PILOT SCALE CONVERSION OF CELLULOSE TO ETHANOL by bestt571

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									                      PILOT SCALE CONVERSION OF CELLULOSE TO ETHANOL

                   Dana K. Becker, Paul J. Blotkamp, George H. Emert

                                      Biomass Research Center
                                       U n i v e r s i t y o f Arkansas
                                    415 A d m i n i s t r a t i o n B u i l d i n g
                                       F a y e t t e v i l l e , Arkansas
INTRODUCTION

       I n t e r e s t i n c e l l u l o s e as a renewable source o f a l c o h o l f u e l s and o t h e r
chemicals has increased as the p r i c e o f petroleum products continues t o r i s e .
Extensive research has been conducted i n t h e area o f c e l l u l o s e u t i l i z a t i o n
f o r a number o f years (1, 2, 3, 4, 5). However, w i t h t h e exception o f The
U. S. Army N a t i c k Research Command which has operated a p r e p i l o t program f o r
the enzymatic conversion o f c e l l u l o s e t o glucose since 1976 (6, 7 ) , these
i n v e s t i g a t i o n s have been confined t o t h e l a b o r a t o r y .

        The importance o f p i l o t i n g a complete process f o r t h e conversion o f
c e l l u l o s e t o ethanol was recognized by t h i s l a b o r a t o r y i n 1974. The complexity
of combining t h e m a t e r i a l handling o f b u l k y s l u r r i e s such as a i r c l a s s i f i e d
municipal s o l i d waste (MSW) and p u l p m i l l waste (PMW) w i t h t h e a s e p t i c opera-
t i o n o f an enzyme p r o d u c t i o n f a c i l i t y posed a unique s e t o f problems which
could n o t adequately be addressed on a l a b o r a t o r y scale. I n o r d e r t o address
these problems, i t was b e l i e v e d t h a t t h e design o f a p i l o t p l a n t should i n -
clude t h e f l e x i b i l i t y o f handling feedstocks o f w i d e l y v a r y i n g composition
and moisture content. Operation o f a p i l o t p l a n t would a l l o w t h e i d e n t i f i c a t i o n
and t e s t i n g o f equipment f o r t h e p r e p a r a t i o n and t r a n s f e r o f s l u r r i e s , s t e r i l i -
zation, and l i q u i d / s o l i d separation.

      The economic f e a s i b i l i t y o f a c a p i t a l i n t e n s i v e process such as t h e c e l l u l o s e
t o ethanol process r e q u i r e s t h a t t h e use o f h i g h l y s p e c i a l i z e d e x o t i c equipment
be kept t o a minimum. As a r e s u l t o f t h i s , low c o s t chemical r e a c t o r s would
be evaluated as fermentation vessels. The vessels f i r s t t e s t e d as " o f f the
s h e l f items" c o u l d then be modified as necessary t o accommodate the i n d i v i d u a l
requirements o f each s e t o f fermentation c o n d i t i o n s . I n t h i s way parameters
such as a g i t a t i o n , a e r a t i o n , temperature and pH c o n t r o l , and s t e r i l i t y c o u l d be
evaluated and adjusted as needed. Using these c r i t e r i a the biochemical conversion
o f c e l l u l o s e t o ethanol was scaled-up approximately 100 f o l d from 1OL l a b o r a t o r y
fermenters t o lOOOL vessels i n a p i l o t f a c i l i t y capable o f processing 1 ton p e r
day o f c e l l u l o s i c feedstock.

METHODS AND MATERIALS

      Three s t r a i n s o f y e a s t were used d u r i n g t h e p i l o t i n v e s t i g a t i o n s o f simultaneous
s a c c h a r i f i c a t i o n fermentation (SSF). (8, 9). These were Saccharomyces cereuisiae
ATCC 4132, obtained from t h e American Type C u l t u r e C o l l e c t i o n , R o c k v i l l e , Maryland;
candida brassicae IF0 1664, obtained from t h e I n s t i t u t e f o r Fermentation, Osaka,
Japan ( 2 ) ; and a s t r a i n o f Saccharomyces obtained from Budweiser, J o p l i n , Missouri.




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Stock c u l t u r e s were s t o r e d on O i f c o YM a g a r s l a n t s a t 4OC. Seed c u l t u r e s o f
each y e a s t were prepared b y t h e a d d i t i o n o f a p o r t i o n o f a s t o c k c u l t u r e i n t o
a shake f l a s k c o n t a i n i n g a medium shown i n T a b l e 1.'          Shake f l a s k s were i n c u -
l a t e d a t 28oC f o r 18 h o u r s . The shake f l a s k c u l t u r e was used t o i n o c u l a t e a
130L f e r m e n t e r made by F e r m e n t a t i o n Design, I n c . , c o n t a i n i n lOOL of t h e medium
                                                                                       ?
i n T a b l e 11. T h i s c u l t u r e was i n c u b a t e d f o r 18 hours a t 30 C, pH 5.0, w i t h a n
a g i t a t i o n speed o f 120 RPM. The y e a s t seed c u l t u r e was h a r v e s t e d i n t o s t e r i -
l i z e d 15 g a l l o n aluminum b a r r e l s p r i o r t o use i n SSF. I f t h e y e a s t was n o t
used immediately t h e b a r r e l s were s t o r e d i n a c o l d room a t 4OC f o r no l o n g e r
t h a n 48 hours.

        The mold Trichoderma r e e s e i QM 9414 was o b t a i n e d f r o m ATCC. T h i s organism
was grown on p o t a t o d e x t r o s e agar a t 29OC u n t i l s p o r u l a t i o n o c c u r r e d . The spore
p l a t e s were s t o r e d a t 4oC u n t i l use. 2'. r e e s e i seed c u l t u r e s were prepared b y
i n o c u l a t i n g shake f l a s k s w i t h a p o r t i o n o f a spore p l a t e . The c u l t u r e medium
used i n the shake f l a s k s i s shown i n T a b l e 111. The 1 l i t e r shake f l a s k s were
scaled-up t o 100 l i t e r f e r m e n t e r s . P h y s i c a l parameters c o n t r o l l e d i n t h e f e r -
menters were a e r a t i o n a t 0.5 v/v/m and a g i t a t i o n speed a t 300 RPM (1OOL fermenter).
The seed c u l t u r e s were i n c u b a t e d f o r 24 hours and t h e n h a r v e s t e d a s e p t i c a l l y
i n t o 15 g a l l o n aluminum b a r r e l s t o be t r a n s p o r t e d t o t h e p i l o t f a c i l i t y where
i t was used as inoculum f o r enzyme p r o d u c t i o n .

        A 10% v / v inoculum was used f o r i n i t i a t i o n o f c e l l u l a s e i n d u c t i o n stage i n
b o t h batch and c o n t i n u o u s phases o f enzyme p r o d u c t i o n . The medium used i n
enzyme p r o d u c t i o n i s d e s c r i b e d i n Table I V . A v i c e l PH 105, comparable t o MSW
i n i n d u c i n g c e l l u l a s e enzymes, was chosen as a model s u b s t r a t e because o f i t s
ease o f h a n d l i n g and u n i f o r m i t y . A v i c e l PH 105 was o b t a i n e d f r o m American
Viscose Co., D i v i s i o n o f FMC, Marcus Hook, Pennsylvania. The l e n g t h o f i n c u b a t i o n
o f t h e c u l t u r e depended on t h e mode o f enzyme p r o d u c t i o n b e i n g used. Batch enzyme
p r o d u c t i o n l a s t e d 96 t o 120 hours whereas c o n t i n u o u s enzyme p r o d u c t i o n had a
r e s i d e n c e t i m e o f 50 hours (D=.02). Batch SSFs were r u n f o r 24 hours u n l e s s
experimental d e s i g n d i c t a t e d o t h e r w i s e . Semi-continuous SSFs were r u n f o r 96 t o
120 hours w i t h t h e r e s i d e n c e t i m e v a r y i n g from 24 hours t o 48 hours. Three
m a j o r types o f f e e d s t o c k s were used, 1 ) p u r i f i e d c e l l u l o s e ( S o l k a f l o c . ) , 2 ) PMW
( d i g e s t e r r e j e c t s , p r i m a r y sludges, and d i g e s t e r f i n e s ) , 3) MSW. None of t h e
feedstocks r e c e i v e d any t y p e o f p r e t r e a t m e n t b e f o r e use i n t h e SSFs. However,
MSW was a t t i m e s p a s t e u r i z e d depending on e x p e r i m e n t a l c o n d i t i o n s . The MSW used
i n t h e SSFs had been shredded so t h a t i t would pass a 4" screen and t h e n a i r
classified p r i o r t o a r r i v a l a t the p i l o t plant.

       Assays f o r measurement o f enzyme a c t i v i t y and p r o t e i n c o n c e n t r a t i o n were
conducted as d e s c r i b e d by Blotkamp, e t a1 ( 9 ) . Glucose measurements were made
w i t h t h e use o f a Yellow S p r i n g s I n s t r u m e n t Company Model 23A glucose a n a l y z e r .
T o t a l r e d u c i n g sugars were measured by t h e d i n i t r o s a l i c y l i c a c i d method ( 1 0 ) .
Ethanol was analyzed u s i n g a Perkin-Elmer Model 3920 B gas chromatograph o r
a Hewlett-Packard Model 5730 A gas chromatograph equipped w i t h f l a m e i o n i z a t i o n



  Chemicals used i n media f o r m u l a t i o n s were m o s t l y t e c h n i c a l o r r e a g e n t grade,
however i n t h e p a s t y e a r many o f t h e compounds used were e i t h e r f e r t i l i z e r o r
food grade.



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d e t e c t o r s , an e l e c t r o n i c i n t e r a t o r , and a 6 f t . column o f Porapak Q.                Isothermal
a n a l y s i s was performed a t 150 C.      %
        Yeast p o p u l a t i o n s were m o n i t o r e d by u s i n g d i l u t i o n p l a t i n g . C e l l u l o s e
c o n c e n t r a t i o n o f samples used i n SSF was determined b y u s i n g a m o d i f i e d
v e r s i o n o f t h e Van Soest procedures (11, 1 2 ) . M o i s t u r e d e t e r m i n a t i o n s were
performed on an Ohaus m o i s t u r e balance.

EQUIPMENT

       The v e s s e l s used f o r enzyme p r o d u c t i o n and SSF were 330 g a l (1250 l i t e r )
c a p a c i t y manufactured by P f a u d l e r (L/D=.78).         Four o f t h e f i v e v e s s e l s were
capable o f a s e p t i c o p e r a t i o n . The v e s s e l s were c o n s t r u c t e d o f s t a i n l e s s s t e e l
w i t h carbon s t e e l j a c k e t s . The vessels were f u l l y j a c k e t e d f o r adequate
temperature c o n t r o l and s t e r i l i z a t i o n .

       A l l process p i p i n g was s t a i n l e s s s t e e l w i t h welded c o n n e c t i o n s except
where p i p i n g e n t e r e d t h e v e s s e l . Flanged f i t t i n g s w i t h t e f l o n gaskets were
used a t these p o i n t s . No pumps were used as a p r e c a u t i o n a g a i n s t c o n t a m i n a t i o n ,
t h e l i q u i d s and s l u r r i e s were moved w i t h p r e s s u r e ( s t e r i l e a i r o r steam) o r
g r a v i t y . The a g i t a t o r s h a f t s were equipped w i t h double mechanical s e a l s f i l l e d
w i t h o i l . Enzyme p r o d u c t i o n v e s s e l s used two f l a t b l a d e i m p e l l e r s , each h a v i n g
f o u r blades ( D i / D t = . 4 5 6 ) . A g i t a t i o n speed was 120 RPM, a e r a t i o n was 0.5 V / V / m
a t which t h e kLa was 84 h r - ' ' v s 330 h r - I on a l a b s c a l e ( w i t h w a t e r ) .

         The b a f f l e t r a y s t r i p p i n g column was c o n s t r u c t e d f r o m 9 " ( I . D . ) g l a s s p i p e
w i t h t r a y s made o f monel t o r e s i s t c o r r o s i o n . A s s o c i a t e d process l i n e s on t h e
s t r i p p e r were s t a i n l e s s s t e e l . Pumps were used on t h e beer feed l i n e s on t h e
s t r i p p i n g column and r e c i r c u l a t i o n l o o p s t o m a i n t a i n s o l i d s i n suspension.

         A b r i e f process f l o w diagram i s p r e s e n t e d i n F i g . 1. A f t e r t h e enzyme
p r o d u c t i o n vessels were f i l l e d w i t h n u t r i e n t s and s t e r i l i z e d , t h e seed inoculum
was t r a n s f e r r e d a s e p t i c a l l y f r o m t h e aluminum b a r r e l s t o t h e v e s s e l s u s i n g
n i t r o g e n t o p r e s s u r i z e t h e b a r r e l s . From t h i s p o i n t t h e enzyme p r o d u c t i o n c o u l d
be r u n i n e i t h e r a b a t c h o r c o n t i n u o u s mode. When enzyme was ready t o be h a r -
vested a p o r t i o n o f t h e whole c u l t u r e enzyme b r o t h was t r a n s f e r r e d t o t h e SSF
vessel i n t o which t h e c e l l u l o s i c feedstock (PMW o r MSW) would be added, a l o n g
w i t h t h e y e a s t . The SSF c o u l d be r u n i n e i t h e r b a t c h o r semi-continuous modes
i n w h i c h one h a l f o f m a t e r i a l was t r a n s f e r r e d o u t e v e r y one h a l f r e s i d e n c e time.
As t h e SSF was h a r v e s t e d t h e r e s u l t i n g beer s l u r r y was moved t o t h e beer s t o r a g e
t a n k where i t c o u l d be pumped i n t o t h e s t r i p p e r column f o r e t h a n o l r e c o v e r y .

RESULTS

Enzyme P r o d u c t i o n

      Performance of b a t c h enzyme p r o d u c t i o n s can be t y p i f i e d b y t h e d a t a p r e -
sented i n F i g u r e s 2 and 3. R e l a t i v e l y h i g h l e v e l s o f p r o t e i n and ~-gZucosidase
a r e p r e s e n t i n t h e c u l t u r e b r o t h . These r e s u l t s compare f a v o r a b l y w i t h those
obtained i n laboratory studies.

   The p i l o t p l a n t was m o d i f i e d t o produce enzyme c o n t i n u o u s l y i n o r d e r t o
demonstrate f e a s i b i l i t y on a l a r g e s c a l e . The economical advantages o f a


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c o n t i n u o u s process l i e i n reduced c a p i t a l i n v e s t m e n t due t o i n c r e a s e d e f f i c i e n c y
o f v e s s e l use. R e s u l t s f r o m c o n t i n u o u s enzyme p r o d u c t i o n s a r e shown i n F i g s . 4
and 5. From these graphs c a n be seen t h a t t h e B-gZucosidase i s somewhat l o w e r
b u t t h e p r o t e i n and FPRS r e m a i n a l m o s t as h i g h as i n b a t c h c u l t u r e . Use o f t h e
enzyme from batch as w e l l as c o n t i n u o u s enzyme p r o d u c t i o n i n s m a l l s c a l e f l a s k
s a c c h a r i f i c a t i o n and SSFs i n d i c a t e o n l y small d i f f e r e n c e s between t h e two
enzyme p r e p a r a t i o n s under t h e same c o n d i t i o n s .
                                                                                                                                  I
SIMULTANEOUS SACCHARIFICATION FERMENTATION

       Batch SSFs       were p e r f o r m e d u s i n g a v a r i e t y o f s u b s t r a t e s . T y p i c a l r e s u l t s
f o r Solka f l o c .     and p u l p m i l l wastes a r e i l l u s t r a t e d i n F i g . 6 and F i g . 7
respectively.           I n b o t h cases o v e r 50% o f t h e t h e o r e t i c a l y i e l d from c e l l u l o s e
t o e t h a n o l was   achieved. B a t c h SSFs were r u n w i t h c e l l u l o s e c o n c e n t r a t i o n s                 1

r a n g i n g from 5    t o 15%.
                                                                                                                                  1
        Semi-continuous SSFs u t i l i z e d p u l p m i l l wastes and m u n i c i p a l s o l i d waste
as p r i m a r y feedstocks. E t h a n o l p r o d u c t i o n can be seen i n F i g . 8. Both MSW
and PMW showed t h e same t r e n d ( F i g . 9 ) c o n c e r n i n g e t h a n o l y i e l d , base u t i l i -
z a t i o n f o r pH c o n t r o l , and b a c t e r i a l contaminant p o p u l a t i o n . The presence o f
contaminants and i n c r e a s e d base usage i n d i c a t e s t h e p r o d u c t i o n o f o t h e r a c i d i c
p r o d u c t s . Lab s c a l e c o n t i n u o u s SSF o p e r a t i o n has proved t o be s i g n i f i c a n t l y
b e t t e r t h a n batch SSF p e r u n i t t i m e .

STRIPPING OPERATIONS

         A f t e r t h e SSFs were completed t h e r e s u l t a n t beer s l u r r y was p r e s s u r e d
t o t h e b e e r s t o r a g e t a n k ( F i g . 1 ) . From t h e b e e r s t o r a g e t a n k t h e s l u r r y was
pumped t o t h e t o p o f t h e b a f f l e t r a y column ( 1 3 ) w h i l e steam was i n j e c t e d
i n t o t h e bottom o f t h e column. As t h e b e e r s l u r r y cascaded down t h e column
t h e h o t vapor from below c o n t a c t e d t h e descending l i q u i d and effected t h e
s t r i p p i n g o f t h e e t h a n o l f r o m t h e b e e r f e e d . The column was designed t o
h a n d l e b e e r s l u r r i e s w i t h s o l i d s c o n t e n t as h i g h as 10% and d e l i v e r a p r o d u c t
s t r e a m o f a p p r o x i m a t e l y 25% w/v e t h a n o l from a feed c o n t a i n i n g 2.0 t o 3.5%
e t h a n o l . The s t i l l bottoms e t h a n o l c o n c e n t r a t i o n remained as low as 0.04%.
I n a l a r g e - s c a l e p l a n t t h e p r o d u c t from t h e s l u r r y s t r i p p e r w i l l be r e c t i f i e d
f u r t h e r t o y i e l d 95-100% i n d u s t r i a l o r m o t o r grade e t h a n o l as necessary.

D I S C U S S I O N AND CONCLUSION

        Many p i e c e s o f equipment used f o r m a t e r i a l s h a n d l i n g were t e s t e d i n t h e
p i l o t p l a n t . An example i s a 750 g a l l o n p u l p e r which worked w i t h some wood
p r o d u c t s b u t n o t v e r y w e l l w i t h MSW because o f t h e p l a s t i c s and metal cans i n
t h e m a t e r i a l . A r o t a r y vacuum f i l t e r was used f o r d e w a t e r i n g some s l u r r i e s
b u t f o r t h e m a j o r i t y o f f e e d s t o c k s i t was n o t a c c e p t a b l e . For these reasons
t h e feedstocks used a t t h e p i l o t p l a n t , as o u t l i n e d i n t h i s paper, r e c e i v e d no
p r e t r e a t m e n t and were used i n t h e process j u s t as t h e y were r e c e i v e d .

        The o p e r a t i o n o f t h e p i l o t p l a n t i n b o t h a b a t c h and c o n t i n u o u s mode u s i n g
p o t e n t i a l i n d u s t r i a l f e e d s t o c k s demonstrated t h e enzymatic c e l l u l o s e t o e t h a n o l
t e c h n o l o g y on a s u b s t a n t i a l l y l a r g e r s c a l e t h a n had p r e v i o u s l y been r e p o r t e d .
The s i z e of t h e p l a n t e n a b l e d t h e use o f b u l k y m a t e r i a l s , such as MSW, which
was d i f f i c u l t on a l a b o r a t o r y s c a l e . The r e s u l t s f r o m t h e p i l o t p l a n t enzyme



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I
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I

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        p r o d u c t i o n compared v e r y f a v o r a b l y w i t h t h e l a b o r a t o r y r e s u l t s , however i n t h e
        case of t h e SSFs t h e d a t a from t h e p i l o t p l a n t and t h e l a b o r a t o r y a r e only com-
        p a r a b l e f o r a p p r o x i m a t e l y t h e f i r s t 24 hours a f t e r which t h e p i l o t p l a n t r e s u l t s
        lagged behind. F o r example, on b a t c h SSFs t h a t r a n l o n g e r t h a n 24 hours a t the
        p i l o t p l a n t t h e p e r c e n t c o n v e r s i o n t o e t h a n o l d i d n o t c o n t i n u e t o r i s e as i n
        t h e l a b o r a t o r y . W i t h p u l p m i l l wastes i n l a b o r a t o r y s t u d i e s , SSFs o f 85 t o
        90% O f t h e o r e t i c a l c o n v e r s i o n t o e t h a n o l was achieved i n 48 hours compared t o
        55 t o 60% c o n v e r s i o n a t t h e p i l o t p l a n t . The reasons f o r t h e d i f f e r e n c e i n
        r e s u l t s can be e x p l a i n e d i n p a r t by t h e l a c k o f adequate environmental c o n t r o l s
        such as temperature and pH due t o poor h e a t and mass t r a n s f e r i n t h e h i g h s o l i d s
\       s l u r r y o f t h e SSFs. Contamination was a l s o a problem i n SSFs t h a t r a n f o r
        extended p e r i o d s as evidenced by t h e i n c r e a s e i n base u t i l i z a t i o n f o r pH c o n t r o l
        and t h e concomitant decrease i n e t h a n o l y i e l d s ( F i g s . 8, 9 ) .
I
                The data g a t h e r e d from t h e o p e r a t i o n o f t h e p i l o t p l a n t was used f o r e x t e n -
I       s i v e economic a n a l y s i s o f t h e c e l l u l o s e t o e t h a n o l t e c h n o l o g y ( 1 4 ) . The r e s u l t s
        o f t h i s a n a l y s i s a l o n g w i t h t h e problem areas mentioned above i n d i c a t e f u r t h e r
t       s c a l e - u p o f t h e process f r o m t h e 1 ton/day t o a 50 t o n l d a y f a c i l i t y s h o u l d be
        c a r r i e d o u t i n o r d e r t o i d e n t i f y s p e c i f i c equipment t o be used on a commercial
        s c a l e and execute process m o d i f i c a t i o n s toward enhancing t h e economic v i a b i l i t y
,       o f the technology.




                                    NOMENCLATURE

                                           a                 area

    \                                      D                 dilution rate

                                           Di                 i m p e l l e r diameter

                                           Dv                 vessel d i a m e t e r

                                           I.D.               i n t e r n a l diameter

                                                             mass t r a n s f e r c o e f f i c i e n t
                                           kL
                                           L                  vessel l e n g t h

                                           m                  minute

                                           V                  volume




                                                                           301
 (1)   Dhawn, S. and J. K. Gupta "Enzymic H y d r o l y s i s o f Comon C e l l u l o s i c
         Wastes by C e l l u l a s e . " J o u r n a l o f General and A p p l i e d M i c r o b i o l o q y ,
         23, 155-161 (1977).

 (2)   Karube, I., t . a l . , " H y d r o l y s i s o f C e l l u l o s e i n a C e l l u l a s e Bead
                           e
         F l u i d i z e d Bed Reactor" B i o t e c h n o l o q y and B i o e n q i n e e r i n g Vol X I X.
         Pages 1183-1191 (1977).

 (3)   Ladisch, M., C. M. Ladisch, and George T. Tsao. " C e l l u l o s e t o Sugars:
         New Path Gives Q u a n t i t a t i v e Y i e l d . " Science V o l . 201, 23 pp. 743-
         745. August 1978.

 (4)   Wilke, C. R., U. V. S t o c k a r , and R. D. Yana, "Process Design B a s i s
         For Enzymatic H y d r o l y s i s o f N e w s p r i n t " , AIChE Symposium S e r i e s
         Vol. 72, 158 pp. 104-114.

 (5) Spand, L. A.,             J. Medeiros, and M. Mandels, "Enzymatic H y d r o l y s i s o f
          C e l l u l o s i c Wastes t o Glucose" Resource Recovery and C o n s e r v a t i o n ,
          1 (1976) pp. 279-294, E l s e v i e r S c i e n t i f i c P u b l i s h i n g Company,
          Amsterdam.

 (6)   Nystrom, J. M. and R. K. Andren, " P i l o t P l a n t Conversion o f C e l l u l o s e
         t o Glucose", Process B i o c h e m i s t r y , December 1976.

 (7)   Nystrom, J. M. and A l f r e d L. A l l e n , " P i l o t S c a l e I n v e s t i g a t i o n s and
         Economics o f C e l l u l a s e P r o d u c t i o n , " B i o t e c h n o l o g y and B i o e n g i n e e r i n g
         Symp. No. 6, 55-74 (1976). John W i l e y and Sons, I n c .

 (8)   Takagi, M., S. Suzuki, and W. F. Gauss, "Manufacture o f A l c o h o l from
         C e l l u l o s i c M a t e r i a l s Using P l u r a l Ferments", U n i t e d S t a t e s P a t e n t
         No. 3,990,944.               (November 9, 1976).

 (9)   Blotkamp, P. J., M. Takagi, M. S. Pemberton, and G. H. Emert, "Enzymatic
         H y d r o l y s i s o f C e l l u l o s e and Simultaneous F e r m e n t a t i o n t o A l c o h o l .I'
         AIChE Symposium S e r i e s , p . 85-90 (181), V o l . 74, 1978.

(10)   M i l l e r , G. L., "Use o f D i n i t r o s a l i c y l i c A c i d Reagent f o r D e t e r m i n a t i o n o f
          o f Reducing Sugars," Analytical, 31 ( 3 ) , March, 1959.

(11)   Von Soest, P. J., and R. H. Wine, J o u r n a l o f A s s o c i a t i o n o f O f f i c i a l
         A q r i c u l t u r a l Chemists, 51 (4) 780-85 (1968).

(12)   U p d e r g r a f f , D. M.,   A n a l y t i c a l B i o c h e m i s t r y , 32, 420-24 (1969).

(13)   Katzen, R., V. B. D i e b o l d , G. D. Moon, J r . , W. A . Rogers, and K. A.
         LeMesurier, "A S e l f - D e s c a l i n g D i s t i l l a t i o n Tower", Chemical E n g i n e e r i n g
                              .
         Process, Vol 64, ( 1 ) January 1968.

(14)   Emert, G. H. and Raphael Katzen, "Chemicals From Biomass b y Improved
         Enzyme Technology", ACS/CSJ J o i n t Chemical Congress, Honolulu, Hawaii,
         A p r i l 1-6, 1979.




                                                                    302
Table I

    Yeast growth medium     (flask)

                                       g/l
    0 -glucose                        20.0
    yeast e x t r a c t                5.0
    malt extract                       5.0
    bacto-peptone                      5.0


Table I1
    Yeast growth medium     (fermenter)

                                       g/l
    0 -glucose                        20.5
    (NH4)2 so4                         1.5
    MgSOb.7HpO                         0.11
    CaCl                               0.06
    Cornsteep L i q u o r              7.5


Table 111

    2'. reesei growth medium

                                       g/l
                                      20.0
                                       2.0
                                       1.23
                                       1.o
                                          3.0
                                       0.05
                                       0.014
                                       0.016
                                       0.04
                                       2.62
                                       1.7
                                       7.5


Table I V

    2'. reesei enzyme production medium

                                       g/l
    Cellulose (Avicel 105)            20.0
    KH2P04                             2.0
    (NH4)2HP04                         1.23
    MgSO, *7H,O                        1 .o
    CaC1,                              3.0
    FeSO,                              0.05
    ZnSO,                              0.014
     nO
    MS ,                               0.016
    COCl                               0.04
    ( N H 4 L SO,                      2.62
    (NH2 ) O
           ,
           C                           1.72
    Cornsteep L i q u o r              7.5
    Tween 80                           0.2%
                                                303
           r
           I




      'c
      0




      a
      L




304
                  Figure 2.   Batch Enzyme Production FPRS A c t i v i t y

                                      and P r o t e i n Concentration



X




!    I
    :1
-   120                                           ,/




    30

          L
          10                                           70     80   90
                                                                        L
                                                                        100
                                          hours
               Figure 3.   Batch enzyme produc t i o n 6-glucosidase

                                 Ac t i v i t y
                                                       311.
     ao-                 I
            batch        I       continuous
                         I
     70 .                I
                         I


                                                                                     I
                                                                                         -
                                                                                             0
F 4ot                                                                                -4
                                                                                             a
                                                                                     -3

                                                                                     -2      .E
                                                                                        *
                                                                                        4)


-    1u                  I
                                                                                        9
                                                                                     -1 k
                         I




c




-
X



.-
w
0
0
3




                         I   .
            20      40       60     a0    100       120 140   160   iao    200 220 240
                                               hours
                 Figure 5.        Continuous Enzyme Production 6-gZucosidase

                                           Ac t i v i t y                 mfi
     32 -
                    batch       SSF (solka floc)
     28-

     24-                                                                                   - 40
                                                                                                  -
                                                                                                  0
                                                                                                  C
     20 -                                                                                         m

     16.
0
                                                                                                  C
y12.                                                                                       - 20 2
-
00                                                                                              c
f     8-
                                                                                                  P
                                                                                                  C
                                                                                                  0
r
CI
                                                                                         -10
0     4-                                                                                          x

                                                                                            ;
              2       4        6     8        10     12    14   16   18    20   22     24
                                                   hours
              Figure 6.       Batch SSF Using Solka Floc. Ethanol Production
                              and % Conversion t o Ethanol (6%Cellulose)
     32   -                                                                            A
               batch
               batch          SSF ( P M W )
                              SSF ( P M W )                                                - 50
     28-


     24 -                                                                                  -40
                                                                                                  0

     20   -                                                                                       C
                                                                                                  m
                                                                                                  c
                                                                                           - 30%
     16-                                                                                          0
                                                                                                  c
0
-
a    12-                                                                                   - 20.5
                                                                                                  C


-                                                                                                 L
                                                                                                  P
ro
m
      8-                                                                                          C
c
w
                                                                                           -10    8
Q)    4.                                                                                          x

          I   2       4         6    8        10     12    14   16   18    20   22
                                                                                        . I
                                                                                       24
                                                   hours
                  Figure 7.     Batch SSF Using Pulp Mill Wastes, Ethanol Production
                                and % Conversion t o Ethanol (7% Cellulose)
                                                                                            337
       batch          semi-continuous


                                                                          -5



                                                                          -4




                                                                          -3
                                                                               L
                                                                               3
                                                                               0
                                                                               c
                                                                          -2   1
                                                                               fs
                                                                          -1 3
                                                                               8



               Municipal Solid Waste, Ethanol Production (8% Cellulose)
20-               I
       batch      I semi-continuous
                  I
                  I
                  I         0
15 -
               ,,y-

								
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