AN ENGINEERING STUDY OF BACTERIAL KINETICS AND ENERGETICS

A . A . Esener

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Delft University of Technology

A N ENGINEERING STUDY OF BACTERIAL KINETICS AND ENERGETICS

Proefschrift

ter verkrijging van de graad van doctor in de technische wetenschappen aan de Technische Hogeschool Delft, op gezag van de rector magnificus prof. ir. B.P.Th. Veltman, voor een commissie aangewezen door het college van dekanen te verdedigen op donderdag 1 oktober te 14.00 uur door
A l i A y d i n Esener

Chemical Engineer B.Sc. M.Sc. geboren te Ankara

Delft University Press, 1981

DR.b a t c h growth ( p a r t o f F i g . 3 o f Chapter 4) .Dit proefschrift is goedgekeurd door de promotoren PROF. ROELS On the f r o n t cover d e v i a t i o n s between the u n s t r u c t u r e d model p r e d i c t i o n s and t h e e x p e r i m e n t a l r e s u l t s a r e shown f o r oxygen uptake and carbon d i o x i d e p r o d u c t i o n r a t e s d u r i n g f e d . J. N. IR. KOSSEN PROF.W. IR.A.F.

Dedicated to my parents .

J.C. Bolmann and C. Chapter 5 and Chapter 7.L. A. G. van Suijdam f o r many d i s c u s s i o n s and t r a n s l a t i n g the summary t o Dutch I r . respectively G i s t Brocades N. Roozenburg.C.I n the c o m p l e t i o n of t h i s t h e s i s I g r a t e f u l l y acknowledge: P r o f e s s o r s Kossen and R o e l s f o r t h e i r e x c e p t i o n a l guidance and encouragement as my s u p e r v i s o r s Dr. van Eybergen f o r t r o u b l e s h o o t i n g i n many computer programs Messrs. J . B o l and T.Ph. van der Steen f o r c h e m i c a l a n a l y s i s of samples Messrs. Ras and G. de Graaf and B.J. of D e l f t f o r f i n a n c i a l l y s u p p o r t i n g me d u r i n g t h i s work K r a u s . J. Veerman f o r t h e i r c o n t r i b u t i o n s t o A p p l i c a t i o n 3.T. C. B r o n k h o r s t . Warnaar f o r drawings and photographs My s t u d e n t s .M.U i t h o f Fonds f o r t h e i r f i n a n c i a l c o n t r i b u t i o n towards the p r i n t i n g c o s t s of t h i s t h e s i s .V. K e r k d i j k f o r h e l p i n t h e h a n d l i n g and maintenance o f the b i o r e a c t o r s and a u x i l i a r y equipment Messrs. G. F. I r .

T A B L E OF CONTENTS CHAPTER 1 INTRODUCTION I II Aim and scope O r g a n i z a t i o n of t h i s thesis CHAPTER ON THE THEORY AND APPLICATIONS OF UNSTRUCTURED GROWTH MODELS I II III IV V VI VII Development of m i c r o b i a l e n e r g e t i c s Macroscopic methods i n the study o f e n e r g e t i c s An i n t r o d u c t i o n t o the m o d e l l i n g o f m i c r o b i a l growth A s i m p l e u n s t r u c t u r e d model f o r m i c r o b i a l growth D i s c u s s i o n o f u n s t r u c t u r e d models and e n e r g e t i c s with reference to experimental r e s u l t s A d i s c u s s i o n on the concept o f maintenance Nomenclature and r e f e r e n c e s 5 7 9 10 13 21 22 CHAPTER 3 MATERIALS AND METHODS I II III IV D e s c r i p t i o n o f t h e e x p e r i m e n t a l system and a n a l y t i c a l methods Developed and used t o o l s and methods A p p l i c a t i o n o f the s t a t i s t i c a l t e c h n i q u e s i n t h e study o f m i c r o b i a l k i n e t i c s and e n e r g e t i c s Nomenclature and r e f e r e n c e s 25 27 32 39 CHAPTER 4 FED-BATCH CULTURE . MODELLING AND APPLICATIONS IN THE STUDY OF MICROBIAL ENERGETICS I II III IV V VI VII VIII IX 41 Summary Introduction 41 42 Model D e t e r m i n a t i o n o f b i o k i n e t i c and e n e r g e t i c parameters 44 45 M a t e r i a l s and Methods 46 R e s u l t s and d i s c u s s i o n 55 Conclus i o n s 56 Appendix Nomenclature and r e f e r e n c e s 57 CHAPTER 5 GROWTH OF MONO AND MIXED CULTURES IN SALINE ENVIRONMENT I II III IV V VI VII Abstract Introduction M a t e r i a l s and Methods Results Discussion Conclusions References 59 59 60 61 63 66 67 .

d e t e r m i n a t i o n o f the maximum s p e c i f i c growth r a t e On t h e s t a t i s t i c a l a n a l y s i s o f b a t c h d a t a Carbon d i o x i d e hold-up as a source o f e r r o r i n batch c u l t u r e c a l c u l a t i o n s II III 89 95 IV V 101 109 117 121 123 125 SUMMARY SAMENVATTING OZET VI .CHAPTER 6 THE INFLUENCE OF TEMPERATURE ON THE KINETICS AND ENERGETICS I II III IV V VI Introduction Model M a t e r i a l s and Methods R e s u l t s and D i s c u s s i o n Nomenclature and r e f e r e n c e s Addendum i n f l u e n c e o f temperature on k i n f l u e n c e of temperature on e n e r g e t i c parameters consequences f o r e n g i n e e r i n g o p e r a t i o n s and design References s 69 69 70 71 73 75 75 77 78 VII CHAPTER 7 A STRUCTURED MODEL FOR BACTERIAL GROWTH I II III IV V VI VII Introduction T h e o r e t i c a l development o f t h e g e n e r a l s t r u c t u r e d model D e s c r i p t i o n o f t h e two compartmental system D e r i v a t i o n o f the b a l a n c e e q u a t i o n s E v a l u a t i o n o f the v a l i d i t y o f the model Discussion Nomenclature and r e f e r e n c e s 79 80 81 83 84 85 88 CHAPTER 8 APPLICATIONS I Comments on t h e d e s c r i p t i o n o f maintenance metabolism d u r i n g a n a e r o b i c growth w i t h product formation B i o e n e r g e t i c c o r r e l a t i o n o f COD t o BOD D e s c r i p t i o n o f m i c r o b i a l growth b e h a v i o u r d u r i n g the wash-out phase.

P.4 idem 4 line 8 + line 4 eq(3) t 1ine.869-4.698(0.631) 10 t i m e s ^x» dX/dt = (2x) K s t line 5 10 times Mg 22 44 higher » Kg p - P.ERRATA s h o u l d read 19 19 P.10" lower 2 0. P. f line 4 the l a s t term of eq(5) s h o u l d be eq(8) u n s u b s c r i p t e d ¥ s s h o u l d be Y / .6 eq(16) line 4 0.689-4. P.698(0. P. P. 81 82 83 83 110 112 113 dx/dt = 7 K s h o u l d be r e p l a c e d by G 2 nd term of eq(19) s h o u l d be d i v i d e d by ( k and Y S7> K Y^q) 2 s h o u l d be Ygjj cannot can 0Lj)2} s. P.631). and not T a b l e 1.

1 I s .

O f t e n t h e r e a r e i n t e r a c t i o n s between these processes as i n d i c a t e d by the two way arrows i n F i g . i ) b i o s y n t h e t i c p r o c e s s d u r i n g which p r e c u r s o r s are formed from the s u b s t r a t e f o l l o w e d by t h e p o l y m e r i z a t i o n of them i n t o biomass. 1: Distribution and Kossen.CHAPTER 1 INTRODUCTION I AIM AND SCOPE I n t h i s t h e s i s some a s p e c t s of b a c t e r i a l k i n e t i c s and e n e r g e t i c s a r e s t u d i e d with reference to engineering a p p l i c a t i o n s . 1. For e n g i n e e r i n g a p p l i c a t i o n s the v e r b a l model presented i n F i g u r e 1 p r o v i d e s a good a p p r o x i m a t i o n t o r e a l i t y i n the d e s c r i p t i o n of b a c t e r i a l growth and primary product metabolism. An a m b i t i o n o f the b i o t e c h n o l o g i s t i s t o understand how the i n p u t energy and 1 . metabolism (from Roels Here b a s i c a l l y t h r e e processes a r e i d e n t i f i e d . product synthesis use of substrate synthesis of biomass precursors biomass synthesis ATP pool maintenance Fig.16). of substrate energy in microbial 19 78. i i ) product f o r m a t i o n and i i i ) maintenance p r o c e s s e s . ref. The energy i n p u t i n t o t h e system i n t h e form of c h e m i c a l energy i s d i s t r i b u t e d between these p r o c e s s e s . see Chapter 2.

b a t c h u n s t r u c t u r e d model presented e a r l i e r . The l a s t s u b . The f i r s t d e s c r i b e s the e x p e r i m e n t a l system and the a n a l y t i c a l methods used.s e c t i o n shows how the use of s t a t i s t i c a l p r o cedures can improve the e f f i c i e n c y of e x p e r i m e n t a t i o n and the r e l i a b i l i t y of the d a t a o b t a i n e d . F i n a l l y .b a t c h c u l t i v a t i o n i n a l s o shown and d i s c u s s e d . The d i s c u s s i o n s are i l l u s t r a t e d . the i n f l u e n c e of temperature i s s t u d i e d i n f e d . 1. The second s u b . s t a r t s w i t h an i n t r o d u c t i o n t o the c u r r e n t s t a t e of m i c r o b i a l e n e r g e t i c s . c o n s i s t s of t h r e e s u b . The c o n c e n t r a t i o n range i s from 0 to 40 kg/m3.s e c t i o n o u t l i n e s some of the techniques developed f o r o b t a i n i n g more o p t i m a l i n f o r m a t i o n from the e x p e r i m e n t a l d a t a .mass i n the form of s u b s t r a t e are d i s t r i b u t e d between these p r o c e s s e s . d e s c r i b e s b a c t e r i a l growth i n f e d . How can models c o n s i d e r i n g i n t e r n a l changes i n the microorganisms be formulated? What are t h e i r p r o s p e c t s ? II ORGANIZATION OF THIS THESIS Contents of the c h a p t e r s are b r i e f l y o u t l i n e d i n the f o l l o w i n g .s e c t i o n s . 2. i n an attempt to m a n i p u l a t e t h i s d i s t r i b u t i o n as to m i n i m i z e the o v e r a l l c o s t of the d e s i r e d product.b a t c h . T h i s can b e s t be a c h i e v e d by d e s c r i b i n g the whole p r o c e s s by a m a t h e m a t i c a l model and o p t i m i z i n g i t a c c o r d i n g to an o b j e c t i v e function.b a t c h c u l t u r e s . 2 . c o n t i n u o u s ) e n e r g e t i c b e h a v i o u r of the system? i n f l u e n c e the 4. The d a t a o b t a i n e d i s a l s o compared w i t h those r e p o r t e d f o r a c t i v a t e d sludge c u l t u r e s under the same c o n d i t i o n s . breaks down e x p o n e n t i a l t o s u b s t r a t e l i m i t e d growth phase. The c h o i c e of k i n e t i c and e n e r g e t i c r e l a t i o n s i s d i s c u s s e d . Temperature e f f e c t s on e n e r g e t i c parameters are a l s o p r e s e n t e d . I n Chapter 6. the i n f l u e n c e s of s e l e c t e d environmental changes on the s u b s t r a t e energy d i s t r i b u t i o n are s t u d i e d . W i t h these c o n s i d e r a t i o n s i n mind the f o l l o w i n g q u e s t i o n s were s e t and s t u d i e d i n an e f f o r t t o develop a sound s t r a t e g y f o r r e s e a r c h . Chapter 2. The t h e o r i e s to be used l a t e r on. How can a m i c r o b i a l system be modelled based on the e x i s t i n g knowledge? data How can the e x p e r i m e n t a l and c o m p u t a t i o n a l methods be improved and processed i n o r d e r to o b t a i n more o p t i m a l i n f o r m a t i o n ? 3. Chapter 4. supported and/or t e s t e d w i t h the b a t c h and continuous c u l t u r e d a t a o b t a i n e d d u r i n g t h i s study. 5. a s h o r t comment on the concept of maintenance i s g i v e n . To what e x t e n t are the k i n e t i c and e n e r g e t i c parameters i n f l u e n c e d by s e l e c t e d environmental changes ( s a l i n i t y and temperature)? Does the mode of c u l t i v a t i o n ( b a t c h . K i n e t i c d a t a are used f o r the e s t i m a t i o n of thermodynamic parameters i n an A r r h e n i u s type of model extended t o d e s c r i b e a l s o the s u p e r o p t i m a l temperature range. Chapter 3. I t i s shown t h a t the d u r i n g the t r a n s i t i o n from of f e d . d e s i g n and c o n t r o l of m i c r o b i a l processes. F i r s t (Chapter S) the i n f l u e n c e of the presence of NaCl i s e v a l u a t e d at d i f f e r e n t c o n c e n t r a t i o n s . are developed here. Use the study of m i c r o b i a l k i n e t i c s and e n e r g e t i c s i s mode. M i c r o b i a l e n e r g e t i c s and k i n e t i c s are d i s c u s s e d i n c o n n e c t i o n w i t h the f o r m u l a t i o n of u n s t r u c t u r e d models. f e d . In Chapters 5 and 6.

s i n c e t h i s i s a more fundamentel u n i t then molar or mass u n i t s . c o n s i s t s of f i v e s h o r t p u b l i c a t i o n s which are a l l more or l e s s a p p l i c a t i o n s of the c o n s i d e r a t i o n s p r e s e n t e d i n Chapter 2 and 3. SBR. Klebsiella pneumoniae NCTC 418 f o r m e r l y known as Klebsiella aerogenes i s chosen as the e x p e r i m e n t a l organism. The Netherlands 3 . The s i m p l e two compartmental model developed i s shown to d e s c r i b e biomass and s u b s t r a t e p r o f i l e s w e l l . a model d e s c r i b i n g the i n t e r n a l s t r u c t u r e of the organism i n a d d i t i o n t o macroscopic v a r i a b l e s .Chapter 7. Biotechnology Group. an e q u i v a l e n t of biomass i s the same as one mole of biomass. Delft 2600. i s an attempt to f o r m u l a t e and t e s t a simple s t r u c t u r e d model i . A l l u n i t s i n v o l v i n g biomass dry weight are expressed on a s h . An e q u i v a l e n t of any compound i s d e f i n e d as t h a t amount c o n t a i n i n g 12 grams of e l e m e n t a l carbon. For the biomass formulae used h e r e . E x t e n s i v e t e s t s w i t h i n t e r n a l c o m p o s i t i o n d a t a i n d i c a t e s the weakness of the model.. Postal address: Department of Chemical Engineering.f r e e b a s i s . Chapter 8. . This thesis has been carried out within the Biotechnology Group of the delft University of Technology. The f i r s t two i l l u s t r a t e the use of macroscopic methods i n d a t a a n a l y s i s and c o r r e l a t i o n . e s t i m a t i o n of the maximum s p e c i f i c growth r a t e by the wash-out t e c h n i q u e and e s t i m a t i o n of the carbon d i o x i d e r e t a i n e d i n b r o t h d u r i n g b a t c h c u l t i v a t i o n . The o t h e r s are on the a p p l i c a t i o n of s t a t i s t i c s on b a t c h d a t a . s i n c e i t i s a t y p i c a l s o i l b a c t e r i u m o f t e n a l s o p r e s e n t i n waste waters and i s capable of growing a e r o b i c a l l y and anaerobically. P r o s p e c t s of these type of models and the c o r r e c t approach to t h e i r f o r m u l a t i o n and v e r i f i c a t i o n are stressed. Jaffalaan 9. The y i e l d of biomass on s u b s t r a t e i s sometimes expressed as C-equiv/C-equiv ( same as C-mole/C-mole ) . TH. I t i n d i c a t e s d i r e c t l y the f r a c t i o n a l c o n v e r s i o n of s u b s t r a t e carbon to biomass carbon. e .

.

i . Monod has d e f i n e d the m a c r o s c o p i c y i e l d o f b i o mass on s u b s t r a t e as the r a t i o o f the biomass produced t o s u b s t r a t e consumed.U e (1) Here i s t h e r a t e of endogeneous metabolism. When C >> K . endogeneous metabolism proceeds a t c o n s t a n t r a t e a t a l l p o s s i b l e growth r a t e s . He has produced h i s w e l l known k i n e t i c e x p r e s s i o n d e s c r i b i n g the dependence o f growth r a t e on the c o n c e n t r a t i o n o f the growth l i m i t i n g s u b s t r a t e ' . Then p h y s i o l o g i c a l cons i d e r a t i o n s were attached t o t h i s o b s e r v a t i o n . the m a c r o s c o p i c growth yield. . This i s e q u i v a l e n t t o s e l f d e s t r u c t i o n .U g { C s / (K s + C g )} . The observed y i e l d f o r a continuous c u l t u r e system can now be shown t o be g i v e n by. .CHAPTER 2 ON THE THEORY AND APPLICATIONS OF UNSTRUCTURED GROWTH MODELS: KINETIC AND ENERGETIC ASPECTS I DEVELOPMENT OF MICROBIAL ENERGETICS Development o f q u a n t i t a t i v e b a c t e r i a l e n e r g e t i c s can be assumed t o have commenced w i t h the work of Monod'. Y was n o t c o n s t a n t b u t d e c r e a s e d as t h e d i l u t i o n r a t e d e c r e a s e d . p . In e f f e c t the f i r s t attempt was a c u r v e f i t t i n g e x e r c i s e . The i n t r o d u c t i o n o f t h i s concept m o d i f i e d Monod's e x p r e s s i o n to a new form: s x U .P > and hence Vmax Pg ~ Ve • When C = 0 \i e g u a l s t o . * H e r b e r t a t t r i b u t e d t h i s e f f e c t t o what he c a l l e d the endogeneous metabolisrn. e .l i m i t e d c o n t i n u o u s c u l t u r e s .3 s s m a x = s e y sx = y m a X sx {D / ( D + u e )} (2) P i r t (1965) c o n s i d e r e d t h e s u b s t r a t e requirement f o r growth a s s o c i a t e d and nona s s o c i a t e d f u n c t i o n s s e p a r a t e l y and p o s t u l a t e d h i s w e l l known r e c i p r o c a l / l i n e a r relation^: 1 / Y SX = 1 / Y m a x + m s / y S X v (3) ' He has f o r m a l l y i n t r o d u c e d the maintenance c o e f f i c i e n t m .p . n e g a t i v e growth i s achieved. I t was suggested t h a t .and a t t r i b u t e d i t t o s 5 . F o l l o w i n g the i n t r o d u c t i o n of c o n t i n u o u s c u l t i v a t i o n t e c h n i q u e s . H e r b e r t (1958) has p r e sented evidence t h a t i n C .

the below r e l a t i o n has been proposed6. the s t a t e of m i c r o b i a l e n e r g e t i c s i s s t i l l not advanced enough a t the fundamental l e v e l to a l l o w e n g i n e e r i n g a p p l i c a t i o n s to be based on them. R e c e n t l y van V e r s e v e l d ^ has reviewed the methods a v a i l a b l e f o r d e t e r m i n i n g the P/0 r a t i o i n b a c t e r i a l s y s tems. Thus.(3) by c o n s i d e r i n g the g e n e r a l energy c u r r e n c y . From h i s account and l i t e r a t u r e i t becomes c l e a r t h a t t h e r e i s y e t no r e l i a b l e method f o r the e s t i m a t i o n of the P/0 r a t i o s . f o r a e r o b i c systems one must know the s o .(3) p r e d i c t s a s t r a i g h t l i n e f o r 1/Y v s . 9 .e n e r g e t i c b e h a v i o u r . osmotic work to m a i n t a i n c o n c e n t r a t i o n g r a d i e n t s . q. t u r n over of c e l l m a t e r i a l s . These a u t h o r s have demonstrated t h a t the presence of u n c o u p l e r s . s t r a i g h t l i n e s c o u l d not be o b t a i n e d and t h i s was shown to be due to the i n f l u ence of the growth r a t e on the f e r m e n t a t i o n p a t t e r n and ATP y i e l d of the p a r t i c u l a r organism.c a l l e d maintenance f u n c t i o n s which i n c l u d e . The r e s p i r a t o r y c h a i n of b a c t e r i a d i f f e r w i d e l y and depend on the growth c o n d i t i o n s . 6 . S i n c e these s t u d i e s r e l y on b a l a n c i n g methods and p r i n c i p l e s of thermodynamics. Eq. 1/u . ATP. c e l l m o t i l i t y e t c . Thus more o f t e n than n o t .1 4 Although an overwhelming body of i n f o r m a t i o n e x i s t s i n l i t e r a t u r e .the s o . T h i s approach may not be v a l i d s i n c e a e r o b i c and a n a e r o b i c sysems are q u i t e d i f f e r e n t e n e r g e t i c a l l y . I n some cases ^ATP l o b t a i n e d from a n a e r o b i c s t u d i e s were used f o r the c a l c u l a t i o n of P/0 r a t i o s and Y ^ p v a l u e s . however. and o b t a i n e d the f o l l o w i n g form^: sx q ATP = y 1 ATP + m ATP ( A ) L a t e r on. M i c r o b i a l e n e r g e t i c s b e i n g at an impasse at the fundamental l e v e l . Stouthamer and Bettenhaussen m o d i f i e d eq. anabolism and c a t a b o l i s m are sometimes not i n tune w i t h each o t h e r and c o n s i d e r a b l e amount of ATP produced c o u l d be wasted. or v i c e v e r s a . one has t o work i n terms of m a c r o s c o p i c y i e l d s and hence eq. i n an attempt to account f o r the d i s c r e p a n c y between the t h e o r e t i c a l and e x p e r i m e n t a l growth y i e l d s .(3) s t i l l f i n d s wide a p p l i c a t i o n . Based on t h i s o b s e r v a t i o n . the Y ^ p v a l u e s can o n l y be c a l c u l a t e d f o r anaerobic systems i f the m e t a b o l i c pathway and the a s s o c i a t e d s t o i c h i o m e t r y are e x a c t l y known. as has been p o i n t e d out by Stouthamer'3 enough a t t e n t i o n has not been p a i d by many workers to t h e i r e n e r g e t i c c a l c u l a t i o n s and t h i s c o u l d be one of the reasons f o r the a c c u m u l a t i o n of i n c o n s i s t e n t d a t a over the y e a r s . In a number of c a s e s . Furthermore.c a l l e d P/0 r a t i o i n order to c a l c u l a t e the Y^ p v a l u e s . R e c e n t l y . e T v a u e s Under a v a r i e t y of c o n d i t i o n s the growth y i e l d s observed were much lower than expected. A d d i t i o n a l l y . Senez^ s t u d i e d t h i s phenomenon and i n t r o d u c e d the term unbalanced growth i m p l y i n g t h a t the two major processes i n the m i c r o o r g a n i s m s . they are favoured f o r q u a n t i t a t i v e t e c h n o l o gical applications. r e c e n t l y much work has been done on the m a c r o .™ ATP - M U / ( YIJ1 ATP ) . p a r t i c u l a r l y f o r a e r o b i c growth w i t h no by-product f o r m a t i o n . excess energy and C-source and f o r c e d t r a n s i e n t s enhance the extent of energy s p i l l a g e . . N e i j s s e l and Tempest^i'O have demonstrated the occurence of t h i s phenomenon i n a number of systems and c o n s i d e r e d energy s p i l l i n g r e a c t i o n s as an i n t e g r a l p a r t of the e v o l u t i o n a r y c o m p e t i t i o n c a p a b i l i t i e s of m i c r o o r g a n i s m s . 'theoretical + m g \i + m e (5) T h i s e q u a t i o n has found l i m i t e d a p p l i c a t i o n s i n c e no means were o f f e r e d f o r the d e t e r m i n a t i o n of the growth a s s o c i a t e d (nig) and independent (m ) maintenance c o e f f i c i e n t s .1 1 For a comprehensive a n a l y s i s of the c u r r e n t s i t u a t i o n i n m i c r o b i a l e n e r g e t i c s the reader i s r e f e r red t o r e c e n t r e v i e w s ^ .

E r i c k s o n et l l 8 . the f o l l o w i n g s t o i c h i o m e t r i c growth e q u a t i o n can be w r i t t e n f o r growth on a s i n g l e C and energy source. y s Y a r e p d e f i n e d by the f o l l o w i n g : 7 . N and 0 are the o n l y elements exchanged i n n o n . 3 a^ b c d^ Q 3 3 + $. bj Cj + <I>0„ 5 2 + $. Y x . Recent advancement of the s u b j e c t i s due to Roels and Kossen'^.C0„ 6 2 + $ H.n e g l i g i b l e amounts i n the system. e . Thus s p e c i f i c a t i o n of any 3 flows f i x e s the system a l g e b r a i c a l l y .II MACROSCOPIC METHODS IN THE STUDY OF MICROBIAL ENERGETICS I n f o r m a t i o n o b t a i n e d by the a p p l i c a t i o n of e l e m e n t a l and energy b a l a n c e s and entropy i n e q u a l i t i e s can be c l a s s i f i e d as m a c r o s c o p i c i n f o r m a t i o n . These t e c h n i q u e s . = .0 7 2 7 (6) product The macroscopic y i e l d f a c t o r i s now Y d e f i n e d as: (C-equiv/C-equiv) (7) sx = I $ 2 I ^ 2 a Y s i m p l y i n d i c a t e s the degree of t r a n s f o r m a t i o n of the s u b s t r a t e carbon i n t o biomass. The s u b j e c t has been advanced by many workers i n the r e c e n t y e a r s . The above e q u a t i o n has 7 f l o w s i n v o l v i n g 4 elements. T h i s source i s assumed to be growth l i m i t i n g . biomass substrate $C H. These r e l a t i o n s w i l l be a p p l i e d to e x p e r i m e n t a l and t h e o r e t i c a l a n a l y s e s . 0„C 2 H. I f structured i n f o r m a t i o n i s supplemented and checked f o r c o n s i s t e n c y by the a p p l i c a t i o n of m a c r o s c o p i c methods v e r y u s e f u l i n f o r m a t i o n can be o b t a i n e d w i t h q u a n t i t a t i v e confidence.d . Hence i t seems t o be a more fundamental parameter than y i e l d v a l u e s expressed on mass or molar b a s i s . i . R o e l s ' ^ . H. 0 N. a Assuming t h a t C. d.r x ( y .NH„ 4 3 >.v r ) 'x x 'p p d 3 r p 's (8) (9) (10) r.2 0 and H e i j n e n and Roels21. n e v e r t h e l e s s . r + d r + N 2 s 1 x Here. l a t e r on i n t h i s work. any 4 unknown f l o w s can be c a l c u l a t e d from the knowledge of any 3 f l o w s at steady s t a t e . no m i c r o s c o p i c d e t a i l s are p r o v i d e d .y r . 0 Nj 1 b. .r p r s . A l t h o u g h such i n f o r m a t i o n p r o v i d e s u s e f u l t o o l s i n e n g i n e e r i n g a p p l i c a t i o n s . p r o v i d e the t e c h n o l o g i s t with a strong s t a r t i n g point i n i n d u s t r i a l a p p l i c a t i o n s . c. I n t h i s s e c t i o n o n l y the r e l e v a n t r e l a t i o n s from these p u b l i c a t i o n s w i l l be g i v e n w i t h o u t p r o o f . s x S t a r t i n g from the p r i n c i p l e of the c o n s e r v a t i o n of atomic b a l a n c e s can be shown to h o l d f o r the system d e s c r i b e d : s p e c i e s the f o l l o w i n g r c r o T = r s = 1/4 . 0 N.C H.

For growth w i t h NH3 as the ammonia s o u r c e . ..Y ) r + Y m C } p p s s x (16) From eqs.1) r + (1/ Y x sp m 3 X m a x . A few a r e shown below. . there are no s i g n i f i c a n t d i f f e r e n c e s between the v a r i o u s r e l a t i o n s . Humphrey and J e f f e r i s * ^ and l a t e r on Roels and Kossen'^ have i n c l u d e d the c o n t r i b u t i o n o f product f o r m a t i o n process and m o d i f i e d eq. F o r the system d e s c r i b e d the f o l l o w i n g u s e f u l l i m i t s have been shown t o h o l d : Y 8 sx < for Y (21) .= 4 = 4 Y + b l " 2 C . (14) + m C s x S i m i l a r forms o f the above e q u a t i o n can be d e r i v e d f o r the c o n v e r s i o n r a t e s o f carbon d i o x i d e and oxygen. Another advantage of these t o o l s l i e s i n t h e f o r m u l a t i o n o f t h e o r e t i c a l l i m i t s t o the e f f i c i e n c y of c o n v e r s i o n proc e s s e s .3d 3 d P = 4 + b 2 c 3 " 3 Y i s a l i n e a r combination o f e l e m e n t a l b a l a n c e s . 2 3 d . 2 (11) (12) (13) + b 2 " 3 " 2 c . l ^ h ' ^ growth p r o c e s s . Roels'? has i n t r o d u c e d the g e n e r a l i z e d degree o f r e d u c t i o n concept which can be a p p l i e d t o growth w i t h any N source. „max r = r / Y + r / Y s x sx p sp .Y ) r + (Y / Y X x s sp .. I t c a n a l s o be d e r i v e d from a degree o f r e d u c t i o n b a l a n c e as d e f i n e d by E r i c k s o n and coworkers.1) r + m C p S X m a X (15) r„ 1/4 { ( Y / Y S S X . o r t le \h = sx / f Y U • ( 2 0 ) Other n parameters have a l s o been d e f i n e d based on oxygen and e l e c t r o n b a l a n c e s and i n e q u a l i t i e s .'8>'* I t must a l s o be noted t h a t Y d e f i n e d here o n l y h o l d s f o r NH3 b e i n g the N-source. By combining e q s . ( 8 ) .21 1 / Y ox m m c o = m m a x = Y M S (1/ Y m a x ) -7/4 X (17) (18) (19) S X s = (y /4) m s s E r i c k s o n e t a l . (9) and (14) t h e f o l l o w i n g can be g i v e n : r c = (1/ Y m a x sx . C a l c u l a t i o n of t h i s a l l o w s the d e f i n i t i o n o f the thermodynamic e f f i c i e n c y .max .(15) and (16) a number of u s e f u l r e l a t i o n s can be o b t a i n e d . A more comprehensive l i s t has been g i v e n r e c e n t l y by H e i j n e n and Roels.. however. R o e l s ' ' has c a l c u l a t e d the maximum p o s s i b l e y i e l d v a l u e s a l l o w e d by the second Law of Thermodynamics. Eq.(3) to : . ' ^ have shown methods f o r data a n a l y s i s and c h e c k i n g t h e cons i s t e n c y by u s i n g r e l a t i o n s o f t h i s s o r t .(3) i n t r o d u c e d p r e v i o u s l y does n o t c o n s i d e r product f o r m a t i o n .

The macroscopic t o o l s were a p p l i e d to the e x p e r i m e n t a l d a t a o b t a i n e d d u r i n g t h i s work f o r c h e c k i n g d a t a c o n s i s t e n c y and f o r the e s t i m a t i o n of e n e r g e t i c parameters. system b e h a v i o u r may 9 . v e r y l a r g e and complex f o r a complete system d e s c r i p t i o n . S i g n i f i c a n t s i m p l i f i c a t i o n s become p o s s i b l e v i a a study of the r e l a x a t i o n times of the v a r i o u s p r o c e s s e s t a k i n g p l a c e i n s i d e and the o u t s i d e of the b i o t i c phase.b i o t i c phase) as w e l l as i n the organism i t s e l f ( b i o t i c phase). The r e s u l t i n g s e t of d i f f e r e n t i a l equations would be. i d e n t i f i c a t i o n of r e a c t i o n s between m o l e c u l e s . compared w i t h t h a t of the growth p r o c e s s . ooli has more than 2000 d i f f e r e n t p r o t e i n s . do not change s i g n i f i c a n t l y . I n p r a c t i c e . ii. F i r s t l y . e s t i m a t i o n of biomass c o n c e n t r a t i o n i n b r o t h w i t h suspended p a r t i c u l a t e subs t r a t e .g. u n i v e r s a l l y .(22) Y < Y / Y sx s x (23) A p p l i c a t i o n of macroscopic p r i n c i p l e s can a l s o p l a y an important r o l e i n p r o cess c o n t r o l . Thus. a l t h o u g h b i o c h e m i c a l k i n e t i c s has b a s i c a l l y the same t a s k s as c h e m i c a l k i n e t i c s i . however.. t h i s approach i n e v i t a b l y f a i l s due to two r e a s o n s : i. i t has to c o n s i d e r more complex k i n d of i n t e r a c t i o n s . the mechanism and thus the c o n c e n t r a t i o n of the compounds i t r e g u l a t e s . l i k e s t a r c h . C o n s i d e r i n g t h a t E. however. m o l e c u l e s and o r g a n e l l e s . based on the measurement of a few o n . Ill AN INTRODUCTION TO THE MODELLING OF MICROBIAL GROWTH In g e n e r a l . A w e a l t h of i n f o r m a t i o n e x i s t s on c h e m i c a l k i n e t i c s and dynamics.s e t s of c h e m i c a l systems. c h e m i c a l systems. f o r an e x a c t d e s c r i p t i o n of m i c r o b i a l metabolism one has t o c o n s i d e r a l l the c o n c e n t r a t i o n s of the c h e m i c a l substances i n the immed i a t e environment of microorganisms ( a . Thus these mechanisms and t h e i r e f f e c t s on the t o t a l . Moreover.. Savageau has g i v e n a g e n e r a l growth e q u a t i o n t h a t i s based upon the n a t u r e of the e l e m e n t a l mechanisms i n complex systems. . as shown by P r i g o g i n e 2 6 these type of e q u a t i o n s are not o n l y s p e c i f i c to b i o l o g i c a l systems but are a p p l i c a b l e to any system.l i n e determined parameters. Secondly. c e l l s and c e l l s e t c . E s t i m a t i o n of heat o u t p u t . can a l s o be done. where the c o n t r o l parameter cannot be determined d i r e c t l y e. I n two cases s i m p l i f i c a t i o n become p o s s i b l e : i. b i o l o g i c a l k i n e t i c s i s not r e s t r i c t e d to the study of r e a c t i o n s between e n t i t i e s b e l o n g i n g to a s i n g l e l e v e l of o r g a n i z a t i o n but a l s o b e l o n g i n g to d i f f e r e n t l e v e l s . A d d i t i o n a l a p p l i c a t i o n s are p r e s e n t e d i n Chapter 8. b i o l o g i c a l systems are s u b . Thus one has to c o n s i d e r and compare the time c o n s t a n t s o f the e n v i r o n m e n t a l changes and those of mechanisms i n s i d e the organism which f a c i l i t a t e the a d a p t a t i o n of the organism to these e n v i r o n m e n t a l changes. S i n c e t h i s type of complete d e s c r i p t i o n has proven t o be not p o s s i b l e . expect to be a b l e to d e s c r i b e b i o l o g i c a l systems i n terms of the dynamic b e h a v i o u r of i t s c o n s t i t u e n t s . 2 4 25 • Recently. I n o t h e r words. For p r o c e s s e s which are c h a r a c t e r i z e d by v e r y l a r g e r e l a x a t i o n t i m e s . one can a t l e a s t i n t h e o r y . t h i s becomes an i m p o s s i b l e t a s k even a t the age of f a s t computers. such as r e a c t i o n s between m o l e c u l e s and c e l l s . one aims f o r s i m p l i f i c a t i o n s through j u s t i f i a b l e assumptions. d e t e r m i n a t i o n of the r a t e s of c h e m i c a l r e a c t i o n s and the development of the r e l e v a n t t h e o r i e s . a v e r y i m p o r t a n t t a s k i n p r o cess d e s i g n and c o n t r o l . e .

t h i s approach i s d i f f i c u l t t o a p p l y and p a r t i c u l a r l y i n t h e c h o i c e o f f u n c t i o n a l s . i n terms o f p o p u l a t i o n s . be c o n v e n i e n t l y n e g l e c t e d . That i s . Model based on t h i s assumption i .b i o t i c and b i o t i c phases. one can t r y t o p r o v i d e a m e c h a n i s t i c f o u n d a t i o n by r e a s o n i n g t h a t one enzymic r e a c t i o n t a k i n g p a r t i n a long sequence might be t h e 10 . IV A SIMPLE UNSRUCTURED MODEL FOR MICROBIAL GROWTH The most popular k i n e t i c e x p r e s s i o n used today i s the Monod r e l a t i o n .b i o t i c phase. . Qcowth i s the p r o d u c t i o n o f new biomass by a p o p u l a t i o n when i t consumes a s u i t a b l e l i v i n g o r non l i v i n g s u b s t r a t e from i t s environment and i n c o r p o r a t e s some o f t h i s substance i n t o i t s own. Based on these c o n s i d e r a t i o n s i . The s t a t e of the mechanism can be r i g o r o u s l y d e s c r i b e d u s i n g o n l y environmental c o n c e n t r a t i o n i . most changes among the components o f a system occur much f a s t e r than the r a t e o f t h e growth f o r the system as a whole.b i o t i c c o n c e n t r a t i o n s . . h i s r e l a t i o n p r o v i d e d good f i t f o r h i s experiment a l d a t a .b i o t i c phase seen by the b i o t i c phase. e . Savageau25 concluded t h a t t h i s m a t h e m a t i c a l l y i m p l i e s a s m a l l number of r e l a t i o n s r e p r e s e n t i n g t h e slowest phenomena determine t h e temporal response o f t h e e n t i r e system.b i o t i c phase. Monod a r r i v e d at i t e m p i r i c a l l y . however. Growth and r e p r o d u c t i o n are o b v i o u s l y coupled p r o c e s s e s .ii. I n p r a c t i c e . T h i s was t h e case f o r experiments t o be r e p o r t e d i n t h i s work.b i o t i c phase b u t a l s o on the e n t i r e h i s t o r y of t h e a . . the degree o f c o u p l i n g may be d i f f e r e n t f o r each case.M e n t e n e q u a t i o n . e . I t i s . I n o t h e r words P o w e l l expressed the s p e c i f i c growth r a t e a t any i n s t a n t t o be a ' f u n c t i o n a l ' o f t h e s t a t e of t h e a . t h a t i s .24 Reproduction i s the increase i n the number of d i s c r e t e independent c e l l s o f a p o p u l a t i o n . I n h i s approach. The most r i g o r o u s s i m p l i f i c a t i o n done i n the development o f p o p u l a t i o n models i s t h e assumption t h a t t h e t o t a l amount of the biomass i n t h e c u l t u r e i s s u f f i c i e n t t o s p e c i f y t h e a c t i v i t i e s o f the m i c r o o r g a n i s m s . Although t h i s r e l a t i o n i s an homologue o f the M i c h a e l i s . important t o note t h a t t h i s approach i s o n l y v a l i d when the number o f organisms i n t h e system i s v e r y l a r g e . however. t h e c u r r e n t s p e c i f i c growth r a t e o f a p o p u l a t i o n i s assumed t o depend n o t o n l y on the c u r r e n t s t a t e o f t h e a . F o r processes which a r e c h a r a c t e r i z e d by v e r y s h o r t r e l a x a t i o n t i m e s . was p r o v i d e d by Powell27. I n t h i s study these two processes w i l l n o t be c o n s i d e r e d s e p a r a t e l y b u t t h e t o t a l e f f e c t i s summed w i t h the d r y weight measurements. A l t e r n a t i v e l y . A s i m p l e r approach would be t o assume t h a t t h e c u r r e n t growth r a t e s a r e f u n c t i o n s o f t h e c u r r e n t s t a t e o f t h e a . P r a c t i c a l growth models are u s u a l l y expressed i n terms of a r a t h e r a b s t r a c t u n i t s o f l i f e . b i o t i c mechanisms f o l l o w and respond t i g h t l y t o t h e environmental changes and a g a i n t h e c o n c e n t r a t i o n s o f the b i o t i c components t h a t a r e a s s o c i a t e d w i t h these p a r t i c u l a r mechanisms can be c a l c u l a t e d from the a . however. and thus a v o i d s c o m p l i c a t i o n s t h a t might a r i s e due t o t h e s t o c h a s t i c phenomena a s s o c i a ted w i t h t h e e x i s t e n c e o f i n d i v i d u a l organisms. i n w h i c h the v a r i a t i o n i n the biomass c o m p o s i t i o n i s t o t a l l y i g n o r e d . e . One o f the most g e n e r a l approaches f o r d e s c r i b i n g growth. A l l o t h e r r e l a t i o n s r e p r e s e n t i n g t h e f a s t e r phenomena can be assumed t o have reached a pseudo-steady s t a t e w i t h time d e r i v a t i v e s equal t o z e r o . c o n c e n t r a t i o n i n t h e a . a r e c a l l e d UNSTRUCTURED WDELS. T h i s approach c o n s i d e r s the p o p u l a t i o n as an e n t i t y homogeneously d i s t r i b u t e d i n space and time.

(24) (25) and (26) w i l l be s u f f i c i e n t t o d e s c r i b e simple systems such as b a t c h . f o r continuous c u l t i v a t i o n $ i s d e s c r i b e d by the f o l l o w i n g : D (C * = D C (27) (28) x For b a t c h c u l t i v a t i o n . the model reduces down t o : so s 1 1 .b a t c h . Thus the f o l l o w i n g p a i r o f eqs.g.bottleneck and thus r a t e l i m i t i n g . $ a r e z e r o . s i n c e i t i s a c l o s e d system as f a r as the non-gaseous phases a r e concerned. Most o f t h e time d u r i n g b a t c h growth organisms grow a t o r near U • S i n c e the e f f e c t of maintenance requirements a r e e f f e c t i v e l y m i n i m i z e d a t n i g h u . To d e s c r i b e the system. / {(K + C ) Y } s max s x s s sx m a X (29) .4 ( f o r the no-product c a s e ) : s r x /Y sx m s C x (26) Eqs. c o n s i d e r a constant volume c u l t i v a t i o n system i n which t h e t o t a l m i c r o b i a l a c t i v i t y i s q u a n t i f i e d by the amount o f biomass (biomass w i l l imply d r y weight throughout t h i s work) and there i s a s i n g l e l i m i t i n g s u b s t r a t e (C and energy s o u r c e ) . Now. i f C >> C Y and C >> K .y C C . continuous and f e d . F u r t h e r s i m p l i f i c a t i o n s a r e p o s s i b l e by c o n s i d e r i n g v a r i o u s e x p e r i m e n t a l c o n d i t i o n s .m C (30) s x No a n a l y t i c a l s o l u t i o n i s p o s s i b l e f o r t h i s s e t and hence n u m e r i c a l methods ' were used f o r s i m u l a t i o n purposes d u r i n g t h i s study.g. $ i s the n e t f l o w term t o the system and i s f i x e d by the mode o f o p e r a t i o n .. e. d e s c r i b e the system: dC /dt = u C C / (K + C ) x max s x s s dC / d t = . e. F o r the g e n e r a l case the f o l l o w i n g balances can be f o r m u l a t e d : s x d C / dt d C / dt s r s consumption + (24) (25) i s chosen f o r r e l a t i n g r t o x I f the r e l a t i o n o f l i n e a r s u b s t r a t e r 2. changes of C and C and their interdependence have t o be e v a l u a t e d . a convenient s i m p l i f i c a t i o n can be i n t r o d u c e d by n e g l e c t i n g the m term i n the above model.. I n t h i s case an a n a l y t i c a l s o l u t i o n i s p o s s i b l e and can be shown to be g i v e n by: m a x s ln(C x /C ) + K Y /(Y C + C ) I n {(C /C ) / ( l + C / (Y C ) xo s sx sx so xo X xo xo sx so C/(Y C ))} = y t x sx so max (31) The i m p l i c i t n a t u r e o f t h i s e x p r e s s i o n g i v e s problems i n parameter e s t i m a t i o n from e x p e r i m e n t a l data by n o n l i n e a r r e g r e s s i o n .

.C ) the h i g h c e l l c o n c e n t r a t i o n r a p i d l y u t i l i z e s the remaining substrate. When C = 0. C d a t a o b t a i n e d under s u b s t r a t e l i m i t a t i o n s u f f e r from l a r g e u n c e r t a i n i t i e s .g. Two o t h e r k i n e t i c e x p r e s s i o n s w i l l be compared w i t h t h a t of Monod : 12 . i t can be shown t h a t the s t a t e e q u a t i o n f o r C is o n l y d e f i n e d by the e n e r g e t i c and e x p e r i m e n t a l parameters and not by the k i n e t i c r e l a t i o n . successfully. As has been shown by Roels28 the d e t a i l e d n a t u r e of the k i n e t i c equation i s o n l y of s l i g h t improtance f o r s u b s t r a t e l i m i t e d growth. However. the concept of endogeneous metabolism becomes handy. time. l i k e s a t u r a t i o n k i n e t i c s . C . Thus the c h o i c e of the k i n e t i c e x p r e s s i o n remains to be r a t h e r a r b i t r a r y . i s low ( K . Since C v s . Under these c o n d i t i o n s .. g e n e r a l l y the r a t e of growth i n c r e a s e s w i t h i n c r e a s i n g C up to a p o i n t . E x p r e s s i o n (1) p r e d i c t s zero growth r a t e when: s C or. T h i s i s p r e s e n t e d i n Chapter 4.b a t c h c u l t u r e s can a l s o be d e s c r i b e d by t h i s u n s t r u c t u r e d model. s i n c e at the p o i n t when the r e s i d u a l s u b s t r a t e . C . may w e l l p r o v i d e u s e f u l i n f o r m a t i o n f o r the v e r i f i c a t i o n or r e j e c t i o n of the r a t e e x p r e s s i o n .s u b s t r a t e l i m i t e d systems e. Roels and Kossen'6 s t u i e d a number of u n s t r u c t u r e d models and have shown t h a t almost any o b s e r v a t i o n can be m o d e l l e d by any of them. a n d f o r s u b s t r a t e l i m i t e d growth i n continuous c u l t u r e s . The d i f f e r e n c e between P i r t and H e r b e r t r e l a t i o n s stem from t h e i r d i f f e r e n t ways of i n t e r p r e t i n g the f u n c t i o n i n g of the maintenance p r o c e s s e s . however. throughout t h i s work Monod r e l a t i o n w i l l be used w i t h o u t any comparative j u s t i f i c a t i o n . Thus d i s c r i m i n a t i o n between the v a r i o u s k i n e t i c models become a d i f f i c u l t t a s k . P a r t i c u l a r l y i n n u m e r i c a l s i m u l a t i o n s the experimenter must c o n s i d e r this p o i n t c a r e f u l l y as t h i s might l e a d t o the c a l c u l a t i o n of n e g a t i v e s u b s t r a t e c o n c e n t r a t i o n v a l u e s . T h e r e f o r e . I n n o n . a n a t u r a l phenomenon which can be observed e x p e r i m e n t a l l y . particularly f o r b a t c h growth where the growth i s not l i m i t e d by s u b s t r a t e . T h i s i s because of the v e r y low C under s u b s t r a t e l i m i t i n g c o n d i t i o n s . p r o f i l e i s not i n f l u e n c e d much by the r a t e e q u a t i o n . t h e r e a f t e r r remains c o n s t a n t e. C s s a = K K s u y e / ( y / y g ~ P e ) (33) (34) s e g T h i s e x p r e s s i o n can a l s o a l l o w f o r n e g a t i v e growth.(33) . The s i m p l e u n s t r u c t u r e d model has been a p p l i e d t o some p r a c t i c a l systems. I n t h i s case o n l y data from the t r a n s i e n t phase from e x p o n e n t i a l to s t a t i o n a r y phase can be used f o r model d i s c r i m i n a t i o n . At such low C v a l u e s a pseudo-steady s t a t e h y p o t h e s i s w i t h r e s p e c t t o C h o l d s . U n f o r t u n a t e l y . g s s x x s s g x g s s In t h e i r r e v i e w . One wonders i f the Monod' r e l a t i o n i s the o n l y s u i t a b l e k i n e t i c e x p r e s s i o n f o r m o d e l l i n g .C x / C xo = exp ( U max t ) (32) general Growth behaviour i n f e d . one s h o u l d not expect to o b t a i n a c c u r a t e i n f o r m a t i o n on the n a t u r e of the r a t e e x p r e s s i o n from the biomass-time d a t a . t h i s t r a n s i t i o n i s u s u a l l y q u i t e a b r u p t . b a t c h systems. when Cs i s s m a l l e r than the r i g h t hand s i d e of eq.g. 0 I t i s important to note t h a t an u n r e a l i s t i c f e a t u r e ^ f the l i n e a r r e l a t i o n is t h a t i t p r e d i c t s s u b s t r a t e uptake even a f t e r s u b s t r a t e has been exhausted.

For a comprehensive l i s t r e c e n t r e v i e w s can be consulted!6.T i e s s i e r Model where Blackman Model: u = U max K = K s u { 1 .b a t c h models.24. s x Fig. •pneumoniae (aerogenes) was c u l t i v a t e d i n b a t c h mode.25. Blackman and T i e s s i e r models i n combin a t i o n w i t h the l i n e a r r e l a t i o n f o r s u b s t r a t e consumption(eq. I n some c a s e s .31 n I n v i e w of these c o n s i d e r a t i o n s most workers f a v o u r Monod r e l a t i o n and do not g i v e any f u r t h e r a t t e n t i o n to o t h e r r e l a t i o n s . A d d i t i o n a l l y t h e s i m u l a t i o n p r o f i l e s by u s i n g the Monod. time p r o f i l e s and simulataion. a r e presented ( s o l i d l i n e s ) . A t y p i c a l experiment i s shown i n F i g .31 i g e n e r a l a l l the proposed models a r e e m p i r i c a l o r semie m p i r i c a l and have more o r l e s s t h e same p r o p e r t i e s . w h i l e t h i s i s not p o s s i b l e w i t h Monod k i n e t i c s . I t has r e c e n t l y been shown i n l i t e r a t u r e t h a t most of these models can i n f a c t be g e n e r a l i z e d i n t o one form. 1: A typical batch experiment. I n o c u l a used were always a c t i v e l y growing and conseq u e n t l y no l a g s were encountered. each model h a v i n g d i f f e r e n t parameters. 13 . o t h e r e q u a t i o n s might be p r e f e r r e d from the p o i n t of m a t h e m a t i c a l c o n v e n i e n c e .exp(-C s / K ) } (35) / In 2 for / A for C > s — < s — u u A A max C s max max (36) C Many more e x p r e s s i o n s have been r e p o r t e d and c l a i m e d t o be s u p e r i o r under c e r t a i n c a s e s . however. V DISCUSSION OF UNSTRUCTURED MODELS AND MICROBIAL ENERGETICS WITH REFERENCE TO EXPERIMENTAL RESULTS Batch Cultures K.^5. C^ and C g vs.(26)). F o r i n s t a n c e . 1 where C and C p r o f i l e s a r e shown as f u n c t i o n s o f t i m e . t o study t h e k i n e t i c and e n e r g e t i c b e h a v i o u r . the use of Blackman k i n e t i c s a l l o w s a n a l y t i c a l s o l u t i o n of f e d .

p r o v i d e d a l l parameters are e s t i m a t e d w i t h e q u a l c a r e . Y ™ . d e s c r i b e s the e a r l y decay phase w e l l . Monod + l i n e a r r e l a t i o n model w i l l now be s u b j e c t e d to a s e n s i t i v i t y a n a l y s i s w i t h r e s p e c t to i t s parameters.3. Even then. 14 . 1 shows t h a t a l l t h r e e models can d e s c r i b e the e x p e r i m e n t a l o b s e r v a t i o n s i n a more o r l e s s i d e n t i c a l way. F i g . H e r b e r t ' s model seems to p r o v i d e a more comprehensive d e s c r i p t i o n of the r e a l b e h a v i o u r . S: Sensitivity of the batch model to changes in m .The broken l i n e becomes a c o n t i n u a t i o n of l i n e 'a' i f the l i n e a r r e l a t i o n i s r e p l a c e d by H e r b e r t ' s endogeneous metabolism d e s c r i p t i o n . The endogeneous metabolism model. I n F i g s . i n a d d i t i o n to p r e d i c t i n g e x a c t l y the same b e h a v i o u r • as Monod+linear r e l a t i o n model. are shown. The parameters used f o r s i m u a l t i o n s were o b t a i n e d from continuous and b a t c h c u l t u r e d a t a . Thus as f a r as t h i s system i s concerned. x s m a x x g Fiq. P . Having shown the r e l a t i v e s i m i l a r i t y of the p r e s e n t e d models. T h i s i s a f o u r parameter model ( K . 2. m ). Parameters f o r Blackman and T i e s s i e r models were e s t i m a t e d r o u g h l y .and 4 r e s u l t s of s i m u l a t i o n s c a r r i e d out by changing one parameter at a t i m e . 2 : Sensitivity v J J of the batch model to variations in u max t(min) Fig.

use o f C p r o f i l e would be more a c c u r a t e . i f the e x p e r i m e n t a l b a t c h d a t a i s p l o t t e d on l o g .32 Thus when growth i s balanced i t i s e x p o n e n t i a l too. i t can be concluded t h a t m v a l u e s cannot be determined from b a t c h d a t a a c c u r a t e l y . As shown i n F i g . n o t t r u e i . i .l i n e a r axes. the k i n e t i c parameter. Hence they are expected t o be s u c c e s s f u l a t steady s t a t e s o r d u r i n g t r a n s i e n t s t a t e s where t h e c e l l u l a r c o m p o s i t i o n remains t h e same. u n s t r u c t u r e d models do n o t c o n s i d e r changes i n c e l l u l a r c o m p o s i t i o n . Moreover. i s t o be e s t i m a t e d from b a t c h data. That i s . . The r e v e r s e i s however. y a x shown t o be of g r e a t importance. i n which case i m p l i c i t n o n l i n e a r r e g r e s s i o n i s n e c e s s a r y . For b a t c h growth i t has been shown t h a t f o r the c o m p o s i t i o n t o remain the same. 5.(32) i s s u f f i c i e n t t o d e s c r i b e most of the b a t c h growth. (30) to (31) ) has no s i g n i f i c a n t drawbacks. steady s t a t e w i t h r e s p e c t t o weight f r a c t i o n s of the components. e x p o n e n t i a l growth need n o t be b a l a n c e d . i f Y . t h i s might be a s i m p l i f i e d p i c t u r e . However. In an attempt t o check the v a l i d i t y of t h i s mathematical statement. Consequently. f o r i n s t a n c e the p r o t e i n 15 . i s not i n f l u e n c e d by c o n s i d e r a b l e changes i n e n e r g e t i c parameters. 4: Sensitivity of the batch model to variations in ^ s x The parameter v a l u e s were v a r i e d around the e s t i m a t e d t r u e v a l u e s .(31) may i n t r o d u c e unnecessary c o m p l i c a t i o n s p a r t i c u l a r l y i n the e s t i m a t i o n of model parameters. e . .From these p l o t s one can c l e a r l y conclude t h a t the k i n e t i c d e s c r i p t i o n o f b a t c h growth i n terms o f C v s . each c o n s t i t u e n t compartment i n the c e l l must grow e x p o n e n t i a l l y a t the same r a t e as t h e t o t a l biomass. Another c o n c l u s i o n can be drawn from F i g 4 i n r e l a t i o n t o parameter e s t i m a t i o n . The case of K i s d i s c u s s e d i n Chapter 4. t h i s parameter i s the most important f o r b a t c h growth as i t r i g i d l y f i x e s the growth b e h a v i o u r . e . ( 2 9 ) . As p r e v i o u s l y d i s c u s s e d . one can see t h a t a f a i r l y good s t r a i g h t l i n e i s o b t a i n e d . g As p o i n t e d out p r e v i o u s l y . s i n c e . When t h i s c o n d i t i o n i s s a t i s f i e d growth i s c a l l e d balanced. time p r o f i l e . s x m a x s l s m ma x s S i n c e m has no s i g n i f i c a n t i n f l u e n c e on t h e outcome o f b a t c h s i m u l a t i o n s one can see t h a t the s i m p l i f i c a t i o n of the g e n e r a l u n s t r u c t u r e d model ( e q s . However. Thus the use o f c o m p l i c a t e d e x p r e s s i o n s l i k e t h a t g i v e n by eq. That i s even a v e r y simple e x p r e s s i o n l i k e t h a t g i v e n by eq. m and Y . macrom o l e c u l a r c o m p o s i t i o n was determined during e x p o n e n t i a l growth.J71CUC Fig. v i s u a l a n a l y s i s cannot r e j e c t the h y p o t h e s i s of b a l a n c e d growth.

a d i s c r e p a n c y e x i s t s between t h e s i m u l a t e d and e x p e r i m e n t a l b e h a v i o u r towards t h e end of the e x p o n e n t i a l phase. o x c x Fig. 5: Macromolecular composition during batch growth. No s a t i s f y i n g e x p l a n a t i o n f o r t h i s behaviour c o u l d be o f f e r e d . In F i g .c o m p o s i t i o n might change w h i l e the t o t a l measurable amount remains the same. T h i s i s f u r t h e r d i s c u s s e d i n Chapter 4. Fig. 16 . 6. solid line simulation. Here. the gas exchange d a t a f o r a d i f f e r e n t b a t c h experiment i s shown t o g e t h e r w i t h the s i m u l a t i o n p r e d i c t i o n s ( s o l i d l i n e s ) . The e x p e r i m e n t a l d a t a i n d i c a t e s i n c r e a s i n g Y and Y v a l u e s . 6: Gas exchange profiles during batch growth.

C H „N ->oO . as shown i n F i g s .3. by : C 1 H b N c d 0 where b c = d z = (7.Fig.16. Based on t h i s a n a l y s i s the e l e m e n t a l f o r m u l a of biomass can be approximated as a f u n c t i o n of t h e growth r a t e . Moreover. Y ™ > X m X s Data o b t a i n e d from a c o n t i n u o u s c u l t u r e experiment were f i t t e d by t h e l i n e a r r e l a t i o n . As shown i n F i g . Statistical a n a l y s i s c a r r i e d out f o r 9 e l e m e n t a l c o m p o s i t i o n d e t e r m i n a t i o n s r e v e a l e d t h a t v a r i a t i o n i n C. i s r i g i d l y f i x e d by the e n e r g e t i c parameters. Fig. changes as a f u n c t i o n o f t h e growth r a t e . However.33 + 3.8 shows a good s t r a i g h t l i n e f i t .(y x = 4.16. p a r t i c u l a r l y t h e RNA f r a c t i o n .065 3 V n W aJ Ta of the growth rate. 17 . However. These p l o t s i n d i c a t e t h a t growth i n continuous c u l t u r e can indeed be d e s c r i b e d by the p r e s e n t e d model.11 w 3 Protein = 0. Hence an u n s t r u c t u r e d model has a good chance of s u c c e s s . The c o n c l u s i o n w i l l s i m p l y be s t a t e d as: growth behaviour d e s c r i b e d by t h e g e n e r a l u n s t r u c t u r e d model. the e l e m e n t a l c o m p o s i t i o n of biomass a l s o changes.Continuous cultures D u r i n g growth i n continuous mode a t steady s t a t e the biomass c o m p o s i t i o n remains t h e same. The s e n s i t i v i t y a n a l y s i s p r e s e n t e d f o r b a t c h growth model w i l l n o t be r e p e a t e d f o r c o n t i n u o u s c u l t i v a t i o n .40 y ) / ( 1 4 z ) = 26.5 h r m o l e c u l a r weight = 23. 8 and 9. H and N c o n t e n t s are s i g n i f i c a n t .61 . such a model a l s o assumes the b i o t i c c o m p o s i t i o n t o remain t h e same at d i f f e r e n t d i l u t i o n r a t e s .~te = 0.71 ]i~0 ' C b°Hld.0. s m a l l b u t d i s t i n c t d e v i a t i o n s can be seen f o r d a t a a t low growth r a t e s .50 y ) / z (12. ) • 9 n i s taken. „ . except near the washout r e g i o n .97/(16z) = (53.74 y)/12 _ 1 For most c a l c u l a t i o n s an average f o r m u l a a t y = 0.33 . 7: Micvomolecular composition as a function w RNA = 0. 7 the macromolecular c o m p o s i t i o n .

y p l o t . The d i f f e r e n c e might stem from the f a c t t h a t q i s n o t a d i r e c t l y measurable q u a n t i t y . The dangers o f t h i s e x e r c i s e i . oxygen. I f t h e r e s i d u a l s a r e examined. The r e s u l t s a r e p r e s e n t e d i n Table I . Here the r e s i d u a l s change t h e i r s i g n c 18 . Fig. Thus i t may have a d i f f e r e n t e r r o r s t r u c t u r e . both methods should g i v e e x a c t l y t h e same parameters. y p l o t one has y i m p l i c i t l y i n both axes and t h i s may be q u i t e u n d e s i r a b l e from a mathematical p o i n t o f view. b u t i s c a l c u l a t e d from q = y / Y . i n c l u d i n g the same v a r i a b l e i n both axes i s d i s c u s s e d by Himmelblau.9). T h i s i s p a r t i c u l a r l y apparent i n the q v s . e . 8: 9: Specific Specific rate of svbstrate consumption as a fuction rate.33 s s s x s The e x p e r i m e n t a l d a t a have f i r s t been i n d i s c r i m a n e n t l y processed by l i n e a r and n o n l i n e a r r e g r e s s i o n procedures. 8. carbon d i o x i d e d a t a v s . i n a q v s . . one can d e t e c t a t r e n d ( F i g . growth r a t e ) n o n l i n e a r r e g r e s s i o n gave a b e t t e r f i t f o r t h e e x p e r i m e n t a l d a t a . I f t h e r e a r e a s s o c i a t e d e r r o r s these approaches may r e s u l t i n d i f f e r e n t parameter e s t i m a t e s . Moreover. T h i s has been assessed by c a l c u l a t i n g the scaled sum of residuals (see Table I ) .Fig. of the growth rate. An a n a l y s i s of the two procedures was c a r r i e d out and i t was found out t h a t i n a l l t h r e e cases ( f o r s u b s t r a t e . OUR and CPR as fuations of the growth The e n e r g e t i c parameters may be o b t a i n e d from c o n t i n u o u s forming l i n e a r r e g r e s s i o n v i a t h e use of e q u a t i o n : c u l t u r e d a t a by p e r - q s = y I Y m a X sx + m s (38) equation: or by p e r f o r m i n g n o n l i n e a r r e g r e s s i o n v i a the use of the f o l l o w i n g Y sx = y Y m a x sx / (y + m Y s sx m a x ) (39) I f t h e e x p e r i m e n t a l measurements a r e e r r o r f r e e .

0. n=21 0.650) 4.065 (3. y > 0.720) 1.012). 8 5 5 ) .803).493) 3. 1 0 2 0.0. F o r the second s e t . y r e l a t i o n changes s i g n 11 t i m e s .623). 3 0 5 ) .613 (1.517 .365 .869 .367 . 1 0 4.710 (0. Be r r r t t l J X g Batch C u l t u r e d a t a Y™£ x (average of t h r e e e x p e r i m e n t s ) : all Y a l l m i m a x 0. Note t h a t t h e r e a r e s i g n i f i c a n t d i f f e r e n c e s i n t h e m v a l u e o b t a i n e d by t h e two p r o c e d u r e s . 6 7 5 ) .320 (3.5 .570 . a n o s Q c c 19 .997 (3.4 .1.4 . i n t h e time sequence o n l y t h r e e t i m e s .701).2. 1 0 -2 sx m a x 0.5.289 .701) 2.1 r e s i d u a l s of the q v s .Table I : Parameter e s t i m a t e s o b t a i n e d from c o n t i n u o u s and b a t c h c u l t u r e d a t a .627 .10~ 4.740 (3.1.513 (2.9 times and q v s .705 1.241 (3.0* 6.10~ 4.583 (1.719 (0.2. y.4 .627 .4 . 1 0 3.313 i n C-eq/C-eq.709) 4.1.6 .698 (0. y. 6 times ( ( n . 1 0 3.391 (2.059 .0.631).1.570) 4.544 2.465 (2.10~ 2 -2 2 c Thermodynamic e f f i c i e n c y v e r s u s growth r a t e d a t a .732) 1.5* 5.8* -2 ox Y™ m m m s a x 0 4.7* 2.1.542 .4 .835 .342 (3. y> 0.l ) / 2 = 10 ) .721) 1. C-eq/mole c-eq/C-eq/hr. F o r d a t a above y=0.998 (3. 1 1 5 ) .846 . v i a b i l i t y i s more than 95 %^4 t h e r set of parameters were o b t a i n e d o n l y by p r o c e s s i n g d a t a c o l l e c t e d above y=0.706 .4 .321 .530 (3. Continuous C u l t u r e d a t a : Nonlinear Regression A l l d a t a p o i n t s .2. mole/C-eq/hr Y™|x Y™ a x n * s c a l e d sum of r e s i d u a l s x 1 0 f i g u r e s i n p a r e n t h e s e s a r e the 95 % c o n f i d e n c e 2 limits.2. 1 0 1.570 .072 (3. 8 5 2 ) . lö~1.561 (2. 1 0 2 4.668 (3.5. 1 0 -2 a x m a x -2 -2 _2 -2 Data Y Y m a x c o l l e c t e d above .624) 2.290 .4* 3. the goodness o f f i t i s a l s o shown f o r l i n e a r and n o n l i n e a r r e l a t i o n s .4.701 .428 .1.609 (3.707) 1. see Table I ) . Whereas i f they had been randomly d i s t r i b u t e d the expected number of s i g n change would have been (n-I)/2= 13.1.146 (3. (n=21.640 (1. Thus w i t h the e x c e p t i o n of q d a t a .376 .0.1.710 (0.661 .563 . n=27 / ( Zie errntiirn L i n e a r Regresión Y m a x sx Y™ Y m m o m cx s 0. 7 5 7 ) .3* 0.819 (3.693 . 1 5 9 ) .5.373 .677) 2.177 .699 . 1 4 8 ) .756 . q v s .707) 4.3. n=63 Y m m a x 0. The d i s t i n c t d e v i a t i o n a t low y can be thought t o be due t o the reduced v i a b i l i t y of the organisms.700 (0.0. S i n c e above y=0.659) 2.620 (1.10" 3.

. 10: The loons of the joint confidence limits for energetic parameters as determined by the linear relation for substrate consumption. S i n c e these parameters a r e determined s i m u l t a n e o u s l y . T h i s can be done by p r o c e s s i n g I] v s . F r o m t h i s d i s c u s s i o n i t can be concluded t h a t the v a l u e o f Y g can be determined w i t h r e a s o n a b l e c e r t a i n i t y . when every measurement c o n t r i b u t e s to the r e s u l t . s i m u l t a n e o u s l y i . w h i l e t h a t of m can o n l y be d e t e r mined w i t h a l a r g e u n c e r t a i n i t y . A b e t t e r p i c t u r e can be o b t a i n e d about t h e a c c u r a c y o f these parameters by c a l c u l a t i n g t h e i r approximate l o c u s o f the j o i n t c o n f i d e n c e l i m i t s ( F i g . one can a l s o conclude t h a t t h e r e i s no s i g n i f i c a n t d i f f e r e n c e between the e n e r g e t i c s of m i c r o b i a l growth i n b a t c h and continuous modes.e x c l u s i o n of data c o l l e c t e d a t v e r y low growth r a t e s . Y and Y d a t a (n=63) . Fig. Such wide c o n f i d e n c e l e v e l s may be one o f t h e reasons f o r the wide range o f m v a l u e s r e p o r t e d i n l i t e r a t u r e .(S&). as f a r as t h e maximal y i e l d s a r e concerned. f o r the same o r s i m i l a r s y s t e m ( s ) . Wide c o n f i d e n c e l e v e l s r e s t r i c t s one t o draw f i r m c o n c l u s i o n s from e x p e r i m e n t a l work c o n c e r n i n g maintenance metabolism. From t h i s t a b l e i t can be seen t h a t t h e 95 % c o n f i d e n c e l i m i t s of m v a l u e s a r e q u i t e l a r g e when compared w i t h those o f Y v a l u e s . by t h e use o f macroscopic methods Thus t h e most o p t i m a l e s t i m a t e o f the parameters.8) 20 . Thus i t can be concluded t h a t above p=0. t h e i r e s t i m a t e s can be c o r r e l a t e d .1 the l i n e a r r e l a t i o n i s a r e a s o n a b l e d e s c r i p t i o n o f the c o n t i n u ous c u l t u r e e n e r g e t i c s . e . The parameters o b t a i n e d i n t h i s way a r e a l s o g i v e n i n Table I . where r| i s c a l c u l a t e d from Y . From t h i s f i g u r e i t can be seen t h a t the estimates of Y ™ and m are s l i g h t l y c o r r e l a t e d . H d a t a . 10). I t has t o be emphasized t h a t the e s t i m a t e s of Y and m may l i e o u t s i d e t h e i r i n d i v i d u a l c o n f i d e n c e l e v e l s . Y and m can be o b t a i n e d by c o n s i d e r i n g data o b t a i n e d from a l l r e s p o n s e s . s x o x c x m a x x x g m x x s m x s With r e f e r e n c e t o Table I . the p r i n c i p a l axes of the e l l i p s e a r e a t an angle to the c o o r d i n a t e axes. sq. r e s t o r e d t h e l i n e a r i t y of the data i n r e l a t i o n t o t h e l i n e a r law. As d e s c r i b e d p r e v i o u s l y Y o r m v a l u e s c a l c u l a t e d from one e x p e r i m e n t a l response c a n be c o n v e r t e d t o one based on a n o t h e r . (for data shewn in Fig.

membrane e n e r g e t i z a t i o n e t c . The r e a l p i c t u r e may a l s o i n c l u d e dormant c e l l s . c o n s t a n t maintenance h y p o t h e s i s i s d i f f i c u l t to r e j e c t . e. s s An o b s e r v a t i o n shared by many workers i s the s i g n i f i c a n t s y s t e m a t i c d e v i a t i o n s observed from the l i n e a r r e l a t i o n a t low y ( t h i s work.g.. y i n F i g . the q v a l u e s can be c o r r e c t e d for v i a b l e c e l l s .. T h i s e f f e c t can be a t t r i b u t e d to p h e n o t y p i c changes ( s i n c e t h e time to reach steady s t a t e a t low y i s v e r y l o n g ) and/or l o s s of v i a b i l i t y . cannot account f u l l y f o r the observed d e v i a t i o n s at low growth r a t e s . m i s a l s o assumed to be c o n s t a n t . i f the e f f i c i e n c y of o x i d a t i v e p h o s p h o r y l a t i o n or the degree of c o u p l i n g are a l s o f u n c t i o n s of the growth r a t e . I t i s a l s o i n t e r e s t i n g t h a t the d e v i a t i o n from the l i n e a r r e l a t i o n does not o c c u r over the e n t i r e y range but develops i n a s m a l l range. w h i c h show up v i a b l e when c u l t u r e d i n r i c h media. a l l non-growth a s s o c i a t e d energy e x p e n d i t u r e i s a u t o m a t i c a l l y assumed t o have been c h a n n e l l e d t o maintenance metabolism. i n a number of systems the l i n e a r r e l a t i o n seems to g i v e a good a p p r o x i m a t i o n over a wide range of growth r a t e s . S i n c e the v i a b i l i t y d a t a were a l s o r e p o r t e d .34 The d a t a was p r o c e s s e s and p l o t t e d as q v s . osmotic work t o m a i n t a i n c o n c e n t r a t i o n g r a d i e n t s . When the l i n e a r r e l a t i o n i s assumed to be v a l i d .35) _ j f Y can be assumed to be a b i o l o g i c a l l y m e a n i n g f u l parameter and as a c o n s t a n t .g. However. The p r e sented d a t a are not s u f f i c i e n t f o r an a c c u r a t e assessment of t h i s o b s e r v a t i o n . i n l i t e r a t u r e a s e t of d a t a c o l l e c t e d at v e r y low y has been reported. At low growth r a t e s the r a t i o i s h i g h . 11. That i s . i t i s known t h a t the s u r f a c e a r e a to volume r a t i o of b a c t e r i a i s a f u n c t i o n of the growth r a t e . T h e r e f o r e such maintenance r e q u i r e m e n t s may be expected to be growth r a t e dependent.06. the above mentioned d e v i a t i o n s i m p l y reduced maintenance r e q u i r e m e n t s a t low growth r a t e s . 21 . one must note t h a t s t r a i g h t l i n e s can be o b t a i n e d even i f the m i s a f u n c t i o n of y but the o v e r a l l e f f e c t i s too s m a l l or the a c t u a l phenomenon i s interacting w i t h o t h e r s . From t h i s f i g u r e i t i s c l e a r t h a t v i a b i l i t y a l o n e . i t would be i m p o s s i b l e to f i l t e r out c o n c l u s i o n s r e g a r d i n g the v a r i a t i o n of maintenance demands. the f o l l o w i n g a n a l y s i s can be c a r r i e d o u t : Assuming t h a t o n l y v i a b l e c e l l s consume s i g n i f i c a n t q u a n t i t i e s of s u b s t r a t e . as shown by h o l l o w c i r c l e s i n the F i g . 11. some of the maintenance f u n c t i o n s may w e l l be i n f l u e n c e d by the growth r a t e e. F o r t u n a t e l y .34. 11: q versus V data:(recalculated from ref: 34). thus the organism i s expected to spend more energy t o keep the u n d e s i r a b l e s o l u t e s out.VI A DISCUSSION ON THE CONCEPT OF MAINTENANCE S p e c i f i c maintenance f u n c t i o n s are now b e l i e v e d t o i n c l u d e : t u r n o v e r of c e l l 1 m a t e r i a l s . However. m a x s s Fig. A s i g n i f i c a n t d e v i a t i o n can be seen below y=0. However. c e l l m o t i l i t y .

S c h r o d i n g e r wrote ( c i t ) " l i v i n g m a t t e r evades e q u i l i b r i u m by f e e d i n g upon i t s n e g a t i v e entropy produced by i t s metabolism (Greek word f o r exchange)" or l e s s p a r a d o x i c a l l y " the organism succeeds i n f r e e i n g i t s e l f from the entropy i t cannot h e l p p r o d u c i n g to remain a l i v e " . In 1970 Gaudy et a l . An o b s e r v a t i o n s i m i l a r to t h a t of P i p y n and V e r s t r a e t e was a l s o made d u r i n g t h i s study. . T h i s Zie erratumvalue i s about 10 times h i g h e r than t h a t o b t a i n e d from a continuous c u l t u r e study w i t h a mono c u l t u r e ( 4 . a s i m p l e experiment a g a i n s t z e r o maintenance c l a i m s . For i n s t a n c e .36 s s _ In l i t e r a t u r e sometimes remarkable c l a i m s c o n c e r n i n g the maintenance concept are made. i t has been c l a i m e d t h a t maintenance requirements c o u l d be made zero by merely m a n i p u l a t i o n of the medium composition. For a c t i v a t e d sludge c u l t i v a t i o n i n f i l l and draw mode. I n thermodynamics i t has l o n g been known t h a t energy i s needed to keep an open system i n i t s o r d e r e d s t a t e . 1 5 x l 0 ~ 2 ) . VII NOMENCLATURE c o n s t a n t i n Blackman e q u a t i o n adenosinetriphosphate c o n c e n t r a t i o n (kg/m3) (C-eq/m^) biomass e l e m e n t a l f o r m u l a s u A ATP C C HkjO^Njj < -'a2^b2^C2 d2 N b s t r a t e elemental formula formula C a ^ H ^ O ^ N ^ product e l e m e n t a l K K D m r^ s qi Y^ ymax Yp W£ x S Y Monod s a t u r a t i o n c o n s t a n t (kg/m^) (C-eq/m-*) constant i n T i e s s i e r equation dilution rate (hr ') maintenance c o e f f i c i e n t (C-eq/C-eq/hr) (mole/C-eq/hr) r a t e of consumption or p r o d u c t i o n of the i ' t h component (C-eq/m3/hr) (mole/m3/hr) s p e c i f i c r a t e of consumption o r . c o n t i n u o u s l y . S i m i l a r o b s e r v a t i o n s have a l s o been r e p o r t e d by Gaudy and Gaudy. p r o d u c t i o n of the i ' t h component (C-eq/C-eq/hr) (mole/C-eq/hr) y i e l d of biomass on the i ' t h component (C-eq/C-eq) (C-eq/mole) £ c o r r e c t e d f o r maintenance (C-eq/C-eq)(C-eq/mole) y i e l d of product on s u b s t r a t e (C-eq/C-eq) weight f r a c t i o n of the i ' t h component x subscripts x 22 biomass . Based on t h i s o b s e r v a t i o n and experiments they have reasoned that the e x p l a n a t i o n must be sought i n the f a c t t h a t m decreases w i t h decr e a s i n g \l v a l u e s . no growth but s u b s t r a t e uptake.Ox 10 3 (C-eq/C-eq/hr) was c a l c u l a t e d .R e c e n t l y P i p y n and V e r s t r a e t e 3 5 have r e p o r t e d t h a t i n waste water systems m v a l u e s determined are about 10 times lower than those f o r l a b o r a t o r y pure c u l t u r e systems. T h i s f a c t was i t e r a t e d S c h r o d i n g e r 39 f o r b i o l o g i c a l systems as e a r l y as 1944. During the 2 nd and 3 r d y e a r s of o p e r a t i o n the amount of biomass i n the system and i t s c o m p o s i t i o n remained more or l e s s c o n s t a n t and the system r e t a i n e d i t s 90 % COD removal c a p a c i t y i . I n f a c t one need not to perform experiments to c o n f i r m the presence of nongrowth a s s o c i a t e d energy e x p e n d i t u r e . ^ r e p o r t e d an e x p e r i m e n t a l a c t i v a t e d sludge system i n which a l l the b i o l o g i c a l s o l i d s were r e c y c l e d back to the a e r a t i o n tank a f t e r b e i n g s e p a r a t e d by a c e n t r i f u g e . e . However. a maintenance c o e f f i c i e n t of 5. w i l l be c i t e d here. T h i s type of s u b s t r a t e e x p e n d i t u r e i s by d e f i n i t i o n f o r maintenance metabolism.37 T h i s c l a i m i s d i s c u s s e d i n d e t a i l i n Chapter 8.

48(1979) . Rev. B i o c h e m i c a l E n g i n e e r i n g .s p N g o c substrate product n i t r o g e n source growth a s s o c i a t e d oxygen carbon d i o x i d e Greek symbols degree of r e d u c t i o n as d e f i n e d by eq.53(I 979). Ph.. B u l l . Gen. 16. 1 1 . (Hermann. A..29. P o w e l l . Recherches s u r l a C r o i s s a n c e des C u l t u r e s B a c t e r i e n n e s . 24. ed. Biochem. J. . F r e d e r i c k s o n and H.H. Bioeng. Symp. 163. 2 8 5 ( 1 9 7 7 ) . E r i c k s o n . A r c h . Roels and N. Free U n i v e r s i t y of Amsterdam. Durham)1976.O. B a c t e r i o l .E. i n Chemical Reactor Theory. 25. D. S.20. 26.M. . I .W. Symp. Stouthamer.H. 18. L. O. M i c r o b i o l . Gen. E r o s h i n .23 .H.A.K.A.J. 2 7 . 12. Bioeng.301 . Y i e l d S t u d i e s i n M i c r o o r g a n i s m s . J H e i j n e n and J.M. M i c r o b i o l . Bioeng. 6. Soc.A. M i c r o b i o l . Stouthamer and C. 2. D.. Kossen. Thermodynamics of i r r e v e r s i b l e p r o c e s s e s ( J o h n W i l e y .95(1 962) .. H e r b e r t . O. A. . England (1981).W. Amsterdam.22. k i n e t i c s and e n e r g e t i c s . 3 7 6 ( I 973).M.1595 (1978). B i o t e c h n o l . e d s . Stouthamer. N e i j s s e l and D. P i r t . R o e l s . A. Congr. 20. B i o t e c h n o l .E. S e l g a and U.M.W. 1 8 .34. J e f f e r i s . Continuous Culture. E r i c k s o n . 8. Amsterdam 1978)vol. N e i j s s e l . ( P r e n t i c e Hall. t h e s i s . 13. J. Bioeng.H. P o w e l l et a l eds.14.New J e r s e y .D t h e s i s . Bioeng.26. Senez. 7. M i n k e v i c h and V. J . 4 8 . B r a z i l ( 1 9 7 3 ) . Tempest. Symp. 15. Advances i n B i o c h e m i c a l Engineering.C. Bettenhaussen. Rev. Londob B. H. 17. V i e s t u r s . Stouthamer.(1 977).. N a g a i .E.W.W. 23 . .H. B i o t e c h n o l .D. ( E l s e v i e r . Soc.F. L. 2 6 7 ( 1 9 8 0 ) .95.. M. on B i o t e c h n o l .E.(1979).2457(1980). E r o s h i n . 21.53 (1 973) . P r o c .G.739(1 981) . B i o t e c h n o l . Humphrey and P. I. . 27.. i n M i c r o b i a l P h y s i o l o g y and Continuous c u l t u r e . New York 1955).K. A. 14.E. L. Biochem. F o l i o M i c r o b i o l . A. Sao P a u l o . Math... a book t o be p u b l i s h e d by E l s e v i e r .. Biochim-Biophys .20. Paper p r e s e n t e d to the 2 nd Eur.G. P a r i s ) ( 1 9 4 2 ) . Roy. B i o s c i . Bettenhaussen. R o e l s . Savageau. S. 1967)p. 10. 9. Monod.G.21. Symp. S. A. Tempest.J. .W.1623 (1978). M.E. van V e r s e v e l d . Ph. Lapidus and Amundson.405. M i c r o b i o l . M i n k e v i c h and V. Acta. 21(1979). h e l d at Eastbourne. 11. A. P r i g o g i n e . B i o t e c h n o l .1(1976) . Soc. 2 nd ed.R.381(1958). 28. I n t .A. P r o g r e s s i n I n d u s t r i a l M i c r o b i o l o g y .A. M i c r o b i o l . 6.725(1979) . E. 3. ( M e a d o w f i e l d . IV GIAM meeting.p. J . U n i v e r s i t y of Amsterdam. T s u c h i y a . Stouthamer and C. 22.11. R o e l s .(H. 19. 2 1 ( 1 9 7 6 ) . 1977)p. J. D.(11)—(13) thermodynamic e f f i c i e n c y of the growth process ( d i m e n s i o n l e s s ) net f l o w of component i to the system (kg/m3/hr) (C-eq/m^/hr) maximum p o s s i b l e y i e l d f o r the d e f i n i t i o n of n (C-eq/C-eq) s p e c i f i c growth r a t e (hr~1) endogeneous metabolism r a t e c o n s t a n t ( h r ' ) maximum growth r a t e i n the absence of endogeneous metabolism ( h r ' ) - VIII REFERENCES 1. I n t . S t a t i o n a r y O f f i c e London.6. E r i c k s o n . J. J. 5. 23. H e r b e r t . 4.224(1965).

A p p l . Gaudy and E.. 39. G. (1971). P h i p p s . A. 1970). Bot.281(1905). S t a t i o n a r y O f f i c e . Bioeng. 1 3 .Gaudy . C o n t r o l Fed. F.240. F.W. 32. 33. . Bioeng.19. 419(1970). 35. M i c r o b i o l . H e r b e r t and P. Tsuchiya. Lett. What i s L i f e ? (Cambridge U n i v e r s i t y P r e s s .1883(1978).209(194-2). A.Adv. 38. 34. 31. Megee and H.W. K a r g i and M. P.. Rev. P. D. Obayashi. D.D. Cromie and H. S. D. Biotechnol. Process A n a l y s i s by S t a t i s t i c a l Methods.. i n M i c r o b i a l P h y s i o l o g y and Continuous C u l t u r e (H. London.J. Wat.1871(1979).21. Gaudy.75.F Blackman.. T i e s s i e r . F r e d e r i c k s o n . Himmelblau. 30. Tempest. 1980). Ann.29.2(8).M.G. B i o t e c h n o l .M. V e r s t r a e t e . R.357(1980). S c h r o d i n g e r . P o l l u t . Yang and A.. Cambridge.43. B i o t e c h n o l . J . A.L. 1967)p. S c h u l e r . M i c r o b i o l o g y f o r E n v i r o n m e n t a l S c i e n t i s t s and E n g i neers . 24 . E. 37. S e i . (John W i l e y .Y. 36. E x t r a i t du 3208.W. New Y o r k .M.Pipyn and W. Doelle.20.1944) p. N e w York.40. (McGraw H i l l .

Experiments w i t h a c t i v a t e d sludge were performed m o s t l y i n a l e s s s o p h i s t i c a t e d 8. I n a l l c a s e s . 10 3 m3 working volume New Brunswick fermentor o p e r a t e d i n b a t c h . The e r r o r i n t r o d u c e d by the mass f l o w meter was determined by c h e c k i n g i t a g a i n s t a p r e c i s i o n wet gas f l o w meter and was found t o be l e s s than 3%.CHAPTER 3 MATERIALS AND METHODS I DESCRIPTION OF THE EXPERIMENTAL SYSTEM AND ANALYTICAL METHODS set-up Experimental A l l b a t c h and f e d .10 3 3 w o r k i n g volume. e l i m i n a t e d the n e c e s s i t y f o r c o r r e c t i o n s due to changes i n the atmospheric p r e s s u r e . F o r t r a n s i e n t experiments t h e o u t l e t gas stream t o the carbon d i o x i d e a n a l y s e r c o u l d n o t be passed through s i l i c a g e l d r i e r s due to t h e a f f i n i t y o f CO2 t o s i l i c a g e l ( a d s o r p t i o n / d e s o r p t i o n ) which d i s t o r t s the observed dynamic response o f the system. which measured the c o n c e n t r a t i o n i n the o u t l e t gas stream as a f r a c t i o n o f t h e i n l e t c o n c e n t r a t i o n . Carbon d i o x i d e c o n c e n t r a t i o n was measured by an i n f r a r e d a n a l y s e r (Beckman 865).5 K.8 ± 0. except o t h e r w i s e s t a t e d . pH and temperature were s e t and c o n t r o l l e d a t 6. r e s p e c t i v e l y . the o u t l e t gas stream was d r i e d by Permeation Distillation t e c h n i q u e which i n t r o d u c e d v i r t u a l l y no time l a g s o r o t h e r i n t e r f e r i n g e f f e c t s (Perma Pure D r i e r PD-750-24P). T h e r e f o r e .b a t c h e x p e r i m e n t s . _ m _ A c i d / a l k a l i added f o r pH c o n t r o l was monitored by a Servo-chem Dose m o n i t o r DM1 and r e c o r d e d . _ 25 .b a t c h experiments w i t h mono c u l t u r e s were performed i n a B i o l a f i t t e 15 S b i o r e a c t o r w i t h 11. T h i s c a p a b i l i t y . 3 Gas phase oxygen c o n c e n t r a t i o n was measured by a t w i n channel paramagnetic oxygen a n a l y s e r ( T a y l o r Servomex OA 184) c o u p l e d t o a r a t i o box. Data o b t a i n e d a s s i s t e d the v e r i f i c a t i o n of s t e a d y s t a t e s and the c o r r e c t i o n o f t h e d i l u t i o n r a t e d u r i n g c o n t i n u o u s c u l t i v a t i o n and p a r t i c u l a r l y f o r the c o r r e c t i o n of volume b a l a n c e s i n b a t c h and f e d . 10 3 m3 w o r k i n g volume. t o a v o i d i n t e r f e r e n c e s by d i f f u s i o n of gases i n and/or o u t . The gas stream out o f t h e fermentor was passed through a condensor which had r e f r i g e r a n t c i r c u l a t i o n a t 268 K on t h e c o o l i n g s i d e . A l l tubes used f o r gas t r a n s p o r t a t i o n were made o f e i t h e r b u t y l rubber o r aluminium. These experiments were c a r r i e d o u t a t a temperature of 293 K and pH o f 6. A i r f l o w r a t e t o the fermentor was c o n t r o l l e d by a thermal mass f l o w meter (Brooks 5811) a t about 60 m /m3/hr at STP. f i l l and draw o r c o n t i n u o u s modes.8 .05 and 308 ± 0. Continuous c u l t u r e experiments were c a r r i e d out i n a New Brunswick r e a c t o r w i t h 3 .

T h i s circumvented c o m p l i c a t i o n s causedby heat s t e r i l i z a t i o n l i k e p r e c i p i t a t i o n .l i n e exchanger designed and manufactured i n o u r department. Dry Weight: Dry weights were determined as d e s c r i b e d by de V r i e s and Stouthamer. I n dynamic e x p e r i ments. Water a c t i v i t y of the s t a n dard medium was about 0. T0C : O c c a s i o n a l l y T o t a l carbon (TC) and T o t a l o r g a n i c carbon (TOC) measurements were performed to check the a c c u r a c y of the enzymic assay and a l s o to l o o k f o r the presence of unexpected C . T y p i c a l r e s i d e n c e time i n the exchanger was about 5-10 seconds.c o n t a i n i n g compounds. T h i s v a l u e i n c l u d e d e r r o r c o n t r i b u t i o n s of sampling and interferences from o t h e r components p r e s e n t i n the c u l t u r e s u p e r n a t a n t . I n g e n e r a l e r r o r s of the o r d e r of 5 % or l e s s were e x p e r i e n c e d . Nitrogen : N i t r o g e n content of the feed/medium and the c u l t u r e supernatant was determined by an auto K j e l d a h l . x 26 . by an o n .c u l t u r e experiments Klebsiella pneumoniae NCTC 418. f o r m e r l y known as Klebsiella aerogenes.996. A t t e n t i o n was p a i d to o b t a i n an a c t i v e l y growing inoculum and i n most cases the inoculum used was v e r y s m a l l to a v o i d the p o s s i b i l i t y of unbalanced growth? A n a l y t i c a l Methods Substrate : Reagent q u a l i t y g l y c e r o l (BDH Chemicals) was used and assayed e n z y m i c a l l y . For a c t i v a t e d sludge c u l t u r e s the b a s a l medium r e c i p e was taken from Harder.* I n continuous c u l t u r e experiments w i t h C i n the order of 3-5 kg/m3. ( B o e h r i n g e r UV method. p a r t i c u l a r l y i n b a t c h r u n s . 14270) D e t e c t i o n l i m i t of the assay was e s t i m a t e d to be about 10 . A f t e r e i g h t weeks of a d a p t a t i o n a w e l l s e t t l i n g y e l l o w i s h . T h i s sludge was f i r s t a c c l i m a t i z e d to g l y c e r o l as the s o l e carbon and energy source.10"^ kg/m3. Cultivation Methods Growth medium was prepared a c c o r d i n g to the f o r m u l a t i o n g i v e n by Evans et a l ' .^ I n i t i a l s u b s t r a t e c o n c e n t r a t i o n was 10 kg/m3 f o r b a t c h and continuous c u l t u r e experiments and a d j u s t e d a c c o r d i n g to the f i n a l d e s i r e d biomass c o n c e n t r a t i o n and f e e d i n g p r o f i l e i n f e d . the e r r o r s i n v o l v e d were c o n s i d e r a b l y higher.b r o w n sludge was obtained.2 m i c r o n pore diameter membrane ( S a r t o r i u s 11307).b a t c h experiments. G l y c e r o l was used as the l i m i t i n g s u b s t r a t e i n a l l cases (except f o r wash-out experiments where l a c t i c a c i d was the s u b s t r a t e ) . TC. e v a p o r a t i o n e t c .N i t r o g e n a n a l y s e r . For mixed c u l t u r e experiments a c t i v a t e d sludge was o b t a i n e d form the P i l o t P l a n t Operated by the Department of C i v i l E n g i n e e r i n g of the D e l f t U n i v e r s i t y of Technology. Samples were c o o l e d down to about 278-280 K w h i l e b e i n g t a k e n . I n t e g r i t y of the s t e r i l i z e d f i l t e r was t e s t e d p r i o r to each f i l t r a t i o n by the Bubble point test. When v e r y low c o n c e n t r a t i o n s have to be e s t i m a t e d the method of Standard Additions was used to i n c r e a s e the r e l i a b i l i t y of the a n a l y s i s .Organism For mono . was used throughout t h i s study. pH d r i f t . The medium was s t e r i l i z e d by membrane f i l t r a t i o n through a 0. the average e r r o r i n d r y weight d i d not exceed 2 %.

Oxygen c o n t e n t was c a l c u l a t e d from t h e d i f f e r e n c e . II DEVELOPED AND USED TOOLS AND METHODS of Gas Exchange Quantification Assumptions: a. because of the d i f f i c u l t i e s w e l l documented elsewhere. 5 was used. as d e s c r i b e d by H e r b e r t e t a l . Between s e t s . sample gas l i n e r. gas processing and analysis flow diagram. 5 Protein : C e l l p r o t e i n content was e s t i m a t e d by t h e B i u r e t method as d e s c r i b e d by Herbert e t a l . fermentor condensor mass f l o w meter a i r i n p u t pump perma pure d r i e r h u m i d i t y measurement s i l i c a g e l column dry gas g e n e r a t i o n cycle O2 a n a l y s e r r e f e r e n ce channel O2 a n a l y s e r sample channel s i l i c a gel driers CO2 analyser heat exchanger sampling p o r t s. by t h e o r c i n o l and diphenylamine procedures. however.-* T h e r e f o r e these r e s u l t s must be t r e a t e d w i t h c a r e . Total Carbohydrate : Anthrone method. RNAj DNA : RNA and DNA c o n t e n t s of t h e f r e e z e d r i e d biomass were determined a f t e r e x t r a c t i o n w i t h p e r c h l o r i c a c i d . The c o n s i s t e n c y of the method was checked and found to be good by comparing w i t h the r e s u l t s of an independent l a b o r a t o r y s p e c i a l i z e d i n these a n a l y s e s . c. No CO2 and/or N2 f i x a t i o n . P r e c i s i o n was good w i t h i n t h e samples a n a l y s e d i n one s e t . b. 27 . as d e s c r i b e d by H e r b e r t e t a l . 1: Experimental set-up. f o r t h e same samples. Only O2 and CO2 exchanged. Steady s t a t e o p e r a t i o n w i t h r e s p e c t t o the concerned gases. t h e same p r e c i s i o n c o u l d not be a c h i e v e d . r e f e r e n c e gas l i n e Fig. r e s p e c t i v e l y . N. A n a l y s i s of the macromolecular c o m p o s i t i o n of f r e e z e d r i e d biomass was one of the most t e d i o u s and l e a s t a c c u r a t e a n a l y s i s r e p o r t e d i n t h i s work. H ) . ( P e r k i n Elmer 240). Ash c o n t e n t was determined s e p a r a t e l y and a l l r e s u l t s r e p o r t e d i n t h i s t h e s i s a r e expressed on Ash Free Basis.Biomass Analysis Elemental Composition : Three elements were determined i n t h e t w i c e washed and d r i e d biomass powder by a computer coupled element a n a l y s e r (C.

and CO^ can be taken as 0. 28 .0224 }. For a b i o l o g i c a l system. The program a l s o i n t e g r a t e d the OUR and CPR p r o f i l e s n u m e r i c a l l y t o f i n d the c u m u l a t i v e s r e q u i r e d f o r b a l a n c e s and y i e l d c a l c u l a t i o n s .033. From eq. I n the f o l l o w i n g .C O " < ) ) / 0. volume changes can be expected t o be s i g n i f i c a n t and t h e r e f o r e an e q u a t i o n d e s c r i b i n g the change i n the volume of the c u l t u r e i s r e q u i r e d .21 and 0. OUR. I f t h i s cannot be assumed. 6 . I n the v i c i n i t y of our l a b o r a t o r y COj was found t o be around 0. 0. N|n . t h e r e f o r e ( J ) can be c a l c u l a t e d i f the exhaust gas stream i s a n a l y s e d f o r O2 and CO2 .°2 > u C (2) i c l a t e d. a mass b a l a n c e reads: E N„ . U s u a l l y such changes are cons i d e r e d t o be i n s i g n i f i c a n t f o r b a t c h and continuous c u l t i v a t i o n systems. 1. i . the g e n e r a l case. when p r o v i d e d w i t h the raw gas exchange d a t a .(2) i t f o l l o w s t h a t a t each moment ^ o u t w i l l depend on t h e e x t e n t of gas exchange and f o r t h e sake of a c c u r a c y i t has t o be c a l c u l a t e d f o r each measurement p o i n t . T h e r e f o r e . 11 r o r e a c n In t h e a n a l y s i s p r e s e n t e d above t h e gas f l o w s were assumed t o be p r a c t i c a l l y dry.out 2 A and A N^ 2 n (b. the water vapour c o n t e n t of t h e f l o w s can be e s t i m a t e d by measuring the gas d r y and wet b u l b temperatures and u s i n g standard c o r r e l a t i o n s .21 6.0224 out (mole 0„/m /hr) 2 3 3 (3) CPR = ( CO^ < f > . c a l c u l a t e d tyout p o i n t and OUR. a b a l a n c e on gaseous N2 . = *in ^ '('out ' " " ' s c a ( 1 " ° 2 .0360. CPR and RQ. D u r i n g t h i s study a s i m p l e computer program was developed which. 2 n = *out n ( 1 . capable o f d e s c r i b i n g any mode of o p e r a t i o n . n out *out Once t h e o u t f l o w expressions . 7 Volume Balancing for Fermentation Processes Volume changes do occur i n f e r m e n t a t i o n systems. r e s p e c t i v e l y .79.b a t c h systems. = N ° in 2 U t i> out . the f o l l o w i n g diagram d e f i n e s the major i n p u t and output f l o w s .°2-°2>/*in C ( 1 ) Here note t h a t <|>i and '('out r e f e r to d r y v o l u m e t r i c gas f l o w s a t STP and t h a t they a r e not n e c e s s a r i l y e q u a l t o each o t h e r except when RQ equals one. where t h e gas f l o w s a r e i n d i c a t e d . which passes through the system unchanged.With r e f e r e n c e t o F i g . in 2 4 > ) / 0. p r o v i d e s the s t a r t i n g p o i n t . s u b s e q u e n t l y . 0^ . I n f e d . e . w i l l be c o n s i d e r e d . I n p r a c t i c a l systems u s u a l l y <j>£ i s measured and/or c o n t r o l l e d . r e l e v a n t t o t h i s s t u d y .i n = 2 or N Z N„ . 2 out 2 in Then RQ can be g i v e n by : RQ = CPR / OUR n 1 (mole C0„/m /hr) (4) 2 (5) For f r e s h a i r . CPR can be e a s i l y c a l c u l a t e d by the OUR = ( 0.

032/RQ . dV /dt .032 r o - 0.$ s s F e 3 + 0.044 r c (8) where $ i s the mass f l o w (kg/m^/hr) r a t e and r . one may t h i n k of a c i d / a l k a l i a d d i t i o n assoc i a t e d w i t h the pH c o n t r o l system.0. 2 t h i s equation gives. i t i n c l u d e s t h e most s i g n i f i c a n t f a c t o r s and c l e a r l y shows t h a t the volume change d u r i n g a c u l t i v a t i o n p r o c e s s i n any mode. Fig.$ .$ t ie c a r o n d (mass)/ d t = (6) For t h e system d e s c r i b e d d (mass)/ d t = $ p e C 0 2 ( ? ) Since <PQ2 . can be m i n i m i z e d t o l e v e l s which would i n s i g n i f i c a n t compared w i t h the s o .{$ . t o a v e r y good a p p r o x i m a t i o n .Of feed oxygen O Ckg/m /hr) 3 c a r b o n dioxide oxygen effluent 1 <l>c <Dg ut • <De process.V (dp / d t ) } / p s s s RQ = r (9) (10) A l t h o u g h t h i s e q u a t i o n does not i n c l u d e a l l of the p o s s i b l e c o n t r i b u t i o n s t o volume change. s s / r c o we o b t a i n the r e l a t i o n .$ + r s F e c (0. t h e magnitude of which depends e n t i r e l y on the m e t a b o l i c s t a t e ' o f t h e b i o l o g i c a l 29 .c a l l e d major f l o w s .g. s u b s t i t u t i n g p V f o r t h e t o t a l mass the f o l l o w i n g form i s o b t a i n e d : s s d ( p V )/ dt = * . b u t a l s o by exchange i n gas phase. These f l o w s . + . Mass. however. P (kg/m ) and V (m3) a r e the system d e n s i t y and v o l u m e . n e t water vapour f l o w and l o s s of o r g a n i c vapours v i a the gas phase. t h e n e t molar f l o w r a t e (mole/ m-Vhr).$ § 2 ' d e f i n e s the n e t oxygen uptake and ®C02 ' ^ dioxide p r o d u c t i o n r a t e (kg/m^/hr). f o r these c o n t r i b u t i o n s the more g e n e r a l l y used molar f l o w s can be s u b s t i t u t e d i f the a c c u m u l a t i o n of CO2 and O2 i n t h e l i q u i d phase can be assumed t o be n e g l i g i b l e compared w i t h t h e i r p r o d u c t i o n and consumption t e r m s . r e s p e c t i v e l y . . 2: Major input and output flaws for a fermentation In a d d i t i o n to the f l o w s i n d i c a t e d . Expanding t h e d i f f e r e n t i a l and s u b s t i t u t i n g t h e r e l a t i o n . e. Moreover. volume of d i l u t i o n e t c . i s a conserved q u a n t i t y i n the absence of n u c l e a r t r a n s f o r m a t i o n s . r e s p e c t i v e l y . T h e r e f o r e . a t o t a l mass b a l a n c e f o r t h e system p r o v i des a c o n v e n i e n t s t a r t i n g p o i n t f o r t h e p r e s e n t d e r i v a t i o n . under c a r e f u l l y d e s i g n e d and conducted e x p e r i m e n t s . i s not o n l y i n f l u e n c e d by the t r a n s p o r t of m a t t e r i n l i q u i d phase as u s u a l l y f i x e d by the e x p e r i m e n t e r .044) . The g e n e r a l mass b a l a n c e reads: n Z Net t r a n s p o r t o f mass i i n F i g .

C o n s i d e r i n g the case f o r continuous c u l t i v a t i o n . T h i s assumption i s j u s t i f i a b l e i n most p r a c t i c a l cases. S i n c e the time c o n s t a n t of the b i o s y n t h e t i c process i s much l a r g e r than t h a t of the s u b s t r a t e g 30 . Fig. S t a r t as b a t c h or m a i n t a i n e x p o n e n t i a l growth and determine q . iv. Fed-Batch Technique. . 3: Schematic representation of the Declining Feed. i s as f o l l o w s : s m iii. However.b a t c h mode l e d to the development of a p o w e r f u l n o v e l technique f o r the d e t e r m i n a t i o n of maintenance c o e f f i c i e n t . Continue as f e d . P ax and Y f o r the e x p o n e n t i a l phase. The procedure i.) Having determined q . y .b a t c h . both time d e r i v a t i v e s can be s e t to zero f o r steady s t a t e o p e r a t i o n . dM /dt can a l s o be assumed t o be z e r o .f i x e d p a t t e r n . T h i s w i l l b r i n g the system to zero growth r a t e s t a t e . F u r t h e r s i m p l i f i c a t i o n of the above equat i o n can be o b t a i n e d by assuming the time d e r i v a t i v e of p as z e r o . the c o n t r i b u t i o n of the gas exchange can be s i g n i f i c a n t f o r some a n a e r o b i c p r o c e s s e s ? System s The Declining Feed Fed-Batch System M o d e l l i n g and e x p e r i m e n t a l study of m i c r o b i a l growth i n f e d . decrease the r a t e of s u b s t r a t e a d d i t i o n a c c o r d i n g to a p r e . say l i n e a r l y . ii. at l e a s t momentarily.m s ) (11) The i m p l i c i t assumption i n t h i s c o n s i d e r a t i o n i s t h a t when dMj^/dt =0. T h i s assumption i s q u i t e j u s t i f i a b l e i f one c o n s i d e r s the time c o n s t a n t s of the major processes i n v o l v e d . e . T h i s method w i l l be r e f e r r e d to as the Declining Feed.as r e f l e c t e d by i t s RQ v a l u e . C a l c u l a t e m from a mass b a l a n c e d i r e c t l y . T h i s a c t u a l l y means t h a t at steady s t a t e the incoming and o u t g o i n g f l o w s would not be e q u a l t o each o t h e r on volume b a s i s and t h a t the d i f f e r e n c e would be a f u n c t i o n of the m e t a b o l i c s t a t e . from F ( t ) = m M when dM /dt = 0 (see the model p r e s e n t e d i n Chapter 4.9 The method can be d e s c r i b e d w i t h the a i d of the f o l l o w i n g figure (Fig. For a e r o b i c growth w i t h moderate RQ v a l u e s the gas exchange term i s u s u a l l y a s m a l l f r a c t i o n of the l i q u i d f l o w s and hence can be n e g l e c t e d f o r most purposes. FedBatch Technique. Y and m calculate Y from : s x s g x x m x s m a x s x g x Y * sx m x = y max / ( q s . 3). i .

*% . . The p r a c t i c a l use of t h i s method. i . I f . v. then the net f l o w of s u b s t r a t e to the m i c r o b i a l system must be determined. s x s The technique has f i r s t been t r i e d w i t h c o n s t a n t f e e d i n g i . Determination c a n e of the Maximum Specific Growth Rate 'max ^ c o n v e n i e n t l y determined by many t e c h n i q u e s . . ii. I n p r a c t i c e however. b e s t to perform i n l a r g e s c a l e to m i n i m i z e e r r o r s i n t r o d u c e d by sampling. sampling i n the s u b s t r a t e l i m i t e d phase can be reduced to a minimum by f o l l o w i n g the RQ p r o f i l e . f o r i n s t a n c e . i s p r o v i d e d i n Chapter 4. i i i . t h i s t e c h n i q u e may be expected to r e s u l t i n more r e a l i s t i c e s t i m a t e s of m f o r most i n d u s t r i a l f e r m e n t a t i o n s as these are m a i n l y c a r r i e d out i n b a t c h and f e d . . problems a s s o c i a t e d w i t h v i a b i l i t y and a d a p t a t i o n can be reduced t o t h e i r minima. s F u r t h e r i n v e s t i g a t i o n s i n t o the use of t h i s t r a n s i e n t t e c h n i q u e seems t o be d e s i r a b l e . m a x m a x s s g Advantages of the new method: i.consumption p r o c e s s . . r e q u i r e s c a r e f u l e x p e r i m e n t a l d e s i g n and more s o p h i s t i c a t e d equipment (a programmer). however. s e n s i t i v e to sampling. dM /dt can be taken as zero whenever dM /dt approximates to z e r o . cannot check i f m = f ( l i ) . I f performed as d e s c r i b e d the experimenter can t h e o r e t i c a l l y determine y .b a t c h modes. C u l t u r e s f e d w i t h c o n s t a n t s u p p l y kept on growing at a v e r y s m a l l but s i g n i f i c a n t growth r a t e f o r l o n g p e r i o d s of time d u r i n g which the c e l l morphology and v i a b i l i t y changed c o n s i d e r a b l y . e . . m i s determined o n l y a t zero growth r a t e . i i i . p r o v i d e d the c u l t u r e i s s u b s t r a t e l i m i t e d . m ) can be o b t a i n e d from a r e l a t i v e l y s h o r t experiment. Many p o s s i b i l i t i e s f o r computer a p p l i c a t i o n s . Even f o r c o n s e r v a t i v e changes i n the growth r a t e . S u p p o r t i n g evidence f o r t h i s r e a s o n i n g has been p r o v i d e d by M i n k e v i c h and Utkina'O. m and Y from a s i n g l e experiment. F ( t ) = c o n s t a n t . no m„ c o n s t a n t h y p o t h e s i s . Hence d i f f e r e n c e s may be expected as the c u l t u r e s are e s s e n t i a l l y under d i f f e r e n t e n v i r o n m e n t a l c o n d i t i o n s and thus have d i f f e r e n t p h y s i o l o g i e s . t h e r e i s s t i l l a p p r e c i a b l e s u b s t r a t e l e f t i n the f e r m e n t o r . growth ceases due to the a c c u m u l a t i o n of i n h i b i t o r s i n the system. Theoretically U can a l s o be determined from the OUR and CPR curves i f the m a x 31 . e . and was found to be u n s a t i s f a c t o r y . would be u s e f u l . t h r e e parameters ( y . e v a l u a t i o n of m independent of Y . g g O b v i o u s l y t h i s methods determines the maintenance v a l u e s under t r a n s i e n t c o n d i t i o n s whereas the v a l u e s determined i n continuous c u l t u r e s t u d i e s r e f e r t o steady s t a t e c o n d i t i o n s . as p o i n t e d out by H a r r i s o n . k . dM /dt w i l l be n e g l i g i b l e . A p p l i c a t i o n of the c o n t r o l t h e o r y and p a r t i c u l a r l y a study of whether or not the r a t e of approach to the zero growth s t a t e has any e f f e c t on the maintenance v a l u e determined. the method results i n k v a l u e s h i g h e r than those r e p o r t e d i n l i t e r a t u r e . max i l . Y . T h i s might happen when. s g g x m a x g x g Disadvantages : i. The most c o n v e n t i o n a l one i s t o determine i t i n b a t c h c u l t u r e s d u r i n g the e x p o n e n t i a l growth phase. iv. as i l l u s t r a t e d w i t h e x p e r i m e n t a l and s i m u l a t i o n data . Under these c o n d i t i o n s i t becomes e x t r e m e l y d i f f i c u l t t o i n t e r p r e t the d a t a o b t a i n e d and a p p l y the c o n v e n t i o n a l r e l a t i o n s . ' ' However. .

e . by many a u t h o r s . e . Based on t h i s data the curve i . I n o t h e r words s m a l l v a l u e s of C i n f l u e n c e the outcome o f the e x e r c i s e most.05 kg/m3 .r e s p o n s e data e t c . V a l i d i t y of r e l a t i o n s were t e s t e d whenever p o s s i b l e . To t h e knowledge of the a u t h o r . t h e d r y weight p r o f i l e i s shown. however. One of the most c h a l l e n g i n g one i s t o e s t i m a t e s e v e r a l parameters from m u l t i . t h e r e a r e s t i l l problems t o be s o l v e d .'2.n e g l i g i b l e e r r o r s a t t a c h e d t o the measured v a r i a b l e s . T h i s d i f f e r e n c e stems from the f a c t t h a t . i n our b a t c h exper i m e n t s s i g n i f i c a n t s y s t e m a t i c d i f f e r e n c e s were observed i n the U values o b t a i n e d f r o m the t h r e e responses determined i . time data was p l o t t e d . The nonl i n e a r r e g r e s s i o n program used was developed i n t h e Chemical E n g i n e e r i n g Department of the D e l f t U n i v e r s i t y of Technology and was based on Marquart's method. some complex f u n c t i o n of the e r r o r s r e s u l t r a t h e r than the e r r o r b e i n g added t o the transformed v a r i a b l e .The a s s o c i a t e d e r r o r i s assumed t o be a =0. where p o s s i b l e use of n o n l i n e a r r e g r e s s i o n should be p r e f e r r e d i n o r d e r t o a v o i d v a r i a b l e t r a n s f o r m a t i o n . C v s . However. proved t o be s u p e r i o r t o l i n e a r r e g r e s s i o n . OUR and CPR d a t a . a n a l y s i s for c o r r e l a t i o n . I f the e r r o r s i n v o l v e d are e x c e e d i n g l y s m a l l t h e r e should be no d i f f e r e n c e between the two procedures. parameter e s t i m a t i o n from m u l t i . Here. .r e s p e c t i v e y i e l d s a r e assumed t o be constant. 4 i s a s i m u l a t i o n of an e x p o n e n t i a l growth phase i n a b a t c h experiment w i t h parameters s i m i l a r t o those r e p o r t e d i n t h i s study ( p =1. These r e s u l t s a r e p r e s e n t e d and d i s c u s s e d i n Chapter 4. . the o n l y r i g o r o u s attemp i n t h i s d i r e c t i o n has come from de Kwaadsteniet e t a l . o f t e n t r a n s f o r m a t i o n s of v a r i a b l e s a r e made. p a r t i c u l a r l y by Johnson and Berthouex' >' and B o y l e and Berthouex17 w i t h waste water processes i n mind. The use of n o n l i n e a r r e g r e s s i o n d u r i n g t h i s study. i n p e r f o r m i n g l i n e a r r e g r e s s i o n . F i g .13 However. r e s u l t e d i n c o n s i s t e n t u v a l u e s from v a r i o u s responses. T h e r e f o r e . The wash-out t e c h n i q u e . Most of the parameters r e p o r t e d i n t h i s work were c a l c u l a t e d by standard l i n e a r or n o n l i n e a r r e g r e s s ion procedures. i t i s q u i t e d i s s a p p o i n t i n g t h a t t h e a p p l i c a t i o n s of these a v a i l a b l e t o o l s a r e s t i l l s c a r c e i n the study o f m i c r o b i a l e n e r g e t i c s . The u n c e r t a i n i t i e s of data p o i n t s are assumed to be equal a s i n d i c a t e d on the l i n e a r s c a l e b u t appear t o be unequal on the l o g a r i t h m i c s c a l e . I n some cases t h i s i s q u i t e d i f f i c u l t to perform due t o t h e h i g h l y n o n l i n e a r and i m p l i c i t n a t u r e o f t h e d i f f e r e n t i a l e q u a t i o n s d e s c r i b i n g the b i o l o g i c a l systems. m a x x x x 32 . a f t e r t r a n s f o r m a t i o n . When t h e r e are n o n . by s u b j e c t i n g the e x p e r i m e n t a l d a t a t o s t a t i s t i c a l a n a l y s i s .0). max m a x x III APPLICATION OF STATISTICAL TECHNIQUES IN THE STUDY OF MICROBIAL KINETICS AND ENERGETICS Notes on the use of Statistical Techniques. C . ' ' However.r e s p o n s e d a t a i n t r a n s i e n t range. regression analysis One can determine the c o n f i d e n c e t o be p l a c e d on the e x p e r i m e n t a l r e s u l t s . 2 3 T h i s can b e s t be i l l u s t r a t e d by a r e a l i s t i c example. ^ 2 F o r the k i n e t i c s and m o d e l l i n g s i d e much more work has been done and r e p o r t e d .'^ In l i t e r a t u r e e x c e l l e n t accounts of e f f i c i e n t e x p e r i m e n t a l d e s i g n and model b u i l d i n g processes have been g i v e n . By f i t t i n g the l i n e a r e q u a t i o n i n s t e a d of the expon e n t i a l r e l a t i o n the experimenter assumes t h a t t h e u n c e r t a i n i t i e s a r e equal on the e x p o n e n t i a l s c a l e and t h e r e f o r e u n d e r e s t i m a t e s the e r r o r s f o r s m a l l v a l u e s of C . Advanced s t a t i s t i c a l t e c h n i q u e s have a l s o been made a v a i l a b l e f o r d a t a r e d u c t i o n .

c. random v a r i a b l e s . From a s t a t i s t i c a l p o i n t of view this procedure i s p e s s i m i s t i c . I t must be reminded t h a t both methods. 8. f o r s m a l l changes i n X's i s g i v e n b y 2 4 : AW = |a| AX + |b| A X 2 + |c| A X 3 + (13) where AW i s the maximal e r r o r i n W. e . 33 . . F o r a l i n e a r f u n c t i o n of the form . The double r e c i p r o c a l p l o t i . a Xj + b X + c X + (12) 2 3 where a. of t h e s y s t e m a t i c e r r o r a n a l y s i s i s p r o v i d e d i n Ch. X . A more d e t a i l e d d i s c u s s i o n and a p p l i c a t i o n to e x p e r i m e n t a l systems. b. one can e v a l u a t e how t h e v a r i a t i o n i n v a r i o u s components a r e t r a n s m i t t e d t o the f i n a l r e s u l t . t h e maximal n u m e r i c a l e r r o r i n W. s u f f e r s b a d l y from the uneven w e i g h t i n g of t h e data p o i n t s and the d a t a c o l l e c t e d a t moderate y v a l u e s tend t o c l u s t e r near the o r i g i n . a r e such as t o produce the maximum p o s s i b l e e r r o r i n W i s v e r y s m a l l . ... Continuous c u l t u r e d a t a o b t a i n e d d u r i n g t h i s study were found t o be f i t t e d best by n o n l i n e a r r e g r e s s i o n technique when assessed by t h e s c a l e d sum of r e s i d u a l s . Assessment of Error Propagation. 4: Simulation nonlinear of exponential growth.. X^. A n a l y s i s o f r e s i d u a l s can be v e r y u s e f u l i n t h i s c o n t e x t . I t i s important t o r e a l i z e t h a t the n a t u r e of some b i o t e c h n o l o g i c a l experiments are such t h a t s i g n i f i c a n t s y s t e m a t i c e r r o r may be i n t r o d u c e d . as w e l l as t h e e r r o r s t r u c t u r e o f these components a r e known. the combined p r o b a b i l i t y t h a t the e r r o r s i n X^. 1/y p l o t . These t o o l s have been p r o v i d e d by Smith and Draper'" among o t h e r s . T h e r e f o r e . l i n e a r and n o n l i n e a r . . 1/Y v s . for linear and The c o n v e n t i o n a l l i n e a r r e g r e s s i o n procedure was a l s o found t o be i n f e r i o r t o the n o n l i n e a r r e g r e s s i o n procedure i n t h e d e t e r m i n a t i o n of e n e r g e t i c paramet e r s . X^. X^. parameters o b t a i n e d by n o n l i n e a r r e g r e s s i o n were used i n t h e c a l culations .Fig. . a r e c o n s t a n t s and X j . a r e founded upon the assumption o f the randomness of e r r o r s . . T h i s may n o t be j u s t i f i e d always. Calculation of the Maximal and Probable Errors When the f u n c t i o n a l r e l a t i o n s h i p c o n n e c t i n g the f i n a l r e s u l t w i t h t h e v a r i a b l e components. error structure regression procedures (see text).

w + S. The a n a l y s i s t o be r e p o r t e d here c o n s i d e r s t h e case o f c o n t i n u o u s c u l t i v a t i o n w i t h one l i m i t i n g s u b s t r a t e . X„ ) } X x_ )/ St } Xj. t h i s approach w i l l o n l y be a b l e t o p r o v i d e an i n d i c a t i o n o f t h e o r d e r of the magnitude o f t h e e r r o r s . C o n s t r u c t i o n o f C balances i s t h e r e f o r e o f prime importance i n such s t u d i e s .f ( X j . t h e procedure can be extended and a p p l i e d t o any system a t steady o r pseudo-steady s t a t e . No C O 2 o r N 2 f i x a t i o n iii. x^. I n t h i s case t h e f o l l o w i n g r e l a t i o n s h i p h o l d s between the v a r i a n c e s o f t h e r e s u l t and t h e components:23.4 . X . in s i s in 2 out | „ CO^ > out 2 34 12/22.A more r e a l i s t i c procedure i s t o c o n s i d e r t h e p r o b a b i l i t y d i s t r i b u t i o n of the t o t a l e r r o r i n W. V X x° x° 2' V ' " ' 0 n where t h e s u p e r s c r i p t '0' r e f e r s t o t h e r e f e r e n c e s t a t e . x . L i n e a r i z a t i o n can be a c h i e v e d by expansion i n t o a T a y l o r s e r i e s about a r e f e r e n c e p o i n t and by neg l e c t i n g t h e second and h i g h e r o r d e r terms.l i m i t e d c u l t u r e s where o n l y one s u b s t r a t e i s t h e source of b o t h carbon and energy. t h e t r u n c a t e d T a y l o r s e r i e s i s g i v e n by Himmelblau23 . x^. x^. t h e v a r i a n c e i s g i v e n by: n E { 3f( 1=1 Var {f( X . Error analysis Most e n e r g e t i c and k i n e t i c s t u d i e s a r e c a r r i e d out i n C . the f u n c t i o n must f i r s t be l i n e a r i z e d i n o r d e r t o use e q . by assuming t h a t t h e random v a r i a b l e s a r e independent. x° E {3f( X i=l X X . C . 3 C o n s i d e r i n g a continuous c u l t u r e system o f u n i t volume (1 m ) and t h e f o l l o w i n g assumptions: i.24 Var (W) = a 2 Var ( X j ) + b 2 Var(X ) + c 2 2 Var(X > + 3 (14) I f t h e f u n c t i o n a l r e l a t i o n s h i p between t h e v a r i a b l e s i s n o t l i n e a r .4 = F ( C w + C w + C w ) + s s x x p p (17) F. However. > x^ ) . ( 1 4 ) . F o r a f u n c t i o n of s e v e r a l v a r i a b l e s . E f f i c i e n t c o n d e n s a t i o n and d r y i n g o p e r a t i o n f o r f e r m e n t a t i o n gases For t h i s system a t o t a l C-balance s h o u l d r e a d : E C ECout 12/22. Steady s t a t e o p e r a t i o n ii. a s EC x . CO. 2 (15) }(X.c o n t a i n i n g p r o d u c t s i n gas phase o t h e r than C O 2 and o n l y one m e t a b o l i c product iv. Carbon and Nitrogen Balances .^2'• Var(X ) i I f t h e v a r i a b l e s a r e c o r r e l a t e d however. Carbon l i m i t e d c u l t u r e and no C . X ) / 3X. F u r t h e r m o r e .

c | > . C 0 ^ in 2 12/22.90 37. i ° l a v 1 experiment. Table I : E r r o r assessment i n C-balance f o r a continuous c u l t u r e „ % error igCRI|. and hence can be assumed t o be c o n s t a n t f o r compounds of known formulae.04 0.94 16. 1 0. SX. 3. r e s p e c t i v e l y .6 s a CRI . C .2 . were e v a l u a t e d and the maximal and p r o b a b l e e r r o r i n CRI were c a l c u l a t e d .4 CRI = f ( F. the above e q u a t i o n does not always h o l d and we can t h e r e f o r e d e f i n e the Carbon Recovery Index. 5 — } Var(X. CRI. I f . <> .84 24.) ). C0^. The raw d a t a a t the p o i n t of l i n e a r i z a t i o n and the c o r r e s p o n d i n g e s t i m a t e s of e r r o r s i n v o l v e d are l i s t e d i n Table I . C . 0130 0 0194 0 0 0062 0 0156 0 0480 0 0001 2 0 0006 0 1 165 0 0 P 67 70 <tWt COA 0 0187 C C .Due t o e x p e r i m e n t a l e r r o r s .46 1 . CO". % total . CRI can be w r i t t e n as (see s e c t i o n on volume b a l a n c i n g f o r the j u s t i f i c a t i o n of t h i s assumption): FCRI (C w + C + w x x + C w ) + < „ C0_ 12/22.4 j > out 2 l n s s P n p (19) F. w in s i s Therefore . w .). maximal error 8 4 1 1 17 0 5 13 41 0. F £ . however. C . t o assess the c o n s i s t e n c y of the C-balances: CRI = I C -out / Ic . p a r t i a l d e r i v a t i v e s of CRI w i t h r e s p e c t t o i t s v a r i a b l e s i n d i c a t e d above.10 aX. To e s t i m a t e V a r ( C R I ) . . • =—AX.in n o u t (18) or the % C-recovery as 100 x CRI. in s x p x out in 2 2 si w and w are not i n c l u d e d i n the above e x p r e s s i o n s i n c e . The f o l l o w i n g example r e f e r s t o continuous c u l t u r e d a t a o b t a i n e d d u r i n g t h i s study (sample no 9).00324xl0~ 5 F C C w X 0 54 0 09 s 5 02 0 51 X 3 5 2 3 0 3 5 5 3 4 0 0092 0 0044 0. C .34 230.5 99. t o g e t h e r w i t h t h e i r c a l c u l a t e d c o n t r i b u t i o n s t o the t o t a l maximal e r r o r i n CRI and the t o t a l variance. they are the carbon f r a c t i o n ! of s u b s t r a t e and p r o d u c t . C . 10 20 si 68 10 $in C0in 3 6x10~ 10 Sum 35 .036 0.0014 0. l l 8.64 0. .F . A.

e . the o p t i m a l number of a n a l y t i c a l d e t e r m i n a t i o n s n e c e s s a r y and the o p t i m a l e x p e r i m e n t a t i o n range t o keep t h e t o t a l d i s c r e p a n c y i n C o r any o t h e r r e l e v a n t b a l a n c e ( s ) below the maximum a c c e p t a b l e l e v e l . d u r i n g a n a e r o b i c growth w i t h product f o r m a t i o n . was c a l c u l a t e d t o be 1. As can be seen C.01 i . nitrogen recovery index d e f i n e d s i m i l a r l y . CRI was c a l c u l a t e d t o be. however. The l a t t e r p o s s i b i l i t y was found t o be p a r t i c u l a r l y i m p o r t a n t f o r t r a n s i e n t e x p e r i m e n t s . A l s o most of the a n a l y t i c a l d e t e r m i n a t i o n s were performed i n d u p l i c a t e s and gas c o n c e n t r a t i o n measurements were time averaged over l o n g p e r i o d s of time.Using t h e v a l u e s l i s t e d . r e s p e c t i v e l y .95. (see Chapter 8) N .b a t c h experiments. I n b o t h cases a s m a l l f r a c t i o n of t h e i n p u t c o u l d n o t be accounted f o r . i s p r e s e n t e d i n Table I I . 0. The apparent l o s s o f C might be due t o the f o r m a t i o n of u n n o t i c e d by-products or l o s s of C as d i s s o l v e d carbon d i o x i d e o r c a r b o n a t e .g. 6 %. I t i s a l s o apparent. 95 % C-recovery. The r e s u l t s show b e t t e r r e c o v e r i e s than those p r e d i c t e d by the e r r o r a n a l y s e s .and N-balances f o r the continuous c u l t u r e experiment r e p o r t e d i n t h i s s t u d y . A s i m i l a r a n a l y s i s was a l s o c a r r i e d out f o r N-balances. S i m i l a r o b s e r v a t i o n s were made f o r b a t c h and f e d . e . The c o n t r i b u t i o n s of the major sources of e r r o r s t o t h e t o t a l maximal e r r o r are shown d i a g r a m m a t i c a l l y i n F i g . T h i s type of e s t i m a t i o n a n a l y s e s can be v e r y u s e f u l i f c a r r i e d out p r i o r t o e x p e r i m e n t a t i o n as t h i s would a l l o w t h e experimenter to choose the r i g h t equipment.r e c o v e r i e s were good. T h i s was q u i t e expected because most e x p e r i m e n t a l d a t a p o i n t s were r e p e a t e d and averaged. . i . 5. The d i s t r i b u t i o n s of the e r r o r s . The c o r r e s p o n d i n g t o t a l maximal and p r o b a b l e e r r o r s were 11 % and 6 %. 101 % r e c o v e r y . can be q u i t e d i f f e r e n t i n o t h e r s i t u a t i o n s e. Fig.. I n t h i s case NRI.l o s s e s may be due t o v o l a t i l i z a t i o n of ammonia and e x i t v i a the gas phase.and N. to the total maximal error in F i n a l l y an assessment of t h e o r i g i n a l C. The p r e s e n t a n a l y s i s shows t h a t d u r i n g t h e course of t h i s work s u b s t r a t e a n a l y s e s c o n t r i buted most t o the t o t a l e r r o r . . f o r i n s t a n c e t h a t any improvement i n t h e c o n t r o l of a i r f l o w r a t e o r feed i n l e t stream i s h a r d l y necessary.b i c a r b o n a t e i n t h e l i q u i d phase. R e c o v e r i e s i n these cases were n o t as good. 5: Contribution of the major sources C. The r e s u l t s f o r the same s e t of d a t a a r e summarized d i a g r a m m a t i c a l l y i n F i g . 36 . T h e r e f o r e every d i f f e r e n t s i t u a t i o n s h o u l d be s t u d i e d on i t s own m e r i t s . 5.and N-balances. The maximum e r r o r i n v o l v e d was e s t i m a t e d t o be 12 % w h i l e the p r o b a b l e e r r o r .

92. more f l o w s were measured than were m i n i m a l l y needed to c a l c u l a t e the r e m a i n i n g ones. biomass.08 2. as f o u r e q u a t i o n s can be w r i t t e n f o r the f o u r elements i n v o l v e d and t h e r e are s i x f l o w s a l l t o g e t h e r .and N-balances Average r e c o v e r i e s Cbalance no of d a t a 27 23 Nbalance 27 + f o r continuous culture. Such a s u r p l u s of d a t a were not wasted b u t used t o o b t a i n the more o p t i m a l e s t i m a t e s of a l l measured and unknown f l o w ( s ) ( w a t e r ) . a B y 2 D + eH„ 0 2 (20) For t h i s system. Table I I : Assessment of the o r i g i n a l C. 1 98 78 5.90 * i f 3 d a t a p o i n t s are excluded as o u t l i e r s : NRI of 0.59 24 + i f 4 d a t a p o i n t s are e x c l u d e d as o u t l i e r s : CRI of 0.84. F i g s . oxygen. e . 1 * 91.83. D u r i n g the experiments however.2 8 C o n s i d e r i n g the system s t u d i e d i n t h i s work the f o l l o w i n g s t o i c h i o m e t r i c e q u a t i o n can be w r i t t e n i . 6. 0. aC.90. 37 . I n a sense a number of i n e x a c t and sometimes c o n f l i c t i n g i n f o r m a t i o n becomes a s u b s t i t u d e of a few p e r f e c t r e s u l t s and one has to f i l t e r out b e t t e r e s t i m a t e s from the i n c o n s i s t e n c i e s . Here o n l y a b r i e f i l l u s t r a t i o n w i l l be p r o v i d e d as an example s i n c e a l l t h e steady and pseudo-steady s t a t e f l o w s and hence the y i e l d v a l u e s have been c o r r e c t e d by the use of t h i s p o w e r f u l s t a t i s t i c a l t e c h n i q u e . 8.0. G e n e r a l l y the a p p l i c a t i o n of t h i s procedure to c o n t i n u o u s c u l t u r e y i e l d d a t a r e s u l t e d i n l e s s d r a m a t i c c o r r e c t i o n s than those shown i n F i g .77 83.25-32 ^he procedure used i n t h i s work has a l r e a d y been d e s c r i b e d i n the l i t e r a t u r e 3 2 j j_ s i m i l a r to t h a t r e p o r t e d by Madron et £1.H 0 383 o o + b0„ + cNH — C 2 3 0 H 0N + dC 0.5 max) 104.6 2 6.9 ) i s shown. The mathematical background to the problem has been g i v e n by a number of authors.A l t e r n a t i v e l y s y s t e m a t i c e r r o r s might have been i n t r o d u c e d by one or more of the components and/or a n a l y s i s i n v o l v e d i n the e x p e r i m e n t a l system. a n s I n F i g .91 . . RQ was chosen f o r t h i s i l l u s t r a t i o n as i t i s one of the most s e n s i t i v e response v a r i a b l e s and hence s i g n i f i c a n t c o r r e c t i o n s may be i n t r o d u c e d . 2 5 . the knowledge of any two steady s t a t e f l o w s i s s u f f i c i e n t to e s t i m a t e the r e s t . 0.85 a i s the s t a n d a r d e r r o r of the average r e c o v e r y System Overdetermination in Elemental Balancing Overdetermined systems a r i s e i n e x p e r i m e n t a l and c o m p u t a t i o n a l work where more r e s u l t s are generated than would be r e q u i r e d i f p r e c i s i o n were a t t a i n a b l e .29-31 The r e a d e r i s r e f e r r e d t o the o r i g i n a l a r t i c l e s f o r a d e t a i l e d d e s c r i p t i o n .97 (min 90. 0.0 108.6 104. . e . carbon d i o x i d e and sometimes the ammonia f l o w s were measured i . 0. f o r f u l l y a e r o b i c growth w i t h no by-product formation. CRI x l O 97 94 99 15 NRI x l O 97 61 2 a 4.8 108. 6 c o r r e c t i o n s brought about by the a p p l i c a t i o n of t h i s t e c h n i q u e to the raw gas exchange d a t a of a c o n t i n u o u s c u l t u r e run (see Chapter 2. s u b s t r a t e .0 94.

8 2.6 5. . biomass and carbon d i o x i d e f l o w . the average c o r r e c t i o n f o r each f l o w should approach t o zero f o r a l a r g e number of samples.2 (27 p o i n t s ) d e v i a t i o n of the average c o r r e c t i o n F i n a l l y a word of c a u t i o n must be s a i d about the r e l i a b i l i t y of t h i s procedure. 6: Rao and statistically corrected RQ data for a continuous culture run. e . C o r r e c t i o n s i n t r o d u c e d f o r b a t c h and f e d batch ( t r a n s i e n t ) data were l a r g e r than the c o n t i n u o u s c u l t u r e d a t a . I n s p e c t i o n of t h i s Table r e v e a l s t h a t w i t h the e x c e p t i o n of CO2 f l o w .An i m p r e s s i o n of the r e l a t i v e magnitude of the c o r r e c t i o n s can be o b t a i n e d from Table I I I . The method i s founded on the assumption of the randomness of the e r r o r s . 38 . I f through i g n o r a n c e o r o t h e r w i s e the above method i s used f o r c o r r e c t i n g data c o n t a i n i n g l a r g e s y s t e m a t i c e r r o r s . I n an i d e a l case i . a l l f l o w c o r r e c t i o n s show some b i a s but these can be n e g l e c t e d when compared w i t h the standard d e v i a t i o n s o f the concerned measurements.7.4 17.3 and 5% f o r s u b s t r a t e . ammonia. Thus the e x p e r i m e n t e r must always be aware o f the l i m i t a t i o n s and the i m p l i c a t i o n s o f the method used. oxygen. which were a p p r o x i m a t e l y 5. when a l l e r r o r s are random. t h e method may b r i n g about s e r i o u s d e v i a t i o n s from the r e a l i t y . Table I I I : C o r r e c t i o n s a p p l i e d t o raw c o n t i n u o u s c u l t u r e d a t a Average c o r r e c t i o n a p p l i e d to the net f l o w of : % substrate oxygen ammonia biomass carbon d i o x i d e a standard -0 78 1 AÍ -1 23 -0 44 -0 04 a 2 04 2 46 9 22 1 76 1 40 min -5 6 -3 2 -16 5 -4 0 -3 1 max 2. ÂRQ 0. r e s p e c t i v e l y .9• c 1 raw data corrected data Fig.10.2 2. t o a v o i d b i a s e d c o n c l u s i o n s through what he regards as l e g i t i m a t e s t a t i s t i c a l procedures.

(McGraw H i l l .W. 2 ( 8 ) . N o r r i s and D. 9. 5. B i o t e c h n o l . Bioeng.W. 1971)vol.A.Cromie amd H. eds. 3 5 7 ( 1 9 8 0 ) . 1963)p. 1970)vol. E a s t b o u r n e ( 1 9 8 1 ) . P e r r y . R.G. 1968)p. 39 . 8.(McGraw H i l l .15. U n i v e r s i t y o f Wageningen.21. Bioeng.209.21.in Methods i n M i c r o b i o l o g y . New Y o r k . D o e l l e .H. J. H a r r i s o n .J. J. Stouthamer. B a c t e r i o l . 6. London. T r e y b a l .E. A. 4 7 2 ( 1 9 6 8 ) . H e r b e r t and D. New Y o r k . R i b b o n s . J .2. M i n k e v i c h and L . 7. N o r r i s and D. D. H a l l . 10. 2. Mass T r a n s f e r O p e r a t i o n . Chemical E n g i n e e r s Handbook. 4 t h ed.F.2. Ribbons.. Evans.E. I . W. N. i n Methods i n M i c r o b i o l o g y .T. D. 3. d i s c u s s i o n d u r i n g t h e open forum s e s s i o n o f t h e 2 nd European Congress on B i o t e c h n o l o g y . S.(Academic.P. Tempest. B i o t e c h n o l .609. B i o t e c h n o l .E.R. U t k i n a .313. R o e l s . 2 nd ed. I. E a s t b o u r n e ( 1 9 8 1 ) . P. 11.G. 9 6 . London.F. E s e n e r .J. t h e s i s . L e t t .eds.W. B a r f o r d and R.W. paper p r e s e n t e d t o t h e 2 nd European Congress i n B i o t e c h n o l o g y .(1979).H.(1979). 4.R.357(1979). D.W. J. .D.IV NOMENCLATURE c o n c e n t r a t i o n (kg/m3) CO2 mole f r a c t i o n i n t h e i n l e t gas (—) CO2 mole f r a c t i o n i n t h e d r y o u t l e t g a s ( a n a l y s e r ) ( — ) CO2 p r o d u c t i o n r a t e (mole/m3/hr) carbon r e c o v e r y i n d e x (—) v o l u m e t r i c l i q u i d f l o w (m3/m3/hr) r a t e o f s u b s t r a t e a d d i t i o n (kg/hr) Monod s a t u r a t i o n c o n s t a n t (kg/m3) maintenance c o e f f i c i e n t on s u b s t r a t e (kg/kg/hr) t o t a l amount of j i n t h e fermentor (kg) N2 mole f r a c t i o n i n gas streams (—) n i t r o g e n r e c o v e r y index (—) O2 mole f r a c t i o n i n d r y o u t l e t gas ( a n a l y s e r ) (—) O2 mole f r a c t i o n i n t h e i n l e t gas (—) oxygen uptake r a t e (mole/m3/hr) s p e c i f i c r a t e o f s u b s t r a t e consumption (kg/kg/hr) OUR CPR r e s p i r a t o r y q u o t i e n t (—) c u l t u r e volume (irw) c a r b o n weight f r a c t i o n (—) y i e l d o f biomass on s u b s t r a t e (kg/kg) maximal Y s p e c i f i c growth r a t e ( h r ~ l ) c u l t u r e d e n s i t y (kg/m3) standard d e v i a t i o n mass f l o w of the j ' t h substance (kg/m3/hr) v o l u m e t r i c f l o w o f i n and o u t g o i n g d r y gas streams a t STP (m3/m3/hr) s x C C02 in C02 A CPR CRI F F(t) k m Mj N2 NRI O2-A 02~in OUR q r r RQ V w ^sx Y -max u p a $j <> j _ _ s s s Q c g sx s subscripts x s p i F biomass substrate product inlet feed V REFERENCES 1.192. C. .(Academic. Ph. J. Kossen and J.5b. H e r b e r t . de V r i e s and A. S t r a n g e .. Harder. A.. P h i p p s and R.A.

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a u t h o r s r e s t r i c t e d the use and a p p l i c a b i l i t y of t h e i r models by imposing s e v e r e r e s t r i c t i o n s on the b e h a v i o u r of the system under c o n s i d e r a t i o n . n m I n an attempt to f i l l t h i s gap. however.CHAPTER 4 FED-BATCH CULTURE: MODELLING AND THE APPLICATIONS IN STUDY OF MICROBIAL ENERGETICS * F. i t i s shown t h a t f e d .1-12 i o s t c a s e s . Moreover i n many c a s e s . Furthermore. (1981) 41 . S e v e r a l workers a p p l i e d mathematical and modeling techniques f o r d e s c r i b i n g t h i s process.s t e a d y s t a t e e t c . SUMMARY M i c r o b i a l growth i n f e d . these s t u d i e s were c o n f i n e d to t h e o r e t i c a l c o n s i d e r a t i o n s w i t h l i t t l e or no e x p e r i m e n t a l d a t a . A s i m p l e u n s t r u c t u r e d model w i t h a minimum number of assumptions based on Monod k i n e t i c s ' ^ a n d the l i n e a r law of s u b s t r a t e consumption'^ i s a l s o developed as an e x t e n s i o n of the model. These i n c l u d e the assumptions of c o n s t a n t volume. Bioeng'. s u b s t r a t e l i m i t a t i o n . Methods f o r d e t e r m i n i n g the maintenance requirements are shown and e v a l u a t e d . Kossen A.b a t c h c u l t i v a t i o n i s a p o w e r f u l experiment a l t o o l i n the study of m i c r o b i a l k i n e t i c s and e n e r g e t i c s s i m u l t a n e o u s l y . I t i s shown t h a t the f e d . r e p o r t e d e a r l i e r by R o e l s and Kossen.b a t c h mode i s d e s c r i b e d by a simple u n s t r u c t u r e d model.b a t c h c u l t i v a t i o n of the b a c t e r i u m K l e b s i e l l a pneumoniae are p r e s e n t e d i n t h i s paper. except under h i g h l y t r a n s i e n t c o n d i t i o n s . E x t e n s i v e e x p e r i m e n t a l d a t a were c o l l e c t e d and the e n e r g e t i c s of the b a c t e r i u m K l e b s i e l l a pneumoniae i s e v a l u a t e d . hence one i s not a b l e t o check the v a l i d i t y of the models and to get an a c c u r a t e i n s i g h t i n t o the growth phenomena under these c o n d i t i o n s . Esener. INTRODUCTION The t h e o r y of f e d . e x t e n s i v e d a t a and m a t e r i a l b a l a n c e s f o r the f e d . Roels and N.b a t c h c u l t u r i n g t e c h n i q u e can be used as a * Accepted f o r p u b l i c a t i o n i n B i o t e c h n o l . J . q u a s i .b a t c h d a t a are s y s t e m a t i c a l l y s m a l l e r than those r e p o r t e d f o r c o n t i n u o u s c u l t u r e systems. R e s u l t s suggest a decrease i n maintenance demands at low s p e c i f i c growth r a t e s . The model i s found to be i n good agreement w i t h the e x p e r i m e n t a l o b s e r v a t i o n s .'^ The outcomes of model s i m u l a t i o n s are compared w i t h the e x p e r i m e n t a l r e s u l t s . A.b a t c h c u l t i v a t i o n of microorganisms has been s t u d i e d q u i t e e x t e n s i v e l y i n l i t e r a t u r e . W. A. The maintenance c o e f f i c i e n t s determined from f e d .

b a t c h process b a s i c a l l y t h r e e r e l a t i o n s a r e needed. and C the l i m i t i n g s u b s t r a t e c o n c e n t r a t i o n . O f t e n .^ and t h e l i n e a r law of s u b s t r a t e consumption'^ a r e assumed t o be a p p l i c a b l e t o the system under c o n s i d e r a t i o n . T h i s i s p o s s i b l e i f t h e t o t a l mass o f b i o m a t e r i a l and s u b s t r a t e a r e c o n s i d e r e d i n s t e a d of t h e i r c o n c e n t r a t i o n s . however. an e f f e c t i v e method f o r volume b a l a n c i n g d e r i v e d from a mass b a l a n c e i s i n t r o d u c e d . F i n a l l y . i f Monod k i n e t i c s ' .p o w e r f u l t o o l f o r the d e t e r m i n a t i o n of t h e b i o k i n e t i c parameters and y i e l d and maintenance c o e f f i c i e n t s w i t h m i n i m a l e f f o r t compared w i t h continuous c u l t u r e methods.b a t c h experiments w i t h o u t t h e use of t r a n s f o r m a t i o n of v a r i a b l e s . which enabled the e x p e r i m e n t e r t o put the developed model i n j e o p a r d y . These a r e balance e q u a t i o n s f o r biomass and the l i m i t i n g s u b s t r a t e and an e q u a t i o n f o r the volume o f the c u l t u r e . By d e f i n i t i o n s R x = y(s) M x (3) where y i s the s p e c i f i c growth r a t e and i s a f u n c t i o n of the l i m i t i n g s u b s t r a t e c o n c e n t r a t i o n . T h e r e f o r e . s i g n i f i c a n t changes i n c u l t u r e volume occur i n a l e s s c o n t r o l l a b l e way. MODEL To d e s c r i b e a f e d . C biomass c o n c e n t r a t i o n . Balance f o r the l i m i t i n g s u b s t r a t e can be expressed by x d(M )/dt = F ( t ) . one can w r i t e ^ and R ( S ) = V x M s / ( M s + + K s M } ( 4 ) s = (V C * ) ™s * (5) 42 . the b a l a n c e f o r biomass can be g i v e n by d(M )/dt = R (1) x x where R i s t h e r a t e o f biomass p r o d u c t i o n and i s assumed t o be a f u n c t i o n o f the l i m i t i n g s u b s t r a t e c o n c e n t r a t i o n . one o f t h e experiments to determine maintenance c o e f f i c i e n t s a l s o p r o v i d e d severe e x p e r i m e n t a l c o n d i t i o n s ( r a t e o f s u b s t r a t e a d d i t i o n decreased l i n e a r l y ) . I n t r o d u c i n g them. ^ I n such a case two s t a t e v a r i a b l e s w i l l s u f f i c e t o d e s c r i b e the system. due t o e v a p o r a t i o n . I t i s e s p e c i a l l y important t o note t h a t t h e maintenance c o e f f i c i e n t s can be o b t a i n e d d i r e c t l y and a c c u r a t e l y from w e l l designed f e d .R g (2) i s t h e r a t e of where F ( t ) i s the s u b s t r a t e f e e d i n g r a t e t o the fermentor and R s u b s t r a t e consumption i n the fermentor. Here. i n some cases i t i s more convenient t o a v o i d the volume e q u a t i o n . a c i d a n d / a l k a l i a d d i t i o n e t c . F u r t h e r m o r e . they are M and M s = V C s x s x = V C x where V i s the c u l t u r e volume.

r s . one can w r i t e s s equations. m nance c o e f f i c i e n t on s u b s t r a t e . ( l ) and U MM s / (M+K s m a X ( 2 ) .(escape r a t e of o r g a n i c vapors) fraction where 9(t) i s the r a t e of feed s o l u t i o n a d d i t i o n . the c o n t r i b u t i o n of h u m i d i t y .10. and K i s d e f i n e d as s st :le 011 s the m a i n t e - K s = V k s s where k i s the Monod s a t u r a t i o n c o n s t a n t . one obtains (6) )/dt = max x s ) d ( M s )/dt = F(t) .( R / Y x sx + m s M x ) (7) E q u a t i o n s (6) and (7) cannot be s o l v e d a n a l y t i c a l l y except f o r s p e c i a l cases. however. i t i s known t h a t the d e n s i t y of the b r o t h changes l i t t l e d u r i n g the course of f e r m e n t a t i o n . balance. and F ( t ) = (weight of s u b s t r a t e i n feed s o l u t i o n ) x 9 ( t ) .Here Ygx i ' yield s u b s t r a t e c o r r e c t e d f o r maintenance. (dW/dt) = 6 ( t ) + (0™ . I f they can be n e g l e c t e d e q . and a c i d / a l k a l i c o r r e c t i o n s can be assumed or made t o be i n s i g n i f i c a n t under p r a c t i c a l o p e r a t i n g c o n d i t i o n s .(6) and (7) can be expanded and reduced to d(C x )/dt = u max C s C x /( C s + k s )-C/V x (dV/dt) (8) d ( C s )/dt = F ( t ) / V . R s and y ( s ) i n b a l a n c e e q s . one wishes to work i n c o n c e n t r a t i o n s r a t h e r than mass. S i m p l i f i c a t i o n s based on v a r i o u s assumptions can be found i n the l i t e r a t u r e . l o s s of o r g a n i c v a p o r s .C /V s (dV/dt) (9) where R = V r . To a f i r s t a p p r o x i m a t i o n K was taken as the p r o d u c t of Monod s a t u r a t i o n c o n s t a n t and the average volume of the c u l t u r e d u r i n g the f e r m e n t a t i o n . ( l l ) p r o v i d e s the r e q u i r e d volume b a l a n c e : 43 . However. 1-5 .15 I f . g s Substituting for R d ( M„ x x . 1 the f l o w diagram of the mathematical model).0 ° ) + (CC> U t .H 0 ° ) + ( r a t e of a c i d / a l k a l i a d d i t i o n ) 2 U t (10) . T h i s assumption d i d not cause any s i g n i f i c a n t d i f f e r e n c e s i n the s i m u l a t i o n r e s u l t s . Furthermore. In t h i s case one needs to know dV/dt to s o l v e the The r e q u i r e d volume b a l a n c e can b e s t be d e r i v e d from a t o t a l mass C o n s i d e r i n g a mass b a l a n c e f o r the c u l t u r e . d i f f e r e n t i a l s i n eqs. I t must be s t r e s s e d t h a t K was d e f i n e d i n t h i s manner o n l y f o r convenience and t r e a t e d as a parameter i n the model (see f i g .C0° ) Ut + (H 0 2 i n .

(6) and (7) when erratum and hence M » _Xj K s u max jj . dV/dt > 9(t)/p„ D T h i s d e r i v a t i o n shows t h a t the volume change d u r i n g the f e d .b a t c h experiment y 44 .(13) can be regarded as i n s i g n i f i c a n t when compared w i t h the v a l u e of 9 ( t ) . when { 32/(44 RQ) . T h i s causes d i s c r e p a n c i e s i n m a t e r i a l b a l a n c e s .h a n d s i d e of eq.(11) f o r the a e r o b i c case becomes p„(dV/dt) = 9 ( t ) + 44 V r B c T h e r e f o r e . b i o k i n e t i c parameters and y i e l d and maintenance c o e f f i c i e n t s can be determined from a s i n g l e f e d .1 } / 1000 (13) RQ > 32/44 . For l a b o r a t o r y s c a l e f e r m e n t a t i o n s the t o t a l volume of the samples taken can a l s o be a s i g n i f i c a n t f r a c t i o n of the i n i t i a l c u l t u r e volume.b a t c h f e r m e n t a t i o n i s not o n l y dependent on the v o l u m e t r i c f e e d i n g r a t e but a l s o on the m e t a b o l i c s t a t e of the c u l t u r e as d e s c r i b e d by RQ. In most a e r o b i c f e r m e n t a t i o n s the second term on the r i g h t . r oxygen uptake. however.p„(dV/dt) = 6 ( t ) + (32 r .b a t c h experiment. and when dV/dt < 9 ( t ) / p RQ < 32/44 .44 r ) V / B o c 1000 (11) Here pg i s the d e n s i t y of the b r o t h . S i n c e the r e s p i r a t o r y q u o t i e n t i s d e f i n e d by Q c RQ = r c / r o (12) Eq. DETERMINATION OF BIOKINETIC PARAMETERS AND AND MAINTENANCE COEFFICIENTS YIELD I f designed c o r r e c t l y . m a t e r i a l exchange v i a the gas phase can be more s i g n i f i c a n t and hence may have to be a l l o w e d f o r . A method f o r c o r r e c t i n g m a t e r i a l b a l a n c e s i s p r o v i d e d i n the appendix. I n a n a e r o b i c f e r m e n t a t i o n s . and r carbon d i o x i d e p r o d u c t i o n r a t e per u n i t volume of the c u l t u r e . C o n s i d e r i n g eqs.• ]i » max M x m a x d(M )/dt x (14) can be T h e r e f o r e d u r i n g the e x p o n e n t i a l p a r t of the f e d .

x m a x s m a x s s x x s x s s s x 0 0 x c c x x V a r i o u s y i e l d c o e f f i c i e n t s can a l s o be o b t a i n e d from f e d . ' ^ G l y c e r o l was used as the l i m i t i n g s u b s t r a t e . the experimenter must be aware of the f a c t t h a t a f e d .o b t a i n e d from a p l o t of l n ( M ) v s . A t t e n t i o n was p a i d t o o b t a i n an a c t i v e l y growing inoculum and i n most experiments the inoculum used was v e r y s m a l l ( i n i t i a l c o n c e n t r a t i o n l e s s than 0.''' By t h i s method v a r i o u s maintenance c o e f f i c i e n t s can be o b t a i n e d d i r e c t l y . I n i t i a l s u b s t r a t e c o n c e n t r a t i o n was a d j u s t e d a c c o r d i n g to the d e s i r e d f i n a l biomass c o n c e n t r a t i o n .2 ymmembrane filter i n t o a s t e r i l e fermentor. Cultivation Methods aerogenes .b a t c h c u l t u r e s d u r i n g the d e c r e a s i n g growth r a t e p e r i o d .b a t c h c u l t u r e i s never i n a t r u e steady s t a t e and s t a t e of the c u l t u r e at any i n s t a n t i s i n f l u e n c e d by i t s h i s t o r y . What i s important here i s t h a t v a r i a t i o n s i n y i e l d due to changes i n growth r a t e can a l s o be o b s e r v e d . The pH was c o n t r o l l e d at 6. time. e x p e r i m e n t a l l y . i f the c u l t u r e i s a l l o w e d to grow w i t h c o n s t a n t feed of l i m i t i n g s u b s t r a t e . when d i f f e r e n t i a t e d and made equal t o y / 2 y i e l d s the time where c = k . At t h i s s t a g e . S i m i l a r l y . I f t h i s new v a l u e i s l e s s than V / 2 > at one stage d u r i n g t h i s t r a n s i t i o n p e r i o d . F o r i n s t a n c e .was Growth medium was prepared a c c o r d i n g t o the f o r m u l a t i o n g i v e n by Evans et a l . w i t h a c a r e f u l l y planned experiment v a r i a t i o n i n v a r i o u s y i e l d s from y = 0 to p = U can be determined. The medium was s t e r i l i z e d by membrane f i l t r a t i o n through a 0.b a t c h d a t a . During the t r a n s i t i o n from e x p o n e n t i a l to s u b s t r a t e l i m i t e d phase. ' " T h i s assumption was checked and j u s t i f i e d l a t e r on.05 % of the f i n a l biomass c o n c e n t r a t i o n s ) to a v o i d the p o s s i b i l i t y of an unbalanced growth of the organism. s i n c e the s u b s t r a t e removal p r o c e s s has a much s m a l l e r time c o n s t a n t than t h a t f o r biomass p r o d u c t i o n . When R = dM /dt = 0. A i r f l o w to 45 . the Monod s a t u r a t i o n c o n s t a n t . I n s e r t i n g z e r o s f o r R and dM /dt i n e q . i s i n the d e t e r m i n a t i o n of maintenance c o e f f i c i e n t s . m = R /M and m = R /M . w h i c h . the assumption of c o n s t a n t maintenance (independent of the s p e c i f i c growth r a t e ) are not n e c e s s a r y . m can now be g i v e n by m = F ( t ) / M . i t has been observed i n our l a b o r a t o r y t h a t RNA l e v e l s l a g s i g n i f i c a n t l y b e h i n d the growth r a t e i n f e d . dM /dt may be assumed to be equal to z e r o . U s u a l l y the biomass curve has a smooth t r a n s i t i o n i n t h i s p e r i o d and a p o l y nomial can be f i t t e d to dry weight d a t a . m a x MATERIALS AND METHODS Organism Klebsiella pneumoniae NCTC 418. however. y w i l l assume a v a l u e of y /2. The major advantage of f e d b a t c h c u l t u r m g t e c h n i q u e . The s u b s t r a t e c o n c e n t r a t i o n at t h i s moment i s by d e f i n i t i o n equal to the v a l u e of k .8 ± 0. f o r m e r l y known as Klebsiella used throughout t h i s study. the t o t a l biomass M w i l l reach a l e v e l at which the feed i n p u t can o n l y s a t i s f y the maintenance requirements. ( 7 ) .5 K. T h e r e f o r e the b e h a v i o u r of the c u l t u r e may not f o l l o w the e x a c t t r e n d shown i n steady s t a t e continuous c u l t u r e s at the same growth r a t e s .'9 Equipment A l l experiments were c a r r i e d out i n an 11 x 10"^ m^ working volume fermentor m a i n t a i n e d at 308 ± 0. T r a n s f o r m a t i o n of v a r i a b l e s . e x t r a p o l a t i o n of d a t a . i .05. however. There i s a l r e a d y some evidence f o r s i g n i f i c a n t l a g s i n the c e l l metabolism. T h e r e f o r e . the s p e c i f i c growth r a t e w i l l decrease from i t s maximum v a l u e to i t s new v a l u e as determined by the r a t e of s u b s t r a t e a d d i t i o n .y. e . and above a l l .

l i n e heat exchanger manufactured i n the workshop of t h i s department.07 0 29. have been c o r r e c t e d s t a t i s t i c a l l y by a computer program which c a l c u l a t e d t h e most p r o b a b l e v a l u e s of m a t e r i a l f l o w s i n t h e system as d e s c r i b e d e a r l i e r .71 6. W i t h i n o c u l a t i o n the feed pump was a l s o s t a r t e d . and d r i e d t o c o n s t a n t weight a t 378 K.44 14. Experiment FB 830 FB 21 1 C 50. ^ ' The method used f o r t h e computer program i s s i m i l a r t o t h a t r e p o r t e d r e c e n t l y by Madron and others. The s u b s t r a t e f e e d i n g r a t e was kept c o n s t a n t 3 46 . Formula 1. Computation of the Results A l l v a l u e s used and r e p o r t e d i n t h i s paper a r e expressed on a s h . Feed f l o w t o the fermentor was r e a l i z e d by a p r e c i s i o n p e r i s t a l t i c (LKB 2120) pump.23°0.2 um pore d i a m e t e r f i l t e r ( S a r t o r i u s 11370).44 1.42 N N CH In p e r c e n t a s h . A l l samples were c o o l e d d u r i n g sampling down t o about 278-280 K by an o n . A m o d i f i e d r e c t a n g u l a r method was used f o r i n t e g r a t i o n . Dry w e i g h t s were determined by the method of de V r i e s and Stouthamer. T y p i c a l r e s i d e n c e time i n the heat exchanger was about 5-10 seconds.148270). The d e t e c t i o n l i m i t of t h e assay was e s t i m a t e d to be 10 mg g l y c e r o l / 1 . For v a r i a b l e feed r a t e experiments the feed f l o w was decreased a c c o r d i n g t o a predetermined l i n e a r f u n c t i o n by an a n a l o g g r a d i e n t programmer (Joens type PG) coupled t o the p r e c i s i o n pump. Table I : Avarage e l e m e n t a l c o m p o s i t i o n and f o r m u l a of K.22-26 ^-Q s i m u l a t i o n s were c a r r i e d out w i t h an IBM 360/65 computer system u s i n g the c o n t i n u o u s systems modeling program (CSMP). Gas phase oxygen and carbon d i o x i d e c o n c e n t r a t i o n s were determined by a t w i n channel paramagnetic oxygen a n a l y z e r ( T a y l o r Servomex OA 184) and an i n f r a r e d carbon d i o x i d e a n a l y z e r (Beckman 864). Y i e l d v a l u e s p l o t t e d .24°0.20 Biomass was c o l l e c t e d on a 0.average ash c o n t e n t was 8 %.f r e e b a s i s . S p e c i a l a t t e n t i o n was p a i d f o r the a c c u r a t e d e t e r m i n a t i o n of gas f l o w s and c o n c e n t r a t i o n s .f r e e weight . the i n i t i a l s u b s t r a t e was about 3. Ash c o n t e n t o f biomass was determined s e p a r a t e l y and the c o m p o s i t i o n was e x p r e s s e d on ash f r e e b a s i s (Table I ) . Analytical Methods A 99 % pure reagent q u a l i t y g l y c e r o l was used and assayed e n z y m a t i c a l l y ( B o e h r i n g e r UV method.5 kg/m .35 CH pneumoniae.b a t c h e x p e r i ment.75 H 6.64 28. A l l f l o w s were c o r r e c t e d f o r h u m i d i t y and v o l u m e t r i c changes. Here. The feed b o t t l e was p l a c e d on a b a l a n c e and a continuous read-out of t h e decrease of i t s weight was o b t a i n e d .22 50.60 0.p r o d u c t was p r e s e n t a t a l e v e l t h a t c o u l d be s i g n i f i c a n t . The average ash c o n t e n t o f d r y biomass was about 8 %.the f e r m e n t o r was c o n t r o l l e d by a thermal mass f l o w meter(Brooks 5811) a t about 0. r e s p e c t i v e l y .84 dry N 13.77 kg d r y a i r / h . washed w i t h d i s t i l l e d w a t e r .62 0. C o r r e c t i o n s f o r s a m p l i n g was i n c o r p o r a t e d i n t o the program as d e s c r i b e d i n the Appendix. Chromatographic a n a l y s i s of the c u l t u r e s u p e r n a t a n t r e v e a l e d t h a t no b y . E l e m e n t a l c o m p o s i t i o n of biomass was determined by a computer-coupled element a n a l y z e r ( P e r k i n Elmer 240). RESULTS AND DISCUSSION Experiment FB 830 was chosen as r e p r e s e n t a t i v e of a c l a s s i c a l f e d .

34.807 x l O ^ kg g l y c e r o l / h r . x s x Fig.S. t o g e t h e r w i t h the s i m u l a t i o n r e s u l t s c a r r i e d out p r i o r t o experimentat i o n w i t h parameters e s t i m a t e d from our p r e v i o u s b a t c h experiments and l i t e r a t u r e . V .10''5 kg. (M) substrate.t M =M„„ + rdM S s rzi [Tmâx m S • . M and s u b s t r a t e .0 kg. biomass and substrate profiles : (9) biomass. F i t ) = 8. the t o t a l biomass.1. 2: Experiment FB 830.10~ kg/kg/hr 3 s xo 2 2 so s When the OUR (oxygen uptake r a t e ) and CPR (carbon d i o x i d e p r o d u c t i o n r a t e ) p l o t s ( F i g . T h i s f i n d i n g i s not v e r y s u r p r i s i n g as 47 . 2 7 Here. I n F i g u r e 2. M .0. m = 7 .62 . i t w i l l be seen t h a t the concerned d e v i a t i o n s i n these v a r i a b l e s a r e much l a r g e r . throughout the experiment a t 8. ( ) model simulation.X M M t r dt M +K = Q 1 1x = x o / M + _ i t fdM ^ d x — • Fig. The most s i g n i f i c a n t d e v i a t i o n occurs d u r i n g t h e t r a n s i t i o n from the e x p o n e n t i a l t o s u b s t r a t e l i m i t e d phase where t h e growth i s expected to be h i g h l y unbalanced. Even f o r t h i s p e r i o d t h e maximum d e v i a t i o n observed between the e x p e r i m e n t a l and s i m u l a t e d M p r o f i l e s remain w i t h i n the l i m i t of a c c e p t a b l e v a r i a t i o n o f o p e r a t i o n f o r most i n d u s t r i a l a p p l i c a t i o n s . 1: Simplified block diagram of the mathematical - model used for simulations. 3) a r e examined. Parameters: Wiax . X dM _ x Mma«.4 . good agreement can be observed w i t h the experiment and the model developed.807. .05 hr~l. 10~ kg/hr. however. M .54 kg/kg.1CT kg. M = 7. K = 1. a r e p l o t t e d as f u n c t i o n s of t h e f e r m e n t a t i o n time.

w i t h d e c r e a s i n g growth r a t e . the fermentation. These f i n d i n g s a r e i n good agreement w i t h t h e g e n e r a l l y accepted p a t t e r n of change throughout the f e r m e n t a t i o n . x x Fig. These p l o t s make i t c l e a r t h a t d u r i n g t h i s h i g h l y t r a n s i e n t phase the u n s t r u c t u r e d model f a i l s t o h o l d .54 mole/kg/hr.these v a r i a b l e s respond t o changes much f a s t e r than M . e . (—) model simulation.. m = 1. 2. Yield of I n F i g . 10~ kg/mole. m = 1.12 . s i n c e M i s an i n t e g r a t e d q u a n t i t y . The o t h e r f e d . other parameters same as shewn in Fig. t h e medium was i n o c u l a t e d and the c u l t u r e was a l l o w e d t o grow i n the b a t c h mode. 3: Experiment FB 830. A decrease of y i e l d v a l u e s w i t h t h e c u l t i v a t i o n time. i s observed. (O) oxygen.b a t c h experiment FB 211. 4: Experiment FB 830: change of yields during biomass on (0) substrate.32 mole/kg/nr. (0) carbon dioxide production rate. i . B e f o r e the t e r m i n a t i o n of b a t c h growth the feed pump was switched on. The r a t e of feed a d d i t i o n was decreased l i n e a r l y v i a a programmer. gas exchange rate profiles: (9) oxygen uptake rate. X 36 15 l c r 2 2 0 c Fig. Here.• kg/mole. Y^E° = 54. the y i e l d s on s u b s t r a t e and oxygen a r e p l o t t e d . was performed under u n c o n v e n t i o n a l c o n d i t i o n s . Parameters ^ox . r e p o r t e d f u l l y i n t h i s paper. The behaviour of t h e c u l t u r e d u r i n g the 48 . 4.

The i n t e r s e c t i o n of these must have a p h y s i o l o g i c a l l y i m p o r t a n t meaning. F i t ) varied according to F i t ) (85 .0.). z In F i g .b a t c h mode i s shown i n F i g . I n f a c t the p r e s e n t e d model should not be a p p l i e d to t h i s decay phase. the above e q u a t i o n p r o v i d e s the t h e o r e t i c a l r e l a t i o n between the RQ and Y . These f i n d i n g s suggest a change i n maintenance requirements around t = 865 min. The observed RQ d u r i n g the f e d . t o g e t h e r w i t h the outcome of the s i m u l a t i o n of the p r e s e n t e d model. 6: Experiment FB 211. 6. 5.0. ( ) model simulation of biomass p r o f i l e Parameters same as given in Fig.508. time).( a / a ) Y ^ } b s / {(Y /4). up t o about t = 865 min.064. The RQ p r o f i l e observed can i n f a c t be f i t t e d w i t h two s t r a i g h t l i n e s ( F i g . 2.(. biomass and RQ profiles during substrate limited phase: (*) biomass.391. the use of a k i n e t i c e x p r e s s i o n which a l l o w s f o r maintenance requirements through c e l l a u t o l y s i s i s p r e f e r a b l e .06.b a t c h growth mode i n c r e a s e d almost linearly. i f the model i s t o be v a l i d . Ys 4. the incoming s u b s t r a t e c o u l d not even supply the maintenance requirements of the c u l t u r e and the c e l l s s t a r t u s i n g up t h e i r i n t e r n a l s t o r a g e polymers and a u t o l y s e . T h e r e a f t e r . Based on t h i s c r i t e r i a . = 4. T h i s p l o t a l s o s e r v e s as a check of c o n s i s t e n c y of the raw and processed d a t a . Here. The r e s u l t s of the s i m u l a t i o n can be s a i d to be i n f a i r l y good agreement w i t h the e x p e r i m e n t a l d a t a . RQ i s g i v e n by RQ = {1 .^° Using t h e i r n o t a t i o n and s i m p l i f y i n g f o r the no-product case. s <Vb'Vs «x» = s )Y ° 5 ) F o r t h i s experiment. The raw d a t a shows some s c a t t e r about the t h e o r e t i c a l l i n e . S t a t i s t i c a l l y c o r r e c t e d d a t a of course g i v e s a b e t t e r f i t . 10~ kg/hr.67 and Yt. y i e l d and RQ v a l u e s o b t a i n e d from raw and s t a t i s t i c a l l y c o r r e c t e d d a t a are p l o t t e d t o g e t h e r w i t h the t h e o r e t i c a l r e l a t i o n determined as d e s c r i b e d by E r i c k s o n et al. ^ 7 Fig. 5. P o s s i b l y up to t h i s p o i n t the s u b s t r a t e a d d i t i o n r a t e was enough f o r s a t i s f y i n g b i o s y n t h e s i s and maintenance demands. A s i g n i f i c a n t s y s t e m a t i c d e v i a t i o n i s observed t h e r e a f t e r . p r o c e s s e d d a t a can be assumed to have no s i g n i f i c a n t s y s t e m a t i c discrepancies. sx 49 . = 0. ($) RQ. H e r b e r t ' s model based on the concept of 'endogeneous metabolism' can be used f o r t h i s s i t u a t i o n .f e d . u s i n g the v a l u e s of aj. a . where t > 49 2 min. The r a t e of i n c r e a s e i n RQ changed suddenly a f t e r the biomass peak was reached.

50 . of c o u r s e . whereas a f t e r the peak. The computer program used was developed i n the Department of Chemical E n g i n e e e r i n g .) f o r experiment FB 830 a r e t o be reexamined and compared w i t h the s i m u l a t i o n r e s u l t s . oxygen uptake (OUR) and carbon d i o x i d e p r o d u c t i o n (CPR) r a t e d a t a are p r e s e n t e d i n T a b l e I I t o g e t h e r w i t h the 95 % c o n f i d e n c e l e v e l s .29. Consistency check for data obtained from experiment FB 211: ( ) theoretical relation between RQ and Y . one can see t h a t d e v i a t i o n s between t h e p r e d i c t e d and e x p e r i m e n t a l l y observed v a l u e s get h i g h e r d u r i n g the e x p o n e n t i a l p a r t of the experiment. 3. Estimation of the Maximum Specific Growth Rate From t h e e x p e r i m e n t a l d a t a p r e s e n t e d . assuming c o n s t a n t y i e l d s on oxygen and carbon dioxide i t i s also possible to c a l c u l a t e U based on t h e r a t e s of oxygen uptake and carbon d i o x i d e p r o d u c t i o n . Moreover. 6. t h e d e v i a t i o n s a r e p o s i t i v e .Fig. Lower y v a l u e s from OUR and CPR d a t a imply e i t h e r m m a x 1) y i e l d s on oxygen and carbon d i o x i d e a r e not c o n s t a n t but change d u r i n g the course o f t h e f e r m e n t a t i o n .31. The e x p e r i m e n t a l curves a r e found t o be somewhat t i l t e d and b e f o r e the peak they show n e g a t i v e d e v i a t i o n s from the s i m u l a t e d c u r v e s . D e l f t U n i v e r s i t y of Technology and based on Marquart's method. A n o n . raD experimental data. As can be seen c l e a r l y from t h i s t a b l e t h e r e a r e s i g n i f i c a n t s y s t e m a t i c d i f f e r e n c e s i n the measured v a l u e s of y a x depending on the v a r i a b l e upon which t h e d e t e r m i n a t i o n was based. (*) statistically corrected experimental sx f •) data. however s l i g h t . an attempt was made t o determine t h e maximum s p e c i f i c growth r a t e of t h i s organism. between the s i m u l a t e d and e x p e r i m e n t a l peak t i m e s .30 m a x m a x The maximum s p e c i f i c growth r a t e s f o r d i f f e r e n t experiments as determined from d r y w e i g h t . (15) in text. A s i g n i f i c a n t hold-up of carbon d i o x i d e has r e c e n t l y been reported. o r 2) some h o l d i n g mechanism d e l a y s t h e o u t p u t ( r e l e a s e ) of oxygen and carbon d i o x i d e from t h e system a t the r a t e w i t h which they a r e p r o c e s s e d i n t h e system. t h e r e a r e a l s o d i f f e r e n c e s .32 A c o m b i n a t i o n of the above two mechanisms i s . Furthermore. I f t h e p l o t s of OUR and CPR ( F i g . eq.l i n e a r r e g r e s s i o n t e c h n i q u e was used f o r M c a l c u l a t i o n s . D u r i n g the e x p o n e n t i a l p a r t of the experiments the growth r a t e may be assumed t o be e q u a l t o i t s maximum as shown p r e v i o u s l y . a l s o p o s s i b l e .

Table I I : Maximum s p e c i f i c growth r a t e of K. pneumoniae different variables. ^max Experiment FB 830 FB 21 1 B AV
b a a

c a l c u l a t e d from

c a l c u l a t e d from d a t a (hr" OUR CPR 0.710(0 705-0 716) 0.745(0 661-0 829)

1 .064(1 .020-1.108) 0.811(0.809-0 813) 0.848(0.788-0 908) 1.070(1 .061-1.076)

F i g u r e s i n parentheses are the 95 % c o n f i d e n c e ^B AV - average v a l u e o b t a i n e d from three batch

limits. experiments.

T h i s c l e a r l y i m p l i e s the presence of a b i o l o g i c a l and/or p h y s i c a l d e l a y mechanism. However, one cannot accept t h i s h y p o t h e s i s r i g h t away s i n c e the amount of these h y p o t h e t i c a l l y delayed q u a n t i t i e s are not found equal t o the ones r e l e a s e d a f t e r the peak, when i n t e g r a t e d . One can, t h e r e f o r e , assume a combination of v a r i o u s phenomena t a k i n g p l a c e . The c e l l s seem to be g e t t i n g more e f f i c i e n t throughout the f e r m e n t a t i o n . One might, of c o u r s e , propose a h y p o t h e s i s of the p r o d u c t i o n of an i n t e r m e d i a t e e n e r g y - r i c h compound d u r i n g the i n i t i a l p a r t of the e x p o n e n t i a l phase, which i s f u r t h e r m e t a b o l i z e d , r e q u i r i n g l e s s oxygen, e t c . f o r c e l l b i o s y n t h e s i s . The p o s s i b i l i t y of the e x i s t e n c e of such a phenomenon has a l s o been s t u d i e d but c o u l d not be accepted. Based on these c o n s i d e r a t i o n s , the maximum s p e c i f i c growth r a t e can b e s t be determined from d r y weight d a t a . However, one should not f o r g e t t h a t biomass c o n c e n t r a t i o n (dry weight) i s an i n t e g r a t e d q u a n t i t y and i s a f f e c t e d by the h i s t o r y o f i t s p r o d u c t i o n and the course of the f e r m e n t a t i o n . T h e r e f o r e i t i s much l e s s s e n s i t i v e to changes d u r i n g the f e r m e n t a t i o n compared w i t h oxygen uptake and carbon d i o x i d e p r o d u c t i o n r a t e s .

Determination

of

k

s

From F i g u r e 2 , the growth r a t e can be c a l c u l a t e d based on d r y weight d a t a . When growth r a t e y i s p l o t t e d a g a i n s t e x p e r i m e n t a l l y measured s u b s t r a t e c o n c e n t r a t i o n , a v a l u e of 1.43 kg g l y c e r o l / m has been c a l c u l a t e d f o r k , i . e . g l y c e r o l c o n c e n t r a t i o n a t y = P / 2 . I t has a l r e a d y been r e p o r t e d t h a t k v a l u e s measured i n b a t c h c u l t u r e s may be ten times h i g h e r than those measured i n continuous c u l t u r e s . 3 3 Even then t o check the s i g n i f i c a n c e of t h i s determined parameter, s i m u l a t i o n s were c a r r i e d out f o r the same e x p e r i m e n t a l c o n d i t i o n s w i t h the e x p e r i m e n t a l l y determined v a l u e of k . The r e s u l t s are shown i n F i g u r e 7. From t h i s f i g u r e , i t becomes obvious t h a t what has been measured as k has no b i o l o g i c a l and/or q u a n t i t a t i v e s i g n i f i c a n c e . T h e r e f o r e , i t can be concluded t h a t the Monod r e l a t i o n by which k i s d e f i n e d i s not v a l i d f o r t h i s h i g h l y t r a n s i e n t p e r i o d d u r i n g which the observed y passes through the v a l u e of y / 2 . Thus, f e d - b a t c h c u l t u r e d a t a , when based on a simple u n s t r u c t u r e d Monod type model, do not p r o v i d e a m e a n i n g f u l v a l u e of k . Yamane and H i r a n o have a l s o expressed t h e i r doubts about the a p p l i c a b i l i t y of Monod model i n f e d - b a t c h systems.' The v a l u e of k they determined was 28 times h i g h e r than t h a t o b t a i n e d from continuous c u l t u r e d a t a .
3 s m a x s s s s m a x s s

Determination

of Maintenance

Coefficients

From the r e s u l t s of experiment FB 211 mined by the f o l l o w i n g methods.

c o e f f i c i e n t s of maintenance were d e t e r -

A) Biomass peak. As p r e v i o u s l y e x p l a i n e d , when the amount of biomass reaches i t s maximum,i.e. when the growth r a t e i s z e r o , a l l s u b s t r a t e i n p u t must be going to 51

M 60 50 40

x

»10-

(kg)

k

s

Ckg/m )
3

30 20
10

® ® © ®

0.1 0.5 1.0 '5

0

0

300

600

Fig.

7: Experiment Parameters

FB 830, ks evaluation (*) biomass; other than k same as in Fig. 2.
s

(

)

simulation.

maintenance metabolism. T h e r e f o r e , m can be o b t a i n e d from a p o i n t d e t e r m i n a t i o n o f the t o t a l biomass i n t h e fermentor and the r a t e of s u b s t r a t e i n p u t a t t h a t i n s t a n t . I f the gas exchange d a t a a r e a l s o a v a i l a b l e m and m can a l s o be o b t a i n e d s i m i l a r l y .
s 0 c

B) RQ. I f a l l the incoming s u b s t r a t e i s used f o r maintenance p r o c e s s e s , w i t h no p r o d u c t s b e i n g produced, the f o l l o w i n g s t o i c h i o m e t r i c e q u a t i o n h o l d s

+

7/2 0

2

3C 0

2

+

4H 0
2

(16)

From t h i s i t f o l l o w s t h a t a t t r u e maintenance the RQ o f t h e system must be equal t o 0.86. Hence from F i g . 5. the time a t which RQ becomes 0.86 can be determined and t h e v a l u e s of m , m and m can be c a l c u l a t e d from the c o r r e s ponding d a t a . Furthermore, i f the RQ d a t a i s f i t t e d by two s t r a i g h t l i n e s , i t w i l l be seen t h a t the i n t e r s e c t i o n l i e s a p p r o x i m a t e l y on the RQ = 0.86 l i n e (Fig. 5.).
s 0 c

The above mentioned p o i n t d e t e r m i n a t i o n methods have some drawbacks f o r l a b o r a t o r y systems. F i r s t , the f e d - b a t c h system i s always i n a t r a n s i e n t s t a t e and i t i s d i f f i c u l t t o r e a l i z e a s t a t e a t which the growth r a t e i s e x a c t l y equal t o z e r o . As the growth r a t e decreases i t takes l o n g e r t o r e a c h a steady s t a t e . A second problem i s caused by f r e q u e n t s a m p l i n g of the c u l t u r e . I t must be r e a l i z e d t h a t , whenever a sample i s taken from the f e r m e n t o r , the growth r a t e would i n c r e a s e and some o f t h e incoming s u b s t r a t e w i l l be used f o r b i o s y n t h e s i s . With the RQ method another major d i s a d v a n t a g e i s i n t r o d u c e d due t o the p o s s i b l e carbon d i o x i d e hold-up i n b r o t h . D i f f e r e n c e s can e x i s t between the measured and t r u e RQ v a l u e s . C) Balancing. To overcome t h e d i f f i c u l t i e s concerned w i t h the above mentioned methods, i t i s b e s t to c o n s t r u c t a m a t e r i a l b a l a n c e f o r t h e r e l a t i v e l y steady s t a t e , observed as the biomass peak i n F i g u r e 5. By t h i s method the s u b s t r a t e spent f o r b i o s y s t h e s i s to compensate f o r the biomass l o s t i n samples can a l s o be a l l o w e d f o r . T h i s i s done i n t h e f o l l o w i n g way. I f d u r i n g a time i n t e r v a l of A t , the i n c r e a s e i n M i s AM , where AM << M , m can be c a l c u l a t e d by
x X X x s

52

m

=

{ ( s u b s t r a t e f e d d u r i n g At) - (AM /Y

)} / {At(M + AM /2)}

(17)

For an a c c u r a t e d e t e r m i n a t i o n o f m the second term i n the nominator s h o u l d be s m a l l compared w i t h the f i r s t . I t i s important t o note here t h a t Y ™ ^ i s assumed t o be c o n s t a n t and known. T h i s method has been a p p l i e d t o the e x p e r i m e n t a l d a t a o f FB 211 f o r the p e r i o d o f 825 < time(min) < 1105.
s x

Table I I I : Maintenance v a l u e s c a l c u l a t e d from the e x p e r i m e n t a l Experiment Method m .10 kg/kg/hr
s 2

data. m mole/kg/hr
c

° , mole/kg/hr 1.37 1.55 1.00 1.13 1.14 1.38 1.25 1.34 (1.19) (2.86) (1.35) (1.20) (2.08) (2.58) (1.72) (1.24)

ra

FB 830 FB 0 1 6

b

C) B a l a n c i n g B) RQ = 0.86 O Balancing A) Biomass peak B) RQ = 0.86 C) B a l a n c i n g

FB 21 1

SC RP Rd SC RP RP R SC

a

C

3.13 7.52 3.56 3.15 5.47 6.78 4.53 3.53

1.10 1.33 0.99 0.96 1.23 1.18 1.12 1.16

(1.02) (2.45) (1.16) (1.03) (2.43) (2.21) (1.48) (1.06)

References 34 35 36
a

(values reported i n l i t e r a t u r e ) 9.21 7.55 7.68 2.67 (3.50) 3.46 (2.87) 2.54 (2.92)

SC- c a l c u l a t e d from s t a t i s t i c a l l y c o r r e c t e d d a t a . ^Experiment FB 016 was a r e p l i c a t e o f FB 211 and n o t r e p o r t e d i n t h i s paper. RP- c a l c u l a t e d from raw p o i n t d a t a . R- c a l c u l a t e d from raw d a t a . The v a l u e s i n parentheses a r e c a l c u l a t e d from s t o i c h i o m e t r y (eq.(16)) and the e x p e r i m e n t a l l y determined m v a l u e s .
C s

In Table I I I , m , m and m v a l u e s determined by the above l i s t e d methods a r e p r e s e n t e d and compared w i t h those r e p o r t e d i n l i t e r a t u r e as c i t e d by H e i j n e n and Roels.34 When the d a t a p r e s e n t e d i n Table I I I a r e examined f o r c o n s i s t e n c y , e.g., i f the e x p e r i m e n t a l l y determined m and m v a l u e s a r e compared w i t h those c a l c u l a t e d from m and s t o i c h i o m e t r y ( i n parentheses) i t w i l l be c l e a r l y noted t h a t the methods A and B y i e l d e d i n c o n s i s t e n t e s t i m a t e s o f maintenance c o e f f i c i e n t s . Method C, however, gave r e s u l t s which show good c o n s i s t e n c y . The d i f f e r e n c e between the maintenance v a l u e s o b t a i n e d from raw and c o r r e c t e d d a t a can stem from t h e f a c t t h a t carbon r e c o v e r i e s o f 95, 93 and 93 % were o b t a i n e d f o r experiments FB 830, FB 016 and FB 211, r e s p e c t i v e l y . The m i s s i n g carbon was p r o b a b l y r e t a i n e d i n b r o t h as c o 2 b e i n g trapped i n c e l l s and/or p a r t i c i p a t i n g i n t h e c a r b o n a t e - b i c a r b o n a t e b u f f e r system. The s t a t i s t i c a l l y c o r r e c t e d d a t a y i e l d e d the most c o n s i s t e n t and t h e r e f o r e r e l i a b l e e s t i m a t e s o f maintenance coefficients.
s 0 c 0 c s

The m v a l u e s c a l c u l a t e d and found t o be more c o n s i s t e n t a r e much s m a l l e r than those r e p o r t e d i n l i t e r a t u r e . 3 4 1 3 5 , 3 6 j p i g g ^ d a t a f o r FB 211 i s g i v e n t o g e t h e r w i t h the r e s u l t s of s i m u l a t i o n s c a r r i e d out w i t h d i f f e r e n t m v a l u e s . As can be seen, up t o t = 865 min (sample 7 ) , t h e model w i t h an m v a l u e taken from l i t e r a t u r e f i t s t h e e x p e r i m e n t a l d a t a w e l l ; t h e r e a f t e r ,
g n u r e j s s

53

r e s p e c t i v e l y . Whether the a c t u a l r e d u c t i o n of the apparent maintenance requirements at low growth r a t e s i s a consequence of a d a p t a t i o n or not has s t i l l to be s t u d i e d . with various m s values. a s i m i l a r behaviour has a l s o been observed i n experiment FB 016.524 kg/kg and 35. These were c a l c u l a t e d from from our p r e v i o u s b a t c h d a t a . p o s s i b l y t r i g g e r e d by a c o n t r o l mechanism which i s capable of i d e n t i f y i n g a p o t e n t i a l s t a r v a t i o n . simulation of the experiment Other parameters same as before.34 mole/kg/hr. T h i s may be the reason f o r the s i g n i f i c a n t d i f f e r e n c e s observed i n maintenance v a l u e s (see Table I I I ) g x s - The e x p e r i m e n t a l m v a l u e c a l c u l a t e d by b a l a n c i n g around the r e l a t i v e l y Mj^ peak a l s o f a i l s to g i v e a good f i t .53 x 1 0 kg/kg/hr and 1. no s i g n i f i c a n t changes i n the e l e m e n t a l c o m p o s i t i o n or c a r b o h y d r a t e content of the biomass c o u l d be d e t e c t e d .Consumption of i n t e r n a l c a r b o h y d r a t e s t o r a g e m a t e r i a l may p r o v i d e an e x p l a n a t i o n f o r t h i s s h i f t . I n view of t h i s s t a t e of the a c c u m u l a t i n g i n f o r m a t i o n .05 h r ' a f t e r a hazardous e x t r a p o l a t i o n .^ has a l r e a d y s p e c u l a t e d about the p o s s i b i l i t y of maintenance b e i n g a f u n c t i o n of the growth r a t e . (see F i g u r e 8) s steady Fig. The new v a l u e s of Y and Y used f o r these balances were 0.a s m a l l e r m v a l u e g i v e s a b e t t e r f i t .57 x 10 kg/mole. To i n v e s t i g a t e the b e h a v i o u r of the system f u r t h e r . m a t e r i a l b a l a n c e s were c o n s t r u c t e d f o r e i g h t time i n t e r v a l s and m and m were c a l c u l a t e d as d e s c r i b e d p r e v i o u s l y by method C. P i p y n and V e r s t r a e t e ^ S have r e p o r t e d lower m v a l u e s f o r the a c t i v a t e d sludge growing at v e r y low growth r a t e s compared w i t h those grown at h i g h e r growth r a t e s . The i n t e r e s t i n g t h i n g to note i s t h a t m v a l u e s r e p o r t e d i n l i t e r a t u r e were a l l o b t a i n e d from continuous c u l t u r e data c o l l e c t e d at U > 0. m and m v a l u e s c a l c u l a t e d f o r these time i n t e r v a l s are p l o t t e d i n F i g u r e 9. A l t h o u g h t h e r e i s c o n s i d e r a b l e s c a t t e r . assuming t r u e v a l u e s of m and m as 3. I f samples 6 and 7 can be regarded as o u t l i e r s . R e c e n t l y . The r e s u l t s r e p o r t e d i n t h i s work add to the newly a c c u m u l a t i n g s p e c u l a t i o n s of reduced maintenance requirements at v e r y low growth r a t e s . A s t a t i s t i c a l t e s t r e j e c t s the h y p o t h e s i s of zero s l o p e at 95 % l e v e l f o r both p l o t s . However. s 54 . s Q m a x m a x J s . a smooth curve f i t s the M d a t a . r e s p e c t i v e l y . the t r e n d s i n these p l o t s suggest d e c r e a s i n g maintenance r a t e s . T h i s almost abrupt change suggests a s h i f t i n c e l l metabolism. U n f o r t u n a t e l y . 8: Experiment FB 211. van Verseveld39 has mentioned t h i s p o s s i b i l i t y and s p e c u l a t e d t h a t maintenance i s most p r o b a b l y a l i n e a r f u n c t i o n of the s p e c i f i c growth r a t e .2 0 g 0 N e i j s s e l . The c e l l s seem to become more e f f i c i e n t .

f o r a p a r t i c u l a r a p p l i c a t i o n the model demands a mass t r a n s f e r c a p a c i t y h i g h e r than a c t u a l l y r e q u i r e d . (0) oxygen. .b a t c h c u l t u r e s were found t o be l e s s than those r e p o r t e d i n the l i t e r a t u r e which were o b t a i n e d from c o n t i nuous c u l t u r e d a t a . i . CONCLUSIONS 1) The f e d . 55 . e . 9: Experiment FB 211: Maintenance coefficients as calculated by balancing for different time intervals during the fed-batch growth phase. 4) Maintenance requirements determined i n f e d . F u r t h e r r e s e a r c h i n t h i s area i s needed. s i m u l t a n e o u s l y . However. 3) The model p r e s e n t e d can s a f e l y be used f o r i n d u s t r i a l d e s i g n purposes as i t p r e d i c t s c o n s e r v a t i v e e s t i m a t e s f o r oxygen uptake and carbon d i o x i d e p r o d u c t i o n r a t e s .b a t c h c u l t i v a t i o n t e c h n i q u e i s a u s e f u l t o o l f o r t h e d e t e r m i n a t i o n of b i o k i n e t i c parameters and t h e study of b i o e n e r g e t i c s . Fed-batch c u l t u r i n g t e c h n i q u e l e n d s i t s e l f f o r t h i s purpose q u i t e s u c c e s s f u l l y . 2) The u n s t r u c t u r e d model p r e s e n t e d d e s c r i b e s t h e b e h a v i o u r o f the system f a i r l y w e l l d u r i n g t h e e x p o n e n t i a l phase and t h e pseudo-steady s t a t e . Coefficients of maintenance on (O) substrate.Fig. A s t r u c t u r e d model s h o u l d be t r i e d f o r a b e t t e r d e s c r i p t i o n of t h e system d u r i n g t h i s period. as t h e r e i s a c c u m u l a t i n g s p e c u l a t i v e evidence about maintenance requirements b e i n g lower a t low growth r a t e s . f u r t h e r i n v e s t i g a t i o n s i n t o the growth phenomenon a t low growth r a t e s under a c c u r a t e l y c o n t r o l l e d e n v i r o n m e n t a l c o n d i t i o n s a r e n e c e s s a r y . the model f a i l s t o h o l d d u r i n g the t r a n s i t i o n p e r i o d . T h i s f i n d i n g may have i m p o r t a n t p h y s i o l o g i c a l and economical i m p l i c a t i o n s . 5) F u r t h e r i n v e s t i g a t i o n s i n t o t h e growth b e h a v i o u r of slow growing c u l t u r e s are n e c e s s a r y .

i t i s t h e r e f o r e b e t t e r t o s u b s t r a c t the c o r r e c t i o n curves o b t a i n e d from t h e curves p r e d i c t e d by model s i m u l a t i o n w i t h no sampling. Method 1: Say a t t = t i . can amount up t o 15 % o f the i n i t i a l volume. C ] .V (I). c . However. I—I S I S I i These c u m u l a t i v e s can be p l o t t e d as a f u n c t i o n o f f e r m e n t a t i o n time.C (I) s s x r EH M (t) = M (t). T h i s method of c o r r e c t i o n i s p a r t i c u l a r l y u s e f u l f o r m a t e r i a l b a l a n c i n g . a sample o f volume V ] and c o m p o s i t i o n C j and C ] i s taken.V (I).C (I) s s S S x x S X Fig. 56 . the "no s a m p l i n g " case can be o b t a i n e d as a f i r s t a p p r o x i m a t i o n . C ] and V ] . one o f t h e s e t s o f d a t a has t o be c o r r e c t e d . T h e r e f o r e at any time t= t . F o r comparison purposes. such a c o r r e c t i o n w i l l d i s t o r t the k i n e t i c s shown by the e x p e r i m e n t a l d a t a .APPENDIX Volume changes d u r i n g a f e d . changes i n c u l t u r e volume due t o feed a d d i t i o n . samples taken e v a p o r a t i o n . I f the curves o b t a i n e d i n t h i s manner a r e added/subtracted t o the e x p e r i m e n t a l d a t a . 10: Correction procedure for sampling in the simulation program. the t o t a l amount o f biomass and s u b s t r a t e removed from the fermentor a r e V i .V (I). I n the f o l l o w i n g two procedures f o r c o r r e c t i o n a r e d e s c r i b e d . r e s p e c t i v e l y .C (U M ( t ) = M ( t ) . Read t U).b a t c h experiment can o f t e n be s i g n i f i c a n t . A t l a b o r a t o r y s c a l e . A t t = t j . When the r e s u l t s o f such experiments a r e t o be compared w i t h t h e outcomes o f t h e mathematical s i m u l a t i o n s . t h e t o t a l amount of biomass taken out of the fermentor t i l l then can be g i v e n by s s x s s s x S S g n n Similarly for substrate n v . a c i d / a l k a l i a d d i t i o n e t c . t h e r e f o r e .

6. T. 7 8 4 ( 1 9 7 8 ) S. J . The s i m u l a t i o n s p r e s e n t e d i n t h i s study have been e x e c u t e d i n t h i s manner. E. Chem. B i o t e c h n o l . Ferment. 5 0 8 ( 1 9 7 8 ) R. B i o t e c h n o l . Dunn. J . J .55. Yamane. B i o t e c h n o l . Ferment. Chem. Yamane. B i o t e c h n o l .380(1977) T. Dunn. Technol. F. B i o t e c h n o l . Yamane and K. K e l l e r and I . 8.111(1979) 57 . A i b a . T h i s would y i e l d the most a c c u r a t e r e s u l t s s i n c e the i n t e g r a t i o n s are c a r r i e d out w i t h the c o r r e c t e d v a l u e s a f t e r each s a m p l i n g .21. Kume. Sada and T. Technol.55. H i r a n o . Technol. 3. E. . NOMENCLATURE C C F(t) k K m m m M M r r r r R R R R RQ V W Y Y Y x s s s s D c x s s D c x x s 0 c s x o x c x biomass c o n c e n t r a t i o n ( d r y w e i g h t ) (kg/m^) l i m i t i n g substrate concentration(kg/m3) r a t e of s u b s t r a t e i n p u t (kg/hr) Monod s a t u r a t i o n c o n s t a n t (kg/m3) p r o d u c t of k and c u l t u r e volume (kg) maintenance c o e f f i c i e n t on s u b s t r a t e (kg/kg/hr) maintenance c o e f f i c i e n t on oxygen (mole/kg/hr) maintenance c o e f f i c i e n t on carbon d i o x i d e (mole/kg/hr) t o t a l mass of biomass i n the f e r m e n t o r (kg) t o t a l mass o f s u b s t r a t e i n the f e r m e n t o r (kg) r a t e of s u b s t r a t e consumption (kg/m3/hr) r a t e of oxygen uptake (mole/m^/hr) r a t e of carbon d i o x i d e p r o d u c t i o n (mole/m3/hr) r a t e o f biomass p r o d u c t i o n (kg/m3/hr) t o t a l biomass p r o d u c t i o n i n the f e r m e n t o r (kg/hr) t o t a l s u b s t r a t e consumption i n the f e r m e n t o r (kg/hr) t o t a l oxygen consumption i n the f e r m e n t o r (mole/hr) t o t a l carbon d i o x i d e p r o d u c t i o n i n the f e r m e n t o r (mole/hr) r e s p i r a t o r y quotient (dimensionless) volume of the c u l t u r e (m3) t o t a l mass o f the c u l t u r e (kg) biomass y i e l d on s u b s t r a t e (kg/kg) biomass y i e l d on oxygen (kg/mole) biomass y i e l d on carbon d i o x i d e (kg/mole) s OT Y Y \i Pmax 8(t) a O p D s D s B ax maximal y i e l d of biomass on i (kg/kg) (kg/mole) degree of r e d u c t i o n of b i o m a s s ( e q u i v a v a i l a b l e e l e c t r o n s / g . K e l l e r and I . J . J . Bioeng.15. 1 0 0 1 ( 1 9 7 6 ) T. Nakamoto. B i o t e c h n o l . H i r a n o . A p p l ..55.Method 2: A more a c c u r a t e approach i s to a l l o w f o r sampling w i t h i n the s i m u l a t i o n program. atom) degree of r e d u c t i o n o f s u b s t r a t e (equiv a v a i l a b l e e l e c t r o n s / g .. 5. N i s h i z e w a . S.J. J . Sada and T. 2 8 . Chem. . 7.. Y o s h i d a . 24. T. .415(1974). atom) s p e c i f i c growth r a t e ( h r ' ) maximum s p e c i f i c growth r a t e ( h r ' ) r a t e of feed s o l u t i o n i n p u t (kg/hr) carbon weight f r a c t i o n i n biomass ( d i m e n s i o n l e s s ) carbon weight f r a c t i o n i n s u b s t r a t e ( d i m e n s i o n l e s s ) d e n s i t y of the b r o t h (kg/m3) - REFERENCES 1. 2. J . S. T.156(1977) T.587 (1977) 9. 2 8 . A s i m p l i f i e d schemat i c f l o w diagram o f t h i s c o r r e c t i o n procedure i s shown i n F i g u r e 10. Takamatsu. Bioeng.257(1973) R. A p p l . Takamatsu. A p p l . 1 8 . 4. J . B i o e n g . . Yamane and S. Yamane and S. Nagai and Y.. P i r t .. Ferment.

T h e s i s .482(1977) 24.357(1979) 17. I n d . Dev. R o e l s .23. K e l l e r . B i o t e c h n o l . Shioya and R. S. t h e s i s . Ann.42.246(1973) 26. N. Creagen. I n t . Dunn. P h .0077-8923/03260127(1979) 12.F. i n Methods i n M i c r o b i l o g y . London. Bacteriol.Ribbons. . v o l . H.E. C o l l e c t .E. E s e n e r . B i o t e c h n o l . 6 . J .J. M a r r . " i n Progress i n I n d u s t r i a l M i c r o b i o l o g y .A.53(1973) 15. Ann. 1 9 7 9 58 . P r o c . H e r b e r t .609(1979) 33. A p p l .22. B i o t e c h n o l . Ph.6. Dev. 19. Murthy."On the m o d e l l i n g o f m i c r o b i a l m e t a b o l i s m . Paris.23. J. Czech. I..T. A.347(1974) 27.M.1883(1978) 39. Madron. H a l l . L. I n d . Eng. de Kok and J. . H e r b e r t and D. Res. D..10. Amsterdam.H. D.(Academic p r e s s . N. Vanecek. B i o t e c h n o l . H. Stouthamer. Res. E d . J.1942) 14. 2 7 .. Bioeng. Proc.K. Symp. J.G. Vanecek. Bettenhaussen. H a l l .I.. E x p .M. I . B i o p h y s ..96. Kossen. M a r q u a r t . Congr.S..38(1958) 36. E r o s h i n . N o r r i s and W. Gen.A.2. M i n k e v i c h and L .425(1977) 13.20. P i r t . A. Math. Bioeng.W. Evans.1979) 30. F i t z p a t r i c k . Himmelblau. C e l l . U t k i n a . F.313.. Sei.102. Ind.T. J.W. A.22. A. P i p y n and W. 1978) 16.. D. Ph. Acad.431(1963) 31. . B i o t e c h n o l .Y. t h e s i s . N e i j s s e l .A. de V r i e s and A. Roels and N.1478(1979) 25. Acad. Chen and C. S e i .. P. Microbiol. I. A. Lim.20.21. D . F. D. B a r f o r d and R.K. J .536(1963) 18. 102. Bioeng.. Chem. S. W. Chem..G.C. Acad. P .W. B i o t e c h n o l .F. V.H. O. Chem.276(1976) 20.J. B i o t e c h n o l .1097 (1980) 22. E r i c k s o n . Bioeng. N i l s o n and D. D.K. Res. van V e r s e v e l d . H...S. H e i j n e n and J. 19. E. J ..1805(1977) 23.Y. Continuous C u l t u r e .1595 (1978) 29. Stouthamer. 2 8 5 ( 1 9 7 7 ) 37.13. Murthy.. 1970)vol. Madron.A. Bioeng.H... Ann.M.G. R o e l s .472(1968) 21. J .21. Madron and V.W. 1976 38. B a r f o r d and R. H e r b e r t . AIChE J. I . M.D. B.301.W.1977 34.H. ( E l s e v i e r . R o e l s and N.S. S.A.p. Bioeng. Amsterdam U n i v e r s i t y .Y.P.2. Amsterdam F r e e U n i v e r s i t y . N. C l a r k .0077-8223/0326-119(1979) 11. B i o e n g . Sei.W. B u l l . Tempest. Eds. J .739(1981) 35. Eng.J...12. Soc.J. Commun. U.1979(1980) 32. Symp. J . B i o t e c h n o l . Veverka and V. P r o c e s s A n a l y s i s by S t a t i s t i c a l Methods(Wiley-Interscience New York. Acta. A. Bioeng.D. J .R.J.J. F. V e r s t r a e t e . Kossen. C. Soc. M i c r o b i o l .G. Recherches sur l a c r o i s s a n c e des c u l t u r e s b a c t e r i e n n e s (Hermann. Monod. ( 1 9 7 5 ) 28.21. Stouthamer and C. B i o t e c h n o l . Bioeng. M i n k e v i c h and V. B i o c h i m .

mono c u l t u r e .F. KEYWORDS S a l i n i t y . Canada(June 1980) 59 . r e v e r s e osmosis. S a l i n i t y was shown t o be an i m p o r t a n t parameter i n f l u e n c i n g t h e k i n e t i c s and e n e r g e t i c s o f b i o l o g i c a l systems. D i s c h a r g e o f waste water i n t o seas and s a l t l a k e s i s a l s o common. k i n e t i c s . These p r o c e s s e s i n c l u d e d i s t i l l a t i o n o f sea w a t e r . Kossen and J. I t was shown t h a t the response o f b o t h mono and mixed c u l t u r e s t o i n c r e a s e d s a l i n i t y f o l l o w e d a s i m i l a r p a t t e r n b u t the magnitudes o f t h e e f f e c t s d i f f e r e d s i g n i f i c a n t l y . water s o f t e n i n g by i o n exchange. e l e c t r o d i a l y s i s . Roels ABSTRACT The e f f e c t s o f s a l i n i t y on t h e k i n e t i c s and e n e r g e t i c s o f mono c u l t u r e s were s t u d i e d i n b a t c h . p i c k l i n g . Some o f the wastes from these p r o c e s s e s and/or combinat i o n o f them w i t h domestic waste waters p r e s e n t a s p e c i a l case f o r t h e convent i o n a l b i o l o g i c a l waste water t r e a t m e n t f a c i l i t i e s . S a l i n e wastes a r e a l s o g e n e r a t e d and has t o be t r e a t e d on board o f marine v e s s e l s and o f f .W. c a n n i n g . b i o l o g i c a l water t r e a t m e n t . mixed c u l t u r e . The major l o a d o f s a l i n e waste w a t e r . waste INTRODUCTION There a r e s e v e r a l p r o c e s s e s t h a t produce b r i n e s . e n e r g e t i c s .CHAPTER 5 GROWTH OF MONO AND MIXED CULTURES IN SALINE ENVIRONMENT * A. however. a r i s e from the u s e o f s e a water f o r domestic purposes and as a c a r r i e r o f domestic and i n d u s t r i a l waste a t c o a s t a l l o c a t i o n s w i t h l i m i t e d s u p p l y o f f r e s h water.A. h e l d a t W a t e r l o o . * Paper p r e s e n t e d t o the Second I n t e r n a t i o n a l Symposium on Waste Treatment and U t i l i z a t i o n .s h o r e i n s t a l l a t i o n s .A. p r o d u c t i o n o f s a l t s . Study o f m i c r o b i a l growth i n s a l i n e environment i s t h e r e f o r e o f p r a c t i c a l importance. N. cheese and f i s h meal m a n u f a c t u r i n g e t c . E s e n e r . R e s u l t s were compared w i t h those r e p o r t e d f o r a c t i v a t e d s l u d g e .

From these s t u d i e s i t can be concluded t h a t a d d i t i o n of s a l t s .1957. p a r t i c u l a r l y f o r the f o r m u l a t i o n of s u i t a b l e growth media f o r e x a c t i n g organisms and mammalian c e l l (Ingram. a l l s u f f e r e d from the same phenomena . Kincannon. i n most cases N a C l . Ludzack and Noran. W o d z i n s k i and F r a z i e r . Endoh and K o b a y a s h i . In waste water f i e l d the e f f e c t of NaCl on a c t i v a t e d sludge and t r i c k l i n g f i l t r a t i o n p r o c e s s e s have been s t u d i e d ( B u r n e t t . been adopted i n t h i s s t u d y . however. Kincannon and Gaudy. P i r t and Tackeray. E l e m e n t a l c o m p o s i t i o n of biomass was determined by an element a n a l y s e r . The r e s u l t s of most of these s t u d i e s c o n f i r m t h a t h i g h c o n c e n t r a t i o n s and/or shocks of NaCl have adverse e f f e c t s on the performance of the treatment p l a n t s .8. 1960) . 1979b. Oxygen c o n t e n t was found by d i f f e r e n c e . P r a c t i c a l i m p l i c a t i o n s of these f i n d i n g s a r e d i s c u s s e d . 1979a). R e s u l t s were t r e a t e d and c o r r e c t e d by a s t a t i s t i c a l procedure as r e p o r t e d elsewhere (de Kok and R o e l s . No carbon c o n t a i n i n g b y . 1965. a b a c t e r i u m commonly p r e s e n t i n s o i l and waste w a t e r s . 1966. Small shocks or g r a d u a l i n c r e a s e s i n s a l t l e v e l s had l i t t l e e f f e c t on the system performance and u s u a l l y the system r e t a i n e d i t s o r i g i n a l a c t i v i t y a f t e r some a d a p t a t i o n time. 1939. S c o t t . W i t h t h i s approach the pure response of one of the p o s s i b l e s p e c i e s p r e s e n t i n a c t i v a t e d sludge communities was determined. Inoculum was p r o v i d e d by an e x p o n e n t i a l l y growing c u l t u r e i n NaCl f r e e medium. 1968. Gaudy and Gaudy.g.Imai. I t i s shown t h a t a l t h o u g h the responses of mono and mixed c u l t u r e s f o l l o w e d a s i m i l a r p a t t e r n . MATERIALS AND METHODS Klebsiella pneumoniae (aevogenes) NCTC 418 was c u l t i v a t e d a e r o b i c a l l y i n s i m p l e s a l t s medium w i t h g l y c e r o l b e i n g the o n l y carbon s o u r c e . Ingram (1939)concluded t h a t c a t i o n of the s a l t s was of importance i n d e t e r m i n i n g the r e s p i r a t i o n r a t e . Moreover. S p e c i a l a t t e n t i o n was p a i d f o r the a c c u r a t e d e t e r m i n a t i o n of gas f l o w s and c o n c e n t r a t i o n s . The r e s u l t s o b t a i n e d were then compared w i t h those r e p o r t e d f o r a c t i v a t e d s l u d g e growing under s i m i l a r c o n d i t i o n s ( I m a i .The e f f e c t s of the presence of i n o r g a n i c s a l t s on m i c r o b i a l growth have f i r s t been s t u d i e d by m i c r o b i o l o g i s t s . K e s s i c k and Manchen. 1966. I t was g e n e r a l l y agreed t h a t maximum a d a p t a t i o n was o b t a i n e d when the s a l i n i t y of the medium was r a i s e d g r a d u a l l y . but t h i s f i n d i n g was not c o n f i r m e d by o t h e r s .p r o d u c t c o u l d be d e t e c t e d a t s i g n i f i c a n t q u a n t i t i e s . r e s p e c t i v e l y . 1 9 8 0 ) . 1964. was s t u d i e d i n b a t c h c u l t u r e s . Kincannon and Gaudy (1968) have observed a tremendous i n c r e a s e i n biomass y i e l d (75 %) i n the presence of 8 kg/m3 NaCl but were not a b l e to i d e n t i f y whether t h i s i n c r e a s e was due t o a change i n the e f f i c i e n c y of m i c r o b i a l m e t a b o l i s m or s e l e c t i o n of s a l t t o l e r a n t s p e c i e s . 1974. t h e r e i s l i t t l e d a t a on the k i n e t i c s of m i c r o b i a l growth in s a l i n e waters. Temperat u r e and pH were s e t and c o n t r o l l e d a t 308 K and 6. Endoh and Kobayashi 1979a. Tokuz and E c k e n f e l d e r . 1979). t h e r e f o r e . 60 . Ash c o n t e n t was determined s e p a r a t e l y . s e l e c t i o n and predominance of s p e c i e s p r e v e n t e d the e x p e r i m e n t e r to draw pure causeresponse r e l a t i o n s h i p s from t h e i r experiments e. P r e d i c t i o n of the amount of biomass which i s to be expected due to breakdown of o r g a n i c m a t t e r and the c o r r e s p o n d i n g oxygen demand i s of g r e a t importance i n the d e s i g n and o p e r a t i o n of treatment p l a n t s . A more fundamental approach has. 1976. the magnitude of responses changed c o n s i d e r a b l y . Amount of biomass c o n t a i n i n g 12 grams of carbon was d e f i n e d as 1 mole of b i o mass. 1953. Lawton and Eggert. a d a p t a t i o n . i n c r e a s e d the r e s p i r a t i o n r a t e up to a s p e c i f i c s a l t c o n c e n t r a t i o n . t h e r e a f t e r a decrease was observed. S t u d i e s w i t h mixed c u l t u r e s . F i r s t the growth b e h a v i o u r of Klebsiella pneumoniae (aevogenes). T h e r e f o r e v a r i o u s y i e l d s were e v a l u a t e d as f u n c t i o n s of NaCl c o n c e n t r a t i o n i n the growth environment. A l l experiments were performed i n a 11 x 10~3 m3 w o r k i n g volume fermentor i n b a t c h mode.

t h e r e f o r e been assumed t o be o n l y a f u n c t i o n o f the NaCl c o n c e n t r a t i o n . t h e i r r e s u l t s were c o n v e r t e d by us i n o r d e r to o b t a i n NaCl dependence. i t must be noted t h a t the f i r s t two d a t a p o i n t s have o v e r lapping confidence i n t e r v a l s . Formula C H l . m a x m a x m a x Table Is Average e l e m e n t a l c o m p o s i t i o n of K. y was determined from the d r y weight d a t a by n o n l i n e a r r e g r e s s i o n . The observed l a g times have. t h e r e f o r e . R e s u l t s o f a c t i v a t e d s l u d g e . the n o r m a l i z e d U . 16 pneumoniae. These a u t h o r s s t u d i e d the response of a c t i v a t e d s l u d g e t o i n c r e a s i n g c o n c e n t r a t i o n s of NaCl i n a r e s p i r o m e t e r . were o b t a i n e d from a p u b l i c a t i o n by I m a i . I n F i g . The experiements were performed i n 0 . I n F i g . 2. 1.5 0 0 10 20 30 »[NaCl] 40 Ckg/m ) 3 Fig. Endoh and Kobayashi (1979a).AO kg/m3 NaCl range which a l s o covered the sea water case (approx. The inoculum c o n c e n t r a t i o n was not d e t e r mined f o r each experiment but was of the same o r d e r o f magnitude. I m a i . NaCl concentration.RESULTS E x p e r i m e n t a l r e s u l t s of mono c u l t u r e s were o b t a i n e d i n t h i s l a b o r a t o r y . Endoh and 61 .71 N 13. 6 2 0 . Endoh and K o b a y a s h i observed a maximum y i n the low NaCl range. Such a maximum was n o t observed i n t h i s study. The same assumption was a l s o adopted by I m a i .68 H 6. c 49. 33 kg/m3) . 2 3 °0.63%. However. 1: Maximum specific growth rate vs. l a g time as d e f i n e d by Dean and Hinshelwood (1966) i s shown as a f u n c t i o n of the NaCl c o n c e n t r a t i o n . i s shown as a f u n c t i o n of NaCl c o n c e n t r a t i o n i n the growth medium t o g e t h e r w i t h the 95 % c o n f i d e n c e l e v e l s (the s u p e r s c r i p t 0 denotes the v a l u e o b t a i n e d i n the NaCl f r e e medium.45 0 30. the maximum s p e c i f i c growth r a t e as d e f i n e d by Monod k i n e t i c s . named as mixed c u l t u r e h e r e a f t e r . Average ash c o n t e n t 8. Mmax Hmax 0.46 N % ash f r e e d r y w e i g h t . They used c h l o r i d e c o n c e n t r a t i o n as a parameter.

mixecC*^ / / • ^ • I l l i i i ) 10 20 30 40 [NaCl](kg/m ) 3 Fig. 3. NaCl concentration. 2: Observed lag time vs. q . Y . NaCl concentration. The presence of a c o r r e s p o n d i n g minimum i n the r e s p i r a t o r y q u o t i e n t . S l i g h t l y d i s t i n c t optima i n y i e l d v a l u e s can be observed a t about 5 kg/m3 NaCl. oxygen Y and carbon d i o x i d e . No s i g n i f i c a n t change i n the e l e m e n t a l c o m p o s i t i o n of biomass c o u l d be d e t e c t e d i n the e x p e r i m e n t a l range and an average f o r m u l a as shown i n Table I . (1979a) was n o t observed i n t h i s study. RQ. 1 1— 1 1 - L a gT i m e Chr) - mono / - A / Imai et. 4. Q 0 Fig. 3: Oxygen uptake rate vs. A l l y i e l d s a r e expressed as mole/mole. was used for y i e l d calculations. are p r e s e n t e d i n F i g . ensures the e x i s t e n c e s x o x c x 62 . Y i e l d s of biomass on s u b s t r a t e .Kobayashi (1979a). Y . A maximum q r e p o r t e d by Imai e t a l . i s p l o t t e d i n F i g .al. R e s p i r a t o r y a c t i v i t y as the t o t a l oxygen uptake.

DISCUSSION Comparison of the Behaviour of Mono and Mixed C u l t u r e s From d a t a p r e s e n t e d i n F i g s . 5: Respiratory quotient as affected by NaCl concentration.Mono c u l t u r e s . y of the mono c u l t u r e decreased by about 10 t i m e s . P o s s i b l y above t h i s l e v e l s i g n i f i c a n t damage i s done to the c e l l membrane by the osmotic p r e s s u r e of i t s e x t e r i o r . Respiratory Quotient |RQ Fig.3) .mixed c u l t u r e s i n m a x 63 . however. Lag time and r e s p i r a t i o n r a t e d a t a a l s o showed a s i m i l a r p a t t e r n . 5. are shown to be much more s e n s i t i v e t o i n c r e a s i n g NaCl c o n c e n t r a t i o n s .of an o p t i m a l y i e l d range ( F i g . m Fig. l . The f u n c t i o n s a d v e r s e l y a f f e c t e d by the presence of NaCl showed a smooth l o s s of a c t i v i t y i n mixed c u l t u r e s .). i t can be concluded t h a t b o t h mono and mixed c u l t u r e s show s i m i l a r b e h a v i o u r i n s a l i n e environment.3. throughout the e x p e r i m e n t a l range. I t t h e r e f o r e becomes e v i d e n t t h a t the o p t i m a l y i e l d i s not a c h i e v e d a t the h i g h e s t growth r a t e but a t about 0. seemed to have a c r i t i c a l s a l i n i t y range above which the c u l t u r e a c t i v i t y slowed down d r a s t i c a l l y (15-20 kg/m. From 0 to 40 kg/m3 N a C l . Mono c u l t u r e s . The r e l a t i v e success of . whereas t h a t of mixed c u l t u r e by 5. 4: Influence of NaCl concentrations on biomass yields.9u where the NaCl c o n c e n t r a t i o n i s 5 kg/m. however. 2 and 3.

A l t h o u g h d e t a i l e d b i o c h e m i c a l and p h y s i o l o g i c a l reasons f o r t h i s decrease a r e s t i l l n o t c l e a r . efficiency of the growth process as a function of NaCl T\ i s g i v e n by Roels n = Y (1980) a s : (. i n t h i s work w i t h mono c u l t u r e s o n l y a s l i g h t i n c r e a s e i n the y i e l d was observed (< 5 %) a t the same NaCl range. i t i s b e l i e v e d t h a t the c e l l s i n s a l i n e environment have t o do e x t r a work t o m a i n t a i n c o n c e n t r a t i o n g r a d i e n t s and v i a b i l i t y . 6 . These authors have r e p o r t e d an i n c r e a s e i n biomass y i e l d (about 75 %) a t 8 kg/m3.c a l l e d "maintenance energy" r e q u i r e m e n t s . I t i s important t o note t h a t t h e knowledge o f n a l l o w s the straightforward estimation o f the oxygen demand by : g x s x Y ox = ( 4 / Y x } • n /(i . T h i s means t h a t i n c r e a s e i n y i e l d observed by Kincannon and Gaudy (1968) was m a i n l y due t o changes i n the predominance o f s p e c i e s . a term which i n c l u d e s energy 64 . n d e s c r i b e s the e f f i c i e n c y o f t h e growth process by c o n s i d e r i n g i t s r e v e r s i b i l i t y . 6: Thermodynamic concentration. R o e l s . Fig.s a l i n e environment i s most p o s s i b l y due t o a d a p t a t i o n and predominance o f s a l t t o l e r a n t s p e c i e s .) g x /Y< where Y i s t h e biomass y i e l d on s u b s t r a t e (mole/mole) and Y j i s t h e maximum p o s s i b l e v a l u e o f Y c o n s i s t e n t w i t h the second law o f thermodynamics. Based on these o b s e r v a t i o n i t i s u n l i k e l y t h a t any o t h e r s p e c i e s c o u l d i n c r e a s e i t s y i e l d a t such amounts. n . 1978. However. T h i s p o s s i b i l i t y has a l r e a d y been p o i n t e d a t by Kincannon and Gaudy (1968). the thermodynamic e f f i c i e n c y o f the growth p r o c e s s . by t h e s o . i s shown as a f u n c t i o n o f the s a l t c o n c e n t r a t i o n i n the medium. 1980) x From the d a t a p r e s e n t e d i t i s c l e a r t h a t b i o s y n t h e s i s gets l e s s e f f i c i e n t a t h i g h NaCl l e v e l s . T h i s type o f energy e x p e n d i t u r e i s c u r r e n t l y accounted f o r .n ) (2) where y i s the degree o f r e d u c t i o n o f biomass ( E r i c k s o n . M i n k e v i c h and E r o s h i n . Energetic Considerations I n F i g .

7 s x a x s 65 . i t must be noted t h a t the v a l u e of m o b t a i n e d i n t h i s study is i n f l u e n c e d most by data c o l l e c t e d at low growth r a t e s i .e x p e n d i t u r e f o r o t h e r f u n c t i o n s too. S i g n i f i c a n t i n c r e a s e s i n m r e q u i r e m e n t s i n h i g h l y s a l i n e media has a l r e a d y been r e p o r t e d i n l i t e r a t u r e (Stouthamer and B e t t e n h a u s s e n . The maintenance v a l u e determined i s about 50 % h i g h e r than t h a t r e p o r t e d i n l i t e r a t u r e by H e r b e r t (1958) f o r the same organism i n s a l t f r e e medium. Rewriting equation r S / r sx + m s / y (4) where r and r are the net f l o w s of s u b s t r a t e and biomass t o and from the system. o n l y a rough a n a l y s i s w i l l be r e p o r t e d here. 1973.97 . i s g i v e n by: s (mole/mole q m a x s = V I Y m 3 X sx + m s s (3) where Y i s the maximal y i e l d on s u b s t r a t e (mole/mole) and m i s the maintenance c o e f f i c i e n t (mole/mole/hr). Here. 7: Effect of NaCl level (3) as : = 1 / Y X m a x on the distribution of substrate carbon. r e s p e c t i v e l y ( 95 % c o n f i d e n c e l i m i t s i n p a r a n t h e s i s ) . e . However.2. both of these parameters may be i n f l u e n c e d by s a l i n i t y . T h e r e f o r e .040) f o r Y and m . I f they are assumed c o n s t a n t s . I f Y ™ i s c o n s t a n t . thus the p o r t i o n of s u b s t r a t e used f o r non-growth a s s o c i a t e d f u n c t i o n s get h i g h e r . s Assuming the l i n e a r r e l a t i o n f o r s u b s t r a t e consumption i s v a l i d . e l e v a t i o n of NaCl content i n c r e a s e s m and decreases y . r e s p e c t i v e l y . one can see the e f f e c t of i n c r e a s i n g NaCl l e v e l on the d i s t r i b u t i o n of the s u b s t r a t e i n p u t . s t a t i s t i c a l a n a l y s i s of the e x p e r i m e n t a l data y i e l d s the v a l u e s of 2. U n f o r t u n a t e l y the c u r r e n t s t a t e of m i c r o b i a l e n e r g e t i c s does not a l l o w a p r e c i s e assessment of the d i r e c t c o n t r i b u t i o n of energy spent f o r o s m o t i c work to maintenance r e q u i r e m e n t s . s p e c i f i c consumption r a t e . q / h r ) . at h i g h salinities. T h i s i s a l s o apparent from F i g .09 (1.21) and 0.023 . 1970). Watson.0. m a x s s s s % Carbon input I ICOn II 100- biomass 50- NaCl (kg/m ) 3 5 10 15 20 25 30 40 Fig.032 (0. A l t h o u g h m i s expected to be a f u n c t i o n of NaCl c o n c e n t r a t i o n the present d a t a does not a l l o w the r e j e c t i o n of c o n s t a n t m hypothesis. .

6 one must remember t h a t the thermodynamic e f f i c i e n c y i s g r e a t l y reduced a t h i g h s a l i n i t i e s . I n F i g . t h e r e f o r e . Removal and d i s p o s a l of sludge may p r e s e n t f i n a n c i a l and e n v i r o n m e n t a l problems. I t i s . CONCLUSIONS 1. Design c a l c u l a t i o n s based on d a t a o b t a i n e d from mono c u l t u r e s s h o u l d a l l o w for the d i f f e r e n c e s observed between the s e n s i t i v i t y of mono and mixed cultures. as p r e d i c t e d by eq.) . P r a c t i c a l Aspects G e n e r a l l y h i g h s l u d g e p r o d u c t i o n i s u n d e s i r a b l e i n treatment i n s t a l l a t i o n s . i s the amount of oxygen taken up per mole of s u b s t r a t e consumed. S a l i n i t y has s i g n i f i c a n t e f f e c t s on the e n e r g e t i c s and k i n e t i c s of p r o c e s s and t h e r e f o r e i s an i m p o r t a n t parameter. 9: Graphical representation of the optimization problem. from F i g . 9. From t h i s p o i n t of v i e w . A consequent decrease i n Y .where the d i s t r i b u t i o n of the s u b s t r a t e carbon i n p u t i n the system i s shown. Y shows the amount of s l u d g e formed per mole of s u b s t r a t e consumed and Y / Y . growth 3. d e s i r a b l e to o p e r a t e an a c t i v a t e d sludge p l a n t a t low growth r a t e s as to i n c r e a s e maintenance requirements o f the c u l t u r e . h i g h s a l i n i t y i s a desirable p r o p e r t y . thereby r e d u c i n g the amount of sludge formed. (2. w i l l then c a l l f o r h i g h e r a e r a t i o n c a p a c i t y . Thus the e n g i n e e r w i l l be f a c e d w i t h an o p t i m i z a t i o n problem. depends on the r e l a t i v e c o s t s i n v o l ved. o x s x s x o x Fig. the s o l u t i o n of which. Mono and mixed c u l t u r e s show s i m i l a r responses t o i n c r e a s e i n s a l i n i t y i n t h e i r environments. T h e r e f o r e i t i s d e s i r a b l e to m i n i m i z e b o t h . However. however. of c o u r s e . 66 . 2. Mono c u l t u r e s . are much more s e n s i t i v e and t h e r e f o r e l e s s e f f i c i e n t i n such environments.

and C. c e r e v i s i a e . C o n t r o l . W (1974).J.K. (1980) B i o e n g i n e e r i n g r e p o r t .K. M.371-378. and V. 2131-2136. 6.57(4) 333-340. Bioeng.R. . 99-104.Y. and C.. . H.. H. de Kok. c e r e u s . (1970) E f f e c t s of sodium c h l o r i d e on steady s t a t e growth and metabolism of S. 572-578. D. Dean. and W. Endoh. 2 9 ( 1 1 ) .F. Gen. Gaudy and A. aeureus a t 3 0 O C . Bioeng. Tunewall ( E d ) . 64. Wastes. . Gaudy (1966) Some e f f e c t s o f h i g h s a l t c o n c e n t r a t i o n on a c t i v a t e d s l u d g e . B i o l .F. A l m q u v i s t and W i n k s e l l . Gaudy (1968) Response o f b i o l o g i c a l waste water t r e a t ment systems to changes i n s a l t c o n c e n t r a t i o n s . .. Wat.G. B i o e n g . E n v i r o n m e n t a l i n f l u e n c e s on the growth o f e r k mammalian c e l l s i n mono l a y e r c u l t u r e s .38(7).J. J .C. A.. 4 8 ( 9 ) . I m a i .4. B a c t e r i o l . K e s s i c k .C. J . R. C l a r e n d o n P r e s s . 5 7 ( 4 ) ... Recent P r o g r e s s i n M i c r o b i o l o g y .. Exp. 3 8 ..381. Endoh and J .. R o e l s . S c i . J . 3 0 1 . 1228. pp. Ferment. G. . C e l l Res. A c t a . Bioeng. B i o t e c h n o l .Kobayashi (1979b) R e s p i r a t o r y a c t i v i t y and sludge volume index o f a c t i v a t e d sludge d u r i n g a c c l i m a t i o n t o s a l i n e w a t e r . REFERENCES B u r n e t t . Water Res. T h e c h n o l . J..p. J . and A. 1148-1159. F . M. E f f e c t s of h i g h s a l i n i t y on the r e s p i r a t i o n c h a r a c t e r i s t i c s of a c t i v a t e d s l u d g e . C o n t r o l . . A. W. C o n t r o l . F o r e f f i c i e n t o p e r a t i o n an o p t i m i z a t i o n must be c a r r i e d o u t t o m i n i m i z e sludge p r o d u c t i o n w h i l e m a x i m i z i n g y i e l d on oxygen. S. B i o t e c h n o l . . Fed.G.. Stouthamer. Fed. E c k e n f e l d e r (1978) The e f f e c t of i n o r g a n i c s a l t s on the a c t i v a t e d sludge p r o c e s s performance. W o d z i n s k i . Tokuz. P o l l u t .E. H e r b e r t . Stockholm. A. .20. Kincannon. B i o t e c h n o l . and A. Technol. Lawton. D. J . A u s t . K. Watson.33.W.. F e d . B i o t e c h n o l .. Hinshelwood(1966).. The e f f e c t of s a l i n i t y v a r i a t i o n s on the a c t i v a t e d s l u d g e p r o c e s s . Growth F u n c t i o n and R e g u l a t i o n i n B a c t e r i a l C e l l s .F. Bioeng. E r i c k s o n . (1953) Water r e l a t i o n s o f S. P o l l u t . D.. B i o c h i m Biophys. Ingram. J .. F r a z i e r (1960) M o i s t u r e r e q u i r e m e n t s of b a c t e r i a .. 67 . . Wat. 613-629. . Sewage and Ind. D.. 1097-1104. Wat.13.91-98. M i c r o b i o l . L. . and C. R o e l s (1980) Method f o r the s t a t i s t i c a l treatment o f e l e m e n t a l and energy b a l a n c e s ..483-496. I n G. 1595-1621. P i r t . J . and J. . and E g g e r t (1957) E f f e c t o f h i g h sodium c h l o r i d e c o n c e n t r a t i o n on t r i c k l i n g f i l t e r s l i m e s .A.22. 1404-1416. Noran (1965) T o l e r a n c e of h i g h s a l i n i t i e s by convent i o n a l waste water treatment p r o c e s s e s . Kincannon. 79. Ferment. 549-564. I m a i .8. Ludzack.. (1958) Some p r i n c i p l e s of c o n t i n u o u s c u l t u r e ..A. B e t t e n h a u s s e n (1973) U t i l i z a t i o n o f energy f o r growth and maintenance i n c o n t i n u o u s and b a t c h c u l t u r e s o f m i c r o o r g a n i s m s ..G. J . I. T.J. 10. Gaudy (1966) S e q u e n t i a l s u b s t r a t e removal by a c t i v a t e d s l u d g e . 22. E r o s h i n (1978). A p p l i c a t i o n o f mass and energy b a l a n c e r e g u l a r i t i e s i n f e r m e n t a t i o n . Sewage Wks. P o l l u t .F. . . B a c t e r i o l . 396-405. J . A p p l i c a t i o n o f m a c r o s c o p i c p r i n c i p l e s to m i c r o b i a l m e t a b o l i s m . B i o t e c h n o l . M i n k e v i c h .L.E. and K.G. 37-55. J . . 3 7 ( 1 0 ) .G. and W. R. . Wat. H.H. Kincannon. Manchen (1976) S a l t water domestic waste t r e a t m e n t . and D. Kobayashi ( 1 9 7 9 a ) . O x f o r d .A. 453-459. K. and Thackeray (1964). (1939)The endogeneous r e s p i r a t i o n o f B. 2457 S c o t t .W. 54-69. 55-68.

.

F.max = A exp(AH*/RT) E C I x r x (1) where r i s the r e a c t i o n r a t e . T h i s has a l r e a d y been s t u d i e d by many a u t h o r s and A r r h e n i u s type e x p r e s s i o n s have been d e r i v e d but found t o be a p p l i c a b l e w i t h i n o n l y a l i m i t e d range. changing the r e a c t i o n r a t e s .b a t c h mode. i s the a c t i v a t i o n e n t h a l p y o f x * P u b l i s h e d i n B i o t e c h n o l . a l l the b i o c h e m i c a l r e a c t i o n s t a k i n g p l a c e i n the c e l l a r e a f f e c t e d by the temper a t u r e .A. u n l i k e h i g h e r organisms l i k e mammals. PNEUMONIAE * E s e n e r . AH. Kossen INTRODUCTION Temperature i s an i m p o r t a n t e n v i r o n m e n t a l parameter f o r m i c r o b i a l growth. I f the maximal s p e c i f i c growth r a t e o f microorganisms i s l i m i t e d by the r e a c t i o n r a t e of one s p e c i f i c enzymatic r e a c t i o n i n a complex sequence as p o s t u l a t e d by the Monod^ model o f growth. MODEL Assuming t h a t the b a c t e r i a l growth i s an end p r o d u c t of a number o f enzymatic r e a c t i o n s and t h a t one s p e c i f i c r e a c t i o n determines the o v e r a l l r e a c t i o n r a t e . temperature dependence o f the maximum growth r a t e can be assumed t o f o l l o w an A r r h e n i u s r e l a t i o n o f the f o l l o w i n g type : r x. I t has l o n g been known t h a t the temperature i n f l u e n c e s the n a t u r e o f metabolism. C i s the biomass c o n c e n t r a t i o n . . E i s the weight f r a c t i o n o f the s p e c i f i c enzyme i n biomass.1 401 (1 981 > 69 .4-7 In t h i s study the i n f l u e n c e o f the temperature on the maximum s p e c i f i c growth r a t e o f the b a c t e r i u m Klebsiella pneumoniae was s t u d i e d i n f e d . t h e n u t r i t i o n a l requirements and the biomass c o m p o s i t i o n i n a d d i t i o n t o i t s p r i m a r y e f f e c t . J. Bioeng.CHAPTER 6 I THE INFLUENCE OF TEMPERATURE ON THE MAXIMUM SPECIFIC GROWTH RATE OF KLEBSIELLA A. T h e r e f o r e . M i c r o o r g a n i s m s . R o e l s and N. a m a t h e m a t i c a l r e l a t i o n between the a b s o l u t e temp e r a t u r e and the maximal growth r a t e can be sought a f t e r . R e s u l t s were used f o r the d e t e r m i n a t i o n of the thermodynamic parameters i n an A r r h e n i u s type model extended t o d e s c r i b e a l s o the h i g h temperature range where the maximum s p e c i f i c growth r a t e d e c l i n e s w i t h i n c r e a s i n g temperature. 23 .W. do not possess the c a p a b i l i t y o f r e g u l a t i n g t h e i r i n t e r n a l temperature.A. I n m i c r o b i a l c u l t u r e s the c e l l temperature must become e q u a l t o the e n v i r o n m e n t a l temperature.

Klebsiella pneumoniae NCTC 418. I t was assayed e n z y m a t i c a l l y . one o b t a i n s : A A f A = {1 + K exp(-AH 2 / RT)}"' (4) Furthermore. s i n c e f j + f = 1. an a c t i v e and an i n a c t i v e form i n e q u i l i b r i u m w i t h each o t h e r . A H 2 i s the e n t h a l p y change of the i n a c t i v a t i o n r e a c t i o n and K i s a c o n s t a n t . The 70 _ .' A t temperatures h i g h e r than optimum a n e g a t i v e c o r r e l a t i o n has been observed between the temper a t u r e and y . E C 1 A x (5) then from eqs. ( 3 ) . A i s a c o n s t a n t . the e f f e c t of temperature on enzyme a c t i v i t y can be e v a l u a t e d by c o n s i d e r i n g the a c t i v a t i o n .2 ym membrane f i l t e r and i n o c u l a t e d w i t h an inoculum a c t i v e l y growing a t the e x p e r i m e n t a l temperature to e l i m i n a t e any lag phase and the p o s s i b i l i t y of unbalanced growth. was c u l t i v a t e d i n s y n t h e t i c medium as d e s c r i b e d by Evans et a l . From eq. from eq. max ing can be written: A' exp (-AH* V m a X / RT) (6) / RT) = 1 + K exp(-AH MATERIALS AND METHODS The organism. .the r a t e l i m i t i n g r e a c t i o n .'0 Biomass was c o l l e c t e d on a 0. below the s o . r e s p e c t i v e l y . 9 G l y c e r o l was used as the o n l y carbon and energy source. I f the maximum s p e c i f i c growth r a t e . ( l ) : y max = A exp(-AH* / RT) E ] (2) T h i s r e l a t i o n s h i p has been shown to d e s c r i b e w e l l the e x p e r i m e n t a l o b s e r v a t i o n s i n a l i m i t e d range.2 ym pore d i a m e t e r membrane f i l t e r ( S a r t o r i u s SM 11307). Medium was s t e r i l i z e d by membrane f i l t r a t i o n through a 0. y is d e f i n e d i n the u s u a l way i t f o l l o w s from e q .c a l l e d o p t i m a l temperature. T h i s phenomenon can be a l l o w e d f o r i n a g e n e r a l i z e d model i f the i n f l u e n c e of temperature on the a c t i v i t y of the enzyme i n v o l v e d i n the growth l i m i t i n g r e a c t i o n i s t a k e n i n t o a c c o u n t .(4) and (5) i n c o m b i n a t i o n w i t h the d e f i n i t i o n of u the f o l l o w . I f the i n a c t i v a t i o n r e a c t i o n i s f a s t . i f o n l y the a c t i v e f r a c t i o n of the enzyme i s engaged i n the growth l i m i t i n g r e a c t i o n . Experiments were c a r r i e d out i n a 11 x 10 3 m^ w o r k i n g volume f e r m e n t o r . R i s the u n i v e r s a l gas c o n s t a n t and T i s the a b s o l u t e temperature. Dry w e i g h t s were determined i n d u p l i c a t e s by the method of de V r i e s and Stouthamer.(3) i t f o l l o w s t h a t : r x. max = A exp(-AH* / RT) f. washed w i t h d i s t i l l e d water and d r i e d to c o n s t a n t weight a t 378 K. the f o l l o w i n g e q u i l i b r i u m r e l a t i o n s h i p can be formulated: m a x f x = f A K exp(-AH 2 / RT) (3) where f and f j a r e the f r a c t i o n s of the t o t a l amount of the enzyme b e i n g a c t i v e and i n a c t i v e . ^ Assuming t h a t t h i s enzyme can e x i s t i n two p o s s i b l e c o n f i g u r a t i o n s .i n a c t i v a t i o n r e a c t i o n .

T 1 1 r Fig. t o g e t h e r w i t h the 95 % c o n f i d e n c e l e v e l s . eq.8 ± 0.y model. temperature. A l l samples were c o o l e d d u r i n g sampling to about 278 K by an o n . ) I n F i g u r e 2 the n a t u r a l l o g a r i t h m of y i s p l o t t e d a g a i n s t the r e c i p r o c a l of temperature.77 kg dry a i r / h r . ' ' The same computer program was used f o r e v a l u a t i n g the parameters of the temperature. but none c o u l d be d e t e c t e d a t any s i g n i f i c a n t l e v e l . A s u p p o r t i n g o b s e r v a t i o n f o r t h i s was the absence of m e t a b o l i c p r o d u c t s a t a l l temperatures.(6). S i n c e t h e r e a r e no abrupt changes i n the s l o p e of t h i s b e l l shaped c u r v e . m a x m a x m a x w a s RESULTS AND DISCUSSION E x p e r i m e n t a l r e s u l t s a r e shown i n F i g u r e 1. model equation (6) evaluated with parameters (•) given experiment. i . Then y was determined by p e r f o r m i n g n o n l i n e a r r e g r e s s i o n based on Marquarts a l g o r i t h m . The a i r f l o w r a t e to the fermentor was c o n t r o l l e d by a thermal mass f l o w meter (Brooks 5811) a t about 0.05. The t y p i c a l r e s i d e n c e time i n the exchanger was about 5-10 sees.l i n e heat exchanger manufactured i n our workshop. ( in Table I.pH was c o n t r o l l e d at 6. one can conclude t h a t t h e r e has been no s i g n i f i c a n t changes i n the c e l l metabolism and t h a t the same enzymatic r e a c t i o n remains the r a t e l i m i t i n g one throughout the e x p e r i m e n t a l range. COMPUTATIONS The maximum s p e c i f i c growth r a t e U determined from the d r y weight d a t a c o l l e c t e d d u r i n g the e x p o n e n t i a l phase. . 1: Maximum specific growth rate vs. The upper boundary of the e x p o n e n t i a l phase was determined by p l o t t i n g the oxygen uptake r a t e d a t a and evaluating the time a t which the maximum i s reached. The c o n t i n u o u s l i n e r e p r e s e n t s the model e v a l u a t e d w i t h the parameters g i v e n i n Table I . The c u l t u r e s u p e r n a t a n t was checked f o r the presence of p r o d u c t s o t h e r than biomass. m a x From F i g u r e 2 i t i s e v i d e n t t h a t a s i m p l e A r r h e n i u s type e x p r e s s i o n can o n l y 71 . carbon d i o x i d e and w a t e r . e .

may be underestimated s i g n i f i c a n t l y . M -.40 287.7°C was c a l c u l a t e d by e v a l u a t i n g the 72 . the reciprocal of absolute temperature. AH*. 2: Natural logarithm of V> max vs. 1 0 48 1. 95 % c o n f i d e n c e AH * AH 2 levels 86.10 1 4 kJ/mole kJ/mole 1/hr (—) (44. ' 2 From eq. d e s c r i b e a p a r t of the e x p e r i m e n t a l o b s e r v a t i o n s . t h e magnitude of the a c t i v a t i o n e n t h a l p y . J O I n s e r t i n g the parameters o b t a i n e d one gets 36. I f such a simple e x p r e s s i o n i s assumed t o h o l d up t o the o p t i m a l temperature f o r f a s t e s t growth.64) (188.9°C as the o p t i m a l temperature f o r t h i s system.78 5.387.1))} (7) with M A X A r e s u l t which i s o b t a i n e d by s e t t i n g the f i r s t d e r i v a t i v e of y r e s p e c t to T equal t o z e r o .19 . average f o r 20 p r o t e i n s .38 .128.69 . Fig.Table I : C a l c u l a t e d and r e p o r t e d model parameters. An i n f l e c t i o n p o i n t of 31.(6) the o p t i m a l c u l t i v a t i o n temperature ( i f the f a s t e s t growth i s the o n l y c o n s i d e r a t i o n s ) can be shown t o be g i v e n by: T = AH 2 / {R l n ( K (AH^AH* .23) A' K Range AH^ a 343 kJ/mole 147 828 From M o r o w i t z .33 . T h i s i s why workers who d i d n o t a l l o w f o r thermal enzyme i n a c t i v a t i o n process have r e p o r t e d low AH] v a l u e s .

i . limiting to the t h i s p l o t . 287 kJ/mole. e . t h e o p t i m a l o p e r a t i o n temperature f o r an i n d u s t r i a l p r o c e s s can be chosen i n t h i s range f o r f a s t biomass p r o d u c t i o n . T h i s can b e s t be v i s u a l i z e d from F i g u r e 3 where the a c t i v e f r a c t i o n of the enzyme i s p l o t t e d as a f u n c t i o n of the growth temperature. Computed according model equation (4). These r e s u l t s i n d i c a t e t h a t the model can be used as a u s e f u l a p p r o x i m a t i o n t o d e s c r i b e the t e m p e r a t u r e . t h e nominator of e q . i n c r e a s e i n V^ax i. As can be seen from m a x s Fig. A t h i g h e r temperatures t h e c o n t r i b u t i o n o f t h e thermal enzyme i n a c t i v a t i o n p r o c e s s becomes s i g n i f i c a n t . changes r e l a t i v e l y l i t t l e as the c o n t r i b u t i o n s o f the two processes b a l a n c e each o t h e r .r e a c t i o n r a t e r e l a t i o n s h i p of m i c r o b i a l growth.temperature a t which the second d e r i v a t i v e ( d 2 y / d T 2 ) becomes z e r o . Up t o t h i s p o i n t . compares w e l l w i t h h i s average of 343 kJ/mole (Table I ) .m a i n l y determined by the s i m p l e A r r h e n i u s type of e x p r e s s i o n . x h e v a l u e of A H j c a l c u l a t e d i n t h i s s t u d y . . ( 6 ) . I n the range 33 < T < 38°C. 3: Active fraction of the enzyme taking part in the growth reaction as a function of temperature. NOMENCLATURE A. a t low temperatures almost a l l of the enzyme remains a c t i v e w h i l e a f t e r about 32°C a d r a m a t i c decrease i n the a c t i v i t y i s c a l c u l a t e d . Morowitz has compiled a l i s t of e n t h a l p y change v a l u e s f o r the thermal i n a c t i v a t i o n of some 20 p r o t e i n s . A' Cx E *A A H * AH 2 K x r constants(hr~') c o n c e n t r a t i o n (kg/m3) weight f r a c t i o n o f the s p e c i f i c enzyme i n jiomass ( d i m e n s i o n l e s s ) a c t i v e f r a c t i o n of the s p e c i f i c enzyme a c t i v a t i o n e n t h a l p y f o r the growth l i m i t i n g r e a c t i o n ( k J / m o l e ) e n t h a l p y change f o r enzyme i n a c t i v a t i o n r e a c t i o n (kJ/mole) a constant(dimensionless) r a t e of r e a c t i o n (kg/m3/hr) 73 . T h e r e f o r e .

R

T max

gas c o n s t a n t (kj/kg.mole) temperature (K) maximum s p e c i f i c growth r a t e

(hr ')

-

REFERENCES 1. S.J. P i r t , P r i n c i p l e s of Microbe and C e l l C u l t i v a t i o n , ( B l a c k w e l l , London, 1975) 2. D.W. Tempest, i n ' M i c r o b i a l Growth' 19 t h Symp. Soc. Gen. M i c r o b i o l . , (Cambridge U.P., Cambridge,1969),p. 87. 3. J . Monod, Recherches s u r l a C r o i s s a n c e des C u l t u r e s Bacteriennes(Hermann, P a r i s , 1942). 4. H.Topiwala and C. G. S i n c l a i r , B i o t e c h n o l . Bioeng.,13,795(1971) . 5. F. Watanable and S. Okada, J . C e l l . B i o l . , 3 2 , 3 0 9 ( 1 9 6 7 ) . 6. A.C.R. Dean and C. Hinshelwood, Growth F u n c t i o n and R e g u l a t i o n i n B a c t e r i a l C e l l s , ( C l a r e n d o n , O x f o r d , 1966). 7. A. Prokop and A. E. Humphrey,'Kinetics o f D i s i n f e c t i o n ' , i n D i s i n f e c t i o n , M.A. Benarde, ed.,(Macel Dekker, New York, 1970). 8. H. E y r i n g and D.W. U r r y , 'Thermodynamics and Chemical K i n e t i c s ' , i n T h e o r e t i c a l and M a t h e m a t i c a l B i o l o g y , T.H. Waterman and H.J. M o r o w i t z , e d s . , ( B l a i n d e l l , New York, 1965). 9. C.G.T. Evans, D.Herbert and D.W. Tempest,in Methods i n M i c r o b i o l o g y , J.R. N o r r i s and W.W. Ribbons, eds.,(Academic, London, 1970), vol.2.,p.313. 10. de V r i e s and A.H. Stouthamer, J . B a c t e r i d ., 96,472, (1 968) . 11. D.W. M a r q u a r t , J . Soc. I n d . A p p l . Mat.,2,431(1963). 12. J.H. L e e , D. W i l l i a m s o n and P.L. Rogers, B i o t e c h n o l . L e t t r . , 2 ( 4 ) , 8 3 ( 1 9 8 0 ) . 13. H.J. M o r o w i t z , Energy Flow i n B i o l o g y , ( A c a d e m i c , New York, 1 9 6 8 ) , p . l l 4 .

74

Addendum t o Chapter 6: Influence of Temperature on fe

g

Very l i t t l e has been p u b l i s h e d on the i n f l u e n c e o f e n v i r o n m e n t a l f a c t o r s on the v a l u e o f k ; Monod s a t u r a t i o n c o n s t a n t . There i s by no means an agreement between t h e r e p o r t e d f i n d i n g s as t o whether k i n c r e a s e s o r decreases w i t h temperature.
s s

Topiwala and S i n c l a i r have r e p o r t e d an i n v e r s e r e l a t i o n f o r k w i t h t h e e n v i r o n m e n t a l temperature.'^ They have o b t a i n e d an a s s o c i a t e d a c t i v a t i o n energy of -49.4 kJ/mole by p l o t t i n g - I n k v s . 1/T(K). I n c o n t r a s t t o t h e i r f i n d i n g s Marr e t a l ' 5 have assumed k t o i n c r e a s e w i t h temperature i n accordance w i t h the A r r h e n i u s r e l a t i o n and e s t i m a t e d an E v a l u e o f 20.9 k j / m o l e . I n t h e waste water f i e l d Novakl6 has reviewed the t e m p e r a t u r e - s u b s t r a t e i n t e r a c t i o n i n f o r m a t i o n ; here k - t e m p e r a t u r e r e l a t i o n s h i p was a l s o expressed i n the A r r h e n i u s form f o r a r e s t r i c t e d temperature range. For a e r o b i c systems, k i n c r e a s e d w i t h temperature. I n a n a e r o b i c systems, however, an i n v e r s e r e l a t i o n was observed (see Table I I ) .
s s s s g

Table I I : Temperature Organism E. coli A. aerogenes A c t i v a t e d sludge A c t i v a t e d sludge A c t i v a t e d sludge A c t i v a t e d sludge A n a e r o b i c sludge A n a e r o b i c sludge

dependence of k
s

s

; the a c t i v a t i o n energy v a l u e s . T°C range 15 - 30 25 - 40 20 - 40 15 20 25 20 26 30 35 35 ref. 15 14
#

substrate

E (k ) kJ/mole

20.9 glucose -49.4 glucose glucose 22.3 61 .2 waste water s y n t h e t i c waste 71.1 l i n o e l i c acid 83.7 acetic acid - 130.5 -51.5 complex waste

*
#

* C a l c u l a t e d from d a t a c o l l e c t e d and r e p o r t e d by Novak'6. I f the Monod s a t u r a t i o n c o n s t a n t i s assumed t o be a r a t i o of t h e k i n e t i c c o n s t a n t s , as i s assumed i n the homologous M i c h e a l i s - M e n t e n r e l a t i o n , i t may i n c r e a s e . o r decrease w i t h i n c r e a s i n g temperature depending on the s p e c i f i c reaction.

Influence

of lemperature

on the Energetic

Parameters

From a m a c r o - e n e r g e t i c p o i n t of v i e w , temperature may b a s i c a l l y be e x p e c t e d to i n f l u e n c e the observed y i e l d s through i t s e f f e c t on t h e maximal y i e l d s and maintenance c o e f f i c i e n t s . P r o v i d e d t h a t no s i g n i f i c a n t changes i n t h e metabol i c r o u t e s take p l a c e , Y cannot be expected to be a s t r o n g f u n c t i o n o f temp e r a t u r e . The r e s u l t s o b t a i n e d i n t h i s study r e a f f i r m s t h i s s t a t e m e n t ( s e e F i g . 4) f o r t h e range 25 - 39° C. C o n s i d e r i n g t h e e x p e r i m e n t a l e r r o r s ( r e f e r back t o the j o i n t c o n f i d e n c e a r e a f o r Y ™ and m ; Chapter 2, F i g . 1 0 ) Y can be assumed t o be c o n s t a n t and independent o f temperature. S i m i l a r f i n d i n g s have a l s o been r e p o r t e d by s e v e r a l workers f o r m e s o p h i l i c organisms.'4,17-20 M a i n z e r and Hempling'^ w i t h E. coli have found o n l y s l i g h t changes i n Y i n the range 17.5 - 32°C. S i m i l a r l y Y v a l u e s o f 52.3 and 50.9 g.dry weight/mole g l y c e r o l , were r e p o r t e d f o r E. coli a t 20.3 and 30 °C,
m a x a x m a x g m a x m a x

75

r e s p e c t i v e l y . " At 40 °C however, Y was 35.3.'8 A s i m i l a r drop i n Y was a l s o observed i n t h i s study as shown i n F i g . 4. T h i s may be due t o some s i g n i f i c a n t change i n the b i o s y n t h e t i c pathways or due t o u n c o u p l i n g of the energy p r o d u c i n g and consuming processes. '» Based on t h e i r e x p e r i m e n t a l s t u d i e s and l i t e r a t u r e survey Farmer and Jones '8 g e n e r a l i z e d t h a t i n E.coli and s e v e r a l o t h e r m e s o p h i l i c b a c t e r i a , decreases i n growth e f f i c i e n c y occur p r i n c i p a l l y at temperatures which are r o u g h l y e q u a l or h i g h e r than those which support the f a s t e s t growth. R e s u l t s r e p o r t e d i n t h i s c h a p t e r g e n e r a l l y support t h i s statement.
2 22

1

m a x

m a x

/naze

Fig. Fig.

4: Variation 5: Normalized absolute

of 1^

with

the growth coefficient

temperature. versus the reciprocal of the

maintenance temperature.

F o l l o w i n g the above d i s c u s s i o n , i t can be concluded t h a t the v a r i a t i o n of the apparent y i e l d b a s i c a l l y i s a consequence of the s t r o n g dependence of m a i n t e nance r e q u i r e m e n t s on temperature. I n F i g . 5 maintenance c o e f f i c i e n t s normal i z e d f o r t h a t a t 25°C are shown as a f u n c t i o n of the c u l t i v a t i o n temperature. These e x p e r i m e n t a l ( f e d - b a t c h experiments as d e s c r i b e d i n Chapter 4) f i n d i n g s can w e l l be d e s c r i b e d by an A r r h e n i u s type of r a t e - t e m p e r a t u r e model as shown by the s t r a i g h t l i n e . Energy of a c t i v a t i o n a s s o c i a t e d w i t h maintenance f u n c t ions was c a l c u l a t e d to be 56.9 k j / m o l e (combined e s t i m a t e ) w i t h a 95 % c o n f i d e n c e l e v e l of(50.2 - 63.6).From t h i s p l o t i t can c l e a r l y be seen t h a t m i s a s t r o n g f u n c t i o n of temperature and i n c r e a s e s f o u r f o l d f o r a 20°C i n c r e a s e i n temperature. Values of E (m ) are p r e s e n t e d i n Table I I I f o r comp a r i s o n . These d a t a i n d i c a t e t h a t i n a l l cases m i s a f u n c t i o n of T, however, the degree of dependence changes c o n s i d e r a b l y f o r d i f f e r e n t systems. Another r e a s o n f o r such a wide range of r e s u l t s c o u l d be the e r r o r s i n t r o d u c e d i n the c a l c u l a t i o n of m v a l u e s . U n f o r t u n a t e l y i n l i t e r a t u r e no i n f o r m a t i o n was p r o v i d e d about the a c c u r a c y or the 95 % c o n f i d e n c e l e v e l s of the e s t i m a t e s r e p o r t e d . In t h e i r r e c e n t a r t i c l e H e i j n e n and R o e l s ^ c o l l e c t e d a v a s t number of m d a t a and a l i n e a r r e g r e s s i o n a n a l y s i s y i e l d e d an E(m ) v a l u e of 39 kJ/mole w i t h 95 % c o n f i d e n c e l e v e l of (21 - 57). T h e i r v a l u e i s s m a l l e r than t h a t r e p o r t e d here but the c o n f i d e n c e l e v e l s are s t i l l o v e r l a p p i n g . F r o m t h i s d i s c u s s i o n and Table I I I one can conclude t h a t the E(m ) v a l u e s r e p o r t e d can o n l y be used to o b t a i n e s t i m a t i o n s of the o r d e r of magnitude of the m v a l u e s a t d i f f e r e n t temperatures.
s s s 2 s s s s

76

lipolytica C. Bakers y e a s t p r o c e s s e s . where t h e f e r m e n t o r volume i n c r e a s e s i n p r o p o r t i o n t o the cube of i t s r a d i u s . p r o cesses i n which t h e d e s i r e d end p r o d u c t i s c l o s e l y a s s o c i a t e d w i t h t h e mainm 77 . caIdotenax substrate glycerol hexane hexane glucose glucose glycerol glycerol me t h a n o l glucose E (m ) kJ/mole s T°C range 20 . polymovpha B. caldotenax. I n t h e s u b o p t i m a l temperature range Umax i n c r e a s e w i t h T b u t the y i e l d drops due t o i n c r e a s e d maintenance r e q u i r e m e n t s .From a p r o c e s s o p e r a t i o n view p o i n t . whereas t h e a v a i l a b l e heat t r a n s f e r a r e a i n c r e a s e s i n p r o p o r t i o n t o t h e square of the f e r m e n t o r r a d i u s . e .. d i f f e r e n t from t h a t r e p o r t e d above. .25 a x Consequences for Engineering Processes and Design Temperature i s an i m p o r t a n t e n g i n e e r i n g parameter which can be c o n t r o l l e d q u i t e a c c u r a t e l y i n some p r o c e s s e s e. O \ • « mole.g. t h e r m o p h i l i c b a c t e r i a seem to d i s p l a y temperature dependent b e h a v i o u r . Recent work of Kuhn e t a l .I n c o n t r a s t t o the p r o c e s s e s w i t h p r i m a r y aim of biomass p r o d u c t i o n . Organism E. With mixed c u l t u r e s . I n a l l of t h e s e p r o cesses i t s i n f l u e n c e i s i m p o r t a n t and o f t e n have i n t e r a c t i n g consequences.I n p r o d u c t i v e f e r m e n t a t i o n s c a r r i e d out i n b a t c h mode. p r o d u c t i v e f e r m e n t a t i o n . t h e a c t i v a t i o n energy d a t a . e. Y and Y ™ a r e a l s o f u n c t i o n s of temperature w i t h E's of 8.g. T h e r e f o r e the o p t i m a l o p e r a t i n g p o i n t has t o be o b t a i n e d by performing a cost o p t i m i z a t i o n a n a l y s i s . as the growth r a t e w i l l e q u a l t o the d i l u t i o n r a t e below t h i s range.45 18 18 15 25 25 25 35 50 30 30 30 40 43 43 43 60 ref. which i s a f u n c t i o n of the p r o d u c t o f t h e maximum spec i f i c growth r a t e and t h e y i e l d v a l u e . T h i s i s because of the temperature c o n s t r a i n t brought out by t h e c o o l i n g water. where the d e s i r e d product i s biomass e.I n c o n t i n u o u s f e r m e n t a t i o n o p e r a t i o n s . coli E. p a r t i c u l a r l y f o r a e r o b i c a c t i v a t e d sludge an a n a l y s i s of temperature dependence i s n o t so c l e a r c u t due to u n a v o i d a b l e changes i n the c u l t u r e . aerogenes K.8 k J / m a x a x OX. lipolytica E. SCP . p r o v i d e d t h a t the p r o c e s s i s o p e r a t e d below y a x I n f l u e n c e of temperature i n c o n t i n u o u s c u l t u r e w i l l be e v i d e n t near the wash-out range.3 and 23. 18 18 19 19 15 14 t h i s study t h i s study 20 24 101 284 82 86 83 37 58 54 117 66 3 6 5 2* 7 7 2 0* 2 5 * o b t a i n e d from oxygen y i e l d d a t a . i . Few w i l l be c i t e d here : . . pneumoniae K. two s p e c i a l cases have t o be p o i n t e d o u t . coli A.g. C o o l i n g problems may become d i f f i c u l t t o s o l v e p a r t i c u l a r l y when s c a l i n g . one aims t o maximize the p r o d u c t i v i t y . coli C. the h i g h e r the f e r m e n t a t i o n temperature the e a s i e r to remove t h e heat produced by metabolism and thus t o c o n t r o l the b i o r e a c t i o n . amount of biomass produced per u n i t s u b s t r a t e consumed p e r u n i t t i m e . .60 C ) . A l t h o u g h t h e r e i s wide agreement on t h e temperature dependence of m f o r mixed c u l t u r e s .Table I I I : Temperature dependence of m v a l u e s . the economics w i l l m a i n l y be d i c t a t e d by the y i e l d . I n most r e p o r t s some s o r t of maximum i s reached a t about 20°C. l i k e changes i n t h e p r e dominance of s p e c i e s e t c . . b i o l o g i c a l waste water treatment p r o c e s s e s . F i n a l l y .. r e s p e c t i v e l y (T range of 50 . pneumoniae 11. 2 4 i n d i c a t e s t h a t f o r t h e r m o p h i l i c B. and n o t i n o t h e r s . d a t a on Y ™ shows no d e f i n i t e t r e n d of change.u p .t e m p e r a t u r e r e l a t i o n .

M e l l i n g .P. 21.T. a h i g h fermentor temperature may facilitate easier s e p a r a t i o n of e t h a n o l from the b r o t h . Farmer and C. 19.L. H e i j n e n and J. A p p l . A.tenance m e t a b o l i s m . B i o t e c h n o l . 1975)p. H. F i e c h t e r . eds. C o l l i n s . N i l s o n and D.303(1980). C.G. 2 5 1 ( 1 9 6 6 ) .. T h e r e f o r e . T h e o r e t i c a l l y . Durand.. 6 7 ( 3 ) . Senez. 1 2 6 ( 1 ) . h i g h temperatures w i l l f a v o u r f o r m a t i o n o f l e s s s l u d g e thus r e d u c i n g the c o s t s and problems a s s o c i a t e d w i t h the d i s p o s a l of i t . J . I n c r o p e r a and J.1984(1974). 20. M o l e t t a . e. Cometta and A. 18. 1 0 2 ( 3 ) . 15. Eur. M i c r o b i a l ... e t h a n o l p r o d u c t i o n by Z. J .S.. M a i n z e r and W. A d d i t i o n a l l y . C. F. i n r e c e n t y e a r s c o n s i d e r a t i o n has been g i v e n t o the f e a s i b i l i t y o f u s i n g waste heat from power s t a t i o n s f o r c o n t r o l l i n g o r a t l e a s t i n c r e a s i n g the temperatures of waste streams. Fed. 10(4). J. A . 17. 1 1 8 . Stouthamer. 23. R . S. I n t . C . The i n f l u e n c e o f t h e treatment temperature on t h e s e t t l i n g p r o p e r t i e s and r a t e s have y e t t o be s t u d i e d . i n Continuous c u l t u r e . 22. ^ 5 r e p o r t e d a comprehensive study o f the i n f l u e n c e o f i n c r e a s e d temperatures on the performance and e f f i c i e n c y o f the a c t i v a t e d s l u d g e p r o c e s s as a whole. J .. I. Hempling.J. J.23. Acad. Grady.T.46.G. B i o t e c h n o l .E. . Rev.P. Bioeng. Jones. 5 3 6 (1963). 2 9 3 ( 1 9 7 8 ) .. N. 16.C. J . much a t t e n t i o n has t h e r e f o r e been focused on t h i s a s p e c t and p a r t i c u l a r l y on t h e s e l e c t i o n and use of t h e r m o p h i l i c organisms. C o l l i n s e t a l . .12.795(1971).95(1962)..A. However. T o p i w a l a and C. 3 5 9 ( 1 9 7 6 ) . REFERENCES 14.739(1981). Y.13. Water Res.G. Cont.26. C h i c h e s t e r . C. R o e l s . mob His.C. Biochem. 24. S i n c l a i r . J .I n t h e f i e l d of waste water t r e a t m e n t . 25. temperature has r e c e i v e d l i t t l e a t t e n t i o n . S c i . M i c r o b i o l .21.g.146. . ( E l l i s Horwood.1(1979). Marr. Water P o l l u t . G.P. D.J.547(1978). Kuhn. Evans and J .H. Novak. M i c r o b i o l . p r o b a b l y because of the l a c k o f an e c o n o m i c a l method f o r chang i n g o r c o n t r o l l i n g waste water t e m p e r a t u r e s . Elwood. 6 .H. Bioeng. due t o i n c r e a s e d maintenance demands. H.E. B a c t e r i o l . C l a r k . Biochem. B a c t e r i d . Ann. M a k i g u c h i .W.L. Rev. Cooney and A. S. E. Dean. I n c r e a s e d temperature i n turn w i l l c a l l f o r increased aeration capacity to s a t i s f y the increased r e s p i r a t i o n demands and due t o the reduced s o l u b i l i t y o f oxygen. R e c e n t l y . . A r c h . R. 78 . FEBS L e t t e r s . Goma and G. A. B i o t e c h n o l . h i g h temperatures would be advantageous. the problem w i l l a g a i n be one o f o p t i m i z a t i o n f o r t h e d e s i r e d o b j e c t i v e .

' I t has t o be n o t e d . no i n f o r m a t i o n can be o b t a i n e d about the g e o m e t r i c a l s t r u c t u r e of the c e l l s nor on the dependence of d i f f u s i o n a l p r o c e s s e s on such s t r u c t u r e s . t h a t even the c o n s i d e r a t i o n of these f o u r r e l e v a n t components i s not s u f f i c i e n t to d e s c r i b e the a c t i v i t i e s of the organisms f u l l y e. I n t h i s way the s t r u c t u r e d models not o n l y p r o v i d e i n f o r m a t i o n on the q u a n t i t y of the c e l l s but a l s o on t h e i r q u a l i t y . I n s t r u c t u r e d model b u i l d i n g one has t o s e l e c t the parameters which a r e the most r e l e v a n t f o r the d e s c r i p t i o n of the p h y s i o l o g i c a l s t a t e of the organism. A l l these v a r i a b l e s can be e x p e r i m e n t a l l y determined and t h e i r dependence on the growth r a t e i s w e l l documented at l e a s t f o r the steady s t a t e c o n t i n u o u s c u l t i v a t i o n . 79 . These models. p r o v i d e good a p p r o x i m a t i o n s f o r d e s c r i b i n g growth i n e x p o n e n t i a l phase o r at steady or pseudo steady s t a t e s . DNA.g. The u n s t r u c t u r e d models are expected to f a i l p a r t i c u l a r l y i f the time c o n s t a n t s f o r e n v i ronmental changes are of the same o r d e r of magnitude as those a s s o c i a t e d adapt a t i o n mechanisms i n s i d e the b i o t i c phase. which c o n s i d e r one b i o t i c v a r i a b l e . For a comprehensive d e s c r i p t i o n too many parameters would have t o be i n c o r p o r a t e d i n t o the model. e . Hence i t w i l l be f o r m a l l y c o r r e c t t o use s t r u c t u r e d models f o r d e s c r i b i n g s i t u a t i o n s i n which the change i n biomass c o m p o s i t i o n i n response t o e n v i r o n m e n t a l changes. I n f o r m a t i o n from m o l e c u l a r c h e m i s t r y and m i c r o b i o l o g y i s r e q u i r e d f o r t h i s purpose. A l o g i c a l f i r s t c h o i c e would be to s e l e c t RNA. i s s i g n i f i c a n t . These models i d e a l l y c o n s i d e r the p h y s i o l o g i c a l s t a t e of the organism and express i t not o n l y as a f u n c t i o n of the p r e s e n t e n v i r o n m e n t a l s t a t e but a l s o on the e n t i r e past h i s t o r y of the system i . I t has been shown t h a t u n s t r u c t u r e d models. 3 Thus a s t r u c t u r e d model should be c o n s t r u c t e d such t h a t i t o n l y g i v e s i n f o r m a t i o n about the most r e l e v a n t p r o c e s s e s and v a r i a b l e s .. the s t a t e of the environment the c e l l s have seen t i l l the p r e s e n t s t a t e . as an e x t e n s i o n of the u n s t r u c t u r e d models. however.CHAPTER 7 A STRUCTURED MODEL FOR BACTERIAL GROWTH I INTRODUCTION I n Chapters 2 and 4. as f o r the system d e s c r i b e d . 2 . t h e o r y and a p p l i c a t i o n s of u n s t r u c t u r e d models have been d i s c u s s e d . Simpler models can be o b t a i n e d by c o n s i d e r i n g a few v a r i a b l e s . c a r b o h y d r a t e and p r o t e i n c o n t e n t s of the c e l l s f o r d e s c r i b i n g t h e i r p h y s i o l o g i c a l s t a t e . S t r u c t u r e d models by d e f i n i t i o n are those models which d e s c r i b e the a c t i v i t y of the organisms by more than one v a r i a b l e . Such c o m p l i c a t e d models are m a t h e m a t i c a l l y v e r y complex t o m a n i p u l a t e and most of the parameters o f t e n l o s e t h e i r b i o l o g i c a l meaning. I n Chapter 2 i t has a l s o been shown t h a t b o t h the e l e m e n t a l and macromolecular c o m p o s i t i o n of the organisms are f u n c t i o n s of the growth r a t e . .

b i o t i c phase : A l l r e a c t i o n s t a k i n g p l a c e i n the a . a complete d e r i v a t i o n the r e a d e r i s r e f e r r e d to the o r i g i n a l work. e . a r e c a l l e d Compartmental Models. Since i n most cases the volume o c c u p i e d by the b i o t i c phase i s v e r y s m a l l compared w i t h t h a t of the a . D e f i n i n g a new v e c t o r . For The g e n e r a l d e s c r i p t i o n to be p r e s e n t e d here f o l l o w s the work by R o e l s ^ . mass f r a c t i o n s i n the b i o t i c phase. these c o n c e n t r a t i o n s may be expressed per u n i t c u l t u r e volume. Two phases can now be i d e n t i f i e d : a. Work t h a t has been r e p o r t e d i n the l i t e r a t u r e i n c l u d e the m o d e l l i n g of d i a u x i c growth^.i n which the a c t i v i t y of the biomass i s s p e c i f i e d by more than one and up to 3 or 4 v a r i a b l e s . x . on a kg/m3 of c u l t u r e volume b a s i s . Y . Let C be the c h e m i c a l s t a t e v e c t o r of dimension n. B i o t i c phase : C o n c e n t r a t i o n s of a l l the components i n the b i o t i c phase are c o l l e c t e d i n an m d i m e n s i o n a l v e c t o r . have c o n t r i b u t e d g r e a t l y to the t h e o r e t i c a l a s p e c t s of s t r u c t u r e d models. C =[ x Y ] (1) However. the r a t e s of r e a c t i o n s t a k i n g p l a c e i n the b i o t i c phase are c o n t r o l l e d by the i n t r i n s i c c o n c e n t r a t i o n of the r e a c t a n t s i . A . Advantages. 7 5 . i-1 1 b.b i o t i c phase. . »14 I n t h i s c h a p t e r a s i m p l e two compartmental model developed by R o e l s ^ w i l l be c r i t i c a l l y e v a l u a t e d w i t h e x p e r i m e n t a l d a t a . f o r these i n t r i n s i c c o n c e n t r a t i o n s . Recent reviews on the s t a t e of the a r t of c o n s t r u c t i n g s t r u c t u r e d models and c r i t i c a l l i t e r a t u r e s u r v e y s can be c o n s u l t e d f o r f u r t h e r information. p o s t u l a t i o n of the r e l e v a n t k i n e t i c e x p r e s s i o n s f o r r e a c t i o n s t a k i n g p l a c e w i t h i n the b i o t i c phase and a t i t s b o u n d a r i e s . one o b t a i n s : 80 . These models have moderate mathematical complexity and are e a s i e r to v e r i f y e x p e r i m e n t a l l y . lioheniformis^^. Based on t h i s d e f i n i t i o n note t h a t C i s g i v e n by : x C X = m E x.b i o t i c phase are c o n t r o l l e d by the c o n c e n t r a t i o n s of the r e a c t a n t s i n t h a t phase. W i l l i a m s has shown t h a t even a s i m p l e model w i t h two compartments c o u l d d e s c r i b e most of the e x p e r i m e n t a l l y observed phenomena.m. e v a l u a t i o n of the c o n s t r a i n t s imposed by e l e m e n t a l b a l a n c e s and by the a p p l i c a t i o n of thermodynamic p r i n c i p l e s . n u t r i e n t dynamics of p h y t o p l a n k t o n growing i n n i t r a t e l i m i t e d environment'3 e t c . Ramkrishna et a l . 0 1 II The i. The f i r s t compartmental model i s due to W i l l i a m s ^ . Some i n c o n s i s t e n c i e s of h i s model were c o r r e c t e d and been m o d i f i e d by R o e l s and KossenS and R o e l s ^ . Thus. Very l i t t l e has so f a r been done w i t h e x p e r i m e n t a l systems. at l e a s t q u a l i t a t i v e l y . The v e c t o r . f o r the system i n c o n s i d e r a t i o n . ii. d e s c r i b i n g these c o n c e n t r a t i o n s i s of dimension n . product f o r m a t i o n by B. X . f u n g a l alpha-amylase production""*. d i s a d v a n t a g e s and f u t u r e p r o s p e c t s of s t r u c t u r e d m o d e l l i n g w i l l be d i s c u s s e d . ' and F r e d r i c k s o n et a l . iii. THEORETICAL DEVELOPMENT OF THE GENERAL STRUCTURED MODEL b u i l d i n g of c h e m i c a l l y s t r u c t u r e d models c o n s i s t s of ^ : s e t t i n g up mass b a l a n c e s f o r r e l e v a n t components of the b i o m a t e r i a l over an a p p r o p r i a t e l y chosen system. growth i n a c t i v a t e d sludge system''.

e t c .(6) d e s c r i b e s t h e i n t e r n a l s t a t e of t h e biomass and i s independent o f the mode o f o p e r a t i o n . Ill DESCRIPTION OF THE TWO COMPARTMENTAL SYSTEM With r e f e r e n c e t o F i g . 7 i n Chapter 2. T h e r e f o r e i n t h e model to be p r e s e n t e d . i=l 1 In a homogeneous c u l t u r e o f c o n s t a n t volume. e . The o t h e r compartment i s c a l l e d the G-compartment and c o n t a i n s t h e s t r u c t u r a l and g e n e t i c m a t e r i a l i . o^) .( { r . .a — —x r . G m a t e r i a l back t o K m a t e r i a l ( s m a l l m o l e c u l e s ) . the r a t e of biomass p r o d u c t i o n . a s t a t e e q u a t i o n f o r the i n t r i n s i c c o n c e n t r a t i o n s can be d e r i v e d as : x dX / d t = {r .b i o t i c phase. / E x . F o r t h e t o t a l biomass i n the system the f o l l o w i n g b a l a n c e e q u a t i o n can be shown t o h o l d : dC x / dt = (r . Furthermore.(4) can be used t o d e s c r i b e the time e v o l u t i o n of the s t a t e of t h e a . A t h i r d r e a c t i o n i n v o l v e s the t u r n over of macromolec u l e s i . J_) X} / C x (6) Eqs(4). Ox . c a l l e d the K-compartment. i n o r d e r to o b t a i n a workable form. i t can be concluded t h a t RNA and c a r b o h y d r a t e f r a c t i o n s change most i n response t o changes i n growth r a t e . K may be expected to be p r o p o r t i o n a l t o t h e RNA c o n t e n t . Here. however. r e l a t i o n s have t o be f o r m u l a t e d f o r J ^ . e . T h i s a l l o w s f o r 81 . A l s o note t h a t eq. t h a t t h e d e r i v a t i o n depends on the r e s t r i c t i o n t h a t o n l y biomass o f the same composit i o n can l e a v e o r e n t e r i n t o the system. S i n c e RNA i s t h e main component. t h e a .b i o t i c and b i o t i c components and the biomass c o n c e n t r a t i o n . 1 — — — x A + $ — v (5) K Thus ( r .(5) and (6) g i v e t h e t o t a l d e s c r i p t i o n of t h e system i . a ) . . carbohydrate and o t h e r s m a l l molecules a r e lumped i n t o one compartment.a + 0 (2) or s p l i t t i n g f o r the two phases : 7<o erratum d X / dt ' — dY / dt - = = r . e . P r o t e i n content changes o n l y s l i g h t l y over t h e same range. F i g . p r o t e i n s . J>y and r . a -y + $ —x (3) (4) i + -y Eq. 1 can be i d e n t i f i e d as r . a g e n e r a l mass b a l a n c e reads : dC / d t = r . 1 and 1-1 1 £ X. from which G forms w i t h a c o n s t a n t y i e l d . i t would be d i f f i c u l t t o c o n s i d e r c a r b o h y d r a t e compartment s e p a r a t e l y as i t s a b s o l u t e i n t r i n s i c c o n c e n t r a t i o n i s v e r y low (about 5 % d r y w e i g h t ) and hence v a r i a t i o n s i n i t a r e d i f f i c u l t t o d e t e c t a n a l y t i c a l l y . 1 g i v e s the b l o c k diagram of the model. Moreover. RNA. 1 m x. Ox). e x t e r n a l s u b s t r a t e i s assumed t o be c o n v e r t e d t o K.X. Thus i t might be d e s i r a b l e t o c o n s i d e r these components e x p l i c i t l y i n a s t r u c t u r e d m o d e l . DNA. Note.

r a t e of s u b s t r a t e uptake i s assumed t o be of s a t u r a t i o n type: r s ' k s S x C C 1 ( K S + C S > (10) Here kg r e p r e s e n t s the maximum r g .\ The r a t e o f t u r n o v e r of G m a t e r i a l back t o K i s tought t o be a f i r s t r e a c t i o n w i t h a y i e l d of 1. = 0 i .K ) C x s i n c e K + G = 1. e . 1: Block diagram of the model.) K K ( 1 . e . when G = 0 t h e r e a r e no enzyme and DNA n e c e s s a r y f o r K f o r m a t i o n . .K ) C x Here mg i s the maintenance f a c t o r f o r the G compartment. . order r m = = m„ G C G x m (12) ( 1 .K G Fig. T h i s has a b i o l o g i c a l e x p l a n a t i o n i . no " iK f o r m a t i o n . ( l l ) p r e d i c t s r^. B e erratum J * . The maintenance process i s accounted f o r o n l y by t h e d e p o l y m e r i z a t i o n r e a c t i o n of the macromolecules w i t h no mass l o s s i n t h e b i o t i c phase. r ^ and r . r ^ i s suggested t o be d e s c r i b e d by r R = = k k K K G C x (1. t h e r a t e e q u a t i o n s The r m (9) have t o be p o s t u l a t e d f o r r g . Thus t h e f o l l o w i n g react i o n s can be f o r m u l a t e d : Y SK K (7) K — Y KG G rate r„ (8) rate Now. Here note t h a t when G =0 e q . the maintenance requirements t o be i n c o r p o r a t e d i n t o t h e model. Such s a t u r a t i o n type r e a c t i o n s a r e c h a r a c t e r i s t i c of enzymic o r m i c r o b i a l r e a c t i o n s . Lj 82 .

} / ^ (1 * ( Y SK S K m x x dynamics of G. s i n c e (15) A s i m i l a r e q u a t i o n need n o t be d e r i v e d f o r the dG/dt = -dK/dt. e. e . C and K by p u t t i n g t h e i r r e s p e c t i v e time d e r i v a t i v e s equal t o z e r o .b i o t i c s t a t e v e c t o r and the s t o i c h i o m e t r y m a t r i x read : a S i m i l a r l y f o r t h e b i o t i c phase : Y X = [ K G ] [ -1 0 i T 0 ] SK 0 KG ( 1 -1 Thus f o r t h e two compartment system d e s c r i b e d .. When the mode o p e r a t i o n i s known t h e b a l a n c e s culture operation : J) = D ( C .K) Y -r S Y SK S K +< P S r s r g (13) + (Y KG r K x K R G x . i s now g i v e n by : r = r r„ L S r„ K r m 1 J The a .IV DERIVATION OF THE BALANCE EQUATIONS The v e c t o r of r e a c t i o n r a t e s r .g. For i n s t a n c e K* can be shown t o be g i v e n by: x K* = (D + m ) / ( k G R Y ) R G (18) Another i n t e r e s t i n g r e l a t i o n t o be d i s c u s s e d l a t e r on. t h e f o l l o w i n g can be d e r i v e d : dC dC dK / d t = g / dt / dt {(1 . e q s ( 1 3 ) . .C ) Yx< si s D C a r e f i x e d . . i . f o r continuous (16) (17) Steady s t a t e r e l a t i o n s can be o b t a i n e d f o r Cg.1) K) + (14) r.V Y sx + ( V Y SK ) ( D + m G ) ( k K \ G - ( D + m G ) ) ( 1 " KG> °x Y ° 9 ) 83 . (14) and ( 1 5 ) . can be g i v e n f o r t h e steady s t a t e a s : r S .

3 r a t e c o n s t a n t s (kg.b a t c h and b a t c h d a t a and used f o r t h e s i m u l a t i o n o f c o n t i n u o u s c u l t u r e d a t a . 2 ) .. m ) and one M i c h e a l i s . steady s t a t e c o n t i n u o u s c u l t u r e b e h a v i o u r was s i m u l a t e d w i t h t h e two compartment model ( F i g . Model output kg Biomass Substrate K compartment (++) very s e n s i t i v e k K Parameters m G Y S K Y K G Kg - + + + . and i s t h e r e f o r e s p e c i f i c t o i t .b a t c h d a t a presented p r e v i o u s l y . the model p r e d i c t s wash-out a t y = i. x - In o r d e r to check whether the model c o u l d p r e d i c t o v e r s h o o t phenomenon d u r i n g a d a p t a t i o n phase f o l l o w i n g a s h i f t . I n F i g . kj^ . However. The model w i l l now be t e s t e d w i t h the continuous and f e d .+ + + + + + + + (-) i n s e n s i t i v e I t must be reminded t h a t t h i s a n a l y s i s was c a r r i e d o u t f o r f e d . A s l i g h t s y s t e m a t i c d e v i a t i o n of 2 .3 % i s e v i d e n t a t the p l a t e a u of the C p r o f i l e . t h e s l o p e s and i n t e r c e p t s are markedly d i f f e r e n t .V EVALUATION OF THE VALIDITY OF THE MODEL K The model presented has s i x parameters. K G Model parameters were e s t i m a t e d from f e d .M e n t e n type c o n s t a n t Kg .. R e s u l t s a r e shown i n Table I I . 2 s t o i c h i o m e t r i c c o e f f i c i e n t s ( Y Q . There seems to be a v e r y good agreement between the model and the experiment. Both s e t s o f d a t a a r e w e l l d e s c r i b e d by s t r a i g h t l i n e s .05 h r ' which i s i n v e r y good agreement w i t h the e x p e r i m e n t a l l y determined Umax v a l u e s . Table I I : Model s e n s i t i v i t y t o i t s parameters. With parameters l i s t e d i n Table I .b a t c h e x p e r i ment FB 830. Parameter k s G 1 96 5 0 0 06 0 73 0 66 0 07 hr-> hr H K m Y k hr" 1 SK YKG K kg/kg kg/kg kg/m 3 S A rough s e n s i t i v i t y a n a l y s i s o f the model t o i t s parameters was c a r r i e d o u t i n a manner s i m i l a r t o t h a t e x p l a i n e d i n Chapter 2 f o r b a t c h growth m o d e l l i n g . Moreover. a wash-out experiment was s i m u l a t e d . Table I : Model parameters. The parameters a r e l i s t e d i n Table I . 3 the p r e d i c t e d K and e x p e r i m e n t a l l y determined RNA f r a c t i o n s a r e shown as f u n c t i o n s o f y. 84 . Y g ) .+ + + + + .

b a t c h experiment FB 830 i s shown t o g e t h e r w i t h the e x p e r i m e n t a l d a t a . VI DISCUSSION From the f o r e g o i n g one can i n g e n e r a l conclude t h a t the p r e s e n t e d s i m p l e structured model can d e s c r i b e most of the phenomena observed i n p r a c t i c e ..C x (kg/m ) 3 Fig. In f a c t s t r u c t u r e d models would be expected t o be s u p e r i o r to u n s t r u c t u r e d models under h i g h l y t r a n s i e n t c o n d i t i o n s . the s t r u c t u r e d model p r o v i d e s a b e t t e r d e s c r i p t i o n than the u n s t r u c t u r e d model (compare w i t h F i g .-) K profile and experimentally determined RNA straight line fitted to RNA data by linear profile. 2 of Chapter 4 ) . 2: Simulation of continuous culture behaviour with the model.1 to D = 1. step-up and down experiments or s i n u s o i d a l f e e d i n g schemes. Here y i n c r e a s e s v e r y f a s t and o v e r s h o o t s the y v a l u e b e f o r e r e a c h i n g the new steady s t a t e v a l u e . 3: Predicted (. 5 the model s i m u l a t i o n f o r f e d . m a x I n F i g . Thus i t would be much b e t t e r t o study and t e s t them under p r e c i s e l y c o n t r o l l e d t r a n s i e n t e n v i r o n m e n t a l c o n d i t i o n s e..g. 4 ) . 85 . Here. U n f o r t u n a t e l y . Fig. A step i n d i l u t i o n r a t e from D = 0. such d a t a i s l a c k i n g f o r model t e s t i n g .2 was a p p l i e d ( F i g . regression.

D 6. at l e a s t q u a l i t a t i v e l y .2 .05 . The model f a i l s to g i v e an a c c u r a t e d e s c r i p t i o n of the RNA f r a c t i o n . t h i s i s h a r d l y s u r p r i s i n g as no c o n s i d e r a t i o n has been g i v e n to any r e g u l a t o r y mechanism o p e r a t i n g i n s i d e the biomass. The model a l s o a l l o w s f o r maintenance t u r n o v e r to be a f u c t i o n of the growth r a t e as can be seen from eq. I t can be seen t h a t the model would p r e d i c t d e c r e a s i n g maintenance requirements a t low growth r a t e s which might be expected on t h e o r e t i c a l grounds.03 0. - 86 .(19) i f i t i s compared w i t h the l i n e a r r e l a t i o n f o r s u b s t r a t e consumption g i v e n i n Chapter 2. 4: Response of the growth from D=0. i n a d d i t i o n to q u a n t i t a t i v e l y p r e d i c t i n g e v e r y t h i n g an u n s t r u c t u r e d model can. The system has simply been modelled i n analogy w i t h c h e m i c a l k i n e t i c s i . rate to a step change in the dilution rate Fig. The model.1. ( 1 8 ) ) . e .11 (see e q . Thus one can conclude t h a t the exact q u a n t i t a t i v e n a t u r e of the dependence of RNA f r a c t i o n to the growth r a t e i s not d e s c r i b e d by the model. 5: Model simulation of the fed-batch experiment FB 830.2- Ol 10° I I I 1 1 1 1 1 I I M I 1 2 1 1 1 I I I I I to 3 10' io Fig. p r o v i d e s i n f o r m a t i o n about the i n t e r n a l s t r u c t u r e of the biomass and on a d a p t a t i o n and o v e r s h o o t i n g phenomena. a v a l u e of 0. As R o e l s ^ has noted. T h i s can be f u r t h e r analysed by e v a l u a t i n g the r a t i o of the i n t e r c e p t t o the s l o p e of the r e l a t i o n f o r K* v s . .5 h r ' i s o b t a i n e d which i s f a r too h i g h . T h i s r a t i o equals mg .1 to 1. assumed to be only chemically s t r u c t u r e d . I f W.Q i s determined from the e x p e r i m e n t a l RNA d a t a i n t h i s manner. A r e a l i s t i c v a l u e should be 0.

a n a l y t i c a l t e c h n i q u e s and c o m p u t a t i o n a l procedures get b e t t e r . Veerman f o r h i s c o n t r i b u t i o n to this 87 . The e x p e r i m e n t e r must aim f o r a comprimise between b i o l o g i c a l sense and m a t h e m a t i c a l c o m p l e x i t y . S t r u c t u r e d m o d e l l i n g i s s t i l l i n i t s infancy.. A s t r u c t u r e d model s h o u l d b e s t be t e s t e d f o r i t s p r e d i c t i o n c a p a c i t y of the i n t e r n a l c o m p o s i t i o n i n a d d i t i o n t o k i n e t i c and e n e r g e t i c b e h a v i o u r . from the K compartment and RNA d a t a a n a l y s i s i t was concluded t h a t the model was not j u s t after a l l . x U n f o r t u n a t e l y . Acknowledgement : The author wishes to thank s t u d e n t chapter. s t r u c t u r e d models w i l l r e p l a c e the u n s t r u c t u r e d ones.Another shortcoming of the model i s t h a t i t p r e d i c t s maintenance r e q u i r e m e n t s as the t u r n o v e r of o n l y the G compartment i . T. In l i t e r a t u r e some a u t h o r s proposed s t r u c t u r e d models w i t h compartments of a b s t r a c t f u n c t i o n s . These models c o u l d never be t e s t e d and t h e r e f o r e remain as m a t h e m a t i c a l e x e r c i s e s . no maintenance f u n c t i o n i s a s s o c i a t e d w i t h the p r e s e r v a t i o n of the K compartment. has a p p e a r e d ^ I t has '72' parameters and a s i z a b l e p r o p o r t i o n of the parameters were o b t a i n e d by curve f i t t i n g procedures or e s t i m a t e d a r b i t r a r i l y . e . R e c e n t l y i n l i t e r a t u r e a model c l a i m e d to be one of the most comprehensive so f a r .g. I f i n t e r n a l c o m p o s i t i o n i s not checked q u i t e m i s l e a d i n g c o n c l u s i o n s may be drawn. e. check mechanisms and o b t a i n parameters from independent e x p e r i m e n t s .However. However. . the model p r e s e n t e d can be accepted as a good a p p r o x i m a t i o n t o r e a l i t y based on the C p r o f i l e s . t h e r e i s no doubt t h a t as the u n d e r s t a n d i n g of m i c r o b i o l o g i c a l s y s t e m s . more b i o l o g i c a l l y m e a n i n g f u l models c a l l f o r e x t r a compartments and t h i s u s u a l l y b r i n g s about m a t h e m a t i c a l c o m p l e x i t y . whenever p o s s i b l e . He s h o u l d . There seems to be g r e a t scope f o r the development of these models much e f f o r t i s needed i n t h i s d i r e c t i o n . From a m a t h e m a t i c a l p o i n t of v i e w i t i s v e r y hard not to assume the whole e x e r c i s e as one of curve f i t t i n g and programming.

. . J . S. t o be p u b l i s h e d i n Advances i n B i o c h e m i c a l E n g i neering. M i c r o b i o l . Gen. Theor. A. Y. A. Ann. 15.M. Leung and C. T h e s i s . Kossen. U. 13. Advances i n A p p l i e d Microbiology.T.G.Moo-Young. B i o c h e m i c a l E n g i n e e r i n g .b i o t i c phase y VIII 1.5. . 5.M. .L. H e r b e r t . F. Ph. B e l l a and H. 1 I.F. N. 3 2 6 . Gen. S c i . Ph.18.W.J. 95(1978). Symp. Grenney.G. 2 9 . T s u c h i y a . 3 2 7 ( 1 9 7 9 ) . Kossen. F r e d r i c k s o n and H.13. F r e d r i c k s o n . 129(1967) . Acad. 14. 2. R. A g r i c u l t u r a l U n i v e r s i t y o f Wageningen.15. R. J.190(1967). Soc. E n e r g e t i c s and K i n e t i c s . Soc.(1977)..A. T s u c h i y a . B i o t e c h n o l ..VII C c NOMENCLATURE chemical s t a t e vector c o n c e n t r a t i o n (kg/kg) d i l u t i o n rate (hr~l) mass f r a c t i o n o f G compartment i n d r y biomass (kg/kg) mass f r a c t i o n o f K compartment i n d r y biomass (kg/kg) s a t u r a t i o n c o n s t a n t (kg/m^) r a t e constant ( h r ' ) maintenance f a c t o r f o r G compartment ( h r ) t o t a l s u b s t r a t e (kg) t o t a l biomass (kg) r e a c t i o n r a t e (kg/m3/hr) maintenance r a t e (kg/m3/hr) mass f r a c t i o n o f b i o t i c component (kg/kg) y i e l d o f G compartment on K compartment (kg/kg) y i e l d o f K compartment on s u b s t r a t e (kg/kg) . Bioeng. B i o l .M. 12. 10.. Ramkrishna. 15.177 (1978) . D i c k . F i t z p a t r i c k . A p p l . v a n Dedem and M. 4. B i o t e c h n o l .391 (1 961 ) .(1979).W. A. 11. S.1 D G K K k m M Mx r S G S m X Y G K r ot $ u stoichiometry matrix f l o w r a t e (kg/m3/hr) s p e c i f i c growth r a t e ( h r ' ) - subs c r i p t s S substrate initial i x biomass ( o r b i o t i c phase) a .T.17.1481(1976). G. W.331(1973). B i o t e c h n o l .F. Biotechnol. 3. E u r . 9.A. S c h u l e r .D. M i c r o b i o l . 6. A.I.A. J.D.S. N. 14. J . R o e l s .A. T h e s i s . . Symp.. 8. REFERENCES D. W i l l i a m s . M. 3 5 ( 1 9 7 9 ) . D. M i c r o b i o l . Harder and J. i n P r o g r e s s i n I n d u s t r i a l M i c r o b i o l o g y . Bioeng.G. K j a e r g a a r d . R o e l s .M.C. Bioeng.. L a r s e n and L.D. 88 . A. a book t o be p u b l i s h e d by E l s e v i e r .419(1970). C u r l . Bioeng. Harder. B i o t e c h n o l .1301(1975). Megee and H. D.9. F r e d r i c k s o n .C. Roels and N. 7.

t r e a t e d system i n o r d e r t o promote a c o n s t r u c t i v e d i s c u s s i o n which may be u s e f u l f o r f u r t h e r r e s e a r c h and a b e t t e r u n d e r s t a n d i n g o f the maintenance metabolism. F.. b c d 3 3 3 + $ C 0„ + * H. c. 3 . examples o f m i s i n t e r p r e t a t i o n o f d a t a from such systems w i l l be p o i n t e d out. J . 1 9 8 0 . Based on i n c o r r e c t t h e o r e t i c a l t r e a t m e n t s and b i a s e d e s t i m a t e s . a l . R o e l s and N. i n our o p i n i o n have drawn a v e r y m i s l e a d i n g p i c t u r e o f t h e e n e r g e t i c s t a t u s o f a n a e r o b i c systems (Cromie and D o e l l e .b a l a n c i n g p r i n c i p l e s . We t h e r e f o r e f e e l o b l i g e d to express our o p i n i o n about t h i s a p p a r e n t l y i l l . In t h i s work.0 2 2 6 7 (1) * P u b l i s h e d i n B i o t e c h n o l o g y L e t t e r s . a g e n e r a l s t o i c h i o m e t r i c e q u a t i o n can be w r i t t e n f o r m i c r o b i a l growth on a s i n g l e carbon and energy source w i t h ammonia as the n i t r o g e n source. . + b. Esener. No. The r e a d e r i s r e f e r r e d t o t h e o r i g i n a l a r t i c l e s f o r a d e t a i l e d a n a l y s i s S t a r t i n g from m a c r o . 2 3 5 $iC H. Kossen SUMMARY P r o d u c t f o r m a t i o n d u r i n g a n a e r o b i c growth i n t h e absence o f e x t e r n a l e l e c t r o n a c c e p t o r s has been shown t o be l i n k e d t o t h e energy p r o d u c t i o n p r o c e s s e s .C H A P T E R 8/APPLICATION 1 COMMENTS ON THE DESCRIPTION OF MAINTENANCE METABOLISM DURING ANAEROBIC GROWTH WITH PRODUCT FORMATION * A. i t has been c l a i m e d t h a t the maintenance r e q u i r e m e n t s c o u l d be reduced e f f e c t i v e l y t o z e r o b y merely v a r y i n g t h e c h e m i c a l c o m p o s i t i o n of t h e c u l t i v a t i o n medium (Cromie and D o e l l e . d. Goma e t . f i r s t . E>C H 0 N a b c d 2 2 2 2 2 + $ 0„ + <KNH. 15-20 (1981) 89 . energy g e n e r a t i o n i s coupled t o t h e product f o r m a t i o n p r o c e s s and s e c o n d l y .]-. 1 9 7 9 ) . 1 9 8 0 ) . e x p e r i mental d a t a r e s u l t s i n an i n c o r r e c t d e s c r i p t i o n o f the maintenance metabolism RESULTS AND DISCUSSION Maintenance metabolism d u r i n g a n a e r o b i c growth has r e c e n t l y r e c e i v e d much a t t e n t i o n i n t h e l i t e r a t u r e . A. 0 N. i t w i l l be shown t h a t i n a n a e r o b i c systems w i t h product f o r m a t i o n i n t h e absence o f e x t e r n a l e l e c t r o n a c c e p t o r s . I t has been demonstrated t h a t when t h i s f a c t i s n o t t a k e n i n t o account. $ C 3 a 3 PL 0 N. A. V o l . F o r s i m p l i c i t y o n l y one product w i l l be considered. W. The m a t h e m a t i c a l t r e a t ment t o be used f o l l o w s from the work o f Roels(1980) and R o e l s and Kossen (1978). Some o f these a r t i c l e s .

C x sx s x (6) (Erickson et. 1958).1978). r x + ( m s . I 9 8 0 . T h e r e f o r e . can be g e n e r a l i z e d to account f o r the product f o r m a t i o n d u r i n g growth (Humphrey and J e f f e r i s . r s = (1/ Y sx .m . r = Y . i n t r o d u c e d a f t e r the p o s t u l a t i o n of the maintenance mechanism ( H e r b e r t . i f the above d e s c r i p t i o n h o l d s . of the form : Y . which i s a s p e c i a l case .For a system at steady s t a t e . Y px 90 (8) and (9) : (10) = Y • Y' p SX / ( Y -Y S X • Y' SX ) . i n the s u b s t r a t e consumption e q u a t i o n . the f i r s t p r o p o r t i o n a l to the biomass p r o d u c t i o n . the f o l l o w i n g e x p r e s s i o n i s o b t a i n e d f o r the s u b s t r a t e consumption r a t e .( 3 ) d e s c r i b e the g e n e r a l case. T h i s i m p l i e s t h a t the energy g e n e r a t i o n i s d i r e c t l y coupled t o the product f o r m a t i o n .. r = r / Y + r / Y + m . can a l s o be d e s c r i b e d by t h i s set of e q u a t i o n s when the oxygen uptake r a t e i s put equal to z e r o . m' . when r ^ = 0.1978. For the case of a n a e r o b i c growth w i t h product f o r m a t i o n .Y -Y' )/( Y -Y' ) ) . 1 9 7 3 . T h i s type of e x p r e s s i o n s have a l r e a d y been used f o r some processes (Roels. (2) be shown to h o l d f o r the oxygen consumption r a t e (3) r = r / Y + r / Y + m . f o r t h i s s p e c i a l case ( a n a e r o b i c growth w i t h the e x c r e t i o n of one product or a m i x t u r e of them i n c o n s t a n t r a t i o ) no separate term f o r rp appears i n the r e x p r e s s i o n .Y sp )). i .al. The a n a e r o b i c case. and the second to the amount of biomass p r e s e n t . e . f l o w s of a l l components can be expressed i n terms of any t h r e e known f l o w s . r + ( y / y ).m .Y / Y ). C s x sx p sp S A s i m i l a r e x p r e s s i o n can now X . Roels and Kossen.(3) one o b t a i n s : r p = Y op ( -r / Y X OX . r can hence be expressed by : s r s = r / Y' + m' . Combining t h i s r e s u l t w i t h a degree of r e d u c t i o n balance R o e l s . C o x ox p op o x Equations ( l ) . R o e l s . 1 9 8 0 ) . C (8) p S X sx p SX X s p S X E q u a t i o n (6) shows t h a t . C O X ) (4) S u b s t i t u t i n g t h i s r e s u l t i n eq(2) and r e a r r e n g i n g . E q u a t i o n (8) shows t h a t the r a t e of product f o r m a t i o n thus c o n t a i n s two c o n t r i b u t i o n s . from eq. C o op sp (5) X The terms g i v e n i n p a r a n t h e s i s i n the above e q u a t i o n are a l l c o n s t a n t s and hence i t becomes c l e a r t h a t t h e r e remains no s e p a r a t e c o n t r i b u t i o n f o r r _ .r + Y . The l i n e a r law of s u b s t r a t e consumption. C (9) p x px p X s the f o l l o w i n g r e l a t i o n s h i p s can be o b t a i n e d by comparing eqs.r s s x x p p (7) an e x p r e s s i o n f o r the r a t e of product r f o r m a t i o n can be f o r m u l a t e d : = (( y .Y op /(Y ox .1980). i f rp i s expressed s i m p l y as : r = r / Y + m .

see F i g . i t was found o u t t h a t a l l t h e i r parameter e s t i m a t i o n s w i t h the e x c e p t i o n o f one (experiment no:10.02 0. t h e i r a n a l y s i s o b v i o u s l y takes no account o f t h e energy produced d u r i n g product f o r m a t i o n .010 fig. g e n e r a l l y t h e above presented argument seems t o h o l d f o r t h i s system.0.e q / C . Energy produced i n t h e form o f ATP d u r i n g e t h a n o l p r o d u c t i o n can n a t u r a l l y be c h a n n e l l e d t o s a t i s f y the maintenance demands. m and nipWere determined by p e r f o r m i n g l i n e a r r e g r e s s i o n a n a l y s i s on the data r e p o r t e d by Cromie and D o e l l e (1980) u s i n g e q u a t i o n s (6) and ( 9 ) . assuming average v a l u e s o f = 4. -) eq. fig.(10). 3) i l l u s t r a t e s the p o s s i b l e mechanisms f o r the d i s t r i b u t i o n o f the s u b s t r a t e energy. a n a e r o b i c a l l y . Yp . I t can be n o t i c e d t h a t t h e s o l i d l i n e i n f i g . an a n a l y s i s o f t h e i r o r i g i n a l d a t a by t h e above presented procedure r e v e a l e d t h a t t h e i r c o n c l u s i o n s were i n c o r r e c t . product f o r m a t i o n i s indeed a s s o c i a t e d w i t h the energy g e n e r a t i o n i n the system.m p = ( Y / Y ) • m' s p s Y (11) Y These r e l a t i o n s h i p s a r e shown as s o l i d l i n e s i n f i g u r e s 1. and 2. f o r the case o f e t h a n o l p r o d u c t i o n from g l u c o s e a n a e r o b i c a l l y . i .005 0. a n a e r o b i c a l l y .Y « Y (see eq. a l (1979) who have c a l c u l a t e d zero maintenance d u r i n g e t h a n o l p r o d u c t i o n by Saceharomyoes oerevisiae growing on g l u c o s e .e q ) sx i | 0. A s i m p l e b l o c k diagram ( f i g . m vs.03 0. m' : (p s s x x s ) eq. F o r the two cases f o r which Cromie and 91 . When t h e i r data were s u b j e c t e d t o l i n e a r r e g r e s s i o n a n a l y s i s . e . A l t h o u g h t h e r e i s c o n s i d e r a b l e s c a t t e r i n f i q . These a u t h o r s c l a i m e d t h a t they have succeeded i n r e d u c i n g the maintenance requirements e f f e c t i v e l y t o zero by changing the c h e m i c a l c o m p o s i t i o n o f t h e c u l t i v a t i o n medium they have used f o r e t h a n o l p r o d u c t i o n by Zymomonas mobilis.01 Y' ( C . However. However. 4. i . i s w e l l approximated by t h e s t r a i g h t l i n e px P • sx x. x = ( Y 1 Y s ) Y s i n c e Y s x s m . U s i n g eq. ( • ) experimental ( • ) experimental data data (Cromie (Cromie and and Doelle) Doelle) Parameters Y .0 (C-eq/C-eq/hr) 2.(10)) . Table I ) were i n g r e a t e r r o r s .. 1 .(11). (2) these workers have c a l c u l a t e d m to be zero towards t h e end o f the f e r m e n t a t i o n . T h i s type o f energy g e n e r a t i o n was n o t taken i n t o account by Goma e t . g R e c e n t l y another c l a i m o f z e r o maintenance came from Cromie and D o e l l e (1980).10 P Y 0.04 px (C-eq/C-eq) 5. 1' :( pec sec 2. e . J 1:1 vs. 2 . When f u r t h e r a n a l y s e s were c a r r i e d out i t became obvious t h a t t h e i r c o n c l u s i o n s were e n t i r e l y based on t h e i r b i a s e d parameter e s t i m a t i o n t e c h n i q u e .5 - 0.2 and t h e m o l e c u l a r weight o f t h e organism as 25.

as one mole o f ATP i s produced f o r every two moles o f e t h a n o l produced v i a the Enthner-Doudorof pathway ( f o t the case o f Z. A n e g a t i v e v a l u e f o r m o b v i o u s l y does n o t make any sense. g s product synthesis use of substrate —*~ synthesis of biomass precursors biomass synthesis ii i ATP pool maintenance fig.D o e l l e (1980) c l a i m e d zero maintenance a l i n e a r r e g r e s s i o n procedure y i e l d e d the v a l u e s o f 1. Mg m s (Cromie & D o e l l e 1980) g/g/hr.85 and -1.8 1 .8 0. ATP consumption. 5. e i t h e r the model does n o t h o l d f o r t h i s s i t u a t i o n or/and the e x p e r i m e n t a l data a r e n o t a c c u r a t e . 3: Distribution of the substrate and Kossen. no: N and c a l c u l a t e d maintenance c o e f f i c i e n t s . 1978) energy in microbial metabolism (Roels Table I : Reported Exp.9 3. mobilis ) : 92 . i t does n o t a l l o w the experimenter to assume and r e p o r t i t z e r o .9 2.4 m g (linear regression)* g/g/hr ( t h i s study) 1 1 2 5 2 1 6 4 -1 3 5 55 05 35 80 95 85 75 05 25 00 00 1 2 3 4 5 6 7 8 9 10 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 05 05 05 10 10 10 20 20 0 0 0 0 0 0 0 0 0 0 0 15 30 45 15 30 45 15 30 45 30 45 N = (NH ) SO. i s g i v e n by ( Stouthamer and Bettenhaussen.1973) : q ATP = " I ATP Y + m ATP ( 1 2 ) Moreover.0 1 .9 5. A l l t h a t can be s a i d i s t h a t . Mg = MgSO % .0 5. *From the o r i g i n a l d a t a r e p o r t e d by Cromie and D o e l l e (1980). S i n c e i t has been shown t h a t the product f o r m a t i o n was d i r e c t l y coupled t o energy g e n e r a t i o n an energy balance f o r t h i s system can now be g i v e n .0 3. %.2 3.25 g/g/hr f o r m (experiment no: 6 and 9 i n Table I ) .0 0. C o n s i d e r i n g ATP as the energy c u r r e n c y . i n terms o f t h e l i n e a r law. however.

9 0. The statement of Cromie and D o e l l e (1980) " In v a r y i n g not o n l y the d i l u t i o n r a t e but a l s o the ammonium 93 . AT ^ATP (mmole/g. i s a good e t h a n o l producer because of i t s h i g h m a i n t e nance requirements and s i n c e i t cannot generate s u f f i c i e n t energy v i a any o t h e r m e t a b o l i c r o u t e . fig.25 0.biomass/hr can be c a l c u l a t e d ( F i g .5 6. mobilis .biomass/hr) 0. oerevisiae c. paraplosis z. i s l i n k e d to the energy p r o d u c i n g p r o c e s s e s .8 9. mobilis glucose g l u c o s e (minimal) g l u c o s e (complex) tryptophan glucose glucose glucose glucose glucose glucose * References as quoted by Stouthamer (1977) * * C a l c u l a t e d from d a t a r e p o r t e d by Cromie and D o e l l e (1980) f o r exp.70 0. to s a t i s f y them. Z.10 fig. I n f a c t i t i s q u i t e p r o b a b l e t h a t t h e i r organism.5 0.3** E. determination for experiment Table I I : Organism L. casei E.05 0. no:6 by eq. Growth l i m i t i n g substance n* ATP 1 . T h i s f a c t must be taken i n t o account when d a t a o b t a i n e d from such systems are to be i n t e r p r e t e d . aerogenes v a l u e s f o r some organisms grown a n a e r o b i c a l l y i n chemostat .21 10. cerevisiae S. coli S. T h i s v a l u e i s q u i t e h i g h when compared w i t h some of the d a t a c o l l e c t e d and r e p o r t e d by Stouthamer (1977) as shown i n Table I I . One can t h e r e f o r e conclude t h a t the maintenance demands were never reduced t o z e r o as c l a i m e d by Cromie and D o e l l e (1980). no:6. CONCLUSIONS Product f o r m a t i o n (a s i n g l e product or a c o n s t a n t r a t i o m i x t u r e ) d u r i n g a n a e r o b i c growth i n the absence of e x t e r n a l e l e c t r o n a c c e p t o r s . 4: Regression 5: m ATP line for experiment no:l.9 38.(12).7 18.q Ethanol ( M / Y ATP + m ATP } (13) I f t h i s e q u a t i o n i s f i t t e d to the d a t a of experiment no:6 ( f o r which a z e r o maintenance c l a i m has been made) an m p of 10 mmoles ATP/g. 5 ) .

Stockholm.G.. M.301. J. . the maintenance c o e f f i c i e n t m.3d s p e c i f i c growth r a t e oxygen substrate biomass REFERENCES Cromie. Sao P a u l o . M i c r o b i o l . A l m q v i s t . 0 N ^3 3 * 3 3 " C d 2 2 2 biomass e l e m e n t a l formula substrate elemental formula product e l e m e n t a l formula 2 m x C. A. G. (1958) i n 'Recent P r o g r e s s i n M i c r o b i o l o g y ' Symp..E.J. B i o p h y s . E r i c k s o n .W.H. (1979) B i o t e c h n o l .. M o l e t t a . and D o e l l e . . 287. (1977) Symp. Stouthamer. 2 . B u l l . A. H e r b e r t .W.A.4. vol. 1 ( 1 0 ) .s u l p h a t e ( n i t r o g e n source) and magnesium s u l p h a t e c o n c e n t r a t i o n s .14. and Bettenhaussen. 3 5 7 . Humphrey.eq. R o e l s . ( 8 ) . L e t t . I n f a c t i t can be shown t h a t a h i g h maintenance v a l u e f o r an e t h a n o l p r o d u c i n g organism v i a t h i s r o u t e . (1973) B i o c h i m .E. 4 1 5 . R.. Ed. 27.K.p..j H Yi Y C y subscripts 0 s X biomass c o n c e n t r a t i o n carbon e q u i v a l e n t maintenance c o e f f i c i e n t o f the i ' t h component maximal y i e l d o f j on t h e i ' t h component f l o w o f t h e i ' t h component degree o f r e d u c t i o n o f the i ' t h component Y i = 4a + b .2c . 20. S.F. R o e l s .Bioeng.53. (1078) B i o t e c h n o l . L e t t . H. M.H. B i o e n g .A.. N. (1980) B i o t e c h n o l .7 t h I n t . M i n k e v i c h . Amsterdam. and Novak.K.22. A.2457.767. Goma. Congr. J. Stouthamer. P. . was s u c c e s s f u l l y reduced t o z e r o a t a l l d i l u t i o n r a t e s " i s based on t h e i r i n c o r r e c t c o n c e p t u a l and mathematical t r e a t m e n t .p95..(1980) Biotechnol.1595.(1973) GIAM m e e t i n g .381. D. and Kossen. 0 N.. Soc. and J e f f e r i s . i s a d e s i r a b l e p r o p e r t y as t h i s w i l l i n c r e a s e t h e e f f i c i e n c y o f c o n v e r s i o n o f the s u b s t r a t e t o the p r o d u c t . G. I.. V.. . and E r o s h i n . C. L. T u n e w a l l .. . Gen. i i. 94 . Acta. M i c r o b i a l .p. E d . NOMENCLATURE C H. ^1 ! l C H. E l s e v i e r . B r a s i l . 0 Nj a b c d C H. (1978) i n P r o g r e s s i n I n d u s t r i a l M i c r o b i o l o g y .

S i n c e COD i s c o n s t a n t and BOD v a r i a b l e . J.A. Prot. the r a t i o i s a f u n c t i o n o f t h e e f f i c i e n c y o f m i c r o b i a l growth. The i n f l u e n c e of a s e l e c t e d change i n t h e e n v i r o n m e n t a l c o n d i t i o n s on t h e r a t i o . The COD o f any waste i s a constant since i t s determination involves a c a r e f u l l y c o n t r o l l e d chemical r e a c t i o n . INTRODUCTION BOD and COD a r e t h e most commonly used parameters f o r t h e c h a r a c t e r i z a t i o n o f waste w a t e r s .A. Roels and N. However. S e v e r a l workers i n t h e waste water f i e l d have r e a l i z e d t h a t t h e r e a c t i o n s t o i c h i o m e t r y was n o t c o n s t a n t .3 No. b u t v a r i e d . Of t h e s e . F o r sewage o f t e n a r a t i o o f 2 . 1977).'. Esener. f o r * P u b l i s h e d i n B i o t e c h n o l o g y L e t t e r s . f o r a b a c t e r i a l system. has been e v a l u a t e d e x p e r i m e n t a l l y .W.Brd. the BOD v a l u e determined f o r t h e same sample may v a r y under d i f f e r e n t e n v i r o n m e n t a l c o n d i t i o n s because t h e e f f i c i e n c y of m i c r o b i a l growth p r o c e s s may be a f f e c t e d s i g n i f i c a n t l y by d i f f e r e n t c o n d i t i o n s . 1972).A 193-198 (1981) 95 . BOD i s the most w i d e l y used measure o f t h e b i o d e g r a d a b l e o r g a n i c content and i s u s u a l l y expressed as t h e 5-day/20 C(B0D5) v a l u e (Schroeder. q u a n t i f i e s t h e t o t a l amount o f o x i d i z a b l e m a t e r i a l i n t h e sample. Swedish Env.The use o f such c o n s t a n t s however. COD on t h e o t h e r hand. l o c a t i o n o f t h e t e s t . As w i l l be shown i n t h e f o l l o w i n g .F. e t c . Vol. The e f f i c i e n c y o f m i c r o b i a l growth i s r e f l e c t e d i n the s t o i c h i o m e t r i c c o e f f i c i e n t s o f the growth e q u a t i o n .2. An a n a l y s i s o f d a t a r e p o r t e d i n l i t e r a t u r e i n d i c a t e s t h a t t h i s r a t i o can assume a wide range v a l u e s f o r d i f f e r e n t s u b s t r a t e s . Kossen SUMMARY A simple e x p r e s s i o n has been d e r i v e d t o demonstrate t h a t t h e r a t i o o f COD t o BOD i s n o t c o n s t a n t b u t a f u n c t i o n of t h e thermodynamic e f f i c i e n c y o f t h e growth p r o c e s s .5 i s used as an a p p r o x i m a t i o n t o COD/BOD (Nat. can be v e r y hazardous under c e r t a i n c o n d i t i o n s . t h e r a t i o COD/BOD cannot be regarded as a constant.APPLICATION 2 BIOENERGETIC CORRELATION OF COD TO BOD * A. S i n c e both o f these u n i t s a r e used and r e p o r t e d i n l i t e r a t u r e i t would be v e r y u s e f u l i f one c o u l d c o r r e l a t e one t o t h e o t h e r . Both o f these u n i t s have advantages and d i s a d v a n t a g e s and t h e c h o i c e u s u a l l y depends on many f a c t o r s such as t h e r e p r o d u c i b i l i t y o f t h e d e t e r m i n a t i o n s . time p e r i o d r e q u i r e d . COD/BOD.

f o r t h e m i c r o b i a l growth process has been approximated by the f o l l o w i n g : ( E r i c k s o n e t a l . F o r s i m p l i c i t y by-product f o r m a t i o n w i l l n o t be i n c l u d e d . 1975. THEORY A g e n e r a l s t o i c h i o m e t r i c r e l a t i o n f o r a e r o b i c m i c r o b i a l growth on a s i n g l e carbon and energy source w i t h ammonia as t h e n i t r o g e n s o u r c e . 0 N.s t a t e f l o w s of the r e s p e c t i v e components. i s known. S h e r r a r d . a g e n e r a l i z e d r e l a t i o n has so f a r n o t been r e p o r t e d . 1980) a r e l a t i o n f o r t h e r a t i o COD/BOD i s developed i n terms of t h e thermodynamic e f f i c i e n c y of the growth p r o c e s s . U s i n g the concept o f the degree of r e d u c t i o n . by: BOD = (Y s BOD / 4) (1 -n ) (6) Furthermore. change i n r e a c t i o n s t o i c h i o m e t r y i s accounted f o r . a c c o r d i n g to e q u a t i o n ( 8 ) . BOD. E r i c k s o n e t a l . E r i c k s o n et a l . They have completed balances a t d i f f e r e n t d i s c r e t e growth r a t e s ( S h e r r a r d and Schroeder. COD. However. the thermodynamic e f f i c i e n c y . u t i l i z i n g t h e r e c e n t l y developed m a c r o . Here.i n s t a n c e . The s t o i c h i o m e t r i c c o e f f i c i e n t shown by $.2c . l the r a t i o .n e g l i g i b l e q u a n t i t i e s . 1978. i . d e f i n e d as t h e amount o f oxygen r e q u i r e d f o r the complete combustion o f u n i t s u b s t r a t e . F o r u n i t amount of s u b s t r a t e consumption. i t i s assumed t h a t c a r b o n .(2) and ( 4 ) . Y> i s the degree of r e d u c t i o n and i s d e f i n e d by the r e l a t i o n : Y and Y" S X Y = 4a + b . the r e a d e r i s r e f e r r e d t o the o r i g i n a l p u b l i c a t i o n s ( M i n k e v i c h and E r o s h i n . 0 N 4 2 3 3 1 b] c j d] + $ C0„ + 5 2 C $. C0D/B0D i s p l o t t e d a g a i n s t the thermodynamic e f f i c i e n c y . 2 a2 D2 C2 d2 + $ 0 + < NH J > — • $ CH. r e p r e s e n t the n e t s t e a d y . C0D/B0D can now be g i v e n s i m p l y as : C0D/B0D = 1 / (1 -n ) (8) In f i g . y. can r e a d i l y be g i v e n by t h e e x p r e s s i o n : COD = (1/4) Y s (7) Thus t h e r a t i o .b a l a n c i n g p r i n c i p l e s and e n e r g e t i c s o f m i c r o b i a l processes ( M i n k e v i c h and E r o s h i n . w i t h the growth r a t e . w i l l be c o n s i d e r e d . R o e l s . e . 1980). I n t h i s work.n . T]. 1980). oxygen and n i t r o g e n c o n s t i t u d e a l l the elements p r e s e n t i n the system i n n o n . a t n=0 C0D/B0D becomes equal t o 1. oxygen demand can be e s t i m a t e d by ( R o e l s . R o e l s . 1978. $„C H. 1980) n = Y" (y /y ) (2) sx X s where. R o e l s . i . 1973. 1978. For a d e t a i l e d d e s c r i p t i o n of the above mentioned t o o l s t o be used h e r e . no 96 . 1973. e . by c o n s i d e r i n g t h e thermodynamic e f f i c i e n c y . hydrogen. t h e b i o c h e m i c a l oxygen demand can be shown t o be g i v e n by : = Y" / Y sx ox o r from eq.3d (3) i s the y i e l d o f biomass on s u b s t r a t e (C-equiv/C-equiv) and i s g i v e n by: sx= *1 1 ( *2 ' 2> a <> 4 Moreover i t has been shown t h a t once.H„0 6 2 (1) Here. 1980) : Y o x Q X = ( 4 / Y ) (0 / (1 -0 )) x (5) Where Y i s the y i e l d o f biomass on oxygen (C-equiv/mole).

U s i n g eqs.46 has been r e p o r t e d w i t h v a r i o u s organisms ( R o e l s .57. E l e m e n t a l c o m p o s i t i o n o f the biomass was determined by a computer c o u p l e d element a n a l y s e r .7. n =0. o x a l a t e . < l RESULTS AND DISCUSSION In a s e t o f experiments c a r r i e d out i n b a t c h mode. F o r growth on methanol f o r example. by u t i l i z i n g the concept o f " g e n e r a l i z e d degree o f r e d u c t i o n " . A l t h o u g h n can t h e o r e t i c a l l y assume any v a l u e between 0 and 1.23 . 1980). A l l experiments were performed i n a 11 x 10~3 m3 w o r k i n g volume b i o r e a c t o r i n b a t c h mode.0. Temperature and pH were s e t and c o n t r o l l e d a t 308 K and 6. The c o m p o s i t i o n o f s u b s t r a t e . i n t r o d u c e d by Roels (1980) i n s t e a d o f e q u a t i o n ( 3 ) . ( g l y c e r o l . Furthermore a l l o x i d i z a b l e m a t e r i a l has been assumed t o be b i o d e g r a d a b l e .26) Roels (1980) has compiled a number o f l i t e r a t u r e d a t a and computedn f o r v a r i o u s s u b s t r a t e s and organisms. which brought a change i n the thermodynamic e f f i c i e n c y o f t h e growth p r o c e s s . Average e l e m e n t a l c o m p o s i t i o n o f the biomass was c a l c u l a t e d to be C H]_62No. ( i .t h e r a t i o COD/BOD 97 . (2) and (8) t h e r a t i o . and 3..01 . I t i s known t h a t t h e e l e m e n t a l c o m p o s i t i o n o f biomass i s u s u a l l y n o t v e r y s e n s i t i v e t o e n v i r o n m e n t a l changes. corresponds t o a s i t u a t i o n where a l l the incoming c h e m i c a l energy s t o r e d i n t h e s u b s t r a t e i s i n c o r p e r a t e d i n t o the biomass.8. I n an a c t u a l waste water s i t u a t i o n however. i t i s important t o e v a l u a t e the most i m p o r t a n t f a c t o r s which i n f l u e n c e the fj. The v a l u e s ranged from 0.7 which r e p r e s e n t s the p r a c t i c a l maximum f o r n. 1. A l l o t h e r e x p e r i m e n t a l c o n d i t i o n s were kept i d e n t i c a l t o e l i m i n a t e ri b e i n g e f f e c t e d by any parameter o t h e r than t h e i n h i b i t o r ( N a C l ) .f a c t o r s can f u r t h e r i n f l u e n c e the y i e l d and c o n s e q u e n t l y the growth e f f i c i e n c y . t h e e n v i r o n m e n t a l c o n d i t i o n s were changed by adding sodium c h l o r i d e i n t o the growth medium. The presence o f i n h i b i t o r s o r t h e absence o f e s s e n t i a l growth c o . From these p l o t s i t i s c l e a r t h a t . r e v e r s i b l y . t h a t t h e treatment does not c o n s i d e r t h e c o n t r i b u t i o n o f t h e p o s s i b l e oxygen demand f o r the n i t r i f i c a t i o n p r o c e s s .biomass i s produced and a l l t h e incoming s u b s t r a t e carbon i s c o n v e r t e d i n t o carbon d i o x i d e . e . r e s p e c t i v e l y . e . Furthermore i t i s known t h a t d i f f e r e n t organisms d i s p l a y d i f f e r e n t thermodynamic e f f i c i e n c i e s when grown on t h e same s u b s t r a t e .23 t o 0. X] as d e f i n e d by e q u a t i o n (2) i s a f u n c t i o n o f the c o m p o s i t i o n o f the biomass and s u b s t r a t e as w e l l as t h e molar growth y i e l d . a range o f n .67 f o r g l u c o n a t e . 2. As the r a t i o o f COD/BOD i s e n t i r e l y dependent on n. COD/BOD was e v a l u a t e d f o r each experiment and p l o t t e d a g a i n s t the NaCl c o n c e n t r a t i o n and the maximum s p e c i f i c growth r a t e determined f o r f o r each b a t c h . i n f i g s . MATERIALS AND METHODS K l e b s i e l l a pneumoniae (aerogenes) NCTC 418 was c u l t i v a t e d a e r o b i c a l l y i n simple s a l t s medium w i t h g l y c e r o l b e i n g the o n l y carbon source. r\ = 1 . correspond t o a change i n COD/BOD o f more than two f o l d . n =0. I t i s t o be n o t e d . I t must be emphasized t h a t these r e s u l t s were o b t a i n e d w i t h mono c u l t u r e s u s u a l l y o p e r a t e d a t optimum c o n d i t i o n s f o r maximum p r o d u c t i v i t y . r e s p e c t i v e l y . from 0. however. however. The o t h e r end o f t h e s c a l e i . Growth on some s u b s t r a t e s i s more e f f i c i e n t than o t h e r s . These v a l u e s as i n d i c a t e d i n f i g . i s o f prime importance. A d d i t i o n o f sodium c h l o r i d e reduced t h e growth r a t e and y i e l d s . a p o l l u t e d r i v e r ) t h e c o n d i t i o n s may be f a r from optimum and hence n much lower. The above p r e s e n t e d treatment can a l s o be extended t o cases w i t h N-sources o t h e r than ammonia.46 » y i e l d i n g a degree o f r e d u c t i o n o f 4. i n p r a c t i c e the maximum e f f i c i e n c y determined has never been found t o exceed 0. The p l o t has t h e r e f o r e been drawn t o n =0.26 f o r o x a l a t e t o 0.


Fig. 1: COD'/BOD versus the thermodynamic efficiency

Na CI (kg/m )
process.

3

of the growth

Fig.

2:

COD/BOD versus

the Sodium

Chloride

concentration

in growth medium.

COD BOD

Fig.

3:

COD/BOD versus

the growth rate

as influenced

by the NaCl

concentration.

changed c o n s i d e r a b l y throughout t h e e x p e r i m e n t a l range. The growth r a t e s determined were much h i g h e r than those n o r m a l l y a t t a i n e d i n waste water treatment i n s t a l l a t i o n s t h e r e f o r e t h e r a t i o may change l e s s d r a m a t i c a l l y i n those a p p l i c a t i o n s . N e v e r t h e l e s s t h e r a t i o COD/BOD must be c o n s i d e r e d as a f u n c t i o n o f t h e growth e f f i c i e n c y which can be a f f e c t e d s i g n i f i c a n t l y by t h e e n v i r o n m e n t a l c o n d i t i o n s . Presence o f an i n h i b i t o r , f o r i n s t a n c e NaCl ( i n t h i s c a s e ) , can t h e r e f o r e a l t e r t h e r a t i o . T h i s may have i m p o r t a n t consequences when i n d u s t r i a l waste waters a r e d i l u t e d t o d i f f e r e n t e x t e n t s p r i o r t o l a b o r a t o r y BOD d e t e r m i n a t i o n s . Hence i t can be c o n c l u d e d t h a t a c o n s t a n t v a l u e f o r COD/BOD cannot be assumed u n l e s s under i d e n t i c a l e n v i r o n m e n t a l c o n d i t i o n s .

REFERENCES Schroeder, E.D., Water and waste water treatment,p.219, McGraw H i l l , New York (1977) N a t i o n a l Sweedish E n v i r o n m e n t a l P r o t e c t i o n Board, S t a t i s t i c a l C o r r e l a t i o n Between A n a l y s i s Data f o r COD and BOD, (1972) as quoted by Johnson COD meter pamphlet. S h e r r a r d , J.H. and S c h r o e d e r , E.D.,(1975) P r o c e e d i n g s o f Purdue conference,p- 14 M i n k e v i c h , I.G. and E r o s h i n , V.K., (1973) F o l i o M i c r o b i o l . , 1 8 , p • 3 7 6 . E r i c k s o n , L.E., M i n k e v i c h , I.G. and E r o s h i n , V.K.,(1978) B i o t e c h n o l . B i o e n g . , 20, p. 1595. R o e l s , J.A.,(1980) B i o t e c h n o l . B i o e n g . , 22, p. 2457. Esener, A.A., Kossen, N.W.F. and R o e l s , J.A. (1980) Paper p r e s e n t e d t o t h e second i n t e r n a t i o n a l symposium on Waste Treatment and U t i l i z a t i o n , h e l d a t W a t e r l o o , Canada.

NOMENCLATURE BOD C H
C 2 b ]

b i o c h e m i c a l oxygen demand 0
2 C l

N
2

d l

biomass e l e m e n t a l formulae s u b s t r a t e e l e m e n t a l formulae
2

a Hb °c Nd COD Y" sx Y ox 0. l
Y

c h e m i c a l oxygen demand biomass y i e l d on s u b s t r a t e biomass y i e l d on oxygen f l o w o f t h e r e s p e c t i v e component degree o f r e d u c t i o n o f t h e i t h component thermodynamic e f f i c i e n c y f a c t o r f o r m i c r o b i a l growth

i n

V subscripts s
X

s p e c i f i c growth r a t e

substrate biomass

99

1

P i r t . i i i . 1980). Roels and N. 1960. 1975. The procedure f o r t h i s method i s as f o l l o w s : i . such t h a t D + AD > U where t h e organism i s expected t o grow a t i t s p o s s i b l e maximum r a t e .(1981). A. growth b e h a v i o u r d u r i n g the wash-out phase has been s t u d i e d w i t h two o b j e c t i v e s i n mind: a. step-up D t o D + AD a t time t=0. as f i r s t d e s c r i b e d by P i r t and C a l l o w (1960). INTRODUCTION The maximum s p e c i f i c growth r a t e i s an important parameter i n the d e s c r i p t i o n of m i c r o b i a l growth. m *Submitted for publication. A. i i .T h e r e f o r e the y has t o be e s t i m a t e d r o u g h l y t o p r i o r t o e x p e r i m e n t a t i o n . Esener. Tempest. u s u a l l y t h e d r y weight oxygen uptake and carbon d i o x i d e p r o d u c t i o n r a t e s as f u n c t i o n s o f time. F. The o p t i m a l e x p e r i m e n t a t i o n range and procedure have been e v a l u a t e d f o r a c c u r a t e e s t i m a t i o n o f the maximum s p e c i f i c growth r a t e s . m a m a x m a x A l t h o u g h the method has been r e p o r t e d and used w i d e l y .APPLICATION 3 DESCRIPTION OF MICROBIAL GROWTH BEHAVIOUR DURING THE WASH-OUT PHASE . a d e t a i l e d t h e o r e t i c a l and e x p e r i m e n t a l e v a l u a t i o n of i t has not been undertaken ( P i r t and C a l l o w . Kuhn e t a l . measure r e s p o n s e ( s ) o f the system. t o t e s t t h e v a l i d i t y o f Monod model under these c o n d i t i o n s . W. V axb. 101 . 1970. t o e s t a b l i s h a s u i t a b l e e x p e r i m e n t a l range and procedure f o r the o p t i m a l e s t i m a t i o n of the b i o k i n e t i c parameter. b r i n g a continuous c u l t u r e system t o steady s t a t e a t D < u x . J . A convenient method of i t s d e t e r m i n a t i o n i n v o l v e s washingout o f a c u l t u r e . I n t h i s work. Kossen SUMMARY M i c r o b i a l growth d u r i n g the wash-out phase has been d e s c r i b e d by an u n s t r u c t u r e d model based on Monod k i n e t i c s and the r e l a t i o n o f l i n e a r s u b s t r a t e consumpt i o n . DETERMINATION OF THE MAXIMUM SPECIFIC GROWTH RATE * A.

r can x I f . r .C ) . A l s o note t h a t t h i s a n a l y s i s assumes n e g l i g i b l e time cons t a n t s f o r the a d a p t a t i o n o f t h e growth r a t e and the subsequent a d a p t a t i o n o f the s u b s t r a t e c o n c e n t r a t i o n .D) t x xo max (5a) Thus Vmax c a n e a s i l y be c a l c u l a t e d by p e r f o r m i n g n o n l i n e a r o r l i n e a r r e g r e s s i o n on the C (In C ) v e r s u s time d a t a o b t a i n e d .THEORY For a constant volume continuous c u l t u r e system the f o l l o w i n g balance equations can be f o r m u l a t e d f o r biomass and the l i m i t i n g s u b s t r a t e c o n c e n t r a t i o n s : d C / d t X = r .D ) t} (6) (7) = CPR exp o {(y .(5) and ( 5 a ) . the r a t e o f biomass p r o d u c t i o n may be g i v e n by the u s u a l Monod relation : r = y {C / (C + k )} C max s s s x g x (3) d u r i n g the wash-out phase. by the use o f eqs.r S S I s s x Here.D ) t} max m a x OUR = ( y / Y o max ox CPR + m o ) C xo ) C xo (8) = (y / Y o max cx m a X + m c (9) 102 . r e s p e c t i v e l y . 1958 . C becomes much g r e a t e r than k be approximated by the f o l l o w i n g : g r x = y C max x f o r biomass. however. P i r t . 1965) can be assumed to be a p p l i c a b l e .D ) t} x xo max or when transformed: (5) In C = I n C + (y . Moreover. . t h e f o l l o w i n g r e l a t i o n s can be d e r i v e d f o r the oxygen uptake r a t e (OUR) and carbon d i o x i d e p r o d u c t i o n r a t e (CPR). exp {(y . i f the l i n e a r s u b s t r a t e consumption r e l a t i o n ( H e r b e r t . p r o v i d e d the r e s p e c t i v e a c c u m u l a t i o n f o r both gases i n the system can be n e g l e c t e d . x x OUR = OUR CPR where. a f t e r i n t e g r a t i o n y i e l d s : (4) I n t h i s case the b a l a n c e C = C exp {( y .DC X X (1) (2) d C / d t = D (C .

e r r o r a n a l y s i s can f a c i l i t a t e the d e t e r m i n a t i o n o f a s u i t a b l e e x p e r i m e n t a l range. OUR. T h i s assumption seems t o be r e a s o n a b l e f o r o n l y s m a l l s t e p s i n D ( P i r t . F o r b i g s t e p s the organism needs a c o n s i d e r a b l e a d a p t a t i o n time to reach b a l a n c e d growth and e x e r t maximum growth r a t e . . 1 9 7 5 ) . C o n s i d e r i n g the s i m p l e s t case o f a wash-out experiment w i t h one response v a r i a b l e . s s m a x A p a r t from p h y s i o l o g i c a l c o n s i d e r a t i o n s . g m I t must be noted t h a t i n the above p r e s e n t e d a n a l y s i s t h e organism i s assumed to adapt to the new d i l u t i o n r a t e i n s t a n t a n e o u s l y and b e g i n t o grow a t i t s maximum r a t e . e .C . D u r i n g such an adaptat i o n p e r i o d the c e l l s w i l l wash-out f a s t e r than expected r e s u l t i n g i n t h e c a l c u l a t i o n o f a Umax v a l u e s m a l l e r than a c t u a l . T h e r e f o r e . si (9) . the e x p e r i m e n t a l c o n d i t i o n s must be chosen w i t h c a r e .(5) as d e s c r i b e d by Himmelblau (1970). + z ) .Furthermore. C .z so si . f o r a c o n s i d e r a b l e p e r i o d o f time f o l l o w i n g the step-up. e . C .(2) and (5) i n c o m b i n a t i o n w i t h the l i n e a r r e l a t i o n f o r s u b s t r a t e consumption: g dC /dt = D (C .l n C i s maximized) i n a d d i t i o n to ' m i n i m i z a t i o n o f the m m a x X 0 s 103 .C ) . „ . . + m ) C /y s xo max The s u b s c r i p t o r e f e r s t o s t a t e a t time t=0. eq.( y /Y + m ) C exp { ( y . To a v o i d t h i s c o m p l i c a t i o n Tempest (1970) recommends the use o f a l a r g e step i n c r e a s e i n D t o a c u l t u r e which has been kept a t steady s t a t e v e r y c l o s e to the t r u e U . commencement o f t h e s t e p i n c r e a s e i n the d i l u t i o n r a t e . simultaneously. Too a s m a l l step i n D. and under these c o n d i t i o n s C p r o f i l e can be g i v e n as a f u n c t i o n o f time by t h e f o l l o w i n g e x p r e s s i o n which can be d e r i v e d from eqs. From a s t a t i s t i c a l p o i n t o f view the o p t i m a l range can be determined by e v a l u a t i n g t h e t o t a l v a r i a n c e o f Umax d f i n d i n g the c o n d i t i o n s under which i t can be m i n i m i z e d . on the o t h e r hand might r e s u l t i n a prolonged s t a t e o f s u b s t r a t e l i m i t a t i o n o r u n j u s t i f y the assumption o f C » k . {(C . Hence f o u r responses can be monitored i n a w e l l designed experiment (C . a t the maximum s p e c i f i c growth r a t e t h e i n f l u e n c e o f the m a i n t e nance terms w i l l be n e g l i g i b l e i n comparison w i t h the c o n t r i b u t i o n o f the growth terms. the probable e r r o r i n t h e e s t i m a t e d Umax be approximated by c a l c u l a t i n g i t s t o t a l v a r i a n c e a f t e r l i n e a r i z i n g the r e l a t i o n used i .In C ) } max x xo Var(C XO 2 Var(C ) / C + Var(D) x x 2 2 (10) l f ) = Var(C ) X and 1/C « 1/C 2 X O X T h i s e x p r e s s i o n r e v e a l s t h a t V a r ( u a x ) w i l l be m i n i m i z e d i f ( y x ~ D) i minimized (or l n C . . i .D ) / ( l n C .D) t } s si s max sx s xo max An a n a l y t i c a l s o l u t i o n f o r t h i s e q u a t i o n can be shown t o be g i v e n by: C = s where. a n x c a n Var(u max ) = {(y . . The s u b s t r a t e a c c u m u l a t i o n term cannot be i g n o r e d however. CPR) and the most o p t i m a l e s t i m a t e o f y a x can be o b t a i n e d by f i t t i n g the e x p e r i m e n t a l d a t a t o the above p r e s e n t e d r e l a t i o n s . Hence the maintenance terms can be c o n v e n i e n t l y dropped out.„max z = (y /Y max sx exp(y max t ) } / exp(D t ) + C .

628 0. T h i s means t h a t wash-out c a r r i e d out c l o s e t o Umax . Table I : Umax Run no: 1 2 3 4 5 6 v a l u e s c a l c u l a t e d from v a r i o u s responses.86 0. i n o t h e r words an experiment which takes a l o n g time f o r s i g n i f i c a n t decrease i n biomass c o n c e n t r a t i o n would be more a c c u r a t e . S i n c e the f i n a l D was s i m i l a r f o r each experiment the s t e p s i z e i n D can be regarded as a measure of p h y s i o l o g i c a l shock. From t h i s 1. (5) . However.(5) immediately a f t e r t h e step-up i n the d i l u t i o n r a t e . A l l o t h e r e x p e r i m e n t a l d e t a i l s were as d e s c r i b e d p r e v i o u s l y (Esener e t a l .922 0. Temperature and pH were s e t and c o n t r o l l e d a t 35°C and 6.023 1 .71 0.e r r o r s i n v o l v e d i n the measurement of C and D.966 0. Batch experiments were c a r r i e d out i n an 11 l i t e r working volume fermentor.095 1 . D u r i n g the i n i t i a l p e r i o d the organism seems t o wash-out f a s t e r . the s m a l l e r the p h y s i o l o g i c a l shock the h i g h e r the v a l u e o f Umax > c a l c u l a t e d .16 0.976 1 . S i m i l a r response p a t t e r n s can be seen f o r both C and OUR. An extreme s i t u a t i o n i s r e p r e s e n t e d by r u n n o : l where the a p p l i c a t i o n o f a v e r y l a r g e s t e p r e s u l t e d i n the c a l c u l a t i o n o f a Umax v a l u e much s m a l l e r than a c t u a l . which l a s t e d about t h r e e h y d r a u l i c r e s i d e n c e t i m e .985 0. A f t e r t h i s p e r i o d .038 Raw data f o r a t y p i c a l wash-out experiment a r e shown i n F i g .939 0. 1.97 0. RESULTS AND DISCUSSION S i x experiments were performed w i t h d i f f e r e n t s t e p s i z e s i n the d i l u t i o n r a t e . The v a l u e s o f the wash-out D were n o t s i g n i f i c a n t l y d i f f e r e n t f o r these experiments w i t h t h e e x c e p t i o n o f experiment no. r e s p e c t i v e l y .966 0.963 0.8. x o r x x MATERIALS AND METHODS Wash-out experiments were c a r r i e d out i n a one l i t e r w o r k i n g volume fermentor ( B i o l a f i t t e ) w i t h a mono c u l t u r e o f Klebsiella pneumoniae (aerogenes) NCTC 418.087 Umax c a l c u l a t e d from ( h r ~ ' ) : C data OUR d a t a CPR x data 0.944 0. c a l c u l a t e d Umax v a l u e s a r e shown as f u n c t i o n s o f the s t e p s i z e a p p l i e d t o a c h i e v e wash-out.78 0. Step i n D ( h r 1) from to 0.748 0.(10) one can a l s o deduce t h a t i t would be d e s i r a b l e to o p e r a t e a t h i g h C v a l u e s as the v a r i a n c e o f Umaxis i n v e r s e l y p r o p o r t i o n a l to the square o f the biomass c o n c e n t r a t i o n .968 0.948 0.959 1 .918 0.390 0.036 1 . both responses c a n be r e a s o n a b l y w e l l d e s c r i b e d by eq. 2 . x In F i g . a p r a c t i c a l c o n s t r a i n t i s u s u a l l y imposed on the v a l u e o f C by t h e oxygen t r a n s f e r c a p a c i t y o f t h e e x p e r i m e n t a l system. A l s o f o r 104 . From eq. T h i s was p r o b a b l y due to the s u b s t r a t e l i m i t a t i o n c o m p l i c a t i o n mentioned e a r l i e r and/or due t o the i n a b i l i t y of the organism t o adapt t o the new c o n d i t i o n s i n s t a n t e n e o u s l y . I n a l l cases the same g e n e r a l t r e n d has been observed.934 0. A s y n t h e t i c medium w i t h l a c t i c a c i d as the s o l e carbon and energy source was used.050 1 . 1980).966 1 . The Umax v a l u e s c a l c u l a t e d from d i f f e r e n t responses a r e p r e s e n t e d i n Table I .038 0. f i g u r e i t can be seen t h a t the system cannot be d e s c r i b e d by eq.88 0. That i s .

2 . They compare w e l l w i t h t h e y v a l u e c a l c u l a t e d from t h e n o r m a l i z e d data m a x m a x m a x m a x m a x m a x 105 . Here..5 10 . 1: Wash-out experiment no:4. By t h i s procedure many experiments c a r r i e d out a t d i f f e r e n t s t e p s i z e s c o n t r i b u t e t o the c a l c u l a t i o n o f t h e t r u e p . C v data 03 . A D Chr"') 10 . Such an i n f i n i t e s i m a l s t e p i s n o t p o s s i b l e i n p r a c t i c e however. u v a l u e s o b t a i n e d by t h i s procedure a r e shown i n Table I I . C and OUR profiles. CPR data 05 . OUR.OUR data 0. as a f i r s t a p p r o x i m a t i o n s t r a i g h t l i n e s a r e f i t t e d t o d a t a presented i n f i g . from the e x p e r i m e n t a l d a t a p r e s e n t e d a good a p p r o x i m a t i o n t o t h a t i d e a l s t a t e seems t o be o b t a i n a b l e by e x t r a p o l a t i n g t h e determined y v e r s u s step s i z e d a t a . 10 . a 2: Calculated y values max as a function J J of the r step-size. 10 . 0 0 F OUR Cmole/m /hr) 3 50- 20 L C (kg/m ) 2V à x 3 _1 1 I I I I I L_ Fig. 05 . v a l u e s c a l c u l a t e d from t h i s case t h e r e was no good agreement between t h e y d i f f e r e n t responses ( C . CPR). Fig. t o zero step s i z e . x m a x From these r e s u l t s i t seems t h a t the most o p t i m a l e s t i m a t e o f y can be o b t a i n e d by a p p l y i n g an i n f i n i t e s i m a l step i n D a t steady s t a t e a c h i e v e d a t almost t h e a c t u a l y .

1. A l o g i c a l e x p e r i m e n t a l sequence would be t o perform the f i r s t experiment w i t h a l a r g e step i n d i l u t i o n r a t e ( s i n c e no e s t i m a t e o f Umax a v a i l a b l e a t t h i s stage) and g r a d u a l l y t o reduce the s t e p s i z e as much as p r a c t i c a b l e i n an attempt t o a v o i d e x t r a p o l a t i o n over a l a r g e s t e p range.060 1.046 (see F i g . 3 ) : nonlinear regression linear regression 1.106) A e x t r a p o l a t i o n w i t h a l l data p o i n t s B e x t r a p o l a t i o n e x c l u d i n g data of run n o : l F i g u r e s i n p a r a n t h e s i s a r e the 95 % c o n f i d e n c e l e v e l s Fig.029 A B C data x 1.987 From b a t c h experiments 1.987 . Once a r e a s o n a b l y a c c u r a t e l s 106 .087 (0.o b t a i n e d from s i x independent b a t c h experiments and a l l a r e w i t h i n the 95 % c o n f i d e n c e l e v e l o f the b a t c h r e s u l t s ( F i g . Table I I : Umax v a l u e s c a l c u l a t e d by e x t r a p o l a t i o n t o AD = 0. I n view o f the above p r e s e n t e d r e s u l t s i t can be concluded t h a t t h e o p t i m a l e s t i m a t i o n o f Umax w i t h t h e wash-out method s h o u l d i n v o l v e more than one experiment c a r r i e d o u t w i t h d i f f e r e n t p h y s i o l o g i c a l s t e p s i z e s . 3: Normaized data for six batch experiments. u c a l c u l a t i o n based on (hr ) : OUR d a t a CPR d a t a 1. 3 ) .088 1.223) 1.121 0.047 (0.950 .1.

i f C p r o f i l e i s t o be determined t h i s can be done by sampling v i a t h e fermentor o u t l e t stream and hence w i t h o u t d i s t u r b i n g t h e system. C p r o f i l e can a l s o be measured c o n v e n i e n t l y by c o n n e c t i n g an o n . the wash-out method i s a p o w e r f u l t e c h n i q u e i n the study o f m i c r o b i a l b e h a v i o u r and p a r t i c u l a r l y f o r the e s t i m a t i o n o f the maximum s p e c i f i c growth r a t e s . S i n c e OUR and CPR can be r e c o r d e d o n . would be n e c e s s a r y . Himmelblau D H (1970)Process A n a l y s i s by S t a t i s t i c a l Methods. H e r b e r t D (1958) Some p r i n c i p l e s o f c o n t i n u o u s c u l t u r e . Kossen N W F (1980) Carbon d i o x i d e hold-up as a source o f e r r o r i n b a t c h c u l t u r e c a l c u l a t i o n . Kuhn H J . i n Recent P r o g r e s s i n M i c r o b i o l o g y .381. SYMBOLS AND ABBREVIATION C CPR D k mi OUR i t max ix U s r I c o n c e n t r a t i o n (kg/m^) carbon d i o x i d e p r o d u c t i o n r a t e (mole/m^/hr) d i l u t i o n rate (hr ') Monod s a t u r a t i o n c o n s t a n t (kg/m^) maintenance c o e f f i c i e n t on the i t h substance ( k g / k g / h r ) o r ( m o l e / k g / h r ) oxygen uptake r a t e (mole/m-Vhr) r a t e o f consumption o r p r o d u c t i o n o f i . Cometta S. A l m q v i s t and W i n k s e l Stockholm. maintenance and death r a t e i n g l u c o s e l i m i t e d 107 . B i o t e c h n o l . mentioned e a r l i e r . Moreover. T h i s l a r g e step i n D w i l l s t i l l be a s m a l l p h y s i o l o g i c a l step b u t w i l l a v o i d d i s t u r b a n c e s due t o p o s s i b l e s u b s t r a t e l i m i t a t i o n . New York. x x I t can t h e r e f o r e be concluded t h a t .l i n e spectrophotometer t o t h e o u t l e t l i n e . thus no c o r r e c t i o n s f o r changes i n c e l l s i z e . F i e c h t e r A (1980) E f f e c t o f growth temperature on maximal s p e c i f i c growth r a t e . p r o v i d e d t h e p h y s i o l o g i c a l . Bioeng 22:19791983. Roels J A . c o l o u r due t o pigment p r o d u c t i o n e t c . y i e l d .l i n e and t h a t t h e r e i s good agreement between t h e Umax v a l u e s o b t a i n e d from d i f f e r e n t responses the whole experiment can be c a r r i e d o u t w i t h o u t a s i n g l e sample b e i n g taken out. John W i l e y . (kg/m3/hr) time ( h r ) maximal y i e l d on i (kg/kg)or(kg/mole) s p e c i f i c growth r a t e ( h r ' ) - subscripts i o s x inlet i n i t i a l s t a t e (t=0) substrate biomass REFERENCES Esener A A . p.e s t i m a t e of M i s o b t a i n e d i t might a l s o be u s e f u l t o p e r f o r m one e x t r a experiment i n which a l a r g e step i n D i s a p p l i e d t o a c u l t u r e m a i n t a i n e d a t steady s t a t e v e r y c l o s e t o t h i s e s t i m a t e . m a x conditions. F o r s m a l l s t e p s no s i g n i f i c a n t p h y s i o l o g i c a l changes a r e e x p e c t e d . The wash-out method has major e x p e r i m e n t a l advantages. e x p e r i m e n t a l and s t a t i s t i c a l c o n s i d e r a t i o n s a r e g i v e n a t t e n t i o n t o . m a x The f a c t t h a t d i f f e r e n t U v a l u e s o b t a i n e d under d i f f e r e n t i n d i c a t e s t h a t Monod model f a i l s t o h o l d i n these i n s t a n c e s .

B l a c k w e l l . P i r t S J (1975) i n P r i n c i p l e s of Microbe and C e l l C u l t i v a t i o n . i n Methods i n M i c r o b i o l o g y . P i r t S J . J Appl B a c t e r i o l 2 3 ( 1 ) : 87-98. p. Oxford. C a l l o w D S (1960) S t u d i e s of the growth of P e n i c i l l i u m chyrogenum i n c o n t i n u o u s f l o w c u l t u r e w i t h r e f e r e n c e to p e n i c i l l i n p r o d u c t i o n . 108 . Proc Roy Soc B 163:224-234. P i r t S J (1965)The maintenance energy of b a c t e r i a i n growing c u l t u r e s . 2 . Eur J A p p l M i c r o b i o l B i o t e c h n o l 10: 303-315. N o r r i s J R Ribbons D W (eds) v o l . London. Academic. Tempest D W (1970) Theory of chemostat.268.continuous c u l t u r e of the t h e r m o p h i l i c B a c i l l u s c a l d e t o n a x .

A. Examples i n c l u d e b a t c h c u l t i v a t i o n o f microorganisms. the maximum s p e c i f i c growth r a t e . Roels and N. T h e r e f o r e . Rate c o n s t a n t s c a l c u l a t e d from r e p e a t e d experiments r e s u l t e d i n a range o f v a l u e s sometimes e x t e n d i n g o u t s i d e the 95 % c o n f i d e n c e i n t e r v a l s determined f o r i n d i v i d u a l experiments. i t has to be e x p o n e n t i a l t o o . THEORY I t has a l r e a d y been shown t h a t when growth i s balanced i n b a t c h mode. t o overcome these d i f f i c u l t i e s w i l l a l s o be p r e s e n t e d and i t s use and r e s u l t s w i l l be compared w i t h those of the c o n v e n t i o n a l l i n e a r r e g r e s s i o n technique. The l i n e a r form of the e x p o n e n t i a l growth e q u a t i o n can be o b t a i n e d by i n t e g r a t i o n and then t r a n f o r m a t i o n : 2 In C = c o n s t a n t + u .APPLICATION 4 ON THE STATISTICAL ANALYSIS OF BATCH DATA * A. we have n o t i c e d t h a t the a p p l i c a t i o n of the l i n e a r r e g r e s s i o n procedure r e s u l t e d i n a s e r i o u s o v e r e s t i m a t i o n of t h e p r e c i s i o n of the r a t e c o n s t a n t (the maximum s p e c i f i c growth r a t e ) . t x max (1) * Accepted f o r p u b l i c a t i o n i n B i o t e c h n o l . (1981) 109 . l i n e a r r e g r e s s i o n a n a l y s i s can be performed to o b t a i n the b e s t e s t i m a t e of the r e q u i r e d parameter. Kossen INTRODUCTION In B i o t e c h n o l o g y . a v a r i e t y o f p r o c e s s e s a r e s t u d i e d i n b a t c h mode. enzymatic h y d r o l y s i s and convers i o n s . In a study of k i n e t i c s and e n e r g e t i c s of b a c t e r i a l growth i n b a t c h mode. S t a r t i n g from t h i s o b s e r v a t i o n f u r t h e r a n a l y s i s o f the c o l l e c t e d d a t a was i n i t i a t e d and i t was found out t h a t i n most cases t h e e r r o r s were not independent but h i g h l y c o r r e l a t e d .W.F. A simple method developed by Mandel .A. Bioeng. In t h i s paper consequences o f these f i n d i n g s w i l l be shown. The r a t e s of these p r o c e s s e s a r e u s u a l l y determined by a p p l y i n g the c o n v e n t i o n a l s t a t i s t i c a l t e c h n i q u e s and p a r t i c u l a r l y the r e g r e s s i o n t e c h n i q u e s . T h i s o b v i o u s l y v i o l a t e s the assumption of the independence o f e r r o r s upon which t h e o r d i n a r y l i n e a r r e g r e s s i o n t e c h n i q u e s a r e based. Esener. J.

i . For measurements to be a c t u a l l y independent. i . t h e r e w i l l always be d e v i a t i o n s from the set p o i n t s . s e v e r a l b a t c h runs must be performed s i m u l t a n e o u s l y . I t i s important to note t h a t p r o c e s s e r r o r s are c u m u l a t i v e i . I f the d a t a are p r e d o m i n a n t l y c u m u l a t i v e . Measurement. i T h i s suggests the use of d i f f e r e n c e s between the s u c c e s s i v e measurements i n d a t a a n a l y s i s . These are u s u a l l y independent.+ e 9 ) . these new o b s e r v a t i o n s w i l l no l o n g e r be homoscedastic i n g e n e r a l . w n E n n = E i e. i n c l u d e s a l l p r o c e s s errors preceeding i t . Data o b t a i n e d at p r o g r e s s i v e stages of a p r o c e s s c a r r i e d out on the same s u b j e c t of e x p e r i m e n t a t i o n are s u b j e c t to e r r o r s of two t y p e s : a. i n s t r u m e n t a l r e a d i n g s e t c . T h i s i n v a l i d a t e s the assumption of the independence of the e r r o r s . .i . e . a l l samples are withdrawn and a n a l y s e d f o r the d e s i r e d components. u n c e r t a i n i t i e s can a r i s e due t o f l u c t u a t i o n s i n the p r o c e s s of f e r m e n t a t i o n . at d i f f e r e n t run t i m e s . No matter how a c c u r a t e l y the e n v i r o n m e n t a l c o n d i t i o n s .}.e r r o r .E }. {( e.DOT e t c . i n t r o d u c e d by a n a l y t i c a l methods. . These e r r o r s are always p r e s e n t because of the f l u c t u a t i o n s i n the e x p e r i m e n t a l c o n d i t i o n s . one measurement b e i n g made from each. . ' In g e n e r a l p a r t i t i o n i n g can be made i n t o known p r o p o r t i o n s of independent and c u m u l a t i v e components and one has to c o n s i d e r the d a t a as b e l o n g i n g to one or the o t h e r type. I n a d d i t i o n to e r r o r s of type a. the s i t u a t i o n i s q u i t e d i f f e r e n t and the a p p l i c a t i o n of the c o n v e n t i o n a l r e g r e s s i o n procedures can r e s u l t i n a s e r i o u s o v e r e s t i m a t i o n of the p r e c i s i o n of the e s t i m a t e d s l o p e . a . o r i g i n a t e s from the same b u l k .where C x is the biomass c o n c e n t r a t i o n at time t . I t i s w e l l known t h a t the assumption of independence of the p r o c e s s e r r o r s i s by f a r the most c r i t i c a l one i n the a n a l y s i s of d a t a by r e g r e s s i o n procedures. . "' . each e r r o r d u r i n g the course of ferment a t i o n not o n l y a f f e c t s the next measurement but a l l the subsequent measurements. are c o n t r o l l e d . b. These type of e r r o r s are c a l l e d Process errors. L i k e many o t h e r s i t u a t i o n s . When parameter and e r r o r e s t i m a t i o n are c a r r i e d out on d a t a o b t a i n e d from a s i n g l e b a t c h . I n o t h e r words each i n d i v i d u a l e r r o r e . as t h i s w i l l a v o i d the i n c l u s i o n of process e r r o r s .{(E + e ) -( e )} l or E I £ l 2 £ I n n-l n n-1 I' However. e x p e r i m e n t a l measurements taken a t p r o g r e s s i v e stages of b a t c h c u l t u r e f e r m e n t a t i o n s are e s s e n t i a l l y c a r r i e d out on the same s u b j e c t of e x p e r i m e n t a t i o n . . s i n c e the v a r i a n c e of each d i f f e r e n c e w i l l depend on the c o r r e s p o n d i n g interval.. e . I n such a ( c o s t l y ) experiment the e r r o r s can be regarded as independent and the l i n e a r r e g r e s s i o n procedure can be used s a f e l y . convent i o n a l l i n e a r r e g r e s s i o n t e c h n i q u e can no l o n g e r be used. temperature. S i n c e i n t h i s case e r r o r s w i l l be : {e. f 1 10 . e . l i k e pH. .

J i J c = constant i . . R e g r e s s i o n f o r d a t a w i t h predominant process e r r o r s ( c u m u l a t i v e e r r o r s ) The s t a t i s t i c a l treatment f o l l o w s the work of Mandel'. 1.How to determine which of the two type of e r r o r s . the o r d i n a r y l i n e a r Y.) f o r non-overlapping test intervals Furthermore._. the below model h o l d s f o r the s i m p l e case of a s t r a i g h t l i n e through the o r i g i n : n I l Y. The r e s u l t i n g e s t i m a t o r i s of the form: n H.i J B = ( )/( J (3) where or Zj = Y j - Z and = 6 (Xj - X j _ j ) + e. For d a t a w i t h independent e r r o r s the a u t o c o r r e l a t i o n c o e f f i c i e n t . Once the d a t a i s concluded to c o n t a i n predominant p r o c e s s e r r o r s . e q u a t i o n ( 3 ) becomes s i m p l y : 111 . - *.) A more q u a n t i t a t i v e d i s c r i m i n a t i o n procedure can be o b t a i n e d i f one computes the a u t o c o r r e l a t i o n f u n c t i o n f o r the r e s i d u a l s o b t a i n e d by the a p p l i c a t i o n of the o r d i n a r y r e g r e s s i o n procedure. should o s c i l l a t e above and below r ^ = 0. e . i t i s i n t e r e s t i n g t o note t h a t . however. For data w i t h p r e d o m i n a n t l y c u m u l a t i v e e r r o r s (Process e r r o r s ) the s i g n i f i c a n c e of the dependence of r e s i d u a l s can r e a d i l y be assessed a t v a r i o u s l a g s from the r ^ p l o t . L. (j = 1. i s predominant ? For l a r g e n (number of o b s e r v a t i o n s ) the e x p e r i m e n t a l p o i n t s tend t o be s c a t t e r e d above and below the f i t t e d o r d i n a r y r e g r e s s i o n l i n e . n : X Q = 0) L. 1 (2a) The b e s t u n b i a s e d l i n e a r e s t i m a t o r of the s l o p e f o r model (2) i s o b t a i n e d by m i n i m i z i n g the weighted sum of squares. measurement or p r o c e s s e r r o r . E. = (x. i f the t e s t i n t e r v a l s r e p r e sented by L j form an u n i n t e r r u p t e d sequence. + l l . a t random. e . i s assumed to be independent and : V a r ( e . i (2) The c o r r e s p o n d i n g model f o r independent r e g r e s s i o n model. . ) = c. Here. ) = f ( L . i f the e r r o r s are of p r e d o m i n a n t l y independent type. e. i s g i v e n by : e r r o r s . the v a r i a n c e of i s assumed to be a f u n c t i o n of the c o r r e s p o n d i n g t e s t i n t e r v a l . The r e a d e r i s r e f e r r e d t o the o r i g i n a l a r t i c l e f o r the d e r i v a t i o n of the r e l a t i o n s to be used h e r e . F o r p r o c e s s e r r o r s .. + l 1 e. 5-i n 2 L. the e x p e r i mental p o i n t s tend to remain on one s i d e of the r e g r e s s i o n l i n e f o r l o n g sequences of p o i n t s ( F i g . i . = g x. r ^ . = g x. ) i .

1 r j J J Note t h a t the e s t i m a t i o n i n v o l v e s a l l measurements d e s p i t e the f a c t t h a t the e s t i m a t i o n o f s l o p e r e q u i r e s o n l y the f i r s t and the l a s t . Mandel' has shown t h a t i f the experiment i s b e s t r e p r e s e n t e d by a c u m u l a t i v e model but through ignorance o r o t h e r w i s e i s t r e a t e d as b e i n g r e p r e s e n t e d by an independent model. e .e. . and the s t a n d a r d e r r o r of the B i s to be e s t i m a t e d . . . a l a r g e e r r o r i s i n t r o d u c e d when data w i t h p r e d o m i n a n t l y c u m u l a t i v e e r r o r s are t r e a t e d by l i n e a r r e g r e s s i o n as i f independent. i . I n t h i s case the r a t i o becomes: e s t .(Z. however. w h i c h i s g i v e n by : n (6) = c / Z 5=1 Var when c L. . 2 i s not known.P. J (4) of the v a r i a n c e i s g i v e n by : a-. the unbiased e s t i m a t e ( Jy~ ) ( —r ) Z f . n-1 L. i t i s 1. + the r a t i o approaches to 1. Furthermore.2 or f o r n= 10. e . ) } (5) I L.6 = Y n / x n i . the b e s t e s t i m a t e of the s l o p e of the c u m u l a t i v e model i s made by t a k i n g the l a s t v a l u e of the dependent v a r i a b l e and d i v i d i n g i t by the l a s t v a l u e of the independent v a r i a b l e . L . The i n t e r m e d i a t e r e s u l t s might seem u s e l e s s . e s t i m a t i o n of the s t a n d a r d e r r o r of the s l o p e . -• . However. the r a t i o of v a r i a n c e s i s g i v e n by : e s t . s t a n d a r d e r r o r of (B) = 1/19. As n •• °°. (6) by 20 t i m e s . Thus the i n c o r r e c t e s t i m a t e of B i s o n l y s l i g h t l y d i f f e r e n t than the c o r r e c t e s t i m a t e . Var (B) = l Var Var (%) _ (B) 6 5 2 n + 2n + 1 (n + 1) (2n + 1) 2 (6) where the t i l d e (y) denotes an e s t i m a t e i n c o r r e c t l y c a l c u l a t e d . b. d e c i d i n g whether the p r o c e s s can b e s t be d e s c r i b e d by an independent model or n o t .i . they are indeed e s s e n t i a l f o r : a. the s t a n d a r d e r r o r of the s l o p e i s u n d e r e s t i m a t e d u n d e r e s t i m a t i o n f a c t o r can c o n v e n i e n t l y be approximated 112 .9 s.15 . Var (&) Var (B) n + 2 2(2n2 + 2n + 100 1) (7) This r a t i o i n d i c a t e s that f o r n = e s t . T h i s by 2/n f o r n > 10.

.RESULTS AND DISCUSSION Dry weight measurements a r e g e n e r a l l y time consuming and t h e r e f o r e o n l y a l i m i t e d number of them can be made d u r i n g the e x p o n e n t i a l growth phase. 1 t o g e t h e r w i t h the c o n v e n t i o n a l l y f i t t e d r e g r e s s i o n l i n e (n = 4 0 ) .k n / Z(Y. F o r independent data a l l r ^ a r e expected t o be o f s i m i l a r i n s i g n i f i c a n t v a l u e because t h e presence of predominant measurement e r r o r s would have i n t r o d u c e d randomness i n t o the d i s t r i b u t i o n o f r ^ . The d i f f e r e n t i a l a n a l y s i s i s a l s o i n c l u d e d s i n c e the m a x 113 .) we can conclude t h a t the c u m u l a t i v e e r r o r s a r e predominant. the oxygen uptake r a t e (OUR) w i l l be c o n s i d e r e d f i r s t s i n c e a continuous readout of t h i s v a r i a b l e i s a v a i l a b l e . a c c o r d i n g t o the f o r m u l a .2. F o r the experiment r e p o r t e d f u l l y h e r e . S i n c e t h i s i s n o t the case w i t h t h e r e p o r t e d data.Y ) i r k + k . ( F i g . n-k = {( £ (Y.). which i s more than f o u r times h i g h e r than the s t a n d a r d d e v i a t i o n o f the expected mean.Y ) ( Y . 3. 3.63. 2 . At l a g 1. r ^ i s 0. . fitted straight line for When the r e s i d u a l s a r e e v a l u a t e d and p l o t t e d a g a i n s t the same x .In Table I y v a l u e s c a l c u l a t e d by assuming c o n s t a n t y i e l d on oxygen a r e shown f o r t h r e e methods o u t l i n e d e a r l i e r . ) .a x i s ( F i g . 1: Oxygen uptake rate data and the conventionally a batch fermentation. When t h e a u t o c o r r e l a t i o n o f these r e s i d u a l s i s approximated f o r l a g up t o 30. The o r i g i n a l OUR data i s p l o t t e d a g a i n s t time as shown i n F i g . the t r e n d indeed shows c o n s i d e r a b l e c o r r e l a t i o n a t l a g s 1 and 2. A p p r o x i m a t e l y d u r i n g the f i r s t and l a s t 1/4 o f the e x p e r i m e n t a l range the r e s i d u a l s were p o s i t i v e and f o r t h e r e s t n e g a t i v e . 1 10 20 30 40 Fig. T h i s suggests t h a t the c o n s e c u t i v e r e s i d u a l s were c o r r e l a t e d . a c l e a r t r e n d can be observed.Y)J Z (8) k = 1. i . i f t h e d a t a were independent. ( F i g .

the model o u t l i n e d above s h o u l d be used. t h a t f o r experiments w i t h v e r y i n a c c u r a t e a n a l y t i c a l measurements.5 can be c a l c u l a t e d f o r n = 40 . which i s c l o s e t o the c a l c u l a t e d v a l u e of 10 (see Table I ) . enzyme a c t i v i t y e t c m a x m a x 114 .35 3.75 m a x 0 0032 0 0338 0 0346 a = standard d e v i a t i o n From Table I .c a l l e d "process e r r o r s " . Residual ( V o b s e r v e d . r e s u l t e d i n s i m i l a r v a l u e s o f P .913 0. however.. 2: Behaviour of the residuals obtained by the application conventional linear regression procedure.protein. RNA.921 hr Method o f a n a l y s i s independent differential cumulative ') a 0 % of y 0. n 40 39 40 Mmax( 0. however. I t i s important t o note t h a t i n t h i s a n a l y s i s .939 0.d a t a p o i n t s are e q u a l l y spaced i n time.g. were a l s o t r e a t e d by the independent and dependent models and the outcomes compared i n Table I I . I f the e r r o r s a r e found t o be c u m u l a t i v e . I n our e x p e r i e n c e most b a t c h f e r m e n t a t i o n s are indeed s i g n i f i c a n t l y i n f l u e n c e d by the presence of the s o . i t can be observed t h a t t h e t h r e e methods used a l l r e s u l t e d i n s i m i l a r v a l u e s of the s l o p e ( U ) . S i n c e o n l y a few d a t a p o i n t s c o u l d be o b t a i n e d a r e s i d u a l a n a l y s i s cannot be c a r r i e d out. e.60 3. Table I : P m a x c a l c u l a t i o n by v a r i o u s methods based on OUR d a t a . however. 2. I t must be emphasized. the f i r s t method (independent) r e s u l t e d i n a s e r i o u s o v e r e s t i m a t i o n of the p r e c i s i o n of the s l o p e . I t can be n o t i c e d from Tables I and I I t h a t as n i n c r e a s e s the d i f f e r e n c e between the e s t i m a t e s of the s t a n d a r d e r r o r s c a l c u l a t e d by the two methods becomes much more s e r i o u s .v r e g r e s s i o n )» 1 Û 3 10 B 20* 30 —r- n 40 Fig. I t can hence be concluded t h a t i f d a t a from b a t c h p r o c e s s e s are to be a n a l y s e d one must f i r s t t e s t the e x p e r i m e n t a l d a t a f o r p o s s i b l e v i o l a t i o n o f the h y p o t h e s i s of independence. Using r e l a t i o n (7) an u n d e r e s t i m a t i o n f a c t o r of 12. e r r o r s i n the transformed form a r e a l s o assumed t o be n o r m a l l y d i s t r i b u t e d and independent of the l e v e l of the measurement. i n t h i s case by about 10 t i m e s . 3: Autocorrelation function for the resiuduals of the Fig. Dry weight d a t a not r e p o r t e d h e r e . Both methods. shewn in Fig.

968 1 . I a 1 a 3.996 0. S t a t . A. 1 2. Mandel.the independent e r r o r s may be p r e d o m i n a t i n g . no : n independent I u 0 max 0.0 0530 0517 0405 6 6 6 REFERENCES 1.552(1957). As the assumption of a c u m u l a t i v e model f o r an a c t u a l l y independent case w i l l a l s o r e s u l t i n s e r i o u s e r r o r s . J . T s u c h i y a .0267 0.106 c 0 0 0494 0 0826 0 0807 C. 52. J . Symp.098 0. Chem. Amer.0160 1. S e r i e s . the s a f e s t procedure w i l l be t o e v a l u a t e and t e s t the dependence/independence of each p r o c e s s on i t s own m e r i t s . 108(67).970 1 . Assn. 1 3. Ramkrishna and H.G.53(1971). max Method of a n a l y s i s exp. 2.. 1 15 .M.0404 cumulative V max 0. P r o g . Eng. D.011 0. F r e d e r i c k s o n . Table I I : y c a l c u l a t i o n f o r t h r e e batches based on d r y weight d a t a .

.

was c u l t u r e d i n a s y n t h e t i c medium as d e s c r i b e d by Evans et a l G l y c e r o l was used as s u b s t r a t e and assayed e n z y m a t i c a l l y (Boehringer UV method No. F. Esener. Klebsiella pneumoniae NCTC 418. are known. where the c o n t r o l parameter i s o b t a i n e d from the i n s t a n t a n e o u s gas exchange d a t a ^.7 . Kossen and J . T h i s s u b j e c t has r e c e n t l y r e c i v e d much a t t e n t i o n i n the l i t e r a t u r e ^ . Biomass was c o l l e c t e d on a 0. T h e r e f o r e the amount of carbon d i o x i d e remaining i n the b r o t h was determined d u r i n g the e x p o n e n t i a l growth phase and the measured o v e r a l l RQ was c o r r e c t e d . Measured and c o r r e c t e d RQ v a l u e s were a l s o compared w i t h the t h e o r e t i c a l RQ which can be c a l c u l a t e d from b i o e n e r g e t i c c o n s i d e r a t i o n s i f the e x p e r i m e n t a l y i e l d on s u b s t r a t e . V o l . T h i s d i s c r e p a n c y was then t r a c e d to the presence of carbon d i o x i d e i n the c u l t u r e b r o t h at c o n c e n t r a t i o n s much h i g h e r than a n t i c i p a t e d . p a r t i c u l a r l y f o r c o m p u t e r . Roels INTRODUCTION C o n s r u c t i o n of m a t e r i a l balances i s of prime importance i n the study of b i o e n e r g e t i c s .c o n t r o l l e d b a t c h or any o t h e r system o p e r a t i n g at unsteady s t a t e . B i o e n g . N. A s i m i l a r o b s e r v a t i o n has r e c e n t l y been r e p o r t e d f o r baker's y e a s t by B a r f o r d and H a l l ^ . I n b a t c h c u l t u r e s of Klebsiella pneumoniae s t u d i e d i n t h i s l a b o r a t o r y .APPLICATION 5 CARBON DIOXIDE HOLD-UP AS A SOURCE OF ERROR IN BATCH CULTURE CALCULATIONS * A. 1979-1983 (1980) 1 17 . s y s t e m a t i c l o s s e s i n carbon balances were observed. T h i s phenomenon has t h e r e f o r e important i m p l i c a t i o n s f o r the i n t e r p r e t a t i o n and use of m e t a b o l i c d a t a . A. washed w i t h d i s t i l l e d water and * P u b l i s h e d i n B i o t e c h n o l . However. MATERIALS AND METHODS The organism. 2 2 . degree of r e d u c t i o n and the carbon c o n t e n t s of biomass and s u b s t r a t e . 1 - An attempt was t h e r e f o r e made to q u a n t i f y the amount of m i s s i n g carbon d i o x i d e and t o e v a l u a t e the i n f l u e n c e of t h i s d i s c r e p a n c y on the c a l c u l a t i o n of r e s p i r a t o r y q u o t i e n t (RQ). A. Dry w e i g h t s were determined by the method of de V r i e s and Stouthamer ^. The c o n s i s t e n c y of e x p e r i m e n t a l data should always be checked v i a the known r e g u l a r i t i e s of f e r m e n t a t i o n processes and e l e m e n t a l b a l a n c e s .2 pm pore diameter f i l t e r ( S a r t o r i u s ) . The c o r r e c t e d RQ was found to be c l o s e to the t h e o r e t i c a l l y c a l c u l a t e d RQ whereas the measured RQ d e v i a t e d s i g n i f i c a n t l y . e x t e n s i v e d a t a have so f a r appeared o n l y f o r continuous c u l t u r e systems o p e r a t i n g at steady s t a t e .148270). . W.

TOC) (kg/m ) 3 2 (2) TC and TOC d e t e r m i n a t i o n s were c a r r i e d out i m m e d i a t e l y a f t e r s a m p l i n g . p r o c e s s i n g . A l l f l o w s were c o r r e c t e d f o r h u m i d i t y and v o l u m e t r i c changes. ash f r e e b a s i s .80 ± 0. The maximum s p e c i f i c growth r a t e of t h i s organism i s q u i t e h i g h (1.92 N 14.24°0. Few 118 .07 ± 0.d r i e d to c o n s t a n t weight a t 378 K. No p r o d u c t s o t h e r than biomass.02 h r ~ ' ) . TIC i s the t o t a l i n o r g a n i c carbon and TOC i s the t o t a l o r g a n i c carbon. RESULTS AND DISCUSSION E x p e r i m e n t a l r e s u l t s are shown i n f i g u r e s 1 and 2.86 C H Formula 1. e .96 * I n % d r y weight . Of these TC and TOC were measured by a Dohrmann DC-50 carbon a n a l y z e r . CARBON DIOXIDE DETERMINATION For the system used carbon d i o x i d e can be assumed to be the o n l y source of i n o r g a n i c carbon.l i n e heat exchanger manufactured i n our workshop.41 N 50. by the gas phase a n a l y s i s .05 . r e s p e c t i v e l y .27 0 27. and water c o u l d be d e t e c t e d at l e v e l s t h a t can be s i g n i f i c a n t . m Table I : E l e m e n t a l c o m p o s i t i o n * and Formula of K.2 ym f i l t e r and i n o c u l a t e d by an a c t i v e l y growing inoculum to e l i m i n a t e any l a g phase and the p o s s i b i l i t y of unbalanced growth. From f i g u r e 2 i t can be seen t h a t the amount of unaccounted carbon d i o x i d e i n c r e a s e s d u r i n g the e x p o n e n t i a l growth phase. t h e r e f o r e i t was not p o s s i b l e to o b t a i n more e x t e n s i v e d a t a due to s a m p l i n g . Complete a e r o b i c growth was ensured by m a i n t a i n i n g the d i s s o l v e d oxygen t e n s i o n w e l l above the l i m i t i n g range. pneumoniae at i t s maximum growth r a t e . and a n a l y s i s times r e q u i r e d . Medium was s t e r i l i z e d by membrane f i l t r a t i o n through a 0. The a i r f l o w r a t e to the fermentor was c o n t r o l l e d by a thermal mass f l o w meter (Brooks 5811) a t about 0. T h e r e f o r e f o r the c u l t u r e b r o t h a t any i n s t a n t TC = TIC + TOC (1) where TC i s the t o t a l carbon (kg/m3). i . Carbon d i o x i d e not accounted f o r . . carbon d i o x i d e r e m a i n i n g i n the b r o t h .77 kg d r y a i r / h r . The e l e m e n t a l c o m p o s i t i o n of the biomass (Table I ) was determined by a computer coupled element a n a l y z e r ( P e r k i n Elmer 240) . S p e c i a l a t t e n t i o n was p a i d f o r the a c c u r a t e d e t e r m i n a t i o n of gas f l o w s and c o n c e n t r a t i o n s . A l l samples were c o o l e d d u r i n g sampling down to about 278 K by an o n .63 0. The t y p i c a l r e s i d e n c e time i n the heat exchanger was about 5 seconds. (TC . can then be c a l c u l a t e d by the f o l l o w i n g e x p r e s s i o n : C0 ( b r o t h ) =44/12 . Experiments were c a r r i e d out i n a 11 x 10~3 3 w o r k i n g volume fermentor m a i n t a i n e d a t 308 K. c grown on g l y c e r o l H 6. Gas phase oxygen and carbon d i o x i d e c o n c e n t r a t i o n s were determined by a t w i n channel paramagnetic oxygen a n a l y z e r (Servomex OA 184) and an i n f r a r e d carbon d i o x i d e a n a l y z e r (Beckman 864). pH was c o n t r o l l e d a t 6. carbon d i o x i d e .

or p a r t i c i p a t i o n i n the carbon d i o x i d e . carbon d i o x i d e and w a t e r . per m3 of c u l t u r e S u b s t r a t e carbon used up Carbon r e c o v e r e d i n biomass Carbon r e c o v e r e d i n gas phas e Carbon r e c o v e r e d i n b r o t h Y i e l s on s u b s t r a t e (ash f r e e b a s i s ) without correction C r e c o v e r y (%) RQ 92 0.69 (%) (kg) RQ c a l c u l a t e d i n d e p e n d e n t l y from y i e l d on s u b s t r a t e by eq.66 1 . T h e r e f o r e o n l y a macro a n a l y s i s i s r e p o r t e d here. Table I I : O v e r a l l c a r b o n b a l a n c e and RQ c a l c u l a t i o n s f o r the e x p o n e n t i a l growth of K. pure p h y s i c a l a b s o r p t i o n by s u p e r n a t a n t .57 with correction 99 0.71 4. 1: Biomass vs. the r e l a t i v e importance of these mechanisms i s not c l e a r . fermentation 2: Carbon dioxide retained time. Assuming complete a e r o b i c growth w i t h no p r o d u c t s o t h e r than biomass.05 2. T h i s r e s u l t s i n a l a r g e e r r o r i n the measured v a l u e of RQ.27 0. U s i n g t h e i r n o t a t i o n and r e a r r e n g i n g . fig. RQ can a l s o be c a l c u l a t e d from the e x p e r i m e n t a l l y determined y i e l d on s u b s t r a t e by the mass-energy b a l a n c e method f i r s t developed by M i n k e v i c h and E r o s h i n 3. These i n c l u d e entrapment by c e l l s . A t t h i s stage however. in the broth during exponential growth.50 Difference 7 18 0.fig.(3) I n Table I I the o v e r a l l carbon b a l a n c e f o r the e x p o n e n t i a l growth phase i s p r e s e n t e d . mechanisms t h a t can m a i n t a i n t h i s carbon d i o x i d e i n the b r o t h can be thought o f . These c a l c u l a t i o n s i n d i c a t e t h a t carbon d i o x i d e c o r r e s p o n d i n g t o 7% of the t o t a l s u b s t r a t e carbon i n p u t remained i n the b r o t h a t the end of e x p o n e n t i a l phase.06 0. pneumoniae.c a r b o n a t e b u f f e r system. RQ i s g i v e n by: 119 .

W. E r o s h i n . Bioeng. S. Tempest. R o e l s .J. B a r f o r d and R. Wang and D.2. N i s h i z a w a .RQ = {1 . Nagai and Y.C. T h i s can l e a d to s e r i o u s problems i n i n d u s t r i e s where b a t c h . 6 7 and Y =0.F.18. 21.W.50 the above e x p r e s s i o n g i v e s an RQ v a l u e of 0.Y ( a / a ) } / { ( / 4 ) . i n Methods i n M i c r o b i o l o g y .609(1979) 6. H a l l . W. s s g = NOMENCLATURE RQ TC T0C TIC Y 0 a Yfo Y s D s s respiratory quotient(dimensionless) t o t a l carbon i n b r o t h ( k g / m ) t o t a l o r g a n i c carbon i n b r o t h ( k g / m ) t o t a l i n o r g a n i c carbon i n b r o t h ( k g / m ) substrate yield(dimensionless) carbon weight f r a c t i o n i n b i o m a s s ( d i m e n s i o n l e s s ) carbon weight f r a c t i o n i n s u b s t r a t e ( d i m e n s i o n l e s s ) r e d u c t a n c e degree of b i o m a s s ( e q u i v e l e c t r o n s / g . J . a t o m carbon) reductance degree of s u b s t r a t e ( e q u i v e l e c t r o n s / g .A. B i o t e c h n o l . 2.22.39 . Wang. i n P r o g r e s s i n I n d u s t r i a l M i c r o b i o l o g y . Kossen. (Academic P r e s s .472(1968) 120 . These r e s u l t s i n d i c a t e t h a t u n l e s s carbon b a l a n c e s f i t t i g h t l y or b r o t h phase C O 2 measurements are made. C. B i o e n g . A i b a . C. Wang. measured RQ v a l u e s can be i n g r e a t e r r o r . s b s Y s ( 1 . 3. L. Amsterdam. Wang.R. Cooney and D.K.P..313. B i o t e c h n o l .p.19.1001(1976) 8. 5 5 ( 1 9 7 7 ) .I. Cooney. J.H.2457(1980) 5.. . de V r i e s and A.E.1595 (1978) 4. Y 4 . Stouthamer.A. N o r r i s and W. B i o e n g . H e r b e r t and D.69(1977) 7. however.W. B u l l .20. Bioeng."In the m o d e l l i n g of m i c r o b i a l m e t a b o l i s m " . C. Bacteriol.. Bioeng. Evans. H. M i n k e v i c h and V. R o e l s and N. B i o t e c h n o l .I. R i b b i n s .C.Y.. ( E l s e v i e r .L.69 which i s i n c l o s e agreement w i t h the RQ v a l u e c o r r e c t e d f o r the carbon d i o x i d e r e t a i n e d i n the c u l t u r e and hence unaccounted f o r i n the gas phase (Table I I ) . L. E d s . Bioeng.G. B i o t e c h n o l .51. S. D. E d .b a t c h or any o t h e r f e r m e n t a t i o n e s s e n t i a l l y not at steady s t a t e i s monitored and c o n t r o l l e d by o n . E r i c k s o n . 1 9 . H..T. a t o m carbon) 3 3 3 REFERENCES 1.Y ( s V b /a Y ))} s s (3) For 0^=0. M. B i o t e c h n o l .J. The measured RQ.G. 9. J. London. I. .1970). 1978).vol. f e d . J. d e v i a t e s s i g n i f i c a n t l y from the c o r r e c t e d and i n d e p e n d e n t l y c a l c u l a t e d RQ v a l u e s . a =0. B i o t e c h n o l . J.l i n e measurement of the RQ.Y.96.

The model f a i l s t o h o l d d u r i n g the t r a n s i t i o n phase. m x x In view of the l i m i t e d p r e d i c t i o n c a p a b i l i t y of the u n s t r u c t u r e d model.SUMMARY I n t h i s t h e s i s an attempt i s made to e v a l u a t e the c u r r e n t s t a t e of m i c r o b i a l e n e r g e t i c s from an e n g i n e e r i n g p o i n t of view. E x t e n s i v e d a t a and m a t e r i a l b a l a n c e s are p r e s e n t e d . Of the e n e r g e t i c parameters. e . The o t h e r parameter more r e l e v a n t to i n d u s t r i a l o p e r a t i o n s i s temperature. Above 40 °C i t decreased a b r u p t l y .i n a c t i v a t i o n model extended to d e s c r i b e the s u p e r o p t i m a l temperature range i s used to c o r r e l a t e the change i n the maximum s p e c i f i c growth r a t e w i t h temperature. The work i s aimed t o h e l p the b i o t e c h n o l o g i s t i n the t r a n s l a t i o n and f i l t r a t i o n of m i c r o b i o l o g i c a l d a t a to a q u a n t i t a t i v e form w i t h s p e c i f i e d c e r t a i n i t y . parameter e s t i m a t i o n and s t a t i s t i c a l a n a l y s i s are developed. An A r r h e n i u s type enzyme a c t i v a t i o n . The maintenance c o e f f i c i e n t i n c r e a s e d e x p o n e n t i a l l y over the same range.b a t c h and continuous c u l t u r e d a t a the model seems to p r e d i c t e v e r y t h i n g an 121 . I t i s a l s o shown t h a t f e d . I n Chapters 5 and 6. R e s u l t s are a l s o compared w i t h those r e p o r t e d f o r mixed c u l t u r e s ( a c t i v a t e d s l u d g e ) . I t i s shown t h a t the s i g n i f i c a n t d e v i a t i o n s at low growth r a t e s cannot be f u l l y accounted f o r by the l o s s of v i a b i l i t y . maximal Y and m . s x s I n Chapter 3 e n g i n e e r i n g t o o l s are a p p l i e d to the study of b a c t e r i a l k i n e t i c s and e n e r g e t i c s . the u n s t r u c t u r e d model developed i s extended f o r the d e s c r i p t i o n of growth behaviour i n f e d . A simple u n s t r u c t u r e d model based on Monod k i n e t i c s and the l i n e a r s u b s t r a t e consumption r e l a t i o n i s developed. S t r a t e g i e s f o r more e f f i c i e n t e x p e r i m e n t a t i o n .b a t c h c u l t i v a t i o n technique i s a u s e f u l t o o l i n the study of b i o k i n e t i c s and e n e r g e t i c s s i m u l taneously. a simple s t r u c t u r e d model i s developed i n Chapter 7. The t h e o r y and a p p l i c a t i o n s of u n s t r u c t u r e d models are d i s c u s s e d i n Chapter 2. the maximum s p e c i f i c growth r a t e . I t i s a l s o shown t h a t the c h o i c e of the k i n e t i c e x p r e s s i o n i s not c r i t i c a l and t h a t almost any of the r e l 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 . Chapter 6 p r e s e n t s r e s u l t s o b t a i n e d i n f e d . When t e s t e d w i t h f e d .40 °C. L i n e a r r e l a t i o n f o r s u b s t r a t e consumption i s t e s t e d w i t h continuous c u l t u r e d a t a . Y i s found to be r o u g h l y the same over the temper a t u r e range of 25 . upon which e n g i n e e r i n g d e s i g n and o p e r a t i o n s can be based. I t i s shown t h a t the mixed c u l t u r e s can adapt much f a s t e r to changes i n s a l i n i t y .b a t c h c u l t u r e s . i s capable of d e s c r i b i n g the e x p e r i m e n t a l l y observed b e h a v i o u r . In Chapter 4. e f f e c t s of two s e l e c t e d environmental changes on the parameters of the u n s t r u c t u r e d model are s t u d i e d .b a t c h mode. The u n s t r u c t u r e d model i s found to p r o v i d e a r e a s o n a b l y good d e s c r i p t i o n f o r the e x p o n e n t i a l and pseudo-steady s t a t e s . g i v e n the same a t t e n t i o n f o r parameter e s t i m a t i o n . The i n f l u e n c e s of s a l i n i t y are s t u d i e d i n Chapter 5. The e n e r g e t i c parameters have m i n i m a l i n f l u ence. Some shortcomings of the c u r r e n t e x p e r i m e n t a l and t h e o r e t i c a l p r a c t i c e are s t r e s s e d and improvements developed are d e s c r i b e d . Growth c o u l d not be s u s t a i n e d at 50 °C. the b e h a v i o u r i s f i x e d by the e n e r g e t i c parameters. I t i s shown t h a t d u r i n g growth i n b a t c h mode the b e h a v i o u r of the system i s r i g i d l y f i x e d by the k i n e t i c parameter. . I n continuous c u l t i v a t i o n the r e v e r s e i s t r u e i .

T e s t s i n v o l v i n g e x p e r i m e n t a l l y o b t a i n e d RNA d a t a i n d i c a t e t h a t the model i s not r e a l i s t i c i n t h i s r e s p e c t and thus has to be r e j e c t e d . a number of t o p i c s are d e a l t w i t h i n s h o r t u n i t s ( a p p l i c a t i o n s ) . i s on the s t a t i s t i c a l treatment of b a t c h d a t a . A d d i t i o n a l l y . growth d u r i n g the wash-out phase i s d e s c r i b e d . I t i s shown t h a t b a t c h data may have c o r r e l a t e d e r r o r s and t h a t t h i s r e s u l t s i n the o v e r e s t i m a t i o n of the p r e c i s i o n of the e s t i m a t e d parameters. The i n f l u e n c e of carbon d i o x i d e hold-up on the e n e r g e t i c c a l c u l a t i o n s and C-balances are a s s e s s e d q u a n t i t a t i v e l y . F i n a l l y . 122 . I n A p p l i c a t i o n 1. a simple e x p r e s s i o n r e l a t i n g COD to BOD i s d e r i v e d i n terms of the thermodynamic e f f i c i e n c y of the growth p r o c e s s . A p p l i c a t i o n 1 and 2 demonstrate the use of m a c r o s c o p i c p r i n c i p l e s i n the study of e n e r g e t i c s . i t i s shown t h a t p r o d u c t f o r m a t i o n i n the absence of e x t e r n a l e l e c t r o n a c c e p t o r s i s l i n k e d to energy g e n e r a t i o n p r o c e s s . an i m p o r t a n t c o n c l u s i o n i s drawn from t h i s e x e r c i s e . N e v e r t h e l e s s . A p p l i c a t i o n 5 g i v e s e v i d e n c e on the hold-up of carbon d i o x i d e i n b r o t h d u r i n g b a t c h c u l t i v a t i o n .the c r i t i c a l t e s t s f o r the v e r i f i c a t i o n of s t r u c t u r e d models should i n c l u d e the i n t e r n a l c o m p o s i t i o n d a t a o b t a i n e d from c a r e f u l l y planned t r a n s i e n t e x p e r i m e n t s . I n Chapter 8. t h a t i s .u n s t r u c t u r e d model can. i t p r o v i d e s i n f o r m a t i o n about the time dependent b e h a v i o u r of the i n t e r n a l c o m p o s i t i o n of the c e l l s . These are a l l a p p l i c a t i o n s of the c o n s i d e r a t i o n s developed i n the p r e v i o u s c h a p t e r s . I n A p p l i c a t i o n 2. O p t i m a l e x p e r i m e n t a t i o n range and procedure f o r the e s t i m a t i o n of the maximum s p e c i f i c growth r a t e are e s t a b l i s h e d . I n A p p l i c a t i o n 3. A p p l i c a t i o n 4.

V e r v o l g e n s wordt gedemonstreerd dat de keuze van de b e s c h r i j v i n g van de k i n e t i e k n i e t van e r g g r o o t b e l a n g i s . De l i n e a i r e g r o e i w e t toegepast op subs t r a a t v e r b r u i k werd g e t o e t s t aan gegevens d i e z i j n v e r k r e g e n i n een k o n t i n u k u i t u u r . De Hoofdstukken 5 en 6 handelen over h e t e f f e k t van w i s s e l e n d e omgevingskondit i e s . I n d i t Hoofdstuk worden de r e s u l t a t e n ook v e r g e l e k e n met d i e i n de l i t e r a t u u r worden vermeld v o o r mengkultures ( a k t i e f s l i b ) . t e weten het z o u t g e h a l t e en de temperatuur. Tevens wordt een eenvoudig o n g e s t r u k t u r e e r d model o n t w i k k e l d .b a t c h k u i t u r e een z e e r b r u i k b a a r h u l p m i d d e l i s v o o r h e t t e g e l i j k e r t i j d b e s t u d e r e n v a n zowel de k i n e t i e k a l s de e n e r g e t i s c h e a s p e k t e n van mikrobiële g r o e i . I n H o o f s t u k 6 wordt ingegaan op de i n v l o e d van de temperatuur a l s m o g e l i j k e s t u u r v a r i a b e l e v o o r 123 . d i e n i e t v o l l e d i g konden worden v e r k l a a r d door een v e r l i e s aan l e v e n s v a t b a a r h e i d van de c e l l e n . te weten de maximale ' y i e l d ' van biomassa op s u b s t r a a t Y en de 'maintenance' koëfficient m . d i e b r u i k b a a r i s v o o r h e t o n t w i k k e l e n en u i t v o e r e n van mikrobiële p r o c e s s e n .SAMENVATTING Met d i t p r o e f s c h r i f t wordt beoogd v a n u i t de o p t i e k v a n de i n g e n i e u r s w e t e n schappen een o v e r z i c h t t e geven op h e t t e r r e i n van de e n e r g e t i s c h e a s p e k t e n van mikrobiële g r o e i . v o l d o e n b i j n a a l l e i n de l i t e r a t u u r vermelde r e l a t i e s aan h e t e x p e r i m e n t e e l v a s t g e s t e l d e gedrag. E r wordt aangetoond dat i n een b a t c h k u i t u r e h e t gedrag van het systeem s t e r k wordt b e p a a l d door één k i n e t i s c h e parameter: de maximale s p e c i f i e k e g r o e i s n e l h e i d u . Het t e g e n d e e l b l i j k t waar t e z i j n i n een k o n t i n u k u i t u r e . Tot s l o t wordt i n d i t h o o f d s t u k aangetoond dat een f e d . Gegeven een goede paramet e r s c h a t t i n g . B i j l a g e g r o e i s n e l h e d e n z i j n s i g n i f i k a n t e a f w i j k i n g e n van deze wet gevonden. Van de o n t w i k k e l i n g e n t o t op heden worden zowel op exp e r i m e n t e e l a l s op t h e o r e t i s c h g e b i e d enige t e k o r t k o m i n g e n o n d e r s t r e e p t en v e r b e t e r i n g e n h i e r v o o r aangedragen. V e r v o l gens wordt e r een s t r a t e g i e o n t w i k k e l d voor een efficiëntere p r o e f o p z e t . Het o n g e s t r u k t u r e e r d e model b l i j k t een adequate b e s c h r i j v i n g t e z i j n voor h e t gedrag t i j d e n s exponentiële g r o e i en gedurende een pseudo s t a t i o n a i r e t o e s t a n d . De e n e r g e t i s c h e parameters b l i j k e n een m i n i m a l e i n v l o e d u i t t e oefenen. p a r a m e t e r s c h a t t i n g en s t a t i s t i s c h e a n a l y s e . dat gebaseerd i s op een k i n e t i e k v o l g e n s Monod en de l i n e a i r e g r o e i w e t . Om d i t model t e t o e t s e n worden u i t g e b r e i d meetgegevens en massabalansen g e p r e s e n t e e r d . Het model i s e c h t e r n i e t meer g e l d i g t i j d e n s o v e r g a n g s t o e standen. De i n v l o e d v a n h e t z o u t g e h a l t e wordt i n Hoofdstuk 5 beschreven. In Hoofdstuk 2 worden de t h e o r e t i s c h e a c h t e r g r o n d e n en de t o e p a s s i n g e n v a n ong e s t r u k t u r e e r d e g r o e i m o d e l l e n besproken. h i e r i n wordt h e t gedrag j u i s t b e p a a l d door de e n e r g e t i s c h e p a r a m e t e r s .b a t c h ' k u i t u r e t e b e s c h r i j v e n . H i e r u i t kan de konk l u s i e worden g e t r o k k e n dat mengkultures z i c h v e e l s n e l l e r aanpassen aan v e r a n d e r i n g e n i n h e t z o u t g e h a l t e van de k u i t u u r v l o e i s t o f . Het i n Hoofdstuk 2 beschreven o n g e s t r u k t u r e e r d e model wordt i n H o o f s t u k 4 u i t g e b r e i d t e n einde de g r o e i i n een ' f e d . op de parameters v a n h e t ong e s t r u k t u r e e r d e model. m g x g In Hoofdstuk 3 worden enige t e c h n i e k e n aangegeven om de k i n e t i e k en de energ e t i s c h e aspekten van de g r o e i van mikro-organismen t e b e s t u d e r e n . De s t u d i e poogt v e r d e r een o n d e r s t e u n i n g t e z i j n v o o r een b i o t e c h n o l o o g t e n behoeve van h e t v e r t a l e n en s e l e k t e r e n van m i k r o b i o l o g i s c h e gegevens i n een k w a n t i t a t i e v e vorm.

Een model van h e t type A r r h e n i u s v e r g e l i j k i n g word g e b r u i k t om de exyme a c t i v a t i e . De derde t o e p a s s i n g b e s c h r i j f t de g r o e i t i j d e n s een zogenaamd'wash-out' exper i m e n t . d i e h e t chemisch z u u r s t o f v e r b r u i k COD k o p p e l t aan h e t b i o l o g i s c h z u u r s t o f v e r b r u i k BOD. Van de e n e r g e t i s c h e parameters bleek Y™ J l k o n s t a n t t e b l i j v e n b i n n e n het temperatuurgebied van 2540°C. De i n v l o e d van deze C O 2 ophoping op de e n e r g e t i s c h e b e r e k e n i n g e n en k o o l s t o f b a l a n s e n wordt op k w a n t i t a t i e v e w i j z e aangegeven. H i e r u i t a f g e l e i d worden de g r e n z e n aangegeven van het o p t i m a l e p r o e f o p z e t g e b e i d en een procedure v o o r de b e p a l i n g van de maximale s p e c i f i e k e groeisnelheid. Er worden i n d i t Hoofdstuk r e s u l t a t e n weergegeven d i e z i j n v e r k r e k e n i n een f e d . Boven de 50°C t r a d er z e l f s helemaal geen g r o e i meer op.industriële p r o c e s s e n . die ten grondslag l i g t aan de t e m p e r a t u u r a f h a n k e l i j k h e i d van de maximale g r o e i s n e l h e i d b i j s u p e r o p t i m a l e temperaturen.b a t c h k u i t u r e . Bovendien v e r s c h a f t het model i n f o r m a t i e over h e t t i j d a f h a n k e l i j k e gedrag van de s a m e n s t e l l i n g van de c e l i n h o u d . hetgeen t o t g e v o l g kan hebben dat de n a u w k e u r i g h e i d van de g e s c h a t t e parameters wordt o v e r s c h a t . Een t o e t s van d i t model u i t g e v o e r d met behulp van gegevens u i t f e d .b a t c h k u l t u r e s toont aan dat het g e s t r u k t u r e e r d e model a l l e s v o o r s p e l t wat h e t ong e s t r u k t u r e e r d e model kan v o o r s p e l l e n . T e n s l o t t e wordt i n de l a a t s t e t o e p a s s i n g het ophopen van C O n i n h e t b e s l a g t i j d e n s een b a t c h kweek gememoreerd. E r wordt aangetoond dat b a t c h gegevens o n d e r l i n g g e k o r r e l e e r d e f o u t e n kunnen hebben. In de tweede t o e p a s s i n g wordt met behulp van het thermodynam i s c h rendement van een g r o e i p r o c e s een eenvoudige r e l a t i e a f g e l e i d . In Hoofdstuk 8 worden een a a n t a l t o e p a s s i n g e n behandeld van de i n de v o o r g a a n de H o o f d s t u k k e n gegeven beschouwingen. Boven de 40°C nam de maximale s p e c i f i e k e g r o e i s n e l h e i d abrupt a f .d e a k t i v a t i e te b e s c h r i j v e n . De e e r s t e twee t o e p a s s i n g e n b e t r e f f e n het g e b r u i k van m a k r o s k o p i s c h e p r i n c i p e s om de e n e r g e t i s c h e a s p e k t e n van mikrobiële g r o e i t e onderzoeken. N i e t t e m i n kan e r een b e l a n g r i j k e k o n k l u s i e worden verbonden aan d i t werk: k r i t i s c h e t o e t s e n voor de e x p e r i m e n t e l e v e r i f i k a t i e van g e s t r u k t u r e e r d e m o d e l l e n behoren ook de c e l s a m e n s t e l l i n g i n ogenschouw t e nemen d i e wordt gemeten b i j z o r g v u l d i g u i t g e z o c h t e o v e r g a n g s t o e s t a n d e x p e r i m e n t e n . Over h e t z e l f d e temperatuurt r a j e c t nam de maintenance koëfficient e x p o n e n t i e e l t o e . De v i e r d e t o e p a s s i n g h a n d e l t over de s t a t i s t i s c h e v e r w e r k i n g van b a t c h g i s t i n g s g e g e v e n s . X v r l w e X In H o o f d s t u k 7 wordt gepoogd de b e p e r k t v o o r s p e l l e n d e waarde van het onges t r u k t u r e e r d e model te v e r b e t e r e n door een g e s t r u k t u r e e r d model te ontwikkel e n . I 24 . Een t o e t s met e x p e r i m e n t e e l v e r k r e g e n RNA meetgegevens g e e f t e c h t e r aan dat het model wat d i t b e t r e f t n i e t r e a l i s t i s c h i s en d e r h a l v e d i e n t t e worden verworpen. De e e r s t e l a a t z i e n dat p r o d u k t v o r m i n g i n a f w e z i g h e i d van e x t e r n e e l e k t r o n a k s e p t o r e n gekoppeld i s aan de e n e r g i e l e v e r ende p r o c e s s e n .

Yinede b u çalxsmadan. p a r a metre t a y i n etme ve i s t a t i s t i k çbzumleme y b n t e m l e r i g e l i s t i r i l m i s t i r . 40°C n i n üzerxnde b u paramètre b i r d e n a z a l m x s t x r . B e s l e m e l i kültür t e k n i g i n i n b i y o k i n e t i k ve e n e r j e t i k çalxsmal a r x n b i r a r a d a yürütülebilecegi f a y d a l x b i r yontem o l d u g u d a g o s t e r x l m i s t i r . g e l i s t i r i l m i s o l a n y e n i y o n t e m l e r açxklanmxstxr. m o d e l i n b u bakxmdan geçerle olmadxgxnx ve dogrulanamxyacagxnx g b s t e r m i s t i r . Sürekli üretimde i s e b u durumun t e r s i geçerli b u l u m n u s t u r . Yapxsxz model. Düsük üreme h x z l a r x n d a g b z l e nen b e l i r g i n sapmalarxn sadeoe üreme k a p a s i t e s i n d e k i ( v i a b i l i t y ) düsüse b a g l a namxyacagx gbsterxlmi§tir. Bolüm 4 de. 50°C de üreme saglanamamxstxr. B u l g u l a r karma kültürler ( a k t i f çamur) için e l d e e d i l e n l e r i l e k a r s x l a s t x r x l m x s t x r . üssel ve y a l a n c x .ÔZET Bu tezde mikrópsal e n e r j e t i g i n bugünkü durumu mühendislik gbrüs açxsindan i n c e l e n m i s t i r . Baha v e r i m l i deney yapma. sürekli kültur v e r i l e r i i l e sxnanmxstxr. Model geçis doneminde geçerliligini k a y b e t m i s t i r . Aynx zamanda k i n e t i k b a g x n t x seçiminin f a z l a o n e m l i olmadxgx ve aynx d i k k a t l e p a r a m e t r e l e r i saptandxgx t a k d i r d e literatürde v e r i l e n h e r h a n g i b i r bagxntxnxn d e n e y s e l davranxsx t a r i f e d e b i l e c e g i g o s t e r x l m i s t i r . Mühendislik i s l e m l e r i n d e d i g e r o n e m l i b i r paramètre i s e s x c a k l x k t x r . Monod k i n e t i g i n e ve d o g r u s a l s u b s t r a t tüketimi b a g x n t x s x n a dayanan b a s i t b i r y a p x s x z model g e l i s t : L r i l m i s t i r . T/uzlulugun e t k i s i Bolüm 5 de a r a s t x r x l m x s t x r .d e a k t i v a s y o n m o d e l i o p t i m a l üzeri s x c a k l x k aral^xjpxLxda kapsayacak s e k i l d e g e l i s t i r i l m i s t i r . Genis kapsamlx v e r i l e r ve m a t e r y e l d e n k l i k l e r e s u n u l m u s t u r . y a n i s i s t e m d a v r a n x s x e n e r j e t i k parametreler olan and ms t a r a f x n d a n saptanmáktadxr. e n e r j e t i k p a r a m e t r e l e r i n i s e az e t k i l e d i g i g o s t e r x l m i s t i r . D o g r u s a l s u b s t r a t tüketimx bagxnt x s x .y a t x s k x n duruml a r x y e t e r l i b i r s e k i l d e t a r i f e t m i s t i r . E n e r j e t i k p a r a m e t r e l e r d e n 1 25-40 C s x c a k l x k a r a l x g x n d a y a k l a s x k o l a r a k d e g x s m e m i s t i r . Yapxsxz m o d e l i n o n d e y i k a p a s i t e s i n i n k x s x t l x bulunmasx k a r s x s x n d a Bolüm 7 ¿ e L b a s i t b i r y a p x l x model g e l i s t i r i l m i s t i r . Buna ek o l a r a k y a p x l x model hücre i c i b i l e s i m i n i n zamana b a g l x dxnamik durumunu t a r i f e t m e k t e d i r . Model b e s l e m e l i k e s i k l i kültür ve sürekli kültür v e r i l e r i i l e t e s t e d i l d i g i n d e .b'nemli b i r sonuç 125 . Maksimum b'zgül üreme h x z x i l e s x c a k l x k a r a s x n d a k i i l i s k i y i t a r i f etmek üzere A r r h e n i u s t i p i b i r enzim a k t i v a s y o n . seçilmis o l a n i k i çevresel d e g i s i m i n y a p x s x z model parametr e l e r e üzerindeki e t k i s i i n c e l e n m i s t i r . Karma kültürlerin t u z l u l u k d e g i s i k l i k l e r i n e daha çabuk u y a r l a m a ( a d a p t a t i o n ) g o s t e r d i g i i z l e n m i s t i r . s Bolüm 3 de b a k t e r i s e l k i n e t i k ve e n e r j e t i k çalxsmalarxna mühendislik y b n t e m l e r i n i n n a s x l u y g u l a n a b i l e c e g i g b s t e r i l m i s t i r . Yapxsxz ( u n s t r u c t u x e d ) m o d e l l e r i n t e o r i ve u y g u l a m a l a r x Solum 2 de t a r t x s x l m x s t x r . B e s l e m e l i k e s i k l i kültürde e l d e e d i l e n s x c a k l x k d e n e y l e r i n i n b u l g u l a r x Bolüm 6 da sunulmu§tur. Mevcut d e n e y s e l ve t e o r i k u y g u l a m a l a r x n e k s i k l i k l e r i n e i s a r e t e d i l m i s . Çalxsmanxn amacx b i y o t e k n o l o g a m i k x o b i y o l o j i k v e r i l e r d e n mühendislik t a s a r x m ve i s l e m l e r i n i n d a y a n d x r x l a b i l e c e g i n i c e l v e r i l e r i n e l d e s i n d e yardxmcx o l m a k t i r . K e s i k l i üretimde s i s t e m davranxsxnx k i n e t i k b i r paramètre o l a n maksimum bzgül çogalma hxzxnxn k e s i n o l a r a k s a p t a dxgx. Bakxm k a t s a y x s x (maintenance c o e f f i c i e n t ) i s e aynx s x c a k l x k a r a l x g x n d a üssel o l a r a k a r t m x s t x r . Bolüm 5 ve 6 d a . e w e l o e kxorulm'us o l a n y a p x s x z model b e s l e m e l i k e s i k l i kültürdeki d a v r a n x s x d a kapsamak üzere g e n i s l e t i l m i s t x r . Ancak d e n e y s e l RNA v e r i l e r i i l e y a p x l a n t e s t . y a p x s x z model b n d e y i l e r i n i n h e p s i n i v e r e b i l e c e k k a p a s i t e d e bulunmustur.

Burada k e s i k l i kültür v e r i l e r i n d e k i h a t a l a r x n i l i s k i l i ( c o r r e l a t e d ) o l a b i l e c e g i ve bu. Uygulama 5» k e s i k l i süreclerde k a r b o n d i o k s i t b i r i k m e s i n i n olduguna i§axet e t m e k t e d i r . üreme sürecinin termodinamik v e r i m i göz önüne a l x n a r a k . türetilmigtir. Bölüm 8 de b i r k a c konii k x s a u n i t e l e r (uygulamalar) §eklinde t a r t x s x l m x g t x r .c x k a r x l a b i l i r . Uygulama 1 de hücre dxsx e l e k t r o n a l x c x l a r x n x n yoklugunda ürün yapxmxnxn e n e r j i üretim süreci i l e b a g x n t x l x o l d u g u gösterilmx§tir. Bu dönemde maksimum özgül üreme h i z x n i saptamak i c i n en uygun d e n e y s e l a r a l x k ve yöntem geli§tirilmi§tir. B u n l a r x n h e p s i e w e l k i 'bölümlerde g e l i g t i r i l m i s . 126 . Uygulama 4» k e s i k l i kültür v e r i l e r i n i n i s t a t i s t i k i g l e n m e s i üzerinedir. Uygulama 1 ve 2 makroskopik i l k e l e r i n e n e r j e t i k g a l x s m a l a r x n d a k i f a y d a l a r x n x v u r g u l a m a k t a d x r . durumun saptanan p a r a m e t r e l e r i n k e s i n l i g i n i ( p r e c i s i o n ) h a t a l x o l a r a k a r t t x r a b i l e c e g i gösterilmigtir. y a p x l x model dogrulamasx l o i n y a p x l a c a k k r i t i k t e s t i e r d i b k a t l e tasarlanmxs dinamik d e n e y l e r d e n e l d e e d i l e n hücre i c i b i l e g i m i v e r i l e r i i l e olmalxdxr. Uygulama 2 de COD ( k i m y a s a l o k s i j e n g e r e k s i n i m i ) ve BOD ( b i y o k i m y a s a l o k s i j e n g e r e k s i n i m i ) a r a s x n d a b i r b a g x n t x . B u durumun e n e r j e t i k h e s a p l a m a l a r x n ve k a r b o n d e n k l i k l e r i üzerindeki e t k i l e r i n i t e l o l a r a k i n c e l e n m i s t i r . Uygulama 3 de üreme süreci s i l i n m e (wash-out) durumunda i n c e l e n m i s t i r . o l a n düsüncelerin uygulamal a r i d i r .

This thesis. 5. S. Dick. . They f a i l d u r i n g h i g h l y t r a n s i e n t situations. 1 s t October 1981. i . NeV York Acad. these days some academics are i n v e n t i n g problems t h a t they can s o l v e . in Murphy's Law. e . Product f o r m a t i o n d u r i n g a n a e r o b i c growth i n the absence of e x t e r n a l e l e c t r o n a c c e p t o r s . the 2. i s not r e a l i s t i c and seems to be o n l y an e x e r c i s e i n mathematical model b u i l d i n g and computer programming.326. . This thesis. Opening Address to the Sixth Symposium. A. Schuler. . Chapter 8.C. Chapter 2.A. e . Ann. The r a t i o of COD to BOD i s not a c o n s t a n t but a simple f u n c t i o n of thermodynamic e f f i c i e n c y of the growth p r o c e s s . The statement made by Gaden. The procedures employed by A. Erdingston Lao. would be u s e f u l i n B i o t e c h n o l o g y . Los Angeles) 19 78. Gaden.STELLINGEN 1. Sci.L. .L. 7. t h e r e f o r e the computers A. 8. Application 1. International Fermentation 10. Chapter 8. and t h a t by I. Bloch (Price. 4.. U n s t r u c t u r e d models p r o v i d e good a p p r o x i m a t i o n to r e a l i t y d u r i n g b a l a n c e d growth or a t pseudo-steady s t a t e s . i . M. A B i o t e c h n o l o g i c a l model can be s a i d to be r e a l i s t i c when i t i s used by i n d u s t r i a l producers f o r p r o c e s s d e s i g n and c o n t r o l . 9. E. Leung and C. This thesis. Much e f f o r t i s needed f o r the development and v e r i f i c a t i o n of these models. 6. " t h a t .P. E s e n e r . .E. i s l i n k e d to energy p r o d u c t i o n . The 74 parameter model r e p o r t e d by S c h u l e r e t .I. Canada (1980). 35(1979). a s s i g n i n g an o f f i c i a l d i s c u s s e r to every paper p r e s e n t e d .. held at London.W. Much open c r i t i s i s m i s needed i n B i o t e c h n o l o g y . r a t h e r than t a c k l i n g the new problems or the e x i s t i n g ones" can be g e n e r a l i z e d to o t h e r f i e l d s than B i o t e c h n o l o g y and p a r t i c u l a r l y to P o l i t i c s . d e s c r i b i n g the growth of a s i n g l e b a c t e r i a l c e l l . o p e n i n g and p u b l i s h i n g d i s c u s s i o n s f o r e v e r y paper they p u b l i s h i n t h e i r j o u r n a l . Simple s t r u c t u r e d models w i l l no doubt r e p l a c e the p o p u l a r u n s t r u c t u r e d models i n the near f u t u r e . a l . Application 2. A. Man and the computer belong to d i f f e r e n t genus and should not be made to perform c e r t a i n t a s k s .R. The number of d i f f e r e n t h y p o t h e s i s e r e c t e d to e x p l a i n a g i v e n b i o l o g i c a l phenomenon i s i n v e r s e l y p r o p o r t i o n a l to the a v a i l a b l e knowledge. 3.C.

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