fermentation of lignocellulose hydrolysates with yeasts and xylosisomerase
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r a b le t o t h o s e o b t a i n e d i n h e x o s e f e r m e n t a t i o n . W e a r e
w e l l a w a r e t h a t i t i s q u i t e i m p o s s i b l e t o u s e a z i d e i n a n
i n d u s t r i a l p r o c e s s , b u t a t t h is l e v e l it o f f e r s a m e a n s o f
s t u d y i n g th e m e c h a n i s m s o f d e p r e s s i n g b y - p r o d u c t
f o r m a t i o n .
I n t h e p r e s e n t s t u d y w e h a v e c o m p a r e d t h e f e r m e n -
t a t i o n o f u n t r e a t e d s p e n t s u l p f it e l iq u o r ( S S L ) a n d
u n t r e a t e d h y d r o g e n f l u o r i d e - p r e t r e a t e d a n d a c i d -
h y d r o l y s e d w h e a t s t r a w ( H F s t ra w ) w i t h f iv e d i f fe r e n t
n o n a d a p t e d y e a s t s a n d f o u r d if f e r e n t c o m m e r c i a l
x y l o s e i s o m e r a s e s . T h e a i m w a s t o c o m p a r e t h e p e r -
f o r m a n c e o f t h e y e a s t s a n d t h e e n z y m e s i n a n u n -
t r e a t e d l i g n o c e l l u l o s e h y d r o l y s a t e .
a t e r ia l s a n d m e t h o d s
R a w m a t e r i a l s
S p e n t s u if i te l i q u o r ( S S L ) , s o d i u m b a s e d , w a s s u p p l i e d
b y M O D O , O r n s k O l d s v i k , S w e d e n . H y d r o g e n fl uo -
r i d e - p r e t r e a t e d a n d a c i d - h y d r o l y s e d s tr a w ( H F s t r a w )
w a s s u p p l i e d b y t h e I n s t i t u te o f B i o t e c h n o l o g y ,
K o l d i n g , D e n m a r k . T h e o n l y p r e t r e a t m e n t o f t h e
s u b s t r a t e s w a s t h e a d j u s t m e n t o f p H t o 6 .0 w i th K O H
a n d a d d i t io n o f f e r m e n t a t i o n n u t r i e n ts : 0 . 2 5 % ( w / v )
y e a s t e x t r a c t ( D i f c o , D e t r o i t , U S A ) a n d 0 . 0 2 5 % ( w / v )
( N H 4 ) 2 H P O 4 ; M g S O 4 7 H 2 0 w a s a d d e d i n f e r m e n t a -
t i o n s w i t h o u t x y l o s e i s o m e r a s e ( X I ) to a c o n c e n t r a t i o n
o f 0 . 0 0 2 5 % ( w / v ) , o t h e r w i s e t o 0 . 2 % ( w / v ) . T h e s u b -
s t r a t e s w e r e b u f f e r e d w i t h s o l i d 0. 1 M s o d i u m p h o s -
p h a t e a n d , i f n e c e s s a r y , t h e p H w a s r e a d j u s t e d t o 6 . 0.
M i c r o o r g a n i s m s a n d g r o w t h m e d i u m
Candida t ropica l i s
( A T C C 3 2 1 1 3 ) ,
Pichia s t ip i t i s
( C B S 5 7 7 6 ) , a n d P a c h y so l e n t a n n o p h i lu s ( C B S 4 0 4 4 )
w e r e m a i n t a i n e d o n Y M P a g a r w i t h x y l o s e , 1 0 g I ].
S c h i z o s a c c h a r o m y c e s p o m b e
( C B S 3 5 2 ) w a s m a i n -
t a i n e d o n Y M P a g a r w i t h g l u c o s e , l 0 g 1 t. S a c c h a ro -
myces cerev is iae , c o m p r e s s e d b a k e r s ' y e a s t , w a s
o b t a i n e d f r o m a l o c a l d i s t r i b u t o r .
C e l l m a s s w a s p r o d u c e d i n 2-1 b a f f l e d E r l e n m e y e r
f l a s k s, f i ll ed t o 2 5 0 m l a n d s e a l e d w i t h c o t t o n s t o p p e r s ,
o n a r o t a r y s h a k i n g w a t e r b a t h a t 3 0 C .
G r o w t h m e d i u m f o r c el l m a s s p r o d u c t i o n w a s t h e
f o l l o w i n g : y e a s t e x t r a c t , 3 g l t ( D i f c o , D e t r o i t , U S A ) ,
m a l t e x t r a c t b r o t h , 3 g 1 ~ ( D i f c o , D e t r o i t , U S A ) ;
b a c t o - p e p t o n e , 5 g I ~ ( D i f c o , D e t r o i t , U S A ) ; K H 2 P 0 4 ,
19 g l - I , ( NH 4) 2H P O4 , 3 g 1 j , M gS O 4 7 H2 0, 1 .1 g 1 l ,
x y l o s e , 3 0 g I ~ ( S i g m a C h e m i c a l C o m p a n y , S t . L o u i s ,
U S A ) ( f o r C. tropicalis , P. st ipit is , a n d P. tanno-
phi lus ;
g l u c o s e , 3 0 g I ~ ( f o r
S . p o m b e .
X y l o s e a n d
g l u c o s e w e r e a u t o c l a v e d s e p a r a t e l y . T h e y e a s t s w e r e
h a r v e s t e d i n l a t e l o g p h a s e b y c e n t r i f u g a t i o n ( a t
1 4 , 0 0 0 ~ , , , 1 5 m i n , 4 C B e c k m a n m o d e l J 2 - 2 1 ) . T h e d r y
w e i g h t w a s d e t e r m i n e d a n d t h e c e l ls w e r e u s e d f o r t h e
f e r m e n t a t i o n o f th e l i g n o c e l lu l o s e h y d r o l y s a t e s .
E n z y m e s
W e u s e d f o u r d i f f e re n t c o m m e r c i a l e n z y m e p r e p a r a -
t io n s w h i c h w e r e g e n e r o u s l y s u p p l i e d b y t h e p r o -
d u c e r s , X I ( A ) - O p t i s w e e t - P k o n z ( t h r o u g h M i l e s
K a l i - C h e m i e , H a n n o v e r , F R G ) , X I ( B ) - N O V O
S w e e t z y m e Q ( N o v o I n d u s tr i A / S , B a g s v a e r d , D e n -
m a r k ) , X I ( C ) - G I S T B r o c a d e s M a x a z y m e G I -
I m m o b ( G I S T B r o c a d e s , D e l f t, T h e N e t h e r l a n d s ) , a n d
X I ( D ) = T a k a s w e e t ( t h r o u g h M i l e s K a l i - C h e m i e ,
H a n n o v e r , F R G ) . X I ( A ) i s a s o li d w h o l e - c e l l n o n i m -
m o b i l i z e d e n z y m e p r e p a r a t i o n u s e d i n t h e f e rm e n t a -
t i o n s . X I ( A ) i s a l s o a v a i l a b l e i n a l i q u i d f o r m w i t h o u t
c e ll s. T h i s w a s u s e d o n c e f o r d e t e rm i n i n g e n z y m e
a c t i v i t y i n s o d i u m c h l o r i d e s o l u t i o n . T h e o t h e r t h r e e
e n z y m e s a r e i m m o b i l i z e d .
F e r m e n t a t i o n c o n d i t i o n s
A 1 5 0 - m l , s l o w l y s t i r r e d , t h e r m o s t a t e d ( 3 0 C ) , f i ll e d
b e a k e r , s e a l e d w i t h a r u b b e r s t o p p e r , w a s i n o c u l a t e d
w i t h y e a s t , 7 5 g I t d r y w e i g h t . F o r P . t a n n o p h i lu s a n d
S. pornbe , 5 3 a n d 5 2 . 5 g I - I , r e s p e c t i v e l y , w e r e u s e d .
F o r t h e c o - c u l t u r e o f
P . t a n n o p h i lu s
a n d
S. cerev is iae
w e u s e d 3 7 .5 g I t o f e a c h y e a s t . W h e n u s e d , X I w a s
a d d e d i n t h e f o l lo w i n g c o n c e n t r a t i o n s i n o r d e r t o u s e
c o m p a r a b l e e n z y m e ( g l u c o s e i s o m e r a s e ) a c t i v i ty i n all
e x p e r i m e n t s : X I (A ) 1 0 g I ~; i n r e p e a t e d b a t c h f e r m e n -
t a t i o ns XI ( B) 145 g I - ~, XI ( C ) 70 g I - ~, and XI ( D ) 57 .5
g 1 ~ . O t h e r w i s e X I ( C ) w a s u s e d a t a c o n c e n t r a t i o n o f
5 0 g I J. W h e n u s e d , a z i d e w a s a d d e d t o a f i n al
c o n c e n t r a t i o n o f 4 . 6 raM . T h e d a t a p r e s e n t e d a r e t h e
a v e r a g e o f at l e as t t w o e x p e r i m e n t s .
A n a l y s i s
E t h a n o l , s u g a r s , a n d b y - p r o d u c t s w e r e m e a s u r e d w i t h
a V a r i a n 5 0 0 0 l i q u i d c h r o m a t o g r a p h w i t h a T e c a t o r
O p t i l a b 5 9 02 r e f r a c t o m e t e r . F o r e t h a n o l , a c e t i c a c i d ,
g l y c e r o l , x y l it o l , g l u c o s e , a r a b i n o s e , a n d t o t a l c o n t e n t
o f x y l o s e , m a n n o s e , a n d g a l a c t o s e , a B i o -R a d a m i n e x
H P X - 8 7 H c o l u m n a t 4 5 C w a s u s e d w i t h a f l o w r a t e o f
0 . 6 m l r a i n - t a n d 0 . 0 0 5 M H 2 S O 4 a s m o b i l e p h a s e .
C o m p l e t e s u g a r a n a l y s is w a s p e r f o r m e d w i t h a B io -
R a d a m i n e x H P X - 8 7 P c o l u m n a t 8 5 C , fl o w r a t e 0 .6 m l
r a in t , a n d w a t e r a s m o b i l e p h a s e . T h e s a m p l e s f o r t h e
l e a d c o l u m n w e r e d e i o n i z e d w i t h a m i x e d - b e d i o n -
e x c h a n g e r e s i n . G l u c o s e i s o m e r a s e a c t i v i ty w a s d e t e r -
m i n e d a c c o r d i n g t o G o n g et al.14 T h e g l u c o s e f o r m e d
w a s d e t e r m i n e d b y t h e g l u c o s e o x i d a s e m e t h o d . ~5 A l l
c h e m i c a l s w e r e o f re a g e n t g r a d e . T h e e r r o r s i n t h e
a n a l y s is o f t he f e r m e n t a t i o n s h a v e b e e n e s t i m a t e d t o
b e l e s s t h a n 5 % .
R e s u l t s a n d d i s c u s s io n
R a w m a t e r ia l
T h e s u g a r c o m p o s i t i o n o f S S L a n d H F s t ra w v a r i e s
w i t h t h e s o u r c e o f r a w m a t e r i a l a n d s e a s o n .
Figure 1
s h o w s t h e s u g a r c o m p o s i t i o n o f tw o t y p i c a l s u b s tr a t e s .
T h e s e a r e n o t t h e s u g a r c o n t e n t s o f th e o r i g in a l
h y d r o l y s a t e s b u t f o r t h o s e i n o c u l a t e d w i t h 7 5 g I ~ d r y
w e i g h t y e a s t , s i n c e a h ig h y e a s t c o n c e n t r a t i o n c a u s e d
a d i l u t io n o f t h e h y d r o l y s a t e 1 .3 7 t i m e s . S S L a n d H F
s t r a w h a v e q u i t e s i m i la r t o t a l s u g a r a n d x y l o s e c o n -
t e n t s . T h e d i f f e r e n c e li e s w i t h i n t h e h e x o s e f r a c t i o n .
584 Enzyme Microb. Technol. 1989 vol. 11 September
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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 concen-
tration 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 xy-
lose-fermenting yeasts C. tropicalis, P. stipitis, and P.
tannophilus give low yields of around 0.2. The hexose-
and 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-pro-
duct 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 pres-
ence of azide.16 With P. stipitis the sugar consumption
was very rapidly inhibited. Nevertheless, some etha-
nol was produced, probably from stored carbohy-
drates. P. tannophilus consumed sugar well but pro-
duced 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
Fermentation of lignocellulose hydrolysates: T. Linddn and B. Hahn Htigerdal
consumed sugar with P.
stipitis
and S. cereuisiae
(Table I)
is probably due to the breakdown and
fermentation of carbohydrate reserves, such as glyco-
gen and trehalose, or the fermentation of an uniden-
tified 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 fermenta-
tion, 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 condi-
tions we have chosen for the fermentation of nonde-
toxified lignocellulose hydrolysates (75 g 1-l dry
weight cell mass and 4.6 mM azide), there is a contri-
bution 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 signifi-
cantly higher yield than the xylose-fermenting yeasts.
Xylose isomeruses
We then investigated the influence of various prepara-
tions of xylose isomerase (XI) on the ethanol yield in
o.51
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. rannoph ilus
Enzyme Microb. Technol., 1989, vol. 11, September 585
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( / )
O
c-
A :
33
0.5
0 . 4
0.3
0.2
0.1
0 , 0 I
0 1 0 2 0 3 0 4 0 5 0 6 0
T i m e h
F igure 3 F e r m e n t a t i o n o f S S L w i t h S ce rev i s i ae w i t h I ) a n d
w i t h o u t E l) a z i d e
]
YIELD B)
0.4 j [ ] YIELD C) I
~ 0.3
~ 0 2
~ 0 1
0.0
1 2 3 4
B a t c h n o .
F igure 4 E t h a n o l y i e l d b a s e d o n to t a l s u g a r i n r e p e a t e d b a t c h
f e r m e n t a t i o n s o f S S L w i t h S cerevis iae i n t h e p r e s e n c e o f
d i f f e r e n t x y l o s e i s o m e r a s e p r e p a r a t i o n s . B l a c k b a r = XI B ),
s l a n t - l i n e d b a r - X I C ) , a n d g r a y b a r = X I D )
t h e f e r m e n t a t i o n o f S S L . B a s e d o n t h e a b o v e r e s u lt s ,
w e c h o s e
S. cerevisiae
f o r th i s i n v e s t i g a t i o n . T h e
p r e v i o u s r e s u l t s h a d i n d i c a t e d t h a t t h e y i e l d c o u l d b e
i m p r o v e d w i t h a b e t t e r e n z y m e , b e c a u s e (i ) q u i te l a r g e
a m o u n t s o f s u g a r w e r e l e ft u n f e r m e n t e d a n d ( ii) w h e n
u s i n g a l iq u i d f o r m o f X I ( A ) , th e e n z y m e w a s t o t a l l y
i n a c t i v a t e d i n a N a C I s o l u t i o n w i t h a n i o n i c s t r e n g t h
c o m p a r a b l e w i t h t h a t i n S S L . F u r t h e r m o r e , t h e m e t h -
o d s a v a i l a b l e to m e a s u r e t h e x y l o s e i s o m e r a s e a c t i v it y
i n S S L a r e n o t r e l i a b l e . A t t h e p r e s e n t t i m e t h i s c a n
o n l y b e d o n e b y t a k in g a s a m p l e f r o m t h e f e r m e n t a t i o n
a n d m e a s u r i n g t h e g l u c o s e i s o m e r a s e a c t i v i t y i n b u f -
f e r , a c c o r d i n g t o G o n g
et al.14
T h i s m e t h o d d o e s n o t
a c c o u n t f o r re v e r s i b l e d e a c t i v a t i o n in S S L . T h e e n -
z y m e X I ( A ) i s a w h o l e c e l l p r e p a r a t i o n w h i c h c o n t a i n s
e n z y m e t h a t m a y b e in a c t i v a t e d in S S L , b u t w h e n
t r a n s f e r r e d t o b u f f e r g i v e s a s i g n i fi c a n t a c t i v i ty .
B e c a u s e o f t h e s e n s i t i v i t y o f X I ( A ) t o h i g h s a l i n i ty ,
w e i n v e s t i g a t e d t h e p e r f o r m a n c e o f t h r e e o t h e r e n -
z y m e p r e p a r a t i o n s X I ( B , C , D )
Figure 4).
R e p e a t e d
b a t c h f e r m e n t a t i o n s o f S S L w i t h
S. cerevisiae,
w h e r e
w e a d d e d a n e x t r a 1 0 g I t x y l o s e , w e r e c h o s e n a s a n
i n d ir e c t m e a s u r e o f e n z y m e p e r f o r m a n c e b e c a u s e o f
t h e l a c k o f a d i re c t e n z y m e a c t iv i t y a s s a y . A l l e n z y m e
p r e p a r a t i o n s w e r e i m m o b i l iz e d . E v e r y 2 4 h t h e y e a s t
a n d t h e e n z y m e s w e r e s e p a r a t e d b y c e n t r i fu g a t i o n a n d
t h e s u p e r n a t a n t w a s r e p l a c e d w i th n e w S S L . W e f o u n d
a s m a l l d i f f e r e n c e i n y i e ld f a v o r i n g X I ( C ) . T h e c o n -
t i n u o u s l y d e c r e a s i n g y i e l d i s p r o b a b l y d u e t o t h e
d e a c t i v a t i o n o f t h e e n z y m e a n d t h e y e a s t . W e a l s o
e s t i m a t e d t h e e n z y m e d e a c t i v a t i o n u s i n g th e m e t h o d
o f G o n g et al. 14 Figure 5). A l s o a c c o r d i n g t o t h i s
m e t h o d , t h e p r e p a r a t i o n X 1 ( C ) a p p e a r s s l ig h t l y m o r e
s t a b l e t h a n t h e o t h e r t w o .
Fermentat ion o f SSL and HF s traw
In
Figures 6
a n d 7 a n d
Table 2,
w e h a v e s u m m a r i z e d
t h e r e s u lt s f ro m t h e f e r m e n t a t i o n o f S S L a n d H F
s t r a w w i t h
S. cerevisiae
a n d X I ( C ) i n t h e p r e s e n c e o f
4 . 6 m M a z i d e . I n S S L 3 1 g 1 - 1 s u g a r w e r e c o n s u m e d
and 16 .8 g l - ] e t h an o l , 1 .0 g 1-1 xy l i t o l , an d 2 .1 g 1
g l y c e r o l w e r e p r o d u c e d . T h e y i e ld o f e t h a n o l b a s e d o n
t o t a l s u g a r w a s 0 . 4 1 a n d o n c o n s u m e d s u g a r 0. 5 4 .
A f t e r 6 8 . 5 h th e r e m a i n i n g s u g a r s c o n s i s t e d o f 4 . 3 g l -
x y l o s e a n d 3 . 2 g l - ] g a l a c t o s e , w h i c h m e a n s t h a t 5 1 %
o f t h e x y l o s e a n d 5 5 % o f t h e g a la c t o s e h a d b e e n
u t i l i z e d . T h e g a l a c t o s e f e r m e n t a t i o n c a n p r o b a b l y b e
i m p r o v e d w i t h a n a d a p t e d o r r e c y c l e d y e a s t , b e c a u s e
t h e e n z y m e s r e s p o n s i b l e f o r g a l a c t o s e t r a n s p o r t a n d
m e t a b o l i s m a r e i n d u c i b le .
T h e f e r m e n t a t i o n o f t h e h y d r o g e n f l u o r i d e- p r e -
t r e a t e d a n d a c i d - h y d r o l y s e d s t r a w r e s u l t e d i n a y i e ld o f
e t h a n o l o f 0 . 4 0 , b a s e d o n t o t a l s u g a r s , a n d 0 . 4 5 , b a s e d
o n c o n s u m e d s u g a rs . F r o m 3 7.1 g 1 t o f c o n s u m e d
s u g a r s , 1 6 .8 g I ' e t h a n o l , 1 .9 g l - ] x y l i t o l , a n d 1 . 6 g I t
g l y c e r o l w e r e p r o d u c e d . A f t e r 51 h f e r m e n t a t i o n , t h e
1 0 0
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F i g u r e G R e m a i n i n g g l u c o s e i s o m e r a s e a c t i v it y i n p e r c e n t o f
i n i t i a l a c t i v i t y i n b u f f e r a f t e r b a t c h f e r m e n t a t i o n s 1 , 2 , a n d 5 in
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