Immobilized cells for continuous solvent production: IBE and ethanol fermentations

IMMOBILIZED CELLS FOR CONTINUOUS

SOLVENT PRODUCTION

IBE AND ETHANOL FERMENTATIONS

Wilt u dit document g o e d verpakt en voldoende gefrankeerd v o o r de

verval-datum retourneren.

Verlenging kan alleen schriftelijk en v o o r het verstrijken van de vervaldatum

w o rd e n a a n g evra a g d.

Bibliotheek TUDelft

Sectie Leverantie Prometheusplein 1 Postbus 98 2600 MG Delft Tel: (015)2784510 Fax: (015)257 2060

2 0

JUN 2001

IMMOBILIZED CELLS FOR CONTINUOUS

SOLVENT PRODUCTION

IBE AND ETHANOL FERMENTATIONS

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TO v*> 0* O 00 0* OH B I B L I O T H E E K T U Delft P 1685 5366 C 503382

IMMOBILIZED CELLS FOR CONTINUOUS SOLVENT PRODUCTION - IBE and e t h a n o l f e r m e n t a t i o n s - by P.G. K r o u w e l ERRATA Z i e e r r a t u m page 5, M i n e 15: page 7, t a b l e 1 : page 28, M i n e 14: page 29, • H i n e 6 : t e x t above page 36, •Mine 6 : page 37, 4-line 13: page 50, M i n e 8 : page page page page page page page page page page page page page page page 53, - H i n e 3 54, M i n e 15 57, -Hine 6 58, l l i n e 12 " p r o d u c t " s h o u l d r e a d " p r o d u c t s " " - c a r r a g e e n a n " s h o u l d r e a d " K - c a r r a g e e n a n " ( 2 x ) " e q u a t i o n ( 1 8 ) " s h o u l d r e a d " e q u a t i o n ( 2 1 ) " " e q u a t i o n ( 2 6 ) " s h o u l d r e a d " e q u a t i o n ( 2 9 ) " t a b l e 10 : "and 2 " s h o u l d r e a d "and x2 " "R„„ " s h o u l d r e a d " r " C 02 i C 02 I "1/R ( . / ( q C ) )2 " s h o u l d r e a d "1/R ( a , / ( q C ) )5 " or p x o r p x s h o u l d r e a d " . . . . l e a s t s q u a r e s ( t h e programme was deve-l o p e d a t t h e L a b o r a t o r y f o r C h e m i c a deve-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 ) . " " d e c r e a s e s " s h o u l d r e a d " d e c r e a s e d " "C " s h o u l d r e a d "C " (2x) e p " c a u t i o n s " s h o u l d r e a d " c a u t i o u s " "95%" s h o u l d r e a d "59%" 59, e q u a t i o n (1.2) : " R q2" s h o u l d r e a d " r 60, 4-line !""": " v a n " s h o u l d r e a d "and" 65, 4-line 13: "nog" s h o u l d r e a d " n o t " 67, e q u a t i o n (9) : "+ unC *" s h o u l d r e a d "- u,C *" 1 g 1 g 74, -Mine 7 : "u " s h o u l d r e a d " i n c r e a s i n g u " g ..; 6 g

79, -Mine 10: "become" s h o u l d r e a d "may become" 83, 4-line 3 : " h a s " s h o u l d r e a d "had" 91, t e x t b e l o w f i g . 4A : "3950 X" s h o u l d r e a d "2370 X" t e x t b e l o w f i g " . 4B' : "2370 X" s h o u l d r e a d "3950 X" 93, 4-line 5 M i n e 2 118,4-line 1 122,-Mine 10 " v e l o c i t y " s h o u l d r e a d " f l o w v e l o c i t y " "zoned" s h o u l d r e a d " z o n e s " "G,/ " s h o u l d r e a d "C,/C," b b l "how" s h o u l d r e a d "show"

STELLINGEN

1. Het f e i t , d a t Clostridium bakteriën s p o r e n kunnen vormen, v e r e e n v o u d i g t h e t werken met geïmmobiliseerde c e l l e n van d i t g e s l a c h t i n s t e r -ke mate.

Dit proefschrift, Hoofdstuk 5 en 6

2. De s u g g e s t i e van M i n i e r en Goma, d a t e r i n de door hun g e b r u i k t e ge-p u l s e e r d e k o l o m v o o r e t h a n o l ge-p r o d u k t i e (met g e l i j k t i j d i g e e x t r a k t i e ) p r o p s t r o m i n g v o o r de v l o e i s t o f f a s e n mag worden aangenomen w o r d t op geen e n k e l e w i j z e e x p e r i m e n t e e l o n d e r s t e u n d .

M. Minier and G. Goma; Bioteohnol. Lett. 3_, 405-408 (1981)

3. W i l l i a m s en Munnecke z i e n i n hun a r t i k e l o v e r e t h a n o l p r o d u k t i e met geïmmobiliseerde g i s t c e l l e n de p r o b l e m e n , d i e o p t r e d e n b i j h e t s c h a a l -v e r g r o t e n -van een g e p a k t e k o l o m r e a k t o r , o -v e r h e t h o o f d .

D. Williams and D.M. Munnecke; Bioteohnol. Bioeng. 23, 1813-1825 (1981)

4. Een n a d e e l v a n de door Karube e t a l . g e p r e s e n t e e r d e BOD-sensor i s d a t a l l e e n s t o f f e n , d i e door h e t membraan kunnen d i f f u n d e r e n gemeten wor-den.

I. Karube, T. Matsunaga, S. Mitsuda and S. Suzuki; Bioteohnol. Bioeng. 19, 1535-1547 (1977)

5 . De door M u l l e r e t a l . o p g e s t e l d e k o o l s t o f b a l a n s voor de anaërobe om-z e t t i n g van b e t a i n e door Eubacterium limosum i s f u n d a m e n t e e l o n j u i s t , omdat geen r e k e n i n g i s gehouden met de toename i n b i o m a s s a c o n c e n t r a t i e . D i t h e e f t v e r r e g a a n d e c o n s e q u e n t i e s v o o r de aanname t e n a a n z i e n van de h o e v e e l h e i d g e f i x e e r d e CC^.

E. Muller, K. Fahlbush, R. Walther and G. Gottsahalk; Appl. Environ. Microbiol. 42, 439-445 (1981)

6. Aan h e t a s e p t i s c h werken met geïmmobiliseerde m i k r o - o r g a n i s m e n w o r d t doorgaans t e w e i n i g aandacht b e s t e e d .

7. E r wordt i n N e d e r l a n d op u n i v e r s i t a i r n i v e a u t e w e i n i g o n d e r z o e k v e r r i c h t naar s p e c i f i e k e s c h e i d i n g s p r o c e s s e n v o o r de o p w e r k i n g v a n f e r -m e n t a t i e p r o d u k t e n . 8. G e z i e n de k o m p e t i t i e rond de e t h a n o l p r o d u k t i v i t e i t i n b i o r e a k t o r e n i n de l i t e r a t u u r i s h e t t e overwegen h i e r v o o r een w e r e l d k a m p i o e n s c h a p i n t e s t e l l e n . 9. De v o r d e r i n g e n i n de b i o t e c h n o l o g i e z i j n k l e i n e r dan de s n e l h e i d , waarmee h e t a a n t a l t i j d s c h r i f t e n en c o n g r e s s e n op d i t g e b i e d toeneemt, d o e t vermoeden. 10. Het v a l t t e b e t r e u r e n d a t de s c h o o l l o g o p e d i e i n D e l f t n i e t z o d a n i g kan f u n k t i o n e r e n a l s men z o u mogen v e r w a c h t e n g e z i e n de d u i d e l i j k e r i c h t l i j n e n h i e r o m t r e n t .

Nota "Logopedie op scholen, een plaatsbepaling" Vereniging voor Logopedie en Foniatvie, oktober 1979

11. Het p a r a f r a s e r e n van p u b l i k a t i e s i n v e r s c h i l l e n d e w e t e n s c h a p p e l i j k e t i j d s c h r i f t e n met een z e l f d e l e z e r s k r i n g d i e n t vermeden t e worden.

12. I n h e t merendeel v a n de D e l f t s e p r o e f s c h r i f t e n wordt h e t c u r r i c u l u m v i t a e t e n o n r e c h t e w e g g e l a t e n .

13. Het overnemen van de s p e l r e g e l s v a n h e t " G a e l i c f o o t b a l l " zou h e t N e d e r l a n d s e v o e t b a l a t t r a k t i e v e r maken.

P.G. K r o u w e l 28 j a n u a r i 1982

IMMOBILIZED CELLS FOR CONTINUOUS

SOLVENT PRODUCTION

IBE AND ETHANOL FERMENTATIONS

PROEFSCHRIFT

ter verkrijging van de graad van d o c t o r in de t e c h n i -s c h e w e t e n -s c h a p p e n aan de T e c h n i -s c h e H o g e -s c h o o l Delft, op g e z a g van de rector m a g n i f i c u s , prof. ir. B.P.Th. V e l t m a n , voor een c o m m i s s i e a a n g e w e z e n door het c o l l e g e van d e k a n e n te v e r d e d i g e n op

d o n d e r d a g 28 januari 1982 te 14.00 uur door

Petrus Gerardus Krouwel

s c h e i k u n d i g ingenieur geboren te V a l k e n s w a a r d

Dit proefschrift is goedgekeurd door de promotor

Prof. Dr. Ir. N.W.F. Kossen

;

B i j de t o t s t a n d k o m i n g van d i t p r o e f s c h r i f t w i l i k de v o l g e n d e p e r s o n e n i n h e t b i j z o n d e r bedanken: P r o f . i r . J.A. R o e l s en d r . i r . A. H a r d e r ( G i s t - B r o c a d e s ) v o o r de v e l e n u t t i g e d i s c u s s i e s de h e r e n B. K e r k d i j k en A. de G r a a f v o o r de t e c h n i s c h e a s s i s t e n t i e t i j d e n s de e x p e r i m e n t e n

de h e r e n G. van d e r S t e e n en C. Ras v o o r de a n a l y s e van de t a l r i j k e monsters de h e e r G. van d e r Hoeven ( G i s t B r o c a d e s ) v o o r de b e r e i d i n g v a n t a l l o -ze C a - a l g i n a a t / g i s t b o l l e t j e s de h e e r A. S u u r l a n d (vakgroep TM) v o o r de h u l p b i j de t r e k b a n k p r o e v e n de h e e r D. Nelemans ( a f d e l i n g d e r M e t a a l k u n d e ) v o o r h e t maken v a n de e l e c t r o n e n m i c r o s c o o p - o p n a m e n

de s t u d e n t e n , d i e hun a f s t u d e e r p e r i o d e aan h e t onderzoek b e s t e e d hebben: L i e v e n B r a b e r , F r a n s v a n d e r L a a n , P i e t v a n Egmond, Wim G r o o t en

P e t e r D u s s e l j e e

de h e e r F. Bolman v o o r h e t maken van de t e k e n i n g e n en de h e r e n A. B a k k e r , F. Hammers, A. S c h r i e l en C. Warnaar v o o r h e t f o t o g r a f i s c h e werk

Dr. A. E s e n e r v o o r h e t c o r r i g e r e n van h e t E n g e l s en t e n s l o t t e , mw. J . van D i j k v o o r h e t type-werk

Table of contents

CHAPTER 1 INTRODUCTION

I A i m 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

CHAPTER 2 ON THE MECHANICAL STABILITY OF GAS PRODUCING IMMOBILIZED SYSTEMS I I n t r o d u c t i o n 3 I I Theory 8 I I I M a t e r i a l s and methods "gas b u b b l e f o r m a t i o n " e x p e r i m e n t s 17 t e n s i l e s t r e n g t h t e s t 19 IV R e s u l t s "gas b u b b l e f o r m a t i o n " e x p e r i m e n t s 20 t e n s i l e s t r e s s - s t r a i n measurement 25 V D i s c u s s i o n 27 VI C o n c l u s i o n s 29 V I I A p p e n d i x 1 30 V I I I A p p e n d i x 2 32 I X A p p e n d i x 3 34 X N o m e n c l a t u r e and r e f e r e n c e s 35

CHAPTER 3 ETHANOL PRODUCTION BY FREE AND IMMOBILIZED YEAST CELLS UNDER NON-GROWTH CONDITIONS I I n t r o d u c t i o n 39 I I Theory 41 I I I M a t e r i a l s and methods 45 IV R e s u l t s and d i s c u s s i o n 48 V C o n c l u s i o n s 59 VI A p p e n d i x 59 V I I N o m e n c l a t u r e and r e f e r e n c e s 60

CHAPTER 4 ON THE USE OF A PPBR FOR CONTINUOUS ETHANOL FERMENTATION BY Ca-ALGINATE IMMOBILIZED YEAST CELLS

I I n t r o d u c t i o n 63 I I R e a c t o r model 65 I I I M a t e r i a l s and methods 67 IV R e s u l t s and d i s c u s s i o n 69 V S c a l e - u p a s p e c t s 74 V I C o n c l u s i o n s 75

V I I A p p e n d i x

V I I I N o m e n c l a t u r e and r e f e r e n c e s

7 6 7 6

CHAPTER 5 CONTINUOUS IBE-FERMENTATION BY IMMOBILIZED Clostridium

beijerinckii CELLS IN A PACKED BED FERMENTOR

I I n t r o d u c t i o n 79 I I L i t e r a t u r e - P r o c e s s p o t e n t i a l s 80 I I I E x p e r i m e n t a l a p p r o a c h 83 IV M a t e r i a l s and methods 84 V R e s u l t s and d i s c u s s i o n q u a l i t a t i v e e x p e r i m e n t s 87 b a t c h f e r m e n t a t i o n s 88 c o n t i n u o u s f e r m e n t a t i o n s 89 V I C o n c l u s i o n s 96 V I I Nomenclature and r e f e r e n c e s 97

CHAPTER 6 CONTINUOUS IBE-FERMENTATION BY IMMOBILIZED Clostridium

beijerinckii CELLS IN A STIRRED TANK FERMENTOR

I I n t r o d u c t i o n 99 I I Model 100 I I I M a t e r i a l s and methods 105 IV R e s u l t s and d i s c u s s i o n 107 V P r o c e s s o u t l o o k 1 16 V I C o n c l u s i o n s 117 V I I N o m e n c l a t u r e and r e f e r e n c e s 1 17 SUMMARY 121 SAMENVATTING 125 CURRICULUM VITAE ' 128

This thesis has been carried out within the Biotechnology Group of the Delft University of Technology.

Postal address: Biotechnology Group, Dept. of Chemical Engineering, Delft University of Technology, Jaffalaan 9, 2628 BX Delft, the Netherlands Delft University of Technology.

Chapter 1

INTRODUCTION

Aim and scope

The o b j e c t i v e o f t h e work d e s c r i b e d i n t h i s t h e s i s was t w o f o l d :

1) To s t u d y some a s p e c t s o f gas p r o d u c i n g i m m o b i l i z e d c e l l s y s t e m s , s u b j e c t e d t o p r o d u c t i n h i b i t i o n u s i n g the e t h a n o l f e r m e n t a t i o n as a model r e a c t i o n . 2) To i n v e s t i g a t e the p o t e n t i a l o f c o n t i n u o u s l - b u t a n o l p r o d u c t i o n by

immobi-l i z e d Cimmobi-lostridium c e immobi-l immobi-l s .

A l t h o u g h the s u b j e c t of t h i s t h e s i s can be c l a s s i f i e d under the g e n e r a l theme o f s o l v e n t p r o d u c t i o n f r o m r e n e w a b l e r e s o u r c e s , i t has t o be e m p h a s i z e d t h a t t h e achievement o f a h i g h p r o d u c t i v i t y was not t h e p r i m a r y o b j e c t i v e o f t h e f o r m e r p a r t o f the work. The a c c e n t was r a t h e r on s o l v i n g some g e n e r a l p r o -b l e m s , e n c o u n t e r e d i n work w i t h a n a e r o -b i c , gas p r o d u c i n g f e r m e n t a t i o n s u s i n g i m m o b i l i z e d c e l l s :

1) F r a c t u r e phenomenon i n the i m m o b i l i z a t i o n m a t r i x due t o i n t e r n a l gas b u b b l e f o r m a t i o n .

2) Consequences o f gas b u b b l e f o r m a t i o n i n t h e l i q u i d phase and p r o d u c t i n h i -b i t i o n f o r r e a c t o r d e s i g n .

I n a d d i t i o n t o t h e s e s u b j e c t s the k i n e t i c s of e t h a n o l p r o d u c t i o n by n o n - g r o w i n g y e a s t c e l l s was s t u d i e d .

The r e s e a r c h m e n t i o n e d under the f i r s t o b j e c t i v e was c a r r i e d out i n c l o s e c o -o p e r a t i -o n w i t h G i s t B r -o c a d e s NV D e l f t . D r i e d b a k e r s ' y e a s t , i m m -o b i l i z e d i n Ca-a l g i n Ca-a t e wCa-as used t h r o u g h o u t t h i s s t u d y . E t h Ca-a n o l f e r m e n t Ca-a t i o n by y e Ca-a s t wCa-as chosen as a model r e a c t i o n , because y e a s t i s easy t o h a n d l e and much i s known about t h e p r o c e s s .

The b u t a n o l f e r m e n t a t i o n shows s i m i l a r phenomena: w h i l s t CO^ i s formed d u r i n g e t h a n o l p r o d u c t i o n , a m i x t u r e of C 02 and i s formed d u r i n g b u t a n o l f e r m e n

-t a -t i o n . A p p r o x i m a -t e l y 50% o f -the m e -t a b o l i z e d g l u c o s e i s -t r a n s f o r m e d i n -t o CO^ i n b o t h c a s e s . F u r t h e r m o r e , b o t h f e r m e n t a t i o n s a r e s u b j e c t t o p r o d u c t i n h i b i

-t i o n .

The s t a r t i n g p o i n t f o r the b u t a n o l f e r m e n t a t i o n was q u i t e t r o u b l e s o m e : 1) I n f o r m a t i o n about the k i n e t i c s of t h i s f e r m e n t a t i o n was i n s u f f i c i e n t and

n o t easy t o o b t a i n .

2) L i k e w i s e , c e r t a i n m i c r o b i o l o g i c a l a s p e c t s were n o t w e l l u n d e r s t o o d and spe-c i f i spe-c i m m o b i l i z a t i o n a s p e spe-c t s had not been i n v e s t i g a t e d .

M o r e o v e r , b a c t e r i a o f t h e genus Clostridium a r e ( s p o r e f o r m i n g ) o b l i g a t e anae-r o b e s , anae-r e q u i anae-r i n g s p e c i a l h a n d l i n g t e c h n i q u e s . Howeveanae-r, the f e anae-r m e n t a t i o n was o f p a r t i c u l a r i n t e r e s t , b e c a u s e o n l y o l d f a s h i o n e d t e c h n o l o g y was a v a i l a b l e so t h a t a p p l i c a t i o n o f modern b i o t e c h n o l o g i c a l knowledge m i g h t be w o r t h w i l e .

I t w i l l be c l e a r t h a t an a p p r o a c h from the s o - c a l l e d ' f i r s t p r i n c i p l e s ' was not p o s s i b l e i n t h i s c a s e . T h e r e f o r e , the r e s e a r c h was p r i m a r i l y aimed a t t h e p r a c t i c a l development of a c o n t i n u o u s p r o c e s s u s i n g i m m o b i l i z e d c e l l s .

Clos-tridium beijerinokii was chosen f o r i t s h i g h s o l v e n t p r o d u c t i o n c a p a c i t y ' . The

p r o c e s s was c a l l e d I B E - f e r m e n t a t i o n , as t h e main f e r m e n t a t i o n p r o d u c t s a r e : i s o p r o p a n o l , n - b u t a n o l and e t h a n o l . Entrapment i n C a - a l g i n a t e was a l s o now

— . . . . . . . . 2

s e l e c t e d as t h e i m m o b i l i z a t i o n method, because i t i s a s i m p l e and m i l d method The f o l l o w i n g q u e s t i o n s w i l l be a n s e r e d i n t h e t h e s i s :

1) I s t h e r e a r e l a t i o n between t h e f r a c t u r e phenomenon i n gas p r o d u c i n g im-m o b i l i z e d s y s t e im-m s , the im-m e c h a n i c a l p r o p e r t i e s of the im-m a t r i x and the gas

f o r m a t i o n k i n e t i c s of the system?

2) How can t h e k i n e t i c s o f e t h a n o l p r o d u c t i o n by non-growing ( i m m o b i l i z e d ) y e a s t c e l l s be d e s c r i b e d ?

3) How can a s i m p l e r e a c t o r f o r gas p r o d u c i n g i m m o b i l i z e d c e l l s , s u b j e c t to p r o d u c t i n h i b i t i o n , be d e s i g n e d ?

4) I s a p a c k e d bed f e r m e n t o r s u i t a b l e f o r c o n t i n u o u s I B E - f e r m e n t a t i o n by i m m o b i l i z e d Clostridium beijerinokii c e l l s ?

5) How does a c o n t i n u o u s s t i r r e d tank f e r m e n t o r compare w i t h a p a c k e d bed f e r m e n t o r f o r t h i s f e r m e n t a t i o n ?

A t t h i s p o i n t t h e r e a d e r may s t i l l wonder how t h e s e v a r i o u s q u e s t i o n s i n t e r -a c t . T h i s w i l l be e l u c i d -a t e d i n t h e f o l l o w i n g p -a r -a g r -a p h on the b -a s i s o f f i g . 1

2l§fEi5§£ion_of _ t h ^ s _ t h e ^ i s

T h i s t h e s i s has been w r i t t e n i n the form of more o r l e s s i n d e p e n d e n t s t u d i e s , a l t h o u g h c h a p t e r s w i l l be c r o s s r e f e r e d sometimes. The 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.

A phenomenon, o f p a r t i c u l a r i m p o r t a n c e i n homogeneously i m m o b i l i z e d s y s t e m s , where a gas p r o d u c i n g a n a e r o b i c r e a c t i o n t a k e s p l a c e , i s t h a t under c e r t a i n

RE ACM'OK CALCULAT IONS INTERNAL DIFFUSION DEACTIVATION KINETICS MICRO-KINETICS MACRO-KINETICS REACTOR MODEL EXTERNAL MASS TRANSPORT MICRO-KINETICS MACRO-KINETICS REACTOR MODEL EXTERNAL MASS TRANSPORT GAS PRODUCTION KINETICS

I

GAS PRODUCT10 FLOW MODEL (MIXING)

MECHANICAL STABILITY GAS CHOICE OF PROPERTIES CRITERIONS TRANSPORT REACTOR

Fig. 1: Simplified block-diagram, showing the interactions of the subjects, described in this thesis

c o n d i t i o n s ( e . g . s l o w gas d i f f u s i o n compared w i t h gas p r o d u c t i o n ) a gas b u b b l e i s formed i n s i d e t h e m a t r i x . The p r e s s u r e i n s i d e t h e b u b b l e w i l l r i s e and c a n l e a d t o f r a c t u r e o f t h e m a t r i x , depending on i t s m e c h a n i c a l p r o p e r t i e s . I n

Chapter 2 a t h e o r y , d e s c r i b i n g t h e subsequent p r o c e s s e s l e a d i n g t o p a r t i c l e

f r a c t u r e i s d e v e l o p e d . An a l t e r n a t i v e a p p r o a c h , b a s e d on d i m e n s i o n a l a n a l y s i s i s a l s o p r e s e n t e d . I n t h e e x p e r i m e n t a l s e c t i o n two k i n d s o f d a t a a r e c o l l e c t e d : m e c h a n i c a l and k i n e t i c d a t a . The f r a c t u r e s t r e n g t h and t h e u l t i m a t e e l o n g a t i o n o f d i f f e r e n t m a t e r i a l s , f r e q u e n t l y used f o r i m m o b i l i z a t i o n a r e compared. The i n f l u e n c e o f v a r i o u s p r o c e s s p a r a m e t e r s and r e a c t o r c o n d i t i o n s on p a r t i c l e r u p t u r e a r e d e t e r m i n e d . F i n a l l y , t h e m e c h a n i c a l and k i n e t i c d a t a a r e combined i n a s t a b i l i t y c r i t e r i o n . The t h e o r y and methods, a p p l i e d t o CO^ p r o d u c t i o n by C a a l g i n a t e i m m o b i l i z e d y e a s t c e l l s have a more g e n e r a l v a l i d i t y , i n p r i n -c i p l e .

I n Chapter 3 e t h a n o l p r o d u c t i o n by ( i m m o b i l i z e d ) y e a s t c e l l s under non-growth c o n d i t i o n s i s s t u d i e d . An advantage o f a n o n g r o w i n g s y s t e m i s t h a t t h e p r o -duct i s c e l l f r e e . K i n e t i c measurements a r e e v a l u a t e d on t h e b a s i s o f r a t e e q u a t i o n s , r e p o r t e d i n l i t e r a t u r e . These d a t a a r e i n d i s p e n s a b l e f o r t h e

development o f a r e a c t o r model. The c h o i c e o f a r e a c t o r t y p e depends b o t h on i n -h i b i t i o n k i n e t i c s and on t-he e f f e c t s o f gas p r o d u c t i o n . S i n c e gas b u b b l e s can-n o t escape u can-n h i can-n d e r e d from a packed bed r e a c t o r , gas p r o d u c t i o can-n i can-n such a r e a c t o r causes s l u g f o r m a t i o n . These s l u g s r i s e from t i m e t o t i m e , t h e r e b y v i g o r o u s l y m i x i n g the r e a c t o r c o n t e n t . From a v i e w p o i n t o f r e a c t o r p r o d u c t i v i -t y , however, -t h i s phenomenon i s u n f a v o u r a b l e f o r r e a c -t i o n s , showing p r o d u c -t i n h i b i t i o n . T h e r e f o r e , the p e r f o r m a n c e o f a PPBR ( p u l s e d p_acked bed j r e a c t o r ) , a compromise between a p l u g f l o w and an i d e a l l y mixed r e a c t o r i n w h i c h gas escape i s f a c i l i t a t e d , i s s t u d i e d i n Chapter 4. A g a i n i m m o b i l i z e d y e a s t c e l l s a r e used as a model system. A r e a c t o r model i s d e v e l o p e d u s i n g t h e d a t a o f C h a p t e r 3. M e c h a n i c a l s t a b i l i t y o f the b e a d s , as d e f i n e d i n C h a p t e r 2 i s a l s o c o n s i d e r e d .

O r i g i n a l l y , the i d e a t o i n v e s t i g a t e t h i s r e a c t o r t y p e a r o s e when p r o b l e m s were e n c o u n t e r e d i n a packed column f o r c o n t i n u o u s I B E - f e r m e n t a t i o n , the s u b j e c t o f

Chapter 5. T h i s r e a c t o r type was i n v e s t i g a t e d f i r s t because i t i s s i m p l e and

an a x i a l c o n c e n t r a t i o n g r a d i e n t was t h o u g h t t o be n e c e s s a r y f o r c o n t i n u o u s I B E - f e r m e n t a t i o n . I n t h i s c h a p t e r some i n t r o d u c t o r y e x p e r i m e n t s a r e d e s c r i b e d and a comparison i s made between the c l a s s i c a l b a t c h f e r m e n t a t i o n p r o c e s s and the r e s u l t s o b t a i n e d u s i n g the packed bed f e r m e n t o r . However, because o f s e v e r a l r e a s o n s a CSTR ( c o n t i n u o u s _ s t i r r e d _tank £eactor) appeared t o be more s u i t a b l e f o r f u r t h e r r e s e a r c h .

I n Chapter 6 the use o f a CSTR f o r c o n t i n u o u s I B E - f e r m e n t a t i o n by i m m o b i l i z e d g r o w i n g c e l l s i s s t u d i e d . A g e n e r a l r e a c t o r model f o r i m m o b i l i z e d g r o w i n g s y -stems and a s p e c i f i c one f o r the I B E - s y s t e m are d e v e l o p e d . The e f f e c t s o f an i n c r e a s e d d i l u t i o n r a t e are compared w i t h model p r e d i c t i o n s . F i n a l l y , a

compar i s o n i s made between d i f f e compar e n t compar e a c t o compar t y p e s and the p o t e n t i a l o f t h e p compar o -cess i s e v a l u a t e d . I n f a c t t h e r e s u l t s a r e q u i t e r e m a r k a b l e , s i n c e ( u n t i l now) i t appeared t o be i m p o s s i b l e t o c a r r y out t h i s f e r m e n t a t i o n ( u s i n g t h e same

3

m i c r o - o r g a n i s m ) i n a c o n t i n u o u s c u l t u r e w i t h f r e e c e l l s . A s i m i l a r e x p e r i e n c e 4

w i t h Clostridium acetobutylicum has a l s o been r e p o r t e d r e c e n t l y .

REFERENCES

1. J.B. van der L e k , Ph. D. Thesis, D e l f t U n i v e r s i t y o f Technology (1930) 2. M. K i e r s t a n and C. Bucke, Bioteohnol. Bioeng. 387-397 (1977) 3. I.M.L. J o b s e s , u n p u b l i s h e d r e s u l t s (1981)

4. J.C. G o t t s c h a l and J.G. M o r r i s , Bioteohnol. Lett. 13, 525-530 (1981)

Chapter 2

ON THE MECHANICAL STABILITY OF GAS PRODUCING IMMOBILIZED SYSTEMS

INTRODUCTION

The use o f i m m o b i l i z e d c e l l s has s e v e r a l advantages compared w i t h the use o f f r e e c e l l s , b u t a l s o some drawbacks. V a r i o u s e x c e l l e n t r e v i e w a r t i c l e s , d e s -c r i b i n g the advantages and d i s a d v a n t a g e s of i m m o b i l i z e d -c e l l systems have been p u b l i s h e d r e c e n t l y ' ^. Some u n f a v o u r a b l e p r o p e r t i e s o f i m m o b i l i z e d c e l l s , com-monly m e n t i o n e d i n l i t e r a t u r e a r e :

1) Mass t r a n s p o r t l i m i t a t i o n o f s u b s t r a t e and p r o d u c t s ( i n t e r n a l and e x t e r n a l ) 2) A c t i v i t y l o s s due t o i m m o b i l i z a t i o n c o n d i t i o n s

However, a n o t h e r s p e c i f i c p r o b l e m may a r i s e i n a n a e r o b i c , gas p r o d u c i n g f e r -m e n t a t i o n s u s i n g i -m -m o b i l i z e d c e l l s ( e . g . p r o d u c t i o n o f e t h a n o l , b u t a n o l and methane and d u r i n g d e n i t r i f i c a t i o n ) . Because o f t h e r e l a t i v e l y low s o l u b i l i t y

o f gasses compared w i t h o t h e r r e a c t i o n p r o d u c t s a gas can come out o f s o l u t i o n z,. «rtaturn and a b u b b l e can be formed i n the i m m o b i l i z a t i o n m a t r i x under some c o n d i t i o n s .

As a consequence of t h e b u b b l e f o r m a t i o n the b i o c a t a l y s t p a r t i c l e w i l l t e n d t o f l o a t . I n case the m a t r i x cannot r e s i s t the s t r e s s , caused by t h e b u b b l e i t w i l l be "blown up" (see f i g . 1). Of c o u r s e , t h i s phenomenon i s a s e v e r e l i m i

-t a -t i o n -t o -the use of i m m o b i l i z e d , gas p r o d u c i n g s y s -t e m s . More power -t o volume i n p u t i s r e q u i r e d f o r complete d i s p e r s i o n of t h e b i o c a t a l y s t p a r t i c l e s i n a s t i r r e d f e r m e n t o r i f the p a r t i c l e s a r e f l o a t i n g on t h e l i q u i d s u r f a c e . I n expanded bed columns problems are c a u s e d by f l o a t i n g p a r t i c l e s because t h e s e p a r t i c l e s a r e w i t h d r a w n f o r m the r e a c t o r b e d , r e s u l t i n g i n a d e c r e a s e d p r o d u c t i v i -t y . I n case -the m a -t r i x i s d i s r u p -t e d -the o r i g i n a l b i o c a -t a l y s -t i s l o s -t .

The consequent problems o f e x c e s s gas p r o d u c t i o n i n agar and C a - a l g i n a t e have been p u b l i s h e d by K r o u w e l and Kossen^'''. I n t h i s s t u d y the p r o b l e m w i l l be des-c r i b e d i n more d e t a i l .

g

Harremoes e t a l have i n v e s t i g a t e d a r e l a t e d phenomenon i n a f i x e d f i l m r e a c t o r 5

Fig. 1: Ca-alginate beads, containing yeast cells "blown up" by excess gas production

9-11 f o r d e n i t r i f i c a t i o n . O t h e r a u t h o r s have o n l y t o u c h e d upon the s u b j e c t

F u r t h e r m o r e a s t r o n g a n a l o g y e x i s t s w i t h t h e foaming p r o c e s s o f p o l y m e r s ( e . g . p o l y u r e t h a n e ) . D u r i n g t h i s p r o c e s s a gas i s e v o l v e d i n a s u p e r s a t u r a t e d p r e -p o l y m e r s o l u t i o n d u r i n g -p o l y m e r i z a t i o n . However, v e r y l i t t l e i s known about the t h e o r y o f t h e foaming p r o c e s s .

As s t a t e d b e f o r e t h e m e c h a n i c a l p r o p e r t i e s o f the m a t r i x a r e i n v o l v e d i n t h e gas b u b b l e f o r m a t i o n p r o b l e m . From a p p l i e d mechanics i t i s known t h a t the key p r o p e r t y i s t h e b r e a k i n g s t r e n g t h .

A l t h o u g h measurement of the b r e a k i n g s t r e n g t h i s a b a s i c r o u t i n e d u r i n g t e s t i n g of r e s i n s i t has n o t been a p p l i e d t o i m m o b i l i z a t i o n m a t r i c e s .

O t h e r m e c h a n i c a l p r o p e r t i e s however, have been m e n t i o n e d i n l i t e r a t u r e and pro-cedures f o r t h e i r measurement have been more o r l e s s s t a n d a r d i z e d ' . These mea-surements i n c l u d e :

1) C o m p r e s s i o n s t r e n g t h o f b i o c a t a l y s t p a r t i c l e s 2) A b r a s i o n i n a s t i r r e d v e s s e l

3) C o m p r e s s i b i l i t y o f - , and p r e s s u r e drop o v e r a packed bed o f b i o c a t a l y s t s Because t e n s i l e and c o m p r e s s i o n s t r e n g t h s can be supposed t o be r e l a t e d , some l i t e r a t u r e r e s u l t s c o n c e r n i n g t h e l a t t e r m e c h a n i c a l p r o p e r t y w i l l be r e v i e w e d s h o r t l y .

12 13 Q u a n t i t a t i v e r e s u l t s have been p r e s e n t e d by N i s h i d a e t a l , Washausen and

14

K l e i n and Washausen . Q u a l i t a t i v e r e s u l t s have been p u b l i s h e d by T a k a t a e t 15 16

a l and Birnbaum e t a l . A l l t h e s e w o r k e r s have used t h e same methods f o r the measurement. They i n v e s t i g a t e d the c o m p r e s s i o n b e h a v i o u r o f a s i n g l e b i o

-17 18

m e c h a n i c a l s t r e n g t h of C a a l g i n a t e g e l s b a s e d on measurements o f t h e c o m p r e s s i -b i l i t y o f a p a c k e d column. T a b l e 1: L i t e r a t u r e v a l u e s o f K f o r d i f f e r e n t m a t e r i a l s m a t e r i a l shape d (mm) P K 0 (N/ .2) r e f e r e n c e s 1.4%t(-carrageenan/7r '""* " 0.5% l o c u s t bean gum cube 3* 31 .4 . . o3 — 2.6%i - c a r r a g e e n a n 3.0 11 H II II 35 72 3 .5 . 1 03 . . o3 • 13 3.4 it 11 80 .4 . . o3 PAAm E p o x i d e / 75 v o l % c e l l s cube sphere 3 1 .3 79 15 .4 .8 . 1 03 . . o5 IT IT 2. 1 14 4 . . o5 II II 2.2 13 .9 . . o5 II It 3.0 7 6 . . o5 E p o x i d e / 69 v o l % c e l l s sphere 3.0 93 4 . . 04 6 7 11 93 4 . . o4 62 11 11 129 C a - a l g i n a t e / 5 g/ml c e l l s s p h e r e 3.9 14 6 . 1 04 14,15 10 II 13 0 . . o4 15 it II 10 0 . . o4 " 20 " H 11 5 9 . . o4 25 it It 4 2 . 1 04 d r i e d C a - a l g i n a t e / 4 4 g/ml c e l l s s p h e r e 1.8 >39 3 .105 P o l y m e t h a c r y 1 a m i d e / s p h e r e 1.9 77 6 . . o4 J 20 g/ml c e l l s

* I n t h i s case dp means t h e l e n g t h o f t h e edge

Some o f t h e r e s u l t s , o b t a i n e d by t h e f i r s t method have been summarized i n t a b l e 1. The c o m p r e s s i o n s t r e s s Kq has been c a l c u l a t e d , i f n e c e s s a r y from t h e c r i t i

c a l f o r c e t h e b i o c a t a l y s t s p a r t i c l e can r e s i s t and t h e o r i g i n a l s u r f a c e , p e r -p e n d i c u l a r t o t h e f o r c e . As can be c o n c l u d e d from t h e t a b l e , i m m o b i l i z a t i o n m a t e r i a l s d i f f e r c o n s i d e r a b l y i n t h e maximum s t r e s s they can r e s i s t b e f o r e f r a c t u r e t a k e s p l a c e . A n o t h e r c o n c l u s i o n i s t h a t f o r e p o x i d e beads K de-c r e a s e s i f t h e p a r t i de-c l e r a d i u s i n de-c r e a s e s . F u r t h e r m o r e i t can be seen t h a t t h e m e c h a n i c a l s t r e n g t h d e c r e a s e s by b o t h an i n c r e a s i n g c e l l c o n c e n t r a t i o n i n t h e m a t r i x and a d e c r e a s i n g p o l y m e r ( K c a r r a -geen) c o n c e n t r a t i o n . 19 D r y i n g o f b i o c a t a l y s t s ( C a - a l g i n a t e , c h i t o s a n ) i n c r e a s e s t h e m e c h a n i c a l 7

s t r e n g t h c o n s i d e r a b l y . A h a r d e n i n g t r e a t m e n t ( e . g . w i t h g l u t a r a l d e h y d e ) of a g e l a l s o i n c r e a s e s t h e m e c h a n i c a l s t r e n g t h (and t h e o p e r a t i o n a l s t a b i l i t y due

12 15 to d i m i n i s h e d c e l l l e a k a g e ) '

THEORY

As t h e s u b j e c t s t u d i e d i s r a t h e r new no t h e o r y has been f o r m u l a t e d f o r t h e gas b u b b l e f o r m a t i o n i n c o n n e c t i o n w i t h f r a c t u r e of m a t r i c e s i n l i t e r a t u r e u n t i l now.

g

Only Harremoes e t a l g i v e a d e s c r i p t i o n of the f o r m a t i o n o f n i t r o g e n b u b b l e s i n t h e b i o f i l m o f a d e n i t r i f i c a t i o n column. They s t a t e t h a t n i t r o g e n s u p e r s a -t u r a -t i o n causes b u b b l e f o r m a -t i o n a -t -t h e boundary be-tween s u p p o r -t m a -t e r i a l and b i o f i l m . As a consequence t h e b i o f i l m w i l l be s l o u g h e d o f f .

K r o u w e l and K o s s e n ^ d e r i v e d t h e o r e t i c a l c r i t e r i a f o r gas b u b b l e f o r m a t i o n , based on 0t'1 o r d e r k i n e t i c s of t h e gas p r o d u c i n g r e a c t i o n , d i f f u s i o n o f t h e

gaseous p r o d u c t i n the m a t r i x and an unknown c r i t i c a l c o n c e n t r a t i o n , above w h i c h b u b b l e f o r m a t i o n t a k e s p l a c e . In t h i s paper t h i s t h e o r y w i l l be e x t e n d e d . D u r i n g t h e p r o c e s s u l t i m a t e l y l e a d i n g t o f r a c t u r e o f t h e b i o c a t a l y s t p a r t i c l e c e r t a i n s t a g e s can be d i s c e r ned. T h e r e f o r e t h e p r o c e s s w i l l be c o n s i d e r e d as a s e r i e s o f s e q u e n t i a l p r o -c e s s e s , a l l of w h i -c h -can r e s u l t i n a s t a b l e s i t u a t i o n . The s u -c -c e s s i v e s t e p s , w h i c h f i n a l l y r e s u l t i n f r a c t u r e o f a b i o c a t a l y s t p a r t i c l e a r e ( s e e f i g . 2 ) :

Fig. 2: A schematic representation of the processes, leading to particle fracture

I ) gas p r o d u c t i o n and gas t r a n s p o r t I I ) n u c l e u s - and b u b b l e f o r m a t i o n I I I ) f r a c t u r e o f t h e b i o c a t a l y s t p a r t i c l e

These s u b j e c t s w i l l be t r e a t e d i n the f o l l o w i n g s e c t i o n s . The t h e o r y w i l l be r e s t r i c t e d t o s p h e r i c a l b i o c a t a l y s t p a r t i c l e s , because o n l y beads were u s e d i n t h e e x p e r i m e n t s .

A) §§s_£roduction_and_gas_transgort The f o l l o w i n g s y s t e m i s c o n s i d e r e d :

A s p h e r i c a l b i o c a t a l y s t p a r t i c l e ( r a d i u s R = | ^p^> c o n t a i n i n g homogeneously

i m m o b i l i z e d c e l l s ( c o n c e n t r a t i o n C ) . The f o l l o w i n g r e a c t i o n t a k e s p l a c e :

S — • n A + n P /+ n, B (1)

a p b

(One c o u l d r e a d g l u c o s e f o r S, e t h a n o l f o r A and CO^ f o r P; B r e p r e s e n t s a by-p r o d u c t , e.g. g l y c e r o l ) .

S i n c e s u b s t r a t e c o n c e n t r a t i o n i s n o r m a l l y h i g h i n f e r m e n t a t i o n s t h e r e a c t i o n i s assumed t o be z e r o o r d e r . T h i s a s s u m p t i o n i s d e f i n i t e l y v a l i d f o r the s t a r t -i n g - p e r -i o d o f a b a t c h p r o c e s s and the -i n l e t - p a r t o f a column r e a c t o r , where r e a c t i o n v e l o c i t y i s maximal.

The r e a c t i o n r a t e can now be e x p r e s s e d as:

r = q C = r i f C > 0 (2) s s a x s a s

r = 0 i f C = 0

s s

Steady s t a t e c o n c e n t r a t i o n p r o f i l e s f o r (gaseous) p r o d u c t and s u b s t r a t e can be d e r i v e d as shown i n an e a r l i e r p u b l i c a t i o n ^ . A g e n e r a l e x p r e s s i o n f o r t h e s e e q u a t i o n s i s 3 3 r; 9 9 2 Rn 2 R cj(r ) = c j i 1 ? i b ( R "r + - i r i f * >6 <3 ) and cj(r ) = c j i 1 F i b ( r 2 "r2) i f *2 4 6 ( 4 )

F o r j = p the p l u s s i g n i s v a l i d ( r . = n r ) and f o r j = s the minus s i g n ( r . = r ). j s a I n t h e s e e q u a t i o n s R i s the c r i t i c a l r a d i u s , d e f i n e d by the c o n d i t i o n r = R i f C ( r ) = 0 (5) O S and (j) i s the T h i e l e - m o d u l u s , w h i c h i s e q u a l t o (j) = R / ( g ^ - ) (6) s s i 2 I f <p > 6 the r e a c t i o n e f f i c i e n c y f a c t o r n, d e f i n e d as 9

R

2

ƒ 4tt r r (C ) d r a c t u a l s u b s t r a t e c o n v e r s i o n r a t e _ J 0 s s s u b s t r a t e c o n v e r s i o n r a t e at C = C . , ; o _3 s s s i 4/3 7T R r (C .) s s i i s e q u a l to R 3

n = 1 -

(rf)

(8)

2 I f no s u b s t r a t e d i f f u s i o n l i m i t a t i o n o c c u r s (<j> ^ 6 , C ( r ) £ 0 and consequent-l y n = 1) i t w i consequent-l consequent-l be c consequent-l e a r t h a t e q u a t i o n (3) reduces t o e q u a t i o n (4) by sub-s t i t u t i n g = 0.

No n u c l e u s f o r m a t i o n w i l l appear i f the maximal v a l u e of C ( r ) remains below a P c r i t i c a l v a l u e C Pc C (R ) < C i f (f>2 > 6 (9) p o pc C (0) < C i f <f>2 s? 6 (10) P PC

By c o m b i n a t i o n o f e q u a t i o n (9) and the r e l e v a n t e x p r e s s i o n s f o r 0 ( r ) and C ( r ) ( e q u a t i o n ( 3 ) ) , r e s p e c t i v e l y e q u a t i o n (10) and t h e r e l e v a n t e x p r e s s i o n f o r C ( r ) ( e q u a t i o n ( 4 ) ) the c r i t e r i a f o r no n u c l e u s f o r m a t i o n can be d e r i v e d . I n a d i m e n s i o n l e s s form they can be shown t o be e q u a l t o :

C C A / < 1 - / i f *2 > 6 (11) pc pc 2 C . C . pc pc I n w h i c h X i s a d i m e n s i o n l e s s p a r a m e t e r , d e f i n e d by n ID X = ^ (13) P

E q u a t i o n s (11) and (12) are shown g r a p h i c a l l y i n f i g . 3. From t h i s f i g u r e i t ^s i can be c o n c l u d e d t h a t no n u c l e u s w i l l be formed i f the r e l e v a n t v a l u e of X —

^pc l i e s a t the l e f t o r below the c u r v e , d e s c r i b i n g the s p e c i f i c p r o b l e m . F o r o t h e r b i o c a t a l y s t g e o m e t r i e s ( s l a b , c y l i n d e r ) the c r i t e r i a have been d e r i v e d

100

Fig. S: Graphical representation of the nucleus formation criterion for th

sphere geometry (0 order k i n e t i c s )

Fig. 4: Graphical representation of the nucleus formation c r i t e r i a for dif-th

fevent geometries (0 order kinetics; C . = 0): a) slab II (two pi

sides open) b) sphere c) cylinder d) slab I (one side open)

T a b l e 2: C r i t e r i a f o r no n u c l e u s f o r m a t i o n f o r s p h e r e , s l a b I (one s i d e o p e n ) , s l a b I I (two s i d e s open) and c y l i n d e r geometry

Geometry C o n d i t i o n s (r)<l) C o n d i t i o n s ( f l=0 Sphere R S l a b I & S l a b I I 6 0 r 2 s a » C . s s i ID C . S SI C y l i n d e r R 2 s a ID C . s s i A C . s i C pc A C . s i C pc A C . s i C pc A C . s i C pc < 1 < 1 < 1 < 1 C . -21 c p c 21 pc - P i "pc £1 Jp c .2 C . v 1 s i . 6 A a " < ' pc 4_ \ SI < i 2 A C < 1 pc ,2 C "pc C . s i Jp c C . -21 C pc C . c pc c . ^>1 pc C . -21 C PC

i n an a n a l o g o u s way . The r e s u l t s have been summarized i n t a b l e 2 and f i g . 4. The same p r o c e d u r e , as d e s c r i b e d above can be f o l l o w e d t o d e r i v e n u c l e u s f o r m a -t i o n c r i -t e r i a f o r o -t h e r r e a c -t i o n k i n e -t i c s . The s o l u -t i o n of -t h e e q u a -t i o n s i s more c o m p l i c a t e d i n t h o s e c a s e s . T h i s i s worked o u t f o r M i c h a e l i s M e n t e n k i n e -t i c s ( e . g . d i s s o c i a -t i o n o f u r e a i n -t o CO^ and NH by -t h e a c -t i o n o f -the enzyme

u r e a s e ) i n A p p e n d i x 1. The i n f l u e n c e of_^xternal_mass_trans£ort As can be c o n c l u d e d f r o m e q u a t i o n s (3) and (4) ( r ) i s a f u n c t i o n o f C ^ , t h e i n t e r f a c i a l c o n c e n t r a t i o n . The v a l u e o f C.. i s h i g h l y dependent on t h e r a t i o of t h e e x t e r n a l mass t r a n s f e r r a t e and t h e i n t e r n a l d i f f u s i o n a l t r a n s p o r t r a t e , i . e . B i o t - n u m b e r ( B i ) , d e f i n e d as k, . R B i = (14) J I n w h i c h k-^ i s t h e e x t e r n a l mass t r a n s f e r c o e f f i c i e n t .

I n case e x t e r n a l mass t r a n s f e r r e s i s t a n c e c a n n o t be n e g l e c t e d an e x t r a mass b a l a n c e i s n e c e s s a r y t o c a l c u l a t e ^ i n r e l a t i o n t o Cj ^ >tne b u l k c o n c e n t r a -t i o n dC. - D. (-j-J-) = fc. . (C,. - C , ) (15) J d r r m R l , j j i j b dC. th ( ~*) can be c a l c u l a t e d r e a d i l y f o r 0 o r d e r k i n e t i c s f r o m e q u a t i o n s (3) r = R and ( 4 ) . C o m b i n a t i o n o f t h e s e e q u a t i o n s w i t h e q u a t i o n (15) y i e l d s r e s p e c t i v e l y : 3 3 3 r . R r . 2 R 2 R

V

r )

=

+-

T T ^ T

( R

" -5->

+ c

jb *

e ± ^ ~ r +

- T ^

( , 6 ) l . J R J i f d)2 > 6 and

V

r )

=

±

l V r

+ C

jb

4 6 T D7 ( R 2

"

r 2 )

i f *

2

* *

( 1 7 ) By c o m b i n a t i o n o f t h e r e l e v a n t e q u a t i o n s f o r C ( r ) (and C ( r ) ) and r e s p e c t i v e -p s l y e q u a t i o n s (9) and (10) d i m e n s i o n l e s s e x p r e s s i o n s f o r t h e no n u c l e u s forma-t i o n c r i forma-t e r i a can be o b forma-t a i n e d : ^ ' B T - B T ) + A C ^ < ' - C ^ " * 2 > O ('8> s p c p c l 2 c " 3 - (bT + » K 1 " if *2 < 6 (19) P PC where i s d e f i n e d as 12

n r P

P PC

B i o c a t a l y s t p a r t i c l e s , made of C a - a l g i n a t e o r K - c a r r a g e e n a n d i f f e r o n l y l i t t l e i n d e n s i t y f r o m the medium, i n w h i c h t h e y a r e suspended. T h e r e f o r e , the v a l u e o f k1, t o be used i n t h e s e r e l a t i o n s , a p p l i e d t o a s t i r r e d f e r m e n t o r , can be . . . . 20 e s t i m a t e d f r o m the c o r r e l a t i o n d e r i v e d by L e v i n s and G l a s t o n b u r y f o r s t i r r e d s u s p e n s i o n s o f s o l i d p a r t i c l e s i n a l i q u i d , when Ap=0. 4/3 1/3 0,62 0 17 d 1 P 1 D u' " 0.36 Sh = 2 + 0.47 ( p - ) <£) (21)

(the symbols are e x p l a i n e d a t the end of t h i s p a p e r ) .

S i n c e C , was h i g h d u r i n g the e x p e r i m e n t s i t can be e x p e c t e d t h a t B i i s r a t h e r

; 21

u n i m p o r t a n t w i t h r e s p e c t t o s u b s t r a t e . As shown by Metz the r e a c t i o n e f f i -2

c i e n c y n i s 1 a t <J> < 6 f o r B i > 10. However, i t can a l s o be e x p e c t e d t h a t C (0) i s more s e n s i t i v e t o B i , s i n c e C , i s r e l a t i v e l y s m a l l ,

p pb g H _ i n f l u e n c e

I n p r o c e s s e s where CO2 i s formed ( e . g . e t h a n o l p r o d u c t i o n ) the pH has an i n f l u e n c e on the s o l u b i l i t y o f the gas. Gaseous C O 2 , d i s s o l v e d i n w a t e r d i s s o -c i a t e s a -c -c o r d i n g t o the f o l l o w i n g r e a -c t i o n s :

C 02 + H2O ^ H C 03" + H30+ (22)

H C 03~ + H20 ^ C 03 = + H30+ (23)

The e q u i l i b r i u m c o n s t a n t s of t h e s e r e a c t i o n s a t 30°C are K = 4 . 7 1 . 10 ^ and - 1 1 . K_2 = 5.13 . 10 , r e s p e c t i v e l y . T h e r e f o r e a good a p p r o x i m a t i o n f o r the t o t a l C 02 s o l u b i l i t y as a f u n c t i o n o f pH a t pH v a l u e s < 7 can be shown t o be [ C 02]T = [ C 02]L (1 + K j . 1 0P H) (24) I n w h i c h [CO ] i s the c o n c e n t r a t i o n o f u n d i s s o c i a t e d , d i s s o l v e d CO . I n t a b l e z L z 3 v a l u e s o f [ C 02] ^ , / [ C C ^ L a r e p r e s e n t e d as a f u n c t i o n o f pH. As can be con-c l u d e d f r o m t h i s t a b l e d i s s o con-c i a t i o n i s n e g l i g i b l e a t pH = 4.5, b u t s o l u b i l i t y i n c r e a s e s more t h a n 1 0 0 % a t pH = 6.5. T h e r e f o r e C can be e x p e c t e d t o be pc r h i g h e r a t pH = 6.5

I n case C 0 „ p r o d u c t i o n i s accompanied w i t h f o r m a t i o n of o r g a n i c a c i d s and

+

the medium i s n o t b u f f e r e d H 3 O c o n c e n t r a t i o n w i l l i n c r e a s e and thus pH w i l l 13

T a b l e 3: T o t a l CO s o l u b i l i t y as a f u n c t i o n o f pH pH [ c o2]T / [ c o2]L 4.5 1.01 5.0 1.06 5.5 1. 16 6.0 1.46 6.5 2.40

d e c r e a s e towards t h e p a r t i c l e c e n t r e . T h i s means t h a t CO,, s o l u b i l i t y i s m i n i mal i n t h e c e n t r e . Under t h e s e c o n d i t i o n s t h e c a l c u l a t i o n o f t h e n u c l e u s f o r -m a t i o n c r i t e r i a i s e x a c t l y t h e sa-me as d e s c r i b e d b e f o r e . A c o -m p l i c a t i o n a r i s e s i f t h e s p e c i f i c a c t i v i t y i s pH dependent. I n t h i s case a H^O b a l a n c e i s nee-ded t o c a l c u l a t e t h e pH p r o f i l e w i t h i n t h e p a r t i c l e . T h i s p r o b l e m has been

22 . . .

worked o u t by A t k i n s o n , b u t i s n o t o f i n t e r e s t i n t h i s s t u d y , s i n c e a l l e x -p e r i m e n t s have been c a r r i e d o u t a t a c o n s t a n t -pH.

B) N u c l e u s - _ a n d _ b u b b l e _ f o r m a t i o n

I n t h e p r e v i o u s s e c t i o n the n u c l e u s f o r m a t i o n c r i t e r i a have been d e r i v e d . I n t h i s s e c t i o n t h e s i t u a t i o n w i l l be c o n s i d e r e d when C (0) exceeds C .

Nu-P Nu-PC c l e u s - and b u b b l e f o r m a t i o n a r e n o t t r e a t e d as d i f f e r e n t s t e p s , because t h e d i f f e r e n c e i s o n l y a m a t t e r o f b u b b l e d i m e n s i o n . I f no s u p e r s a t u r a t i o n o c c u r s a n u c l e u s w i l l be formed i f C (0) = C (= gas s o l u b i l i t y ) . A minimum p r e s s u r e

P Ps i s r e q u i r e d f o r t h e development o f a n u c l e u s P = P + P, +

—

(25) b o 1 r, b In w h i c h P = a t m o s p h e r i c p r e s s u r e o P^ = l i q u i d head T = s u r f a c e t e n s i o n r. = n u c l e u s r a d i u s b

I t can be c o n c l u d e d from t h i s e q u a t i o n t h a t , i n case t h e i m m o b i l i z a t i o n m a t r i x c o n t a i n s no i r r e g u l a r i t i e s e n h a n c i n g n u c l e a t i o n , s u p e r s a t u r a t i o n e a s i l y de-v e l o p s .

I f a n u c l e u s has been formed a t s a t u r a t i o n t h e p r e s s u r e w i l l r i s e because o f gas p r o d u c t i o n and c o n s e q u e n t l y t h e b u b b l e w i l l grow, u n l e s s n u c l e a t i o n has i n i t i a t e d r u p t u r e . I n t h a t case f r a c t u r e w i l l r e s u l t i m m e d i a t e l y . N e g l e c t i n g t h i s p o s s i b i l i t y , t h e m e c h a n i c a l s t r e s s i n t h e m a t r i x m a t e r i a l w i l l i n c r e a s e at t h e same t i m e . The e q u i l i b r i u m gas c o n c e n t r a t i o n C^ a t the m a t r i x - b u b b l e

i n t e r f a c e , w h i c h i s p r o p o r t i o n a l t o P w i l l a l s o i n c r e a s e so t h a t d i f f u s i o n a l

b

t r a n s p o r t i s enhanced. A s t e a d y s t a t e can be r e a c h e d by t h i s mechanism. I n case s u p e r s a t u r a t i o n t a k e s p l a c e n u c l e a t i o n has o t h e r consequences. A t t h e moment t h e n u c l e u s i s formed i t w i l l a c t as a c a p a c i t y . D i s s o l v e d gas w i l l a c -cumulate i n i t . T h i s phenomenon causes a v e r y r a p i d growth o f t h e b u b b l e . How-e v How-e r , a s t How-e a d y s t a t How-e can a l s o bHow-e r How-e a c h How-e d i n t h i s c a s How-e .

C) F r a c t u r e _ o f _ t h e _ b i o c a t a l y _ s t _ 2 a r t i c l e

In case a b u b b l e has been formed an a n a l o g y o f t h e f r a c t u r e p r o b l e m c a n be f o u n d i n a t h i c k w a l l e d ( l i t t l e d e f o r m a t i o n ) and a t h i n w a l l e d ( l a r g e deforma-t i o n ) s p h e r i c a l v e s s e l ( s e e f i g . 5).The maximum o v e r p r e s s u r e , w h i c h c a n e x i s deforma-t

U |

Fig. 5: Fig. 5A Thick walled sphere Fig. SB Thin walled sphere

i n s i d e t h e s p h e r e b e f o r e t h e w a l l f r a c t u r e s Ap i s i n t h e s e cases max 23 t h i c k w a l l e d s p h e r e Ap 3 (r / r , N3 max (a b) max 1.5 , 3 , (26) t h i n w a l l e d sphere Ap = 2 (1 - r , / r ) a 'max b a max (27) I n w h i c h a i s t h e maximum a l l o w a b l e s t r e s s , t h e m a t e r i a l can r e s i s t . I n max t h e o r y t h e maximum o v e r p r e s s u r e can be a s s e s s e d u s i n g t h e s e e q u a t i o n s , b u t i t i s i m p o r t a n t t o r e a l i z e t h a t t h e s e r e l a t i o n s a r e o n l y v a l i d f o r s t i f f m a t e r i -a l s ( e . g . s t e e l ) . F r -a c t u r e t h e o r y c o n c e r n i n g m -a t e r i -a l s showing l -a r g e deform-a- deforma-t i o n s i s v e r y c o m p l i c a deforma-t e d . T h e r e f o r e one can g e deforma-t o n l y an i d e a o f deforma-t h e o r d e r o f magnitude o f Ap b y s u b s t i t u t i o n o f t h e b r e a k i n g s t r e n g t h a . ( d e f i n e d as max & ° o f the s t r e s s i n a m a t e r i a l a t f r a c t u r e ) f o r a A p p l i c a t i o n o f e q u a t i o n (26) seems most r e a l i s t i c , b e c a u s e " b a l l o o n - l i k e " beads have n e v e r been o b s e r v e d . I n case r, << r t h i s e q u a t i o n r e d u c e s t o b a n Ap = 0.6 7 0 t max o f (28) 15

The b r e a k i n g s t r e n g t h a ^ i s d e t e r m i n e d i n a t e n s i l e t e s t . At the same time the s t r a i n a t b r e a k £ can be measured. T h i s s t a t i c t e n s i o n t e s t has been

max 2 4

d e s c r i b e d i n d e t a i l by M a r i n . T h e r e f o r e o n l y the d e f i n i t i o n s o f the parame-t e r s used i n parame-t h i s s parame-t u d y need parame-t o be d i s c u s s e d . T h i s i s worked ouparame-t i n A p p e n d i x 2. S i n c e e x a c t c a l c u l a t i o n s were n o t p o s s i b l e on t h e b a s i s of t h e p r e s e n t e d t h e o r i e s , a n o t h e r approach has been chosen, i . e . d i m e n s i o n a l a n a l y s i s . T h i s method w i l l be e l u c i d a t e d i n the f o l l o w i n g p a r a g r a p h s .

D i m e n s i o n a l a n a l y s i s i s a t e c h n i q u e w i d e l y a p p l i e d i n c h e m i c a l e n g i n e e r i n g t o reduce the number of v a r i a b l e s i n v o l v e d i n a p r o b l e m , and thus the number o f n e c e s s a r y e x p e r i m e n t s .

By a p p l y i n g d i m e n s i o n a l a n a l y s i s t o the r e l e v a n t p a r a m e t e r s , d e s c r i b i n g the r u p t u r e p r o b l e m (see A p p e n d i x 3) i t can be shown t h a t m e c h a n i c a l s t a b i l i t y i s a f u n c t i o n of many d i m e n s i o n l e s s groups. However, two of t h e s e groups seem t o be of m a j o r i m p o r t a n c e . F i r s t , the group d e f i n e d as

a , a D

n. = f p (29)

IT q C P x

I n t h i s group a l l a s p e c t s o f the r u p t u r e p r o b l e m ( a p a r t from e x t e r n a l mass t r a n s f e r ) : gas p r o d u c t i o n , gas d i f f u s i o n and m e c h a n i c a l s t r e n g t h a r e combined As can be seen from e q u a t i o n (29) the e f f e c t of the p a r t i c l e r a d i u s appears

2 . . .

as R i n I I j . The p h y s i c a l meaning of t h i s group can be d e r i v e d f r o m t h e f o l i o w i n g crude model (see f i g . 5A):

F o r a gas p r o d u c i n g , s p h e r i c a l b i o c a t a l y s t p a r t i c l e ( r = R) w i t h a gas b u b b l i n the c e n t r e ( s t e a d y s t a t e ) the gas p r o d u c t i o n r a t e can be a p p r o x i m a t e d by

3

%

a 2 q C R an the d i f f u s i o n r a t e by — £ — Ap R . A t s t e a d y s t a t e b o t h r a t e s a r e p x R e q u a l so t h a t an e x p r e s s i o n f o r Ap can be d e r i v e d 2 R q C Ap = (30) P

II, i s now o b t a i n e d as the r a t i o o f O . and Ap. T h i s means t h a t t h i s d i m e n s i o n 1 of l e s s number can be seen as the r a t i o of t h e b r e a k i n g s t r e n g t h t o the o v e r p r e s s u r e i n the gas b u b b l e a t e q u i l i b r i u m .

S e c o n d l y , i f e x t e r n a l mass t r a n s f e r r e s i s t a n c e i s i m p o r t a n t a n o t h e r r e l e v a n t group i s B i (= IJ ) .

I t w i l l be shown i n one of the f o l l o w i n g s e c t i o n s t h a t II. i n f a c t can be used as a s t a b i l i t y c r i t e r i o n .

A t t h i s p o i n t i t s h o u l d be s t r e s s e d t h a t because o f t h e v a r i a n c e o f t h e e x p e r i -m e n t a l r e s u l t s and the i n c o -m p l e t e n e s s o f f o r exa-mple t h e f r a c t u r e t h e o r y , the p r e s e n t e d t h e o r i e s cannot be a p p l i e d i n d e t a i l . However, the t h e o r i e s a r e u s e f u l , because t h e y g i v e i n s i g h t i n t o the p a r a m e t e r s , c o n t r o l l i n g the b u b b l e f o r -m a t i o n and r u p t u r e p r o c e s s e s and i n t o t h e i r r e l a t i v e i -m p o r t a n c e . F o r exa-mple, i t w i l l be c l e a r t h a t the p a r t i c l e r a d i u s w i l l have a marked i n f l u e n c e on me-c h a n i me-c a l s t a b i l i t y , s i n me-c e the s q u a r e d form appears i n e q u a t i o n s ( 1 6 ) , (17) and (29).

MATERIALS AND METHODS

Because two t o t a l l y d i f f e r e n t k i n d s o f e x p e r i m e n t s have been p e r f o r m e d t h i s s e c t i o n i s d i v i d e d i n t o two p a r t s .

"Gas bubble formation" experiments

I m m o b i l i z e d _ y _ e a s t _ c e l l s

C a - a l g i n a t e b e a d s , c o n t a i n i n g y e a s t c e l l s ( d r i e d b a k e r s ' y e a s t (tradename " F e r m i p a n " , a p r o d u c t o f G i s t B r o c a d e s N.V. D e l f t ) ) were o b t a i n e d f r o m G i s t Brocades N.V. D e l f t . The beads were p r e p a r e d by d r o p p i n g a y e a s t / N a - a l g i n a t e s u s p e n s i o n i n t o a 10 g/1 C a C l ^ 2 ^ 0 s o l u t i o n . The used N a - a l g i n a t e ( S a t i a l g i n ) i s a p r o d u c t of R a p i d a s e ( F r a n c e ) .

Medium

The c o m p o s i t i o n o f the medium used i n the e x p e r i m e n t s was: 200 g/1 g l u c o s e ( B a k e r C h e m i c a l s N.V., the N e t h e r l a n d s ) 0.05 M C a C l2 2 H20 0.01 M KH.PO, 2 4 d i s t i l l e d w a t e r A p p a r a t u s

The e x p e r i m e n t s were c a r r i e d out i n a 2 l i t e r f e r m e n t o r ( B i o l a f i t t e , t y p e 010). The e x p e r i m e n t a l s e t - u p i s shown s c h e m a t i c a l l y i n f i g . 6.

G l u c o s e c o n c e n t r a t i o n was d e t e r m i n e d u s i n g t h e h e x o k i n a s e / g l u c o s e 6 phospha-te dehydrogenase method ( B o e h r i n g e r ) . E t h a n o l c o n c e n t r a t i o n was d e t e r m i n e d g a s c h r o m a t o g r a p h i c a l l y . ( P a c k a r d B e c k e r gaschromatograph t y p e 428; Porapak

QS 80-100 mesh; 100 cm; i . d . = 2.0 mm; column t e m p e r a t u r e 150°C; c a r r i e r gas N 35 ml/min.; FID d e t e c t o r ) .

CO. D-H_T CO, I.R. a n a l y z e r M © (T) : s i l i c o n e t u b i n g f o r d i s s o l v e d CO^ mea-surement

Fig. 6: Schematic diagram of the experimental set-up for the "gas bubble formation" experiments

CO^ p r o d u c t i o n r a t e was d e r i v e d from e t h a n o l p r o d u c t i o n r a t e assuming t h a t f o r e v e r y mol o f e t h a n o l formed one mol o f CC^ was p r o d u c e d .

The method used t o measure t h e d i s s o l v e d C0„ c o n c e n t r a t i o n i s a n a l o g o u s t o t h e 25

method d e s c r i b e d by A l f o r d . A p i e c e o f s i l i c o n e t u b i n g ( l e n g t h : 6.1 cm, d i a -meter i : 2.7 mm, d i a m e t e r o: 3.5 mm), c o n n e c t i n g two p i p e s i n t h e l i q u i d was i n f a c t t h e s e n s o r (see f i g . 6 ) . A c o n s t a n t N2 f l o w (44 N l / h ) went t h r o u g h t h e

t u b i n g and t h e n t o an I.R. a n a l y z e r (Beckman Model 865) v i a a d r y i n g chamber, f i l l e d w i t h s i l i c a g e l . The m e a s u r i n g s y s t e m was c a l i b r a t e d u s i n g d i s t i l l e d wa-t e r . A s p e c i f i c CO^ c o n c e n wa-t r a wa-t i o n was s e wa-t i n wa-t h e f e r m e n wa-t o r by b u b b l i n g a mix-t u r e o f N2 and C 02 t h r o u g h t h e l i q u i d . (pH: 4.5; T: 3U°C). T h i s c o n c e n t r a t i o n

v a l u e was d e t e r m i n e d by t i t r a t i o n o f a sample w i t h 0.1 M NaOH. The c a l i b r a t i o n curve i s shown i n f i g . 7. As can be c o n c l u d e d from t h i s d i a g r a m e x t e r n a l mass

x N = 2s • N = 1 0 s-'

Fig. 7: Calibration curve for the dissolved COg measurement

t r a n s f e r ( s t i r r e r speed) i n f l u e n c e s t h e measurement a t N = 2 s . An i n c r e a s e of t h e s t i r r e r speed above N = 10 s ' caused no s i g n i f i c a n t change i n t h e r e a -d i n g o f t h e I.R. a n a l y z e r .

Because d i s t i l l e d w a t e r had been used f o r c a l i b r a t i n g t h e method t h e CO^ s o l u -b i l i t y o f medium ( c o n t a i n i n g K H P O , weak a c i d s ) h a d t o -be d e t e r m i n e d . The

me-26

t h o d used h a s a l s o been d e s c r i b e d by S a l o n e n . The s o l u b i l i t y of CO^ i n me-dium appeared t o be i d e n t i c a l w i t h t h e v a l u e i n w a t e r . I n b o t h cases i t was 1.27 ± 0.03 g/1 (30°C). T h i s v a l u e f i t s w e l l w i t h l i t e r a t u r e d a t a .

P r o c e d u r e

A t t h e s t a r t o f an e x p e r i m e n t t h e f e r m e n t o r was f i l l e d w i t h 0.75 1 medium and a s m a l l volume f r a c t i o n o f C a - a l g i n a t e / y e a s t beads ( a p p r o x i m a t e l y 0.02). P a r t i c l e s , d i f f e r i n g i n r a d i u s R, y e a s t c e l l c o n c e n t r a t i o n C and a l g i n a t e c o n -c e n t r a t i o n C . were used. A t a low s t i r r e r speed (N = 2 s ) t h e p e r -c e n t a g e _{a l g * ' v &} of p a r t i c l e s f l o a t i n g on t h e l i q u i d s u r f a c e was e s t i m a t e d as a f u n c t i o n o f the s t e a d i l y i n c r e a s i n g b u l k CO c o n c e n t r a t i o n (and t i m e ) .

-1

A t a h i g h e r s t i r r e r speed (N = 10 s ) a d e f i n i t e CO^ c o n c e n t r a t i o n was s e t . The p e r c e n t a g e o f f l o a t i n g p a r t i c l e s was c o u n t e d a f t e r 1 h o u r , b e c a u s e i t was found t h a t t h e number was more o r l e s s c o n s t a n t t h e n . Next t h e CO^ cone, was i n c r e a s e d and t h e p r o c e d u r e was r e p e a t e d u n t i l CO^ s a t u r a t i o n . The s p e c i f i c e t h a n o l p r o d u c t i v i t y o f e v e r y b a t c h o f p a r t i c l e s used was d e t e r m i n e d i n a s e -p a r a t e e x -p e r i m e n t .

D u r i n g a l l e x p e r i m e n t s t e m p e r a t u r e was 30°C; pH was m a i n t a i n e d c o n s t a n t a t 4.5.

Tensile strength test

C h e m i c a l s

A l g i n a t e s o l u t i o n s and N a - a l g i n a t e / y e a s t s u s p e n s i o n s were o b t a i n e d from G i s t Brocades N.V. D e l f t ; K c a r r a g e e n a n was p u r c h a s e d from Sigma and agar f r o m D i f -co. O t h e r r e a g e n t s were l a b o r a t o r y - g r a d e m a t e r i a l s .

Fi§E§E§ti^n_of_the_slabs

S l a b s o f K - c a r r a g e e n a n , p o l y a c r y l a m i d e (PAAm) and a g a r - a g a r were p r e p a r e d by g e l a t i o n c.q. p o l y m e r i z a t i o n i n a p e t r i - d i s h .

Because a C a - a l g i n a t e g e l s h r i n k s d u r i n g r e a c t i o n w i t h C a+ + i o n s , a method had

to be d e s i g n e d t o p r e p a r e a s l a b o f a c o n s t a n t t h i c k n e s s and a f l a t s u r f a c e . A y e a s t / N a - a l g i n a t e s u s p e n s i o n ( y e a s t was k i l l e d b y a d d i t i o n o f i o d o - a c e t i c a c i d ) was p o u r e d on a s i n t e r e d g l a s s f i l t e r p l a t e on t h e b o t t o m o f a b e a k e r o f the same d i a m e t e r . The g l a s s f i l t e r p l a t e had p r e v i o u s l y been c o v e r e d w i t h a p a p e r f i l t e r , soaked w i t h 10 g/1 C a C ^ ^ R ^ O s o l u t i o n . The s u s p e n s i o n l a y e r was c o v e r e d w i t h a n o t h e r w e t t e d paper f i l t e r . The b e a k e r was f i l l e d w i t h a 10 g/1

C a C l ^ . 21^0 s o l u t i o n . F i n a l l y a n o t h e r s i n t e r e d g l a s s f i l t e r p l a t e was p u t on t o p . Some p r e s s u r e was e x e r t e d by hand upon t h e t o p p l a t e t o smoothen t h e s u r -f a c e s o -f t h e s l a b .

A f t e r 24 h o u r s t h e f i l t e r s were removed. The C a - a l g i n a t e s l a b was s t o r e d i n a 1% C a C ^ . 2 ^ 0 s o l u t i o n . A f t e r a n o t h e r 24 h o u r s specimens were c u t and t e s t e d .

M22su^emen£_of _ t h e J ^ r e a k i j i g ^ s ^ r e

The t e n s i l e s t r e s s - s t r a i n d i a g r a m o f d i f f e r e n t m a t e r i a l s was measured w i t h an a p p a r a t u s f o r t e s t i n g t e n s i l e s t r e n g t h ( I n s t r o n TT/CM). A specimen o f m a t e r i a l was c u t w i t h a mold (see f i g . 8) from a s l a b o f t h e m a t e r i a l t o be measured. The measurements were c a r r i e d out a t ambient t e m p e r a t u r e . To p r e v e n t d r y i n g o f t h e specimens t h e y were w e t t e d d u r i n g t h e measurements.

Fig. 8: Three dimensional diagram of the mold used for the preparation of the specimens. Dimensions in millimetres

RESULTS

"Gas bubble formation" experiments

The main r e s u l t s o f t h e "gas b u b b l e f o r m a t i o n " e x p e r i m e n t s a r e g i v e n i n t a b l e 4. As c a n be c o n c l u d e d from t h e t a b l e (see "% f l o a t i n g " ) t h e b a t c h e s o f p a r t i -c l e s -c a n be d i v i d e d i n t o two g r o u p s : s t a b l e and u n s t a b l e . I n t h i s -c o n t e x t " s t a b l e " means t h a t n o t a s i n g l e p a r t i c l e was f l o a t i n g d u r i n g t h e e x p e r i m e n t s . "Not s t a b l e " means t h a t a c e r t a i n p e r c e n t a g e r o s e t o t h e l i q u i d s u r f a c e . Some of t h e s e p a r t i c l e s r u p t u r e d a f t e r some t i m e .

I2il

u

222i_2^_5

Two e x p e r i m e n t s (no. 2 and 3) were p e r f o r m e d u s i n g p a r t i c l e s , o n l y d i f f e r i n g i n r a d i u s . A s m a l l i n c r e a s e o f R c l e a r l y d i m i n i s h e d t h e s t a b i l i t y .

I n f l u e n c e o f C _x

E x p e r i m e n t s , numbered 4, 5 and 6 d i f f e r e d i n u s i n g a d i f f e r e n t c e l l c o n c e n t r a

T a b l e 4: R e s u l t s o b t a i n e d d u r i n g "gas b u b b l e f o r m a t i o n " e x p e r i m e n t s b a t c h no. R** (mm) C X ( g / D a l g ( g / D qc o2 (g/g dw, h r ) N ( s "1) [ c o2]L (g/1) time (min) % f l o a t i n g 1 1. 1 103 52 0.22 2* 0.99 80 0 2 1.0 94 38 0.22 2 0.51 80 0 3 1.4 90 36 0.24 2 0.42/0 .22/0.40 80 92/72/56 4 1.0 60 23 0.20 2 0.64 80 0 5 0.9 97 24 0.22 2 0.56 80 22 6 1 .0 124 24 0. 18 2 0.51 80 58 u s i n g e q u a t i o n (21) c a n be shown t o be a p p r o x i m a t e l y 4.0 . 1 0 ~5 m/s ( B i = P 70) 1 1 . 1 103 52 0.22 10* 1.26 240 0 2 1 .0 94 38 0. 22 10 1 .26 240 0 3 1.4 90 36 0.24 10 1.20/1 . 1 2 240 34/71 4 1 .0 60 2 3 0. 20 10 1.18 240 0 5 0.9 97 24 0. 22 10 1 .22 240 24 6 1.0 124 24 0. 18 10 1.24/1 , ,18 240 31/20

*

_{u s i n g e q u a t i o n (21) k^ c a n b e shown t o be a p p r o x i m a t e l y} -4 1.0 .10 m/s ( B i = _P 180)

**

_{s t a n d a r d d e v i a t i o n : 0.03 mm}

t i o n . An i n c r e a s i n g v a l u e o f c l e a r l y d e c r e a s e d s t a b i l i t y . I n f l u e n c e o f C ,

al g

I t i s o b v i o u s t h a t b e a d s , p r e p a r e d from a 2.5% and 2% a l g i n a t e s u s p e n s i o n ( b a t c h 1 and 2) a r e s t a b l e compared w i t h b e a d s , p r e p a r e d from a 1% s u s p e n s i o n ( b a t c h 5 ) . A h i g h e r a l g i n a t e c o n c e n t r a t i o n enhances s t a b i l i t y . The a c t u a l c a^ g

i s l a r g e r t h a n 0 f the s u s p e n s i o n due t o s h r i n k i n g o f t h e beads. Of c o u r s e , the same h o l d s f o r C .

x

As can be c o n c l u d e d from t a b l e 5 t h e s t i r r e r speed i n f l u e n c e s t h e o b s e r v e d s t a b i l i t y . A t comparable b u l k CO^ c o n c e n t r a t i o n the p e r c e n t a g e of f l o a t i n g p a r t i -c l e s i s h i g h e r a t low N. A n o t h e r o b s e r v a t i o n was t h a t , when t h e p a r t i -c l e s o f any b a t c h were l e f t s t a n d i n g i n a g l a s s c y l i n d e r they a l l r o s e t o t h e l i q u i d s u r f a c e , f r a c t u r i n g w i t h i n a few m i n u t e s . T a b l e 5: E x p e r i m e n t a l d a t a i l l u s t r a t i n g t h e e f f e c t o f s t i r r e r the p e r c e n t a g e o f f l o a t i n g p a r t i c l e s b a t c h n o . [ c o2] L % f l o a t i n g N (s ') time (min) 3 0 60 21 10 120 0 47 63 2 83 3 0 25 17 10 60 0 22 74 2 80 5 0 73 1 7 10 180 0 52 5 8 2 80 6 1 12 24 10 240 0 99 48 2 113 D u r i n g e x p e r i m e n t s a t N = 2 s ' t h e number o f f l o a t i n g p a r t i c l e s i n c r e a s e d w i t h t i m e . However, a t the same t i m e t h e b u l k CO^ c o n c e n t r a t i o n i n c r e a s e d due to CO^ p r o d u c t i o n (see t a b l e 6 ) . F u r t h e r m o r e , i t has t o be r e a l i z e d t h a t t h e s e numbers a r e o n l y e s t i m a t e s , because c o u n t i n g o f t h e p a r t i c l e s i s i m p o s s i b l e . I n a c c u r a c y i n c r e a s e s w i t h t h e numbers.

The e f f e c t o f [CO ] i s a p p a r e n t from two k i n d s o f e x p e r i m e n t a l r e s u l t s . -1

i ) A t N = 2 s t h e p e r c e n t a g e o f f l o a t i n g p a r t i c l e s i n c r e a s e d f a s t e r w i t h t i m e a t a h i g h e r s t a r t i n g v a l u e o f [ C C ^ L (s e e f i g - 9 ) .

i i ) A t N = 10 s ' t h e number o f f l o a t i n g p a r t i c l e s a l s o i n c r e a s e d w i t h [CO^] ^ (see t a b l e 7 ) . I n t h i s c a s e , however, time dependence a l s o p l a y s a r o l e 2 2