PILOT SCALE CONVERSION OF CELLULOSE TO ETHANOL

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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


                                                               299
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
I
I

I
L
        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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Description: High cellulose content of the diet to reduce the incidence of colon cancer (colon cancer in men susceptible to the third), you can also control diabetes glycemic index, and even help you lose weight.