Facile Methods for the Immobilization of Microbial Cells without

8 Facile Methods for the Immobilization of Microbial Cells

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without Disruption of their Life Processes J. F. KENNEDY Research Laboratory for the Chemistry of Bioactive Carbohydrates and Proteins, Department of Chemistry, University of Birmingham, Birmingham B15 2TT, England

The immobilisation of enzymes by attachment to water insoluble material has now received considerable attention for some time and possible applications have been pursued extensively (1,2). A logical extension of this approach, especially where multi-stage enzymic reactions are being considered, is the immobilisation of microorganisms, which are often the source of many enzyme preparations. The advantages of such an approach are immediately obvious. The tedious and time consuming procedures for enzyme extraction and purification are instantly eliminated, cofactors and coenzymes are readily at hand, the cellular enzymes are often organised into the requisite metabolic pathways and problems associated with enzyme instability may also be avoided. Furthermore, the use of immobilised cells would avoid the problem in industrial processes of separating the product from the enzyme. Hydrous Metal Oxides as Supports Justification. Investigation of a number of gelatinous hydrous metal oxides (frequently called hydroxides, although their full structures are uncertain) has established (3) that hydrous titanium (IV), zirconium (IV), iron (III), vanadium (III) and tin (II) oxides at least are capable of forming with enzymes insoluble complexes which are enzymically active. From the practical viewpoint hydrous titanium (IV) and zirconium (IV) oxides proved the most satisfactory. Comparatively high retentions of enzyme specific activity may be achieved (3, 4, 5). Such hydrous metal oxide materials have also proved to be suitable for the immobilisation of amino acids and peptides (3), antibiotics with retention of antimicrobial activity (6), polysaccharides (7), etc. 0-8412-0508-6/79/47-106-119$05.00/0 © 1979 American Chemical Society Venkatsubramanian; Immobilized Microbial Cells ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

120

IMMOBILIZED

MICROBIAL

CELLS

H y d r o u s t i t a n i u m (IV) a n d z i r c o n i u m (IV) o x i d e s a r e i n s o l u b l e o v e r the n o r m a l p h y s i o l o g i c a l p H r a n g e , as

enzyme immobilisation matrices

enzymic activity,

they thus

and since when acting

they give good retention of

s e e m to h a v e l i t t l e o r n o e f f e c t

the f u n c t i o n of b i o l o g i c a l l y a c t i v e m o l e c u l e s . which is

extremely

enzyme molecule, then there

s e n s i t i v e to c o n f o r m a t i o n a l c h a n g e s i n t h e i s not s e r i o u s l y

affected

by immobilisation,

s e e m s to b e l i t t l e r e a s o n w h y c e l l w a l l s

s h o u l d be

r u p t e d o r d e s t r o y e d b y t h i s p r o c e s s a n d the c e l l s therefore,

have a good chance of remaining

viable.

M e c h a n i s m f o r F o r m a t i o n o f the C e l l - S u p p o r t B o n d . as

i n v o l v i n g the r e p l a c e m e n t

of

the m e t a l h y d r o x i d e b y s u i t a b l e l i g a n d s

The

L-serine

such ligands

the c a r b o x y l s

of L - g l u t a m i c

in Figure

u s i n g h y d r o u s z i r c o n i u m (IV) o x i d e

1,

group of L - l y s i n e

acid

ε-amino

oxygen-containing ligands

containing nitrogen.

In the c a s e o f c e l l s ,

d i v e r s i t y of suitable ligands carbohydrate

moieties

t i t a n i u m (IV) o x i d e as

f r o m both protein,

(illustrated

to

i n F i g u r e 2,

or as

those

the s t r u c t u r a l

com

great

and also using

from

hydrous

example).

E x p e r i m e n t a l M e t h o d f o r F o r m a t i o n of the S u p p o r t . ( e a c h o f 1.3 m m o l ) o f the m e t a l h y d r o x i d e s f o r u s e immobilisation were prepared

f r o m solutions

( t i t a n i u m (IV) c h l o r i d e

chloric acid (BDH, Poole, (BDH)

case

residues,

being p r e f e r r e d

p l e x i t y of the c e l l w a l l e n s u r e s the a v a i l a b i l i t y o f a

chlorides

cell,

In the

a c i d a n d the

(illustrated example),

surface

from enzyme or

c o u l d be the s i d e - c h a i n h y d r o x y l s o f

or L-threonine,

L-aspartic

envisaged

o f h y d r o x y l g r o u p s o n the

r e s u l t i n g i n the f o r m a t i o n o f p a r t i a l c o v a l e n t b o n d s . enzymes,

dis

themselves,

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

of

on

If e n z y m i c a c t i v i t y ,

Samples

in cell

of their

tetra

15% w / v in 15% w / v hydro

E n g l a n d ) a n d z i r c o n i u m (IV) c h l o r i d e

0 . 6 5 M i n 1.0 M h y d r o c h l o r i c a c i d ) b y the s l o w a d d i t i o n o f

2 . 0 M a m m o n i u m h y d r o x i d e to n e u t r a l i t y ( p H 7 . 0 ) . were washed with saline

solution (0.9% w/v,

r e m o v e a m m o n i u m ions and then used for c e l l studies as

The

3x5.0 ml)

immobilisation

below.

E x p e r i m e n t a l M e t h o d for I m m o b i l i s a t i o n of C e l l s . procedure

samples to

The

for c e l l i m m o b i l i s a t i o n is v e r y s i m p l e and is

t r a t e d b y the f o l l o w i n g e x a m p l e . saline) of E s c h e r i c h i a c o l i cells s a m p l e o f the m e t a l h y d r o x i d e a s

A

suspension

(in 1 0 m l

(A^QQ 0 . 2 1 6 ) w a s prepared

as

mixture was

s e t t l e d out,

leaving a clear

5-7)

The

t h e n a l l o w e d to s t a n d a t r o o m t e m p e r a t u r e

the s u s p e n s i o n

0.9w/v

m i x e d with a

above (pH

a n d a g i t a t e d g e n t l y f o r 5 m i n at r o o m t e m p e r a t u r e .

illus

supernatant

Venkatsubramanian; Immobilized Microbial Cells ACS Symposium Series; American Chemical Society: Washington, DC, 1979.

and

8.

0

/

\

0

121

Methods of Immobilization

KENNEDY

0

0^/ 0

0

Figure 1. Projected structures of the chelates!complexes of hydrous zirconiwm (7V,) oxide u;ii/i carboxyl, hydroxyl, amino groups, respectively

0

\yorbohydrote\\\

\ / -Ti

A

0

OH, 2

Ο 0

A Figure 2.

.

0

Λ

— Τι — Ti

H,0

/

2

\

/ Ti

A

0

Τι Ti

/

_

7

\ Ti

A

OH, HO 2

Ο 0

OH, HO /

OH,r Hp 2

. -Ti Τι ··—ΟΗ., * Λ 1 1

2

0

OH, HO

\ / Ti

2

0

A ι

0

ι

\

HO

Ti

/

OK -

'

A ι

0

\WcarDohydrate\W

A

y \

HO 0 1 i I Wear bohyd rat 0

. 0

HO

Projected structure of the chelate/complex of hydrous titanium (IV) oxide with macromolecular carbohydrate


122

IMMOBILIZED MICROBIAL CELLS

which was

p r a c t i c a l l y devoid of

(shown by m i c r o s c o p y ) .

microorganisms

The immobilised cell preparation

c o n s o l i d a t e d b y c e n t r i f u g a t i o n at l o w s p e e d a n d r e m o v e d the s u p e r n a t a n t It w a s pH,

for further

c o u l d b e i m m o b i l i s e d at

h y d r o u s t i t a n i u m (IV) o x i d e b e i n g m o r e e f f e c t i v e

useful

i n the r a n g e s t u d i e d (pH 2-5).

s i n c e not a l l m i c r o o r g a n i s m s

e n v i r o n m e n t (for enabled us as

examination.

a l s o found that c e l l s

purpose,

example,

lower

for

exist in a neutral pH

Lactobacillus and Acetobacter)

o f the L i f e e t c .

Immobilised Cells.

Characteristics

Saccharomyces

cerevisiae

of a n d E . c o l i cells

w e r e i m m o b i l i s e d i n this m a n n e r a n d the p r e p a r a t i o n s examined for continued viability by measurement oxygen uptake 5.0 o r

(at 2 5 ° C

Cambridge,

This

result

(Pye

T h e rate of oxygen

uptake

^ 3 0 % o f that o f the s a m e n u m b e r o f

s h o w e d that r e s p i r a t i o n o f the

c o u l d c o n t i n u e w h e n the c e l l s w e r e i m m o b i l i s e d . rate of oxygen uptake

buffer

of an oxygen electrode

England).

of the i m m o b i l i s e d c e l l s was cells.

were

of their

i n a e r a t e d 0.2 M s o d i u m a c e t a t e

0.9% w / v s a l i n e ) b y u s e

Unicam Ltd., free

and

reactor,

described.

Demonstration

pH

this

T h i s phenomenon is

to p r o d u c e a s m a l l s c a l e i m m o b i l i s e d c e l l

is now

was

from

is p r o b a b l y c a u s e d

cells

The

reduced

by the r e s t r i c t i o n by

the h y d r o u s m e t a l oxide of a c c e s s of a e r a t e d buffer

to the

and

for

a d e c r e a s e o f the a r e a o f c e l l s u r f a c e a v a i l a b l e

cells

oxygen

transfer. To

s h o w that the c e l l s w e r e f i r m l y a t t a c h e d to the s u r f a c e

of

the h y d r o u s m e t a l o x i d e a n d not j u s t l o o s e l y t r a p p e d i n the gelatinous

matrix,

c e l l s of E .

coli are

a n u m b e r of arguments comparatively

are

invoked.

s m a l l and cannot

c e n t r i f u g e d d o w n to a n y s i g n i f i c a n t e x t e n t i n t h e

centrifugation

c o n d i t i o n s u s e d f o r c o l l e c t i n g the i m m o b i l i s e d c e l l s ; not

Free

be they

are

p a r t i c u l a r l y r o b u s t a n d so a n y d i s r u p t i o n p r o c e s s w h i c h h a d

occurred would, u n a b l e to r e s p i r e . fluoride

therefore,

have rendered them inactive and

Solutions of bicarbonate,

( a n d s o o n ) i o n s w h i c h h a v e b e e n s h o w n (3) t o

loosely bound proteinaceous

and other materials

z i r c o n i u m (IV) h y d r o x i d e w e r e attempted To

remove

from

singularly ineffective

r e l e a s e o f the i m m o b i l i s e d c e l l s f r o m the

i n the matrix.

s h o w f u r t h e r that the c e l l s w e r e f i r m l y a t t a c h e d to

hydrous metal oxide, marcescens at 2 5 ° C , t h i s

was

a different m i c r o o r g a n i s m ,

employed.

so

phosphate,

the

Serratia

When incubated i n nutrient m e d i u m

organism produces

a distinctive

w h i c h e n a b l e s the i m m o b i l i s e d c e l l s

red colouration,

to b e d i s t i n g u i s h e d


readily

8.

123


KENNEDY

f r o m those of any other contaminating m i c r o o r g a n i s m s o b v i a t e s the n e e d f o r

sterile experimental

O n adding cultures

o f S.

marcescens

and

to h y d r o u s

zirconium

(IV) a n d t i t a n i u m (IV) o x i d e s , the r e d c o l o r a t i o n (that i s , c e l l s ) b e c a m e a s s o c i a t e d w i t h the i n s o l u b l e m a t r i x , natant and subsequent w a s h i n g s almost

cell free,

metal hydroxide interaction. i l i s e d c e l l s w e r e added as medium,

(with saline

thus d e m o n s t r a t i n g

growth was

solution)

(as

culture

S i n c e at no t i m e t h r o u g h o u t temperature

c o l o u r f r o m the c e l l s

observed,

the c e l l s h a d s u f f e r e d

no d e l e t e r i o u s

It i s a l s o c l e a r

cell-

d e t e c t e d b y the l a r g e i n c r e a s e

this was

that the i m m o b i l i s e d c e l l s at

evidence

can retain their least. Fermentations

Introduction and M e t h o d s of I m p r o v i n g F e r m e n t a t i o n Acetobacter

species,

profit margins in research

of v i n e g a r ,

and this

results i n there being little

or m o d e r n equipment.

m e a n s that the i n e f f i c i e n t semi-continuous

inexpensive first

Quickf

p r o c e s s is

efficient,

conservatism

still widely used

efficient,

the F r i n g ' s

- it is at l e a s t as and simpler

process

efficient as

the

laboratory

i n c o n s t r u c t i o n but the

volume per day/fermenter

conditions).

the i n i t i a l

of 5-10.

volume),

commercially,

High volumetric efficiencies

some yeasts and

efficiencies

(0.5

the

initial capital fer

efficiency which 0.8

is

under

have

been

growing in tower fermenters

moulds

The reason

c a n be g r o w n at for this has

the f l o c c u l e n t n a t u r e of t h e s e o r g a n i s m s , h i g h c o n c e n t r a t i o n of o r g a n i s m

and

fulfils

w o u l d r e q u i r e a r e d u c t i o n i n the s i z e o f the

obtained with other organisms, 10) e . g .

required,

Fringfs

m e n t e r with a c o n c o m i t a n t i n c r e a s e i n its v o l u m e t r i c effluent

would

(8),

What is

s i m p l e to o p e r a t e

T o reduce

l o w f o r the a c e t i f i c a t i o n p r o c e s s

by

continuous

The tower fermenter

still comparatively high.

(V. Ε . ,

of

investment

enforced

f o r t h e m to i n s t a l l .

to i n s t a l l a n d r u n .

s t i r r e d tank reactor expenditure

This

because more

is a s y s t e m w h i c h is

two c r i t e r i a

cost is

f

method e.g.

be prohibitively expensive therefore,

Rates.

w h i c h is b a s e d o n the a c t i o n s

is an industry w h i c h o p e r a t e s with low

many small manufacturers or

that

on immobilisation.

A p p l i c a t i o n s o f L i v i n g I m m o b i l i s e d C e l l s to

The manufacture

of

composition

r e l e a s e of

taken as

effects

of w e e k s / m o n t h s

was

extended

conditions

and m e d i u m

and ultraviolet irradiation-induced mutation,

activity for a matter

being

a s m a l l i n o c u l u m to f r e s h

oxygen tension,

super

the s t r e n g t h of the

s t u d i e s o f the g r o w t h o f S. m a r c e s c e n s , i n v a r i o u s humidity,

the

the

W h e n samples of these i m m o b

observed

in n u m b e r of r e d cells).

also

conditions.

(9,

volumetric

been attributed

to

which results in a

i n the f e r m e n t e r

and

therefore


124


a l l o w s h i g h f l o w r a t e s to b e a c h i e v e d . are

Unfortunately

o p e r a t i o n of the a c e t i f i c a t i o n f e r m e n t e r flow rate,

bacteria

are

!

for,

above a

h e n c e the a m o u n t of a c e t i c

fermenter

found of maintaining an i n c r e a s e d Since,

exceeded, treating

Thus,

the b a c t e r i a

the

time

acid.

Increased

Concentration in a Fermenter.

One

obvious

to s o l v i n g t h i s p r o b l e m w o u l d b e to i n d u c e the

to f l o c c u l a t e to w a s h o u t However,

thus,

hopefully,

giving them a greater

even aggregating

strains of A c e t o b a c t e r

b r i n g this about.

I m m o b i l i s a t i o n of the c e l l s was

required

predicted

c o u l d be expected

of A c e t o b a c t e r

to c o n t a i n m u r e i n s

i n n e r m o s t l a y e r of the w a l l , lipopolysaccharides

i n the o u t e r l a y e r s .

t h e r e m a y be a c a p s u l e o r consisting

seemed ample

The use

or a

polypeptide

o f h y d r o u s t i t a n i u m (IV) o x i d e

cells

oxides. rather

than hydrous

s e e m e d to b e n e c e s s a r y b e c a u s e o f t h e

e t h a n o l t a k e s p l a c e a t a b o u t p H 3,

The acetification

in which region

by c o m p a r i s o n ,

of

hydrous

(IV) o x i d e i s n o t c o m p l e t e l y p r e c i p i t a t e d i . e.

s t i l l m a n y z i r c o n i u m (IV) i o n s i n s o l u t i o n .

t i t a n i u m (IV) o x i d e ,

fore-

o p p o r t u n i t y f o r c h e l a t i o n o f the

c o n d i t i o n s p r e v a i l i n g i n the f e r m e n t a t i o n . zirconium

wall

composition

T h u s o n the b a s i s o f the

to h y d r o u s t i t a n i u m (IV) a n d z i r c o n i u m (IV) z i r c o n i u m (IV) o x i d e

the and

O u t s i d e the c e l l

of either a single p o l y s a c c h a r i d e

going there

as

lipoproteins

s l i m e l a y e r of s i m p l e

containing a single amino acid.

and

(Gram-negative)

(peptidoglycans)

with proteins,

or

to

Bacterial

notable for their c h e m i c a l c o m p l e x i t y

d i v e r s i t y a n d the c e l l w a l l s

to

without h a r m

achieve this d e s i r e d i n c r e a s e i n c e l l concentration. are

cells.

naturally,

d o n o t a g g r e g a t e to a n y

s o m e artificial m e a n s is

a c c o r d i n g to the a b o v e m e t h o d ,

cell walls

bacteria

resistance

i n a n a n a l o g o u s m a n n e r to f l o c c u l a t e d y e a s t w h i l s t flocculent s t r a i n s of y e a s t s o c c u r

m a r k e d extent a n d thus

are

greater

w h i l s t at the s a m e

A n s w e r s to the P r o b l e m o f the n e e d f o r

damage,

be

i n s o m e w a y s u c h that they h a v e a

r e t a i n i n g t h e i r a b i l i t y to c o n v e r t e t h a n o l i n t o a c e t i c

approach

growth

s o l u t i o n w o u l d s e e m to i n v o l v e

t e n d e n c y to r e m a i n i n the f e r m e n t e r ,

Bacterial

be

b a c t e r i a l c o n c e n t r a t i o n i n the

a m a x i m u m value which cannot

the o n l y a l t e r n a t i v e

i f the

some means must

u n d e r a g i v e n set of c o n d i t i o n s ,

r a t e of the o r g a n i s m has

at a f a s t e r

concentration

acid produced fall.

i s to o p e r a t e a t h i g h f l o w r a t e s ,

fermenter.

certain

w a s h e d out1 of the f e r m e n t e r

r a t e than t h e i r g r o w t h r a t e a n d thus the b a c t e r i a l and

bacteria

n o n - f l o c c u l e n t a n d t h i s p l a c e s a c o n s t r a i n t u p o n the s p e e d o f

there

Hydrous

is f u l l y p r e c i p i t a t e d at

p H a n d r e s u l t s o b t a i n e d o n the i n s o l u b i l i s a t i o n o f s t a r c h


by

this

8.

KENNEDY

125


hydrous titanium (IV) oxide indicate that the complexing effect begins to be significant in the region of pH 3 (3·). This is a very useful property since it indicates that there will be an equil ibrium existing between immobilised and free bacteria. One result of this is that when the immobilised cells die, they will eventually be released from the hydrous oxide surface and be replaced by living cells. Thus, the hydrous oxide will not become rapidly exhausted by being covered with dead cells. Another consequence of this is that if the immobilised cells are incapable of reproduction then there will be sufficient free cells to grow normally and replace those bacteria dying or being washed out of the fermenter in the effluent. P r a c t i c a l Demonstrations of the Use of Living Immobilised Cells for Increasing Fermenter Efficiencies. The tower fermenter system used is described in Figure 3. Wort was used as the ethanol source, and an inoculum of an aggregating strain of Acetobacter species was prepared and added to the tower fermenter (2\ litres capacity). When the level of acetic acid i n the fermenter had reached about 3% w/v, the medium delivery pump was started and the flow rate adjusted to a level that gave almost complete conversion of the ethanol available into acetic acid. Undue haste in increasing the flow rate and also serious decrease or stoppage of the a i r flow caused the expected fall in conversion efficiency. Adjustment of the flow and aeration rate showed that a maximum V. E. of 0.82 could be attained (see Table I). Table I. Average Rates of Production of Acetic A c i d for Maximum Efficiencies for Aggregating Acetobacter Strains Average taken over days

Highest efficiency (V. E. x % )

g acetic acid produced per day

21-29

0.82 χ 86 (day 23)

87 t 7

81-88

1.6.4 χ 99 (day 88)

263 ΐ 13

Comment

No hydrous titanium (IV) oxide Hydrous titanium (IV) oxide added, Ξ aver age 0. 75 g T i C l daily 4



Device

Attemporation

Figure 3.

Condenser

The tower fermentation system

ΖΖΓ

Valve

Filter/ Pressure Regulator

Air from Compressor

Product

ο w

Ci

8.

127


KENNEDY

A t t h i s p o i n t the a d d i t i o n of h y d r o u s t i t a n i u m (IV) o x i d e ( p r e p a r e d as

before) began,

efficiency was

increased

slightly increased.

a n d o v e r a p e r i o d the v o l u m e t r i c

( T a b l e I) t h e a e r a t i o n r a t e b e i n g

A t one s t a g e the a m o u n t o f a c e t i c

p r o d u c e d b e g a n to f a l l a n d t h i s w a s rate becoming limiting. increased,

to t e s t t h i s h y p o t h e s i s .

version rose once m o r e , i n c r e a s e d as

well,

Ultimately a V . E .

was

aeration

the a e r a t i o n r a t e

T h a t the p e r c e n t a g e

was con-

e v e n t h o u g h the m e d i u m f l o w r a t e h a d

t a k e n to b e s u f f i c i e n t p r o o f o f t h i s .

o f 1.64 w a s

t h r o u g h o u t the r u n it w a s V . E .

a t t r i b u t e d to t h e

Consequently,

only

acid

a c h i e v e d ( T a b l e I).

possible

to i n c r e a s e

Thus

the f l o w r a t e a n d

w h i l s t m a i n t a i n i n g t h e c o n v e r s i o n o f e t h a n o l to a c e t i c a c i d

a t b e t w e e n 9 0 a n d 100% f o r t h e m a j o r i t y o f t h e

time.

T h e h y d r o u s t i t a n i u m (IV) o x i d e h a d a n i m m e d i a t e l y n o t i c e a b l e effect fermenter,

o n the a p p e a r a n c e

which was

f r o m p u r p l e to b r o w n . performance addition,

T h i s d i d not a p p e a r

of the f e r m e n t e r

another,

o f the b a c t e r i a

i n the

to c a u s e a c o l o u r c h a n g e i n t h e in any way,

organisms

to a f f e c t

the

however,

in

m o r e gradual change was

observed over

a

p e r i o d of about ten days after

the c o m m e n c e m e n t o f h y d r o u s

t i t a n i u m (IV) o x i d e a d d i t i o n .

Before

v e r y little aggregation clumps had formed.

this point,

of the b a c t e r i a ,

After

there had been

although a few

t i t a n i u m (IV) o x i d e m a n y m o r e a g g r e g a t e s f o r m e d , t h r o u g h o u t the f e r m e n t e r . close-up photographs particles,

o f the t o w e r a s

the f e r m e n t e r a n d s u s p e n d e d diameter.

spherical

A sample

to b e s p i k y p e l l e t s ,

removed from mixture,

about 2 m m i n

A single particle fixed by heating and stained

(IV) o x i d e e m b e d d e d i n the a g g r e g a t e , T h i s is not a c o m p l e t e l y true

situation,

diffuse

in a glycerol/water

M e t h y l e n e B l u e r e v e a l e d the p r e s e n c e spots.

dispersed

T h e s e w e r e d i s t i n g u i s h a b l e i n the

surrounded by a i r bubbles.

showed these particles

small

the t r e a t m e n t w i t h h y d r o u s

s i n c e the s a m p l e

has

of particles v i s i b l e as

dense,

dark

r e p r e s e n t a t i o n o f the

to b e d r i e d b e f o r e

t h e r e b y c a u s i n g the h y d r o x i d e p a r t i c l e s p a r t i a l l y c o n v e r t e d to o x i d e .

with

of titanium

staining

to s h r i n k a n d to b e

H o w e v e r , it is c e r t a i n f r o m

this

that h y d r o u s t i t a n i u m (IV) o x i d e i s p r e s e n t i n the a g g r e g a t e d particles,

and therefore

a useful aggregating

that the h y d r o u s m e t a l o x i d e d o e s

effect

o n the b a c t e r i a a s

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

predicted.

it c o u l d be

have Under

seen

that m a n y b a c t e r i a w e r e not a s s o c i a t e d w i t h the a g g r e g a t e d particle

(free bacteria),

c o n f i r m i n g the p r e d i c t e d e q u i l i b r i u m

situation. A

v a r i e t y of conditions of fermentations

with Acetobacter


128


s p e c i e s using i m m o b i l i s e d c e l l s have now b e e n investigated various

s i z e s of f e r m e n t e r .

aggregating whereas

In the c a s e of u s e

s t r a i n of A c e t o b a c t e r s p e c i e s (2j l i t r e

fermenter),

a d d i t i o n of h y d r o u s t i t a n i u m (IV) o x i d e r e s u l t e d i n a n

increased V. E .

( T a b l e II),

this was

a c h i e v e d u s i n g the a g g r e g a t i n g ( T a b l e II) w a s

not so m a r k e d as

strain.

that

a higher V . E .

the s u p p o r t m a t e r i a l f o r c e l l i m m o b i l i s a t i o n .

H y d r o u s t i t a n i u m (IV) o x i d e - c e l l u l o s e mixing equal weights powder

However,

a c h i e v e d u s i n g h y d r o u s t i t a n i u m (IV) o x i d e -

c e l l u l o s e chelate as

chelate was

prepared

(1.2 g) o f c h r o m a t o g r a p h i c g r a d e

by

cellulose

( W h a t m a n C F 1 1 ) a n d t i t a n i u m (IV) c h l o r i d e s o l u t i o n

above) and s t i r r i n g for 2 h o u r s . 45°,

in

of a non-

g r o u n d to a p o w d e r ,

washings aqueous

were neutral, suspension.

T h e mixture was

(as

then d r i e d at

washed with distilled water until

the

a n d t h e n a d d e d to the f e r m e n t e r a s

an

T h e effect o f the c h e l a t e w a s

not p r o d u c e d

b y a m e r e m i x t u r e o f c e l l u l o s e a n d h y d r o u s t i t a n i u m (IV) o x i d e . T h e a d d e d s u c c e s s w i t h the c h e l a t e i s t h e r e f o r e

attributable

to

the a l t e r e d m o d e of p r e s e n t a t i o n o f the i m m o b i l i s i n g t i t a n i u m species. Conclusions.

It m a y b e c o n c l u d e d t h a t t h e a d d i t i o n o f

h y d r o u s t i t a n i u m (IV) o x i d e to a b a c t e r i a l f e r m e n t a t i o n w i l l cause aggregation

o f the b a c t e r i a

c o n c e n t r a t i o n i n the f e r m e n t e r .

a n d thus give a h i g h e r b a c t e r i a l T h i s apparently allows both a

higher aeration rate and a higher dilution rate, rates

(V. E . ~1.6)

at l e a s t t w i c e as

high as

giving dilution

those

previously

obtained with a i r aeration (V. Ε . ^ Ο . δ ) and c o m p a r a b l e t h o s e o b t a i n e d w h e n the f e r m e n t e r w a s (V.E.

aerated

with

with pure

oxygen

1.8).

M o r e generally, metal oxides

are

immobilisation.

it m a y be c o n c l u d e d that h y d r o u s

effective

matrices

for enzyme,

transition

etc.

T h e i r advantages include low cost, convenience

of p r e p a r a t i o n (which m a y be c o n d u c t e d i n any l o c a t i o n without specialised preparation,

facilities),

the a b s e n c e o f a n y n e e d f o r

pre-

a b i l i t y to c o u p l e e n z y m e a t n e u t r a l p H ,

high

r e t e n t i o n o f e n z y m e s p e c i f i c a c t i v i t y o f the e n z y m e o n immobilisation,

a n d t h e a b i l i t y o f m o d i f i c a t i o n to e x e r t

e n v i r o n m e n t a l effects o n a n d t h e r e b y a l t e r the

micro-

characteristics

of the i m m o b i l i s e d e n z y m e . If t h i s s i m p l e m e a n s other m i c r o o r g a n i s m s

o f c e l l i m m o b i l i s a t i o n w e r e a p p l i e d to

it c o u l d w e l l r e s u l t i n f u r t h e r

immobilised cell reactors

of this sort,

f o r the

selective

production of c o m m e r c i a l l y important b i o c h e m i c a l and pharmaceutical compounds.

Magnetic forms

of the h y d r o u s


8.

KENNEDY

Table Average

Highest

taken

efficiency

4-14

g acetic

( V . E . x%)

day

1.03

178

χ 99

1.00 (day

x 95

Comment

No hydrous (IV)

185

17)

0.98 (day

χ 97

oxide added,

191

19)

1.45

χ 97

230

( d a y 31)

Hydrous titanium daily

4

oxide-cellulose added,

1.40

χ 90

( d a y 60)

236

(IV)

1.2 g

Hydrous titanium

1.2 56-75

(IV)

0.6 g

daily

4

oxide added, TiCl

25-31

titanium

oxide

Hydrous titanium TiCl

18-24

Strain

acid

produced per

( d a y 9) 15-17

Maximum

for Non-Aggregating Acetobacter

Average days

II

R a t e s of P r o d u c t i o n of A c e t i c A c i d for

Efficiencies

over

129


g

1.2 g T i C l

4

oxide-cellulose 1.2 g

+

cellulose

Hydrous titanium added,

(IV)

chelate

1.2 g T i C l cellulose


(IV)

chelate 4

+

130


t i t a n i u m (IV) oxide have a l r e a d y b e e n p r o d u c e d (7) a n d c l e a r l y such a r e additionally advantageous where s p e c i a l i s e d r e s t r i c t e d m o v e m e n t o f the i m m o b i l i s e d c e l l p a r t i c l e s i s c a l l e d f o r . A s with i m m o b i l i s e d e n z y m e s , i m m o b i l i s e d c e l l s y s t e m s have b e e n a p p l i e d to v e r y few i n d u s t r i a l p r o c e s s e s , p a r t i a l l y b e c a u s e o f the i n e r t i a o f e s t a b l i s h i n g m e t h o d s a n d p a r t i a l l y b e c a u s e o f the i n c r e a s e d d i f f i c u l t y o f o p e r a t i n g s u c h s y s t e m s . It s e e m s c e r t a i n , h o w e v e r , that the i n h e r e n t a d v a n t a g e s o f these s y s t e m s w i l l e v e n t u a l l y p r e v a i l a n d t h e r e w i l l be a n i n c r e a s i n g u s e o f i m m o b i l i s e d s y s t e m s by the p h a r m a c e u t i c a l , c h e m i c a l a n d food industries.

Abstract The use of immobilised cells for industrial and analytical enzymic processes is prophetically advantageous, the problems of isolation of the enzyme(s) and separation of the enzyme(s) from the product being avoided. However, the majority of the reaction currently used for direct enzyme immobilisation would cause cell death if applied to cells. Our approach has been based on the ability of water-insoluble metal hydroxides to chelate and retain peptides, proteins, etc. including enzymes. From various studies it was concluded that gelatinous titanium and zirconium hydroxide matrices are effective matrices for enzyme etc. immobilisation. Their advantages include low cost, convenient preparation (which may be conducted in any location without specialised facilities), the absence of any need for pre-preparation, ability to couple enzyme at neutral pH, the high retentions of specific activity of the enzyme on immobilisation, and the ability of modification to exert microenvironmental effects on and thereby alter the characteristics of the immobilised enzyme. Using this process, Saccharomyces cerevisiae and Escherichia coli have been immobilised, and the continuation of the living processes of the cells in the immobilised state were demonstrated by oxygen uptake experiments. That the cells become firmly bound to the support was demonstrated by following the course of immobilised coloured cells (Serratia marcescens), no colour being released during continuation of the living processes of the cells. The process of immobilisation on transition metal hydroxides has now been extended to immobilisation of cells of species of Acetobacter on hydrous titanium [IV] oxide and use of the cells in this form for the continuous production of malt


8.

KENNEDY


131

vinegar from wort in tower fermenters. The efficiency of the fermenter was increased well above its normal maximum throughput using only free cells. With other species of Acetobacter, pre-chelation of the titanium species with cellulose further improved the efficiency of the fermenter.

Literature Cited 1. Kennedy, J.F., Chemically reactive derivatives of polysaccharides. Advances Carbohydrate Chem. Biochem., (1974) 29 305. 2. Kennedy, J.F., Macromolecules, Specialist Periodical Reports, Carbohydrate Chemistry, Part II, Vols. 4-11, The Chemical Society, London, 1971-1978. 3. Kennedy, J.F., Barker, S.A. and Humphreys, J . D . , Insoluble complexes of amino acids, peptides and enzymes with metal hydroxides. J. Chem. Soc., Perkin I, (1976) 962. 4. Kennedy, J . F . and Kay, I.Μ., Hydrous titanium oxides-new supports for the simple immobilisation of enzymes. J. Chem. Soc., Perkin, I, (1976) 329. 5. Kennedy, J.F., Barker, S.A. and White, C . A . , Immobilization of α-amylase on polyaromatic and titanium compounds incorporating a magnetic material. Die Starke, (1977) 29 240. 6. Kennedy, J . F . and Humphreys, J.D., Active immobilized antibiotics based on metal hydroxides. Antimicrobial Agents and Chemotherapy, (1976) 9 766. 7. Kennedy, J . F . Barker, S.A. and White, C . A . , The adsorption of D-glucose and glucans by magnetic cellulosic and other magnetic forms of hydrous titanium(IV) oxide. Carbohydrate Res., (1977) 54 1. 8. Frings GmbH, British Pat. 1, 101, 560 (1968). 9. Royston, M . G . , Tower fermentation of beer. Process Biochem., (1966) 1 215. 10. Shore, D.T. and Royston, M . G . , Chemical engineering of the continuous brewing process. Chem. Eng. (London), (1968) 46 No. 218, CE99. RECEIVED

February

15, 1979.


Facile Methods for the Immobilization of Microbial Cells without

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