Foundations of Biochemical Engineering - American Chemical Society

24 Growth Inhibition Kinetics for the Acetone— Butanol Fermentation JEANINE M. COSTA and ANTONIO R.

1

MOREIRA

Downloaded by IOWA STATE UNIV on October 19, 2014 | http://pubs.acs.org Publication Date: January 18, 1983 | doi: 10.1021/bk-1983-0207.ch024

Colorado State University, Department of Agricultural and Chemical Engineering, Fort Collins, CO 80523

The i n h i b i t o r y e f f e c t of each fermentation product on the cell growth r a t e and the k i n e t i c s of product formation was s t u d i e d f o r the acetonebutanol fermentation with C l o s t r i d i u m acetobutylicum ATCC 824. I n h i b i t i o n of cell growth was s t u d i e d by c h a l l e n g i n g c u l t u r e s with v a r y i n g concentrations of each product. There was a t h r e s h o l d c o n c e n t r a t i o n which must be reached before growth i n h i b i t i o n occurred. This c o n c e n t r a t i o n was found to vary with each i n h i b i t o r . Above the t h r e s h o l d c o n c e n t r a t i o n , there was a l i n e a r decrease of the growth r a t e with an increase in product c o n c e n t r a t i o n .

Due t o t h e r i s i n g c o s t s a s s o c i a t e d w i t h p r o d u c i n g c h e m i c a l s f r o m f o s s i l f u e l s , a l c o h o l f e r m e n t a t i o n s b a s e d on r e n e w a b l e r e s o u r c e s a r e b e c o m i n g more and more a t t r a c t i v e a s an a l t e r n a t i v e r o u t e f o r o b t a i n i n g commodity c h e m i c a l s . However, a p r o b l e m common t o many o f t h e s e f e r m e n t a t i o n s i s t h e r e l a t i v e l y l o w c o n c e n t r a t i o n of the d e s i r e d product i n the f i n a l fermented b r o t h . B a c t e r i a l ethanol fermentations stop a t a l c o h o l l e v e l s i n the r a n g e o f 5-8 w e i g h t p e r c e n t C l ) . A more d r a m a t i c t o x i c e f f e c t i s o b s e r v e d i n t h e a c e t o n e - b u t a n o l f e r m e n t a t i o n . The t o t a l s o l v e n t s c o n c e n t r a t i o n i s 2-3 w e i g h t p e r c e n t a t t h e p o i n t where s o l v e n t l e v e l s a r e t o x i c t o t h e p r o d u c i n g C l o s t r i d i u m (2). Butanol, b u t y r i c a c i d , and a c e t i c a c i d a r e t h e m o s t t o x i c p r o d u c t s o f t h e acetone-butanol fermentation. I t has b e e n p r e v i o u s l y shown (3) t h a t a t b u t a n o l c o n c e n t r a t i o n s i n t h e r a n g e o f 0.10-0.15 M, 50% i n h i b i t i o n i s o b s e r v e d s i m u l t a n e o u s l y f o r t h e maximum s p e c i f i c g r o w t h r a t e , t h e n u t r i e n t u p t a k e r a t e , and t h e membrane-bound ATPase a c t i v i t y . This end-product t o x i c i t y r e s u l t s i n l a r g e

1

Current address: International Flavors and Fragrances, Inc., Union Beach, N J 07735

0097-6156/83/0207-0501$06.00/0 ©

1983 American Chemical Society

In Foundations of Biochemical Engineering; Blanch, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

502

BIOCHEMICAL

ENGINEERING

energy e x p e n d i t u r e s f o r p r o d u c t r e c o v e r y as w e l l as i n l a r g e c a p i t a l i n v e s t m e n t s due t o t h e l a r g e s i z e e q u i p m e n t r e q u i r e d t o r e a c h an e c o n o m i c a l l y a t t r a c t i v e s c a l e o f p r o d u c t i o n . Conse q u e n t l y , k i n e t i c d a t a i s needed t o d e v e l o p a b a s i c u n d e r s t a n d i n g of t h e s e f e r m e n t a t i o n p r o c e s s e s and t o p e r m i t an o p t i m a l d e s i g n f o r t h e f e r m e n t o r s y s t e m s t o be u s e d f o r t h e p r o d u c t i o n o f t h e s e chemicals. T h i s paper addresses t h e k i n e t i c s o f end-product i n h i b i t i o n o f c e l l growth i n t h e acetone-butanol f e r m e n t a t i o n .


M a t e r i a l s a n d Methods M i c r o o r g a n i s m and C u l t u r e C o n d i t i o n s . C l o s t r i d i u m a c e t o b u t y l i c u m s t r a i n ATCC 824 was u s e d i n t h i s s t u d y . Maintenance c u l t u r e s were grown i n c o r n mash medium f o r 72 h o u r s a t 37 C a n d then r e f r i g e r a t e d a t 4 t o 5 C The c o r n mash medium c o n s i s t e d o f 5% (w/v) c o r n m e a l w i t h 0.05% c y s t e i n e added t o e n s u r e f o r anaerobic conditions. The m e d i a was b o i l e d w i t h t h e a p p r o p r i a t e volume o f d i s t i l l e d w a t e r f o r one h o u r . The mash was t h e n d i s t r i b u t e d i n t o s c r e w c a p t u b e s (16 χ 125 mm) a n d a u t o c l a v e d f o r 15 m i n u t e s a t 121 C. The m i c r o o r g a n i s m was r o u t i n e l y t r a n s f e r r e d a n a e r o b i c a l l y i n s c r e w c a p t u b e s w i t h 10 m l o f medium c o m p r i s e d o f 5 0 % ( v / v ) t h i o g l y c o l l a t e 135C medium ( D i f c o L a b o r a t o r i e s , D e t r o i t , M i c h i g a n ) a n d 50% ( v / v ) s o l u b l e medium c o n t a i n i n g t h e f o l l o w i n g components i n g/1: K H P 0 , 0.75; Κ 2 Η Ρ Ο 4 , 0.75; MgS04, 0.20; M n S 0 . H 0 , 0.01; FeS04.7H20, 0.01; N a C I , 1.00; c y s t e i n e , 0.50; y e a s t e x t r a c t , 5.00; g l u c o s e , 60; a s p a r a g i n e . H 2 0 , 2.00; ( N H 4 ) S 0 4 , 2.00. 2

4

4

2

2

G r o w t h C h a l l e n g e S t u d i e s . The e f f e c t o f t h e f e r m e n t a t i o n p r o d u c t s o n t h e g r o w t h r a t e o f C I . a c e t o b u t y l i c u m was d e t e r m i n e d by t h e f o l l o w i n g p r o c e d u r e . A 24-hour o l d c u l t u r e was u s e d a s a 5% i n o c u l u m t o f l a s k s c o n t a i n i n g 200 m l o f 2% (w/v) g l u c o s e s o l u b l e medium. A f t e r 10-12 h o u r s , t h e s e c e l l s w e r e u s e d a s a 2 0 % (v/v) i n o c u l u m f o r f l a s k s c o n t a i n i n g s o l u b l e m e d i a w i t h 2% (w/v) g l u c o s e . A f t e r a l a g p h a s e o f 30-45 m i n u t e s , t h e c u l t u r e s were c h a l l e n g e d w i t h v a r i o u s c o n c e n t r a t i o n s o f e t h a n o l , b u t a n o l , and acetone. G r o w t h was m o n i t o r e d b y h o u r l y measurements o f o p t i c a l d e n s i t y a t 560 nm. The p r o c e d u r e f o r d e t e r m i n a t i o n o f g r o w t h r a t e s i n t h e p r e s e n c e o f a c e t i c a n d b u t y r i c a c i d was m o d i f i e d a s f o l l o w s . The i n o c u l u m was p r e p a r e d b y u s i n g c e l l s f r o m a c o r n t u b e a s a 1 0 % (v/v) i n o c u l u m f o r t h i o g l y c o l l a t e / s o l u b l e medium c o n t a i n i n g 20 g/1 MES b u f f e r ( 2 - [ N - m o r p h o l i n o ] e t h a n e s u l f o n i c a c i d ) , (Sigma Chemi c a l Co., S t . L o u i s , MO). A f t e r 10-12 h o u r s , t h e s e c e l l s w e r e used as a 20% (v/v) inoculum f o r f l a s k s c o n t a i n i n g s o l u b l e media (20 g/1 g l u c o s e + 20 g/1 MES b u f f e r ) . A f t e r a l a g p h a s e o f 30-45 m i n u t e s , t h e c e l l s were c h a l l e n g e d w i t h v a r i o u s c o n c e n t r a t i o n s o f a c e t i c and b u t y r i c a c i d .


24.

COSTA AND

MOREiRA

Growth

Inhibition

Kinetics

503


pH C o n t r o l l e d F e r m e n t a t i o n . A 7 - l i t e r New B r u n s w i c k M i c r o f e r m f e r m e n t o r (New B r u n s w i c k s c i e n t i f i c , E d i s o n , NJ) w i t h a w o r k i n g volume o f 4 l i t e r s was u s e d i n t h i s s t u d y . The a g i t a t i o n s p e e d was m a i n t a i n e d a t 2 0 0 rpm d u r i n g t h e f e r m e n t a t i o n . The pH o f t h e f e r m e n t a t i o n s t a r t e d a t 6.2 and was a l l o w e d t o f a l l t o pH 5.0. The pH was t h e r e a f t e r m a i n t a i n e d a t 5.0 by t h e a d d i t i o n o f 1 Ν NaOH on a demand b a s i s . The t e m p e r a t u r e was c o n t r o l l e d a t 37 C Analysis. The c e l l d e n s i t y was m e a s u r e d a t 560 nm u s i n g a B a u s c h and Lomb S p e c t r o n i c 2 0 s p e c t r o p h o t o m e t e r . F e r m e n t a t i o n s a m p l e s were c o l l e c t e d and c e n t r i f u g e d i m m e d i a t e l y u n d e r r e f r i g e r a t i o n a t 15,000 rpm f o r 1 0 m i n u t e s . The s u p e r n a t a n t s were s t o r e d i n p l a s t i c v i a l s and i m m e d i a t e l y f r o z e n f o r s u b s e q u e n t a n a l y s i s . The c e l l p e l l e t was washed t w i c e w i t h d i s t i l l e d w a t e r . The c e l l s were t h e n r e s u s p e n d e d i n d i s t i l l e d w a t e r and p l a c e d i n t a r e d c o n t a i n e r s . The c e l l d r y w e i g h t was d e t e r m i n e d a f t e r d r y i n g f o r 24 h o u r s a t 80 C. The c o n c e n t r a t i o n o f p r o d u c t s i n t h e s u p e r n a t a n t s was d e t e r m i n e d on a V a r i a n m o d e l 2400 gas c h r o m a t o g r a p h e q u i p p e d w i t h a flame i o n i z a t i o n d e t e c t o r . The c h r o m a t o g r a p h had i n s t a l l e d a 6 f t χ 1/8 i n s t a i n l e s s s t e e l t e f l o n l i n e d c o l u m n p a c k e d w i t h C h r o m o s o r b W-AW coated w i t h 1 0 % AT-1000. The t e m p e r a t u r e o f t h e c o l u m n was programmed f r o m 1 0 0 C t o 180 C a t a r a t e o f 2 0 C/min. H e l i u m was u s e d as t h e c a r r i e r gas a t a f l o w r a t e o f 30 m l / m i n . The f l a m e i o n i z a t i o n d e t e c t o r t e m p e r a t u r e was 230 C. A 10% (w/w) n - p r o p a n o l s o l u t i o n c o n t a i n i n g 4% (w/w) H 2 S O 4 was u s e d a s an i n t e r n a l standard. The g l u c o s e c o n c e n t r a t i o n o f t h e f e r m e n t a t i o n s a m p l e s was m e a s u r e d by t h e DNSA ( d i n i t r o s a l i c y l i c a c i d ) method (4) u s i n g g l u c o s e as a s t a n d a r d . Samples were d i l u t e d t o c o n t a i n l e s s t h a n 1 g/1 g l u c o s e . R e s u l t s and

Discussion

Product C h a l l e n g e d Growth S t u d i e s . To s t u d y t h e i n h i b i t o r y f a c t o r s of the acetone-butanol fermentation, the growth r a t e s of CI. acetobutylicum i n the presence of each fermentation product were d e t e r m i n e d . The end p r o d u c t s u s e d i n t h i s s t u d y i n c l u d e d e t h a n o l , b u t a n o l , a c e t o n e , a c e t i c a c i d , and b u t y r i c a c i d . From the slopes of the l e a s t squares r e g r e s s i o n l i n e s of o p t i c a l d e n s i t y v s . t i m e d a t a , t h e maximum s p e c i f i c g r o w t h r a t e s i n t h e p r e s e n c e o f v a r y i n g c o n c e n t r a t i o n s o f e a c h i n h i b i t o r ( y ) were d e t e r m i n e d . The r e s u l t s f o r e a c h f e r m e n t a t i o n p r o d u c t a r e shown in Figures 1 - 3. T h e r e a p p e a r s t o be a t h r e s h o l d c o n c e n t r a t i o n w h i c h must be r e a c h e d b e f o r e g r o w t h i n h i b i t i o n o c c u r s . This c o n c e n t r a t i o n was f o u n d t o v a r y w i t h e a c h i n h i b i t o r s t u d i e d . Above t h e t h r e s h o l d c o n c e n t r a t i o n , t h e g r o w t h i n h i b i t i o n c a n be d e s c r i b e d by a l i n e a r r e l a t i o n s h i p o f t h e f o r m : m


504

BIOCHEMICAL

ENGINEERING

.ο Σ! 2


Ι S a

•s-s δο * ο s; c α i «

t° •8 6 8* ed Ο

_· s:

U

S

si

S "S

•s I â -S Ο «ο

§·§

s-S

sΡ §« •S ?

s .00


24.

COSTA

AND

MOREiRA

Growth

Inhibition

Kinetics

505


.ο

s:

* §

%< 1& u * ° ? s:

M

•S s:

Si 3 00


506

ENGINEERING


BIOCHEMICAL

BUTYRIC Figure 3.

ACID

CONCENTRATION

(9/1)

Maximum specific growth rate of CI. acetobutylicum when challenged with various concentrations of butyric acid.


24.

COSTA AND

μ

1

= μ m m

Growth

MOREIRA

[1 -

κ

Ρ

Inhibition

(Ρ - Ρ ) ] ο

507

Kinetics

C

forΡ > Ρ = ο

C o n c e n t r a t i o n s c a u s i n g a 5 0 % r e d u c t i o n i n g r o w t h r a t e were a l s o determined. A summary o f t h e i n h i b i t i o n d a t a o b t a i n e d i s g i v e n i n Table I . Table


Product

I. K

Summary o f G r o w t h I n h i b i t i o n D a t a

p

(M)

Ρ

(M) —

ο

Cone, a t w h i c h g r o w t h was i n h i b i t e d by 50% M

Butyric Acid Butanol Acetic Acid Acetone Ethanol

0.07 0.15 0.19 0.98 1.01

0.02 0.06 0.05 0.26 0.26

0.07 0.15 0.13 0.88 1.10

2/1 6.0 11.0 8.0 43.5 51.0

The i n h i b i t i o n c o n s t a n t Kp f o r e a c h p r o d u c t was c a l c u l a t e d from t h e s l o p e o f the l e a s t squares r e g r e s s i o n l i n e o f y ^ / V ^ v s . Ρ data. The i n h i b i t i o n c o n s t a n t s f o r e t h a n o l a n d a c e t o n e a r e approximately t e n times g r e a t e r than t h a t f o r b u t a n o l , a c e t i c a c i d , and b u t y r i c a c i d . T h i s i s i n d i c a t i v e o f t h e r e l a t i v e l y low t o x i c i t y o f a c e t o n e a n d e t h a n o l a s compared w i t h t h e o t h e r f e r mentation products. F o r e a c h f e r m e n t a t i o n p r o d u c t , t h e P v a l u e was p l o t t e d a g a i n s t t h e Kp v a l u e a s shown i n F i g u r e 4. The t h r e s h o l d c o n c e n t r a t i o n was f o u n d t o i n c r e a s e l i n e a r l y w i t h a n i n c r e a s i n g Kp value. The e q u a t i o n o f t h i s l i n e was d e t e r m i n e d t o b e : Q

Κ

Ρ

= 3.94 Ρ

ο

- 0.03

(2)

by a l e a s t s q u a r e s r e g r e s s i o n a n a l y s i s . The c o r r e l a t i o n c o e f f i c i e n t was f o u n d t o be 0.997. S i m i l a r growth c h a l l e n g e e x p e r i m e n t s were p e r f o r m e d u s i n g t e r t - b u t y l a l c o h o l and n-hexanol. Although t e r t - b u t y l a l c o h o l and n - h e x a n o l a r e n o t p r o d u c t s o f t h i s f e r m e n t a t i o n , t h e Kp a n d P v a l u e s o b t a i n e d f o r t h e s e two a l c o h o l s w e r e f o u n d t o l i e on t h e s t r a i g h t l i n e shown i n F i g u r e 4. L i n d e n e t a l . (3) h a v e shown t h a t t h e e n d p r o d u c t t o x i c i t y i n t h e a c e t o n e - b u t a n o l f e r m e n t a t i o n o c c u r s b y a l t e r i n g membrane f u n c t i o n a l i t y . The l i n e a r r e l a t i o n s h i p b e t w e e n Kp a n d P may i n d i c a t e t h a t t h e i n h i b i t i o n o f e a c h o f t h e s e v a r i o u s compounds o c c u r s b y t h e same mechanism. The r e s u l t s o f a 39-hour b a t c h f e r m e n t a t i o n a r e shown i n F i g u r e 5. The l e v e l s o f a c e t o n e a n d e t h a n o l t y p i c a l l y o b s e r v e d d u r i n g a f e r m e n t a t i o n (5 g/1 a n d 1.5 g/1 r e s p e c t i v e l y ) w e r e 0

Q



Figure 4.

Relationship between K

p

and ?

0

from the product inhibition studies.



24.

COSTA A N D MOREiRA

Growth

Inhibition

Kinetics

509

f o u n d t o be n o n i n h i b i t o r y t o c e l l g r o w t h . The h i g h e s t c o n c e n t r a t i o n s o f b u t y r i c a c i d and a c e t i c a c i d w h i c h h a v e b e e n o b s e r v e d d u r i n g f e r m e n t a t i o n a r e i n t h e r a n g e o f 3.5 t o 4.0 g/1. These l e v e l s o f a c i d s a p p r o a c h t h o s e c a u s i n g 50% i n h i b i t i o n o f g r o w t h . T y p i c a l butanol concentrations observed d u r i n g a fermentation are 11-12 g/1. Butanol i s the only product t h a t reaches l e v e l s a c t u a l l y c a u s i n g 50% g r o w t h i n h i b i t i o n . R e c e n t r e s u l t s r e p o r t e d by L e u n g and Wang (5) show a c e t i c and b u t y r i c a c i d c o n c e n t r a t i o n s c a u s i n g 50% i n h i b i t i o n o f g r o w t h , w h i c h a r e about t w i c e t h e l e v e l s o b t a i n e d i n t h i s work. The a p p a r e n t d i s a g r e e m e n t b e t w e e n t h e two s e t s o f d a t a c o u l d be due t o t h e d i f f e r e n t methods u s e d i n e a c h c a s e t o s t a b i l i z e t h e pH o f t h e f e r m e n t a t i o n m e d i a d u r i n g an a c i d c h a l l e n g e . The m e t h o d o l o g y u s e d i n t h i s s t u d y i n v o l v e d t h e use o f a b i o l o g i c a l b u f f e r , MES ( 2 - [ N - m o r p h o l i n o ] e t h a n e s u l f o n i c a c i d ) t o s t a b i l i z e t h e pH. C e l l s grown i n two c o n t r o l f l a s k s , one c o n t a i n i n g MES b u f f e r and t h e o t h e r w i t h o u t MES b u f f e r showed no d i f f e r e n c e i n g r o w t h r a t e s . T h i s i n d i c a t e s t h a t MES has no e f f e c t on t h e g r o w t h r a t e o f t h e o r g a n i s m . The m e t h o d o l o g y t o s t a b i l i z e t h e pH o f t h e f e r m e n t a t i o n m e d i a r e p o r t e d by L e u n g and Wang (5) i n v o l v e d t h e p r e p a r a t i o n o f 200 g/1 s o l u t i o n s o f a c e t a t e and b u t y r a t e by t i t r a t i o n w i t h NaOH. I n t h i s manner, t h e c e l l s were e x p o s e d t o Na+ c o n c e n t r a t i o n s i n t h e r a n g e o f 2-7 g/1. The h i g h c o n c e n t r a t i o n s o f Na+ may h a v e an o s m o t i c o r membrane s t a b i l i z i n g e f f e c t w h i c h may a c c o u n t f o r t h e h i g h e r t o l e r a n c e t o a c i d s . However, i n s p i t e o f t h e s e d i s c r e p a n c i e s , t h i s i n v e s t i g a t i o n supports the c o n c l u s i o n t h a t the con c e n t r a t i o n o f b u t a n o l i s an i m p o r t a n t p a r a m e t e r i n t h e a c e t o n e butanol fermentation. Attempts t o model the c e l l growth curve d u r i n g a b a t c h f e r m e n t a t i o n by c o n s i d e r i n g t h e i n h i b i t i o n due t o e a c h p r o d u c t s e p a r a t e l y r e s u l t e d i n much h i g h e r g r o w t h r a t e s t h a n were a c t u a l l y observed. Two f a c t o r s c o u l d c o n t r i b u t e t o t h i s d i s c r e p a n c y . On t h e one h a n d , t h e p r o d u c t i n h i b i t i o n e x p e r i m e n t s were c a r r i e d o u t u s i n g e a c h f e r m e n t a t i o n p r o d u c t s e p a r a t e l y . However, i n an a c t u a l fermentation the products are present together. T h i r t e e n hours i n t o t h e f e r m e n t a t i o n , as shown i n F i g u r e 5 , t h e c o n c e n t r a t i o n o f e a c h p r o d u c t i s b e l o w t h e t h r e s h o l d c o n c e n t r a t i o n when g r o w t h i n h i b i t i o n was d e t e r m i n e d t o o c c u r . T h i s i n d i c a t e s t h a t no g r o w t h i n h i b i t i o n s h o u l d be o b s e r v e d . However, t h e c e l l g r o w t h r a t e a t t h i s t i m e was d e t e r m i n e d t o be o n e - h a l f o f t h e maximum s p e c i f i c c e l l g r o w t h r a t e . T h i s i n d i c a t e s t h a t some s y n e r g i s m o c c u r s among t h e s e v e r a l f e r m e n t a t i o n p r o d u c t s and i t i s p r o b a b l y t h e t o t a l concentration of products which i s important i n determining t h e t o x i c e f f e c t on g r o w t h r a t e . The o t h e r f a c t o r c o u l d be t h e d i f f e r e n c e i n t o x i c e f f e c t s o f p r o d u c e d s o l v e n t s as o p p o s e d t o a d d e d s o l v e n t s . Novak e t a l . (6) have shown t h a t e t h a n o l p r o d u c e d d u r i n g a b a t c h f e r m e n t a t i o n o f S a c c h a r o m y c e s c e r e v i s i a e i s more i n h i b i t o r y t h a n e t h a n o l added t o the fermentation. Therefore, i t i s p o s s i b l e t h a t the i n h i b i t i o n


510

BIOCHEMICAL

ENGINEERING


s sa δ· ο

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JD

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24.

C O S T A A N D MOREiRA

Growth

Inhibition

511

Kinetics

p a r a m e t e r s d e t e r m i n e d i n t h i s s t u d y , based on added s o l v e n t s , m i g h t be l e s s s e v e r e t h a n t h e a c t u a l v a l u e s due t o t h e s o l v e n t s produced during the fermentation. Conclusions


From t h e s e s t u d i e s o f g r o w t h i n h i b i t i o n a n d f e r m e n t a t i o n k i n e t i c s i n the acetone-butanol fermentation, the f o l l o w i n g c o n c l u s i o n s may be made: 1.

A t l e v e l s normally observed during fermentation a. B u t a n o l , a c e t i c a c i d , and b u t y r i c a c i d a r e i n h i b i t o r y t o c e l l growth. b. A c e t o n e a n d e t h a n o l show no i n h i b i t i o n e f f e c t s .

2.

F o r each f e r m e n t a t i o n product t h e r e i s a t h r e s h o l d concentra t i o n b e l o w w h i c h no g r o w t h i n h i b i t i o n o c c u r s a n d above w h i c h a l i n e a r d e c r e a s e i n growth r a t e i s observed w i t h an i n c r e a s e i n product concentration.

3.

There i s a l i n e a r r e l a t i o n s h i p between t h e t h r e s h o l d concen t r a t i o n ( P ) a n d t h e i n h i b i t i o n c o n s t a n t (Kp) f o r e a c h fermentation product. Q

4.

P r e l i m i n a r y o b s e r v a t i o n s seem t o i n d i c a t e t h a t t h e g r o w t h i n h i b i t i o n c a u s e d by s o l v e n t s p r o d u c e d d u r i n g f e r m e n t a t i o n i s d i f f e r e n t from t h e i n h i b i t i o n caused by e x t e r n a l l y added solvents.

Legend o f Symbols Κ

i n h i b i t i o n constant

(M) — p r o d u c t c o n c e n t r a t i o n (M) threshold product concentration

Ρ Ρ P^ μ

maximum s p e c i f i c g r o w t h r a t e

m μί

(M)

(hr~l)

maximum s p e c i f i c g r o w t h r a t e i n p r e s e n c e o f i n h i b i t o r (hr" ) 1

Acknowledgments P a r t i a l s u p p o r t f o r t h i s r e s e a r c h was p r o v i d e d b y t h e U.S. D e p a r t m e n t o f E n e r g y u n d e r S u b c o n t r a c t No. ΧΚ-φ-9059-l. The a u t h o r s a l s o w i s h t o a c k n o w l e d g e D r . James C. L i n d e n f o r h e l p f u l discussions. Literature Cited 1.

Reed, G.; Peppier, H. J . "Yeast Technology;" A v i P u b l i s h i n g Co.: Westport, 1973; 185.



512

BIOCHEMICAL

ENGINEERING

2.

S t e e l , R. "Biochemical Engineering: U n i t Processes in Fermentation;" Macmillan: New York, 1958; 125.

3.

Linden, J . C.; Ulmer, D. C.; Moreira, A. R. "A Mechanism f o r A l i p h a t i c Alcohol-Induced T o x i c i t y in C l o s t r i d i u m acetobutylicum, " presented a t the 182nd N a t i o n a l Meeting American Chemical S o c i e t y , D i v i s i o n o f M i c r o b i a l and Biochemical Technology New York, New York. 1981.

4.

M i l l e r , G. C. A n a l . Chem.

5.

Leung, J. C. Y.; Wang, D. I . C. Proc. 2nd World Congress o f Chemical Engineering. 1981, 1, 348.

6.

Novak, M.; Strehaiano, P.; Moreno, M.; Goma, G. Bioeng., 1981, 23, 201.

1959, 31, 426.

Biotechnol.

R E C E I V E D August 2, 1982


Foundations of Biochemical Engineering - American Chemical Society

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