Separation, Recovery, and Purification in ... - ACS Publications

5

Downloaded by UNIV OF CALIFORNIA IRVINE on October 30, 2014 | http://pubs.acs.org Publication Date: July 11, 1986 | doi: 10.1021/bk-1986-0314.ch005

Membrane Processes in the Separation, Purification, and Concentration of Bioactive Compounds from Fermentation Broths Enrico Drioli Instituto di Principi di Ingeneria Chimica, Universitàdegli Studi di Napoli, Piazzale Tecchio, 80125 Napoli, Italy

The potential of membrane separation techniques (such as cross-flow microfiltration(MF), u l t r a f i l t r a t i o n (UF), Reverse Osmosis (RO)and electrodialysis (ED) ) and membrane reactors in the treatment of fermentation broths are huge. The synergistic effects obtainable by designing the overall b i ο t e c h n o l o g i c a l pro cess combining various membrane technique are particularly significant. In this paper experimental results are descri bed which refer to processes of industrial interest studied assuming membrane technolo gies as the best available. The separation,purification and concentration of a thermosensitive bioactive compound from a lysate has been carried out combining UF, ion exchange and RO with significant cost re duction and productivity increase. Enzyme mem brane reactors have been used for triglyceride enzymatic hydrolysis and product separation. Thermophilic,thermostable enzyme u l t r a f i l t r a tion membrane have been prepared, and used in high temperature lactose hydrolysis.

The

term

the

chain

Downstream

tem

f o r t h erecovery,

o fu n i t

Processing

operations

tration

o ft h ep r o d u c t s possible

recovery

step

a r ecombined

purification,

highest The

i nBiotechnology

that

separation

a tt h e l o w e s t

recovery

factor

generally

possible

refers t o into

a

sys

and concen cost and

and quality.

represents

a large

part

0097-6156/86/0314-0052$06.00/0 © 1986 American Chemical Society

In Separation, Recovery, and Purification in Biotechnology; Asenjo, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

o f

the

5. DRIOLI

overall its

capital

cost

investiment

efficiency

biotechnological The

recovery

broth

complicated

considered


systems.

core

that

the

i nthese

b i o -

often un-

processing i s

o f biotechnology.

and particularly

as broad

the fermentation

The downstream

f o r f u r t h e r development

technologies

from

by t h ef a c t

lowconcentration

stable ,non-newtonian brane

plant and

f o r t h ep r o d u c t i o n o f

compounds.

a r ei nvery

key area

i na fermentation

i sa key factor

o f bioactive materials

i sg e n e r a l l y

products a

53

Membrane Processes for Bioactive Compounds

UF,MF

technologies

Mem-

a n d RO c a n b e

i nthis

industrial

segment ( 1 ) . In

Table

I a r esummarized

ducts

o f interest

costs

o f production

t h emarket

on l a r g e

ficantly

and positively

In

Table

I I a r esummarized

of

interest

Those in

cells; tion;

development

Systems

i n methods o f enzyme

fermentation

u t i l i z a t i o n

The

shown

c i a l l y

available

been

of

involves

problems

liquids membranes

and ener-

m i c r o - f i l -

introduced,

significantly. suspended

f i l t r a t i o n .

Commer-

and tubular o f broths f i l t r a t i o n

a n d amino

solved

process

i n terms o f

containing

o f membrane

vaccines been

many

o f product,

correctly

and f o r s t e r i l e

have

immobiliza-

membrane

process

f o r theconcentration

p r o t e i n s , enzymes,

ing

membranes

o f bacteria, o f solutions

a c i d s (3.).

by a p e r i o d i c

backflush-

o f t h e membrane.

Fluids

containing

volume

module from

upwards

c a n be pumped

easily

whole

membranes a

capillary

and moulds,

Fouling

by

used

t h escope

when

this

o f treating

broadens

improvements o r whole

reactors.

cross-flow

t o improve

t o :

and uneconomical

and u l t r a f i l t r a t i o n ,

p o s s i b i l i t y

have

(_2.) .

processes

Processes

generally

Continuous

solids

yeasts

membrane

f o r biocatalyst

o f raw materials,recovery

consumption. been

signi-

technology

o f enzymes

membrane

i nDownstream

areinefficient

which

tration

pro-

Their

be a f f e c t e d

i ngeneral

and reuse

steps

have

w i l l

membrane

t h ev a r i o u s

Traditional

gy

scale

by u s i n g

cancontribute

f o r recovery

development

Membrane

o f various

f o r biotechnology.

systems

methods

values

i nbiotechnological processes.

achieving

fermentation a r eused

microfiltration

cromolecular

very

a well-designed

high

broths

solutes,

w i l l

ning

relatively

liquid-phase

(2).

on t h ebroths stage),

material, only

o f 50% t o 6 0 % suspended

through

When

solids

membrane recoveries

u l t r a f i l t r a t i o n

( o r on t h epermeate

retention o f thes o l u b i l i z e d

as well

be accomplished,

as p a r t i c u l a t e giving

lowmolecular

ma-

and c o l l o i d a l

a f i l t r a t e

weight

from

solutes.


contai-

54

SEPARATION, RECOVERY, AND PURIFICATION IN BIOTECHNOLOGY

Table

I.

Total

duct

market

values

Number Product

f o r the various

of

Current


value χ millions)

category compounds

Amino

acids

($

9

Vitamins Enzymes

$

1,703.0

6

667.7

11

217.7

Steroid

hormones

....

6

376.8

Peptide

hormones

....

9

263.7

Viral

antigens

9

Short

peptides

2

Nucleotides proteins

Antibiotics Gene

preparations

I

A

4.4 72.0

2

a

300.0

4

b

4,240.0

b

100.0

.. .

3

Pesticides Aliphatics

Ν

2

Miscellaneous

Ν

2

I

A

: 1

Methane

12,572.0

Other

24

c

2,737.5

Aromatics

10

c

1,250.9

Inorganics Mineral

leaching

Ν

Only These

two

c

These the

of a

numbers

actual

numbers numbers

largest

2,681.0

5

Ν

I

A

Ν

107

Totals

D

2 ....

Biodégradation

a

number refer of

compounds

to major

are considered

classes

Current SOURCE

value : Genex

d

27,186.7

here.

o f compounds;

not

compounds.

refer

market

of

$

IA JA

only

to those

volume

compounds

i n classes

representing

specified

i n the

thext. d

pro

categories.

excluding Corp.

methane

=

$14,614,700.000

( J j.


5.

DRIOLI

55


Membrane P r o c e s s e s Used Today i n B i o t e c h n o l o g y A r e a of A p p l i c a t i o n

D r i v i n g Force

Symmetric m i c r o porous polymer membrane. Pore s i z e 0.05-10 ι

Hydrostatic p r e s s u r e 15 bar

S i e v i n g mechanism, pore s i z e and p a r t i c l e diameter de termine s e p a r a t i o n characteristics

Sterile filtration, clarification, cell harvesting bacteria, viruses separation.

Ultrafil tration

Asymmetric m i c r o porous polymer membrane. Pore s i z e 1-50 nm

Hydrostatic p r e s s u r e 210 b a r

S i e v i n g mechanism, pore s i z e , and p a r t i c l e diameter de termine s e p a r a t i o n characteristics

S e p a r a t i o n , concen t r a t i o n and p u r i f i c a t i o n o f macromolecular s o l u t i o n s such a s p r o t e i n s , enzymes, p o l y p e p tides, etc.

Reverse Osmosis

Asymmetric mem brane w i t h homo geneous s k i n and microporous sub- o r support structure

Hydrostatic p r e s s u r e ΙΟ Ι 00 b a r

Solution-diffusion mechanism, s o l u b i l i t y , and d i f f u s i v i t y of i n d i v i d u a l components i n t h e homogeneous polymer m a t r i x determine s e p a r a tion character istics.

Concentration of m i c r o s o l u t e s , such as s a l t s , s u g a r s , amino acids, etc., recovery of water from m i c r o b i o l o g i c a l processes.

Membrane Distilla tion

Symmetric o r asymmetric m a i n l y hydro phobic microporous membrane

P a r t i a l vapor pressure gra dient i n t r o duced by a temperature difference

P a r t i a l vapor p r e s sure, separation mechanism i s t h e same a s i n d i s tillation.

Separation v o l a t i l e o r g a n i c s o l v e n t s such as acetone, e t h a n o l , e t c . from aqueous fermentation s o l u t i o n .

Pervaporation

Asymmetric mem brane w i t h homo geneous s k i n and microporous substructure.

P a r t i a l vapor pressure gra d i e n t 0.001 t o 1 bar

Solution-diffusion mechanism, s o l u b i l i t y and d i f f u s i v i t y of i n d i v i d u a l components i n the polymer m a t r i x determine s e p a r a tion character istics.

Separation of organic s o l u t i o n s such a s ethanol, butanol, a c e t i c a c i d , e t c . from aqueous s o l u t i o n s , especially separation of a z e o t r o p i c m i x t u r e s .

Electrodialysis

C a t i o n - and anion-exchange membrane

Electrical potential difference

E l e c t r i c charges of p a r t i c l e

Removing s a l t s , a c i d s , and bases from f e r m e n t a t i o n b r o t h s , sep a r a t i o n o f amino acids, etc.

Chemical potential gradients

p.e. C a r r i e r transport

Microfiltration

μ Π


Mass S e p a r a t i o n Mechanism

Membrane Type

Liquid supported membranes

Symmetric o r asymmetric microporous membranes sup porting l i q u i d phase

S e l e c t i v e removing of s a l t s , b i o a c t i v e compounds, e t c .


SEPARATION, RECOVERY, AND

56

This

f i l t r a t e

osmosis pounds Those

i f of

processes

can

osmosis

tion

antibiotics

of

removal

of

mixtures


v i a

An

interesting

al

scale

for

from

solid

The

study

also

(4).

of

The in

h)

water;

g)

f i l t r a t i o n ; m) on

d)

i)

significantly increasing

obtained,

using

ghof

with

atm 30

FDR)

applied 1/m

with of

organic 97-98%

cut-off

of

and

h

were

normal

Pyrogens

were

completely

At The


and

final

has

been

of

the

obtained

tion raw were

amount step

of

higher

few

in

R0

than

a l l the

hundred

in

UF

of

the

a

orga1).

possibicosts,and

and

process

purification methanol).

membranes

10.000

M.W.

of

order

the

with

was

(Berat

1

of

by

reverse

Figure

recovery

concentration

high.

columns osmosis

2).

regeneration,

High

80

product.

particularly

ion-exchange

(see

column

permeate

was

eluate

and

factor

step,

waste up

with

to final

g/1.

carbon

The

an

activated

the

of

e)

into

state.

the

moreover

acid;

Figure

(e.g.

Fluxes

detail

for

the

on

quality

purity

observed.

treated.

required of

of in

activated

decreased

material

duction

in

order

absent

combining

costs was

precipitation

production

capillary

°C.

product

analyzed

concentrations The

of

treatment

was

the

the

cen-

centri-

S-adenosy1-L-methionine

steady

concentration also

Reduction water 90%

HPLC

at

a)

c)

p i c r i c

showed

place

amino process

of

lysis;

as

solvents

of

20

industri-

transformation

the in


pressure

at

downstream

chemical

such

product


factor

example. biologi-

unstable

consisted

process

decreasing

precipitation

developed

purification

operation,

recovery

an in

l y o p h i 1 i z a t i o n (see this

for

p u r i f i c a -

p r e c i p i t a t i o n s with

of

A

com-

immunocomplexation

and

of

using

RO,is

thermal

agents

l i t y

by

reverse

s u g g e s t e d ( 3_) .

fermentor b)

and

present

particularly

purification

out

and

of

been

a

separation;

f i l t r a t i o n ; carried

by

by

weight

pretreatment

traditional

a

complexing in

UF

use

separation;

f)

ideal

been

time

safety

treated

molecular

concentration

combined has

part

soluble 1)

easily low

contaminants

recovery

cells

solvent;

carbon;

The

a p p l i c a t i o n has

centrifugation; nic

the

the

specific

compound

of

sequential

enrichment

for

for

using

more

considered

by

lysate

yeast

trifuge fuge

a

be

antigenic

and

after

also

processes.

cal

acid

be

interest.

reverse The


might

i f i t s concentration

PURIFICATION IN BIOTECHNOLOGY

used

in

the

1

Kg

to

that

no

organic

from

fact process

dollars

gave per

Kg

an of

final 150

g.

overall final

p u r i f i c a per

Kg

of

solvents cost

re-

bioactive

product.



5. DRIOLI

Figure

1. T r a d i t i o n a l

2)

centrifugation;

5)

chemical

tion 10)

57


downstream

3) t h e r m a l

precipitation;

p r o c e s s :1 ) e n r i c h m e n t ;

lysis;

4)vacuum

6)centrifugation;

f i l t e r ;

7)purifica-

by s o l u b i l i z a t i o n ; 8) r e p r e c i p i t a t i o n ; 9 ) f i l t r a t i o n active

Figure

carbonjll)

2. M o d i f i e d

exchange;

f i l t r a t i o n ;

Process

9) Reverse

11)

;

lyophi1ization.

: 6 ) u l t r a f i 1 t r a t i o n;

8)

Osmosis.


i o n

58


Enzyme

Membrane

Reactors

In

previous

examples

the

red of

g e n e r a l l y as small

the

molecules

separation

solution t i f i e d a

or

as

a

Such

a

true

removal

of

loss

substrate

to

when

an

ous

fermentation

reactor. A

products the

i n

separation

parallel

place system

classical

connected

designed,

(or

of

membrane

or

from

the

to

in

the

may

be

bulk iden-

example

by

a

the

unit.

continuous

bulk or

i s

continuous

dialysis

permits

insoluble

separation

systems

i t i s p o s s i b l e to

increase

duct-inhibited

fermentation, the

removal

of

used

higher

than

a

solution

macromolecular

component i n

the

for

this

productivity

of

pro-

example, weight

of

cellulose

to

inhibited

12%,

might

be

at

continuIn

molecular

are

of

interest.

low

degradation

croorganisms

as

i s growing

ous

tion

the

conside-

the

).

use

zymatic

When

takes

i t s e l f ,

been

for


well

enzyme

ones.

reactor

reaction

of

The case

membrane

have

barriers

reaction

membrane

loop

the

membranes

bigger

enzymatic

system,

without

from

chemical

the

stirred-tank

recirculation


a

i n

the

semipermeable

by

the

alcohol, an

continu-

products.

alcohol

improved

by

The

where

en-

the

mi-

concentra-

the

use

of

this

concept. Recently,

a

similar

the

production

ids

are

of

obtained

produced

acetyl

by

previous

type

ted

acylase,

the

ble

form,

from

the

b i l i z i n g Other from

stage

pyrogens.

might

allow

a

in


This

that the

the

apply

laboratory

(6J . T r a d i t i o n a l

operations

at

ried

out

room

possibility

to

in

step

the

same

i s

The

prepare

and

appears

study

natural olive

matic

membrane

of

o i l was

reactor

in

concepts

and

was

to

i s

at

the

can

be

conti-

obtained

mass

and of

total bacteria.

enzymatic

hydrolysis make

i n

particular used

as

raw

used

based

the

pro-

be

car-

pressure.

g l y c e r i n e and interest. material. on

our

requiring

h y d r o l y s i s can

atmospheric

free

products

the

pressure

immo-

(JL) .

growth to

solu-

enzyme

investigation

separate

Unlike

in

the

toxic

cell for

under

and

enzyme

losses

of

ac-

carrier-loca-

content

removal

enzymatic

temperature

the

solution

chemical

temperature

non-competitive. at

enzyme

those

enzymes.

consumption

increases

triglyceride

of

Degussa f o r L-amino

synthetically-

separating

fermentation

of

of

with

avoids

product

continuous

cess

means

enzyme

hydrolysis

high

by

reactor

by

case,

division

for

batch to

applied this

employs

reduces

are

The

In

approach

solution.

and

been

membrane

significant

yield

acids

fixed-bed

new

and

adjusted

product The

reagent

advantages

nually

of

uses

has

acids.

biocatalytic

DL-amino

the

and

process

L-amino

In An

The

acids our enzy-



5.

DRIOLI

capillary

membranes.

dracea,2975 form

A continuous

the

capillary applied

centration


membrane

and separation

and In

The enzyme

Figure

membranes

pressure

typical

as f u n c t i o n o f time.

in

show

Microporous

be used

emulsion rate

phase

Only

study

where

an high

interface

were o b flow

rate

enzyme

con-

(Figure3 ) .

results

are presen-

o f the t r i g l y c e r i -

g l y c e r i n e was p r e s e n t capillary

t o distribute

t h e enzyme

c a n be c o n t r o l l e d

by changing

o f enzyme

outa t axial

o f conversion

hydrophobic

i nthis

t h ewater

Increase

t o mantein

experimental

t h edegree

cylin-

i na g e l

o f t h eo i l substrate i n

was c a r r i e d

des also

surface.

a t t h emembrane-solution 4 some

Candida

o f t h er e a c t i o n products

useful

which

from

immobilized

recirculation

ted

permeate.

(lipase

U/mg) w a s d y n a m i c a l l y

on t h e i n t e r n a l

s t a b i l i t y tained.

59


small

the

o i l droplets

i sdissolved.

i ndroplets

i n

membranes c a n

size

This

and formation

t h etransmembrane pressure

and axial

flow

c a n be a l s o

pro-

rate .

Enzyme

Membranes

Highly duced

efficient

enzyme

by i m m o b i l i z i n g

fibers.

F o rexample,

support

matrix

enzymes

The dense

permeable through

theinner

spongy

part,

The

development design

could to

denaturation.

In

most

ver,

o f improved without

have

membranes which

been

on a l a r g e

selective

branes require dures. studied

very mass

i scombined

l o w membrane

scale

loss

mobilization

where

exposed

procedures,howei n membrane

and standard

processes, i n

might

mem-

reactions,would

preparation

have

tech-

o f enzyme

thea r t i f i c i a l

chemical

proce-

been r e c e n t l y

accomplish

membranes,involving

a t t h emembrane-solution

per-

biocatalysis

and less

progress

across

specific

enzyme

a r e parameters performance.

The p r e p a r a t i o n

i no u r laboratory, which Gelled

into the

Applied

i nthe effluent

protected

Two i m m o b i l i z a t i o n p r o c e d u r e s

requirements.

rate

f o r industrial

transfer cost

place.

immobilization

limited.

with

flow

reactors

enzyme more

o f thetraditional

bei m -

diffuse

immobilization techniques

flow

a r ealso

should

t o t h e enzyme

takes

while

thefiber l u -

The l a t t e r

o f t h er e a c t o r

thecontributions o f recent

nology

through

o f thefiber and axial

i n t h e porous

membrane,

a t t h elumen w a l l

o f continuous

Enzymes

flows

molecules.

t o control

be a c h i e v e d

stream.

capillary

theconversion

pressure

contribute

mits

layer

wall

where

transmembrane that

skin

or i n hollow

c a n be c o n f i n e d

solution

t o t h e enzyme

reactors

i n membranes

o f an asymmetric

substrate-containing men.

membrane

enzymes

interface,

those

labile imcan r e s u l t


SEPARATION, RECOVERY, A N D PURIFICATION IN BIOTECHNOLOGY


60

FLOW Figure REM,

3. F l o w

capillary

servoir,

Figure pH

OF

sheet enzyme

LABORATORY

EXPERIMENTAL

o f laboratory membrane

reservoir.

4. T r i g l y c e r i d e s

degree

o i l3%,lipase

PLANT

experimental

plant.

reactor ;SS,substrate re-

S P ,permeate

= 6, o l i v e

rate

SHEET

o fconversion

with

3 0 mg i n 5 0 0 m l ; a x i a l

960 ml/min.


time. flow

5.

DRIOLI

from

concentration

unstirred with

or p a r t i a l l y

surized

face

zyme

o f t h emembrane,

membrane

urease,

studied.

that

enzymes

has

From

been

a gel

cantly

forming

membrane trary,

been

behaviour specific

techniques

activity

a r ep a r t i c u l a r l y induced

vironment;

this

The


represent membrane

o f using

technology

f i l l e d

enzymes

vents,

h a s been

cent

limited

i nthecasting

nealing,

which

isolation

thermophile

zymes

a r eg e n e r a l l y

offers

an i n t e r e s t i n g

nes

technique

f i l l e d

with

S.Soifataricus such

for

whole

prepared

membranes

by s e v e r a l

optimally

methods.

with

tech sol

temperature an

properties. (JL),

The r e an

for

denaturating using

ex

enzymes,

en

agents,

t h ephase

o f U F a n d RO

i n

membra

source.

o f industrial

and malic

f i l l e d

membranes

non-aqueous

a s t h e enzyme

enzymes

However, t h e

a t 8 0 °C a n d w h o s e

t o protein

cells

a s β-galactosidase

A r t i f i c i a l

for

t h ep r e p a r a t i o n

contains

favora

traditional

Solfatarieus"

opportunity

immobi

micro-en

b i o c a t a l y s t might

using

t h ec a t a l y t i c

stable

be

i nthegel.

o f U F a n d RO

by t h eneed

growing

transi con

i nt h edevelopment o f

cells,

o f "Solfolobus

the

u l t r a f i l t r a t i o n and

with

engineering.

scale

o f

i sp a r t i c u l a r l y

solutions and high

destroys

treme

version

f i l l e d

o r whole

should

concentration

a n d enzyme

The more

enzymes,

i n t h e enzyme

improvement

a t industrial

technique

the kinetic

t o a decrease

traditional

preparation niques,

on t h e con The

(JL)·

enzymes

t h e enzyme

enzyme

membranes

asignificant

with

not s i g n i f i


o r environmental

changes

mem

polymeric

t o conformational

thesituation

o f t h ehigh

osmosis

does

The

studying

lead

ligands

technique

moreover,

because

The

factors.

for

sensitive

significant

ble

also

retain The method

and tubular

morphology,

allosteric

by s p e c i f i c

With

without

appears

o r t o a d e a c t i v a t i o n o f these

tions

straints.

etc., has

i t

system.

membrane

i nfact,

which

reverse

membranes

s t a b i l i t y .

t o be u s e f u l

o f immobilized

traditional

lized

flat

t o be t h e c o n t r o l l i n g

shown

gelled en

phosphatase,

i sincreased.

recirculating

t h e enzyme

formed

^ c i d

o n a UF m e m b r a n e

c u t - o f f a n d t h e membrane

appear

on t h e d e t a i l e d

results

s t a b i l i t y

c a nbe

on t h e pres

enzymes,1ipase,

t h esupporting

influence

for

layer

o u tusing

i na continuous

material

depending

a dynamically malic

solution

o f enzyme

i n " g e l " form

technique

and t h e i r

carried

amount

theexperimental

forming

activity

branes

has

formation

β-galactosidase,

been their

Such

i n batch processes

o f thesubstrate

immobilized

( JL ) .

Both

a n d i n UF

an appropriate

totally

dynamics

phenomena.

processes

recirculation

t h emembrane,

fluid


polarization


continuous

along

61


for

interest, example.

S . S o i f a t a r i c u s have

Cellulose acetate

been

and poly-


SEPARATION, RECOVERY, AND

62

sulfone phase were

were

used

used

for

crosslinking A

obtain

cell

asymmetric

(10)·

immobilization

hydrophilic polyisocyanate

liquid two

structured

polyurethane

free

porous

suggested

films

containing Physical

and

groups as

an

tubes

and

by

the

glutaraldehyde

membranes

groups

and

used

in

per

thin which

by

the

be

co-

protein

porous

The

use

at

with

has

material,

compounds

r e a c t i o n between

amino-groups

been

Hydrophilic this

active

of

least

molecule, agent.

prepared

specific

prepare

contains

polymer

biologically

and

to

films.

immobilyzing

can

immobilized

entrapment

cyanate

in

was

foams

prepolymer,

isocyanate

recently

membranes

Albumin

method.

polyurethane


to

inversion technique

PURIFICATION IN BIOTECHNOLOGY

(11).

free

contribute

iso

to

the

"immobilization". The

physico-chemical

sidase shown

activity by

the

in

enzyme

the

β-galactosidase

100

°C

and

appeared

rature

and

a

lactosidase nic 9

of

activity cant

for

up

to

was

intact

free by

ne

system

and

than

°C.

p i l l a r y res

of

Those

at

w/v,

was the

Membranes

were

6

membrane

to

decrease

entrapment may

be

a

were a

The

the

dope

used

the

the of

in

as

the

the

non-solvent

The

range in a

N-N

of ca

mixtu dimethyl-

bacteria,

5).

phase

the

in

conversion,

lyophilized

in

of

polyuretha

prepared

(Figure

to

dope

s i g n i f i with

microorganism

c o n s i s t i n g of

mixture

according

a

preparations.

also

8-

enzymatic

increase

for

studied

dope

of

the

degree

been

orga

consequence

of

membrane

B-ga-

After

comparison

greater

have

tempe

with

imparted

in

procedures.

other

the

room

activity.

p o l y v i n y l p y r r o l i d o n and

of

on

wall

membranes

the

the

the

indications The

capillaries

yer

typical

ger

structure

t i a l l y

at

pH

about

inversion

equipment agent

to

in F i

promote

formation.

Microphotographs of

no

activity

spinning

was

°C,

those

trapped-cell

of

system

formed

: water

hr

at

loss

35-fold

t e c h n i q u e ^ (_12.) s p i n n i n g gure

24

of

β-galactosidase

the

added

to

to

optimal

any

effect

membranes

Before

were

up

the

activity

permeabi1ization

polysulphone,

acetamide.

At

temperature

4

c o n f i g u r a t i o n from

of

c e l l .

B-galacto-

similar

room

entrapment

in

were

at

Cell

This

rate,

s t a b i l i t y

70-85

3%

the

flow

hr

enzymatic

activity

permeate

for

cause

membrane

by

enzymatic

24

storage

cells.

cytoplasmic caused

not

in

trapped-cell

Incubation

observed.

increase

free

stable 3-8.

wet

the

of

models

e x h i b i t e d maximal

of

did

above

in

pH

solvents months

properties the

of

membranes,

distribution

s t i l l

and

(1

=

10-20

Figure

the where

the

dense

membranes, c\m)

7,

give

b a c t e r i a both

underneath

exhibit

asymmetric

of

and

dense internal a

clear in

skin l a

supporting

b a c t e r i a are

the

layer. f i n

preferen

allocated.


DRIOLI


Τ-ο

O*

w/foie ceils of ο £ SolfaÎar/cus qq fiiacrotnolecules


*

^

00

g-

Suhsiraèe

° J Proc/ucis • )

Figure red

Figure ry

5. A s y m m e t r i c

by phase

6. F l o w

membrane

enzyme

invertion

sheet

capillary

membranes

prepa-

method.

o f thespinning

system

f o r capilla

formation.



SEPARATION, RECOVERY, A N D PURIFICATION IN BIOTECHNOLOGY

F i g u r e 7 a . SEM p i c t u r e o f t h e d e n s e s k i n o f a n a s y m m e t r i c c a p i l l a r y membrane w i t h e n t r a p p e d c e l l s . Reproduced w i t h p e r m i s s i o n f r o m R e f . 14.

F i g u r e 7 b . SEM p i c t u r e o f t h e p o r o u s s u b l a y e r o f a n a s y m m e t r i c c a p i l l a r y membrane w i t h e n t r a p p e d c e l l s . Reproduced w i t h p e r m i s s i o n f r o m R e f . 14.


5. DRIOLI

Mechanical tested

fibres.

properties

strain The a

average factor

o f t h emembranes

t o those

The Young carried

2.5 r e l a t i v e

t o t h eaverage

fibres. activity

evaluating

as permeate

rate

was found

entrapped

o f glucose

times

lactose

Tester.

lower

value

cells

by

o f

was

production,defi

glucose

permeate

concentration,

concen

and transmem

pressures. t o product

cytoplasmatic

concentration

β-galactosidase

Michaelis-Menten

parent

Michaelis

as

t h ep r e s s u r e

70

°C r a n g e s

In

flow

a t different

Referring

atm,

o f membrane

t h er a t e

poly-

by a s t r e s s -

Universal

o f about

cell-free

ent

estimated

o u ton a Instrom modulus

catalytic

brane

were

by c e l l - f r e e

Ε wet, and t h e ultima

o f t h eYoung

the

tration

modulus,

were p r e l i m i n a r l y

value

The ned

exhibited

o f t h emembranes

analysis

assessed


properties

and compared

sulphone te

65


kinetic

i n t h epermeate

i ncells

behaviour,Figure

constant

increases

from

increases.

Maximum

glucose

from

2 0 . 4 t o 34 m o l e s / h r ,

stream,

e x h i b i t s an

appar

8. T h e

ap

3 . 7 t o 1 9 . 2 mM production

a t

a t 0.04 a n d 0.055

respectively. terms

ganisms

o f s t a b i l i t y shows

preciable months,

loss

t h e β-galactosidase

t o be s t a b l e o f activity,

a t least,

during

up t o o n e y e a r when

stored,

continuous Τ = 78'C

o f the microor without

any

ap

a n d up t o t h r e e

operation.

Qox = 3 . 8 U / h

50 mM acetate buffer pH 5

0.35 .

t

F i g u r e 8. G l u c o s e p r o d u c t i o n r a t e v s . l a c t o s e f e e d c o n c e n t r a t i o n . Τ = 70 *C. R e p r o d u c e d w i t h p e r m i s s i o n f r o m R e f . 1 3 .



66 From

the point

mances most

comparable

resting the

o f view

o f capillary

t o those

conversions

remarkable

tosidase

o f mechanical

membranes

i nl a c t o s e

o f immobilized

encourage

further

studies

membrane

reactor

oriented

an

enzyme

al

applications.

with

o f bacteria-free

observed

s t a b i l i t y

properties,

charged

perfor

cells ones.

area l The i n t e

hydrolysis and bacterial

B-galac-

f o rt h e d e v e l o p m e n t o f t o possible

industri


Literature Cited 1. 2. 3.

"Impact of Applied Genetics",OTA NR-132 April 1981. Michaels,A.S. Desalination 1980,35,329. Michaels,A.S.; Matson.S.L. Desalination 1985,53,231258. 4. Drioli,E.; Serafin,G.; Rigoli.A. presented at First Engineering Foundation Conference " Advances in Fer mentation Recovery Process Tech." Banff June 6-12 (1981) unpublished results. 5. Leuchtenberger,W.;Karrenbauer,M.;Picker,U. "Scale up of an Enzyme Membrane Reactor Process for the Manu facture of L-enantiomeric Compounds" Report from De gussa AG, D-6450 Hanau I, FDR. 6. Molinari,R.; Drioli,Ε. Proc.Nat.Congr.Ind.Chem.Div. Sci., Siena 10-12 June 1985 7. Drioli,E.;Scardi,V. J.Mem.Sci. 1976,1,237-248. 8. Rossi,M.;Nucci,R.;Raia,C.A.;Molinari,R.;Drioli,Ε. J.of Mol.Cat. 1978,4,233. 9. De Rosa,M.;Gambacorta,A.;Esposito,Ε.; Drioli,Ε.;Gaeta, S. Biochimie 1980,62, 517 10. Drioli,Ε.;Iorio,G.; De Rosa,M.; Gambacorta,A.;Nicola us,B. J.Mem.Sci. 1982,11,365-370 11. Drioli,Ε.;Iorio,G.;Santoro,R.; De Rosa,M.;Gambacorta, A.;Nicolaus,R. J . Mol.Cat.1982,14,247. 12. Catapano,G.;Iorio,G.;Drioli,Ε.;Filosa,M. "Capillary Membrane Bioreactors with Entrapped Whole Cells : a Theoretical Model" submitted for publication. 13. Drioli,Ε.;Iorio,G.; Catapano,G.; De Rosa,M.; Gamba corta, Α. J.οf Mem.Sci. in press. 14.

Drioli, E.; et al. La Chimica Ε L'Industria 1985, (67)11, 617-622.

Received March 26, 1986


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