MYCELIAL PELLET SUSPENSIONS
B I B L I O T H E E K T U Delft P 1627 4 2 8 9
MYCELIAL PELLET SUSPENSIONS
Biotechnological aspects
Proefschrift ter verkrijging van
de graad van doctor in de
technische wetenschappen
aan de Technische Hogeschool Delft,
op gezag van de rector magnificus,
voor een commissie aangewezen
door het college van dekanen
te verdedigen op
woensdag 1 oktober 1980
te 14.00 uur door
Johannes Christinus van Suijdam
scheikundig ingenieur,
geboren te 's-Gravenhage
Dit proefschrift is goedgekeurd door de promotor
PROF.DR.IR. N.W.F. KOSSEN
On the front cover a scanning electron micrograph of hyphae and conidia of Pénicillium chrysogenum made by Dr. R.A. Samson of the Centraal Bureau voor Schimmelcultures, Baarn© (magnification 5200 x)
To those who are familiar with
Murphy's law
I am most grateful to those who helped to realize this thesis.
Linguistic advice: A . A . Esener
Drawings: F. Bolman and C. Warnaar Typing: J.H. van der Lee-van der Wei
CONTENTS
CHAPTER 1 INTRODUCTION
I Aim and scope 1
I I O r g a n i z a t i o n o f t h i s t h e s i s 2
I I I R e f e r e n c e s 5
CHAPTER 2 AN INOCULUM TECHNIQUE FOR THE PRODUCTION OF FUNGAL PELLETS
I I n t r o d u c t i o n 7 I I M a t e r i a l s and methods 8 organisms 8 c o n i d i a 9 growth media 9 a d d i t i v e 10 a p p a r a t u s 10 p e l l e t c o u n t i n g and s i z e m e a s u r i n g t e c h n i q u e 12 I I I R e s u l t s and d i s c u s s i o n 12 shake f l a s k e x p e r i m e n t s 12 b u b b l e column e x p e r i m e n t s 13 IV C o n c l u s i o n s 18 V R e f e r e n c e s 18
CHAPTER 3 UNSTRUCTURED MODEL FOR GROWTH OF MYCELIAL PELLETS IN
SUBMERGED CULTURES
I I n t r o d u c t i o n
I I Survey o f growth models f o r p e l l e t s
I I I I n t e g r a t e d model f o r p e l l e t growth biomass b a l a n c e s u b s t r a t e b a l a n c e oxygen b a l a n c e s e f f e c t i v e n e s s f a c t o r f o r p e l l e t s V I I 21 22 24 24 25 25 26
CHAPTER 3 ( c o n t i n u e d ) IV A u x i l i a r y r e l a t i o n s 29 e f f e c t i v e d i f f u s i v i t y 29 mean p e l l e t d e n s i t y 29 g a s - l i q u i d mass t r a n s f e r c o e f f i c i e n t 30 l i q u i d - s o l i d mass t r a n s f e r 31 V M a t e r i a l s and methods 32 V I R e s u l t s and d i s c u s s i o n 32 V I I N o m e n c l a t u r e 35 V I I I R e f e r e n c e s 36 A p p e n d i x 37
CHAPTER 4 DENSITY OF FUNGAL PELLETS
I I n t r o d u c t i o n 39 I I P e l l e t d e n s i t y AO I I I P e l l e t d e n s i t y : d e f i n i t i o n s and r e l a t i o n s 42 IV M a t e r i a l s and methods 45 p e l l e t d e n s i t y measurements 45 V R e s u l t s and d i s c u s s i o n 48 V I Consequences f o r p r o c e s s d e s i g n 52 V I I C o n c l u s i o n s 54 V I I I N o m e n c l a t u r e 54 I X R e f e r e n c e s 55
CHAPTER 5 ENERGETIC EFFICIENCY OF A MICROBIAL PROCESS WITH AN EXTERNAL POWER INPUT : THERMODYNAMIC APPROACH
I I n t r o d u c t i o n 57
I I Thermodynamic e f f i c i e n c y o f a m i c r o b i a l p r o c e s s 57
I I I A p p l i c a t i o n o f t h e thermodynamic e f f i c i e n c y on
the growth o f moulds 60
IV R e s u l t s 62
V C o n c l u s i o n s 64
V I N o m e n c l a t u r e 64
V I I R e f e r e n c e s 65
CHAPTER 6 THE RHEOLOGY OF PELLET SUSPENSIONS OF Pénicillium
ohrysogenum
I I n t r o d u c t i o n 67
CHAPTER 6 ( c o n t i n u e d ) I I Theory 68 r h e o l o g i c a l model f o r mould s u s p e n s i o n s 69 I I I M a t e r i a l s and methods 70 IV R e s u l t s and d i s c u s s i o n 72 v e r i f i c a t i o n of t h e Casson model 72 'Casson' v i s c o s i t y K 76 ' c the y i e l d s t r e s s x 79 V I n t e r p r e t a t i o n o f t h e r h e o l o g y measurements 80 shear r a t e s i n a f e r m e n t o r 80 a p p l i c a t i o n t o r h e o l o g y d a t a 82 c o m p a r i s o n o f t h e a p p a r e n t v i s c o s i t y of a f i l a m e n t o u s and a p e l l e t b r o t h 82 VI N o m e n c l a t u r e 84 V I I R e f e r e n c e s 84
CHAPTER 7 FUNGAL PELLET BREAKUP AS A FUNCTION OF SHEAR I N A
FERMENTOR I I n t r o d u c t i o n 87 I I T h e o r e t i c a l model 88 c o m p a r i s o n w i t h p r e v i o u s l y r e p o r t e d models and r e s u l t s 89 I I I M a t e r i a l s and methods 91 IV R e s u l t s and d i s c u s s i o n 92 V N o m e n c l a t u r e 94 VI R e f e r e n c e s 94
CHAPTER 8 THE SPECIFIC RESISTANCE TO FILTRATION OF A Pénicillium
chrysogenum BROTH I I n t r o d u c t i o n 97 I I F i l t r a t i o n t h e o r y 97 s p e c i f i c f i l t e r a b i l i t y r e s i s t a n c e 97 f i l t e r cake c o m p r e s s i b i l i t y 99 f i l t r a t i o n e f f i c i e n c y 99 I I I E x p e r i m e n t s 101 IV R e s u l t s and d i s c u s s i o n 102 V C o n c l u s i o n s 105 VI N o m e n c l a t u r e 106 IX
CHAPTER 8 ( c o n t i n u e d )
V I I R e f e r e n c e s
SUMMARY
SAMENVATTING
1. INTRODUCTION I A I M AND SCOPE The a i m o f t h i s s t u d y i s t o a s s e s s and d e v e l o p t h e p o t e n t i a l o f a f e r m e n t a t i o n p r o c e s s u s i n g t h e p e l l e t growth form. I n i n d u s t r i a l f e r m e n t a t i o n p r o c e s s e s w i t h moulds one u s u a l l y d e a l s w i t h a h i g h v i s c o s i t y o f t h e s u s p e n s i o n ( b r o t h ) . T h i s causes problems i n a c h i e v i n g t h e
n e c e s s a r y m i x i n g , oxygen and heat t r a n s f e r i n a f e r m e n t o r . Use o f t h e p e l l e t
growth f o r m has t h e advantage o f e l i m i n a t i n g t h e p r o b l e m o f s u s p e n s i o n v i s c o
-s i t y t o a g r e a t e x t e n t .
However, a n o t h e r p r o b l e m a r i s e s , b e i n g t h e p o s s i b l e d i f f u s i o n a l l i m i t a t i o n on
t h e t r a n s p o r t o f n u t r i e n t s i n t o a p e l l e t .
I n t h i s t h e s i s s o l v i n g t h e f o l l o w i n g q u e s t i o n s was aimed:
1. i s t h e r e a c o n v e n i e n t method t o o b t a i n p e l l e t s ?
2. I f s o , how can t h e c o u r s e o f t h e growth o f p e l l e t s be d e s c r i b e d i n a model, t o e n a b l e an e n g i n e e r t o p e r f o r m d e s i g n c a l c u l a t i o n s ?
3. What i s t h e maximum a t t a i n a b l e biomass c o n c e n t r a t i o n i n a f e r m e n t o r ,
i f t h e p e l l e t growth form i s a p p l i e d ?
4. Can t h e d i f f e r e n c e s between t h e e f f i c i e n c i e s o f a p e l l e t and f i l a m e n -t o u s p r o c e s s on m i c r o s c a l e ( s u b s -t r a -t e ) and macro s c a l e (power i n p u -t ) be e x p r e s s e d i n one s i n g l e measure? 5. Can a q u a n t i t a t i v e e v a l u a t i o n be g i v e n o f t h e r e d u c t i o n o f t h e v i s -c o s i t y ? These q u e s t i o n s a r e s u b s e q u e n t l y t r e a t e d i n c h a p t e r 2 t o 6. In a d d i t i o n t o t h e s e i n v e s t i g a t i o n s t h e e f f e c t o f s h e a r i n g f o r c e s upon p e l l e t s and t h e f i l t r a t i o n p r o p e r t i e s o f a p e l l e t b r o t h were s t u d i e d . H o p e f u l l y a f u r t h e r c o n t r i b u t i o n i s g i v e n towards t h e s o l u t i o n o f t h e problems c o n c e r n i n g t h e growth o f f u n g i i n t h e p e l l e t form. I
Mycelial pellets, a characterization
At t h i s s t a g e t h e r e a d e r may a l r e a d y have r a i s e d t h e q u e s t i o n o f what e x a c t l y
i s a p e l l e t . The c o l l e c t i v e noun f o r t h e biomass o f f u n g i i s m y c e l i u m . T h i s
m y c e l i u m c o n s i s t s o f l o n g f i l a m e n t s o f c e l l s c a l l e i l ' ' h y p h a e (shown t o g e t h e r w i t h
c o n i d i a on t h e f r o n t c o v e r ) . When t h e hyphae a r e a r r a n g e d i n such a way t h a t
the m y c e l i a l b r o t h a p p e a r s s t r u c t u r a l l y homogeneous t o t h e u n a i d e d eye and t h e
i n d i v i d u a l hyphae c a n o n l y be i d e n t i f i e d by m i c r o s c o p i c e x a m i n a t i o n , we speak
of a t r u e f i l a m e n t o u s t y p e o f growth. When t h e hyphae a g g l o m e r a t e t o s t a b l e
s p h e r i c a l a g g r e g a t e s t h a t c a n e a s i l y be i d e n t i f i e d w i t h t h e u n a i d e d eye we
speak o f p e l l e t s . T h i s t y p e o f a g g r e g a t e s must be d i s t i n g u i s h e d from t h e
non-permanent a g g r e g a t e s caused by r a p i d f l o c c u l a t i o n - d e f l o c c u l a t i o n phenomena i n
a f e r m e n t o r . The d i f f e r e n c e between t h e p e l l e t and t h e f i l a m e n t o u s growth form
i s c l e a r l y d e m o n s t r a t e d by t h e p h o t o g r a p h s on page 104, a l t h o u g h o f t e n , as 2
Solomons s a i d : 'the c h a r a c t e r i s t i c s o f l o o s e f l o c c o s e p e l l e t s a t some p o i n t
b e g i n t o merge w i t h t h o s e o f a t r u e f i l a m e n t o u s t y p e o f growth'.
I I O R G A N I Z A T I O N OF T H I S T H E S I S
The s t u d y p r e s e n t e d i n t h i s t h e s i s has been c a r r i e d o u t w i t h i n t h e
B i o t e c h n o l o g y Group o f t h e D e l f t U n i v e r s i t y o f T e c h n o l o g y . * I t i s a c o n t i n u a
-t i o n o f a p r o j e c -t s -t a r -t e d e a r l i e r by M e -t z ' . I -t i s n o -t -t h e purpose o f -t h i s
t h e s i s t o go o v e r t h e work done by Metz a g a i n . T h e r e f o r e t h e r e a d e r i s
f r e q u e n t l y r e f e r r e d t o t h e o r i g i n a l work o f Metz.
The c h a p t e r s i n t h i s t h e s i s have been s e t up as i n d e p e n d e n t s t u d i e s , each
d e a l i n g w i t h a d i f f e r e n t a s p e c t . The s u b j e c t s o f t h e s e c h a p t e r s a r e b r i e f l y
o u t l i n e d below.
Inoculation procedure
A p r e r e q u i s i t e t o s t u d y p e l l e t s i s t h e i r a v a i l a b i l i t y .
P r e v i o u s s t u d i e s on t h e f o r m a t i o n o f p e l l e t s emphasized t h e i m p o r t a n c e o f t h e
c o n c e n t r a t i o n o f s p o r e s i n t h e i n o c u l u m and t h e i n f l u e n c e o f t h e growth medium
c o m p o s i t i o n . An e x t e n s i v e l i t e r a t u r e s u r v e y i s g i v e n by M e t z ' , s l i g h t l y a l t e r e d
3 4 p u b l i s h e d e l s e w h e r e , and e a r l i e r by W h i t a k e r and Long . I n t h i s s t u d y an
e n t i r e l y d i f f e r e n t a p p r o a c h was a p p l i e d . # a d d r e s s : B i o t e c h n o l o g y Group, Dept. o f C h e m i c a l E n g i n e e r i n g , D e l f t U n i v e r s i t y o f T e c h n o l o g y , J a f f a l a a n 9, 2628 BX D e l f t , t h e N e t h e r l a n d s . 2
As r e p o r t e d i n chapter 2 , i n o c u l a t i o n was e f f e c t e d by means of f i l a m e n t o u s m y c e l i u m from a p r e c u l t u r e , e i t h e r f r o m a shake f l a s k or a s m a l l f e r m e n t o r ^ .
D u r i n g t h e c o u r s e of a f e r m e n t a t i o n i n a b u b b l e column t h i s t r u e f i l a m e n t o u s
t y p e of s u s p e n s i o n g r a d u a l l y d e v e l o p e d i n t o t h e p e l l e t growth form. T h i s c o n
-v e n i e n t i n o c u l a t i o n method f a c i l i t a t e s t h e use o f s i m p l e s y n t h e t i c growth media
and saves t h e n e c e s s i t y o f v a s t amounts of s p o r e s .
Model for the growth of mycelial pellets
I n chapter S an u n s t r u c t u r e d model i s p r e s e n t e d f o r growth o f m y c e l i a l p e l l e t s
i n submerged c u l t u r e s * ' . T h i s model i s p a r t l y an e x t e n s i o n and p a r t l y a s i m p l i
-f i c a t i o n o -f t h e model o r i g i n a l l y p o s t u l a t e d by Metz'. The model i n t e g r a t e s
growth k i n e t i c s a t the s c a l e o f t h e hyphae w i t h t h e p h y s i c a l mechanisms o f mass
t r a n s f e r p r o c e s s e s a t t h e s c a l e of the p e l l e t s and t h e f e r m e n t o r . The d i f f e
r e n c e w i t h t h e model g i v e n by Metz i s t h a t the model p r e s e n t e d h e r e i n c o r p o
-r a t e s t h e e f f e c t o f m a i n t e n a n c e -r e q u i -r e m e n t s c o n c e -r n i n g t h e c o n s u m p t i o n o f
g l u c o s e and oxygen. F u r t h e r m o r e the b a l a n c e s a r e s e t up i n a d i f f e r e n t way and
t h e s o - c a l l e d a u x i l i a r y r e l a t i o n s a r e g i v e n i n a more g e n e r a l manner. R e s u l t s
of t h e growth e x p e r i m e n t s a r e i n good agreement w i t h t h e outcome o f computer
s i m u l a t i o n s . However, f u r t h e r s t u d y i n t h i s f i e l d w o u l d be r e w a r d i n g .
Pellet density
I n l i t e r a t u r e , d a t a about t h e d e n s i t y of p e l l e t s a r e r a t h e r s c a r c e . The most
i m p o r t a n t s t u d i e s on the d e n s i t y o f f u n g a l p e l l e t s a r e r e p o r t s by James^ and
M e t z ' . However, t h e e x p e r i m e n t a l method used by t h e s e a u t h o r s t o e s t a b l i s h t h e
d e n s i t y was a r a t h e r time-consuming and h a r a s s i n g j o b . Moreover t h i s method,
u s i n g t h e t e r m i n a l f a l l i n g v e l o c i t y of p e l l e t s , y i e l d e d f r e q u e n t l y a s c a t t e r
of e x p e r i m e n t a l d a t a .
In o r d e r t o overcome t h e s e p r o b l e m s , i n chapter 4 a method i s p r e s e n t e d t o
d e t e r m i n e t h e p e l l e t d e n s i t y based upon an a c c u r a t e and c o n v e n i e n t method t o g
measure t h e number and s i z e ( d i s t r i b u t i o n ) of l a r g e amounts o f p e l l e t s .
Numbers and s i z e s were e v a l u a t e d from p h o t o g r a p h s u s i n g an automated f i e l d
s c a n n i n g t e c h n i q u e . D e s p i t e some e v i d e n c e t o t h e c o n t r a r y ' , t h e e x p e r i m e n t s on
t h e d e n s i t y s u g g e s t e d t h a t p e l l e t s become more dense w i t h i n c r e a s i n g d i a m e t e r
owing t o g r o w t h a c c o r d i n g t o a c o n s t a n t h y p h a l growth u n i t . At p e l l e t s c a l e
mass t r a n s f e r l i m i t a t i o n overwhelms t h i s mechanism f o r l a r g e r p e l l e t s , c a u s i n g
a d e c r e a s e o f the d e n s i t y w i t h i n c r e a s i n g d i a m e t e r .
Efficiency of a p e l l e t process
I n chapter 5 a g e n e r a l i z e d thermodynamic e f f i c i e n c y i s d e r i v e d w h i c h a l l o w s
e s t i m a t i o n o f t h e energy d i s s i p a t i o n i n a f e r m e n t a t i o n p r o c e s s i n c l u d i n g energy . . . 9 r e q u i r e m e n t s f o r a e r a t i o n , m i x i n g o r any o t h e r energy demanding p r o c e s s .
A p p l i c a t i o n o f t h i s e f f i c i e n c y d e m o n s t r a t e s t h a t a f e r m e n t a t i o n p r o c e s s u s i n g p e l l e t s i s more e f f i c i e n t t h a n a comparable p r o c e s s u s i n g t h e f i l a m e n t o u s growth form. O b v i o u s l y , t h e a c t i v i t y l o s s i n t h e c e n t r e o f t h e p e l l e t i s v e r y w e l l compensated on a m a c r o s c o p i c l e v e l by t h e r e d u c e d power r e q u i r e m e n t f o r a e r a t i o n . I n chapter 5 an i m p r e s s i o n i s g i v e n o f t h e ' p o i n t ' e f f i c i e n c i e s , b u t an o v e r a l l e f f i c i e n c y c a n e a s i l y be o b t a i n e d by w e i g h t a v e r a g i n g t h e s e e f f i -c i e n -c i e s w i t h r e s p e -c t t o t i m e , t h e w e i g h i n g f u n -c t i o n b e i n g t h e i n s t a n t a n e o u s t o t a l energy i n p u t . R e s u l t s o f such a p r o c e d u r e y i e l d e d f o r t h e o v e r a l l thermodynamic e f f i c i e n c y o f a p e l l e t p r o c e s s a v a l u e o f 0.18. F o r a f i l a m e n t o u s
t y p e o f p r o c e s s t h i s v a l u e was 0.09. C l e a r l y a p e l l e t p r o c e s s i s much more
e f f i c i e n t when t h e p r o d u c t i o n o f b i o m a s s ' ^ i s c o n s i d e r e d .
A d d i t i o n a l l y t h e g e n e r a l i z e d thermodynamic e f f i c i e n c y p r o v i d e s a u s e f u l e s t i m a t e
of t h e heat p r o d u c t i o n from oxygen c o n s u m p t i o n i n a m i c r o b i a l p r o c e s s and may
even be u s e d f o r a p r e l i m i n a r y e s t i m a t e o f i n v e s t m e n t c o s t s f o r l a r g e s c a l e
t. 11
b i o p r o c e s s e s
Rheology of pellet suspensions
The r h e o l o g y o f m y c e l i a l s u s p e n s i o n s has r e c e i v e d t h e a t t e n t i o n o f o u r group
f o r a c o n s i d e r a b l e p e r i o d o f t i m e , w h i c h i s i l l u s t r a t e d by an e a r l y r e p o r t o f 12 . . .
Bongenaar e t a l . The r h e o l o g y o f f i l a m e n t o u s s u s p e n s i o n s i s t r e a t e d i n d e t a i l 1 13
by Metz and Metz e t a l . I n chapter 6 t h e r h e o l o g y o f p e l l e t s u s p e n s i o n o f
Penicillium chrysogenum i s s t u d i e d ' ^ * . A t t e m p t s t o d e s c r i b e t h e r h e o l o g i c a l b e h a v i o u r o f p e l l e t b r o t h s t a r t i n g from m e c h a n i s t i c p r i n c i p l e s f a i l e d . T h e r e -f o r e an e m p i r i c a l a p p r o a c h i s p r e s e n t e d t o c o r r e l a t e t h e apparent v i s c o s i t y o -f t h e b r o t h t o t h e volume f r a c t i o n and d i a m e t e r o f t h e p e l l e t s . As e x p e c t e d , t h e r e s u l t s i n d i c a t e d a s i g n i f i c a n t d e c r e a s e of t h e a p p a r e n t v i s c o s i t y compared w i t h a f i l a m e n t o u s s u s p e n s i o n . T h i s c a n be r e g a r d e d as t h e most i m p o r t a n t advantage of t h e a p p l i c a t i o n o f t h e p e l l e t growth form.
Influence of shearing forces upon pellets
I t i s w e l l known t h a t s h e a r i n g f o r c e s i n a f e r m e n t o r c a n have a p r o f o u n d e f f e c t
T h i s e f f e c t i s i m p o r t a n t as i t s e t s an u p p e r l i m i t t o t h e power d i s s i p a t i o n i n
a f e r m e n t o r , t o p r e v e n t e x c e s s i v e damage t o t h e m y c e l i a l c e l l s .
Metz d e v e l o p e d a method f o r t h e q u a n t i t a t i v e r e p r e s e n t a t i o n o f h y p h a l m o r p h o l
-ogy, w h i c h p r o v e d t o be v e r y u s e f u l ' ' ' " ' . T h i s method e n a b l e d t h e s t u d y on t h e
i n f l u e n c e o f s h e a r i n g f o r c e s , growth r a t e and oxygen t e n s i o n upon t h e m o r p h o l
-ogy o f t h e f i l a m e n t o u s growth form'''*'.
Chapter 7 d e a l s w i t h t h e i n f l u e n c e o f s h e a r i n g f o r c e s upon p e l l e t s ' ^ . A model
i s p r e s e n t e d based upon t h e a s s u m p t i o n o f v i s c o u s s h e a r i n g f o r c e s b e i n g r e s
-p o n s i b l e f o r t h e a b r a s i o n o f the -p e l l e t s . E x -p e r i m e n t a l r e s u l t s w i t h -p e l l e t s
of P e n i c i l l i u m ohrysogenum show a good agreement w i t h t h i s m o d e l .
Filtration aharaeteristios of a pellet suspension
An a d d i t i o n a l a d v a n t a g e o f a p e l l e t b r o t h i s t h e e a s i e r s e p a r a t i o n o f t h e b i o -mass f r o m t h e b r o t h . To e s t a b l i s h t h i s e f f e c t i n a q u a n t i t a t i v e manner, i n chapter 8 t h e s p e c i f i c r e s i s t a n c e t o f i l t r a t i o n o f a P e n i c i l l i u m ohrysogenum 18 b r o t h i s i n v e s t i g a t e d . The r e s u l t s c o n f i r m e d t h e e x p e c t e d improvement o f t h e f i l t r a t i o n p r o p e r t i e s o f a p e l l e t b r o t h . I n c o m p a r i s o n w i t h a f i l a m e n t o u s b r o t h a d e c r e a s e was o b s e r v e d o f t h e s p e c i f i c f i l t r a t i o n r e s i s t a n c e o f about 60%. In f i l t r a t i o n p r a c t i c e t h i s means c o n s i d e r a b l e s a v i n g s i n energy c o s t s f o r t h e r e c o v e r y p r o c e s s s t e p . I l l R E F E R E N C E S 1. B. M e t z , Ph.D thesis, D e l f t U n i v e r s i t y o f T e c h n o l o g y ( 1 9 7 6 ) .
2. G.L. Solomons, paper p r e s e n t e d a t t h e Symposium Fungal Biotechnology,Glasgow,
September 1978.
3. B. Metz and N.W.F. K o s s e n , Biotechnol. Bioeng., 19, 781 ( 1 9 7 7 ) .
4. A. W h i t a k e r and P.A. L o n g , Process Biochemistry, November, 27 ( 1 9 7 3 ) .
5. J.C. v a n S u i j d a m , N.W.F. K o s s e n and P.G. P a u l , Eur. J. Appl. Microbiol.
Biotechnol., a c c e p t e d f o r p u b l i c a t i o n .
6. J.C. v a n S u i j d a m , H. H o l s and N.W.F. K o s s e n , s u b m i t t e d f o r p u b l i c a t i o n t o
Biotechnol. Bioeng.
7. A. James, Ph.D thesis, U n i v e r s i t y o f A s t o n , Birmingham (1973).
8. J.C. v a n S u i j d a m , N.W.F. Kossen and P.G. P a u l , s u b m i t t e d f o r p u b l i c a t i o n t o
Biotechnol. Bioeng.
9. J.A. R o e l s and J.C. v a n S u i j d a m , Biotechnol. Bioeng., 22, 463 ( 1 9 8 0 ) .
11. J.A. R o e l s and J.C. v a n S u i j d a m , P r o c e e d i n g s Vlth International
Fermen-tation Symposium, London (1980).
12. J.J.T.M. Bongenaar, N.W.F. K o s s e n , B. Metz and F.W. Meijboom, Biotechnol.
Bioeng., 15, 201 (1973).
13. B. M e t z , N.W.F. K o s s e n and J.C. v a n S u i j d a m , Adv. Bio ahem. Eng., 11, 103
( 1 9 7 9 ) .
14. J.C. van S u i j d a m , C.J. H o o i m e i j e r and N.W.F. K o s s e n , s u b m i t t e d f o r p u b l i
-c a t i o n t o Biote-chnol. Bioeng.
15. B. M e t z , E.W. de B r u i j n and J.C. v a n S u i j d a m , Biotechnol. Bioeng., a c c e p
-t e d f o r p u b l i c a -t i o n .
16. J.C. van S u i j d a m and B. M e t z , Biotechnol. Bioeng., a c c e p t e d f o r p u b l i c a t i o n .
17. J.C. v a n S u i j d a m and B. M e t z , J. Ferment. Technol., a c c e p t e d f o r p u b l i -c a t i o n .
18. J.C. van S u i j d a m , H. H o l s and N.W.F. K o s s e n , s u b m i t t e d f o r p u b l i c a t i o n t o
Biotechnol. Bioeng.
2. AN INOCULUM TECHNIQUE FOR THE PRODUCTION OF FUNGAL PELLETS
I I N T R O D U C T I O N
P e l l e t s can be a t t r a c t i v e as a l t e r n a t i v e growth form f o r t h e c u l t u r e o f
f u n g i . The most i m p o r t a n t advantage of a p e l l e t s u s p e n s i o n i s the s i g n i f i c a n t
d e c r e a s e o f t h e v i s c o s i t y i n c o m p a r i s o n t o a f i l a m e n t o u s s u s p e n s i o n . T h i s
enhances t h e d e s i r a b l e m i x i n g and mass t r a n s f e r p r o p e r t i e s o f t h e s u s p e n s i o n
c o n s i d e r a b l y . An a d d i t i o n a l advantage i s t h e e a s i e r s e p a r a t i o n o f t h e biomass
from the b r o t h . On the o t h e r s i d e , however, a p o s s i b l e s e v e r e drawback may be
i n t r o d u c e d , i . e . d e c r e a s e o f growth and a c t i v i t y due t o d i f f u s i o n a l l i m i t a
-t i o n o f oxygen and/or o -t h e r n u -t r i e n -t s i n -t o -t h e i n -t e r i o r p a r -t s o f -t h e p e l l e -t s .
F o r t h e c u l t i v a t i o n o f mushrooms i n submerged f e r m e n t a t i o n s p e l l e t s a r e
i m p o r t a n t f o r t h e f l a v o u r development, p r o b a b l y caused by a u t o l y s i s i n the
c e n t r e of t h e p e l l e t s . A secondary advantage t h e r e b y i s t h e f a v o u r a b l e
t e x t u r e o f p e l l e t s , f o r i n s t a n c e when used i n soup p r o d u c t i o n .
A n o t h e r a p p l i c a t i o n of m y c e l i a l p e l l e t s i s the use as c a r r i e r m a t e r i a l f o r
i m m o b i l i z e d enzymes where t h e y can p r o v i d e a b e t t e r s t a r t i n g m a t e r i a l f o r t h e
p r o d u c t i o n o f i m m o b i l i z e d m y c e l i a l p a r t i c l e s i n o r d e r t o c r e a t e a s t a b l e 1 2 3 . . . .
c o n t i n u o u s p r o c e s s ' ' . To o u r o p i n i o n i t i s a l s o p o s s i b l e t o use m y c e l i a l
p e l l e t s as a s u p p o r t f o r whole c e l l s . The l a r g e v o i d f r a c t i o n makes p e l l e t s
e s p e c i a l l y s u i t a b l e f o r t h i s p u r p o s e .
The i n o c u l u m q u a l i t y i s of prime i m p o r t a n c e f o r the r e s t o f t h e c o u r s e o f 4 5 .
a f e r m e n t a t i o n ' . T h e r e f o r e the o b j e c t i v e of t h i s s t u d y was t o d e v e l o p a s i m p l e s t a n d a r d i n o c u l a t i o n p r o c e d u r e t o o b t a i n p e l l e t s i n f e r m e n t o r s ,
e s p e c i a l l y i n a b u b b l e column. T h i s was p a r t o f a more g e n e r a l s t u d y o f
t h e f e a s i b i l i t y o f t h e use o f f u n g a l p e l l e t s i n i n d u s t r i a l f e r m e n t a t i o n s .
The f o r m a t i o n o f p e l l e t s i n submerged c u l t u r e s may o f t e n have seemed t o o c c u r
somewhat h a p h a z a r d l y . However, two e x t e n s i v e l i t e r a t u r e r e v i e w s have
e l u c i d a t e d much o f t h e i n f l u e n c e s p l a y i n g a r o l e upon t h e f o r m a t i o n o f p e l l e t s .
The f i r s t one has been w r i t t e n by W h i t a k e r and Long*', t h e o t h e r one more
r e c e n t l y by Metz and K o s s e n ^ .
I f one c o n f i n e s h i m s e l f t o a g i v e n chosen o r g a n i s m , s t r a i n and medium c o m p o s i t i o n f a c t o r s m o s t l y f i x e d i n a p r o c e s s f o r r e a s o n s o t h e r t h a n p e l l e t f o r m a -t i o n — -t h e n -t h r e e major v a r i a b l e s r e m a i n -t h a -t can be c o n -t r o l l e d : 1. i n o c u l u m c o n c e n t r a t i o n 2. polymer a d d i t i v e s and 3. s h e a r i n g f o r c e s i n a f e r m e n t o r G e n e r a l l y i t i s s t a t e d t h a t t h e c r i t e r i o n t o be met f o r t h e development o f 11 12 3 p e l l e t s i s an i n i t i a l s p o r e c o n c e n t r a t i o n s m a l l e r t h a n 10 - 10 spores/m ; above t h i s v a l u e f i l a m e n t o u s growth o c c u r s .
The i n f l u e n c e o f s u r f a c e a c t i v e a g e n t s and polymer a d d i t i v e s upon t h e m o r p h o l -ogy o f f u n g i , c a u s i n g a more d i s p e r s e d g r o w t h , has been w e l l documented i n
g
l i t e r a t u r e . E l m a y e r g i o b t a i n e d t h e most s t r i k i n g r e s u l t s out o f a s e r i e s o f
p o l y m e r s w i t h C a r b o p o l - 9 3 4 . T h i s a n i o n i c polymer had a s t r o n g t e n d e n c y t o
change t h e morphology o f Aspergillus niger from p e l l e t s i n t o d i s p e r s e d
mycelium. C a t i o n i c p o l y m e r s were found t o have a r e v e r s e d e f f e c t . The r e a s o n
f o r t h e e f f e c t o f a g e n t s such as C a r b o p o l i s t h a t t h e y p r e v e n t t h e a g g l o m e r a
-t i o n o f s p o r e s i n -t o c l u s -t e r s , -t h u s r a i s i n g -t h e e f f e c -t i v e spore c o n c e n -t r a -t i o n
and hence s t i m u l a t i n g p u l p y g r o w t h . A s i m i l a r e f f e c t was o b s e r v e d by Cimerman 9 . . et a l u s i n g n a t i v e p o l y m e r s l i k e a l g i n a t e s and s t a r c h w i t h t h e c u l t u r e o f Aspergillus niger. A d d i t i o n o f 0.1% a l g i n a t e r e s u l t e d i n a t w e n t y f o l d r e d u c -t i o n o f -t h e s p o r e / p e l l e -t r a -t i o , c a u s i n g -t h e f o r m a -t i o n o f more and s m a l l e r p e l l e t s . C o n c e r n i n g t h e i n f l u e n c e o f s h e a r i n g f o r c e s , i t i s w e l l known t h a t m i l d a g i t a t i o n and s h e a r s t r e s s e s f a v o u r s t h e development o f p e l l e t s . I I M A T E R I A L S AND METHODS Organisms
E x p e r i m e n t s were c a r r i e d out w i t h s t r a i n s o f Penioillium ohrysogenum s t r a i n A,
o b t a i n e d from G i s t Brocades N.V. D e l f t ; Sporotriehum pulverulentum Novobranova
CBS 67171, o b t a i n e d from C e n t r a a l Bureau v o o r S c h i m m e l c u l t u r e s and Aspergillus
niger LMD 7352, o b t a i n e d from t h e L a b o r a t o r y f o r M i c r o b i o l o g y , D e l f t U n i v e r
-s i t y o f T e c h n o l o g y .
The o r g a n i s m s were c u l t u r e d on m a l t - a g a r s l a n t s ( 1 % m a l t - a g a r , O x o i d ) a t 25°C
and were t r a n s f e r r e d t o f r e s h s l a n t s e v e r y 10 t o 14 d a y s . E v e r y two months
new s p o r e s from a d r y sand s t o c k were u s e d .
Conidia
S p o r u l a t i o n was a c h i e v e d i n K o l l e d i s h e s on m a l t a g a r w i t h 1.25% c a l c i u m
-c h l o r i d e . The s p o r e s u s p e n s i o n s were p r e p a r e d by g e n t l e w a s h i n g t h e K o l l e
d i s h e s w i t h a 0.1% T r i t o n X - l 0 0 / d i s t i l l e d w a t e r s o l u t i o n and t r a n s f e r r e d t o
s m a l l serum b o t t l e s w h i c h were k e p t i n t h e r e f r i g e r a t o r . Care was t a k e n n o t
t o use s p o r e s o l d e r t h a n one month.
The c o n c e n t r a t i o n o f s p o r e s was c o u n t e d under a m i c r o s c o p e by means o f a Thoma
c o u n t i n g chamber. C h a i n s and c l u s t e r s o f s p o r e s ( s e e f i g . 1) were c o u n t e d as
one, assuming t h e y g i v e r i s e t o t h e f o r m a t i o n o f o n l y one n u c l e u s g r o w i n g i n t o
a p e l l e t .
fig. 1: photograph of spores of Pénicillium chrysogenum (magnification 5S0x).
Growth media
The c o m p o s i t i o n s o f t h e s y n t h e t i c growth media u s e d f o r t h e c u l t u r e o f t h e
o r g a n i s m s a r e g i v e n i n t a b l e I . Medium M10 and M33 were used f o r Pénicillium
chrysogenum i n shake f l a s k s and f e r m e n t o r s r e s p e c t i v e l y . Temperature was k e p t
a t 25°C; pH was c o n t r o l l e d a t 6.8 ± 0 . 1 . F o r Sporotriahum pulverulentum a l w a y s
M20 was u s e d ; t e m p e r a t u r e 40°C; pH = 5.0. The c o n d i t i o n s f o r Aspergillus niger
were: medium M31 i n shake f l a s k s , M33 i n f e r m e n t o r s ; t e m p e r a t u r e 25°C; pH=5.0.
T a b l e I; c o m p o s i t i o n s of the growth media. C o n s t i t u e n t C o n c e n t r a t i o n kg/m MIO M20 M31** M33 g l u c o s e 10/20 10/60 10/20 10/60 l a c t i c a c i d - - 3.5
-y e a s t e x t r a c t (Oxoid) - 5 - -( N H4)2 s o4 10-
- 10 K H2P 04 7.5 13.7 1.5 K N 03 --
2.0 -MgS04. 7 aq. 1 .6 - 1 .2 0.5 FeSO.. 4 7 aq. 0.18 - 0.01 0.18 CuSO^. 0 aq. 0.008-
- 0.008 ZnSO^. 7 aq. 0.05 0.002 0.05 MnS04. 4 aq. 0.05-
0.01 0.05 C a C l2. 0 aq. 0.05 - - 0.05 NaEDTA 0.60-
-
1 .3* g l u c o s e was added a c c o r d i n g t o t h e r e q u i r e d f i n a l biomass c o n c e n t r a t i o n
** medium M31 a c c o r d i n g t o T o s o n i and G l a s s ' " .
G l u c o s e , the l i m i t i n g n u t r i e n t i n t h e s e m e d i a , was added a c c o r d i n g t o t h e
r e q u i r e d f i n a l biomass c o n c e n t r a t i o n . The media were s t e r i l i z e d a t 121°C f o r
20 m i n u t e s w i t h t h e e x c e p t i o n o f g l u c o s e and l a c t i c a c i d w h i c h were s t e r i l i z e d
s e p a r a t e l y a t 110°C f o r 30 m i n u t e s .
Additive
As p o l y m e r i c a d d i t i v e C a r b o p o l - 9 3 4 was u s e d (B.F. G o o d r i c h Chem. Comp.).
A l t h o u g h C a r b o p o l a c t s as a w e t t i n g agent w i t h an a f f i n i t y f o r w a t e r due t o
i t s h y d r o p h i l i c n a t u r e , c a r e was t a k e n w h i l e d i s p e r s i n g t h e polymer t o a v o i d
clumps b e i n g formed. A f t e r d i s s o l v i n g t h e pH was a d j u s t e d .
Apparatus
The f e r m e n t a t i o n s were c a r r i e d out i n a 2 0 - l i t r e f e r m e n t o r ( B i o l a f i t t e , t y p e
S20L) and a 3 0 - l i t r e w o r k i n g volume b u b b l e column.
P r e c u l t u r e s were c a r r i e d out i n shake f l a s k s (0.5 o r 2.0 1 E r l e n m e y e r f l a s k s )
o r i n a 2 - l i t r e B i o l a f i t t e f e r m e n t o r .
The b u b b l e column was s p e c i a l l y d e s i g n e d and c o n s t r u c t e d f o r t h i s p r o j e c t i n
t h e m e c h a n i c a l workshop o f o u r l a b o r a t o r y . A s c h e m a t i c d i a g r a m i s g i v e n i n
f i g . 2.
Q
fig. 2 : schematic diagram of fig- 3: schematic diagram of
bubble column. Dimensions sparger (top view), in meters. Dimensions in mm.
The f e r m e n t o r c o n s i s t e d o f a g l a s s c y l i n d e r e n c l o s e d between a s t a i n l e s s s t e e l
bottom and t o p . A d o u b l e w a l l c o o l i n g j a c k e t was p l a c e d a t t h e b o t t o m o f t h e
c y l i n d e r t o c o n t r o l t h e t e m p e r a t u r e . Through t h i s j a c k e t p r o b e s were i n s e r t e d
f o r d i s s o l v e d oxygen, pH and t e m p e r a t u r e c o n t r o l systems as w e l l as a s a m p l i n g
p o r t w i t h an i n n e r d i a m e t e r o f 10 mm. T h i s d e s i g n e l i m i n a t e d e x c e s s i v e growth
o f m y c e l i u m on o b j e c t s s t i c k i n g t h r o u g h t h e s u r f a c e o f t h e l i q u i d , and e n s u r e d
v i s u a l o b s e r v a t i o n o f t h e p r o c e s s .
A s p e c i a l f e a t u r e o f t h e b u b b l e column was t h e s p a r g e r ( s e e f i g . 3 ) . I t was
made o f e i g h t s i n t e r e d s t a i n l e s s s t e e l p i p e s , w i t h a mean p o r e s i z e o f 3
m i c r o n .
Vellet counting and size measuring technique
P e l l e t c o n c e n t r a t i o n s and s i z e s were d e t e r m i n e d by t a k i n g a l a r g e sample out
of t h e f e r m e n t o r . T h i s sample was photographed as a w h o l e u s i n g a g r a p h i c
f i l m t o o b t a i n p h o t o g r a p h s w i t h good c o n t r a s t . The p h o t o g r a p h s were t h e n
a n a l y s e d by means o f a Quantimet 720 system (Image A n a l y s i n g Computer, IMANCO
L t d . ) . T h i s Quantimet 720 system uses an automated f i e l d s c a n n i n g t e c h n i q u e ,
p r o d u c i n g p e l l e t c o u n t s and e n a b l i n g e v a l u a t i o n s o f t h e s i z e d i s t r i b u t i o n .
T h i s method i s v e r y c o n v e n i e n t t o a n a l y s e l a r g e numbers o f p a r t i c l e s . I n t h i s
way v e r y l a r g e amounts o f p e l l e t s can be p r o c e s s e d (of t h e o r d e r o f hundreds
p e r p h o t o g r a p h ) . The d i s a d v a n t a g e s a r e : t h e s a m p l i n g t e c h n i q u e becomes more
c r i t i c a l , the p h o t o g r a p h s have t o be o f h i g h q u a l i t y ( c o n t r a s t ) and a l l t h e
p e l l e t s must be l y i n g s e p a r a t e l y from each o t h e r .
I l l R E S U L T S AND D I S C U S S I O N
Shake flask experiments
I n t a b l e I I t h e r e s u l t s o f shake f l a s k e x p e r i m e n t s w i t h Sporotrichum
pulveru-lentum a r e p r e s e n t e d . Shake f l a s k s were i n c u b a t e d a t 40°C i n an o r b i t a l
i n c u b a t o r . Two t y p e s o f f l a s k s were u s e d : w i t h o u t and w i t h b a f f l e s ( d e t a i l s
a r e g i v e n by van S u i j d a m e t a l ' ' ) . The b a f f l e d f l a s k s were shaken w i t h an
o r b i t i n g speed o f 80 rpm, t h e o t h e r s a t 150 rpm. The i n o c u l u m c o n c e n t r a t i o n 10 3
was 10 spores/m ; measurements were p e r f o r m e d a f t e r 42 h o u r s .
T a b l e I I : shake f l a s k e x p e r i m e n t s w i t h Sporotrichum pulvevumentum showing t h e
i n f l u e n c e o f C a r b o p o l and b a f f l e s . exp. C a r b o p o l kg/m3 b a f f l e s s p o r e / p e l l e t r a t i o mean p e l l e t d i a m e t e r 1 0 "3 m S7/S8 _ _ 8.8 x l O 2.6 S13/S14 3 - 0 . 2 5 x l 03 1.3 S15/S16
-
+ 1.9 x l O3 1.7 S21/S26 3 + 40.0 0.3 12As c a n be seen, t h e r e s u l t s c o n f i r m t h e i n f l u e n c e o f s h e a r i n g f o r c e s and
e s p e c i a l l y o f C a r b o p o l as an a d d i t i v e . A s i g n i f i c a n t r e d u c t i o n i n t h e s p o r e /
p e l l e t r a t i o and t h e mean p e l l e t d i a m e t e r i s o b s e r v e d . The most o u t s p o k e n
r e s u l t s a r e found when a c o m b i n a t i o n o f b o t h b a f f l e s and C a r b o p o l a d d i t i o n i s
a p p l i e d : a d e c r e a s e o f more t h a n 200 t i m e s i n t h e s p o r e / p e l l e t r a t i o and a 80%
d e c r e a s e i n t h e mean p e l l e t d i a m e t e r .
E x p e r i m e n t s w i t h Penioillium chvysogenum and Aspergillus niger showed s i m i l a r 3
r e s u l t s . A d d i n g C a r b o p o l a t a c o n c e n t r a t i o n o f 3 kg/m caused a decrease of a p p r o x i m a t e l y t h i r t y t i m e s i n t h e s p o r e / p e l l e t r a t i o . An a d d i t i o n a l e f f e c t o f
t h e use o f C a r b o p o l was t h e absence o f t h e m o s t l y o b s e r v e d r i n g o f w a l l growth
i n a shake f l a s k . T h i s p r o b a b l y improves t h e u n i f o r m g e r m i n a t i o n of t h e s p o r e s . F i n a l l y , w i t h a l l t h r e e o r g a n i s m s i n o c u l u m c o n c e n t r a t i o n s h i g h e r t h a n l o ' ' spores/m i n c o m b i n a t i o n w i t h t h e a d d i t i o n o f C a r b o p o l i n v a r i a b l y produced
d i s p e r s e d m y c e l i a l growth.
12 . .
I n p r e v i o u s work by Metz media were used c o n t a i n i n g CaCO, a t t h e c o n c e n t r a -3
t i o n o f 3 kg/m to cause p e l l e t f o r m a t i o n . The r e s u l t s of t h i s study i n d i c a t e t h a t t h e p r e s e n c e of s o l i d p a r t i c l e s i n the growth media i s n o t e s s e n t i a l f o r
p e l l e t f o r m a t i o n .
Bubble aolumn experiments
I n o r d e r t o f i n d the most s u i t a b l e i n o c u l a t i o n t e c h n i q u e f o r a f e r m e n t a t i o n
i n a b u b b l e column, t h r e e a l t e r n a t i v e s were i n v e s t i g a t e d , i . e . :
1. i n o c u l a t i o n d i r e c t w i t h s p o r e s i n t o t h e f e r m e n t o r ,
2. from a p r e c u l t u r e c o n s i s t i n g o f s m a l l p e l l e t s and 3 . i n o c u l a t i o n from a p r e c u l t u r e of pulpy mycelium.
I n o c u l a t i o n w i t h s p o r e s d i r e c t i n t o t h e f e r m e n t o r , was t e s t e d w i t h a l l t h r e e
organisms used. W i t h Sporotriahum pulverulentum t h i s r e s u l t e d i n a g r a d u a l
development o f t h e m y c e l i u m i n t o p e l l e t s of f a i r l y u n i f o r m s i z e (see f i g . 4 ) .
T h i s r e s u l t h a r d l y came as a s u r p r i s e , s i n c e i t i s w e l l known t h a t
Sporotri-ahum pulverulentum forms p e l l e t s v e r y e a s i l y . One d i s a d v a n t a g e r e m a i n s ; i t
t a k e s a v e r y l a r g e amount o f s p o r e s t o o b t a i n a d e s i r e d p e l l e t c o n c e n t r a t i o n .
E x p e r i m e n t s t o s t u d y t h e b e h a v i o u r o f Aspergillus niger f a i l e d because o f t h e
v e r y s t r o n g tendency o f t h e s p o r e s t o f l o a t on t h e s u r f a c e o f t h e b r o t h . T h i s
f l o t a t i o n phenomenon i s v e r y common w i t h s p o r e s o f f u n g i . The low a g i t a t i o n
i n t e n s i t y i n a b u b b l e column enhances t h i s e f f e c t , making t h e r e s u l t s o f an
e x p e r i m e n t l i k e t h i s , m e a n i n g l e s s .
fig. 4: pellets of Sporotrichum pulverumentum, cultured in a bubble column. 10 3
I n i t i a l spore concentration 8x10 spores/m . Sample taken after 68 hours; mean p e l l e t diameter ~ 2 mm.
fig. 5." growth of Pénicillium chrysogenum in bubble column, after inoculation direct with spores.
W i t h Penicillium chrysogenwn t h i s e f f e c t a l s o o c c u r r e d , a l t h o u g h l e s s p r o -nounced. A g g l o m e r a t i o n o f s p o r e s w i t h d r o p l e t s o f a n t i - f o a m ( p o l y p r o p y l e n e g l y c o l , P2000) c a u s e d an uneven g e r m i n a t i o n o f t h e s p o r e s and t h e f o r m a t i o n o f i r r e g u l a r shaped p e l l e t s ( f i g . 5 ) . I n o c u l a t i o n w i t h a p r e c u l t u r e c o n s i s t i n g of s m a l l p e l l e t s c u l t u r e d i n shake f l a s k s , r e s u l t e d f o r a l l t h r e e o r g a n i s m s i n t o the f o r m a t i o n o f a p e l l e t t y p e of s u s p e n s i o n . I n f i g u r e 6 t h e r e s u l t s of a p e l l e t s i z e d i s t r i b u t i o n a f t e r
49 h o u r s o f a f e r m e n t a t i o n w i t h Aspergillus niger i n the b u b b l e column a r e
shown. The r e l a t i v e f r e q u e n c y v e r s u s t h e p e l l e t d i a m e t e r i n t e r v a l s a r e g i v e n
i n t h i s f i g u r e . The t o t a l number o f o b s e r v a t i o n s was 118. As can be seen, a
v e r y w i d e s i z e d i s t r i b u t i o n i s o b t a i n e d . 0.075 0 050 0 025 -J _ L 1.5 2.0 2.5 ao a5 4.0 5.0 5.5
fig. 6: size distribution of pellets of Aspergillus niger after 49 hours in a bubble column.
The t h i r d i n o c u l a t i o n t e c h n i q u e examined made use o f a p r e c u l t u r e o f a
f i l a m e n t o u s t y p e of m y c e l i u m . T h i s m y c e l i u m was e i t h e r c u l t u r e d i n a shake
f l a s k o r i n a s m a l l 2 1 w o r k i n g volume s t i r r e d f e r m e n t o r . I t was a s t o u n d i n g
to f i n d t h a t a t r u e f i l a m e n t o u s t y p e o f m y c e l i u m i n t h e b u b b l e column a l w a y s
p. l a d u a l l y d e v e l o p e d i n t o more dense h y p h a l f l o e s u n t i l f i n a l l y becoming
c l e a r l y d i s t i n g u i s h a b l e p e l l e t s . I n t h e f i g u r e s 7 t o 10 an i m p r e s s i o n i s g i v e n
of t h i s phenomenon t a k i n g p l a c e w i t h a c u l t u r e o f Penicillium chrysogenum.
F i g u r e 7 shows t h e l o o s e f l o e s o f t h e p u l p y m y c e l i u m from t h e p r e c u l t u r e ;
f o l l o w i n g t r a n s f e r t o t h e b u b b l e column a f t e r a few h o u r s we a l r e a d y see t h e
i n d i v i d u a l hyphae and l o o s e f l o e s a g g r e g a t e (see f i g . 8 ) . A f t e r 20 - 30 h o u r s
p e l l e t s a r e formed, a t f i r s t as c h a i n - l i k e n u c l e i w i t h i n f l o c c o s e f i l a m e n t o u s
m y c e l i u m , l a t e r on more and more as i n d i v i d u a l p e l l e t s ( f i g . 9 and 10).
fig. 7: pulpy mycelium of Pénicillium fig. 8: developing pellets; 10 h chrysogenum from preculture; after transfer to tower fer— 44 h after inoculation. mentor.
fig. 9: pellets, 17 h after transfer. fig. 10: pellets, 27 h after transfer.
I n f i g u r e s 11a t o 11c t h e p e l l e t s i z e d i s t r i b u t i o n s a r e g i v e n f o r a c u l t u r e o f
Aspergillus niger as a f u n c t i o n o f t i m e . C l e a r l y d i s c e r n a b l e i s t h e i n c r e a s e
o f t h e 'mean' p e l l e t d i a m e t e r w i t h t i m e . A c o m p a r i s o n o f t h e s i z e d i s t r i b u t i o n s
g i v e n i n f i g u r e s 6 and 11c b o t h samples t a k e n a t a s i m i l a r growth phase
-i l l u s t r a t e s t h e o b s e r v a t -i o n t h a t t h e method u s -i n g a p u l p y p r e c u l t u r e p r o d u c e s s m a l l e r p e l l e t s o f a more homogeneous s i z e . 0.25- 0.20- 0.15-0.10 0.05 n = 2 4 4
--rfk^,
0.20- 0.15-0.10 0.05 n = 3 3 6 0.5 1.0 1.5 2.0 2.5 3.0 3.5 -dCIO m)fig. 11: pellet size distribution of Aspergillus niger as a function of time; 24 (fig. a), 29 (fig. b) and 48 (fig. a) hours after inoculation.
T h i s i s o f c o u r s e an advantage from an e n g i n e e r i n g p o i n t o f v i e w . Taken i n t o
a c c o u n t t h e number o f s p o r e s used i n t h e p r e c u l t u r e s , t h e s p o r e / p e l l e t r a t i o
•
d e c r e a s e d i n t h i s way from 2 - 20 t o 0.1 - 0.2 f o r Penicillium ahrysogenum.
T h i s means t h a t from one spore f i n a l l y 5 t o 10 p e l l e t s were formed.
To make s u r e t h e p r e c u l t u r e c o n s i s t e d s o l e l y o f f i l a m e n t o u s m y c e l i u m t h e f o l -3
l o w i n g p r o c e d u r e was h e l d : i n a shake f l a s k 3 kg/m C a r b o p o l was added t o t h e
medium, t h i s t o g e t h e r w i t h a h i g h i n o c u l u m c o n c e n t r a t i o n o f s p o r e s (> l o '1 spores/m^) a l w a y s p r o d u c e d p u l p y g r o w t h . L a t e r on i n t h i s p r o j e c t i t was found
3 3 t h a t l o w e r i n g t h e C a r b o p o l c o n c e n t r a t i o n f i r s t t o 1 kg/m and even t o 0.5 kg/m
d i d not change t h i s e f f e c t . I n a s t i r r e d f e r m e n t o r , a d d i n g C a r b o p o l i n
com-b i n a t i o n w i t h a r e l a t i v e l y h i g h s t i r r e r speed had t h e same r e s u l t s .
I t was f o u n d , however, t h a t one r e s t r i c t i o n i s imposed upon t h i s i n o c u l a t i o n t e c h n i q u e : t h e i n o c u l u m s h o u l d n o t be too h e a v y , i . e . i n i t i a l c o n c e n t r a t i o n s
, 3
e x c e e d i n g about 1 kg/m r e s u l t e d m p u l p y growth.
IV C O N C L U S I O N S
An i n o c u l a t i o n t e c h n i q u e u s i n g f i l a m e n t o u s m y c e l i u m t o p r o d u c e p e l l e t s i n a
b u b b l e column p r o v e d t o be a c o n v e n i e n t method, y i e l d i n g many s m a l l p e l l e t s
w i t h a f a i r l y homogeneous s i z e d i s t r i b u t i o n . I n t h e e a r l y phase o f t h e g e r m i n a t i o n o f f u n g a l s p o r e s t h e p r e s e n c e o f p o l y m e r s l i k e C a r b o p o l i s v e r y i m p o r t a n t , i n t h a t t h i s s t r o n g l y e f f e c t s t h e e l e c t r o s t a t i c f o r c e s among s p o r e s . I n a l a t e r phase of t h e growth o f m y c e l i u m t h e i n f l u e n c e of s h e a r i n g f o r c e s becomes more p r e d o m i n a n t . Acknowledgement
The a u t h o r s acknowledge t h e h e l p o f t h e Department o f C i v i l E n g i n e e r i n g o f
t h e D e l f t U n i v e r s i t y o f T e c h n o l o g y f o r p u t t i n g t o our d i s p o s a l t h e Quantimet
720 system.
V R E F E R E N C E S
1. I . K a r u b e , K . I . H i r a n o and S. S u z u k i ; Biotechnol. Bioeng., 19, 1233-1238
( 1 9 7 7 ) .
2. Y. M o r i k a w a , I . Karube and S. S u z u k i ; Biotechnol. Bioeng., 21, 261-270,
( 1 9 7 9 ) .
3. S. S u z u k i and I . K a r u b e ; P r o d u c t i o n o f a n t i b i o t i c s and enzymes by
i m m o b i l i z e d w h o l e c e l l s , ACS Symposium Series, 106, 59-72, ( 1 9 7 9 ) .
4. C T . Calam; Process Biochemistry, A p r i l , 7-12 (1976).
5. J . M e y r a t h and J . Suchanek; Methods in Microbiology, 7B, 159-209 (1972).
6. A. W h i t a k e r and P.A. Long; Process Biochemistry, November, 27-31 ( 1 9 7 3 ) .
7. B. Metz and N.W.F. K o s s e n ; Biotechnol. Bioeng., 19, 781-799 (1977).
8. H. E l m a y e r g i ; J. Ferment. Technol., 53, 722-729 (1975).
9. A. Cimerman, V. J o h a n i d e s and S. S k a f a r ; Paper p r e s e n t e d a t t h e Fifth
International Fermentation Symposium, B e r l i n (1976).
10. A.L. T o s o n i and D.G. G l a s s , Canadian Patent: 671.647 ( 1 9 6 3 ) .
11. J.C. v a n S u i j d a m , N.W.F. Kossen and A.C. J o h a ; Biotechnol. Bioeng., 2 0 ,
1695-1709 (1978).
12. B. M e t z ; Ph.D thesis, D e l f t U n i v e r s i t y o f T e c h n o l o g y (1976).
3. UNSTRUCTURED MODEL FOR GROWTH OF MYCELIAL PELLETS IN SUBMERGED CULTURES
I I N T R O D U C T I O N
To e v a l u a t e t h e p o t e n t i a l o f a f e r m e n t a t i o n p r o c e s s u s i n g m y c e l i a l p e l l e t s , i t
i s v i t a l t o have an adequate m a t h e m a t i c a l model t o p r e d i c t growth r a t e s ,
sub-s t r a t e c o n sub-s u m p t i o n e t c . The purposub-se o f t h i sub-s c o n t r i b u t i o n i sub-s t o p r e sub-s e n t a model
t h a t i n t e g r a t e s k i n e t i c s and mass t r a n s f e r a s p e c t s t o d e s c r i b e t h e g r o w t h o f
m y c e l i a l p e l l e t s of t h e mould Penicillium ahrysogenym i n submerged c u l t u r e s ,
t h e macro k i n e t i c s .
Growth o f f u n g i has r e c e i v e d wide a t t e n t i o n i n l i t e r a t u r e . I n p r i n c i p l e g r o w t h
of f u n g i can o c c u r i n two m o r p h o l o g i c a l modes, i . e . a form i n w h i c h t h e
m y c e l i a l hyphae appear i n a d i s p e r s e d way, u s u a l l y c a l l e d f i l a m e n t o u s o r p u l p y
g r o w t h ; t h e o t h e r i n w h i c h s p h e r i c a l s t a b l e a g g r e g a t e s a r e formed, c a l l e d
p e l l e t s .
In submerged c u l t u r e s growth i n f i l a m e n t o u s form has been o b s e r v e d t o f o l l o w 1-4 . . .
t h e e x p o n e n t i a l law . I t has been found t h a t i n d i v i d u a l hyphae o n l y grow a t
the t i p s , h a v i n g a l i n e a r e x t e n s i o n r a t e . E x p o n e n t i a l growth i s m a i n t a i n e d by
c o n t i n u o u s b r a n c h i n g of t h e hyphae. T h i s can be e x p r e s s e d by i n t r o d u c i n g t h e
h y p h a l growth u n i t , t h e mean l e n g t h o f hyphae p e r g r o w i n g t i p . I t can e a s i l y be
shown t h a t a l i n e a r e x t e n s i o n r a t e i n c o m b i n a t i o n w i t h a c o n s t a n t h y p h a l growth
5 . . 4 u n i t c o n c e p t r e s u l t s i n e x p o n e n t i a l growth. Metz and van S u i j d a m and Metz
o b s e r v e d e x p e r i m e n t a l l y t h i s h y p h a l growth u n i t t o be independent o f t h e
s p e c i f i c growth r a t e .
Growth i n t h e form o f p e l l e t s has been d i s c u s s e d e x t e n s i v e l y i n r e v i e w s by 6 7
W h i t a k e r and Long and Metz and Kossen . I n g e n e r a l i t has been a c c e p t e d t h a t
p e l l e t s do n o t always grow e x p o n e n t i a l l y because o f mass t r a n s f e r l i m i t a t i o n s
i n t h e p e l l e t . However, t h e r e i s no consensus i n l i t e r a t u r e about w h i c h growth
model can be a p p l i e d b e s t f o r p e l l e t s .
I I S U R V E Y OF GROWTH MODELS FOR P E L L E T S
A p a r t from t h e f a m i l i a r e x p o n e n t i a l growth l a w , w h i c h can be w r i t t e n by:
r = u X (1) x m
t h e w e l l known Monod e q u a t i o n has been a p p l i e d :
rx = u X (2)
where the s p e c i f i c growth r a t e u i s g i v e n by:
y = u {S/(K + S)} (3) m s
g
Koch a p p l i e d t h e common l o g i s t i c biomass r a t e e q u a t i o n t o e x p e r i m e n t a l d a t a
.9 . . . .
of T r i n c i . T h i s e q u a t i o n can be g i v e n i n d i f f e r e n t i a l form by:
r = y (1 - X / X ) X (4)
x m m
C h i u and Z a j i c ' ^ u s e d t h e s o - c a l l e d Gompertz's law t o d e s c r i b e the growth o f
m y c e l i a l p e l l e t s . T h i s law c a n be r e p r e s e n t e d by:
rx = C] X exp (- a t ) (5)
The most g e n e r a l growth model r e p o r t e d i s t h e Cube-root law (see Metz and
K o s s e n ^ ) . U s u a l l y t h i s law i s g i v e n i n t h e form f o r b a t c h growth by:
xl / 3 = x^ / 3 + k u v t ( 6 )
The r a t e e q u a t i o n b e h i n d t h i s form i s :
rx = 3 k 0) y X2/3 (7)
The Cube-root l a w i s i n f a c t a c o m b i n a t i o n of the e x p o n e n t i a l growth law and
mass t r a n s f e r l i m i t a t i o n e x p r e s s e d by t h e a s s u m p t i o n o f a p e r i p h e r a l zone o f w i d t h to t h a t o n l y c o n t r i b u t e s t o growth' ' . F i g u r e 1 summarizes t h e p r e d i c t i o n s by t h e s e e q u a t i o n s o f X ( t ) f o r b a t c h growth i n a p l o t o f t h e d i m e n s i o n l e s s biomass c o n c e n t r a t i o n X/Xq v e r s u s the d i m e n s i o n -l e s s t i m e 6 = y t ( o r a t ) . m
fig. 1: comparison of various reported rate equations in integrated form for the hiomass production in case of pellet growth; X^X^ - 250,
1 /3 Cj = 5.5 a, k
to
-
1.5 kg- /m.On t h e b a s i s of t h i s p l o t i t c a n be e x p e c t e d t h a t e x p e r i m e n t a l d a t a c a n a l w a y s
be f i t t e d by a t l e a s t one o f t h e d e s c r i b e d models. Moreover some o f t h e p a r a
-m e t e r s can be a d j u s t e d a t w i l l ( e . g . c^ and a i n e q u a t i o n ( 5 ) ) .
A n o t h e r p r o b l e m i s t h a t i t i s n o t a l w a y s p o s s i b l e t o d i s c r i m i n a t e between
models due t o e x p e r i m e n t a l e r r o r s m a i n l y a s s o c i a t e d w i t h t h e biomass d e t e r m i -12 . . . • n a t i o n . C a r r o a d and W i l k e showed i n a s t a t i s t i c a l a n a l y s i s t h a t e x p e r i m e n t a l
d a t a on t h e growth i n p e l l e t form o f Polyporus versicolor and Pleurotus
ostreatus c o u l d be e q u a l l y w e l l d e s c r i b e d by t h e e x p o n e n t i a l growth l a w and
t h e Cube-root law.
A l t h o u g h some o f t h e above m e n t i o n e d models i n d i c a t e a k i n d o f l i m i t e d l e v e l
of t h e biomass c o n c e n t r a t i o n i n c a s e o f b a t c h growth ( e q u a t i o n ( 4 ) , (5) and
( 7 ) ) as t h e y s h o u l d do, t h e main o b j e c t i o n a g a i n s t t h e s e models i s t h a t t h e y
a r e autonomous i . e . t h e biomass r a t e e q u a t i o n i s n o t r e l a t e d t o t h e c o n c e n t r a
-t i o n o f -t h e l i m i -t i n g s u b s -t r a -t e . Moreover mass -t r a n s f e r l i m i -t a -t i o n s a r e n o -t
t a k e n i n t o a c c o u n t . T h e r e f o r e i n o r d e r t o i n t r o d u c e t h e s e phenomena, a c o m b i
-n a t i o -n has t o be used o f mass t r a -n s f e r l i m i t a t i o -n e f f e c t s w i t h e.g. t h e Mo-nod
e q u a t i o n , w h i c h has t h e a d d i t i o n a l a d v a n t a g e o f an i n t u i t i v e a s s o c i a t i o n w i t h
a b i o l o g i c a l b a c k g r o u n d .
Metz d e v e l o p e d a model f o r p e l l e t growth based upon t h e s e c o n s i d e r a t i o n s . The
model p r e s e n t e d i n t h i s s t u d y c a n be c o n s i d e r e d t o be an e x t e n s i o n and m o d i f i
-c a t i o n o f t h e model o r i g i n a l l y d e r i v e d by Metz.
I l l I N T E G R A T E D MODEL FOR P E L L E T GROWTH
The main e l e m e n t s o f t h e i n t e g r a t e d model t o d e s c r i b e t h e growth o f m y c e l i a l
p e l l e t s i n submerged c u l t u r e s a r e a biomass b a l a n c e , a s u b s t r a t e b a l a n c e f o r
the l i m i t i n g c a r b o n s o u r c e , an oxygen b a l a n c e f o r t h e l i q u i d phase and an
oxygen b a l a n c e f o r t h e p e l l e t s . F o r b a t c h g r o w t h t h e l i m i t i n g n u t r i e n t f o r t h e i n t e r i o r p a r t s o f t h e p e l l e t s w i l l be oxygen. T h e r e f o r e t h e oxygen b a l a n c e f o r the p e l l e t s i s v e r y i m p o r t a n t , as i t i n c l u d e s t h e e f f e c t o f d i f f u s i o n a l l i m i t a -t i o n i n -t h e p e l l e -t s . The e f f e c -t s o f -t h i s oxygen l i m i -t a -t i o n w i l l be e x p r e s s e d by an e f f e c t i v e n e s s f a c t o r , w h i c h w i l l be d i s c u s s e d i n a f o l l o w i n g s e c t i o n . I n f i g u r e 2 a s i m p l i f i e d b l o c k d i a g r a m i s shown t o d e m o n s t r a t e s c h e m a t i c a l l y t h e i n t e r r e l a t i o n s between t h e d i f f e r e n t e l e m e n t s . Pellet density Pellet diameter Biomass balance Substrate balance Effectiveness Oxygen balances
fig. 2: simplified block diagram of the model for p e l l e t growth.
Biomass balance
For t h e r e a s o n s d i s c u s s e d above, t h e Monod e q u a t i o n has been c h o s e n t o d e s c r i b e
the growth o f t h e m y c e l i u m . As l o n g as oxygen i s p r e s e n t , t h e biomass p r o d u c
-t i o n r a -t e as a f u n c -t i o n o f -t h e l i m i -t i n g c a r b o n s o u r c e c a n be w r i -t -t e n a s :
rx = ym {S/(Ks + S)} X (8)
i n c a s e o f b a t c h growth.
A u t o l y s i s o f m y c e l i a l c e l l s s e t s i n when t h e s u b s t r a t e o r oxygen c o n c e n t r a t i o n
becomes z e r o . The r a t e o f a u t o l y s i s i s assumed t o be o f f i r s t o r d e r , s o :
r = k X (9) aut a u t
For b a t c h growth t h e t i m e dependence o f t h e biomass c a n t h e n be r e p r e s e n t e d by
t h e f o l l o w i n g b a l a n c e :
dX/dt = r - r (10) x a u t
Substrate balance
By a p p l i c a t i o n of t h e f a m i l i a r r e l a t i o n s h i p between t h e s u b s t r a t e c o n s u m p t i o n
r a t e and t h e biomass growth r a t e , t h e s u b s t r a t e b a l a n c e c a n c o n v e n i e n t l y be
w r i t t e n by:
r (U X/Y + m X) (11) s sx s
where a g a i n t h e s p e c i f i c growth r a t e u i s g i v e n by t h e Monod e q u a t i o n as i n
e q u a t i o n ( 8 ) .
Oxygen balances
The oxygen b a l a n c e f o r the l i q u i d phase can be r e p r e s e n t e d by:
d C1/ d t = kxa (C* - C1) - ksap (C^ - Cg) (12)
where t h e f i r s t term of t h e r i g h t hand s i d e o f t h e e q u a t i o n r e p r e s e n t s t h e t r a n s f e r o f oxygen a c r o s s t h e g a s - l i q u i d i n t e r f a c e ; t h e second term s t a n d s f o r
: lie t r a n s f e r o f oxygen a c r o s s t h e l i q u i d - p e l l e t boundary l a y e r .
For t h e oxygen b a l a n c e o v e r t h e p e l l e t s h o l d s :
dC / d t = k a (C. - C ) - R0„ (13) s s p 1 s 2
whore RO., i s t h e macro oxygen consumption r a t e o f t h e p e l l e t s p e r u n i t volume
|U> I. lot b r o t h .
Thus f a r no d i s t i n c t i o n has emerged between t h e d e s c r i p t i o n o f m y c e l i a l growth
i n p e l l e t o r f i l a m e n t o u s form. To d e a l w i t h t h e s p e c i a l s i t u a t i o n e n c o u n t e r e d
i n p e l l e t s an e f f e c t i v e n e s s f a c t o r i s i n t r o d u c e d .
Effectiveness factor for pellets
F o r a d e s c r i p t i o n o f t h e d i f f u s i o n o f oxygen i n t o a p e l l e t t h e f o l l o w i n g w e l l
known d i f f e r e n t i a l e q u a t i o n c a n be u s e d ' , assuming pseudo s t e a d y s t a t e f o r t h e
oxygen p r o f i l e i n a p e l l e t : TO ( d2C / d r2 + 2/r dC /dr) = r„ (14) e p p 02 I n t h i s e q u a t i o n ID i s t h e e f f e c t i v e d i f f u s i v i t y i n a p e l l e t . I n t h e p a s t many a u t h o r s have d e a l t w i t h t h e p r o b l e m o f s o l v i n g e q u a t i o n ( 1 4 ) , e i t h e r n u m e r i c a l -l y , o r f o r s p e c i a -l c a s e s a n a -l y t i c a -l -l y . I n t h i s s t u d y a s i m p -l e g r a p h i c a -l a p p r o a c h i s a p p l i e d . D u r i n g t h e d e r i v a t i o n o f t h e t h e o r i e s o u t l i n e d below, t h e
a s s u m p t i o n has been made t h a t g r a d i e n t s o f t h e l i m i t i n g c a r b o n s u b s t r a t e
( g l u c o s e ) i n a p e l l e t a r e n e g l i g i b l e . T h i s a s s u m p t i o n i s j u s t i f i e d f o r b a t c h
growth where g l u c o s e i s p r e s e n t i n e x c e s s compared t o t h e d i s s o l v e d oxygen
c o n c e n t r a t i o n . I t has been r e p o r t e d i n l i t e r a t u r e t h a t t h e r e l a t i o n between t h e
oxygen c o n s u m p t i o n r a t e and t h e oxygen t e n s i o n f o l l o w s a p a t t e r n as shown i n ,. ,11.13
f i g u r e 3
fig. 3: specific oxygen consumption rate as a function of the oxygen tension.
Below a c e r t a i n c r i t i c a l oxygen c o n c e n t r a t i o n t h e oxygen c o n s u m p t i o n r a t e f a l l s
down d r a s t i c a l l y ; above t h i s v a l u e t h e r a t e i s c o n s t a n t . F o r t u n a t e l y , f o r
Pénicillium ahrysogenum t h e c r i t i c a l oxygen c o n c e n t r a t i o n i s r a t h e r l o w ; t h e
n u m e r i c a l v a l u e b e i n g i n t h e r a n g e o f 0.4 - 0.7 10 ^ kg/m^ '^''^.The oxygen
c o n s u m p t i o n r a t e may f o r t h a t r e a s o n be s a f e l y a p p r o x i m a t e d by z e r o - o r d e r
k i n e t i c s .
As a consequence t h e growth r a t e i n t h e c e n t r e o f a p e l l e t d i s p l a y s a s h a r p d e c r e a s e ( f i g u r e A ) , i n c a s e t h e p e l l e t r a d i u s exceeds a c r i t i c a l r a d i u s , g i v e n by: Rc = \ ( 6 !De C ^ r ^ ) (15) For t h e oxygen c o n s u m p t i o n r a t e r i n t h e g r o w i n g p a r t o f t h e p e l l e t c a n t h e n 2 be w r i t t e n : r„ = y p/Y + m p (16) 0^ m ox o
where p i s t h e volume averaged d e n s i t y .
In c o m b i n a t i o n w i t h e q u a t i o n (15) f o r t h e c r i t i c a l r a d i u s c a n be w r i t t e n :
Rc = \ 6 De Cs/ { ( ym/ Yo x + mo) p } (17)
The e f f e c t i v e n e s s f a c t o r n o f a p e l l e t i s now d e f i n e d as t h e r a t i o o f t h e a c t u a l oxygen c o n s u m p t i o n r a t e r and t h e maximum oxygen c o n s u m p t i o n r a t e
. 2 r _ , w h i c h c a n be r e a l i z e d i n t h e p r e s e n c e o f e x c e s s oxygen: 02,max r Jb ri = r / r (18) O2 t)2,max m a x i m u m g r o w t h rate -Pellet radius
fig. 4: s p e c i f i c growth rate p r o f i l e in a pellet in case of d i f f u s i o n a l growth limitation in the centre.
In a n a l o g y w i t h c a t a l y s t t h e o r i e s e q u a t i o n (14) can t h e n be s o l v e d by u s i n g a
p l o t o f the e f f e c t i v e n e s s f a c t o r n v e r s u s the T h i e l e modulus <j),_ f o r a sphere
w i t h z e r o - o r d e r k i n e t i c s ( f i g u r e 5 ) . For the T h i e l e modulus can be w r i t t e n :
^Th " R V < r o2 / 1 De Cs) (.9)
o r
<J)Th = (R/Rc) /6 (20)
The s p e c i f i c growth r a t e used i n e q u a t i o n (8) can now be c o r r e c t e d f o r t h e
e f f e c t of t h e d i f f u s i o n a l l i m i t a t i o n by i n t r o d u c i n g an e f f e c t i v e s p e c i f i c growth r a t e F o r yg h o l d s : ue= n um ( 2 i ) I n t h e l i g h t o f t h e p r e c e e d i n g , t h e biomass p r o d u c t i o n r a t e can be r e w r i t t e n by: r = u X (22) x e v '
S i m i l a r l y , f o r t h e oxygen c o n s u m p t i o n r a t e o f t h e b r o t h as a whole can now be
w r i t t e n :
Rn = n r . d> = n (u X/Y + m X) (23)
2 2 m ox o
fig. 5: effectiveness factor versus Thiele modulus plot for spheres with zero-order kinetics.
As P e n i c i l l i u m chrysogenum i s a s t r i c t l y a e r o b i c o r g a n i s m , no c a r b o n s o u r c e c o n s u m p t i o n t a k e s p l a c e w i t h o u t t h e p r e s e n c e o f oxygen, hence: r = - n (u X/Y s m (24) sx I V A U X I L I A R Y R E L A T I O N S B a s i c a l l y t h e e q u a t i o n s d e s c r i b e d b e f o r e c o m p l e t e l y c o m p r i s e t h e m a t h e m a t i c a l model f o r p e l l e t growth. So f a r , t h e v a l u e s f o r t h e e f f e c t i v e d i f f u s i v i t y ID^, t h e mean p e l l e t d e n s i t y p, t h e g a s - l i q u i d mass t r a n s f e r c o e f f i c i e n t k^a and the l i q u i d - s o l i d mass t r a n s f e r c o e f f i c i e n t k a have been r e g a r d e d t o be c o n s t a n t .
s p &
However, under p r a c t i c a l c o n d i t i o n s t h e y may v a r y . I t i s t h e r e f o r e u s e f u l t o d e v e l o p m a t h e m a t i c a l r e l a t i o n s t o p r e d i c t t h e v a l u e s o f t h e s e p a r a m e t e r s under c h a n g i n g o p e r a t i n g c o n d i t i o n s .
E f f e c t i v e diffusivity
The e f f e c t i v e d i f f u s i o n c o e f f i c i e n t c a n be assumed t o be p r o p o r t i o n a l t o t h e v o i d f r a c t i o n i n a p e l l e t . R e s u l t s o f a s t u d y o f t h e d e n s i t y o f p e l l e t s o f
Pénicillium chrysogenum r e p o r t e d elsewhere'"' y i e l d e d t h e f o l l o w i n g e s t i m a t e
f o r t h e v o i d f r a c t i o n i n p e l l e t s :
where £, i s t h e v o i d f r a c t i o n o f hyphae (~ 0.75) and p i s t h e d e n s i t y o f d r i e d 3 . S . . m y c e l i u m (- 1400 kg/m ) . So, t h e e f f e c t i v e d i f f u s i o n c o e f f i c i e n t f o r oxygen i n a p e l l e t c a n be c a l c u l a t e d by: whpre t h e v a l u e f o r TO i s about 2.1 10 m /s a t 25°C A l t h o u g h t h e r e i s no consensus i n l i t e r a t u r e about t h e e x a c t v a l u e o f D^, e q u a t i o n (26) p r o d u c e s r e s u l t s w h i c h a r e i n agreement w i t h t h o s e r e p o r t e d i n l i t e r a t u r e ^ .
Mean pellet density
(25)
E = (1 - p/350) H) (26)
R e s u l t s o f t h e p r e v i o u s mentioned s t u d y o f t h e p r e s e n t a u t h o r s a l l o w p o s t u l a -t i o n o f -t h e f o l l o w i n g e m p i r i c a l r e l a -t i o n -t o p r e d i c -t -t h e mean p e l l e -t d e n s i -t y
