. Enzymatic hydrolysis
The effect of inhibitors on EH was evaluated using the same
kind of study as described above for SSF. EH was performed on
the whole slurry as well as on washed slurry with additional
sugars added to compensate for the soluble sugar loss in the
washing procedure. EH of the whole slurry resulted in a glucose
Fig. 2. Ethanol, glucose and acetic acid concentrations during SSF of whole slurry (a) and washed slurry with additional sugars (b), both at 8% WIS.
837
productivity during the first 24 h of 0.95 g/L h and a final
glucose concentration of 36.7 g/L, which corresponds to 65.9%
overall glucose yield (Fig. 3(a)). In EH of washed slurry the
16.7 values were a glucose productivity of 1.46 g/L h and a final
10.6
glucose concentration of 42.4 g/L, which corresponds to 78.5%
overall glucose yield (Fig. 3(b)). Removing the inhibitors seems
52.7 thus to have a positive effect on enzymatic hydrolysis. The
8.8
27.7a
inhibitory effect of steam pretreatment hydrolysate on
enzymatic hydrolysis observed here is in accordance with
earlier studies on steam-pretreated spruce [17] and on steam-
6.6
24.8 pretreated poplar [16].
2.6 The enzymes used in enzymatic hydrolysis are not only
0.6 inhibited by the degradation products present in the steam-
0.7 pretreatment hydrolysate. They are also end-product inhib-
ited. This means that the very product that they produce
inhibits them due to a competitive inhibiting effect.
Consequently, endo- and exoglucanases are inhibited by
cellobiose, and b-glucosidase is inhibited by glucose. The
inhibiting effect of the sugars present in the pretreatment
hydrolysate was investigated by not replacing the sugars
removed in the washing process. EH using washed material
with no addition of additional sugars was performed with the
same WIS content (8%) as in the other experiments resulting
in the same glucose productivity during the first 24 h (1.41 g/
L h, data not shown) as for the EH with washed material and
the addition of additional sugars. It was thus concluded that
the inhibitory effect of the pretreatment hydrolysate on the EH
could be attributed to sugar degradation products and other by-
products produced during the steam pretreatment and not the
sugars present in the liquid fraction. However, the fairly high
sugar concentration at the end of the EH probably caused some
end-product inhibition.
3.4. SSF versus SHF
SSF was compared with SHF for three types of slurry: whole
slurry, washed slurry with additional sugars and whole slurry
with additional xylanases for improved enzymatic hydrolysis.
To be able to compare SHF with SSF, the SHF results were
based on the EH results assuming 90% fermentation yield in a
24 h long separate fermentation (0.46 g ethanol per gram
glucose). SHF will thus take 144 h since the EH takes 120 h and
the fermentation is assumed to take 24 h. Table 3 summarizes
the results of the comparison between the SSF and SHF.
838 K. Öhgren et al. / Process Biochemistry 42 (2007) 834–839

Fig. 3. Glucose and acetic acid concentrations during SHF of whole slurry (a) and washed slurry with additional sugars (b), both at 8% WIS.

Table 3 In EH of whole slurry with additional xylanases, the glucose

Comparison between ethanol yield from SSF (after 120 h) and SHF (after 120 h productivity during the first 24 h was 1.18 g/L h. This is higher
hydrolysis and 24 h fermentation)
than the glucose productivity for the whole slurry but lower than
Ethanol yield Overall ethanol Ethanol concentration for washed slurry. Since adding xylanases to the whole slurry
in SSF/SHF (%) yield (%) after 120/144 h (g/L)
can boost the ethanol productivity in SSF compared with
Whole slurry washed slurry but not in EH, fermentation clearly has an effect
SSF 78.2 72.4 20.5 on the inhibition of the enzymes. The fermentation of
SHF 64.1 59.3 16.8
pretreatment hydrolysate to overcome inhibition in enzymatic
Washed slurry with additional sugar hydrolysis has been demonstrated previously by Tengborg et al.
SSF 69.3 64.1 18.2 [17] using spruce. The glucose concentration after 120 h in the
SHF 76.2 70.6 19.4
EH of whole slurry with additional xylanases was 39.1 g/L
Whole slurry with additional xylanases (Fig. 4(b)), corresponding to an overall glucose yield of 70.2%,
SSF 81.5 75.4 21.4
which is lower than the yield for the washed slurry with
SHF 68.2 63.2 17.9
additional sugars.
The fermentation yield in the separate fermentation was assumed to be 90% SSF was shown to be the preferable process configuration
(0.46 g ethanol/g glucose).
when the whole slurry was used (Table 3), which is in
accordance with the results found by Söderström et al. [13] for
The results for the whole slurry and the washed slurry with softwood. This is due to three factors: (1) lower glucose
additional sugars are based on the results presented above for concentration and thus decreased glucose inhibition of the
SSF and EH. In SSF of whole slurry with additional xylanases, enzymes; (2) less inhibition of the enzymes by degradation
the ethanol productivity during the first 24 h was 0.69 g/L h, products present in the pretreatment hydrolysate since
which was slightly higher than those for the whole slurry and fermentation seems to afford some detoxification; (3) the
the washed slurry with standard enzyme addition. The positive effect of low concentrations of some of the inhibitors
increased enzymatic hydrolysis rate is also evidenced by the on the fermentation yield. When washed slurry was used, the
fact that the glucose concentration did not decrease to zero until ethanol yield in SHF was higher than in SSF (Table 3), probably
after 72 h (Fig. 4(a)). The ethanol concentration after 120 h was since the enzymes in the SHF were allowed to work at optimal
21.4 g/L (Table 3), corresponding to an overall ethanol yield of temperature. Alfani et al. [28] also found that washed steam-
75.4%. The difference in sugar concentration between SSF and pretreated wheat straw gave a higher ethanol yield when SHF
EH at 0 h is again due to the high viscosity, which makes it was used rather than SSF. Adding a washing step in the ethanol
difficult to take a representative sample. production process would, however, increase the production

Fig. 4. Glucose, acetic acid and ethanol concentrations during SSF (a) and SHF (b) of whole slurry with additional xylanases, both at 8% WIS.
 K. Öhgren et al. / Process Biochemistry 42 (2007) 834–839
cost and cause a considerable loss of sugars, which cannot be
replaced in a large-scale process. Another way of decreasing
the concentration of inhibitors before hydrolysis is to add a
detoxification step [29]. However, this may constitute up to
22% of the total ethanol production cost [30] and is therefore
best avoided.
Adding extra xylanases to the process increases the ethanol
yield slightly for both SSF and SHF of the whole slurry
(Table 3). The beneficial effect of xylanases in enzymatic
hydrolysis has been established previously [20]. However, the
increase in ethanol yield due to the addition of xylanase must be
weighted against the cost.
4. Conclusions
The inhibitors present in the liquid fraction of the pretreated
slurry had a significant and negative impact on enzymatic
hydrolysis. This negative impact was decreased when SSF was
used. Due to the reduction of glucose inhibition in the
enzymatic hydrolysis during SSF, the detoxifying effect of
fermentation and the positive effect of inhibitors present in the
pretreatment hydrolysate (for example acetic acid) on the
fermentation, SSF was concluded to be a better process
configuration than SHF when the whole slurry was used.
Adding extra xylanases had a slightly positive effect on the
ethanol yield in both SSF and SHF of the whole slurry.
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