Enzyme and Microbial Technology 37 (2005) 195204

A comparison between batch and fed-batch simultaneous

saccharication and fermentation of steam pretreated spruce
Andreas Rudolf

, Malek Alkasrawi, Guido Zacchi, Gunnar Lid en

Department of Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden
Received 20 September 2004; received in revised form 10 January 2005; accepted 10 February 2005
Abstract
In order to improve the process economy it is important to use as high dry matter content as possible in simultaneous saccharication and
fermentation (SSF). However, too high dry matter content often gives rise to severe inhibition of the yeast metabolism, due to the increased
levels of toxic compounds. The aimof the present work was to increase the brous content in SSFof steampretreated spruce to 10%by adapting
the yeast to the inhibitory substrate and by using a fed-batch process. Both batch and fed-batch approaches were evaluated. The fed-batch
experiments were started with a batch fermentation containing 6% dry matter. Fibrous slurry from the pretreatment was then added four times
during the rst 24 h giving a nal dry matter content corresponding to 10%. The yeast used in the fermentation was produced aerobically on
the hemicellulose hydrolysate obtained from the pretreatment. SSF batch and fed-batch experiments with a cell mass concentration of 2, 3
and 5 g/L were carried out. When adapted yeast was used, the available hexoses were completely converted within 72 h and the nal ethanol
concentrations reached 4044 g/L. No major differences in performance between batch and fed-batch were seen, but the ethanol productivity
during the rst 24 h was higher in the fed-batch SSF experiments, particularly during the experiments with a cell mass concentration of 2 and
3 g/L.
2005 Elsevier Inc. All rights reserved.
Keywords: Ethanol; Yeast; Fermentation; Enzymatic hydrolysis; Spruce
1. Introduction
Reducing the production costs of ethanol produced from
lignocellulose material is crucial in enabling its commercial-
ization. One important factor for the production cost is the
ethanol concentration in the liquid left after the saccharica-
tion and fermentation. This should be as high as possible [1]
in order to minimize the energy costs of evaporation and dis-
tillation. By recirculating some of the liquid fromthe fermen-
tation back into the fermentor vessel, the nal ethanol con-
centration can be increased. However, compounds inhibitory
to the yeast cells and enzymes, formed during the pretreat-
ment of lignocellulose material [214], are thereby further
concentrated, making a high degree of recirculation difcult
[15,16]. Raising the dry matter content is another obvious
way to reach a higher nal ethanol concentration. However,

Corresponding author. Tel.: +46 46 222 82 50; fax: +46 46 14 91 56.

E-mail address: andreas.Rudolf@chemeng.lth.se (A. Rudolf).
the concentrations of compounds inhibitory to yeast and en-
zymes are simultaneously increased. In addition, the rheolog-
ical properties of a very dense brous suspension may cause
mixing and heat transfer problems.
Previously, when using compressed Bakers yeast in Batch
SSF with a dry matter content of 8% it proved difcult to
achieve high ethanol yields (Alkasrawi et al., accepted for
publication). The high concentrations of inhibitors in fermen-
tations with a high dry matter content might be overcome by
applying a fed-batch technique [17]. By adding the slurry to
the fermentor vessel continuously or by scheduled additions,
the inherent abilityof the yeast todetoxifythe substrate canbe
maintained. This approach has proved successful in fermen-
tations of dilute-acid hydrolysates from softwood [18,19].
Furthermore, since added bers are gradually degraded, the
initial viscosity can be kept lower than in a batch process.
Provided that it is possible to reduce the inhibition by ap-
plying a fed-batch it could be possible to decrease the amount
of yeast used in the SSF. Cultivation of yeast will consume
0141-0229/$ see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.enzmictec.2005.02.013
196 A. Rudolf et al. / Enzyme and Microbial Technology 37 (2005) 195204
substrate, which could otherwise be used for ethanol produc-
tion thereby decreasing the over-all ethanol yield. In order to
minimize the yeast concentration in the SSF, it is important
to have as robust yeast as possible. It has been shown that
the yeast can be adapted to the inhibiting medium prior to
the fermentations by cultivating the yeast on liquid from the
pretreatment (Alkasrawi et al., accepted for publication).
The aim of the present work was to enable conversion of
10% dry matter content (the dry matter content only includes
the water insoluble solids) in simultaneous saccharication
and fermentations of steam pretreated spruce by using yeast
cultivated on the liquid from the pretreatment and by using a
fed-batch process. During the fed-batch SSF, the ber slurry,
obtained from the pretreatment, was added ve times to the
fermentor vessel during the rst 25 h. The time between the
additions was varied in order to see how it affected fermen-
tation performance. Batch and fed-batch experiments with
different cell mass concentrations were carried out in order
to study if it was possible to decrease the amount of yeast
added.
The results showed that it was possible to obtain conver-
sion of all available hexose sugars in the fermentation liquid
within 72 h in SSF using 10% dry matter. Ethanol yields of
7984% of the theoretical yield (based on glucan, glucose,
mannose and galactose present in the added substrate) were
achieved.
2. Material and methods
2.1. Raw material
Fresh, chipped spruce was kindly provided by a sawmill
in southern Sweden (Harry Nilssons S agverk, H astveda,
Sk ane). The wood was chipped and sieved to obtain a chip
size of 210 mm. The chips were stored in a plastic bag at
4

C prior to use. The composition was analyzed using the

H agglund method [20] followed by HLPC-analysis.
2.2. Pretreatment
The wood chips were impregnated with SO
2
(3%, w/w
moisture) for 20 min at room temperature. The impregnation
was performed in tightly closed plastic bags to enable pene-
tration of the gas into the wood structure. The amount of SO
2
absorbed was determined by weighing the plastic bags before
and after impregnation. The impregnated material was steam-
pretreated at 215

C for 5 min in a steam-pretreatment unit

equipped with a 10 L-reactor, which has been described pre-
viously [21]. In total about 3.6 kg of impregnated chips were
pretreated. Due to the limited size of the reactor, 600 g was
pretreated at a time. The slurries obtained from the pretreat-
ment runs were mixed together into a single batch and stored
at 4

C for future use. The composition of the dry wood and

the dry bers in the pretreatment slurry was determined by
the H agglund method [20]. The pretreatment slurry consists
Table 1
Composition of the dry wood raw material and the dry washed bers from
the two batches of pretreatment slurry
Components Wood (wt.%) Washed pretreated
material (wt.%)
Batch 1 Batch 2
Glucan 43 48 48
Mannan 12 0 0
Xylan 5.4 0 0
Galactan 2.3 0 0
Lignin 27 47 45
The dry matter content of the wood raw material was 56%.
of a water insoluble part mainly glucan and lignin (Table 1) as
well as a liquid fraction containing the sugars released from
the hydrolysis of the hemicellulose as well as acetic acid and
sugar degradation products (Table 2). The water insoluble
solids content of both pretreatment batches was 11.5%.
2.3. Cell cultivation
Since the more cell mass had to be produced during some
of the initial cultivations, two different reactor set-ups were
used for the cell cultivations. For the rst three, SSF-runs
(AC) the cells were cultivated in a 14 L-bioreactor (NLF
14, Bioengineering AS, Wald, Switzerland). The cell culti-
vations preceding the SSF-experiments DL were performed
in a 2.5 L-reactor (BiostatA, B. Braun Biotech International,
Melsungen, Germany). The nal working volume in the 14 L-
reactor was 4.3 and 2.0 L in the 2.5 L-reactor. However, all
other parameters such as nutrient concentrations, ratio be-
tween added pretreatment liquid and batch volume, relative
feed rate increase were identical in the two cultivation set-
ups. Thus, the cells produced in the two different reactors
should be equally resistant to the harsh conditions in the
SSF-experiments. In the description of the cultivation proce-
dure, the cultivations made in the 2.5 L-reactor will be written
within brackets.
2.3.1. Inoculum cultures
Fiftymilligrams of pure Bakers yeast culture (J astbolaget,
Rotebro, Sweden) from an agar plate was added to a 300 mL
Erlenmeyer-ask, which contained 100 mLof sterile medium
with a pH of 5.5. The medium composition was as follows:
glucose, 16.6 g/L; (NH
4
)
2
SO
4
, 7.5 g/L; KH
2
PO
4
, 3.5 g/L;
Table 2
Composition of the liquid from the two pretreatment batches
Components Concentration (g/L)
Batch 1 Batch 2
Glucose 23.4 22.1
Mannose 19.4 18.5
Xylose 8.9 8.0
Galactose 4.0 4.6
Furfural 1.7 1.6
HMF 3.0 2.4
Acetic acid 3.7 3.4
A. Rudolf et al. / Enzyme and Microbial Technology 37 (2005) 195204 197
MgSO
4
, 0.75 g/L; trace metal solution, 10 mL/L and vita-
min solution, 1mL/L. The trace metal and vitamin solutions
were prepared according to Taherzadeh et al. [22]. The cul-
ture was incubated at 30

C for 24 h. The Erlenmeyer-ask

was closed with a cotton plug.
2.3.2. Aerobic batch cultivation
In order to produce cell mass before the fed-batch cul-
tivation phase, batch cultivation, with a working volume of
2.5 L [1.2 L],
1
was carried out at 30

C under sterile condi-

tions. The mediumcontained the following components: glu-
cose, 17 g/L; (NH
4
)
2
SO
4
, 20 g/L; KH
2
PO
4
, 9.4 g/L; MgSO
4
,
2.0 g/L; trace metal solution, 26 mL/L and vitamin solution,
3 mL/L. The pH was maintained at 5.0 by automatic addition
of 2 M NaOH. The stirrer speed was kept at 700 rpm and the
aeration was maintained at 2.5 L/min [1.3 L/min]
1
. The cul-
tivation was started by adding 80 mL [40 mL]
1
of inocolum.
2.3.3. Aerobic fed-batch cultivation
Subsequent todepletionof the ethanol producedduringthe
batch phase, feeding of the pretreatment liquid was started.
The liquid was enriched with 28 g glucose/L. 1.8 L [0.85 L]
1
of glucose enriched pretreatment liquid was added during
16 h. The feed rate was initially set to 0.07 L/h [0.035 L/h]
1
and was increased linearly to 0.15 L/h [0.07 L/h]
1
after 16 h.
The pH was maintained at 5.0 by automatic addition of 2 M
NaOH. The stirrer speed was kept at 700 rpmand the aeration
was maintained at 2.5 L/min [1.3 L/min]
1
.
2.3.4. Cell harvest
The cultivation liquid was centrifuged (1000 g) in
700 mL containers using a HERMLE Z 513 K centrifuge
(HERMLE Labortechnik, Wehingen, Germany). The pellets
were resuspended in 0.9% NaCl-solution in order to obtain
a cell suspension with a cell mass concentration of about
75 g dw/L. The time lapse between the end of the fed-batch
phase and the addition of the harvested cells to the SSF-
fermentations was always kept below 3 h.
2.4. SSF-experiments
2.4.1. Fed-batch
SSF-experiments with a nal working weight of 1.6 kg
(ca. 1.5 L) were performed in a 2.5 L-reactor (Biostat A,
B. Braun Biotech International, Melsungen, Germany). The
pretreatment slurry was diluted with fresh water in order to
get an initial dry matter content of 6%. The reactor contain-
ing the diluted slurry was autoclaved at 120

C for 20 min.
The fermentation medium was supplemented with nutrients:
NH
4
H
2
PO
4
, 0.87 g/L; MgSO
4
7H
2
O, 0.025 g/Landyeast ex-
tract, 1.0 g/L (based on nal volume). The experiments were
started by adding cell suspension, giving a nal cell concen-
tration, after all slurry additions, of 2, 3 or 5 g/L (Table 3).
1
Refers to the cultivations performed in the 2.5 L-reactor.
Table 3
The complete experimental series
SSF-index Pretreatment
batch
Time between
slurry
additions (h)
N
2
-ushing Cell mass
concentration
(g/L)
A 1 Batch Yes 5
B 1 1.5 Yes 5
C 1 3 Yes 5
D 1 5 Yes 5
E 2 Batch Yes 5
F 2 1.5 Yes 5
G 2 Batch Yes 3
H 2 1.5 Yes 3
I 2 Batch Yes 2
J 2 1.5 Yes 2
K 2 Batch No 5
L 2 1.5 No 5
The enzyme used preparations were Novozyme 188 with a
-glucosidase activity of 362 IU/g and Celluclast 1.5 L with
a cellulase activity of 75 FPU/g and a -glucosidase activity
of 12 IU/g. The enzyme preparations were kindly provided
by Novozymes A/S (Bagsvaerd, Denmark). The amount of
Novozyme 188 added corresponded to 4% (w/w) on the dry
matter, giving a total -glucosidase activity of 36.2 IU/g glu-
can (the combined activity of Celluclast 1.5 L and Novozyme
188) and the amount of Celluclast 1.5 L added corresponded
to 24% (w/w) on the dry matter (a cellulase activity of
37.5 FPU/g of glucan). After the initial batch phase with 6%
dry matter, pretreatment slurry, which had been pH-adjusted
to 4.9 with 6 M NaOH, was added four times giving a total
dry matter content of 10%. Experiments with a time lapse
between the slurry additions of 1.5, 3 and 5 h were carried
out (Table 3). The fermentation pH was maintained at 5.0 by
the addition of 3 M NaOH. All SSF experiments were per-
formed at 37

C for 72 h. The CO
2
evolution rate (CER) was
monitored by ushing N
2
through the reactor and analyzing
the CO
2
amount in the exhaust gas with a gas analyzer (Tan-
Dem, Adaptive Biosystems, Luton, United Kingdom). The
N
2
-ow was 400 mL/min during the rst 24 h and was then
decreased to 200 mL/min.
2.4.2. Batch
Batch experiments with a working weight of 1.6 kg (ca.
1.5 L) anda drymatter content of 10%were alsorun(Table 3).
For the batch fermentations a 2.5 L-bioreactor was used
(BIOFLO III, New Brunswick Scientic, Edison, NJ, USA).
The fermentation conditions, nutrient concentrations and en-
zyme loadings were the same as in the fed-batch experiments.
2.5. Fermentation of sugar-containing solutions without
bers
Fermentations with pulse addition of pure glucose solu-
tions or pretreatment liquid were carried out in order to exam-
ine what caused the decrease in CER response to the slurry
additions during the SSF fed-batch experiments. The glucose
198 A. Rudolf et al. / Enzyme and Microbial Technology 37 (2005) 195204
solution used had a concentration of 36 g/L, which equalled
the sum of glucose and mannose in the pretreatment liquid.
Glucose solution or pretreatment liquid were added every 3 h
during the rst 12 h. After 24 h glucose solution (36 g/L) was
added a last time in both experiments. During the experi-
ments, a glucose solution (188 g/L) was continuously fed at
9 mL/h in order to simulate the slowrelease of glucose during
a SSF experiment. The nal volume was 1.5 L and the cell
mass concentration 5 g/L. The yeast was cultivated exactly in
the same way as prior to the SSF experiments.
2.6. Analysis
2.6.1. Cell mass concentration
The cell mass concentration during the aerobic cell culti-
vation was measured by taking duplicate 10 mLsamples. The
samples were centrifuged(1000 g) for 4 minat 3000 rpm(Z
200 A, HERMLE Labortechnik, Wehingen, Germany). The
supernatants were discarded and the pellets were washed with
deionised water and recentrifuged. The pellets were dried at
105

C for 24 h and subsequently weighed.

2.6.2. Cell viability
Samples for cell viabilitytests were takenve times during
the batch and fed-batch experiments (EJ). One milliliter of
fermentation liquid was diluted 10
4
10
5
times and 75 L of
diluted sample was applied to an agar plate. Five to six plates
were prepared for every sample. The plates were incubated
at 30

C for 24 h. The cell viability was expressed as the total

number of colony forming units.
2.6.3. HPLC-analysis
Samples of the fermentation liquid were centrifuged
(16,000 g) in 2 mL eppendorf tubes at 14,000 rpm for
5 min. (Z 160 M, HERMLE Labortechnik, Wehingen, Ger-
many). The supernatant was ltered with 0.2 m sterile l-
ters and the ltered samples were stored at 20

C. Analysis
of the most common metabolites and substrates was made
using HPLC. The concentrations of glucose, mannose xy-
lose and galactose were determined using a polymer column
(Aminex HPX-87P, Bio-Rad Laboratories, M unchen, Ger-
many) at 85

C. The eluent used was MilliQ-water with a

ow rate of 0.6 mL/min. Ethanol, glycerol, acetate, lactate,
HMF and furfural were analyzed using an Aminex HPX-
87H column (Bio-Rad Laboratories, M unchen, Germany) at
60

C. The eluent used was 5 mM H

2
SO
4
with a ow rate of
0.6 mL/min. The compounds of interest were detected with a
refractive index detector (Waters 2410, Waters, Milford, MA,
USA).
2.6.4. Yield calculations
The ethanol yields, Y
SE
, were calculated based on the total
amount of fermentable sugars added to the fermentations, i.e.
the sum of glucose, mannose and galactose present in the
liquid part of the added pretreatment slurry plus the glucose
in the form of glucan.
The theoretical amounts of glucose released during the
hydrolysis are 1.11 times the amount of glucan (due to the
addition of water when the glycosidic bonds are hydrolysed).
The relative ethanol yield, Y

SE
, i.e. the actual ethanol yield,
Y
SE
, divided by the theoretical ethanol yield is described by
Eq. (1).
Y

SE
=
Y
SE
0.51
(%) (1)
2.6.5. Estimation of ethanol evaporation
N
2
-ushing will strip substantial amounts of the produced
ethanol, even if the exhaust gas passes a condenser. In the
present work, the rate of ethanol evaporation was studied in
experiments performed with the fermentors used for the SSF-
experiments. After a SSF-experiment, the residual cells were
killed by a short elevation of temperature (55

C for 20 min).
Additional ethanol was added in order to get an initial ethanol
concentration of about 30 g/L. The decrease in ethanol con-
centration was then measured during 48 h. The experiments
were run with a N
2
-ow of 400 or 200 mL/min. A relation-
ship between the molar ethanol fraction in the fermentation
liquid and the molar ethanol fraction in the exhaust gas was
estimated based on the ethanol evaporation data for the two
N
2
-ows (Eq. (2)):
y = 0.57x (2)
where y is the mole fraction of ethanol in exhaust gas and x
is the mole fraction of ethanol in the fermentation liquid.
3. Results
3.1. Cell cultivation
The cell yield on glucose was about 0.35 g/g for the aero-
bic batch growth (after consumption of ethanol formed) and
about 0.48 g/g for aerobic fed-batch growth. The lower yields
during the batch phases are due to the well-known less ef-
cient diauxic growth on glucose and subsequently on the
ethanol formed as a result of over-ow metabolism [23]. Ad-
ditionally, ethanol evaporation will lower the yield further.
The nal cell mass concentration after the fed-batch phase
reached 1617 g/L.
3.2. SSF-experiments
Batch and fed-batch fermentations with a dry matter con-
tent of 10% were carried out. In the experiments AD, the
inuence of the feed prole on the fermentation performance
and process dynamics was studied. In experiments EJ, the
effect of cell mass concentration on the fermentation perfor-
mance in batch and fed-batch SSF were evaluated. Addition-
ally, one fed-batch (experiment L) and one batch-experiment
(experiment K) were run without N
2
-ushing in order to ex-
clude that the ushing affected the fermentation performance,
A. Rudolf et al. / Enzyme and Microbial Technology 37 (2005) 195204 199
Table 4
Final ethanol concentrations and ethanol yields on available fermentable
sugars
Experiment Final ethanol
concentration (g/L)
Y
SE
(72 h) (g/g) Y

SE
(72 h) % of
theoretical yield
A 42.6 0.42 82
B 44.0 0.43 84
C 40.0 0.40 78
D 43.5 0.43 84
E 44.5 0.43 84
F 44.5 0.43 84
G 41.9 0.42 82
H 43.7 0.43 84
I 41.6 0.41 80
J 41.0 0.41 80
K 42.2 0.43 84
L 42.2 0.43 84
The nal ethanol concentrations have been compensated for evaporation
(except for experiments K and L).
and also to conrm the estimated ethanol evaporation in ex-
periments AJ. The model used to estimate ethanol evapo-
ration gave nal ethanol yields close to the expected values
(Table 4).
Over-all, the fermentation performance was satisfying.
The nal ethanol concentrations were above 40 g/L (after
compensation for ethanol evaporation) in all experiments and
the ethanol yields, Y
SE
, on the total amount of fermentable
sugars were above 0.40 g/g in all experiments (Table 4). The
nal ethanol yields did not differ signicantly between batch
and fed-batch. The cell mass concentration could be de-
creased to 2 g/L without any signicant deterioration in nal
ethanol yield.
The responses in CER subsequent to the slurry additions
were fast and sharp (Figs. 1 and 2). However, the responses
decreased with time, particularly, when the time between the
slurry additions was 3 or 5 h (Fig. 1). At 24 h, 3 g of glucose
was added to the fermentation medium (marked by arrows
in Figs. 1 and 2) in order to examine the fermentative ca-
pacity of the yeast cells after extended time in the inhibiting
medium. The responses in CER to the pulse additions of glu-
cose strongly suggest that the enzymatic hydrolysis is the
rate limiting process. However, the responses were small in
experiments A and G (Figs. 1 and 2).
The glucose and mannose concentrations during the fed-
batch fermentations were in general below 5 g/L and did not
exceed 1.5 g/L after 72 h (Figs. 35). The measured hex-
ose concentrations during the rst 24 h were higher in the
batchfermentations comparedtothe fed-batchfermentations.
These differences in hexose concentration between batch and
fed-batch were more signicant in the experiments with a cell
mass concentration of 2 and 3 g/L (Fig. 4, experiments GJ).
The total number of colony forming units (CFU) was mea-
sured during some of the fermentations (Table 5). The CFU
decrease during the fermentations suggests that the yeast cells
suffered frominhibition and/or starvation. It is thus likely that
the sugar consumption for cell growth was very low. The CFU
decreased somewhat slower during the fed-batch fermenta-
Fig. 1. CER during SSF-experiments with a dry matter content of 10%. Steam-pretreated spruce (batch 1) was used as substrate. The yeast was cultivated on
the liquid from the pretreatment. The cell mass concentration was 5 g/L. Pulse additions of 3 g of glucose are marked with arrows. (A) SSF batch, (B) SSF
fed-batch with substrate additions every 1.5 h, (C) SSF fed-batch with substrate additions every 3 h and (D) SSF fed-batch with substrate additions every 5 h.
200 A. Rudolf et al. / Enzyme and Microbial Technology 37 (2005) 195204
Fig. 2. CER during SSF-experiments with a dry matter content of 10%. Steam-pretreated spruce (batch 2) was used as substrate. The yeast was cultivated on
the liquid from the pretreatment. The time lapse between the substrate additions during the fed-batch experiments was 1.5 h. Pulse additions of 3 g of glucose
are marked with arrows. (E) SSF batch with a cell mass concentration of 5 g/L, (F) SSF fed-batch with a cell mass concentration of 5 g/L, (G) SSF batch with a
cell mass concentration of 3 g/L, (H) SSF fed-batch with a cell mass concentration of 3 g/L, (I) SSF batch with a cell mass concentration of 2 g/L and (J) SSF
fed-batch with a cell mass concentration of 2 g/L.
tions, suggesting that the pulse wise addition decreased the
inhibition and cell death rate.
The extent of yeast inhibition was also studied by perform-
ing fermentations with pulse addition (every 3 h) of glucose
solution (36 g/L) and liquid from the pretreatment. When a
pure glucose solution was fed the responses in CER did not
decrease signicantly during 24 h (Fig. 6), suggesting that
neither the elevated temperature (37

C) nor the periods of

Table 5
Total number of colony forming units (CFU) in experiments EJ
Experiment 1 h (CFU10
9
) 7 h (CFU10
9
) 24 h (CFU10
9
) 48 h (CFU10
9
) 72 h (CFU10
9
)
E 124 85 73 34 31
F 173 117 91 79 55
G 94 59 44 12 6
H 107 133 110 45 37
I 74 52 41 17 8
J 86 70 50 22 13
The degree of error is 10%.
A. Rudolf et al. / Enzyme and Microbial Technology 37 (2005) 195204 201
Fig. 3. Ethanol (), glucose () and mannose () concentrations during SSF-experiments with a dry matter content of 10%. Steam-pretreated spruce (batch 1)
was used as substrate. The yeast was cultivated on the liquid from the pretreatment. The cell mass concentration was 5 g/L. (A) SSF batch, (B) SSF fed-batch
with substrate additions every 1.5 h, (C) SSF fed-batch with substrate additions every 3 h and (D) SSF fed-batch with substrate additions every 5 h.
carbon limitation had a signicant effect on cell viability.
However, in the experiment with pulse feeding of pretreat-
ment liquid, the responses in CERdecreased steadily (Fig. 6),
indicating cell inhibition most likely caused by certain com-
pounds formed during the pretreatment. The decrease in CER
response to the substrate additions was in agreement with
what has previously been observed during SSF-experiments
C and D.
4. Discussion
Increasing the brous content to 10% worked satisfactory
using the yeast cultivated on the pretreatment liquid. The
ethanol yields on available sugars were in all cases above
0.40 g/g and nal ethanol concentrations above 40 g/L were
attained (Table 4). To reach a high ethanol concentration was
one of the main targets of the present work since the nal
ethanol concentration in the fermentation liquid is critical
when trying to reduce ethanol production costs [1]. In shake
ask fermentations of pretreatment liquid or glucose using
the same yeast preparation and medium composition as in
the SSF-experiments ethanol yields on fermentable sugars
reached 0.450.46 g/g. The slightly lower ethanol yields in
the SSF-experiments could be due to the fact that a small frac-
tion of the glucan was not completely hydrolyzed within 72 h.
The small difference in fermentation performance be-
tween batch and fed-batch was somewhat surprising. In previ-
ous fermentation experiments using dilute-acid hydrolysate
as substrate, fed-batch fermentation proved to be superior to
batch fermentation [19,24]. However, the pretreatment prior
to the SSF is less severe than the hydrolysis used to produce
dilute-acid hydrolysates. Furthermore, the cell cultivation on
the pretreatment liquid probably adapted the yeast to the
inhibitory compounds present in the fermentation medium.
Adaptation by cultivating the yeast on the pretreatment liq-
uid have previously proved to be successful (Alkasrawi et al.,
accepted for publication).
Even when the cell mass concentration was decreased to
2 and 3 g/L the differences in over-all fermentation perfor-
mance between batch and fed-batch remained small. How-
ever, the ethanol productivity during the rst 24 h and es-
pecially during the rst 7 h was considerably higher during
the fed-batch experiments (H and J) compared to the batch
experiments (G and I). This suggests that the fed-batch ap-
proach actually reduces the cell inhibition, but since the rate
of hydrolysis during the entire SSFis lowcompared to the po-
tential sugar uptake by the cells, all hexoses are metabolized
even though the cells initially are severely inhibited and the
cell viability decreases throughout the fermentation. The fact
that the potential sugar uptake rate by the cells exceeds the
hydrolysis rate, suggests that there couldbe a potential for fur-
ther decreasing the amount of yeast added while still maintain
good fermentation performance. When using cell mass con-
centrations below2 g/L, a fed-batch approach is probably the
most feasible option. The glucose concentrations were lower
202 A. Rudolf et al. / Enzyme and Microbial Technology 37 (2005) 195204
Fig. 4. Ethanol (), glucose () and mannose () concentrations during SSF-experiments with a dry matter content of 10%. Steam-pretreated spruce (batch
2) was used as substrate. The yeast was cultivated on the liquid from the pretreatment. The time lapse between the substrate additions during the fed-batch
experiments was 1.5 h. (E) SSF batch with a cell mass concentration of 5 g/L, (F) SSF fed-batch with a cell mass concentration of 5 g/L, (G) SSF batch with a
cell mass concentration of 3 g/L, (H) SSF fed-batch with a cell mass concentration of 3 g/L, (I) SSF batch with a cell mass concentration of 2 g/L and (J) SSF
fed-batch with a cell mass concentration of 2 g/L.
Fig. 5. Ethanol (), glucose (-) and mannose () concentrations during SSF-experiments with a dry matter content of 10%. The SSF experiments were carried
out without N
2
-ushing. Steam-pretreated spruce (batch 2) was used as substrate. The yeast was cultivated on the liquid from the pretreatment. The cell mass
concentration was 5 g/L. (K) SSF batch and (L) SSF fed-batch with substrate additions every 1.5 h.
A. Rudolf et al. / Enzyme and Microbial Technology 37 (2005) 195204 203
Fig. 6. CER during pulse feeding experiments with glucose solution and
liquid from the pretreatment. The yeast was cultivated on the liquid from the
pretreatment. The cell mass concentration was 5 g/L, ( ) pulse feeding of
pretreatment liquid and ( ) pulse feeding of glucose solution (36 g/L).
during the rst 24 h in the fed-batch experiments compared
to the batch experiments. Thus, a fed-batch approach could
reduce the end-product inhibition of the cellulases. However,
since there were no major differences in ethanol yield be-
tween the two methods, end-product inhibition did not seem
to affect the hydrolysis rate considerably.
Inorder toincrease the ethanol productivityfurther, clearly
the rate of enzymatic hydrolysis has to be increased. Due to
the high prize of currently available commercial cellulase
preparations, addition of more enzymes is not an attractive
option. Alternatively, the rate of hydrolysis can be accelerated
by raising the temperature; an increase from 37 to 50

C can
result in a 29% increase in enzymatic activity [25]. At such
temperatures, however, SSF is not a feasible process, since
Saccharomyces cerevisiae cannot tolerate such high temper-
atures. The enzymatic hydrolysis would in such a case be
performed in a separate step, after which fermentation of the
sugar solution can take place (separate hydrolysis and fer-
mentation, SHF). The higher productivity is thus paid for
by an increased investment cost [1]. Speaking against SHF
is furthermore the fact that in previous experiments using
steam-pretreated spruce as a substrate, the ethanol yield on
total hexoses has been higher using a SSF conguration com-
pared to a SHF conguration [26]. A way forward which
combines the advantages of SSF and SHF could be to run a
short hydrolysis at elevated temperature and when the hydrol-
ysis becomes end product inhibited switch to SSF by adding
yeast and lower the temperature. Another option would be to
develop a more thermostable yeast strain. This would have
several benets, not only could the enzymatic hydrolysis be
performed closer to the optimum temperature, but the risk of
contamination would be reduced.
Due to the current high prices of commercial cellulases a
reduction of the amount of cellulases added could improve
the process economy even more than an increase in ethanol
productivity. There is a potential for decreasing the enzyme
loading while still maintaining a high ethanol yield since
approximately 95%of the ethanol is produced during the rst
48 h of the fermentations (Fig. 5). Thus, even if the enzymatic
hydrolysis is slower high ethanol yields could still be reached
within 72 h.
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