World Journal of Microbiology & Biotechnology 18: 857862, 2002.

 2002 Kluwer Academic Publishers. Printed in the Netherlands.

857

Preparation of hydrolysates from tobacco stalks and ethanolic fermentation

by Saccharomyces cerevisiae
Carlos Mart n1,2, Teresa Fernandez1, Ramon Garc a1, Eugenio Carrillo1, Marcelo Marcet1, Mats Galbe3
and Leif J. Jonsson4,*
1
Department of Chemistry and Chemical Engineering, University of Matanzas, 44 740 Matanzas, Cuba
2
Applied Microbiology, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
3
Department of Chemical Engineering I, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden
4
Biochemistry, Division for Chemistry, Karlstad University, SE-651 88 Karlstad, Sweden
*Author for correspondence: Tel.: +46-54-7001801, Fax: +46-54-7001457, E-mail: leif.jonsson@kau.se
Received 22 March 2002; accepted 3 September 2002

Keywords: Ethanol, fermentation inhibitors, lignocellulose hydrolysates, Saccharomyces cerevisiae, tobacco stalks

Summary
Chipped tobacco stalks were subjected to steam pretreatment at 205
C for either 5 or 10 min before enzymatic
hydrolysis. Glucose (15.417.1 g/l) and xylose (4.55.0 g/l) were the most abundant monosaccharides in the
hydrolysates. Mannose, galactose and arabinose were also detected. The hydrolysate produced by pretreatment for
10 min contained higher levels of all sugars than the 5 min-pretreated hydrolysate. The amounts of inhibitory
compounds found in the hydrolysates were relatively low and increased with increasing pretreatment time. The
hydrolysates were fermented with bakers yeast. Ethanol yield, maximum volumetric productivity and specic
productivity were used as criteria of fermentability of the hydrolysates. The fermentation of the hydrolysates was
only slightly inhibited compared to reference solutions having a similar composition of fermentable sugars. The
ethanol yield in the hydrolysates was 0.380.39 g/g of initial fermentable sugars, whereas it was 0.42 g/g in the
reference. The biomass yield was twofold lower in the hydrolysates than in the reference. The fermentation
inhibition caused by the tobacco stalk hydrolysates was less than that caused by sugarcane bagasse hydrolysates
obtained under the same hydrolysis conditions.

Introduction
Ethanol is now recognized as a potential alternative for
petroleum-derived vehicle fuels, and oers environmental advantages. The combustion of biofuels gives no net
contribution of CO2 to the atmosphere, so they do not
contribute to the so-called greenhouse eect. Lignocellulosic materials are the most abundant renewable
resource available for the production of fuel ethanol.
The cellulose and hemicellulose fractions of lignocellulosic materials can be hydrolysed to sugars, which are
then converted to ethanol by fermentation (Olsson &
Hahn-Hagerdal 1996).
Hydrolysis of cellulose can be brought about by using
mineral acids or cellulolytic enzymes. Acid hydrolysis is
a well-established process (Parisi 1989), which gives
good yields within a short reaction time. It has,
however, several drawbacks, such as for example the
requirement of costly corrosive-resistant construction
materials (Nguyen 1993). Furthermore, acid hydrolysis
gives rise to compounds which might inhibit the
ethanolic fermentation (Olsson & Hahn-Hagerdal
1996; Larsson et al. 1999a, b). Therefore, enzymatic

hydrolysis oers advantages. Lignocellulosic materials

must be pretreated prior to enzymatic hydrolysis in
order to make the cellulose macromolecules accessible
for the cellulolytic enzymes. Steam explosion is a simple
and ecient pretreatment method (Saddler et al. 1993).
During steam pretreatment, the lignocellulose is softened and becomes accessible to cellulolytic enzymes. The
hemicellulose fraction is hydrolysed leading to formation of sugars. During the steam pretreatment of
lignocellulosic materials, by-products derived from the
degradation of carbohydrates and lignin are also
formed. Some of these compounds, such as furfural,
5-hydroxymethylfurfural (HMF), phenolic compounds,
as well as acetic, formic and laevulinic acid, inhibit the
metabolism of yeast (Larsson et al. 1999a, b, 2000).
The tobacco plant (Nicotiana sp.) is an important crop
in many areas of the world. Cigars are made from
selected tobacco leaves. The tobacco stalks are the
residue left after separation of the tobacco leaves.
Tobacco stalks can be used as animal feed and for
production of cigarette paper (Kung et al. 1980; Tso
1991), but these uses are of only slight economical
signicance, and the stalks are usually incorporated into

858

C. Martin et al.

the soil or burnt after harvesting. Consequently, tobacco

stalks represent an abundant and cheap renewable
resource, which could potentially be an appropriate
substrate for bioconversion to ethanol. No previous
attempts to perform ethanolic fermentation using tobacco stalks as the raw material have been found in the
literature. In this work, enzymatic hydrolysis of pretreated tobacco stalks was performed, the chemical
composition of the resulting hydrolysate was analysed
and the fermentability using bakers yeast, Saccharomyces cerevisiae, was investigated.

with rubber stoppers and equipped with cannulas for

sampling and CO2 removal. The asks were inoculated
to an initial cell concentration of 4 g (dry weight) per l
and incubated at 30
C. Fermentations were run for
48 h under nonsterile conditions. Samples of 100 ll
were withdrawn at the start and after 6, 12, 24 and
48 h. Fermentations were performed in triplicate and
the mean values are given. The ethanol yield (g/g), the
volumetric productivity of ethanol (g/(lh)), and the
specic productivity (g/(gh)), were used as criteria of
fermentability.

Materials and methods

Analyses

Preparation of the hydrolysates

All hydrolysis samples were analysed by HPLC (Gilson,

Middletown, Wisconsin, USA). The samples were
ltered through a 0.20 lm lter and diluted prior to
analysis. Glucose, xylose, cellobiose, galactose, mannose and arabinose were separated using an ion
chromatograph equipped with pulsed amperometric
detection (PAD) (Dionex, Sunnyvale, California,
USA) and a CarboPac PA 10 column (Dionex). The
mobile phase consisted of NaOH and de-ionized water
applied at a ow rate of 1 ml min 1 using a gradient
pump (GP40, Dionex). Acetic, formic and laevulinic
acid, as well as furfural and 5-HMF were separated
using an Aminex HPX-87H column (Bio-Rad, Hercules, California, USA) operating at 45
C with 5 mM
H2SO4 as the mobile phase and at a ow rate of
0.6 ml min 1. Acetic, formic and laevulinic acid were
detected with an RI-detector (Shimadzu RID-6A, Kyoto, Japan). Furfural and HMF were determined with a
u.v.-detector (Shimadzu SPD-6A, Kyoto, Japan). The
total content of phenolic compounds was determined
colorimetrically by using the Prussian Blue method
(Graham 1992). Catechol was used as the calibration
standard and the measurements were performed with an
Ultrospec 2000 spectrophotometer (Pharmacia Biotech,
Bucks, UK).
Glucose, ethanol, glycerol, lactic acid, acetic acid,
furfural and HMF in all fermentation samples were
analysed by HPLC on a column of cation exchange resin
(LG-KS 0802, Prague, Czech Republic) in the hydrogen
form operating at 45
C with 5 mM H2SO4 as the
mobile phase and at a ow rate of 0.6 ml min 1 using a
high pressure pump (HPP 4001, Laboratorn Pr stroje,
Prague, Czech Republic). Glucose, ethanol, glycerol,
lactate and acetate were detected with an RI detector
(RID 101, Laboratorn Pr stroje). Furfural and HMF
were detected with a u.v.-detector (LCD 2040, Laboratorn Pr stroje).
The cell mass was analysed gravimetrically. Fivemillilitre samples were vacuum ltered through 0.45 lm
preweighed lters (Double Rings, Xinhua Paper Mill,
China). The lters were dried in an oven (WSU 100
MLW Labortechnik, Ilmenau, Germany) at 105
C for
15 min and then weighed with an analytical scale
(Nagema VEB, Growaagen, Berlin, Germany).

Tobacco stalks were fractionated to a particle size

between 2.2 and 10 mm and subjected to pretreatment
by steam explosion. The pretreatment vessel was connected to a boiler, from which saturated steam was
supplied. A computer, ABC 800 (Luxor AB, Motala,
Sweden), was used to control valve operations. Stalks
were heated at 205
C for either 5 min (Hydrolysate 1)
or 10 min (Hydrolysate 2). The pretreated material was
discharged into a cyclone connected to the outlet of the
reactor and collected. A sample for bre yield analysis
was taken.
Pretreated tobacco stalks were hydrolysed by the
cellulase mixture Celluclast 2L (75 FPU g 1 and 12
Cellobiase IU g 1) and the b-glycosidase preparation
Novozym 188 (392 Cellobiase IU g 1) (Novo Industri
A/S, Bagsvaerd, Denmark). The tobacco stalk slurry
was diluted with water, adjusted to pH 4.8 and mixed
with 2.3 g Celluclast and 0.5 g Novozym. Water was
added to a nal weight of 500 g and the dry matter was
5% (w/w). Hydrolysis was performed at 40
C for 96 h
and with stirring. The hydrolysate was subsequently
separated by vacuum ltration. Samples of the hydrolysate were taken for analysis of the chemical composition.
Fermentation
Compressed bakers yeast S. cerevisiae (Hollandia,
DSM, Delft, The Netherlands) was used for fermenting
the hydrolysates. The hydrolysates were supplemented
to nal concentrations of (per l) 1 g yeast extract
(Biocen, Havana, Cuba), 0.5 g (NH4)2HPO4, 0.025 g
MgSO4 7H2O and 1.38 g NaH2PO4 (Merck, Darmstadt, Germany). The pH was adjusted to 5.5 with 2 M
NaOH using a pH-meter (Pracitronic, MV-68, Dresden, Germany). Fermentations of sugar solutions,
consisting of mixtures of glucose and mannose (Merck
Laboratory Supplies, Poole, England) in similar concentrations as in the hydrolysates and supplemented
with the same nutrients as stated above, were performed as references. The fermentations were carried
out in 50-ml asks containing 45 ml medium, sealed

859

Ethanol from tobacco stalk hydrolysates

Calculations
The ethanol yield (g/g) was calculated as the maximum
amount of ethanol formed per gram of initial fermentable sugars (glucose and mannose) present in hydrolysates and references. The volumetric productivity
(g/(lh)) was based on grams of ethanol produced per
litre of culture medium per hour during the rst 6 h of
the fermentation, since the mean maximum volumetric
productivity in the reference fermentations was achieved
within this period. The specic ethanol productivity was
calculated as volumetric productivity divided by initial
cell mass concentration. Anaerobic growth yield, hereafter referred to as biomass yield (g/g), was based on the
amount of biomass formed during 48 h per gram of
initial fermentable sugars.

Results and discussion

Analysis of the composition of the hydrolysates
The bre yield after steam pretreatment of the tobacco
stalks at 205
C was 91.3 and 85.8 g per 100 g dry
matter when the residence time was 5 and 10 min,
respectively. The decrease in bre yield can mainly be
attributed to solubilization of the hemicellulosic fraction
of the raw material. Some of the cellulose could also be
solubilized during steam pretreatment, especially when
the residence time is long (Stenberg et al. 1998).
The chemical composition of the tobacco stalk enzymatic hydrolysates is shown in Table 1. The main
components were the monosaccharides glucose and
xylose, derived from the hydrolysis of cellulose and
hemicellulose xylans, respectively. Other sugars found in
the hydrolysates were cellobiose, mannose, galactose
and arabinose. The concentration of glucose was relatively low compared to sugarcane bagasse hydrolysates
obtained using the same conditions (Mart n et al. 2001).
However, the cellobiose concentration was high, indicating incomplete hydrolysis, probably due to the

inhibition of the b-glucosidase by the released sugars.

This is in agreement with the demonstration of the
competitive inhibition of b-glucosidase by glucose (Lee
& Fan 1983). Increasing the pretreatment time from 5 to
10 min led to an increase in the concentration of all
sugars except cellobiose, which slightly decreased. Evidently, stronger pretreatment conditions led to a more
complete hydrolysis of hemicellulose and favoured a
more ecient enzymatic hydrolysis of cellulose. However, even with 10 min steam pretreatment, some
cellobiose remained unhydrolysed. Stronger pretreatment conditions than used in this work or higher
concentrations of the cellulolytic enzymes could probably improve the enzymatic hydrolysis.
The sugar composition of the hydrolysates shows that
tobacco stalk hemicellulose is very heterogeneous in
composition. Although this hemicellulose contains
mainly xylans, it also contains mannans, galactans and
arabinans. The broad spectrum of sugars found in
tobacco stalk hydrolysates contrasts with bagasse hydrolysate, which only contained glucose, xylose and very
small amounts of arabinose and mannose (Mart n et al.
2001; Mart n et al. 2002). The results of the sugar
analysis indicate that tobacco stalks roughly have the
same carbohydrate composition as common hardwoods.
This is in accordance with previous reports (Tso 1991).
Under the pretreatment conditions used in this work,
the formation of fermentation-inhibiting compounds
was minimal. The furan derivatives furfural and HMF,
produced by dehydration of pentoses and hexoses,
respectively, were formed in detectable amounts only
under the longer pretreatment time (Table 1). Acetic
acid, released from hemicellulose by deacetylation, and
phenolic compounds, derived from degradation of
lignin, were formed in relatively low amounts (Table 1).
Increased residence time in the pretreatment led to an
increase in the concentration of acetic acid and phenols
of 44 and 50%, respectively. Since formic acid was
formed only in very small amounts and laevulinic acid
was not detected at all in the hydrolysates, it is evident
that the pretreatment conditions, even under the longer
pretreatment time, were not harsh enough for the
degradation of furfural and HMF.

Table 1. Composition of the enzymatic hydrolysates.

Component (g/l)

Glucose
Xylose
Cellobiose
Mannose
Galactose
Arabinose
Phenols
Acetic acid
Formic acid
Furfural
HMF

Pretreatment time (min)

Fermentation

10

15.4 (0.40)
4.5 (0.18)
4.5 (0.24)
2.5 (0.17)
2.1 (0.11)
1.5 (0.09)
0.3 (0.04)
1.8 (0)
0 (0)
0 (0)
0 (0)

17.1 (0.46)
5.0 (0.21)
3.9 (0.31)
2.9 (0.23)
2.4 (0.15)
1.9 (0.11)
0.6 (0.05)
2.6 (0.12)
0.1 (0)
0.5 (0.05)
0.2 (0)

Table 2 summarizes the results of the fermentations.

Hydrolysates produced after pretreatment for 5 and
10 min are hereafter referred to as Hydrolysate 1 and
Hydrolysate 2, respectively. Experimental data for
ethanol content were processed with the software
Statgraphics 2.1 for Windows for performing analysis
of variance, which allowed comparison of the results.
The least signicant dierences (LSD) test showed that
Hydrolysates 1 and 2 form a homogeneous group with a
mean ethanol content signicantly lower than that
obtained for the reference at a 95% condence level.
Glucose consumption and ethanol formation during
fermentation of Hydrolysate 2 are shown in Figure 1.

Mean from three replicates. Standard deviations are shown within

parentheses.

860

C. Martin et al.

Table 2. Results of the fermentation.

Medium

Hydrolysate 1

Reference 1

Hydrolysate 2

Reference 2

IS (g/l)
Ethanol (g/l)
YE/IS (g/g)
Q (g/(lh))
Maximum specic productivity (g/(gh))
Biomass increase (g/l)
YX/IS (g/g)

18.0 (0.55)
7.0 (0.35)
0.39
1.13
0.28
0.46 (0.05)
0.025

18.2 (0.15)
7.9 (0.26)
0.43
1.20
0.30
0.67 (0.05)
0.037

20.2 (0.47)
7.6 (0.28)
0.38
1.17
0.29
0.54 (0.06)
0.027

20.1 (0.09)
8.5 (0.33)
0.42
1.28
0.32
0.71 (0.04)
0.035

IS initial fermentable sugars (glucose plus mannose); YE/IS ethanol yield on initial fermentable sugars; Q maximum volumetric
productivity; YX/IS biomass yield on initial sugar. Standard deviations are shown within parentheses.

Figure 1. Ethanol formation and glucose consumption during fermentation of tobacco stalk Hydrolysate 2. Ethanol in the hydrolysate (d),
ethanol in the reference (s), glucose in the hydrolysate (j) and glucose
in the reference (h) are indicated.

Glucose was fermented readily within the rst 6 h in

the hydrolysates as well as in the reference. Ethanol was
formed at a high rate simultaneously with glucose
consumption. After glucose depletion, there were still
small amounts of ethanol formed, probably due to
utilization of mannose. It is not surprising if the
mannose was consumed mainly after depletion of
glucose, since it is well known that in media composed
of mixtures of sugars the utilization of dierent carbon
sources is sequential due to the repression of several
yeast enzymes by glucose (Gancedo & Serrano 1989;
Gancedo 1992). Glucose repression constitutes a problem in several industrial fermentative processes using
feedstocks like molasses or wort, which are composed of
mixtures of sugars (Olsson & Nielsen 2000).
The maximum concentration of ethanol was reached
after 12 h (Figure 1). After that, the ethanol concentration slightly decreased. This could be due to evaporation
and, possibly, assimilation of ethanol. The fermentative
performance of the yeast was comparable in both
hydrolysates (Table 2). No major dierences in ethanol
yield and productivity were observed. The higher
ethanol concentration in the hydrolysate obtained after
the 10 min steam pretreatment can be attributed to its
higher initial sugar content.
The fermentation of the hydrolysates was only slightly
inhibited compared to the reference (Figure 1, Table 2).

In the reference, ethanol was produced at a higher

concentration and at a higher rate than in the hydrolysates. The maximum ethanol concentration achieved
in the hydrolysates was 7.6 g/l, which was around 11%
less than in the reference fermentation. The ethanol yield
in the reference was 0.42 g/g of initial fermentable
sugars, whereas in the hydrolysates it was only a little
less (0.380.39 g/g).
The maximum volumetric ethanol productivity was
around 1.15 g ethanol per litre and hour in the hydrolysates, while the reference fermentation had a value as
high as 1.28 g/(lh). The specic productivity, which
takes into account the cell mass involved in the
fermentation, also had a higher value in the reference
than in the hydrolysates.
Biomass production in the hydrolysates was considerably lower than in the reference. The biomass yield on
initial sugar in the reference showed a twofold increase
compared to the value achieved for the hydrolysates.
The observed inhibition of fermentation in the hydrolysates is due to the presence of inhibitory compounds, such as acetic acid, phenolics and furan
derivatives. However, the inhibition of fermentation
was not as strong as in bagasse hydrolysates (Mart n
et al. 2001). This fact might be due to the relatively low
concentration of inhibitors in the tobacco stalk hydrolysates. Nevertheless, the high inoculum used could
contribute to decreasing the eect of fermentation
inhibition. As can be observed (Figure 2), the furfural

Figure 2. The variations in the concentrations of acetic acid (j),

furfural (m) and glycerol, () during fermentation of tobacco stalk
Hydrolysate 2.

861

Ethanol from tobacco stalk hydrolysates

was consumed rapidly within the rst 2 h of the
fermentation.
The main by-product formed during fermentation
was glycerol (Figure 2). However, the formation of
glycerol in the hydrolysates started only after complete
consumption of furfural (Figure 2). Lower amounts of
glycerol were produced in the hydrolysates compared
to the reference (data not shown). The retardation in
glycerol formation might be linked to transformations
occurring during furfural assimilation by yeast.
S. cerevisiae cells reduce furfural to furfuryl alcohol
(D az de Villegas et al. 1992; Palmqvist et al. 1999) in
a reaction that is probably catalysed by an NADHdependent dehydrogenase (Wahlbom & Hahn-Hagerdal 2002). Glycerol is synthesized by yeast to equilibrate the intracellular redox balance by reoxidizing the
excess of NADH generated during cell biosynthesis to
NAD+. The excess of NADH is reoxidized during the
reduction of dihydroxyacetone phosphate to glycerol
phosphate by glycerol-3-phosphate dehydrogenase
(Van Dijken 1986). The lack of glycerol formation
during the reduction of furfural can be attributed to
the reoxidation of NADH to NAD+ by the reduction
of furfural to furfuryl alcohol by an NADH-dependent
dehydrogenase instead of by the reduction of dihydroxyacetone phosphate to glycerol phosphate by
glycerol-3-phosphate dehydrogenase. The lower
amounts of glycerol produced in the hydrolysates
(data not shown) could be a consequence of the lower
biomass production compared with the reference
fermentation. Furthermore, acetic acid present in the
hydrolysates might also cause the low glycerol concentration. Physiological studies have previously
shown that addition of acetate leads to a decrease in
glycerol formation (Taherzadeh et al. 1996). The concentration of acetic acid remained roughly constant
during the course of fermentation (Figure 2). A small
increase was, however, detected at the end of the fermentation.
In conclusion, the potential of using tobacco stalks as
raw material for fuel ethanol production by fermentation with bakers yeast was demonstrated. High levels of
cellobiose and xylose in the hydrolysates indicated
the importance of further optimization of hydrolysis
conditions and introduction of xylose-utilizing microorganisms for performing ethanolic fermentations, respectively. The level of inhibition of fermentation by
tobacco stalk hydrolysates was low compared with
sugarcane bagasse hydrolysates, as a consequence of less
formation of fermentation-inhibiting compounds during
the pretreatment.

Acknowledgements
The Swedish Institute, the University of Matanzas and
the Swedish National Energy Administration are gratefully acknowledged for nancial support. Dr Fredrik
Wahlbom (Lund University, Sweden) is thanked for

assistance in the analysis of the hydrolysates. Prof Ben

Whitty (University of Florida, USA) is acknowledged
for his comments regarding disposal of tobacco stalks.
The linguistical advice of Ms Maria Elena Pineda is also
appreciated.

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