Industrial Crops and Products 20 (2004) 103110

Simultaneous saccharification and fermentation (SSF) of

industrial wastes for the production of ethanol
Zs. Kdr, Zs. Szengyel, K. Rczey
Department of Agricultural Chemical Technology, Budapest University of Technology and Economics,
Szt. Gellrt tr 4, Budapest H-1521, Hungary
Received 1 April 2002; accepted 22 December 2003

Abstract
During the past decades considerably large efforts have been made to optimize the production of lignocellulose derived fuel
ethanol production in order to develop a process configuration which is economically feasible and competitive with gasoline. One
of the process alternatives uses cellulase enzymes for the conversion of cellulose content of lignocellulosic biomass to fermentable
glucose. Due to the relatively similar process conditions in the enzymatic hydrolysis and ethanol fermentation, the option of
carrying out these two-steps together in one vessel exists. The application of simultaneous saccharification and fermentation
(SSF) for the conversion of lignocellulosics to alcohol would result in a more cost-effective process. In the present study
various lignocellulosic substrates, i.e. Solka Floc, OCC waste cardboard, and paper sludge, were examined in SSF experiments
for the production of ethanol. Two yeast strains were compared, a commercially available bakers yeast and a thermotolerant
Kluyveromyces marxianus, in two types of SSF experiments, i.e. isothermal SSF and SSF with temperature profiling. The results
showed that OCC waste and paper sludge could be used as substrates for ethanol production in SSF. There was no significant
difference observed between Saccharomyces cerevisiae and K. marxianus when the results of SSF were compared. The ethanol
yields were in the range of 0.310.34 g/g for both strains used. SSF resulted in higher ethanol yields compared to non-isothermal
SSF (NSSF; SSF with temperature profiling).
2004 Elsevier B.V. All rights reserved.
Keywords: Simultaneous saccharification and fermentation; Paper sludge; Ethanol

1. Introduction
Since the technical revolution the carbon dioxide
concentration in the atmosphere has been increasing
gradually. However, during the past century the net
carbon dioxide production has increased exponentially
Corresponding author. Tel.: +36-1-463-2843;
fax: +36-1-463-2589.
E-mail address: kati reczey@mkt.bme.hu (K. Reczey).

because of the tremendous expansion in the transportation sector resulting in notably changes in the earths
ecosystem.
In order to prevent irreversible changes and reduce
the impact of greenhouse gases on the earths climate
international collaboration is needed. Several countries have decided that in their energy production, renewable sources are going to play an important role.
The first rather effective step in this process, since
the majority of CO2 is produced by the transportation

0926-6690/$ see front matter 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.indcrop.2003.12.015

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Zs. Kadar et al. / Industrial Crops and Products 20 (2004) 103110

sector, would be using biomass derived so called alternative fuels. One of the candidates, which could substitute fossil fuels, is ethanol. Today ethanol is produced
in USA from cornstarch based process (Claassen et al.,
1999). However, the economic competitiveness with
gasoline still remains an issue. One of the main cost
contributive parameters of biomass originated ethanol
is the cost of the raw material. For the reduction of
overall production, cost cheap raw materials, such as
industrial wastes, have to be used. Further cost reduction can be obtained if the conversion efficiency of the
raw material is increased to the maximum.
In Hungary approximately 50,000 t of paper sludge
is produced annually. Paper sludge is the solid waste
stream of the papermaking industry containing the
short cellulose fibers, which leave the process. Usually this stream is deposed off, which has a significant
cost-increasing factor on the paper production. Another option for utilizing the organic content of this
waste stream is heat and electricity generation by
direct combustion. However, the high water and inorganic matter content, which can be as high as 30 wt.%
dry matter, would result in substantial energy loss.
Producing a value added product, such as fuel ethanol,
from the cellulose present in the paper sludge, could
provide an economically more attractive option. Due
to the high and rather accessible cellulose content
(5060%) of paper sludge, it could be a potential feedstock for fuel ethanol production (Lynd et al., 2001).
However, the high ash content of paper sludge could
be a problem for several reasons. First of all the handling of the raw material can be difficult. Secondly,
the economy of the process highly depends on the utilization of the solid residue i.e. the lignin, in the case
of for instance wood based fuel ethanol production,
which constitutes approximately 25 wt.% of the raw
material (Claassen et al., 1999). Lignin can be burnt
to provide energy required for the processing. When
paper sludge is used, the solid residue will contain
mostly inorganic ash. Obviously, energy cannot be obtained from this waste. Alternatively, this by-product
could be sold as an additive for the construction
industry.
There are several technologies available for the
conversion of lignocellulosics to fuel ethanol. The
main difference between these technologies is the
catalyst used for the brake-down of polysaccharides
in the raw material. In the one-step concentrated acid,

or in the two-step dilute acid technologies, acids are

used to convert the hemicellulose and cellulose fraction to monomeric sugars. In the enzymatic process,
the hemicellulose fraction is hydrolyzed by means
of acids or bases, while cellulase enzymes are used
for the conversion of cellulose. Although the current market price of cellulases makes the process
less favorable compared to technologies using acid
catalysts, working with enzymes, due to the significantly milder processing conditions applied, makes
it possible to combine the cellulose hydrolysis with
the ethanol fermentation. Simultaneous saccharification and fermentation (SSF) technique provides the
possibility to overcome the main disadvantage of
the enzymatic hydrolysis i.e. decreasing the enzyme
loading and therefore the production cost.
In spite of the economical advantage of SSF over
separate hydrolysis and fermentation (SHF), the critical problem with SSF is the difference in temperature
optima of the cellulases and the fermenting microorganism. Saccharomyces strains are well known as
good ethanol producing microorganisms, however
they require an operating temperature of 35 C. Fungal cellulases, which are most frequently applied in
the cellulose hydrolysis have an optimum temperature of 50 C. At lower temperatures, the substantially
lower hydrolysis rates would be unfavorable in terms
of increased processing time. A possible solution
to solve this problem is using thermotolerant yeast
strains instead of Saccharomyces strains, which would
allow higher processing temperatures, thus increased
rates of the hydrolysis.
Szczodrak and Targonski (1988) screened 58 yeast
strains from 12 different genera for their ability
to grow and ferment at temperatures above 40 C.
It was reported that some of the Fabospora and
Kluyveromyces strains were able to grow at as high
temperature as 46 C. Fabospora fragilis CCY51-1-1
cultivated at 43 C produced 56 g/l ethanol from
140 g/l glucose, which corresponds to 0.4 g/g (g
ethanol/g glucose) ethanol yield. However, when the
cultivation temperature was increased to 46 C the
performance of the strain significantly decreased, and
0.25 g/g ethanol yield was obtained.
In another study Saccharomyces, Candida and
Kluyveromyces strains were examined by Ballesteros
et al. (1991) for their ability to ferment glucose at
temperatures above 40 C. Similarly to the results

Zs. Kadar et al. / Industrial Crops and Products 20 (2004) 103110

obtained by Szczodrak and Targonski (1988)

Candida and Saccharomyces strains proved to be less
thermotolerant than Kluyveromyces strains. When
Kluyveromyces marxianus L.G. was cultivated at
42 C on glucose containing medium 37.6 g/l ethanol
concentration was obtained with an ethanol yield of
0.4 g/g.
Bollk and Rczey (2000) evaluated five different
Kluyveromyces strains based on the examination of
their growth on agar slants and in shake flask cultures
at different temperatures. On glucose medium in aerobic cultivation, K. marxianus Y01070 proved to be
the best thermotolerant strain of all examined strains.
In SSF experiments Ballesteros et al. (1991)
achieved best conversion of 10% Solka Floc cellulose
substrate to ethanol using both K. marxianus L.G. and
K. fragilis L.G. at temperatures up to 42 C, where 0.5
and 0.46 ethanol yields were achieved, respectively.
The ethanol yields were reduced at 45 C because of
cell death.
Barron et al. (1995) found that the K. marxianus
IMB3 is capable of producing ethanol at as high temperature as 45 C using milled paper as substrate. The
obtained ethanol yield was 0.11 g/g (21% of the theoretical).
Nilsson et al. (1995) reported that the ethanol yield
could be further increased using the same substrate.
Pretreatment of the milled paper with phosphoric acid increased the accessibility of the substrate
resulting in considerably higher ethanol yield of
0.21 g/g.
SSF experiments on paper sludge were carried
out by Lynd et al. (2001). The results showed wide
variability with respect to the sludge processing. It
was concluded that the prediction of the results of
SSF experiments was a difficult task, however some
general conclusions could be made. Most commercially available cellulase enzyme preparations have
low -glucosidase activity. This enzyme is essential,
because it converts the cellobiose to glucose, which
then can be fermented by the yeast. Supplementing the cellulases with -glucosidase from external
source increased the theoretical ethanol yield from 63
to 84%. It was also shown that neither the composition of growth medium (rich or lean) nor the cellulase
loading had any significant effect on the results and
similar results were obtained at 10 and 20 FPU/g substrate enzyme loading. Using rich medium the ethanol

105

yield was 83% of the theoretical, while performing

the fermentation in lean medium 86% ethanol yield
was obtained. Increasing the enzyme loading from
10 FPU/g cellulose to 20 FPU/g cellulose resulted in
slightly lower, 81% of theoretical, ethanol yield when
compared to the results, 87% of theoretical ethanol
yield, obtained at lower cellulase loading.
Huang and Chen (1988) examined the SSF technique with temperature profiling using Solka Floc
as the substrate. The fermenting microorganism was
Zymomonas mobilis, and culture medium was supplemented with Trichoderma reesei cellulases. Two
different strategies were applied: temperature cycling
and profiling, to enhance the SSF fermentation. In a
cycling study the temperature was changed periodically square-pulse-function wise between the optimal
fermenting temperature, 37 C, and the highest tolerable temperature, 40 C, of Z. mobilis. In the profiling
experiment during the initial phase, the temperature
was controlled between 30 and 37 C to allow optimal condition for the propagation of the cells. After
the cells entered into their active ethanol production phase, therefore the hydrolysis reaction became
the rate limiting step of SSF process, the temperature was increased to 40 C. The results showed that
with temperature cycling the ethanol productivity
(the final ethanol concentration divided by the reaction time) could be increased from 0.49 g/(l h)
to 0.62 g/(l h). However, similar ethanol yield,
0.23 g/g, was obtained as with traditional SSF. In
contrary, with temperature profiling the ethanol yield
obtained was significantly higher, 0.32 g/g, than that
obtained with isothermal SSF. Unfortunately, the
productivity, 0.32 g/(l h), was reduced due to the
increased processing time required for the prehydrolysis.
Since in SSF the rate limiting step is the hydrolysis, it is possible that a pre-hydrolysis at optimum
temperature for enzymes could improve the process.
In the present study, SSF and non-isothermal SSF
(NSSF) experiments with temperature profiling were
carried out. The ethanol production of thermotolerant
yeast (K. marxianus) and ordinary bakers yeast was
also investigated on different cellulosic waste materials, i.e. old corrugated cardboard and paper sludge,
produced in high amount in Hungary. Solka Floc, a
purified spruce cellulose powder, was used in control
experiments.

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Zs. Kadar et al. / Industrial Crops and Products 20 (2004) 103110

2. Materials and methods

2.1. Microorganisms
K. marxianus Y01070 was obtained from the
National Collection of Agricultural and Industrial
Microorganisms, Szent Istvn University (Budapest,
Hungary) and maintained on agar slants containing
5 g/l glucose, 1 g/l peptone, 20 g/l malt extract and
20 g/l agar. After 3 days incubation at 30 C the agar
slants were stored at 4 C. For each experiments, fresh
commercial compressed bakers yeast (Budafok Yeast
and Spirit Factory Ltd., Budapest, Hungary) was used.

SSF experiments. Solka Floc 200 pure cellulose powder (FS&D, Urbana, IL, USA) was used in reference
fermentation tests. All three substrates were analyzed
for cellulose content using the Hgglunds (1951)
method with the modification that the acid hydrolysate
obtained during the assay was analyzed for the concentration of glucose using HPLC, which was then
used to calculate the cellulose content. The estimated
cellulose contents of raw materials for OCC, paper
sludge and Solka Floc 200 were 75, 45, and 95 wt.%.

3. Simultaneous saccharification and

fermentation and non-isothermal SSF

2.2. Inoculum preparation

For the inoculum preparation of K. marxianus
Y01070 a loopful of cells was added to each 750 ml
E-flask containing 150 ml of sterile culture medium
in which the concentration of nutrients in g/l were 50
glucose, 2.5 yeast extract, 5 peptone, 1 KH2 PO4 , 0.3
MgSO4 and 2 NH4 Cl. The E-flasks were incubated
in a rotary shaker at 30 C and 300 rpm for 24 h.
2.3. Enzymes
Commercially available enzyme solutions, Celluclast 1.5 l and Novozym 188, were kind gifts from
Novo Industri A/S (Bagsvaerd, Denmark). Iogen Cellulase was obtained from Iogen Corporation (Ottawa,
Canada). Celluclast 1.5 l and Iogen Cellulase were
analyzed for cellulase and -glucosidase activity,
whereas only -glucosidase activity was determined
in Novozym 188. Celluclast 1.5 l had a cellulase activity (in terms of filter-paper units (FPU) per milliliter of
enzyme solution) of 75.8 FPU/ml and a -glucosidase
activity (in international unit of 1 ml enzyme) of
38.5 IU/ml. The cellulase and -glucosidase activities
in Iogen Cellulase were measured to be 99.8 FPU/ml
and 114.9 IU/ml, respectively. The Novozym 188 had
a -glucosidase activity of 421.0 IU/ml.

The SSF and NSSF experiments were performed in

750 ml E-flasks. Each flask contained 500 ml of culture medium in which the concentrations of nutrients
were the same as used in the inoculum preparation except for the carbon source. Instead of glucose, 6 wt.%
DM Solka Floc 200, OCC or paper sludge were used.
In all cases, the culture medium was supplemented
with 15 FPU of cellulase enzymes (Celluclast 1.5 or
Iogen Cellulase) and 15 IU of Novozym 188 per g
dry substrate. The fermentation was started with addition of yeast. The living cell content in the medium
was 2 109 cells/ml after inoculation, according to a
relationship between optical density of inoculum and
the living cell content (not shown here). The flasks
were incubated in a rotary shaker at 40 C for 96 h.
Samples were withdrawn regularly, centrifuged in a
laboratory desktop centrifuge at 1400 g, and the
supernatants were analyzed for glucose and ethanol
concentrations. The pH of the fermentation broth was
measured at each sampling and if necessary adjusted
between 4.4 and 5.3 by addition of either 10 wt.%
NaOH or H2 SO4 . In case of NSSF experiments, the
same procedure was followed except a 24 h prehydrolysis was performed at 50 C, with the same enzyme
loading written above, prior to inoculation with yeast
cell. After the prehydrolysis each flask was inoculated
with yeast cells and incubated at 30 C.

2.4. Substrates
3.1. Analytical methods
Two industrial wastes, old corrugated cardboard
(OCC) and paper sludge, obtained from Dunapack
Paper and Packaging Ltd. (Budapest, Hungary), were
used in this study for the production of ethanol in

Samples for analysis of glucose and ethanol contents were first filtered through a ME 24 0.2 m
membrane filters (Schleicher & Schuell, Dassel,

Zs. Kadar et al. / Industrial Crops and Products 20 (2004) 103110

Germany) and then analyzed on an HPLC unit.

Glucose and ethanol were separated on an Aminex
HPX-87H (Bio-Rad, Hercules, CA, USA) at 65 C
using 5 mM H2 SO4 solution as the mobile phase at a
flow rate of 0.5 ml/min, and then detected with a refractive index detector. For analysis Gilson UniPoint
software was used.
The enzyme activity of industrial enzymes was determined as filter paper activity (FPA) using Mandels
et al. (1976) procedure and -glucosidase activity using Berghem and Pettersons (1974) method.
3.2. Calculations
Data obtained from the HPLC analysis was used
to calculate ethanol yields, initial ethanol production
rates, ethanol productivities, and cellulose conversions. For the calculation of ethanol yield (YEtOH ),
ethanol concentration in g/l was taken after 72 h of
fermentation and then divided with initial cellulose
concentration in g/l. It has to be noted that caution
must be taken when these ethanol yields are compared with the theoretical 0.51 g ethanol/g glucose
yield, since the residual cellulose content was not
determined in this case, thus the amount of cellulose
consumed could not be calculated. Conversion of cellulose was calculated from the ethanol concentration
measured after 72 h of fermentation. Initial ethanol
production rates and ethanol productivities (r5 , r172 )

107

were calculated from ethanol concentrations measured after 5 and 72 h fermentation. In case of NSSF
an additional productivity (r272 ) was calculated after
72 h of residence time in which the prehydrolysis was
taken into account, i.e. the ethanol concentration was
taken after 48 h of fermentation.

4. Results and discussions

In the present study the ethanol production of K.
marxianus, a thermotolerant yeast strain and commercially available bakers yeast, Saccharomyces
cerevisiae, was investigated in SSF experiments using OCC waste and paper sludge. Solka Floc 200, a
purified cellulose powder, was used in control fermentation tests. As an alternative of SSF, NSSF with
temperature program was also studied in order to
increase ethanol yields.
During the SSF experiment with both K. marxianus and S. cerevisiae, the rate of hydrolysis was
higher than the rate of glucose consumption by the
yeast cell, which resulted in glucose accumulation
in the fermentation broth. Furthermore, there was
no ethanol production observed until the 5th hour
of fermentation. Although, it was expected that after
an initial adaptation phase, the glucose concentration
would be reduced to zero, it stayed at around 35 g/l,
which is considered to be rather high (Fig. 1). It could

20.0

Concentration [g/l]

15.0

10.0

5.0

0.0
0

24

48

72

Time [hours]

Fig. 1. SSF of Solka Floc 200 cellulose powder using S. cerevisiae () and K. marxianus (). Continuous lines: glucose concentration
in g/l. Dotted lines: ethanol concentration in g/l.

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Zs. Kadar et al. / Industrial Crops and Products 20 (2004) 103110

Table 1
SSF and NSSF of various substrates using K. marxianus and S. cerevisiae
K. marxianus

S. cerevisiae

cEtOH

YEtOH

Conversion

cEtOH

YEtOH

Conversion

Solka Floc
SSF
NSSF

17.8
16.0

0.337
0.303

60.4
54.3

16.6
15.1

0.314
0.287

56.2
51.3

OCC
SSF
NSSF

14.1
12.3

0.312
0.273

55.8
48.8

14.2
12.4

0.315
0.276

56.3
49.2

8.8
6.3

0.325
0.246

58.1
41.3

9.0
7.0

0.334
0.259

59.7
46.2

Paper sludge
SSF
NSSF

Ethanol concentrations (cEtOH ) in g/l, ethanol yields (YEtOH ) in g EtOH/g cellulose, and cellulose conversions in percent. Data are taken
after 72 h of fermentation. The experiments were carried out in duplicate, in the table means are shown.

be that the cells suffered from this temperature. It

was especially unexpected with the thermotolerant K.
marxianus, which was thought to be performing much
better at 40 C than S. cerevisiae. It can be also seen
(Fig. 1) that using Solka Floc 200 as a substrate, similar ethanol concentrations, 17 g/l, were obtained with
both strains. In Table 1 the ethanol concentrations,
ethanol yields, and cellulose conversions obtained
after 72 h of fermentation are summarized for the different substrates using both microorganisms. When
the performance of the two yeast strains was compared on the same substrate, there was no significant
difference observed and similar ethanol yields were
obtained (Table 1). It can be stated that the generally
used S. cerevisiae for the ethanol fermentation can be
applied at 40 C in the SSF process.
When Solka Floc substrate in the SSF fermentation
was substituted with OCC or paper sludge the ethanol
concentration obtained after 72 h of fermentation significantly decreased from about 1714 g/l and 9 g/l,
because of the substantially lower cellulose concentration in OCC and paper sludge. However, the cellulose conversions rates calculated from the ethanol
produced were in the same range, 5560%, for both
yeast strains applied (Table 1), which indicates that
both OCC and paper sludge were suitable for substrates for ethanol production. The cellulose conversions and ethanol yields, 0.250.34 g ethanol/g cellulose were considered rather low, since about 40%
of the cellulose was unutilized, even on Solka Floc

substrate. It is assumed that the high initial dry matter content, 6 wt.% substrate, which could cause mass
transfer problems, might be responsible for the low
ethanol yields obtained.
The initial ethanol production rates (r5 ) varied over
a wide range, 00.69 g/(lh), in the SSF experiments.
When Solka Floc 200 supplemented medium was used
for ethanol production the values of r5 was 44% higher
with compressed bakers yeast than with K. marxianus (Table 2). The same value for OCC medium was
148%. However, in the initial phase of fermentation
the production rate of ethanol on paper sludge was
30% higher when K. marxianus was used instead of S.
cerevisiae. Comparison of the ethanol productivities
calculated after 72 h fermentation time did not show
large variation and approximately same values were
obtained with both strains on the same substrate as it
is shown in Table 2. However, when the pure cellulosic substrate, Solka Floc, was replaced with paper
sludge the ethanol productivity decreased to the half
of obtained on Solka Floc, which is consequence of
the lower cellulose content of the raw material used.
In contrary to the results of Huang and Chen (1988),
in the NSSF experiments lower ethanol yields, and
therefore cellulose conversions were reached when
compared to the data obtained with SSF using the
same substrate. In the case of S. cerevisiae, 11%
higher cellulose conversion was obtained with SSF
than with NSSF Solka Floc 200 as the substrate. Considerably higher difference was measured on paper

Zs. Kadar et al. / Industrial Crops and Products 20 (2004) 103110

109

Table 2
Initial ethanol production rates (r5 ) [g EtOH/(l h)] and productivities (r172 , r272 ) [g EtOH/(l h)] of SSF and NSSF experiments on
various substrates
K. marxianus

S. cerevisiae

r5

r172

r272

r5

r172

r272

Solka Floc
SSF
NSSF

0.460
0.032

0.248
0.223

0.189

0.662
0.708

0.230
0.210

0.205

OCC
SSF
NSSF

0.262
0.024

0.195
0.171

0.145

0.694
0.638

0.197
0.172

0.154

Paper sludge
SSF
NSSF

0.348
0.00

0.122
0.092

0.041

0.268
0.274

0.125
0.097

0.053

40.0
35.0

Concentration [g/l]

30.0
25.0
20.0
15.0
10.0
5.0
0.0
-24

24

48

72

Time [hours]

Fig. 2. Comparison of SSF () and NSSF () using S. cerevisiae on Solka Floc 200 cellulose powder. Continuous lines: glucose
concentration in g/l. Dotted lines: ethanol concentration in g/l.

sludge. With SSF operation, the cellulose conversion

was 40% higher than applying the NSSF technique.
Similarly to the results obtained with the S. cerevisiae
on all substrates, the SSF resulted in higher ethanol
yields. As it can be seen on Fig. 2, NSSF operation
did not increase the ethanol yield at all. Furthermore,
when the productivities calculated after 72 h of residence time (r272 ) are compared to the productivities
calculated for SSF after 72 h of fermentation (r172 ),
it can be seen that considerably lower values were
obtained for NSSF operation (Table 2), which indicates that from an industrial point of view there is no
advantage to apply the NSSF operation mode.

5. Conclusions
The main objectives of the present study were to
compare the performance of a non-thermotolerant
yeast, S. cerevisiae, to a thermotolerant K. marxianus
yeast strain, to evaluate two different potential substrates in SSF ethanol production, and to investigate
the possibility of NSSF in order to increase ethanol
yield. The results showed that S. cerevisiae was as
good as K. marxianus in simultaneous saccharification and fermentation at 40 C using both industrial
wastes, i.e. OCC and paper sludge. The results showed
that both OCC and paper sludge could be used for

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Zs. Kadar et al. / Industrial Crops and Products 20 (2004) 103110

ethanol production in SSF. The cellulose conversions

were in the range of 5560% in the SSF experiments
for all substrates. Although, the ethanol yields were
considered rather low, 0.300.34 g ethanol/g cellulose
added, they were comparable with data obtained in
the literature varied between 0.11 and 0.4 g/g. The
NSSF operation mode did not increase the ethanol
yield at all, and slightly lower values were obtained,
which is in contrary to the results published by Huang
and Chen (1988).

Acknowledgements
The authors would like to gratefully acknowledge
the National Research Fund of Hungary (OTKA,
Hungary, T-029382), the Research and Development
Division of the Ministry of Education (OM, Hungary,
NKFP-OM-00231/2001), and the Research Found
of the Ministry of Education (OM, Hungary, FKFP
502-121) for their financial support. We also acknowledge the contribution by . Srdi, M. Csizmadia and
N. Sos.

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