THE CYCLODEXTRINS AND T H E I R A P P L I C A T I O N I N E N V I R O N M E N T A L

BIOTECHNOLOGY

J. SZEJTLI

C y c l o d e x t r i n Res. L a b o r a t o r y o f C H I N O I N Pharm.-Chem. Works, 1026 B u d a p e s t , E n d r o d i S . 38 /40

SUMMARY

C y c l o d e x t r i n s and t h e i r d e r i v a t i v e s enhance t h e s o l u b i l i t y o f comp lexed s u b s t r a t e s i n aqueous med ia , b u t do n o t damage t h e m i c - r o b i a l c e l l s o r t h e enzymes. T h e r e f o r e t h e e n z y m a t i c c o n v e r s i o n o f l i p o p h y l i c s u b s t r a t e s c a n b e i n t e n s i f i e d ( a c c e l e r a t e d , o r p e r f o r m e d a t h i g h e r s u b s t r a t e c o n c e n t r a t i o n ) . Examples a r e t h e h y d r o l y s i s o f t r i g l y c e r i d e s o r t h e c o n v e r s i o n o f h y d r o c o r t i s o n e t o p r e d n i s o l o n . I n t h e p r e s e n c e o f an a p p r o p r i a t e C D - d e r i v a t i v e ( e . 9 . 2 , 6 - d i m e t h y l - 0 C D ) t h e l i p i d l i k e i n h i b i t o r s u b s t a n c e s a r e comp lexed , t h e r e f o r e t h e p r o g a t e i o n o f ' B o r d a t e l l a p e r t u s s i s and t h e p r o d u c t i o n o f p e r - t u s s i s t o x i n i n c r e a s e s up t o 1 0 0 - f o l d . The t o l e r a b l e l e v e l o f t o - x i c compounds a t b i o l o g i c a l d e t o x i c a t i o n o f sewage o f o r g a n i c che- m i c a l i n d u s t r y c a n b e e l e v a t e d by a d m i x i n g a s m a l l amount o f R - c y c - l o d e x t r i n t o t h e sys tem, because t h e comp lexed t o x i c s u b s t a n c e s do n o t k i l l t h e d e t o x i c a t i n g m i c r o b e s .

C H E M I S T R Y OF C Y C L O D E X T R I N S

C y c l o d e x t r i n s a r e c y c l i c , n o n - r e d u c i n g o l i g o s a c c h a r i d e s . Th ree

d i f f e r e n t c y c l o d e x t r i n s a r e known: d. -, R - and 1 ' - c y c l o d e x t r i n .

A l l o f them e r e p r o d u c e d i n d u s t r i a l l y as homogeneous ( p u r i t y o v e r

9 9 , 5 X ) c r y s t a l l i n e s u b s t a n c e s . ( 1 , Z ) .

The s t r u c t u r e o f 0 - c y c l o d e x t r i n and t h e m o l e c u l a r d i m e n s i o n s

o f t h e t h r e e c y c l o d e x t r i n s a r e shown i n F i g . 1.

The c4 - c y c l o d e x t r i n c o n s i s t s o f 6 , t h e R - c y c l o d e x t r i n 7 and

t h e y - c y c l o d e x t r i n 8 g l u c o p y r a n o s e u n i t s . A l l s e c o n d a r y OH-g roups

a r e l o c a t e d on one edge o f t h e t o r u s - l i k e c y c l o d e x t r i n m o l e c u l e ,

w h i l e e l l p r i m a r y OH-groups a r e on t h e o t h e r s i d e . The " l i n i n g " o f

t h e i n t a r n e l c a v i t y i s f o r m e d by H-atoms, and g l u c o s i d i c oxygen-

- b r i d g e atoms, t h e r e f o r e t h i s s u r f a c e i s s l i g h t l y a p o l a r ( F i g 2).

The u n i q u e shape and p h y s i c a l - c h e m i c a l p r o p e r t i e s o f c y c l o d e x -

t r i n s e n a b l e s them t o i n c l u d e t h e m o l e c u l e s o f o t h e r s u b s t a n c e s :

t h i s i s t h e essence o f t h e " m o l e c u l a r e n c a p s u l a t i o n " .

- 3 8 8 -

Ffg. 1. Structure o f 0 -cyclodextrin, and the approximative geomet- rical dimensions o f the a-, 0 - and y -cyclodextrins.

M O L E C U L A R E N C A P S U L A T I O N W I T H C Y C L O D E X T R I N S

A cyclodextrin molecule can be considered as an empty capsule

o f molecular size (Fig. 2 ) . When it is Filled with the molecule o f

another substance, it is called "inclusion complex".

AFQLAR CAVITY SECONDARY HYDROXYLS

PRIMARY HYDROXYLS

Fig. 2 . Functional schema o f a cyclodextrin "cylinder".

Inclusion complexes are entities comprising two or more molecu-

les, in which one o f the molecules, the "host", includes totally o r

in part, only by physical f o r c e s , i.e. without covalent bonding a

- 3 8 9 -

" g u e s t " m o l e c u l e . C y c l o d e x t r i n s a r e t y p i c a l " h o s t " m o l e c u l e s and

may i n c l u d e a g r e a t v a r i e t y o f m o l e c u l e s h a v i n g t h e s i z e o f one o r

two benzene r i n g s , o r e v e n l a r g e r ones w h i c h a r e a s i d e c h a i n o f

comparab le s i z e , t o form c r y s t a l l i n e i n c l u s i o n comp lexes .

I n aqueous s o l u t i o n t h e s l i g h t l y a p o l a r c y c l o d e x t r i n c a v i t y i s

o c c u p i e d by w a t e r m o l e c u l e s w h i c h a r e e n e r g e t i c a l l y u n f a v o u r e d (po -

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

by a p p r o p r i a t e " g u e s t m o l e c u l e s " w h i c h a r e l e s s p o l a r t h a n w a t e r .

The d i s s o l v e d c y c l o d e x t r i n i s t h e " h o s t " m o l e c u l e , and t h e " d r i v i n g

f o r c e " o f t h e complex f o r m a t i o n i s t h e s u b s t i t u t i o n o f t h e h i g h - e n t -

h a l p y w a t e r m o l e c u l e s b y an a p p r o p r i a t e " g u e s t " m o l e c u l e ( F i g . 3 ) .

o o 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 O J

0

F i g . 3 . I n c l u s i o n o f p - x y l e n e by a CD- "capsu le " .

The c y c l o d e x t r i n comp lexes a r e r e l a t i v e l y s t a b l e . T h e i r w a t e r

s o l u b i l i t y compared t o p u r e c y c l o d e x t r i n s a r e s t r o n g l y r e d u c e d s o

t h e y r a p i d l y s e p a r a t e f r o m t h e s o l u t i o n i n c r y s t a l l i n e fo rm. One,

t w o o r t h r e e c y c l o d e x t r i n m o l e c u l e s c o n t a i n one o r more e n t r a p p e d

" g u e s t " m o l e c u l e s . T h i s i s t h e essence o f t h e " m o l e c u l a r e n c a p s u l a -

t i on" . Though t h e p h y s i c o c h e m i c a l p r o p e r t i e s o f t h e m o l e c u l e s t r a p p e d

i n t h e c a v i t y a r e c o n s i d e r a b l y a l t e r e d , t h e s e comp lexes d i s s o c i a t e

e a s i l y u n d e r p h y s i o l o g i c a l c o n d i t i o n s and t h e g u e s t m o l e c u l e s can

t h u s e x e r t t h e i r d e s i r e d e f f e c t s .

A l m o s t a l l i n d u s t r i a l a p p l i c a t i o n s o f c y c l o d e x t r i n s i n v o l v e com-

p l e x a t i o n . I n many c a s e s comp lexes a r e s e p a r a t e d i n more or l e s s pu-

re f o r m and u t i l i z e d as c r y s t a l l i n e s u b s t a n c e s ( d r o g and f l a v o u r

c o m p l e x e s ) , w h i l e i n o t h e r c a s e s c o m p l e x a t i o n i s o n l y a t r a n s i e n t

s t a t e , and becomes a p p a r e n t t h r o u g h t h e f i n a l r e s u l t ( c y c l o d e x t r i n -

c a t a l y s i s , s e p a r a t i o n o f m i x t u r e s , m o d i f i c a t i o n o f t h e r e a c t i o n -

pa tway , e t c . ) (1, 2 ) .

- 390 -

M I C R O B I A L C O N V E R S I O N , FERMENTATION

Enzymes ( e x c e p t a m y l o l y t i c ones) or m i c r o o r g a n i s m s a r e gene-

r a l l y n o t a f f e c t e d by t h e p r e s e n c e o f CDs. T h e r e f o r e m i c r o b i o l o g i c a l

o r enzymic p r o c e s s e s can be p e r f o r m e d i n CD s o l u t i o n s , a t h i g h e r d i -

s s o l v e d s u b s t r a t e c o n c e n t r a t i o n s , b u t a t l o w e r f r e e - s u b s t a n c e ( l e s s

i n h i b i t o r y , or l e s s t o x i c ) c o n c e n t r a t i o n s . F i g u r e 4 i l l u s t r a t e s t h e

s o l u b i l i t y o f h y d r o c o r t i s o n e i n v a r i o u s CD s o l u t i o n s ( 3 ) . The d i -

m e t h y l - R CD i s t o o e x p e n s i v e ( y e t ) f o r i n d u s t r i a l p u r p o s e s , b u t a

v e r y s i g n i f i c a n t improvemen t o f t h e m i c r o b i o l o g i c a l c o n v e r s i o n o f

h y d r o c o r t i s o n e t o p r e d n i s o l o n i s based on t h e s e e m i n g l y s m a l l s o l u -

b i l i t y i n c r e a s e i n 0 CD s o l u t i o n .

The s o l u b i l i t y o f h y d r o c o r t i s o n e i n w a t e r i s o n l y 0 .4 mg/ml.

T h e r e f o r e i n l a r g e vo lumes o n l y a r e l a t i v e l y s m a l l amount o f t h i s

s t e r o i d c o u l d b e c o n v e r t e d . The c o n v e r s i o n p r o c e s s was s l o w , and t h e

e n d p r o d u c t was n o t homogeneous, because o f m i x e d - c r y s t a l f o r m a t i o n

(hydrocortisone-prednisolon).

W 5 20 cn + a 0 0 0

> I

a n

10

F i g . 4. S o l u b i l i t y o f h y d r o c o r t i s o n e i n v a r i o u s C D - s o l u t i o n s a t 2 5 OC.

P e r f o r m i n g t h i s p r o c e s s i n an aqueous ll CD s o l u t i o n , t h e capa-

c i t y i n c r e a s e d by more t h a n 300 %. The s o l u b i l i t y o f h y d r o c o r t i s o n e

i n c r e a s e s , 3 - 4 t i m e s more h y d r o c o r t i s o n e can b e c h a r g e d i n t o t h e

c o n v e r t e r , t h e r e a c t i o n i s f a s t e r , and t h e e n d p r o d u c t i s more homo-

geneous. P r a c t i c a l l y a l l m i c r o b i o l o g i c a l s t e r o i d c o n v e r s i o n p r o c e s s e s

have been t e s t e d i n aqueous CD s o l u t i o n s w i t h u n a n i m o u s l y p r o m i s i n g

- 3 9 1 -

r e s u l t s ( 4 ) . The m e n t i o n e d example i s a l r e a d y p r a c t i c e d i n t h e p h a r -

m a c e u t i c a l i n d u s t r y . R e c e n t l y , examp les have been p u b l i s h e d on t h e

u t i l i z a t i o n o f c y c l o d e x t r i n s i n a n t i b i o t i c f e r m e n t a t i o n . The p r o d u c -

t i o n o f L a n k a c i d i n A and C by S t r e p t o m y c e s r o c h e i v o l u b i l i s was en-

hanced by a d d i n g 11 mM 0 CD t o t h e f e r m e n t a t i o n b r o t h f r o m 0.05 and

0.04 mM t o 0 . 5 5 and 4 . 6 mM, r e s p e c t i v e l y . A new a n t i - t u m o r s u b s t a n -

c e , SPF-1000 i s p r o d u c e d by g r o w i n g a s p e c i f i c s t r a i n o f S t r e p t o -

c c u s , a c c o r d i n g t o t h e p a t e n t a p p l i c a t i o n t h e c u l t u r e med ia a l s o

c o n t a i n s CD ( 6 ) .

ENHANCEMENT OF V A C C I N E P R O D U C T I O N

P e r t u s s i s t o x i n ( = L e u k o c y t o s i s p r o m o t i n g f a c t o r , LPF-hemaglu-

t i n i n ) i s one o f t h e m a i n p r o t e c t i v e a n t i g e n s a g a i n s t whoop ing cough

i n f e c t i o n , and i s a l s o one o f t h e componen ts o f a p e r t u s s i s v a c c i n e .

I t i s p r o d u c e d by B o r d a t e l l a p e r t u s s i s . F o r t h e p r o d u c t i o n o f a l e s s

r e a c t o g e n i c v a c c i n e , a s y n t h e t i c medium i s needed. However t h e p r o -

d u c t i o n o f p e r t u s s i s t o x i n was r a t h e r d i f f i c u l t i n s y n t h e t i c medium,

e s p e c i a l l y i n shaken c u l t u r e s . B o r d a t e l l a p e r t u s s i s i s v e r y suscep-

t i b l e t o a number o f i n h i b i t o r s , e .g . f a t t y a c i d s ( p a l m i t i c or o l e -

i c a c i d ) a l r e a d y a t 10 p M c o n c e n t r a t i o n s t o p s t h e c e l l p r o p a g a t i o n

( 5 ) . A d d i n g however 0 . 5 mg/ml d i m e t h y l - 0 CD ( o r t r i m e t h y l - 0 CD)

an i n c r e a s e d c e l l g r o w t h was o b s e r v e d , w h i c h enhanced t h e p e r t u s s i s

t o x i n p r o d u c t i o n 100 f o l d ( 8 , 9 , 1 0 ) . F i g u r e 5. i l l u s t r a t e s t h e t o -

x i n p r o d u c t i o n a t v a r i o u s d i m e t h y l - 0 C D - c o n c e n t r a t i o n s ( 1 1 ) . Howe-

v e r t h e mechanism o f t h e e f f e c t i s n o t y e t f u l l y u n d e r s t o o d ( s e q u e s -

t r a t i o n o f i n h i b i t o r y f a t t y a c i d s , o r s t a b i l i z a t i o n o f r e d u c e d g l u -

t a t h i o n e , or m o d i f i c a t i o n o f c e l l membrane p e r m e a b i l i t y ) , i t i s an

i m p o r t a n t examp le f o r u t i l i z a t i o n o f CDs i n b i o t e c h n o l o g y ( 1 2 , 1 3 ) .

The enhancement o f p r o d u c t i o n o f f i l a m e n t o u s h e m a g l u t i n i n was even

h i g h e r y e t , w i t h s e v e r a l h u n d r e d t i m e s more b e i n g p r o d u c e d i n t h e

p r e s e n c e o f d i m e t h y l - 0 C D t h a n w i t h o u t i t ( 9 ) .

E N Z Y M I C R E A C T I O N OF L I P I D S

H y d r o l y s i s o f t r i g l y c e r i d e s by l i p a s e s i n aqueous sys tems i s a

v e r y s l o w p r o c e s s . E i t h e r a l i p i d d i s s o l v i n g w a t e r m i s c i b l e o r g a n i c

s o l v e n t has t o be added t o t h e s y s t e m - w h i c h i s t o l e r a t e d o n l y t o

a 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 because o f t h e e n z y m e - p r o t e i n dena-

t u r a t i o n - or an a p p r o p r i a t e d e t e r g e n t - e.g. n a t u r a l b i l e - has t o

b e p r e s e n t . T a b l e 1 i l l u s t r a t e s t h e h y d r o l y s i s o f o l i v e o i l by l i p a -

se w i t h o u t any d e t e r g e n t o f p r e s e n c e o f hog b i l e , or d i m e t h y l - O C D

- 392 -

DlMEB

( 1 4 ) . The h y d r o l y s i s was f o l l o w e d by t i t r a t i n g t h e l i b e r a t e d f a t t y

a c i d s w i t h NaOH. As i t i s seen, t h e d i m e t h y l - 0 CD r e m a r k a b l y acce -

l e r a t e d t h e l i p o l y s i s .

The o b s e r v a t i o n t h a t p h o s p h a t i d e s ( l i g n o c e r i c a c i d , c e r e b r o s i -

de, c e r a m i d e ) can b e s o l u b i l i z e d w i t h C O s p r o b a b l y w i l l b e e x p l o i -

t e d i n l i p i d enzymology ( 1 5 ) .

S t u d y i n g t h e L i g n o c e r y l - C o A l i g a s e a c t i v i t y i n m i c r o s o m a l f r a c

t i o n p r e p a r e d f r o m r a t b r a i n , t h e l i g n o c e r i c a c i d , s o l u b i l i s e d b y

d C D was u t i l i z e d , b u t t h e one s o l u b i l i s e d by T r i t o n WR 1339 was

n o t u t i l i z e d ( 1 6 ) .

r

R e a c t i o n Consumed 0.005 n NaOH m l t i m e [ h ] a b C

c o n t r o l b i l e d i m e t h y l - 0 C O

0.5 0.0 0.82 1 .74

1 0.36 1.17 2.00

2 0.48 1 .37 2.20

19 0.63 2.62 4.77

a c c e l e r a t i o n - 4 x - 7.4 x r e a c t i o n r a t e

1 - 1000 E - \

3 800-

v, 1 w

P 6oo-

3 I- *oo{

HOURS OF INCUBATION

F i g . 5. E f f e c t o f d i m e t h y l - R - c y c l o d e x t r i n on p e r t u s s i s t o x i n p r o - d u c t i o n i n B. p e r t u s s i s Tohama phase I i n s y n t h e t i c medium.

- 3 9 3 -

T I S S U E CULTURES

The CD comp lexes o f u n s a t u r a t e d f a t t y a c i d s can be u t i l i z e d as

serum s u b s t i t u t e s i n mammalian c e l l c u l t u r e s . B o t h o l e i c a c i d - O C D

and l i n o l e i c a c i d - O C D comp lexes showed a g r o w t h e n h a n c i n g e f f e c t

o f human l y m p h o b l a s t c e l l s , up t o 100 mg / l medium. A t h i g h e r concen-

t r a t i o n s t h e f a t t y a c i d O C D comp lex appeared t o b e t o x i c , b u t t h i s

c a n p r o b a b l y b e a t t r i b u t e d t o t h e f a t t y a c i d s . 100 mg o f f a t t y a c i d -

O C D comp lex and 1000 mg f r e e O CD t o g e t h e r r e s u l t e d i n n o t o x i c

e f f e c t s , b u t e x h i b i t e d a s t a b l e and r e p r o d u c i b l e g r o w t h p r o m o t i n g

e f f e c t . I n human d i p l o i d f i b r o l a s t c u l t u r e s g r o w t h , s i m i l a r t o t h a t

i n t h e b o v i n e a l b u m i n , a s u p p l e m e n t e d medium was o b s e r v e d a f t e r t h e

O C D - f a t t y a c i d complex r e s o l v e d t o a f i n a l c o n c e n t r a t i o n o f 10-20

mg/ml. B o v i n e serum a l b u m i n can be p a r t i a l l y o r c o m p l e t e l y s u b s t i -

t u t e d by O C D - f a t t y a c i d comp lexes i n mammalian c e l l c u l t u r e s ( 1 7 , 18)

e . g . , i n human i n t e r f e r o n p r o d u c t i o n ( 1 9 ) .

H i g h d e n s i t y c u l t u r e 0 - l y m p h o b l a s t - l i k e c e l l s t r a i n s c a n b e

p r e p a r e d i n a s e r u m - f r e e medium w h i c h c o n t a i n s d C D . E .g . i n t h e

case o f p r o d u c t i o n o f i n t e r f e r o n s f r o m UMCL c e l l s , t h e amount o f

t h e p r o d u c e d i n t e r f e r o n s may b e i n c r e a s e d ( 2 0 ) . Mouse mammary t u -

mor c e l l s c a n be c u l t u r e d u n d e r serum f r e e c o n d i t i o n s when t h e b o v i -

n e a l b u m i n i s s u b s t i t u t e d by d CD comp lex o f o l e i c a c i d ( 2 1 ) .

The s t e r i l i z a t i o n o f t h e components o f s e r u m l e s s m e d i a f o r c u l -

t i v a t i n g a n i m a l c e l l s i s d e s c r i b e d . Such c o m p o s i t i o n may c o n t a i n

a C D u n s a t u r a t e d f a t t y a c i d i n c l u s i o n comp lexes ( 2 2 ) .

O C D i s a u s e f u l m a t e r i a l as a serum s u b s t i t u t e i n i n d u c i n g

p r i m a r y a n t i b o d y r e s p o n s e i n v i t r o . F e t a l c a l f serum mus t b e p r e s e n t

a t 113 X i n t h e c u l t u r e medium f o r o p t i c a l l y e l i c i t i n g t h e p r i m a r y

a n t i b o d y r e s p o n s e t o sheep e r y t r o c y t e s i n m u r i n e l y m p h o c y t e s . The

r e s p o n s e can n o t be o b s e r v e d w h e n t h e c o n c e n t r a t i o n o f t h e f e t a l c a l f

serum i s l e s s t h a n 1 X. The a d d i t i o n o f 250-500 p g / m l O C D was t h e

mos t e f f e c t i v e , a , T -CD and D I M E B were l e s s e f f e c t i v e ( 2 3 , 24 ) .

a C D c a n be used a s a c h o l e s t e r o l c a r r i e r i n a s e r u m - f r e e c a r -

r i e r p r o t e i n f r e e medium i n t i s s u e c u l t u r e s , f o r example i n t h e p r o -

p a g a t i o n o f a newborn r a t a d r e n o c o r t i c a l c e l l s . The a C D f o r m s a

n e a r l y 1:l comp lex w i t h c h o l e s t e r o l ( 2 5 ) .

The use o f a C 0 i n a s e r u m - f r e e medium o f mammalian c e l l c u l t u -

r e s p r o l o n g e d c e l l l i f e , and i n c r e a s e d t h e p r o d u c t i o n o f m o n o c l o n a l

a n t i b o d y ( 2 6 ) .

B e s i d e s t h e s o l u b i l i t y enhancement , t h e s t a b i l i z a t i o n i s t h e

o t h e r i m p o r t a n t consequence o f c o m p l e x a t i o n o f N y s t e t i n - a p o l y e n e

- 394 -

a n t i b i o t i c - w i t h CD. The N y s t a t i n i s a f r e q u e n t l y u s e d a n t i f u n g a l

a n t i b i o t i c , w i d e l y used i n human m e d i c i n e f o r l o c a l a n t i f u n g a l t r e a t -

men t . T i s s u e c u l t u r e - an i m p o r t a n t t o o l i n b i o t e c h n o l o g y - needs

s o l u b l e , a n t i f u n g a l a g e n t s , w h i c h have t o be d i s s o l v e d i n t h e c u l -

t u r e medium. N y s t a t i n w o u l d b e q u i t e adequa te f o r t h i s p u r p o s e , ho-

wever i t i s p r a c t i c a l l y i n s o l u b l e i n w a t e r and r a p i d l y decomposes

by o x y d a t i o n . The N y s t a t i n y CD comp lex i s a r e l a t i v e l y s t a b l e , ea-

s i l y s o l u b l e powder , w e l l a p p l i c a b l e f o r s u c h p u r p o s e ( 2 7 ) .

S E W A G E D E T O X I C A T I O N

A b i g v a r i e t y o f o r g a n i c t o x i c s u b s t a n c e s ( h y d r o x y - h a l o g e n o - ,

n i t r o - , amino e t c . d e r i v a t i v e s o f a r o m a t i c and a l i p h a t i c compounds)

o c c u r s i n t h e w a s t e w a t e r s o f t h e o r g a n i c c h e m i c a l and p h a r m a c e u t i -

c a l i n d u s t r y . The b i o l o g i c a l was te w a t e r t r e a t m e n t means t h a t t h e s e

t o x i c s u b s t a n c e s a r e d e g r a d e d by c e r t a i n y e a s t s and b a c t e r i a w h i c h

a r e p r e s e n t i n t h e b i o l o g i c a l s l u d g e . These m i c r o o r g a n i s m s can t o -

l e r a t e t h e t o x i c s u b s t a n c e s i f t h e i r c o n c e n t r a t i o n does n o t exceed

a c r i t i c a l c o n c e n t r a t i o n and t r a n s f o r m s them by m e t a b o l i c p r o c e s s e s

t o n o n - t o x i c s u b s t a n c e s . By a p p r o p r i a t e a d a p t a t i o n , t h e t o l e r a b l e

c o n c e n t r a t i o n o f t o x i c s u b s t a n c e s c a n be enhanced, b u t s u r p a s s i n g

t h i s c r i t i c a l c o n c e n t r a t i o n l i m i t - even f o r a s h o r t t i m e - r e s u l t s

i n k i l l i n g t h e m e n t i o n e d m i c r o o r g a n i s m s , i . e . i r r e v e r s i b l e i m p a i r i n g

o f t h e d e t o x i c a t i n g c a p a c i t y o f t h e l i v i n g s l u d g e . R e g e n e r a t i o n o f

t h i s b i o l o g i c a l s y s t e m i s n o t a r a p i d p r o c e s s . T h e r e f o r e p r e s e r v a -

t i o n o f t h e d e t o x i c a t i n g c a p a c i t y o f t h e " l i v i n g s l u d g e " i s a p r i -

mary g o a l o f e n v i r o n m e n t a l p r o t e c t i o n .

To a v o i d t h e c r i t i c a l - i . e . " k i l l i n g " - c o n c e n t r a t i o n o f was te ,

w a t e r s o f s u p e r c r i t i c a l c o n c e n t r a t i o n s have t o b e d i l l u t e d w i t h wa-

t e r , t o r e d u c e t h e c o n c e n t r a t i o n s t o s u b c r i t i c a l v a l u e s . Somet imes

t h i s method w o u l d need t h o u s a n d s o f t o x i c s u b s t a n c e f r e e w a t e r , w h i c h

is n o t a l w a y s p o s s i b l e .

A J d i n g CD, w h i c h o f c o u r s e must n o t be a p u r e one, t h e c o n v e r -

s i o n m i x t u r e w i t h a b o u t 5 0 % CD c o n t e n t i s s a t i s f a c t o r y t o t h e was te

w a t e r , and a c o n s i d e r a b l e p a r t o f t h e m e n t i o n e d o r g a n i c t o x i c compo-

unds w i l l be comp lexed . ( F i g . 6 ) . The comp lexed m o l e c u l e s w h i c h can

n o t p e n e t r a t e t h e c e l l membrane, a r e n o t t o x i c ( 2 8 ) .

The c o n c e n t r a t i o n o f t h e f r e e t o x i c s u b s t a n c e s i s s t r o n g l y redu-

ced , b e l o w t h e c r i t i c a l c o n c e n t r a t i o n . As i t s c o n c e n t r a t i o n d e c r e a s e s

f u r t h e r b y t h e m e t a b o l i c p r o c e s s , t h e CD-complex behaves as a dynamic

- 395 -

P h e n o l p - C h l o r p h e n o l Benzene Oxygen Consumpt ion

d e p o t , r e l e a s i n g t h e i n c l u d e d m o l e c u l e s , and t h i s p r o c e s s i s g o v e r -

n e d by t h e d i s s o c i a t i o n e q u i l i b r i u m ( F i g . 7 ) .

B e f o r e A f t e r 24 h A f t e r 24 h t r e a t m e n t c o n v e n t i o n a l t r e a t m e n t w i t h

t r e a t m e n t a d d i n g 40 m l / l 0 CD

30 10 1 10 5 1

4 3 0 9 0 5 B O O 200 60

Low CONC. HIGH CONC HIGH CONC

NON TOXIC FOR TOXIC FOR NON TOXK: FOR MICROORGANISMUS MICROORGANISMUS MICROORGANISMUS

METABOLISM PROEEDS NO METABOLISM METABOLISM PROCEEDS

F i g . 6 . The t o l e r a b l e l e v e l o f t h e t o x i c compounds i s augmented by a d d i n g CD t o t h e t o x i c sewage.

NON -TOXIC

1 DETOXICATION

NON-TOXIC METABOCITES

F i g . 7. A d d i n g CD t o t h e t o x i c p - n i t r o c h l o r o b e n z e n e c o n t a i n i n g se - wage, a f r a c t i o n o f t h i s compounds w i l l b e comp lexed by t h e C D . The CD-complexes - b e i n g h y d r o p h y l i c - have l o w e r a f f i n i t y t o t h e l i p o p r o t e i n c e l l membranes o f t h e m i c r o b i a l c e l l s , t h e r e f o r e a r e c o n s i d e r e d t o be l e a s t o x i c . On ly t h e non-comp lexed f r a c t i o n w i l l p e n e t r a t e t h r o u g h t h e c e l l membranes.

A m i x e d i n d u s t r i a l and communal w a s t e w a t e r , t h a t c o n t a i n e d

p h e n o l , p - c h l o r o p h e n o l , benzene and f u r t h e r o r g a n i c s u b s t a n c e s was

t r e a t e d b y t h e c o n v e n t i o n a l merhod, w i t h or w i t h o u t a d d i n g 40 m g / l

R C D . The r e s u l t s a r e seen i n T a b l e 2.

- 396 -

The a d d i t i o n o f s o l u b l e n CD-polymer t o p h e n o l c o n t a i n i n g sewa-

ge i n c r e a s e s t h e e f f i c i e n c y o f m i c r o b i o l o g i c a l d e t o x i c a t i o n o f phe -

n o l by C a n d i d a t r o p i c a l i s . The c e l l s f i x e d i n p o l y m e r beds t o g e t h e r

w i t h 0 CO-polymer can b e r e u s e d f o r sewage t r e a t m e n t ( 2 9 ) .

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15 16

17

18

19 2 0 2 1

2 2 23 2 4

25

26

27

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