Fermentation of lignocellulose

hydrolysates with yeasts and xyiose

isomerase
Torbj6rn Linden and Bfirbel Hahn-H~igerdal
Applied Microbiology, Chemical Center, University o f Lund, P.O. Box 124,
S-221 O0 Lund, S w e d e n

Untreated spent sulfite liquor (SSL) was fermented with five yeasts, Candida tropicalis, Pichia stipitis,
Pachysolen tannophilus, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and a co-culture of
P. tannophilus and S. cerevisiae, in the presence of commercial xylose (glucose) isomerases and 4.6
mM azide. The highest yield of ethanol, 0.41 g g ~ total sugar, was obtained with S. cerevisiae. The
yield based on consumed sugars and per gram cell dry weight was also highest with this yeast. C.
tropicalis and P. tannophilus produced considerable amounts of polyoles, mainly xylitol. With P.
stipitis sugar uptake was rapidly inhibited in untreated SSL. The presence of azide contributed to the
yield by about 0.04, mainly due to the fermentation of stored carbohydrates. The fermentation of
hydrogen fluoride-pretreated and acid-hydrolysed wheat straw with S. cerevisiae, xylose isomerase,
and azide gave a yield of O.40 g ethanol g-Z total sugar. In this substrate the xylose utilization was 84%
compared with 51% in SSL, which is discussed in relation to the salt sensitivity of xylose isomerases.

Keywords:Fermentation;lignocellulose;hydrolysate:yeast; xylose isomerase

Introduction
Great attention has been focused on lignocellulose
material as a renewable and " c l e a n " source of energy
in the form of ethanol for fuel and industrial use. One
complication in fermenting lignocellulose hydrolysates
is the effective fermentation of the hemiceilulose-derived sugars xyiose and galactose.
The hemicellulose fraction of wood is around 30%,
depending on the species. In straw it can be as much as
50%. The complete fermentation of xylose, the main
component of hemicellulose, is of vital importance for
the economy in a process for producing ethanol from
wood.
Many yeasts are able to ferment xylose to ethanol
directly. J One problem in the fermentation of lignocellulose hydrolysates is the inhibitory substances in
wood-derived hydrolysates. Inhibition can be reduced
by treatment with ion-exchange resin, 2 adaptation of
yeast to a specific substrate, 3"4 and steam stripping. 5
Pretreatment seems to be especially important for the

Address reprint requests to Dr. Hahn-Hfigerdalat the Universityof

Lund, Dept. of Applied Microbiology,Chemical Center, PO Box
124, S-221 00 Lund, Sweden
Received 9 March 1988; revised 24 August 1988

1989

Butterworth Publishers

xylose-fermenting yeasts. Our approach to the problem of fermenting both the hexose and the pentose
fraction from lignocellulose hydrolysates has been to
use the well-known, highly productive Saccharomyces
cerevisiae, Bakers' yeast, in combination with commercial glucose isomerase, which is a natural xylose
isomerase. This concept was originally presented b y
Wang et al. 6 Our assumption was that S. cerevisiae
might be better suited for an unpretreated technical,
substrate containing inhibitors than most other yeasts.
The reason for using bakers' yeast and xylose
isomerase is that S. cerevisiae does not ferment xylose
but it can metabolize xylulose to ethanol. Xylose
isomerase catalyses the reaction xylose ~ xylulose.
Xylulose is then phosphorylated and metabolized
through the pentose phosphate shunt. However, the
equilibrium of the isomerization step is less than 20%
towards xylulose. 7-1 By using the enzyme and the
yeast in combination, the reaction is continuously
pulled to the right. Xylose isomerase is commercially
available as glucose isomerase used in the high-fructose corn syrup process.
We have also used azide to prevent infection, ~ to
block the respiration, and to repress by-product formation, j2,j3 With this system in pure xylose solutions
we have achieved yields and productivities compa-

Enzyme Microb. Technol., 1989, vol. 11, September

583

Papers
rable to those obtained in hexose fermentation. We are
well aware that it is quite impossible to use azide in an
industrial process, but at this level it offers a means of
studying the mechanisms of depressing by-product
formation.
In the present study we have compared the fermentation of untreated spent sulpfite liquor (SSL) and
untreated hydrogen fluoride-pretreated and acidhydrolysed wheat straw (HF straw) with five different
nonadapted yeasts and four different commercial
xylose isomerases. The aim was to compare the performance of the yeasts and the enzymes in an untreated lignocellulose hydrolysate.

ducers, XI (A) - Optisweet-P konz (through Miles

Kali-Chemie, Hannover, FRG), XI (B) - NOVO
S w e e t z y m e Q (Novo Industri A/S, Bagsvaerd, Denmark), XI (C) - GIST Brocades Maxazyme GIImmob (GIST Brocades, Delft, The Netherlands), and
XI (D) = Takasweet (through Miles Kali-Chemie,
Hannover, FRG). XI (A) is a solid whole-cell nonimmobilized enzyme preparation used in the fermentations. XI (A) is also available in a liquid form without
cells. This was used once for determining enzyme
activity in sodium chloride solution. The other three
e n z y m e s are immobilized.

Fermentation conditions
Materials and methods

R a w materials
Spent suifite liquor (SSL), sodium based, was supplied
by MODO, OrnskOldsvik, Sweden. Hydrogen fluoride-pretreated and acid-hydrolysed straw (HF straw)
was supplied by the Institute of Biotechnology,
Kolding, Denmark. The only pretreatment of the
substrates was the adjustment of pH to 6.0 with K O H
and addition of fermentation nutrients: 0.25% (w/v)
yeast extract (Difco, Detroit, USA) and 0.025% (w/v)
(NH4)2HPO4; MgSO4"7H20 was added in fermentations without xylose isomerase (XI) to a concentration
of 0.0025% (w/v), otherwise to 0.2% (w/v). The substrates were buffered with solid 0.1 M sodium phosphate and, if necessary, the pH was readjusted to 6.0.

Microorganisms and growth medium

Candida tropicalis (ATCC 32113), Pichia stipitis
(CBS 5776), and Pachysolen tannophilus (CBS 4044)
were maintained on YMP agar with xylose, 10 g I ].
Schizosaccharomyces pombe (CBS 352) was maintained on YMP agar with glucose, l0 g 1 t. Saccharomyces cerevisiae, compressed bakers' yeast, was
obtained from a local distributor.
Cell mass was produced in 2-1 baffled E r l e n m e y e r
flasks, filled to 250 ml and sealed with cotton stoppers,
on a rotary shaking water bath at 30C.
Growth medium for cell mass production was the
following: yeast extract, 3 g l t (Difco, Detroit, USA),
malt extract broth, 3 g 1 ~ (Difco, Detroit, USA);
bacto-peptone, 5 g I ~(Difco, Detroit, USA); KH2P04,
19 g l -I, (NH4)2HPO4, 3 g 1-j, MgSO4"7H20, 1.1 g 1 l,
xylose, 30 g I ~ (Sigma Chemical Company, St. Louis,
USA) (for C. tropicalis, P. stipitis, and P. tannophilus); glucose, 30 g I ~ (for S. pombe). Xylose and
glucose were autoclaved separately. The yeasts were
harvested in late log phase by centrifugation (at
14,000~,,, 15 min, 4C Beckman model J2-21). The dry
weight was determined and the cells were used for the
fermentation of the lignocellulose hydrolysates.
Enzymes
We used four different commercial e n z y m e preparations which were generously supplied by the pro-

584

A 150-ml, slowly stirred, thermostated (30C), filled

beaker, sealed with a rubber stopper, was inoculated
with yeast, 75 g I t dry weight. For P. tannophilus and
S. pornbe, 53 and 52.5 g I -I, respectively, were used.
For the co-culture of P. tannophilus and S. cerevisiae
we used 37.5 g I t of each yeast. When used, XI was
added in the following concentrations in order to use
comparable e n z y m e (glucose isomerase) activity in all
experiments: XI (A) 10g I ~; in repeated batch fermentations XI (B) 145 g I-~, XI (C) 70g I-~, and XI (D) 57.5
g 1-~. Otherwise XI (C) was used at a concentration of
50 g I J. When used, azide was added to a final
concentration of 4.6 raM. The data presented are the
average of at least two experiments.

Analysis
Ethanol, sugars, and by-products were measured with
a Varian 5000 liquid chromatograph with a Tecator
Optilab 5902 refractometer. For ethanol, acetic acid,
glycerol, xylitol, glucose, arabinose, and total content
o f xylose, mannose, and galactose, a Bio-Rad aminex
HPX-87H column at 45C was used with a flow rate of
0.6 ml rain -t and 0.005 M H2SO4 as mobile phase.
Complete sugar analysis was performed with a BioRad aminex HPX-87P column at 85C, flow rate 0.6 ml
rain t, and water as mobile phase. The samples for the
lead column were deionized with a mixed-bed ionexchange resin. Glucose isomerase activity was determined according to Gong et al.14 The glucose formed
was determined by the glucose oxidase method. ~5 All
chemicals were of reagent grade. The errors in the
analysis of the fermentations have been estimated to
be less than 5%.

Results and discussion

R a w material
The sugar composition of SSL and H F straw varies
with the source of raw material and season. Figure 1
shows the sugar composition of two typical substrates.
These are not the sugar contents of the original
hydrolysates but for those inoculated with 75 g I ~ dry
weight yeast, since a high yeast concentration caused
a dilution of the hydrolysate 1.37 times. SSL and HF
straw have quite similar total sugar and xylose contents. The difference lies within the hexose fraction.

Enzyme Microb. Technol., 1989, vol. 11, September

Fermentation

of lignocellulose

hydrolysates:

T. Linddn

and B. Hahn-Htigerdal

consumed

sugar with P. stipitis and S. cereuisiae

is probably due to the breakdown and
fermentation of carbohydrate reserves, such as glycogen and trehalose, or the fermentation of an unidentified carbon source in SSL. The use of high cell mass
in fermentations, especially in combination with azide,
can influence the yield. .I* Therefore, we estimated
the ethanol production from stored carbohydrates with
S. cereuisiae (75 g 1-l dry weight) in buffer under the
same conditions as for the fermentation of SSL, but
with no carbon source added. With azide 1.5 g 1-l
ethanol was produced, and 0 g 1-l without. Translating
these numbers to the yield of ethanol in SSL fermentation, we may introduce an error of 0.04, which is in
agreement with the yield based on consumed sugars
(Table I). This does not explain the extremely high
yield obtained for P. stipitis. Because P. stipitis is so
rapidly inhibited in SSL, we did not look further into
this discrepancy.
In order to confirm the influence of azide on the
yield of ethanol, we also fermented SSL with S.
cereuisiae, with and without azide (Figure 3). The
difference in yield is slightly higher than 0.04. This
seems to support our hypothesis that, under the conditions we have chosen for the fermentation of nondetoxified lignocellulose
hydrolysates
(75 g 1-l dry
weight cell mass and 4.6 mM azide), there is a contribution to the yield from stored carbohydrates.
In the comparisons of the different yeasts, we did
not use the same amount of cells in all experiments.
Therefore we have also calculated the yield of ethanol
based on initial cell mass (Table I). Also in this
comparison S. pombe and S. cereuisiae give a significantly higher yield than the xylose-fermenting
yeasts.
(Table

XYL

GAL

MANN ARA

GLU

TOT

Sugar
Figure 1

Sugar content of SSL (filled) and HF straw (shaded)

An important difference between the substrates is

their salinity. SSL has an estimated ionic strength of
about 2 M, mainly due to high amounts of sodium
sulfite added in the pulping process. The salt concentration in HF straw is lower and arises from the acid
hydrolysis and the neutralization.
Fermentation

of SSL

with different

yeasts

First we compared ethanol production with the five

yeasts C. tropicalis, P. stipitis, P. tannophilus, S.
pomhe, and S. cereuisiae.
In these fermentations we
used azide and XI (A). Figure 2 shows the yield of
ethanol per gram of total sugar. Thus, 0.5 is the
theoretical maximum. Under our conditions the xylose-fermenting yeasts C. tropicalis, P. stipitis, and P.
tannophilus give low yields of around 0.2. The hexoseand xylulose-fermenting
yeasts S. cereuisiae and S.
pombe give reasonable yields of about 0.4 and 0.3,
respectively. In a co-culture of P. tannophilus and S.
cereuisiae a yield of 0.3 was achieved.
Sugar consumption,
ethanol production,
by-product formation, and yield are summarized in Table I.
The velocity of the fermentations
can be estimated
from Figure 2. C. tropicalis consumed sugar quite well
but produced by-products and did not produce ethanol
in proportion to the sugar consumption. It has recently
been suggested that this may be due to an excessive
carbon dioxide production with this yeast in the presence of azide.16 With P. stipitis the sugar consumption
was very rapidly inhibited. Nevertheless,
some ethanol was produced, probably from stored carbohydrates. P. tannophilus consumed sugar well but produced large amounts of by-products.
S. pombe
consumed less sugar than the other yeasts, except for
P. stipitis, but produced low amounts of by-products,
and, thus, gave a better yield of ethanol. S. cereuisiae
gave the best yield of ethanol. The co-culture of P.
tannophilus and S. cerevisiae produced high amounts
of by-products which lowered the yield of ethanol.
The more than theoretical yield of ethanol based on

Xylose

I)

isomeruses

We then investigated the influence of various preparations of xylose isomerase (XI) on the ethanol yield in

o.51

0.0
0

10

20

30

40

50

60

Time h
Figure 2 Time course of ethanol yield based on total sugars in
SSL with five yeasts in the presence of Xl (A) and 4.6 mM azide.
(x) C. Wopicalis;(0) P. stipitis; (H)P. tannophilus; (+) S. pombe;
(A) S. cerevisiae; (A) S. cerevisiae + P. rannophilus

Enzyme Microb. Technol.,

1989, vol. 11, September

585

Papers
Because of the sensitivity of XI (A) to high salinity,
we investigated the performance of three other enzyme preparations XI (B,C,D) (Figure 4). Repeated
batch fermentations of SSL with S. cerevisiae, where
we added an extra 10 g I t xylose, were chosen as an
indirect measure of e n z y m e performance because of
the lack of a direct e n z y m e activity assay. All enzyme
preparations were immobilized. Every 24 h the yeast
and the enzymes were separated by centrifugation and
the supernatant was replaced with new SSL. We found
a small difference in yield favoring XI (C). The continuously decreasing yield is probably due to the
deactivation of the e n z y m e and the yeast. We also
estimated the e n z y m e deactivation using the method
of Gong et al. 14 (Figure 5). Also according to this
method, the preparation X1 (C) appears slightly more
stable than the other two.

0.5
0.4
(/)

s 0.3
O
c-

0.2

A:
33

0.1
0,0

"I"

10

20

30
Time h

40

50

60

Figure 3 Fermentation of SSL with S. cerevisiae with ( I ) and

w i t h o u t (El) azide

YIELD (B)

0.4 j

0.3

0.2

0.1

[]

YIELD (C) I

0.0
1

Batch no.

Figure 4 Ethanol yield based on total sugar in repeated batch

f e r m e n t a t i o n s of SSL with S. cerevisiae in the presence of
different xylose isomerase preparations. Black bar = XI (B),
slant-lined bar - XI (C), and gray bar = XI (D)

F e r m e n t a t i o n o f S S L and H F straw
In Figures 6 and 7 and Table 2, we have summarized
the results from the fermentation of SSL and H F
straw with S. cerevisiae and XI (C) in the presence of
4.6 mM azide. In SSL 31 g 1-1 sugar were consumed
and 16.8 g l -] ethanol, 1.0 g 1-1 xylitol, and 2.1 g 1
glycerol were produced. The yield of ethanol based on
total sugar was 0.41 and on consumed sugar 0.54.
After 68.5 h the remaining sugars consisted of 4.3 g lxylose and 3.2 g l -] galactose, which means that 51%
of the xylose and 55% of the galactose had been
utilized. The galactose fermentation can probably be
improved with an adapted or recycled yeast, because
the enzymes responsible for galactose transport and
metabolism are inducible.
The fermentation of the hydrogen fluoride-pretreated and acid-hydrolysed straw resulted in a yield of
ethanol of 0.40, based on total sugars, and 0.45, based
on consumed sugars. From 37.1 g 1 t of consumed
sugars, 16.8 g I ' ethanol, 1.9 gl -] xylitol, and 1.6g I t
glycerol were produced. After 51 h fermentation, the

100

I ~
the fermentation of SSL. Based on the above results,
we chose S. cerevisiae for this investigation. The
previous results had indicated that the yield could be
improved with a better enzyme, because (i) quite large
amounts of sugar were left unfermented and (ii) when
using a liquid form of XI (A), the e n z y m e was totally
inactivated in a NaCI solution with an ionic strength
comparable with that in SSL. Furthermore, the methods available to measure the xylose isomerase activity
in S S L are not reliable. At the present time this can
only be done by taking a sample from the fermentation
and measuring the glucose isomerase activity in buffer, according to Gong et al.14 This method does not
account for reversible deactivation in SSL. The enzyme XI (A) is a whole cell preparation which contains
e n z y m e that may be inactivated in SSL, but when
transferred to buffer gives a significant activity.

586

80 J ~

Xl (B)

[] xl (c)

>,

60

-~ 40
g" 2o

o
start

Batch no.
Figure G Remaining glucose isomerase activity in percent of
initial activity in buffer after batch fermentations 1, 2, and 5 in
Figure 4. Same s y m b o l s as in Figure 4

Enzyme Microb. Technol., 1989, vol. 11, September

oi
G0

O"

<
O

t.o
co
cD

....~

('}
3"

ET

N
-<

133

26
7
31
27
25
32

Consumed sugar
~g 1-1)
7.9
6.3
5
10.3
14.1
12.6

Max E T O H
(g I 1)
3.4
0
10.9
0
1.0
5.8

Xylitol
(g I 1)
1.8
1.5
4.6
2~0
2.0
2.9

Glycerol
~g I 1~
0.21
0.17
0.12
0.30
0.38
0.31

0.3
0.9
0.16
0.38
0.56
0.39

Max yield ETOH

~g g-1 sugar consumed)

0.105
0.084
0.094
0.196
0.188
0.168

Max yield ETOH

Ig g 1 cells)

48
48
48
47.5
47.5
48

Time
(h)

o f S S L w i t h f i v e y e a s t s in t h e p r e s e n c e o f x y l o s e

Max yield ETOH

(g g 1 sugar tot)

a n d p r o d u c t y i e l d in t h e f e r m e n t a t i o n

Consumed sugar
(g I 1)
31
37

Substrate

SSL
HF straw

16.8
16.8

Max E T O H
(g I 1)
1.0
1.9

Xylitol
(g I 1)
2.1
1.6

Glycerol
(g I 1)
0.41
0.40

Max yield ETOH

(g g 1 sugar tot)

0.54
0.45

Max yield ETOH

(g g 1 sugar consumed)

0.224
0.224

Max yield ETOH

(g g 1 cells)

45
51

Time
(h)

51
84

Xylose utilization
(%)

T a b l e 2 S u g a r c o n s u m p t i o n , p r o d u c t f o r m a t i o n , a n d p r o d u c t y i e l d in t h e f e r m e n t a t i o n o f S S L a n d HF s t r a w w i t h S. c e r e v i s i a e , x y l o s e i s o m e r a s e
Xl (C), a n d 4.6 mM azide

Candida tropicalis
Pichia stipitis
Pachysolen tannophilus
Schizosaccharomyces pombe
Saccharomyces cerevisiae
S. cerevisiae + P. tannophilus

Yeast

Table 1 Sugar consumption, product formation,

i s o m e r a s e Xl (A) a n d 4.6 mM azide

,=

b,

=,

q~

5"
4

3
b

C3

Papers
50
40
3O
20
10
0

10

20

30 40 50
Time h

60

70

80

Figure 6 Fermentation of SSL with S. cerevisiae in the presence of XI (C) and 4.6 mM azide in SSL. Total sugar (C]), ethanol
( I ) , by-products (xylitol and glycerol) ( + ) i n g I 1 and yield of
ethanol () in percentage (g ethanol g 1 total sugar) x 100

50
40
30
20
10

"lff

10

20

30
40
Time h

Figure 7 Fermentation of HF straw

presence of XI (C) and 4.6 mM azide.
by-products (xylitol and glycerol) in
percent (g ethanol g 1 total sugar) x
Figure 6

50

60

visualize the relative contributions of the yeast S.

cerevisiae, azide, and the e n z y m e xylose isomerase to
the yield of ethanol based on total sugar. The yield in
the presence of all three components is 0.4. The
presence of the e n z y m e improves the yield by about
0.1. The contribution from azide, which is related to
the suppression of by-product formation ~2'~3 and the
fermentation of stored carbohydrates, jT~ is about
0.05. Thus, an estimated yield of 0.35 based on total
sugar would be achieved with a combination of S.
cerevisiae and the e n z y m e preparation of the xylose
isomerase studied in the present investigation. This is
quite low compared with yields achieved with, e.g.
Candida shehatae and P. stipitis. 4"~J9 Our results have
been obtained in lignocellulose hydrolysates in which
only pH has been adjusted and no further detoxification has been carried out. High yields with P.
stipitis and its anamorph C. shehatae have been
obtained with so-called adapted R strains which have
been recycled a number of times in enzymatically
hydrolysed substrates. 4 Other substrates have undergone steam stripping in order to remove volatile
inhibitors such as acetic acid. 5 These organisms are
very sensitive to acetic acid, especially at low pH.
Furthermore, the above-mentioned R strains, which
have been recycled a number of times in the substrate,
were used in these studies. Acetic acid does not have
such a dramatic effect on S. cerevisiae, even if the
yeast is inhibited in SSL not only by acetic acid but
also by a number of other compounds. 22
In conclusion, we have shown that ordinary
Bakers' yeast, S. cerevisiae, in combination with the
e n z y m e xylose isomerase, may offer an alternative to
the xylose-fermenting yeasts in the fermentations of
lignocellulose hydrolysates. S. cerevisiae has been
naturally selected for thousands of years |br ethanol
production under a variety of conditions. It is thus not
surprising that among the five yeasts investigated in

with S. cerevisiae in the

Total sugar, ethanol, and
g I 1. Yield of ethanol in
100. Same symbols as in

0.5
0.4
Ill

remaining sugars were 1.6 g 1 J xylose and 1.0 g I-t

galactose. This gives a xylose utilization of 84%. Thus
the xylose utilization is much higher in H F straw than
in SSL. This is probably due to the general sensitivity
of the xylose isomerase enzymes to high ionic
strength. Therefore our approach of using S. cerevisiae in combination with xylose isomerase might be
most applicable to low-salinity lignocellulose hydrolysates, such as enzymatically hydrolysed steam-pretreated material.

General discussion
In Figure 8 we have summarized the time course of
three fermentations of nondetoxified S S L in order to
588

0.3
0.2
0.1

0,0

10

20

30
40
Time h

50

60

Figure 8 Ethanol yield based on total sugar in f e r m e n t a t i o n of

SSL. (x) S. cerevisiae only, (+) S. cerevisiae in the presence of
4.6 mM azide, ( I ) S. cerevisiae in the presence of Xl (C) and 4.6
mM azide

E n z y m e M i c r o b . -I-echnol., 1989, vol. 11, S e p t e m b e r

Fermentation of lignocellulose hydrolysates: T. Linden and B. Hahn-H~gerdal

the present study, this yeast performs best in such a
hostile environment as untreated SSL. P. tannophilus,
isolated from tanneries, would also fit in this category,
if not for the severe polyol formation.
The weak link in the combination of S. cerevisiae
and xylose isomerase seems rather to be the stability
of the enzymes, which are generally very salt sensitive
and have a high temperature and pH optimum. 212-"
Therefore, this approach might be most applicable to
low-salinity lignocellulose hydrolysates, such as enzymatically hydrolysed steam-pretreated materials. In
designing a strategy for fermenting biomass pentoses,
various factors have to be considered and optimized.
The cost of pretreatment, e.g steam-stripping, has to
be balanced against the cost of xylose isomerase.
Whether pretreatment is necessary or not depends on
the substrate (method of hydrolysis, source of raw
material, etc.), the choice of yeast, and the cost and
stability of a xylose isomerase suited for a lignocellulose substrate. The choice of Xl could also be linked to
the choice of cellulases, since some organisms produce both enzymes.
Acknowledgement
This study was supported by the National Swedish
Energy Administration.

2
3
4
5
6
7
8
9

10
11
12

13
14
15
16
17
18
19
2O

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