AN ENGINEERING STUDY OF BACTERIAL KINETICS AND ENERGETICS

A . A . Esener

o o o

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

Dit proefschrift is goedgekeurd door de promotoren PROF. DR. IR. N.W.F. KOSSEN PROF. IR. J.A. 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 - b a t c h growth ( p a r t o f F i g . 3 o f Chapter 4)

Dedicated to my parents

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. I r . J.C. 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 . G.C. 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. C. Ras and G. 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. J.Ph. B r o n k h o r s t , A.L. de Graaf and B.J.T. 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. F. Bolmann and C. Warnaar f o r drawings and photographs

My s t u d e n t s , J . Roozenburg, G.M. B o l and T. 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, Chapter 5 and Chapter 7, respectively G i s t Brocades N.V. 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 - 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

T A B L E OF CONTENTS

CHAPTER

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

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

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

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

CHAPTER

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

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; 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

ERRATA s h o u l d read 19 19
P.

and

not

T a b l e 1,4 idem 4 line 8 + line 4 eq(3) t 1ine.6 eq(16)

line 4

0.698(0.689-4.631).10" lower
2

0.698(0.869-4.631) 10 t i m e s ^x dX/dt = (2x)

K s

t line 5 10 times
Mg

22 44

higher

Kg

P. P. 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>

Y^q)

s h o u l d be Ygjj cannot can 0Lj)2} s.

P. P. 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

1 I s

CHAPTER 1

INTRODUCTION

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 . 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.

product synthesis

use of substrate

synthesis of biomass precursors

biomass synthesis

ATP

pool

maintenance

Fig.

1: Distribution and Kossen,

of substrate energy in microbial 19 78, see Chapter 2, ref.16).

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 ; 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, 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 . 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 . 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 . 1. 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

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 , 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. 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. 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 , 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. 1. 2. 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.

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 , f e d - b a t c h , 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.

5.

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 . Chapter 2, 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 . The t h e o r i e s to be used l a t e r on, are developed here. 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. 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 . The d i s c u s s i o n s are i l l u s t r a t e d , 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. F i n a l l y , a s h o r t comment on the concept of maintenance i s g i v e n . Chapter 3, c o n s i s t s of t h r e e s u b - s e c t i o n s . 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. The second s u b - 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 . The l a s t s u b - 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 . Chapter 4, d e s c r i b e s b a c t e r i a l growth i n f e d - 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 , 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. 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. 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 - 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 .

In Chapters 5 and 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 . 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 . The c o n c e n t r a t i o n range i s from 0 to 40 kg/m3. 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 . I n Chapter 6, 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 - b a t c h c u l t u r e s . 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. 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 . 2

Chapter 7, 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 . e . , 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 . 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 . 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. 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. Chapter 8, 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. 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 . 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 , 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 .

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, 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. 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 - f r e e b a s i s . 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 , an e q u i v a l e n t of biomass i s the same as one mole of biomass. 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 ) , 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 . 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.

This thesis has been carried out within the Biotechnology Group of the delft University of Technology. Postal address: Department of Chemical Engineering, Biotechnology Group, SBR, Jaffalaan 9, TH., Delft 2600, The Netherlands

CHAPTER 2

ON THE THEORY AND APPLICATIONS OF UNSTRUCTURED GROWTH MODELS: KINETIC AND ENERGETIC ASPECTS

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'. 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. 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 ' . 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 - 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 , the m a c r o s c o p i c growth yield, 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 . * 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. 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 . 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 . I t was suggested t h a t , 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 . 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 - U

{ C

/ (K

)} - U

(1)

Here i s t h e r a t e of endogeneous metabolism. When C >> K , p - P > and hence Vmax Pg ~ Ve When C = 0 \i e g u a l s t o - p , i . e . , n e g a t i v e growth i s achieved. 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 . 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;3
s s m a x = s e

sx

= y

sx

{D / ( D + u

)}

(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

(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 , .and a t t r i b u t e d i t t o

s

the s o - c a l l e d maintenance f u n c t i o n s which i n c l u d e ; t u r n over of c e l l m a t e r i a l s , 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 , c e l l m o t i l i t y e t c . Eq.(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 . 1/u . In a number of c a s e s , however, 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. Based on t h i s o b s e r v a t i o n , Stouthamer and Bettenhaussen m o d i f i e d eq.(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 , ATP, and o b t a i n e d the f o l l o w i n g form^:
sx

ATP

ATP

ATP

( A )

L a t e r on, 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 , the below r e l a t i o n has been proposed6;

q, ATP

U / ( YIJ1 ATP

) . . 'theoretical

m g

\i

(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 . Furthermore, 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. 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 . Thus, f o r a e r o b i c systems one must know the s o - 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 , or v i c e v e r s a . 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 . 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 . 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. 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 . Thus more o f t e n than n o t , 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.(3) s t i l l f i n d s wide a p p l i c a t i o n , 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 .
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. 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 ; 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. R e c e n t l y , 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 . 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 , 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 . 9 - 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 ^ - 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 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. A d d i t i o n a l l y , 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 . 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 , r e c e n t l y much work has been done on the m a c r o - e n e r g e t i c b e h a v i o u r . 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, 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. 6

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 . 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 , 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 . These t e c h n i q u e s , n e v e r t h e l e s 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 . 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. 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 . Recent advancement of the s u b j e c t i s due to Roels and Kossen'^, R o e l s ' ^ , E r i c k s o n et l l 8 - 2 0 and H e i j n e n and Roels21. 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 . 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 , l a t e r on i n t h i s work.
a

Assuming t h a t C, H, N and 0 are the o n l y elements exchanged i n n o n - n e g l i g i b l e amounts i n the system, 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. T h i s source i s assumed to be growth l i m i t i n g . 0C 2 H, 0 N, bj Cj + <I>0 5 2 + $.NH 4 3 >.C H, 0 Nj 1 b, c, d, biomass

substrate

$C H, 0 N, 3 a^ b c d^
Q 3 3

+ $,C0 6 2

$ H.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
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. 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 . 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. 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 , i . e . , 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 .
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

- r x
( y

- r p r s - y r - v r ) 'x x 'p p d 3 r p 's

(8) (9) (10)

r. = - d , r + d r + N 2 s 1 x Here, Y
x

, y

a r e p

d e f i n e d by the f o l l o w i n g :

= 4 = 4
Y

"

2 C

, 2

3 d

.
2

(11) (12) (13)

2 " 3 "

2 c

- 3d
3 d

= 4

2 c

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 . 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.'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. 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. Eq.(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 . 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.(3) to : , ..max , max r = r / Y + r / Y s x sx p sp ,, .. (14)

+ m C s

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. By combining e q s . ( 8 ) , (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

- 1) r + (1/ Y x sp
m 3 X

m a x

- 1) r + m C p S X
m a X

(15)

1/4 { ( Y / Y
S

S X

- Y ) r + (Y / Y X x s sp

- Y ) r + Y m C } p p s s x

(16)

From eqs.(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 few a r e shown below. 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.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

= (y /4) m s s

E r i c k s o n e t a l . ' ^ 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 . 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 . 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. 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 , l ^ h ' ^ growth p r o c e s s .
o r t le

\h

= sx / f

( 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 . For growth w i t h NH3 as the ammonia s o u r c e , however, 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 . 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)

(22) Y < Y
/ Y

sx

(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 , where the c o n t r o l parameter cannot be determined d i r e c t l y e.g., 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 , l i k e s t a r c h . E s t i m a t i o n of heat o u t p u t , 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 , can a l s o be done, based on the measurement of a few o n - l i n e determined parameters. 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. 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.

Ill

AN INTRODUCTION TO THE MODELLING OF MICROBIAL GROWTH

In g e n e r a l , b i o l o g i c a l systems are s u b - s e t s of c h e m i c a l systems. 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. Thus, one can a t l e a s t i n t h e o r y , 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 ; c h e m i c a l systems. I n p r a c t i c e , 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. F i r s t l y , 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 - 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). C o n s i d e r i n g t h a t E. ooli has more than 2000 d i f f e r e n t p r o t e i n s , 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, ii. Secondly, 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 . e . , 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 , 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 , 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 , 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 , m o l e c u l e s and o r g a n e l l e s , c e l l s and c e l l s e t c . I n o t h e r words, 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 . 2 4 25 Recently,. 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. 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, however, 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 . Moreover, 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, u n i v e r s a l l y . 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 , 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. 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. 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. 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. 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 , compared w i t h t h a t of the growth p r o c e s 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 , do not change s i g n i f i c a n t l y . Thus these mechanisms and t h e i r e f f e c t s on the t o t a l , system b e h a v i o u r may 9

ii.

be c o n v e n i e n t l y n e g l e c t e d , 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 , 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 - b i o t i c c o n c e n t r a t i o n s . 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 . e . , c o n c e n t r a t i o n i n t h e a - b i o t i c phase.

Based on these c o n s i d e r a t i o n s i . e . , 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, 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. 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 . 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 , t h a t i s , i n terms o f p o p u l a t i o n s . 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, 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 t i s , however, 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 . 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. 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.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 . 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 , however, 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. 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. 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, was p r o v i d e d by Powell27. I n h i s approach, 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 - 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 - b i o t i c phase seen by the b i o t i c phase. 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 - b i o t i c phase. I n p r a c t i c e , however, 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 . 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 - b i o t i c and b i o t i c phases. 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 . Model based on t h i s assumption i . e . , 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 , a r e c a l l e d UNSTRUCTURED WDELS.

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 . 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 - M e n t e n e q u a t i o n , Monod a r r i v e d at i t e m p i r i c a l l y . That i 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 . A l t e r n a t i v e l y , 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

bottleneck

and

thus r a t e l i m i t i n g .

Now, 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 ) . To d e s c r i b e the system, changes of C and C and their interdependence have t o be e v a l u a t e d . 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 2,4 ( f o r the no-product c a s e ) :

s

r x

/Y sx

C x

(26)

Eqs. (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 , continuous and f e d - b a t c h . $ 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.g., 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)

For b a t c h c u l t i v a t i o n , $ 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. Thus the f o l l o w i n g p a i r o f eqs. 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 = - y C C . / {(K + C ) Y } s max s x s s sx
m a X

(29) - m C (30)

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. 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 , 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

/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 . 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 , e.g., i f C >> C Y and C >> K , the model reduces down t o : so s 1 1

/ C

xo

exp

( U

max

t )

(32) general

Growth behaviour i n f e d - 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. T h i s i s p r e s e n t e d i n Chapter 4.

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. 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 . When C = 0, the concept of endogeneous metabolism becomes handy. 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, C

s s
a

= K K

s u

/ ( y / y

~ P

(33) (34)

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; 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 , 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.(33) . 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 . 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, successfully, 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 , 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 . 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 . 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. 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 . 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 . Under these c o n d i t i o n s , 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 . Since C v s . time, 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 , 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 . C , however, 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 . U n f o r t u n a t e l y , 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 . 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 . I n n o n - s u b s t r a t e l i m i t e d systems e.g., b a t c h systems, 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 , t h e r e a f t e r r remains c o n s t a n t e.g., l i k e s a t u r a t i o n k i n e t i c s . 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 . However, 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 , 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 , i s low ( K - 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.
g s s x x s s g x g s s

In t h e i r r e v i e w , 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. 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 . T h e r e f o r e , 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 . 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

T i e s s i e r Model where Blackman Model:

u = U max K = K s u

{ 1 - exp(-C

/ K ) }

(35)

/ In 2 for / A for C > s < s u u A A

max C s

max max

(36)

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 . For a comprehensive l i s t r e c e n t r e v i e w s can be consulted!6,24,25,31 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 . 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, each model h a v i n g d i f f e r e n t parameters.^5,31
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 . I n some c a s e s , however, 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 . F o r i n s t a n c e , 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 - b a t c h models, 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 .

DISCUSSION OF UNSTRUCTURED MODELS AND MICROBIAL ENERGETICS WITH REFERENCE TO EXPERIMENTAL RESULTS

Batch

Cultures

K. pneumoniae (aerogenes) was c u l t i v a t e d i n b a t c h mode, 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 . 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. A t y p i c a l experiment i s shown i n F i g . 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 . 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, 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.(26)), a r e presented ( s o l i d l i n e s ) .
s x

Fig.

1: A typical

batch

experiment;

C^ and C

vs, time p r o f i l e s and

simulataion. 13

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 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 . 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 . Even then, F i g . 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, 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 . The endogeneous metabolism model, 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, d e s c r i b e s the e a r l y decay phase w e l l . Thus as f a r as t h i s system i s concerned, 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 . 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, 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. T h i s i s a f o u r parameter model ( K , P , Y , m ). I n F i g s . 2,3,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 , are shown.
x s m a x x g

Fiq. 2 : Sensitivity
v

of the batch

model to variations

in u max

t(min)

Fig.

S: Sensitivity

of the batch

model to changes

in m .

14

J71CUC

Fig.

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 . The case of K i s d i s c u s s e d i n Chapter 4.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 . time p r o f i l e , 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, m and Y . However, the k i n e t i c parameter, y a x shown t o be of g r e a t importance. As p r e v i o u s l y d i s c u s s e d , 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 . 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 . That i s , i f Y . i s t o be e s t i m a t e d from b a t c h data,use o f C p r o f i l e would be more a c c u r a t 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 . ( 2 9 ) , (30) to (31) ) has no s i g n i f i c a n t drawbacks. Consequently, 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 . Moreover, 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 - l i n e a r axes, 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 . 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.(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. 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.(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, 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 .
g

As p o i n t e d out p r e v i o u s l y , 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 . 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. 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, 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. i . e . , 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. 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.32 Thus when growth i s balanced i t i s e x p o n e n t i a l too. The r e v e r s e i s however, n o t t r u e i . e . , 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 . In an attempt t o check the v a l i d i t y of t h i s mathematical statement, 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. As shown i n F i g . 5, 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. However, t h i s might be a s i m p l i f i e d p i c t u r e , s i n c e , f o r i n s t a n c e the p r o t e i n 15

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.

Fig.

5: Macromolecular

composition

during

batch

growth.

In F i g . 6, 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 ) . Here, 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. 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 . 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 . 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.
o x c x

Fig.

6: Gas exchange profiles

during

batch

growth;

solid

line

simulation.

16

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. 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 . However, 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 . As shown i n F i g . 7 the macromolecular c o m p o s i t i o n , p a r t i c u l a r l y t h e RNA f r a c t i o n , changes as a f u n c t i o n o f t h e growth r a t e . Moreover, 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. 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, H and N c o n t e n t s are s i g n i f i c a n t . 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 , by : C
1

H
b

N
c d

where

b c = d z

= (7.33 - 0.50 y ) / z (12.33 + 3.40 y ) / ( 1 4 z ) = 26.97/(16z) = (53.61 - 3.74 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 = 0.5 h r m o l e c u l a r weight = 23.16, C H N ->oO , , )

9 n

i s taken;(y

= 4.16,

Fig.

7: Micvomolecular composition as a function w RNA = 0.11 w 3 Protein = 0.71 ]i~0 ' C bHld- ~te = 0.065
3 V n W aJ Ta

of the growth

rate,

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 . 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, except near the washout r e g i o n , 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, 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 s . 8 and 9. 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.Fig.8 shows a good s t r a i g h t l i n e f i t . However, 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 . 17

Fig. Fig.

8: 9:

Specific Specific

rate

of svbstrate

consumption

as a fuction rate.

of the growth

rate.

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 -

= y I Y

m a X

sx

+ m

(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

sx

y Y

m a x

sx

/ (y +

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 , both methods should g i v e e x a c t l y t h e same parameters. 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 . 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 , oxygen, carbon d i o x i d e d a t a v s . 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 . 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 ) . 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 , b u t i s c a l c u l a t e d from q = y / Y . 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 . Moreover, i n a q v s . 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. The dangers o f t h i s e x e r c i s e i . e . , 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.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. The r e s u l t s a r e p r e s e n t e d i n Table I . I f t h e r e s i d u a l s a r e examined, one can d e t e c t a t r e n d ( F i g . 8,9). 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 . y p l o t . Here the r e s i d u a l s change t h e i r s i g n
c

18

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 . 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 , n=27

/ ( Zie errntiirn

L i n e a r Regresin

m a x

sx Y Y m m o m cx s

0.698 (0.869 - 0.707) 1.583 (1.542 - 1.624) 2.391 (2.289 - 2.493) 3.342 (3.059 - 3.623).10" 3.740 (3.365 - 4 . 1 1 5 ) . 1 0 3.668 (3.177 - 4 . 1 5 9 ) . 1 0
2

0.710 (0.701 - 0.720) 1.613 (1.570 - 1.659) 2.465 (2.367 - 2.570) 4.072 (3.290 - 4 . 8 5 2 ) . 1 0 4.065 (3.373 - 4 . 7 5 7 ) . 1 0 3.998 (3.321 - 4 . 6 7 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 , y > 0.1, n=21 0.710 (0.699 - 0.721) 1.620 (1.563 - 1.677) 2.513 (2.376 - 2.650) 4.241 (3.627 - 4 . 8 5 5 ) . 1 0
-2

sx
m a x

0.7* 2.5* 5.3*

0.719 (0.706 - 0.732) 1.640 (1.570 - 1.701) 2.561 (2.428 - 2.709) 4.997 (3.846 - 6 . 1 4 8 ) . 1 0

1.4* 3.0* 6.8*

-2

ox Y m m m
s a x

4.320 (3.627 - 5.012).10~ 4.530 (3.756 - 5 . 3 0 5 ) . 1 0

4.609 (3.517 - 5.701).10~ 4.819 (3.835 - 5.803).10~

-2

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 , y> 0.1, n=63 Y m

m a x

0.700 (0.693 - 0.707) 4.146 (3.661 - 4.631). l~1,

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

0.705 1.544 2.313

i n C-eq/C-eq, C-eq/mole c-eq/C-eq/hr, mole/C-eq/hr

Y|x Y
a x

* 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

limits.

i n t h e time sequence o n l y t h r e e t i m e s . 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. 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. S i n c e above y=0.1, 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=0.1, (n=21, see Table I ) . 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 . F o r the second s e t , 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 . F o r d a t a above y=0.1 r e s i d u a l s of the q v s . y r e l a t i o n changes s i g n 11 t i m e s , q v s . y,9 times and q v s . y, 6 times ( ( n - l ) / 2 = 10 ) . Thus w i t h the e x c e p t i o n of q d a t a ,
a n o s Q c c

19

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 , 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. Thus i t can be concluded t h a t above p=0.1 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 . 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 , 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, 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 i m u l t a n e o u s l y i . e . , when every measurement c o n t r i b u t e s to the r e s u l t . T h i s can be done by p r o c e s s i n g I] v s . H d a t a , where r| i s c a l c u l a t e d from Y , Y and Y d a t a (n=63) . 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 . 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 . 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 e i r e s t i m a t e s can be c o r r e l a t e d . 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 . 10). 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 ; 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. 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 . 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 , 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 . 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. 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 , f o r the same o r s i m i l a r s y s t e m ( 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 , as f a r as t h e maximal y i e l d s a r e concerned, 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.

Fig.

10: The loons of the joint confidence limits for energetic parameters as determined by the linear relation for substrate consumption; sq.(S&). (for data shewn in Fig.8)

20

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 , 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 , c e l l m o t i l i t y , membrane e n e r g e t i z a t i o n e t c . 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 , 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. m i s a l s o assumed to be c o n s t a n t . However, 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.g., 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 . At low growth r a t e s the r a t i o i s h i g h , 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. 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. However, 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 . That i 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 . However, 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 , e.g., 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 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.
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,34,35) _ 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 , 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 . 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 . 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 . F o r t u n a t e l y , 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.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 . y i n F i g . 11. 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=0.06. 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 , 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 , 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 , as shown by h o l l o w c i r c l e s i n the F i g . 11. 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 , 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 . 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 , 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. 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.
m a x s s

Fig.

11:

versus

data:(recalculated

from ref:

34).
21

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. 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 . 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. 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, a maintenance c o e f f i c i e n t of 5.Ox 10 3 (C-eq/C-eq/hr) was c a l c u l a t e d . 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 . 1 5 x l 0 ~ 2 ) . 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.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. For i n s t a n c e , 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.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. However, 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 , w i l l be c i t e d here. In 1970 Gaudy et a l . ^ 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 , c o n t i n u o u s l y . 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 . e . , no growth but s u b s t r a t e uptake. 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. 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 . 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 . 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. 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 " .

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

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

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 . 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

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.(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. 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 B a c t e r i e n n e s , 2 nd ed., (Hermann, P a r i s ) ( 1 9 4 2 ) . 2. D. H e r b e r t , Symp. I n t . Congr. M i c r o b i o l . , 6,381(1958), 3. D. H e r b e r t , Continuous Culture,6,1(1976) . 4. S.J. P i r t , P r o c . Roy. Soc. Londob B, 163,224(1965). 5. A.H. Stouthamer and C.W. Bettenhaussen, Biochim-Biophys . Acta,301 ,53 (1 973) . 6. A.H. Stouthamer and C.W. Bettenhaussen, A r c h . M i c r o b i o l . , 1 1 , 2 1 ( 1 9 7 6 ) . 7. H.W. van V e r s e v e l d , Ph.D t h e s i s , Free U n i v e r s i t y of Amsterdam,(1979). 8. J.C. Senez, B a c t e r i o l . Rev.,26.95(1 962) . 9. O.M. N e i j s s e l , Ph.D. t h e s i s , U n i v e r s i t y of Amsterdam,(1 977). 10. O.M. N e i j s s e l and D.W. Tempest, Soc. Gen. M i c r o b i o l . Symp.,29,53(I 979). 11. D.W. Tempest, Paper p r e s e n t e d to the 2 nd Eur. Symp. on B i o t e c h n o l . h e l d at Eastbourne, England (1981). 12. A.H. Stouthamer, Symp. Soc. Gen. M i c r o b i o l . , 2 7 , 2 8 5 ( 1 9 7 7 ) . 13. A.H. Stouthamer, I n t . Rev. Biochem. M i c r o b i o l . Biochem., 21(1979). 14. A.H. Stouthamer, 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 , ( M e a d o w f i e l d , Durham)1976. 15. S. N a g a i , Advances i n B i o c h e m i c a l Engineering,11,48(1979) . 16. J.A. Roels and N.W.F. Kossen, 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 , M.J. B u l l , ed. ( E l s e v i e r , Amsterdam 1978)vol.14,p.95. 17. J.A. R o e l s , B i o t e c h n o l . Bioeng.,22,2457(1980). 18. L.E. E r i c k s o n , I.G. M i n k e v i c h and V.K. E r o s h i n , B i o t e c h n o l . Bioeng.,20,1595 (1978). 19. L.E. E r i c k s o n , S.E. S e l g a and U.E. V i e s t u r s , B i o t e c h n o l . Bioeng.,20,1623 (1978). 20. L.E. E r i c k s o n , B i o t e c h n o l . Bioeng.,21,725(1979) . 21. J . J H e i j n e n and J.A. R o e l s , B i o t e c h n o l . Bioeng.,23 ,739(1 981) . 22. J.G. M i n k e v i c h and V.K. E r o s h i n , F o l i o M i c r o b i o l . , 1 8 , 3 7 6 ( I 973). 23. A.E. Humphrey and P.R. J e f f e r i s , IV GIAM meeting, Sao P a u l o , B r a z i l ( 1 9 7 3 ) . 24. A.G. F r e d e r i c k s o n and H.M. T s u c h i y a , i n Chemical Reactor Theory, Lapidus and Amundson, e d s . , ( P r e n t i c e Hall,New J e r s e y , 1977)p.405. 25. M.A. Savageau, Math. B i o s c i . , 4 8 , 2 6 7 ( 1 9 8 0 ) . 26. I . P r i g o g i n e , 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 , New York 1955). 27. E.O. P o w e l l , 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 , P o w e l l et a l eds.,(H.M. S t a t i o n a r y O f f i c e London, 1967)p.34. 28. J.A. R o e l s , B i o c h e m i c a l E n g i n e e r i n g ; k i n e t i c s and e n e r g e t i c s , a book t o be p u b l i s h e d by E l s e v i e r , Amsterdam. 23

29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39.

G. T i e s s i e r , Rev. S e i . E x t r a i t du 3208,209(194-2). F.F Blackman, Ann. Bot.,19,281(1905). F. K a r g i and M.L. S c h u l e r , B i o t e c h n o l . Bioeng.,21,1871(1979). A.G. F r e d e r i c k s o n , R.D. Megee and H.M. Tsuchiya,Adv. A p p l . M i c r o b i o l . , 1 3 , 419(1970). D.M. Himmelblau, Process A n a l y s i s by S t a t i s t i c a l Methods, (John W i l e y , New Y o r k , 1970). D.W. Tempest, D. H e r b e r t and P.J. P h i p p s , 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.M. S t a t i o n a r y O f f i c e , London, 1967)p.240. P.Pipyn and W. V e r s t r a e t e , B i o t e c h n o l . Bioeng.,20,1883(1978). A. Gaudy and E.Gaudy , 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 , (McGraw H i l l , N e w York, 1980). S. Cromie and H.W. Doelle, Biotechnol. Lett.,2(8),357(1980). A. Gaudy, P.Y. Yang and A.W. Obayashi, J . Wat. P o l l u t . C o n t r o l Fed.,43,40, (1971). E. S c h r o d i n g e r , What i s L i f e ? (Cambridge U n i v e r s i t y P r e s s , Cambridge,1944) p.75.

24

CHAPTER 3

MATERIALS AND METHODS

DESCRIPTION OF THE EXPERIMENTAL SYSTEM AND ANALYTICAL METHODS set-up

Experimental

A l l b a t c h and f e d - 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 11.10 3 3 w o r k i n g volume. 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 . 10 3 m3 w o r k i n g volume. I n a l l c a s e s , 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 6.8 0.05 and 308 0.5 K, r e s p e c t i v e l y .
_ 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 . 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 - b a t c h e x p e r i m e n t s . 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. 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%. 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 . 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. T h e r e f o r e , 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).
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, 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 . T h i s c a p a b i l i t y , 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 . 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). 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, 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 . 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. 10 3 m3 working volume New Brunswick fermentor o p e r a t e d i n b a t c h , f i l l and draw o r c o n t i n u o u s modes. These experiments were c a r r i e d o u t a t a temperature of 293 K and pH o f 6.8 .
_

25

Organism For mono - c u l t u r e experiments Klebsiella pneumoniae NCTC 418; f o r m e r l y known as Klebsiella aerogenes, was used throughout t h i s study. 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. 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. 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 - b r o w n sludge was obtained. 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 ' . 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 ) . Water a c t i v i t y of the s t a n dard medium was about 0.996. 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 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 - b a t c h experiments. 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 0.2 m i c r o n pore diameter membrane ( S a r t o r i u s 11307). 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 , pH d r i f t , e v a p o r a t i o n e t c . 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. 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 . ( B o e h r i n g e r UV method, 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 .10"^ kg/m3. 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 . 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 . 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 . 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 , by an o n - l i n e exchanger designed and manufactured i n o u r department. 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.

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 - N i t r o g e n a n a l y s e r .

TC, 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 - c o n t a i n i n g compounds.

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 continuous c u l t u r e experiments w i t h C i n the order of 3-5 kg/m3, the average e r r o r i n d r y weight d i d not exceed 2 %. I n dynamic e x p e r i ments, p a r t i c u l a r l y i n b a t c h r u n s , 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.
x

26

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, N, 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. 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 . 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 , f o r t h e same samples. 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 , by t h e o r c i n o l and diphenylamine procedures, r e s p e c t i v e l y , as d e s c r i b e d by H e r b e r t e t a l . 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 . Total Carbohydrate : Anthrone method, as d e s c r i b e d by H e r b e r t e t a l . 5 was used.

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, because of the d i f f i c u l t i e s w e l l documented elsewhere.-* 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 . 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 . Between s e t s , however, 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 .

II

DEVELOPED AND USED TOOLS AND METHODS of Gas Exchange

Quantification

Assumptions: a. 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. b. No CO2 and/or N2 f i x a t i o n . c. Only O2 and CO2 exchanged. 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. sample gas l i n e r. r e f e r e n c e gas l i n e Fig. 1: Experimental set-up; gas processing and analysis flow diagram.

27

With r e f e r e n c e t o F i g . 1, where t h e gas f l o w s a r e i n d i c a t e d , a b a l a n c e on gaseous N2 , which passes through the system unchanged, p r o v i d e s the s t a r t i n g p o i n t . T h e r e f o r e , a mass b a l a n c e reads:

E N - i n = 2 or
N

Z N - out 2 A

and A

N^ 2

(b. = N in 2

U t

i> out

. 2
n =

*out
n

- 2-2>/*in

( 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. 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 , 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 , i . e ,
n out

*out Once t h e o u t f l o w expressions ;

*in ^ '('out ' " "

'
s c a

" 2 - 2 >
u

(2)

d,

OUR, CPR can be e a s i l y c a l c u l a t e d by the

OUR = ( 0.21 6. in

4 > ) / 0.0224 out

(mole 0/m /hr) 2

3

(3)

CPR = ( CO^ < f > - C O " < ) ) / 0.0224 }. 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 , N|n , 0^ , and CO^ can be taken as 0.79, 0.21 and 0.033, r e s p e c t i v e l y . 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 0.0360. From eq.(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 . D u r i n g t h i s study a s i m p l e computer program was developed which, when p r o v i d e d w i t h the raw gas exchange d a t a , c a l c u l a t e d tyout p o i n t and OUR, CPR and RQ, s u b s e q u e n t l y . 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 .
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. I f t h i s cannot be assumed, 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 . 6 , 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. 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. I n f e d - b a t c h systems, 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 . I n the f o l l o w i n g , the g e n e r a l case, 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 , w i l l be c o n s i d e r e d . For a b i o l o g i c a l system, r e l e v a n t t o t h i s s t u d y , 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 . 28

Of

feed oxygen

O
Ckg/m /hr)
3

c a r b o n dioxide oxygen effluent

1

<l>c

<Dg

ut

<De
process.

Fig.

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 , 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, 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. These f l o w s , however, 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 , 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 - c a l l e d major f l o w s . Mass, t o a v e r y good a p p r o x i m a t i o n , 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 . 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 . 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 . 2 t h i s equation gives; + - $ - $ t ie c a r o n

(mass)/ d t =

(6)

For t h e system d e s c r i b e d

d (mass)/ d t = $

C 0 2

( ? )

Since <PQ2 - $ 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 . Moreover, 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

( p V )/ dt = * - $ s s F e
3

0.032 r o

0.044 r c

(8)

where $ i s the mass f l o w (kg/m^/hr) r a t e and r , t h e n e t molar f l o w r a t e (mole/ m-Vhr). 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 , r e s p e c t i v e l y . 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 ;
s s

/ r c o we o b t a i n the r e l a t i o n ; dV /dt - {$ - $ + r s F e c (0.032/RQ - 0.044) - 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, e.g. volume of d i l u t i o n e t c . , 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, 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 , b u t a l s o by exchange i n gas phase, 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

as r e f l e c t e d by i t s RQ v a l u 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 . 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. 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 , 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 . 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 . 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. However, 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 - 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 . T h i s method w i l l be r e f e r r e d to as the Declining Feed, FedBatch Technique.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. 3).

Fig.

3: Schematic

representation

of the

Declining

Feed, Fed-Batch

Technique.

The procedure i. ii.

i s as f o l l o w s :
s m

iii. iv.

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 , P ax and Y f o r the e x p o n e n t i a l phase. Continue as f e d - b a t c h ; 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 - f i x e d p a t t e r n , say l i n e a r l y . 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 , at l e a s t momentarily. 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 , i . 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.) Having determined q , y , Y and m calculate Y from :
s x s g x x m x s m a x s x g x

Y * sx

y max

/ ( q s

- 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, dM /dt can a l s o be assumed t o be z e r o . 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 . 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

consumption p r o c e s s , dM /dt can be taken as zero whenever dM /dt approximates to z e r o . 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. 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 , 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 , dM /dt w i l l be n e g l i g i b l e . I f , however, 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 , 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. T h i s might happen when, f o r i n s t a n c e , 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.
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 . e . , F ( t ) = c o n s t a n t , and was found to be u n s a t i s f a c t o r y . 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 . 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 . 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 , k , m and Y from a s i n g l e experiment. I n p r a c t i c e however, 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 . The p r a c t i c a l use of t h i s method, 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 , i s p r o v i d e d i n Chapter 4.
m a x m a x s s g

Advantages of the new method: i. no m c o n s t a n t h y p o t h e s i s . , , *% , . , max i l . e v a l u a t i o n of m independent of Y . i i i . 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. iv. t h r e e parameters ( y , Y , 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. v. 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 . 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 .
s g g x m a x g x g

Disadvantages : i. m i s determined o n l y a t zero growth r a t e , i . e . , cannot check i f m = f ( l i ) . ii. 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). i i i . s e n s i t i v e to sampling; 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.
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 . 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 , as p o i n t e d out by H a r r i s o n . ' ' However, 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 - b a t c h modes.
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 . 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, would be u s e f u l .

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 . 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. Theoretically U can a l s o be determined from the OUR and CPR curves i f the
m a x

31

r e s p e c t i v e y i e l d s a r e assumed t o be constant.'2,13 However, 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 . e . , C , OUR and CPR d a t a . 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 wash-out t e c h n i q u e , 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. 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;

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 , 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 . 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. 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 . 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.'^ 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 , 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. 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 , a n a l y s i s for c o r r e l a t i o n , parameter e s t i m a t i o n from m u l t i - r e s p o n s e data e t c . by many a u t h o r s . ' ' However, 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 . To t h e knowledge of the a u t h o r , 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 . ^ 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 . However, t h e r e a r e s t i l l problems t o be s o l v e d . 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 - r e s p o n s e d a t a i n t r a n s i e n t range. 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. 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, 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 . 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 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 , 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. When t h e r e are n o n - 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 , a f t e r t r a n s f o r m a t i o n , 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 . 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; F i g . 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 =1.0). Here, t h e d r y weight p r o f i l e i s shown.The a s s o c i a t e d e r r o r i s assumed t o be a =0.05 kg/m3 . Based on t h i s data the curve i . e . , C v s . time data was p l o t t e d . 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 . 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 . 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. T h e r e f o r e , 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 . 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.
m a x x x x

32

Fig.

4: Simulation nonlinear

of exponential growth; error structure regression procedures (see text).

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 . The double r e c i p r o c a l p l o t i . e . , 1/Y v s . 1/y p l o t , 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 . 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 . T h e r e f o r e , 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 . I t must be reminded t h a t both methods, l i n e a r and n o n l i n e a r , 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. 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 . 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 . 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 . 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, 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. 8. Assessment of Error Propagation; 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, 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, 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 . F o r a l i n e a r f u n c t i o n of the form ; a Xj + b X + c X + (12)

where a, b, c,... a r e c o n s t a n t s and X j , X^, X^, . . . . random v a r i a b l e s , t h e maximal n u m e r i c a l e r r o r i n W, 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

+ |c| A X

(13)

where AW i s the maximal e r r o r i n W. 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 ; 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^, X , X^, 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 .
33

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. 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:23,24 Var (W) = a
2

Var ( X j ) + b

Var(X ) + c
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 , 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 . ( 1 4 ) . 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. 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 , 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 ;
a s

EC x , x^, x^,

> x^ )

- f ( X j , x , x^,
2

(15) }(X. x

E {3f( X i=l

, X

) / 3X.

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 . F u r t h e r m o r e , by assuming t h a t t h e random v a r i a b l e s a r e independent, 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 , X ,

X ) }

x_ )/ St

} Xj,^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, 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 . Carbon and Nitrogen Balances ; 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 - 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. 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 . 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 . However, 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 . 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. Steady s t a t e o p e r a t i o n ii. No C O 2 o r N 2 f i x a t i o n iii. Carbon l i m i t e d c u l t u r e and no C - 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. 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 12/22.4 = F ( C w + C w + C w ) + s s x x p p (17)

F. C . w + S. CO. in s i s in 2

out

| CO^ > out 2 34

12/22.4

Due t o e x p e r i m e n t a l e r r o r s , however, 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, CRI, t o assess the c o n s i s t e n c y of the C-balances: CRI = I C -out / Ic - in
n o u t

(18)

or the % C-recovery as 100 x CRI. I f , F - F , 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

P
n

(19)

F. C . w in s i s Therefore ;

A. C 0 ^ in 2

12/22.4

CRI = f ( F. , C , C , C , w , c | > . <> , C0^, CO", C .) ). 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 , 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 , r e s p e c t i v e l y , and hence can be assumed t o be c o n s t a n t f o r compounds of known formulae. To e s t i m a t e V a r ( C R I ) , 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, 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 . 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). 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 , 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.

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|. =AX. SX. i l

a v 1

experiment.

% total . . maximal error 8 4 1 1 17 0 5 13 41 0. 1 0.5 99.6

a CRI - 2 , 5 } Var(X.).10 aX. l l 8.46 1 .94 16.90 37.64 0. 3.84 24.34 230.04 0.0014 0.036 0.00324xl0~
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. 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 . 10 20 si 68 10 $in C0in 3 6x10~

10 Sum

35

Using t h e v a l u e s l i s t e d , CRI was c a l c u l a t e d t o be. 0.95, i . e . , 95 % C-recovery. 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 , 6 %. 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 . 5. The d i s t r i b u t i o n s of the e r r o r s , however, 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.g., 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 . 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 . 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, 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 . 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 . I t i s a l s o apparent, 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. 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. 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 . 5. I n t h i s case NRI, nitrogen recovery index d e f i n e d s i m i l a r l y , was c a l c u l a t e d t o be 1.01 i . e . , 101 % r e c o v e r y . 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 %, r e s p e c t i v e l y .

Fig.

5: Contribution of the major sources C- and N-balances.

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- 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 , i s p r e s e n t e d i n Table I I . 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 . 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. 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. As can be seen C- and N- r e c o v e r i e s were good. 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 . 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 - b a t c h experiments. R e c o v e r i e s i n these cases were n o t as good. 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 - b i c a r b o n a t e i n t h e l i q u i d phase. 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 , (see Chapter 8) N - 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. 36

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. Table I I : Assessment of the o r i g i n a l C- 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.

CRI x l O 97 94 99 15 NRI x l O 97 61

a 4.08 2.97

(min 90.0 94.5

max) 104.6 104.6

2 6.77 83.0 108. 1

* 91.8 108. 1 98 78 5.59 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 0.90, 0.91 , 0.92,0.90 * 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 0.83, 0.84, 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 . 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 . 2 5 - 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 . e . , f o r f u l l y a e r o b i c growth w i t h no by-product formation. aC,H 0 383
o o

+ b0 + cNH C 2 3
0

H 0N + dC 0. a B y 2
D

+ eH 0 2

(20)

For t h i s system, 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 , 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 . D u r i n g the experiments however, s u b s t r a t e , oxygen, biomass, carbon d i o x i d e and sometimes the ammonia f l o w s were measured i . e . , 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. 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 ) . The mathematical background to the problem has been g i v e n by a number of authors.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 1.29-31 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 . 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 .
a n s

I n F i g . 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, F i g s . 8,9 ) i s shown. 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 . 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 . 6. 37

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 . 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 , 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, which were a p p r o x i m a t e l y 5,10,7,3 and 5% f o r s u b s t r a t e , oxygen, ammonia, biomass and carbon d i o x i d e f l o w , r e s p e c t i v e l y . 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 an i d e a l case i . e . , when a l l e r r o r s are random, 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.

RQ

0.9 c 1 raw data corrected data

Fig.

6: Rao and statistically

corrected

RQ data for a continuous

culture

run.

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 A -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 2.6 5.4 17.2 2.8 2.2

(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. The method i s founded on the assumption of the randomness of the e r r o r s . 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 , 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 . 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, 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.

38

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

REFERENCES

1. C.G.T. Evans, D. H e r b e r t 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 D.W. R i b b o n s , eds,(Academic, London, 1970)vol.2,313. 2. A. Harder, Ph.D. t h e s i s , U n i v e r s i t y o f Wageningen,(1979). 3. J.P. B a r f o r d and R.J. H a l l , B i o t e c h n o l . Bioeng.,21,609,(1979). 4. W. de V r i e s and A.H. Stouthamer, J . B a c t e r i o l . , 9 6 , 4 7 2 ( 1 9 6 8 ) . 5. D. H e r b e r t , P.J. P h i p p s and R.E. S t r a n g e , i n Methods i n M i c r o b i o l o g y , J.R. N o r r i s and D.W. Ribbons,eds,(Academic, London, 1971)vol.5b,209. 6. R.E. T r e y b a l , Mass T r a n s f e r O p e r a t i o n , 2 nd ed.(McGraw H i l l , New Y o r k , 1968)p.192. 7. J.H. P e r r y , Chemical E n g i n e e r s Handbook, 4 t h ed.,(McGraw H i l l , New Y o r k , 1963)p.15.2. 8. S.Cromie amd H.W. D o e l l e , B i o t e c h n o l . L e t t . , 2 ( 8 ) , 3 5 7 ( 1 9 8 0 ) . 9. A.A. E s e n e r , N.W.F. Kossen and J.A. R o e l s , 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 , E a s t b o u r n e ( 1 9 8 1 ) . 10. I.G. M i n k e v i c h and L . I . U t k i n a , B i o t e c h n o l . Bioeng.,21,357(1979). 11. D.E.F. H a r r i s o n , 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 , E a s t b o u r n e ( 1 9 8 1 ) . 39

12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32.

D.T. B o y l e , B i o t e c h n o l . Bioeng.,19,297(1977). D.T. B o y l e , B i o t e c h n o l . Bioeng.,20,1101(1978). D.W. M a r q u a r t , J . Soc. I n d u s t . A p p l . Math.,2,431(1963). D.B. Johnson and P.M. Berthouex, B i o t e c h n o l . Bioeng.,17,557 (1975). D.B. Johnson and P.M. Berthouex, B i o t e c h n o l . Bioeng.,17,571(1975). W.C. B o y l e and P.M. Berthouex, B i o t e c h n o l . Bioeng.,16,1139(1974) G.E.P. Box and N.R. Draper, B i o m e t r i k a . 5 2 , 3 5 5 ( 1 9 6 5 ) . N.R. Draper and H. Smith, A p p l i e d R e g r e s s i o n A n a l y s i s , ( W i l e y , New York, 1967) . P.M. Berthouex and W.G. Hunter, J . S a n i t . Eng. D i v . Amer. Soc. C i v i l Eng., 97,393(1971). P.M. Berthouex and W.C. Hunter, J . S a n i t . Eng. D i v . Amer. Soc. C i v i l End., 97,333(1971) J.W. de K w a a d s t e n i e t , J.C. Jaeger and A.H. Stouthamer, J . Theor. B i o l . , 57,103(1976). D.M. Himmelblau, 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, New Y o r k , 1970)p.36. C.G. P a r a d i n e and B.H.P. R i v e t t , S t a t i s t i c a l Methods f o r T e c h n o l o g i s t s , (The E n g l i s h U n i v . P r e s s , 1972)p.l61. F . S c h e i d , N u m e r i c a l A n a l y s i s , ( S c h a u m O u t l i n e S e r i e s , McGraw H i l l , New York 1968) p.357. A.K.S. Murthy, Ind. Eng. Chem. P r o c e s s Des. Develop.,12(3),246(1973). A.K.S. Murthy, Ind. Eng. Chem. P r o c e s s Des. Develop.,13(4),347(1974). P.M.E.M. van G r i n t e n and J.M.H. L e n o i r , S t a t i s c h e P r o c e s s B e h e r s i n g , (Prisma T e c h n i c a , Utrecht,1973)p.336. F. Madron and V.Vacenek, C l l . Czech. Chem. Commun.,42,1805(1977). F. Madron, V, Veverka and V. Vacenek, J.AIChE.,23(4),482(1977). F. Madron, B i o t e c h n o l . Bioeng.,21,1487(1979). H.E. de Kok and J.A. R o e l s , B i o t e c h n o l . Bioeng.,22,1097(1980).

40'

CHAPTER 4

FED-BATCH CULTURE: MODELLING AND THE

APPLICATIONS IN

STUDY OF MICROBIAL ENERGETICS * F. Kossen

A. A. Esener, J . A. Roels and N. W.

SUMMARY M i c r o b i a l growth i n f e d - 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. 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 , 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 . I t i s shown t h a t the f e d - 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 . 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 . The maintenance c o e f f i c i e n t s determined from f e d - 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 .

INTRODUCTION The t h e o r y of f e d - 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 . 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.1-12 i o s t c a s e s , however, 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 . These i n c l u d e the assumptions of c o n s t a n t volume, s u b s t r a t e l i m i t a t i o n , q u a s i - s t e a d y s t a t e e t c . Moreover i n many c a s e s , 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 ; 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 .
n m

I n an attempt to f i l l t h i s gap, 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 - 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. 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, r e p o r t e d e a r l i e r by R o e l s and Kossen.'^ 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 . Furthermore, i t i s shown t h a t f e d - 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 . Bioeng'. (1981) 41

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. Here, 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 ) , 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 . 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 - 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 . F i n a l l y , 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 .

MODEL To d e s c r i b e a 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. 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 . O f t e n , however, 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, due t o e v a p o r 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 . T h e r e f o r e , 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 . 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 . ^ 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. I n t r o d u c i n g them, they are M and M s = V C s
x s

= V C

where V i s the c u l t u r e volume, C biomass c o n c e n t r a t i o n , 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 , 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)

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 . 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 ) - R

(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. By d e f i n i t i o n

= y(s) M

(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 . F u r t h e r m o r e , i f Monod k i n e t i c s ' - ^ 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 , one can w r i t e

^
and
R

( S )

= V x

/ ( M

s
+

s
M

( 4 )

s = (V

C *

s *

(5)

42

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, m nance c o e f f i c i e n t on s u b s t r a t e , and K i s d e f i n e d as

s

st

:le

011

the m a i n t e -

= 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 . 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 . 1 the f l o w diagram of the mathematical model). 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 . 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 .
g s

Substituting for R d ( M
x

, R

and y ( s ) i n b a l a n c e e q s . ( l ) and U MM s / (M+K s

m a X

( 2 ) , one

obtains (6)

)/dt =

max

d ( M

)/dt =

F(t) - ( R / Y x sx

+ m

(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. 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 . 1-5 ,10,15 I f , however, 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, d i f f e r e n t i a l s i n eqs,(6) and (7) can be expanded and reduced to

d(C

)/dt = u

max

/( C

+ k

)-C/V x

(dV/dt)

(8)

d ( C
s

)/dt = F ( t ) / V - r
s

- C /V
s

(dV/dt)

(9)

where R = V r . 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 , one can w r i t e
s s

equations. balance.

(dW/dt) = 6 ( t )

(0

- 0 ) + (CC>

U t

- C0 )

Ut

+ (H 0
2

i n

- 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)

- (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 , 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 ) .

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 . Furthermore, the c o n t r i b u t i o n of h u m i d i t y , l o s s of o r g a n i c v a p o r s , 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 . I f they can be n e g l e c t e d e q . ( 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

p(dV/dt) = 6 ( t ) + (32 r - 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 , r oxygen uptake, 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 . 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

/ r o

(12)

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 , when

{ 32/(44 RQ)

- 1 } / 1000

(13)

RQ > 32/44 , and when

dV/dt < 9 ( t ) / p

RQ < 32/44 ,

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 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 - h a n d s i d e of eq.(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 ) . I n a n a e r o b i c f e r m e n t a t i o n s , however, 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 . 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. 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 . 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.

DETERMINATION OF BIOKINETIC PARAMETERS AND AND MAINTENANCE COEFFICIENTS

YIELD

I f designed c o r r e c t l y , 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 - b a t c h experiment. C o n s i d e r i n g eqs.(6) and (7) when

erratum and hence

_Xj

K s u max

jj - ]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 - b a t c h experiment y 44

o b t a i n e d from a p l o t of l n ( M ) v s . time. 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,y, 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 . 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 , y w i l l assume a v a l u e of y /2. 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 , the Monod s a t u r a t i o n c o n s t a n t . 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 , w h i c h , 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 . 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 , however, 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 . When R = dM /dt = 0, dM /dt may be assumed to be equal to z e r o , 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 . ' " T h i s assumption was checked and j u s t i f i e d l a t e r on, e x p e r i m e n t a l l y . 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 . ( 7 ) , m can now be g i v e n by m = F ( t ) / M . S i m i l a r l y , m = R /M and m = R /M . T h e r e f o r e , 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 , 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.''' 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 . 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 , and above a l l , 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 .
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 - b a t c h d a t a . 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 , i . e . 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. At t h i s s t a g e , however, the experimenter must be aware of the f a c t t h a t a f e 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 . 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 . 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. F o r i n s t a n c e , 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 - 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 .
m a x

MATERIALS AND METHODS Organism Klebsiella pneumoniae NCTC 418, f o r m e r l y known as Klebsiella used throughout t h i s study. Cultivation Methods aerogenes ,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 . ' ^ 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 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 . 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 0.2 ymmembrane filter i n t o a s t e r i l e fermentor. 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 0.05 % 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.'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 0.5 K. The pH was c o n t r o l l e d at 6.8 0.05. A i r f l o w to 45

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 0.77 kg d r y a i r / h . 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 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. 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), r e s p e c t i v e l y . 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. 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. 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 . 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,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 . Dry w e i g h t s were determined by the method of de V r i e s and Stouthamer.20 Biomass was c o l l e c t e d on a 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), washed w i t h d i s t i l l e d w a t e r , and d r i e d t o c o n s t a n t weight a t 378 K. 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). 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 ) .

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. Experiment FB 830 FB 21 1 C 50.22 50.75 H 6.71 6.84 dry N 13.44 14.07 0 29.64 28.35
CH

pneumoniae.

Formula 1.60 0.230.44 1.62 0.240.42

N N

CH

In p e r c e n t a s h - f r e e

weight ;average

ash c o n t e n t was 8 %.

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 - 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 . 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 - l i n e heat exchanger manufactured i n the workshop of t h i s department. 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. 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 - f r e e b a s i s . The average ash c o n t e n t o f d r y biomass was about 8 %. Y i e l d v a l u e s p l o t t e d , 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 . ^ ' 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.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). 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 . 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.

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 - b a t c h e x p e r i ment. Here, the i n i t i a l s u b s t r a t e was about 3.5 kg/m . 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 . 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

t M =M + rdM
S

rzi
[Tmx
m

, V ,

dM _
x

Mma- S- X
M M

dt

M +K
=

1
1x = x o /
M + _

i
t
fdM ^
d x

Fig.

1: Simplified

block

diagram

of the mathematical
-

model used for

simulations.

throughout the experiment a t 8.807 x l O ^ kg g l y c e r o l / h r . I n F i g u r e 2. the t o t a l biomass, M and s u b s t r a t e , M , 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, 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 . 2 7 Here, good agreement can be observed w i t h the experiment and the model developed. 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 .
x s x

Fig.

2: Experiment FB 830, biomass and substrate profiles : (9) biomass; (M) substrate; ( ) model simulation. Parameters: Wiax - 1.05 hr~l; K = 1.0 kg; F i t ) = 8.807. 10~ kg/hr; M = 7.4 .10''5 kg; M - 34.62 .1CT kg; - 0.54 kg/kg; m = 7 .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 . 3) a r e examined, however, 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 . 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

these v a r i a b l e s respond t o changes much f a s t e r than M , 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 . 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 .
x x

Fig.

3: Experiment FB 830, gas exchange rate profiles: (9) oxygen uptake rate; (0) carbon dioxide production rate; () model simulation. Parameters ^ox - kg/mole; Y^E = 54.12 . 10~ kg/mole; m = 1.54 mole/kg/hr; m = 1.32 mole/kg/nr.; other parameters same as shewn in Fig. 2.
X 36 15 l c r 2 2 0 c

Fig.

4: Experiment FB 830: change of yields during biomass on (0) substrate; (O) oxygen.

the fermentation.

Yield

of

I n F i g . 4. 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 . 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 . 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 . e . w i t h d e c r e a s i n g growth r a t e , i s observed. The o t h e r f e d - b a t c h experiment FB 211, r e p o r t e d f u l l y i n t h i s paper, 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 . Here, 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. 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. The behaviour of t h e c u l t u r e d u r i n g the 48

f e d - b a t c h mode i s shown i n F i g . 5. 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. 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 , up t o about t = 865 min. 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 . These f i n d i n g s suggest a change i n maintenance requirements around t = 865 min, i f the model i s t o be v a l i d . The observed RQ d u r i n g the f e d - b a t c h growth mode i n c r e a s e d almost linearly. 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. 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 . 5.). 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. 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. T h e r e a f t e r , 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 . 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. Here, 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 . 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 . ^ 7

Fig.

6: Experiment FB 211, biomass and RQ profiles during substrate limited phase: (*) biomass; ($) RQ; ( ) model simulation of biomass p r o f i l e Parameters same as given in Fig. 2. F i t ) varied according to F i t ) (85 - 0.064. time). 10~ kg/hr, where t > 49 2 min.
z

In F i g . 6. 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.^ 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, RQ i s g i v e n by

RQ = {1 - ( a / a ) Y ^ }
b s

/ {(Y /4).(. s

<Vb'Vs x
= s

)Y

5 )

F o r t h i s experiment, u s i n g the v a l u e s of aj, = 0.508, a - 0.391, Ys 4.67 and Yt, = 4.06, 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 . 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 . 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 . Based on t h i s c r i t e r i a , 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

Fig.

6. Consistency check for data obtained from experiment FB 211: ( ) theoretical relation between RQ and Y , eq. (15) in text; raD experimental data; (*) statistically corrected experimental
sx

f ) data.

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 , 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. 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 . Furthermore, 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 . A n o n - 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 . The computer program used was developed i n the Department of Chemical E n g i n e e e r i n g , D e l f t U n i v e r s i t y of Technology and based on Marquart's method.29,30
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 , 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 . 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. 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 , 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. 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.31,32 A c o m b i n a t i o n of the above two mechanisms i s , of c o u r s e , a l s o p o s s i b l e . I f t h e p l o t s of OUR and CPR ( F i g . 3.) 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 , 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. Moreover, t h e r e a r e a l s o d i f f e r e n c e s , however s l i g h t , 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 . 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 , whereas a f t e r the peak, t h e d e v i a t i o n s a r e p o s i t i v e . 50

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

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

10-

(kg)

Ckg/m )
3

30 20
10

0.1 0.5 1.0 '5

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

3C 0

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

{ ( 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

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

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

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 . I f samples 6 and 7 can be regarded as o u t l i e r s , a smooth curve f i t s the M d a t a . However, a s i m i l a r behaviour has a l s o been observed i n experiment FB 016. T h i s almost abrupt change suggests a s h i f t i n c e l l metabolism, 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 . The c e l l s seem to become more e f f i c i e n t .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 . U n f o r t u n a t e l y , 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 . 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 > 0.05 h r ' a f t e r a hazardous e x t r a p o l a t i o n . 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 . (see F i g u r e 8)

s

steady

Fig.

8: Experiment FB 211, simulation of the experiment Other parameters same as before.

with

various

values.

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. The new v a l u e s of Y and Y used f o r these balances were 0.524 kg/kg and 35.57 x 10 kg/mole, r e s p e c t i v e l y . 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 , assuming t r u e v a l u e s of m and m as 3.53 x 1 0 kg/kg/hr and 1.34 mole/kg/hr, r e s p e c t i v e l y . 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 , 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 . 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 .
s Q m a x m a x J s - 2 0 g 0

N e i j s s e l - ^ 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 . 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 . R e c e n t l y , 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 . 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 . 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 . 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 ,
s

54

Fig.

9: Experiment FB 211: Maintenance coefficients as calculated by balancing for different time intervals during the fed-batch growth phase. Coefficients of maintenance on (O) substrate; (0) oxygen.

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 . 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 . 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.

CONCLUSIONS 1) The f e d - 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 , s i m u l t a n e o u s 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 . However, 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 . 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. 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 ; i . e . , 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 . 4) Maintenance requirements determined i n f e d - 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 . 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 , 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 .

55

APPENDIX Volume changes d u r i n g a f e d - 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 , 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 , a c i d / a l k a l i a d d i t i o n e t c . can amount up t o 15 % o f the i n i t i a l volume. 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 , 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 . 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 .

Method 1: Say a t t = t i , 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. A t t = t j , t h e r e f o r e , 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 . C ] and V ] . C ] , r e s p e c t i v e l y . T h e r e f o r e at any time t= t , 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

Similarly for substrate n

v . c .

II 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. 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 , 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 . 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 . However, 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 . F o r comparison purposes, 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.

Read t U).V (I).C (I)

s s x

r EH

M (t) = M (t)- V (I).C (U M ( t ) = M ( t ) - V (I).C (I)

s s S S x x S X

Fig.

10: Correction

procedure

for sampling

in the simulation

program.

56

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. 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 . 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. 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.

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 . 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 . 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)
-

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S.J. P i r t , J . A p p l . Chem. B i o t e c h n o l . , 24,415(1974). F. Y o s h i d a , T. Yamane and K. Nakamoto, B i o t e c h n o l . Bioeng.,15,257(1973) R. K e l l e r and I . J . Dunn, J . A p p l . Chem. B i o t e c h n o l . , 2 8 , 5 0 8 ( 1 9 7 8 ) R. K e l l e r and I . J . Dunn, J . A p p l . Chem. B i o t e c h n o l . , 2 8 , 7 8 4 ( 1 9 7 8 ) S. A i b a , S. Nagai and Y. N i s h i z e w a , B i o t e c h n o l . B i o e n g . , 1 8 , 1 0 0 1 ( 1 9 7 6 ) T. Yamane and S. H i r a n o , J . Ferment. Technol.,55,156(1977) T. Yamane and S. H i r a n o , J . Ferment. Technol.,55,380(1977) T. Yamane, T. Kume, E. Sada and T. Takamatsu, J . Ferment. Technol.,55,587 (1977) 9. T. Yamane, E. Sada and T. Takamatsu, B i o t e c h n o l . Bioeng.,21,111(1979) 57

10. S.J. P i r t , Ann. N.Y. Acad. S e i . ,0077-8223/0326-119(1979) 11. I . J . Dunn, S. Shioya and R. K e l l e r , Ann. N.Y. Acad. Sei.,0077-8923/03260127(1979) 12. H.C. Lim, B.J. Chen and C. Creagen, B i o t e c h n o l . B i o e n g . , 19,425(1977) 13. J . Monod, 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, Paris,1942) 14. A.H. Stouthamer and C. Bettenhaussen, B i o c h i m . B i o p h y s . Acta,301,53(1973) 15. J.A. Roels and N.W.F. Kossen,"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 , " 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 , M.J. B u l l , E d . ( E l s e v i e r , Amsterdam, 1978) 16. I.G. M i n k e v i c h and L . I . U t k i n a , B i o t e c h n o l . Bioeng.,21,357(1979) 17. A.G. M a r r , E.H. N i l s o n and D.J. C l a r k , Ann. N.Y. Acad. Sei.,102,536(1963) 18. C.G.T. Evans, D. H e r b e r t and D.W. Tempest, i n Methods i n M i c r o b i l o g y , J.R. N o r r i s and W.W.Ribbons, Eds.(Academic p r e s s , London, 1970)vol.2,p.313. 19. J.P. B a r f o r d and R.J. H a l l , E x p . C e l l . Res., 102,276(1976) 20. W. de V r i e s and A.H. Stouthamer, J . Bacteriol.,96,472(1968) 21. H.E. de Kok and J.A. R o e l s , B i o t e c h n o l . Bioeng.,22,1097 (1980) 22. F. Madron and V. Vanecek, C o l l e c t . Czech. Chem. Commun.,42,1805(1977) 23. F. Madron, V. Veverka and V. Vanecek, AIChE J.,23,482(1977) 24. F. Madron, B i o t e c h n o l . Bioeng.,21,1478(1979) 25. A.K.S. Murthy, Ind. Eng. Chem. P r o c . Res. Dev.,12,246(1973) 26. A.K.S. Murthy, I n d . Eng. Chem. Proc. Res. Dev.,13,347(1974) 27. D. H e r b e r t , Continuous C u l t u r e , v o l . 6 , ( 1 9 7 5 ) 28. L.E. E r i c k s o n , I.G. M i n k e v i c h and V.K. E r o s h i n , B i o t e c h n o l . Bioeng.,20,1595 (1978) 29. D.M. Himmelblau, 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,1979) 30. D.W. M a r q u a r t . J . Soc. I n d . A p p l . Math.,2,431(1963) 31. A.A. E s e n e r , J.A. R o e l s and N.W.F. Kossen, B i o t e c h n o l . Bioeng.,22,1979(1980) 32. J . P . B a r f o r d and R.J. H a l l , B i o t e c h n o l . Bioeng.,21,609(1979) 33. S.W. F i t z p a t r i c k , P h . D . t h e s i s , U.M.I.S.T.,1977 34. J . J . H e i j n e n and J.A. R o e l s , B i o t e c h n o l . Bioeng.,23,739(1981) 35. D. H e r b e r t , Symp. I n t . Congr. Microbiol.,6,38(1958) 36. A.H. Stouthamer, Symp. Soc. Gen. M i c r o b i o l . , 2 7 , 2 8 5 ( 1 9 7 7 ) 37. O.M. N e i j s s e l , Ph.D. T h e s i s , Amsterdam U n i v e r s i t y , 1976 38. P. P i p y n and W. V e r s t r a e t e , B i o t e c h n o l . Bioeng.,20,1883(1978) 39. H. van V e r s e v e l d , Ph.D. t h e s i s , Amsterdam F r e e U n i v e r s i t y , 1 9 7 9

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CHAPTER 5

GROWTH OF MONO AND MIXED CULTURES IN SALINE ENVIRONMENT * A.A. E s e n e r , N.W.F. Kossen and J.A. 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 . 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 . 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 . 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.

KEYWORDS S a l i n i t y ; mono c u l t u r e ; mixed c u l t u r e ; e n e r g e t i c s ; k i n e t i c s ; b i o l o g i c a l water t r e a t m e n t . 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 . 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 , p r o d u c t i o n o f s a l t s , water s o f t e n i n g by i o n exchange, r e v e r s e osmosis, e l e c t r o d i a l y s i s , p i c k l i n g , c a n n i n g , cheese and f i s h meal m a n u f a c t u r i n g e t c . 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 . The major l o a d o f s a l i n e waste w a t e r , 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. 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 - s h o r e i n s t a l l a t i o n s . 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. 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. * 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 , h e l d a t W a t e r l o o , Canada(June 1980)

59

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 , 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, 1939; P i r t and Tackeray, 1964; S c o t t , 1953; W o d z i n s k i and F r a z i e r , 1960) . 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 , i n most cases N a C l , 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. 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 , 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 . 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 . 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 , 1974;Imai, Endoh and Kobayashi 1979a, 1979b; K e s s i c k and Manchen, 1976; Kincannon and Gaudy, 1966, 1968; Kincannon, Gaudy and Gaudy, 1966; Lawton and Eggert,1957; Ludzack and Noran, 1965; Tokuz and E c k e n f e l d e r , 1979). 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 . 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. S t u d i e s w i t h mixed c u l t u r e s , however, a l l s u f f e r e d from the same phenomena ; a d a p t a t i o n , 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.g. 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 . Moreover, 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. A more fundamental approach has, t h e r e f o r e , been adopted i n t h i s s t u d y . F i r s t the growth b e h a v i o u r of Klebsiella pneumoniae (aevogenes); 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 , was s t u d i e d i n b a t c h c u l t u r e s . 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. 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 , Endoh and K o b a y a s h i , 1979a). 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 , the magnitude of responses changed c o n s i d e r a b l y . 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 . 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. 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 .

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 . 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. 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 6.8, r e s p e c t i v e l y . 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. 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 . 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 . Oxygen c o n t e n t was found by d i f f e r e n c e . Ash c o n t e n t was determined s e p a r a t e l y . No carbon c o n t a i n i n g b y - 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 . 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. 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 , 1 9 8 0 ) . 60

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 . R e s u l t s o f a c t i v a t e d s l u d g e , named as mixed c u l t u r e h e r e a f 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 , Endoh and Kobayashi (1979a). 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 . 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, t h e r e f o r e , 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. The experiements were performed i n 0 - AO kg/m3 NaCl range which a l s o covered the sea water case (approx. 33 kg/m3) .

Mmax Hmax

0.5

0 0 10 20 30
[NaCl]

40
Ckg/m )
3

Fig.

1: Maximum specific

growth rate

vs. NaCl

concentration.

I n F i g . 1, the n o r m a l i z e d U , 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 , 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. 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 . I m a i , 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. However, 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 .
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. c 49.68 H 6.71 N 13.45 0 30. 16

pneumoniae. Formula
C H

l . 6 2 0 . 2 3 0.46

% ash f r e e d r y w e i g h t . Average ash c o n t e n t 8.63%. I n F i g . 2, 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 . 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. The observed l a g times have, 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 . The same assumption was a l s o adopted by I m a i , Endoh and
61

Kobayashi

(1979a).

L a gT i m e

Chr)

mono

A /

Imai et.al. mixecC*^

10

20

30

40 [NaCl](kg/m )
3

Fig.

2: Observed

lag time vs. NaCl

concentration.

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, q , i s p l o t t e d i n F i g . 3. A maximum q r e p o r t e d by Imai e t a l . (1979a) was n o t observed i n t h i s study.

Q 0

Fig.

3: Oxygen uptake

rate

vs. NaCl

concentration.

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 , was used for y i e l d calculations. Y i e l d s of biomass on s u b s t r a t e , Y , oxygen Y and carbon d i o x i d e , Y , are p r e s e n t e d i n F i g . 4. A l l y i e l d s a r e expressed as mole/mole. 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. 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 , RQ, ensures the e x i s t e n c e
s x o x c x

62

of an o p t i m a l y i e l d range ( F i g . 5.). 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 0.9u where the NaCl c o n c e n t r a t i o n i s 5 kg/m.3.
m

Fig.

4: Influence

of NaCl concentrations

on biomass

yields.

Respiratory Quotient |RQ

Fig.

5: Respiratory

quotient

as affected

by NaCl

concentration.

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 . l , 2 and 3, 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. Mono c u l t u r e s , however, 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 . From 0 to 40 kg/m3 N a C l , y of the mono c u l t u r e decreased by about 10 t i m e s , whereas t h a t of mixed c u l t u r e by 5. 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 . 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 , throughout the e x p e r i m e n t a l range.Mono c u l t u r e s , however, 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.3) . 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 . The r e l a t i v e success of .mixed c u l t u r e s i n
m a x

63

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 . 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). 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. However, 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. 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. 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 .

Energetic

Considerations

I n F i g . 6 , the thermodynamic e f f i c i e n c y o f the growth p r o c e s s , n , 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.

Fig.

6: Thermodynamic concentration.

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 : (.)

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, 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 . 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

ox

( 4 /

n /(i - 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 , 1978; R o e l s , 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 . 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 , 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 . 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 , by t h e s o - c a l l e d "maintenance energy" r e q u i r e m e n t s ; a term which i n c l u d e s energy
64

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 , 1973; Watson, 1970). 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 . T h e r e f o r e , 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.
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 , q / h r ) , s p e c i f i c consumption r a t e , i s g i v e n by:

(mole/mole

q
m a x

I Y

m 3 X

sx

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, 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 . I f they are assumed c o n s t a n t s , 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 2.09 (1.97 - 2.21) and 0.032 (0.023 - 0.040) f o r Y and m , 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 ) . 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. 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. However, 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 . , at h i g h salinities.
m a x s s s s

% Carbon input

ICOn

II 100-

biomass

50-

NaCl

(kg/m )
3

10

15

20

25

30

40

Fig.

7: Effect

of NaCl level (3) as : = 1 / Y

X
m a x

on the distribution

of substrate

carbon.

Rewriting equation r
S

/ r

sx

/ 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, 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 . I f Y i s c o n s t a n t , 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 ; 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 . T h i s i s a l s o apparent from F i g . 7
s x a x s

65

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.

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 . 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 , t h e r e f o r e , 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 , thereby r e d u c i n g the amount of sludge formed. From t h i s p o i n t of v i e w , h i g h s a l i n i t y i s a desirable p r o p e r t y . However, from F i g . 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 . A consequent decrease i n Y , as p r e d i c t e d by eq. (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, the s o l u t i o n of which, of c o u r s e , depends on the r e l a t i v e c o s t s i n v o l ved. I n F i g . 9, 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 , i s the amount of oxygen taken up per mole of s u b s t r a t e consumed. 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 .
o x s x s x o x

Fig.

9:

Graphical

representation

of the optimization

problem.

CONCLUSIONS 1. 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. Mono c u l t u r e s , however, 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. 2. 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. growth

3. 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. 66

4. 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.

REFERENCES B u r n e t t , W (1974). 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 . , Wat. Sewage Wks., 37-55. Dean, A.C.R., and C. Hinshelwood(1966). 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 P r e s s , O x f o r d , pp. 55-68. E r i c k s o n , L.E., I.G. M i n k e v i c h , and V.K. 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 . , B i o t e c h n o l . Bioeng.,20, 1595-1621. H e r b e r t , D. (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 . I n G. Tunewall ( E d ) , Recent P r o g r e s s i n M i c r o b i o l o g y , A l m q u v i s t and W i n k s e l l , Stockholm,p.381. I m a i , H., K. Endoh and J . Kobayashi ( 1 9 7 9 a ) . 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 . J . Ferment. Technol.,57(4) 333-340. I m a i , H., K. Endoh, and C.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 . , J . Ferment. T h e c h n o l . , 5 7 ( 4 ) , 453-459. Ingram, M. (1939)The endogeneous r e s p i r a t i o n o f B. c e r e u s . , J . B a c t e r i o l . , 3 8 , 613-629. K e s s i c k , M.A., and K.L. Manchen (1976) S a l t water domestic waste t r e a t m e n t . , J . Wat. P o l l u t . C o n t r o l . Fed., 4 8 ( 9 ) , 2131-2136. Kincannon, D.F., and A.G. 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 . J . Wat. P o l l u t . C o n t r o l . Fed.,38(7), 1148-1159. Kincannon, D.F., A.F. Gaudy and A.G. 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 . B i o t e c h n o l . Bioeng.,8,371-378. Kincannon, D.F., and A.G. 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 . B i o t e c h n o l . Bioeng., 10,483-496. de Kok, H.E., and J.A. 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 . B i o t e c h n o l . Bioeng.,22, 1097-1104. Lawton, G.W., 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 . Sewage and Ind. Wastes, 2 9 ( 1 1 ) , 1228. Ludzack, F . J . , and D.K. 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 . , J . Wat. P o l l u t . C o n t r o l . F e d . , 3 7 ( 1 0 ) , 1404-1416. P i r t , S.J., and Thackeray (1964). 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 . Exp. C e l l Res.,33, 396-405. R o e l s , J.A. (1980) B i o e n g i n e e r i n g r e p o r t , 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 . B i o e n g . , 22, 2457 S c o t t , W.J. (1953) Water r e l a t i o n s o f S. aeureus a t 3 0 O C , A u s t . J . B i o l . S c i . 6, 549-564. Stouthamer, A.H. and C. 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 . B i o c h i m Biophys. A c t a . , 3 0 1 , 54-69. Tokuz, R.Y. and W.W. 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. Water Res.,13, 99-104. Watson, T.G. (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. c e r e v i s i a e . , J . Gen. M i c r o b i o l . , 64,91-98. W o d z i n s k i , R.J., and W.C. 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 . J . B a c t e r i o l . , 79, 572-578.

67

CHAPTER 6 I

THE INFLUENCE OF TEMPERATURE ON THE MAXIMUM SPECIFIC GROWTH RATE OF KLEBSIELLA A.A. PNEUMONIAE *

E s e n e r , J.A. R o e l s and N.W.F. 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. M i c r o o r g a n i s m s , u n l i k e h i g h e r organisms l i k e mammals, 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. 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. T h e r e f o r e , 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 . 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, 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 ; changing the r e a c t i o n r a t e s . 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, 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 . 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.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 - b a t c h mode. 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.

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 :

x,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 , 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, AH, 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 . Bioeng. , 23 ,1 401 (1 981 > 69

the r a t e l i m i t i n g r e a c t i o n , A i s a c o n s t a n t , 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. I f the maximum s p e c i f i c growth r a t e , 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 . ( l ) :

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, below the s o - c a l l e d o p t i m a l temperature.' 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 . 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 . ^ 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 , 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 , 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 - i n a c t i v a t i o n r e a c t i o n . 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 , 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

K exp(-AH

/ 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 , r e s p e c t i v e l y , 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 . From eq. ( 3 ) , s i n c e f j + f = 1, one o b t a i n s :
A A

= {1 + K exp(-AH

/ RT)}"'

(4)

Furthermore, 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 , from eq.(3) i t f o l l o w s t h a t :

x, max

= A exp(-AH* / RT) f. E C 1 A x

(5)

then from eqs.(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 , . max ing can be written: A' exp (-AH*
V
m a X

/ RT) (6) / RT)

= 1 + K exp(-AH

MATERIALS AND METHODS The organism, Klebsiella pneumoniae NCTC 418, 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 . 9 G l y c e r o l was used as the o n l y carbon and energy source. I t was assayed e n z y m a t i c a l l y . 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.'0 Biomass was c o l l e c t e d on a 0.2 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), 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. 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 0.2 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. 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 . The 70
_

pH was c o n t r o l l e d at 6.8 0.05. 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 0.77 kg dry a i r / h r . 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, carbon d i o x i d e and w a t e r , 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 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 - l i n e heat exchanger manufactured i n our workshop. 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.

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. 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. 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 same computer program was used f o r e v a l u a t i n g the parameters of the temperature- y model, i . e . , eq.(6).
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. 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 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 .

Fig.

1: Maximum specific growth rate vs. temperature. model equation (6) evaluated with parameters

() given

experiment; ( in Table I.

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. 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 , 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. 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.
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

Table I : C a l c u l a t e d and r e p o r t e d model parameters. 95 % c o n f i d e n c e AH * AH

2

levels

86.40 287.78 5.69 . 1 0 48 1.38 .10

1 4

kJ/mole kJ/mole 1/hr ()

(44.19 - 128.64) (188.33 - 387.23)

A' K

Range

AH^
a

343

kJ/mole

147

828

From M o r o w i t z , average f o r 20 p r o t e i n s .

Fig.

2: Natural

logarithm

of

V>

max

vs. the reciprocal

of absolute

temperature.

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 . 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, t h e magnitude of the a c t i v a t i o n e n t h a l p y , AH*, may be underestimated s i g n i f i c a n t l y . 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 . ' 2 From eq.(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

/ {R l n ( K (AH^AH* - 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 .

M

-,

I n s e r t i n g the parameters o b t a i n e d one gets 36.9C as the o p t i m a l temperature f o r t h i s system. An i n f l e c t i o n p o i n t of 31.7C was c a l c u l a t e d by e v a l u a t i n g the 72

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 , i n c r e a s e i n V^ax 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 , i . e . , t h e nominator of e q . ( 6 ) . 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 . 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. As can be seen from
m a x s

Fig.

3: Active fraction of the enzyme taking part in the growth reaction as a function of temperature. Computed according model equation (4).

limiting to the

t h i s p l o t , 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 32C 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 . I n the range 33 < T < 38C, 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 . T h e r e f o r 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 . 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 . 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 , 287 kJ/mole, compares w e l l w i t h h i s average of 343 kJ/mole (Table I ) . 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 - 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.

NOMENCLATURE A, 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 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

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

; the a c t i v a t i o n energy v a l u e s . TC 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 - 32C. 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

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 25C 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 20C 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

Table I I I : Temperature dependence of m v a l u e s ; t h e a c t i v a t i o n energy d a t a . Organism E. coli E. coli C. lipolytica C. lipolytica E. coli A. aerogenes K. pneumoniae K. pneumoniae 11. polymovpha B. caIdotenax substrate glycerol hexane hexane glucose glucose glycerol glycerol me t h a n o l glucose E (m ) kJ/mole
s

TC range 20 - 45 18 18 15 25 25 25 35 50 30 30 30 40 43 43 43 60

ref. 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 . F i n a l l y , two s p e c i a l cases have t o be p o i n t e d o u t ; 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 , d i f f e r e n t from t h a t r e p o r t e d above. Recent work of Kuhn e t a l . 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. caldotenax, 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 8.3 and 23.8 k J /
m a x a x

OX,

mole, r e s p e c t i v e l y (T range of 50 - 60 C ) . With mixed c u l t u r e s , 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 , 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 . 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 , d a t a on Y shows no d e f i n i t e t r e n d of change. I n most r e p o r t s some s o r t of maximum i s reached a t about 20C.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.g., p r o d u c t i v e f e r m e n t a t i o n , and n o t i n o t h e r s , e.g., b i o l o g i c a l waste water treatment p r o c e s s 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. Few w i l l be c i t e d here : - From a p r o c e s s o p e r a t i o n view p o i n t , 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 . 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. 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 - u p , 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 , 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 . - 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, where the d e s i r e d product i s biomass e.g. SCP , Bakers y e a s t p r o c e s s e s , one aims t o maximize the p r o d u c t i v i t y , i . e . , 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 , 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 . 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 . 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 . - 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 , 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 - t e m p e r a t u r e r e l a t i o n , 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, 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. - 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 , 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

tenance m e t a b o l i s m , e.g., e t h a n o l p r o d u c t i o n by Z. mob His, h i g h temperatures would be advantageous. A d d i t i o n a l l y , 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 . R e c e n t l y , 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. - I n t h e f i e l d of waste water t r e a t m e n t , 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 , 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 . However, 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. T h e o r e t i c a l l y , due t o i n c r e a s e d maintenance demands, 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 . 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. T h e r e f o r e , 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 . 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 . C o l l i n s e t a l . ^ 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.

REFERENCES 14. H. T o p i w a l a 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). 15. A.G. Marr, E.H. N i l s o n and D.J. C l a r k , Ann. N. Y. Acad. S c i . , 1 0 2 ( 3 ) , 5 3 6 (1963). 16. J.T. Novak, J . Water P o l l u t . Cont. Fed.,46,1984(1974). 17. S.E. M a i n z e r and W.P. Hempling, J . B a c t e r i d . , 1 2 6 ( 1 ) , 2 5 1 ( 1 9 6 6 ) . 18. I.S. Farmer and C.W. Jones, FEBS L e t t e r s , 6 7 ( 3 ) , 3 5 9 ( 1 9 7 6 ) . 19. R. M o l e t t a , G. Goma and G. Durand, A r c h . M i c r o b i o l . , 1 1 8 , 2 9 3 ( 1 9 7 8 ) . 20. C.L. Cooney and A. M a k i g u c h i , i n Continuous c u l t u r e , 6 , A . C . R . Dean, D.C. Elwood, C.G.T. Evans and J . M e l l i n g , eds., ( E l l i s Horwood, C h i c h e s t e r , 1975)p.146. 21. J.C. Senez, B a c t e r i o l . Rev.,26,95(1962). 22. A.H. Stouthamer, I n t . Rev. Biochem. M i c r o b i a l . Biochem.,21,1(1979). 23. J . J . H e i j n e n and J.A. R o e l s , B i o t e c h n o l . Bioeng.,23,739(1981). 24. H.J. Kuhn, S. Cometta and A. F i e c h t e r , 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(4),303(1980). 25. C.E. C o l l i n s , F.P. I n c r o p e r a and J.P.L. Grady, Water Res.,12,547(1978).

78

CHAPTER 7

A STRUCTURED MODEL FOR

BACTERIAL GROWTH

INTRODUCTION

I n Chapters 2 and 4, 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 . I t has been shown t h a t u n s t r u c t u r e d models, as f o r the system d e s c r i b e d , 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 . 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 . 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 s s i g n i f i c a n t . 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. 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 . 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 . e . , 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 . 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. 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, DNA, 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 . 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 . ' I t has t o be n o t e d , however, 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.g., 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 . 2 , 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 . 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. 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. 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 , 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, which c o n s i d e r one b i o t i c v a r i a b l e . These models, 79

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 , a r e c a l l e d Compartmental Models. 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 . The f i r s t compartmental model i s due to W i l l i a m s ^ . 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, at l e a s t q u a l i t a t i v e l y . 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 ^ . 7 5 . Ramkrishna et a l . ' and F r e d r i c k s o n et a l . 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. 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. 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^, f u n g a l alpha-amylase production""*, growth i n a c t i v a t e d sludge system'', product f o r m a t i o n by B. lioheniformis^^, 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 . 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. 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 . Advantages, 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 .
0 1

II The i. ii. 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, 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 . 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 . 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 ^ . 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.

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, f o r the system i n c o n s i d e r a t i o n . Two phases can now be i d e n t i f i e d : a. 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 , x , on a kg/m3 of c u l t u r e volume b a s i s . 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.
i-1
1

b. A - 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 - 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. 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 - 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. The v e c t o r , Y , 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 - m. Thus, 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 . e . , mass f r a c t i o n s i n the b i o t i c phase. D e f i n i n g a new v e c t o r , X , 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 , one o b t a i n s : 80

X. 1

m x. / E x .
1

and

1-1

X. i=l
1

In a homogeneous c u l t u r e o f c o n s t a n t volume, a g e n e r a l mass b a l a n c e reads :

dC / d t

r .a +

(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 .a x r . a -y

$ x

(3) (4)

i + -y

Eq.(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 - b i o t i c phase. 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 . a ) . 1
x A

(5)

Thus ( r . o^) . 1 can be i d e n t i f i e d as r , the r a t e of biomass p r o d u c t i o n . Furthermore, 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 . Ox

- ( { r . Ox). J_) X}

/ C

(6)

Eqs(4),(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 . e . 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 . Note, however, 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. A l s o note t h a t eq.(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 , i n o r d e r to o b t a i n a workable form, 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 ^ , J>y and r .

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, 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 . 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 . 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. Moreover, 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 . 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 , RNA, carbohydrate and o t h e r s m a l l molecules a r e lumped i n t o one compartment, c a l l e d the K-compartment. S i n c e RNA i s t h e main component, 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 . 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 . e . , p r o t e i n s , DNA, e t c . F i g . 1 g i v e s the b l o c k diagram of the model. Here, 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, from which G forms w i t h a c o n s t a n t y i e l d . 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 . e . , 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 ) . T h i s a l l o w s f o r
81

Fig.

1: Block

diagram

of the model.

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. 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. 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 :

SK

(7)

KG

rate

(8)

rate Now, 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 , r ^ and r .

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:

'

s S x
C C

S >

(10)

Here kg r e p r e s e n t s the maximum r g . 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 , r ^ i s suggested t o be d e s c r i b e d by

= =

k k

K G C

(1.)

K ( 1 - K ) C

s i n c e K + G = 1. Here note t h a t when G =0 e q . ( l l ) p r e d i c t s r^, = 0 i . e . , no " iK f o r m a t i o n . 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 . 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 .
B e erratum
J

* .\

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.

order

= =

m G C G x m

(12)

( 1 - K ) C x Here mg i s the maintenance f a c t o r f o r the G compartment.

Lj

82

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 , i s now g i v e n by : r =

r L S

r K

1
J

The a - 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

i T 0 ]

SK

0 KG

-1

Thus f o r t h e two compartment system d e s c r i b e d , 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 =

/ dt / dt {(1 - K) Y

-r S Y SK
S K

+< P S r s r
g

(13) + (Y KG r
K

K
R G

x - 1) K)
+

(14) r,} / ^

(1 * ( Y

SK

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. 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 . - C ) Yx< si s D C

a r e f i x e d , e.g., 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, 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 , i . e . , e q s ( 1 3 ) , (14) and ( 1 5 ) . 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

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, can be g i v e n f o r t h e steady s t a t e a s :

S - V

sx

SK

) ( D

) ( k

K \

( D

) ) ( 1

" KG> x

83

EVALUATION OF THE VALIDITY OF THE MODEL

K

The model presented has s i x parameters; 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 , Y g ) , 3 r a t e c o n s t a n t s (kg, kj^ , m ) and one M i c h e a l i s - M e n t e n type c o n s t a n t Kg . The model w i l l now be t e s t e d w i t h the continuous and f e d - b a t c h d a t a presented p r e v i o u s l y .
K G

Model parameters were e s t i m a t e d from f e d - 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 . The parameters a r e l i s t e d i n Table I . Table I : Model parameters.

Parameter
k

s
G

1 96 5 0 0 06 0 73 0 66 0 07

hr-> hr
H

K m
Y

hr"

SK YKG
K

kg/kg kg/kg kg/m

3

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 . R e s u l t s a r e shown i n Table I I . Table I I : Model s e n s i t i v i t y t o i t s parameters. Model output kg Biomass Substrate K compartment (++) very s e n s i t i v e k
K

Parameters m
G

S K

K G

Kg -

+ + + - - + + + + + - - + + + + +

+ + +

(-) 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 - b a t c h e x p e r i ment FB 830, and i s t h e r e f o r e s p e c i f i c t o i t . With parameters l i s t e d i n Table I , 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 . 2 ) . There seems to be a v e r y good agreement between the model and the experiment. 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 . Moreover, the model p r e d i c t s wash-out a t y = i.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 . I n F i g . 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. 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 . However, 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 .
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 , a wash-out experiment was s i m u l a t e d . 84

(kg/m )

Fig.

2: Simulation

of continuous

culture

behaviour

with

the

model.

Fig.

3: Predicted (- - -)

K profile and experimentally determined RNA straight line fitted to RNA data by linear

profile. regression.

A step i n d i l u t i o n r a t e from D = 0.1 to D = 1.2 was a p p l i e d ( F i g . 4 ) . 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 .
m a x

I n F i g . 5 the model s i m u l a t i o n f o r f e d - 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 . Here, 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 . 2 of Chapter 4 ) . 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 . 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., step-up and down experiments or s i n u s o i d a l f e e d i n g schemes. U n f o r t u n a t e l y , 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 .

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 , 85

1.2-

Ol 10

I M

1
2

I I I

I
to
3

10'

io

Fig.

4: Response of the growth from D=0.1 to 1.2 .

rate

to a step

change in the dilution

rate

Fig.

5: Model simulation

of the fed-batch

experiment

FB

830.

at l e a s t q u a l i t a t i v e l y . The model, 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, 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. 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.(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. 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. 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 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 . D 6,11 (see e q . ( 1 8 ) ) . T h i s r a t i o equals mg . 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 v a l u e of 0.5 h r ' i s o b t a i n e d which i s f a r too h i g h . A r e a l i s t i c v a l u e should be 0.03 0.05 . 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. As R o e l s ^ has noted, 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 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 . e . , assumed to be only chemically s t r u c t u r e d .
-

86

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 . e . , 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. 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 . 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 . 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.g., 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 . However, 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 , 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 . 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 . He s h o u l d , whenever p o s s i b l e , check mechanisms and o b t a i n parameters from independent e x p e r i m e n t s . 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 , 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 . 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. 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.However, 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 , 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 , 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. 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 .

Acknowledgement : The author wishes to thank s t u d e n t chapter. T. Veerman f o r h i s c o n t r i b u t i o n to this

87

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)
- 1

D
G K

K k m M Mx r
S G S

m X
Y G
K

ot
$

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 - b i o t i c phase y

VIII 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

REFERENCES D. H e r b e r t , Symp. Soc. Gen. M i c r o b i o l . , 1 I,391 (1 961 ) . N.W.F. Kossen, Symp. Soc. Gen. M i c r o b i o l . , 2 9 , 3 2 7 ( 1 9 7 9 ) . A.G. F r e d r i c k s o n , B i o t e c h n o l . Bioeng.,18,1481(1976). F.M. W i l l i a m s , J . Theor. B i o l . , 15,190(1967). J.A. Roels and N.W.F. Kossen, 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 , 14, 95(1978). J.A. R o e l s , B i o c h e m i c a l E n g i n e e r i n g ; E n e r g e t i c s and K i n e t i c s , a book t o be p u b l i s h e d by E l s e v i e r . D. Ramkrishna, A.G. F r e d r i c k s o n and H.M. T s u c h i y a , B i o t e c h n o l . Bioeng.,9, 129(1967) . A.G. F r e d r i c k s o n , R.D. Megee and H,M. T s u c h i y a , Advances i n A p p l i e d Microbiology,13,419(1970). G. v a n Dedem and M.Moo-Young, B i o t e c h n o l . Bioeng.,17,1301(1975). S.T. F i t z p a t r i c k , Ph.D. T h e s i s , U.M.I.S.T.,(1977). A. Harder, Ph.D. T h e s i s , 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,(1979). R. L a r s e n and L. K j a e r g a a r d , E u r . J . A p p l . M i c r o b i o l . Biotechnol.,5,177 (1978) . W.J. Grenney, D.A. B e l l a and H.C. C u r l , B i o t e c h n o l . Bioeng.,15,331(1973). A. Harder and J.A. R o e l 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.L. S c h u l e r , S. Leung and C.C. D i c k , Ann. N. Y. Acad. S c i . , 3 2 6 , 3 5 ( 1 9 7 9 ) .

88

C H A P T E R 8/APPLICATION 1

COMMENTS ON THE DESCRIPTION OF MAINTENANCE METABOLISM DURING ANAEROBIC GROWTH WITH PRODUCT FORMATION * A. A. Esener, J . A. R o e l s and N. W. F. 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 . 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, 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 . Some o f these a r t i c l e s , 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 , 1 9 8 0 ; Goma e t . a l . , 1 9 7 9 ) . 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 , 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 , 1 9 8 0 ) . 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 - 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. In t h i s work, f i r s t , 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 , 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 , 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. 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). 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 - b a l a n c i n g p r i n c i p l e 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. 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. E>C H 0 N a b c d
2 2 2 2

+ $ 0 + <KNH,, 2 3
5

$iC

H, 0 N, + b, c, d,

$ C
3

PL 0 N, b c d
3 3

+ $ C 0 + * H.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 , V o l . 3 , No.]-, 15-20 (1981) 89

For a system at steady s t a t e , i f the above d e s c r i p t i o n h o l d s , 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 . The l i n e a r law of s u b s t r a t e consumption, 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 , 1958), 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 , 1 9 7 3 ; R o e l s , I 9 8 0 ; Roels and Kossen,1978). r = r / Y + r / Y + m . 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 ,

(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 . C o x ox p op o x

Equations ( l ) - ( 3 ) d e s c r i b e the g e n e r a l case. The a n a e r o b i c case, which i s a s p e c i a l case , 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 . i . e . when r ^ = 0, from eq.(3) one o b t a i n s : r p = Y op ( -r / Y
X

OX

- m . C
O

(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 , 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 . r s = (1/ Y sx - Y op /(Y ox .Y sp )). r x + ( m s - m .Y / Y ). 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 _ , i n the s u b s t r a t e consumption e q u a t i o n . 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 , r can hence be expressed by :
s

r s

= r / Y' + m' . C x sx s x

(6) (Erickson et.al.,1978;

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 , 1 9 8 0 ) , of the form : Y . r = Y - r + Y - 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 -Y' )/( Y -Y' ) ) . r + ( y / y ). m' . 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 , 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 . 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 . 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 ; 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 , and the second to the amount of biomass p r e s e n t . 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,1980). T h e r e f o r e , i f rp i s expressed s i m p l y as : r = r / Y + m . 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. Y px

90

(8) and

(9) : (10)

= Y Y' p SX

/ ( Y -Y
S X

Y'
SX

= ( Y / Y ) m' s p s
Y

(11)

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., assuming average v a l u e s o f = 4.2 and t h e m o l e c u l a r weight o f t h e organism as 25.0, 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 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 . 1 . 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- Y Y (see eq.(10)) .
x = ( Y 1 Y s ) Y s i n c e Y s x s

m .10 P Y 0.04 px (C-eq/C-eq) 5.0

(C-eq/C-eq/hr)

2.5

0.01

Y' ( C - e q / C - e q ) sx i | 0.02 0.03

0.005

0.010

fig. fig. J

1:1

vs. 1' :( pec sec 2. m vs. m' : (p s

s x x s

) eq.(10); -) eq.(11);

( ) experimental ( ) experimental

data data

(Cromie (Cromie

and and

Doelle) Doelle)

Parameters Y , Yp , 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 ) . 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 . 2 , 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, i . e . 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. 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 . 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 , a n a e r o b i c a l l y . U s i n g eq. (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 . However, 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 . 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. A s i m p l e b l o c k diagram ( f i g . 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.
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). 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, a n a e r o b i c a l l y . However, 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 . 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 . 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 , 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, Table I ) were i n g r e a t e r r o r s , i . e . see F i g . 4. F o r the two cases f o r which Cromie and
91

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 1.85 and -1.25 g/g/hr f o r m (experiment no: 6 and 9 i n Table I ) . 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, however, 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 . A l l t h a t can be s a i d i s t h a t , 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 .
g s

product synthesis

use of substrate

*~

synthesis of biomass precursors

biomass synthesis

ii

ATP

pool

maintenance

fig.

3: Distribution of the substrate and Kossen, 1978)

energy

in microbial

metabolism

(Roels

Table I : Reported Exp. 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 . Mg m
s

(Cromie & D o e l l e 1980) g/g/hr. 5.9 2.9 3.2 3.9 5.8 0.0 1 .8 1 .0 0.0 3.0 5.4

(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, %, Mg = MgSO

% , *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 . C o n s i d e r i n g ATP as the energy c u r r e n c y , ATP consumption, i n terms o f t h e l i n e a r law, i s g i v e n by ( Stouthamer and Bettenhaussen,1973) :
q

ATP

" I ATP
Y

ATP

( 1 2 )

Moreover, 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. mobilis ) :
92

Ethanol

( M / Y

ATP

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.biomass/hr can be c a l c u l a t e d ( F i g . 5 ) . 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). 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, Z. mobilis , 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 , to s a t i s f y them.
AT

^ATP

(mmole/g.biomass/hr)

0.05

0.10

fig. fig.

4: Regression 5: m ATP

line

for

experiment

no:l. no:6 by eq.(12).

determination

for experiment

Table I I : Organism L. casei E. 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 . Growth l i m i t i n g substance n* ATP 1 .5 6.8 9.9 38.7 18.9 0.5 0.25 0.70 0.21 10.3**

E. coli S. cerevisiae S. oerevisiae c. paraplosis z. 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.

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 , 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 . 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 . 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

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 , the maintenance c o e f f i c i e n t m, 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 . 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 , 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 . NOMENCLATURE C H, 0 N, ^1 ! l C H, 0 Nj a b c d C H, 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

x C- eq. i i,j H Yi
Y

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 - 3d s p e c i f i c growth r a t e oxygen substrate biomass

REFERENCES

Cromie, S. and D o e l l e , H.W. (1980) B i o t e c h n o l . L e t t . , 2 , ( 8 ) , 3 5 7 . 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. (1078) B i o t e c h n o l . B i o e n g . , 20,1595. Goma, G. M o l e t t a , R., and Novak, M. (1979) B i o t e c h n o l . L e t t . , 1 ( 1 0 ) , 4 1 5 . H e r b e r t , D. (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.7 t h I n t . Congr. M i c r o b i a l . , E d . T u n e w a l l , G.,p.381, A l m q v i s t , Stockholm. Humphrey, A.E., and J e f f e r i s , P.K.,(1973) GIAM m e e t i n g , Sao P a u l o , B r a s i l , vol.4,p.767. R o e l s , J.A.,(1980) Biotechnol.Bioeng.,22,2457. R o e l s , J.A., and Kossen, N.W.F. (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 , Ed. B u l l , M.J.,14,p95, E l s e v i e r , Amsterdam. Stouthamer, A.H. (1977) Symp. Soc. Gen. M i c r o b i o l . , 27, 287. Stouthamer, A.H., and Bettenhaussen, C. (1973) B i o c h i m . B i o p h y s . Acta.,301,53.

94

APPLICATION 2

BIOENERGETIC CORRELATION OF COD TO BOD * A.A. Esener, J.A. Roels and N.W.F. 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 . 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 . 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 , COD/BOD, 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 , f o r a b a c t e r i a l system. 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 . Of t h e s e , 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, 1977). COD on t h e o t h e r hand, 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. 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 , l o c a t i o n o f t h e t e s t , e t c . 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 . F o r sewage o f t e n a r a t i o o f 2 - 2.5 i s used as an a p p r o x i m a t i o n t o COD/BOD (Nat. Swedish Env. Prot.Brd.', 1972).The use o f such c o n s t a n t s however, 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 . As w i l l be shown i n t h e f o l l o w i n g , 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 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 . However, 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 . 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 , t h e r a t i o COD/BOD cannot be regarded as a constant. 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 . 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 , b u t v a r i e d , 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 , Vol.3 No.A 193-198 (1981)

95

i n s t a n c e , w i t h the growth r a t e . 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, 1975; S h e r r a r d , 1980). However, 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 . I n t h i s work, 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 - 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 , 1973; E r i c k s o n et a l , 1978; R o e l s , 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 , i . e . 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 , 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 . 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 , 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 , 1973; E r i c k s o n e t a l , 1978; R o e l s , 1980). 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 , w i l l be c o n s i d e r e d . 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 . $C H, 0 N, 2 a2 D2 C2 d2 + $ 0 + < NH J > $ CH, 0 N 4 2 3 3 1 b] c j d] + $ C0 + 5 2
C

$,H0 6 2

(1)

Here, i t i s assumed t h a t c a r b o n , hydrogen, 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 - n e g l i g i b l e q u a n t i t i e s . 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 $, r e p r e s e n t the n e t s t e a d y - s t a t e f l o w s of the r e s p e c t i v e components. U s i n g the concept o f the degree of r e d u c t i o n , y, the thermodynamic e f f i c i e n c y , T], 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 , 1978; R o e l s , 1980) n = Y" (y /y ) (2) sx X s where, 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 - 2c - 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>

<>

Moreover i t has been shown t h a t once,n , i s known, oxygen demand can be e s t i m a t e d by ( R o e l s , 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). F o r u n i t amount of s u b s t r a t e consumption, BOD, 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.(2) and ( 4 ) , by: BOD = (Y
s

BOD

/ 4) (1 -n )

(6)

Furthermore, COD, 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 , 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 , 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 . l the r a t i o , 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 , a c c o r d i n g to e q u a t i o n ( 8 ) . Here, a t n=0 C0D/B0D becomes equal t o 1. i . e . no 96

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 . The o t h e r end o f t h e s c a l e i . e . r\ = 1 , 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, r e v e r s i b l y . 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, 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 0.7. The p l o t has t h e r e f o r e been drawn t o n =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. As the r a t i o o f COD/BOD i s e n t i r e l y dependent on n, 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. 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 . 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. The c o m p o s i t i o n o f s u b s t r a t e , however, i s o f prime importance. 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 , ( g l y c e r o l ; n =0.57, o x a l a t e ; n =0.26) 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. The v a l u e s ranged from 0.26 f o r o x a l a t e t o 0.67 f o r g l u c o n a t e . These v a l u e s as i n d i c a t e d i n f i g . 1. correspond t o a change i n COD/BOD o f more than two f o l 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 . I n an a c t u a l waste water s i t u a t i o n however, ( i . e . 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 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 - 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 . 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 . F o r growth on methanol f o r example, a range o f n , from 0.23 t o 0.46 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 , 1980). 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, 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 " , 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 ) . I t i s t o be n o t e d , however, 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 . 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 . 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. 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. Temperature and pH were s e t and c o n t r o l l e d a t 308 K and 6.8, r e s p e c t i v e l y . 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 . 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.23 - 0.46 y i e l d i n g a degree o f r e d u c t i o n o f 4.01 .
< 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, 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, 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 . 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 ) . 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 . U s i n g eqs. (2) and (8) t h e r a t i o , 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 . 2. and 3., r e s p e c t i v e l y . From these p l o t s i t i s c l e a r t h a t - t h e r a t i o COD/BOD 97

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

Na CI (kg/m )
process.

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

APPLICATION 3

DESCRIPTION OF MICROBIAL GROWTH BEHAVIOUR DURING THE WASH-OUT PHASE ; DETERMINATION OF THE MAXIMUM SPECIFIC GROWTH RATE *

A. A. Esener, J . A. Roels and N. W. F. 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 . 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 .

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. 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 , 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). The procedure f o r t h i s method i s as f o l l o w s : i . 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 - 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 , i i . step-up D t o D + AD a t time t=0, 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 , i i i . measure r e s p o n s e ( s ) o f the system, 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.
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 , 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 , 1960; Tempest, 1970; P i r t , 1975; Kuhn e t a l , 1980). I n t h i s work, 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. 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, V axb. 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 .
m

*Submitted

for publication,(1981).

101

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

- DC
X X

(1) (2)

d C / d t = D (C . - C ) - r S S I s s
x

Here, r , 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

(3) d u r i n g the wash-out phase, r can

x

I f , however, 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

y C max x f o r biomass, 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 - D ) t} x xo max or when transformed:

(5)

In C = I n C + (y - 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 , by the use o f eqs.(5) and ( 5 a ) , r e s p e c t i v e l y . Moreover, 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 , 1958 ; P i r t , 1965) can be assumed to be a p p l i c a b l e , 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), 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 . 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 .
x x

OUR = OUR CPR where,

exp

{(y

- D ) t}

(6) (7)

= CPR exp o

{(y - D ) t} max
m a x

OUR = ( y / Y o max ox CPR

+ m

) C xo ) C xo

(8)

= (y / Y o max cx

m a X

+ m

(9)

102

Furthermore, 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. Hence the maintenance terms can be c o n v e n i e n t l y dropped out. 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, 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.(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 . - C ) - ( y /Y + m ) C exp { ( y - 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,

{(C - C . + z ) - z so si , ,max z = (y /Y max sx

exp(y

max

t ) } / exp(D t ) + C .

si

(9)

. , + 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, i . e . , 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 . Hence f o u r responses can be monitored i n a w e l l designed experiment (C , C , OUR, 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 , simultaneously.
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 . 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 , 1 9 7 5 ) . 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 . 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 . Too a s m a l l step i n 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 , 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. 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 . T h e r e f o r e , 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 .
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 , 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. 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 . 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 , C , 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 . e . , eq.(5) as d e s c r i b e d by Himmelblau (1970).
a n x c a n

Var(u

max

) = {(y - D ) / ( l n C - In C ) } max x xo Var(C

XO

Var(C ) / C + Var(D) x x
2

(10)

l f

) = Var(C )
X

and

1/C

1/C

X O

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 - 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

103

e r r o r s i n v o l v e d i n the measurement of C and D. 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 . 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 . From eq.(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 . However, 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.
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. 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. Temperature and pH were s e t and c o n t r o l l e d a t 35C and 6.8, r e s p e c t i v e l y . Batch experiments were c a r r i e d out i n an 11 l i t e r working volume fermentor. 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 , 1980).

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 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 . 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. 1.

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.

Step i n D ( h r 1) from to 0.16 0.78 0.71 0.86 0.88 0.97 0.976 1 .095 1 .023 1 .050 1 .036 1 .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

0.390 0.922 0.934 0.918 0.948 0.985

0.628 0.944 0.939 0.966 0.959 1 .038

0.748 0.968 0.963 0.966 0.966 1 .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 . From t h i s 1. 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.(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 . 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 . 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. 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 . A f t e r t h i s p e r i o d , 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 , 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. (5) .
x

In F i g . 2 , 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. 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. I n a l l cases the same g e n e r a l t r e n d has been observed. That i s , 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 . 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 . A l s o f o r 104

, 0 0

OUR

Cmole/m /hr)

50-

20

C (kg/m ) 2V
x

_1

L_

Fig.

1: Wash-out

experiment

no:4. C

and OUR

profiles.

10 .
C
v

data

03 . 10 .OUR data

0.5 10 .
CPR data

05 . 05 .

A D Chr"') 10 .

Fig.
a

2: Calculated

y values max

as a function
J

of the

step-size. 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 , OUR, CPR).

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 . 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, 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 , t o zero step s i z e . 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 . Here, 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 . 2 . 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 . 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

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 . 3 ) .

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. 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 1.088 1.046 (see F i g . 3 ) : nonlinear regression linear regression 1.060 1.029 A B

C data x 1.121 0.987

From b a t c h experiments 1.087 (0.950 - 1.223) 1.047 (0.987 - 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.

3: Normaized

data for six batch

experiments.

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 . 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. Once a r e a s o n a b l y a c c u r a t e
l s

106

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 . 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 , mentioned e a r l i e r .
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 .

m a x

conditions,

The wash-out method has major e x p e r i m e n t a l advantages. S i n c e OUR and CPR can be r e c o r d e d o n - 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. Moreover, 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 - l i n e spectrophotometer t o t h e o u t l e t l i n e . 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 ; 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 , c o l o u r due t o pigment p r o d u c t i o n e t c . would be n e c e s s a r y .
x x

I t can t h e r e f o r e be concluded t h a t , p r o v i d e d t h e p h y s i o l o g i c a l , 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 , 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 .

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 . (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 , Roels J A , 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 . B i o t e c h n o l . Bioeng 22:19791983. 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 , 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 , A l m q v i s t and W i n k s e l Stockholm, p.381. 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, John W i l e y , New York. Kuhn H J , Cometta S, 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 , y i e l d , maintenance and death r a t e i n g l u c o s e l i m i t e d 107

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 . 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. P i r t S J , 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 . J Appl B a c t e r i o l 2 3 ( 1 ) : 87-98. 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 . Proc Roy Soc B 163:224-234. 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 , B l a c k w e l l , Oxford. Tempest D W (1970) Theory of chemostat, i n Methods i n M i c r o b i o l o g y , N o r r i s J R Ribbons D W (eds) v o l . 2 , p.268, Academic, London.

108

APPLICATION 4

ON THE STATISTICAL ANALYSIS OF BATCH DATA * A.A. Esener, J.A. Roels and N.W.F. 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. 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, enzymatic h y d r o l y s i s and convers i o n s . 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 . 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, 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 ) . 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. 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 . 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. In t h i s paper consequences o f these f i n d i n g s w i l l be shown. A simple method developed by Mandel , 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. 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, i t has to be e x p o n e n t i a l t o o . T h e r e f o r e , 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; the maximum s p e c i f i c growth r a t e . 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 . 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 . Bioeng.

(1981)

109

where C

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. L i k e many o t h e r s i t u a t i o n s , 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 , 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, o r i g i n a t e s from the same b u l k . 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 . For measurements to be a c t u a l l y independent, 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 , one measurement b e i n g made from each, at d i f f e r e n t run t i m e s . 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 . 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 , 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 . 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. Measurement- e r r o r , i n t r o d u c e d by a n a l y t i c a l methods, i n s t r u m e n t a l r e a d i n g s e t c . These are u s u a l l y independent. b. I n a d d i t i o n to e r r o r s of type a., 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 . 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 . 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 , l i k e pH, temperature, a ,DOT e t c . are c o n t r o l l e d , 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 . These type of e r r o r s are c a l l e d Process errors. 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 . e . , 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. 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 , i n c l u d e s a l l p r o c e s s errors preceeding i t . i . e . ,
w n

n = E
i

e. 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 , 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 . S i n c e i n t h i s case e r r o r s w i l l be :

{e,}, {( e.+ e

) - E },

,{(E

+ e ) -(

)}

l or
E

l 2

I n

n-l

n-1

I'

However, 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 , 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. "' - i . . . ' 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 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 , 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.
f

1 10

- How to determine which of the two type of e r r o r s ; measurement or p r o c e s s e r r o r , 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 , 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. F o r p r o c e s s e r r o r s , however, 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 . 1.) 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. 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 , r ^ , should o s c i l l a t e above and below r ^ = 0, a t random. 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 . 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'. 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 . 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 , 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. = g x. + l l .

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, i s g i v e n by :

e r r o r s , i . e . , the o r d i n a r y l i n e a r

Y. = g x. + l

e.

(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. 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. 5-i n 2 L. ) i - i
J

B = (

)/(
J

(3)

where or

Zj = Y j -

Z and

= 6

(Xj

X j _ j )

+ e.

(j

1,

n :

0)

L. = (x. -

*,_,) f o r non-overlapping test intervals

Furthermore, E. i s assumed to be independent and : V a r ( e . ) = f ( L . ) = c. L. J i J

c = constant

i . e . , 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 . Here, i t i s i n t e r e s t i n g t o note t h a t , 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, e q u a t i o n ( 3 ) becomes s i m p l y :

111

6 = Y

/ x

i . e . , 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 . 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 , however, they are indeed e s s e n t i a l f o r : a. 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 , b. 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 , w h i c h i s g i v e n by : n (6) = c / Z 5=1

Var when c

L.
J

(4) of the v a r i a n c e i s g i v e n by : a-, - . .

2

i s not known, the unbiased e s t i m a t e

( Jy~ ) ( r ) Z f - i - (Z. - P, L . ) } (5) I L. n-1 L. 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 . Furthermore, 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, 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 . Var

(B) =

Var Var

(%) _ (B)

6 5

2 n + 2n + 1 (n + 1) (2n + 1)

(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 . As n , + the r a t i o approaches to 1.2 or f o r n= 10, i t i s 1.15 . 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 . However, 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, 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 . I n t h i s case the r a t i o becomes:

e s t . 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 . s t a n d a r d e r r o r of (B) = 1/19.9

s.e.

(6) by 20 t i m e s . T h i s by 2/n f o r n > 10.

i . e . , 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

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. F o r the experiment r e p o r t e d f u l l y h e r e , 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 . 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 . 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 ) .

10

20

30

40

Fig.

1: Oxygen uptake rate data and the conventionally a batch fermentation.

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 - a x i s ( F i g . 2 . ) , a c l e a r t r e n d can be observed. 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 . 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 . 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, a c c o r d i n g t o the f o r m u l a ; n-k = {( (Y. - Y ) ( Y . i ,k n / Z(Y. - Y ) i

+ k

- Y)J

(8)

k = 1,2,

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. ( F i g . 3.). At l a g 1, r ^ i s 0.63, 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, i f t h e d a t a were independent. 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 ^ . 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. ( F i g . 3.) 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.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 . 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

d a t a p o i n t s are e q u a l l y spaced i n time. 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 , 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.

Residual ( V o b s e r v e d - v r e g r e s s i o n ) 1
3

10

20*

30

r-

40

Fig.

2: Behaviour of the residuals obtained by the application conventional linear regression procedure. 3: Autocorrelation function for the resiuduals

of the

Fig.

shewn in Fig. 2.

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 . n 40 39 40 Mmax( 0.913 0.939 0.921

hr

Method o f a n a l y s i s independent differential cumulative

')

0 % of y 0.35 3.60 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 . 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 ) , however, 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 n t h i s case by about 10 t i m e s . 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 12.5 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 ) . Dry weight d a t a not r e p o r t e d h e r e , 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 . 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. 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 . Both methods, however, 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 . 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. 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 , the model o u t l i n e d above s h o u l d be used. 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 - c a l l e d "process e r r o r s " . I t must be emphasized, however, 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, e.g., RNA,protein, enzyme a c t i v i t y e t c
m a x m a x

114

the independent e r r o r s may be p r e d o m i n a t i n g . 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 , 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 . 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 , max Method of a n a l y s i s exp. no : n independent I u 0 max 0.0160 1.011 0.0267 0.968 1 .098 0.0404 cumulative V max 0.996 0.970 1 .106
c

0 0 0494 0 0826 0 0807

C. I a 1 a 3. 1 3. 1 2.0

0530 0517 0405

6 6 6

REFERENCES 1. J . Mandel, J . Amer. S t a t . Assn., 52,552(1957). 2. A.G. F r e d e r i c k s o n , D. Ramkrishna and H.M. T s u c h i y a , Chem. Eng. P r o g . Symp. S e r i e s , 108(67),53(1971).

1 15

APPLICATION 5

CARBON DIOXIDE HOLD-UP AS A SOURCE OF ERROR IN BATCH CULTURE CALCULATIONS * A. A. Esener, N. W. F. Kossen and J . A. 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 . 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 . 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 ^ . However, 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 . 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 , 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 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 . 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 ^ .
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). 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 , 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 , are known. 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 . 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 , p a r t i c u l a r l y f o r c o m p u t e r - 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 , 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 .

MATERIALS AND

METHODS

The organism, Klebsiella pneumoniae NCTC 418, 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.148270). Dry w e i g h t s were determined by the method of de V r i e s and Stouthamer ^. Biomass was c o l l e c t e d on a 0.2 pm pore diameter f i l t e r ( S a r t o r i u s ) , 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 . B i o e n g . , V o l . 2 2 , 1979-1983 (1980) 1 17

d r i e d to c o n s t a n t weight a t 378 K. 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 0.2 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. 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) . 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. pH was c o n t r o l l e d a t 6.80 0.05 . 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 0.77 kg d r y a i r / h r . 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 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. 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), r e s p e c t i v e l y . 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. No p r o d u c t s o t h e r than biomass, carbon d i o x i d e , 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. pneumoniae at i t s maximum growth r a t e .

c

grown on g l y c e r o l

H 6.92

N 14.27

0 27.86
C H

Formula 1.63 0.240.41

N

50.96

* I n % d r y weight ; ash f r e e b a s i s .

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. 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), 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. Of these TC and TOC were measured by a Dohrmann DC-50 carbon a n a l y z e r . Carbon d i o x i d e not accounted f o r , by the gas phase a n a l y s i s , i . e . , 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 , 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 . (TC - TOC) (kg/m )
3

(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 . 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 - l i n e heat exchanger manufactured i n our workshop. 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.

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. 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 (1.07 0.02 h r ~ ' ) , 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 , p r o c e s s i n g , and a n a l y s i s times r e q u i r e d . 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. Few 118

fig. fig.

1: Biomass vs. fermentation 2: Carbon dioxide retained

time. in the broth during exponential growth.

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 i n c l u d e entrapment by c e l l s , 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 , 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 - c a r b o n a t e b u f f e r system. A t t h i s stage however, the r e l a t i v e importance of these mechanisms i s not c l e a r . 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. pneumoniae. 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 0.57 with correction 99 0.71 4.05 2.66 1 .06 0.27 0.50 Difference 7 18 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.(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 . 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. 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. 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, carbon d i o x i d e and w a t e r , 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, 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 , RQ i s g i v e n by:

119

RQ = {1 - Y ( a / a ) } / { ( / 4 ) .
s b s Y s

( 1

- Y (
s

V b

/a Y ))}
s s

(3)

For 0^=0.51, a =0.39 , Y 4 . 6 7 and Y =0.50 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 0.69 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 ) . The measured RQ, however, 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 . 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, measured RQ v a l u e s can be i n g r e a t e r r o r . 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 , f 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 - l i n e measurement of the RQ.
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 . 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 t o m carbon)
3 3 3

REFERENCES 1. C. L. Cooney, H.Y. Wang and D.I.C. Wang, B i o t e c h n o l . B i o e n g . , 1 9 , 5 5 ( 1 9 7 7 ) . 2. J.A. R o e l s and N.W.F. Kossen,"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 " , 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 , M.J. B u l l , E d . ( E l s e v i e r , Amsterdam, 1978). 3. L.E. E r i c k s o n , I.G. M i n k e v i c h and V.K. E r o s h i n , B i o t e c h n o l . Bioeng.,20,1595 (1978) 4. J.A. R o e l s , B i o t e c h n o l . Bioeng.,22,2457(1980) 5. J.P. B a r f o r d and R.J. H a l l , B i o t e c h n o l . B i o e n g . , 21,609(1979) 6. H.Y. Wang, C.L. Cooney and D.I.C. Wang, B i o t e c h n o l . Bioeng.,19,69(1977) 7. S. A i b a , S. Nagai and Y. N i s h i z a w a , B i o t e c h n o l . Bioeng.,18,1001(1976) 8. C.G.T. Evans, D. H e r b e r t and D.W. Tempest, i n 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. R i b b i n s . E d s . (Academic P r e s s , London,1970),vol.2,p.313. 9. W. de V r i e s and A.H. Stouthamer, J . Bacteriol.,96,472(1968)

120

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. 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 . 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 , 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. 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. 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. 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; the maximum s p e c i f i c growth r a t e . 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. 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 . e . , 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; maximal Y and m . 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 , 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 , 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 . 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 . 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 .
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 . 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 , 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. In Chapter 4, 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 - b a t c h mode. 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 . 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 . 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. I t i s a l s o shown t h a t f e d - 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. I n Chapters 5 and 6, 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 . 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. 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 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 . 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. 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 - b a t c h c u l t u r e s . An A r r h e n i u s type enzyme a c t i v a t i o n - 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. Of the e n e r g e t i c parameters, 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 - 40 C. Above 40 C i t decreased a b r u p t l y . Growth c o u l d not be s u s t a i n e d at 50 C. 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.
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, a simple s t r u c t u r e d model i s developed i n Chapter 7. When t e s t e d w i t h f e d - 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

u n s t r u c t u r e d model can. A d d i t i o n a l l y , 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 . 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 . N e v e r t h e l 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 ; t h a t i s ,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, 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 ) . 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 . 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 n A p p l i c a t i o n 1, 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 . I n A p p l i c a t i o n 2, 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 . I n A p p l i c a t i o n 3, growth d u r i n g the wash-out phase i s d e s c r i b e d . 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 . A p p l i c a t i o n 4, 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 . 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. F i n a l l y , 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 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 .

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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 mikrobile g r o e i . 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. 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, 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 mikrobile p r o c e s s e n . 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. 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 , 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 . 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 n 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 . 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. 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 ; 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 , 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' kofficient m . 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 . Gegeven een goede paramet e r s c h a t t i n g , 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. 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 . 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, 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 .
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 . 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 efficintere p r o e f o p z e t , 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 . 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 - 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 . 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 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 exponentile 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 . 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. 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 - 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 mikrobile g r o e i . 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 , t e weten het z o u t g e h a l t e en de temperatuur, 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. 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. 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 ) . 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 . 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

industrile p r o c e s s e n . 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 - b a t c h k u i t u r e . 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 - d e a k t i v a t i e te b e s c h r i j v 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. 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 2540C. Boven de 40C 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 . Boven de 50C t r a d er z e l f s helemaal geen g r o e i meer op. Over h e t z e l f d e temperatuurt r a j e c t nam de maintenance kofficient e x p o n e n t i e e l t o e .
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 . 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 . 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 . 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. 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 . 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. 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 mikrobile g r o e i t e onderzoeken. 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 . 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 , 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. 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 . 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. 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 . 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, 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 . 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. 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.

I 24

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Bu tezde mikrpsal e n e r j e t i g i n bugnk durumu mhendislik gbrs axsindan i n c e l e n m i s t i r . 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 , 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 axklanmxstxr. alxsmanxn 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 mhendislik 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 . 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 . 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 tketimi 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 . K e s i k l i retimde s i s t e m davranxsxnx k i n e t i k b i r paramtre o l a n maksimum bzgl ogalma hxzxnxn k e s i n o l a r a k s a p t a dxgx, 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 . Srekli retimde i s e b u durumun t e r s i geerli b u l u m n 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 saptanmktadxr. Aynx zamanda k i n e t i k b a g x n t x seiminin 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 literatrde 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 . D o g r u s a l s u b s t r a t tketimx bagxnt x s x , srekli kltur v e r i l e r i i l e sxnanmxstxr. Dsk reme 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 reme k a p a s i t e s i n d e k i ( v i a b i l i t y ) dsse b a g l a namxyacagx gbsterxlmitir.
s

Bolm 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 alxsmalarxna mhendislik 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 . Baha v e r i m l i deney yapma, p a r a metre t a y i n etme ve i s t a t i s t i k bzumleme 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 . Bolm 4 de, 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 kltrdeki d a v r a n x s x d a kapsamak zere g e n i s l e t i l m i s t x 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 . Yapxsxz model, ssel ve y a l a n c 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 . Model geis doneminde geerliligini k a y b e t m i s t i r . B e s l e m e l i kltr 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 alxsmal a r x n b i r a r a d a yrtlebilecegi 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 . Bolm 5 ve 6 d a , seilmis o l a n i k i evresel d e g i s i m i n y a p x s x z model parametr e l e r e zerindeki e t k i s i i n c e l e n m i s t i r . T/uzlulugun e t k i s i Bolm 5 de a r a s t x r x l m x s t x r . B u l g u l a r karma kltrler ( a k t i f amur) iin 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 . Karma kltrlerin 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 abuk 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 . Mhendislik 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 paramtre i s e s x c a k l x k t x r . B e s l e m e l i k e s i k l i kltrde 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 Bolm 6 da sunulmutur. Maksimum b'zgl reme 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 zere 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 - 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 zeri 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 . 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 . 40C n i n zerxnde b u paramtre b i r d e n a z a l m x s t x r . 50C de reme saglanamamxstxr. 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 ssel o l a r a k a r t m x s t x 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 Bolm 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 . Model b e s l e m e l i k e s i k l i kltr ve srekli kltr 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 , 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. Buna ek o l a r a k y a p x l x model hcre 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 , 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 , m o d e l i n b u bakxmdan geerle olmadxgxnx ve dogrulanamxyacagxnx g b s t e r m i s t i r . Yinede b u alxsmadan- b'nemli b i r sonu
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c x k a r x l a b i l i r ; 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 hcre i c i b i l e g i m i v e r i l e r i i l e olmalxdxr. Blm 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 . B u n l a r x n h e p s i e w e l k i 'blmlerde g e l i g t i r i l m i s , o l a n dsncelerin uygulamal a r i d i r . 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 . Uygulama 1 de hcre dxsx e l e k t r o n a l x c x l a r x n x n yoklugunda rn yapxmxnxn e n e r j i retim sreci i l e b a g x n t x l x o l d u g u gsterilmxtir. 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 , reme srecinin termodinamik v e r i m i gz nne a l x n a r a k , tretilmigtir. Uygulama 3 de reme sreci s i l i n m e (wash-out) durumunda i n c e l e n m i s t i r , Bu dnemde maksimum zgl reme 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 yntem gelitirilmitir. Uygulama 4 k e s i k l i kltr 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 zerinedir. Burada k e s i k l i kltr 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. 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 gsterilmigtir. Uygulama 5 k e s i k l i sreclerde 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 iaxet e t m e k t e d i r . 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 zerindeki 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 .

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STELLINGEN 1. 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 , i s l i n k e d to energy p r o d u c t i o n . This thesis, Chapter 8, Application 1. the

2. 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 . This thesis, Chapter 8, Application 2.

3. 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 . 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. This thesis, Chapter 2.

4. 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 . 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. 5. The 74 parameter model r e p o r t e d by S c h u l e r e t . a l . , 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 , 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. M.L. Schuler, 35(1979). S. Leung and C.C. Dick, Ann. NeV York Acad. Sci.,326,

6. 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 . 7. 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 . The procedures employed by A.I.C.E. , i . e . , 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 , and t h a t by I.A.W.P.R., i . e . , 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 , would be u s e f u l i n B i o t e c h n o l o g y . 8. 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. Erdingston Lao, in Murphy's Law, A. Bloch (Price, Los Angeles) 19 78.

9. The statement made by Gaden, " t h a t , 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 , 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 . E.L. Gaden, Opening Address to the Sixth Symposium, held at London, Canada (1980). International Fermentation

10. 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 .

t h e r e f o r e the computers

A. A. E s e n e r ,

1 s t October

1981.