Biotechnology

EFFECT

AERATION

OF

CULTURES

Nigel

J.

The

ON L A R G E

OF P L A N T

Smart

Wolfson

Letters Vol.3 No.4

University,

(1981)

SCALE

CELLS

and Michael

Institute

171-176

W.

Fowler*

of B i o t e c h n o l o g y

Sheffield

S10

2TN

U.K.

Summary

The

large

sents

scale

problems

culture

of a d i f f e r e n t

micro-organisms.
the m o d e

growth.

problem

through

the

One

of c u l t u r e

on c e l l

both

of p l a n t

Data
the

initial

of C a t h a r a n t h u s

nature

particular

aeration

cells

and

growth

to t h o s e

problem

which

of v a r y i n g

rate

seen w i t h
with

of a e r a t i o n

illustrates

rates

and m a x i m u m

pre-

is c o n c e r n e d

the e f f e c t s

is p r e s e n t e d

effects

in b i o r e a c t o r s

this

of a e r a t i o n

growth

rate

on

of c e l l s

roseus.

INTRODUCTION

A number
plant

of

cells

factors
are

in m a r k e d

micro-organisms
require

(3).

the h i g h

normally

organisms.

Indeed

aeration

viability,
culture
high

activity
plant
time

contrast

plant

of o x y g e n a t i o n

associated
studies

with

in this

to e i t h e r
oxygen

of DO 2 t h e r e b y

is due

(typically

(7)).

leading
The m u c h

171

microthat

growth

resulting

to i n h i b i t i o n

compared

do n o t

and

of CO 2 f r o m the

toxicity

lower

with

high

suggest

to c e l l

removal

to a low m e t a b o l i c

30 - 70 hr)

cultures

respiring

laboratory

due

of

encountered

consequent

rapidly

possibly

culture

cell

and

be d e t r i m e n t a l

or a d i r e c t

scale

to t h o s e

may

(see a l s o

cells

large

rates

broth,

levels

the

In p a r t i c u l a r

rates

KLa values

high

concerning

oxygen

activity

from

of m e t a b o l i c
demand

of

and doubling

with micro-organisms.

Because of other properties

tolerance)

'air lift'

in this and other

biomass

of plant cells

(eg. low shear

systems have come under close attention

laboratories

(8) as a system for mixing the

and nutrient broth rather than a conventional

driven system.

turbine

In consequence the possible conflict between

the needs of achieving good m i x i n g while at the same time

exercising control over aeration,
careful

KLa and DO 2 values requires

study.

This paper details

bioreactors

information gained from work with five

exemplifying

in comparatively

the close interaction between changes

low aeration rates and cell growth.

that the cell cultures are not photosynthetic,

Note

excluding

problems of 02 exchange due to photosynthesis.

MATERIALS AND METHODS

Cell cultures
established

(line no. CIIC)

roseus were

from leaf explants on Gamborg's B5 m e d i u m

with sucrose
(i.O mg 1 -I)

(20 g I-i)~2,
and kinetin

sterilisation
was 5.8;

of Catharanthus

4, - d i c h l o r o p h e n o x y a c e t i c

(O.i mg i-i).

(working volume

acid

M e d i u m pH prior to

(by autoclaving at 12C 20 p.s.i,

cells were cultivated

(4)

for 15 min)

in glass bioreactors

(9)

4 i) at 25C.

Aeration was through a No. 3

-i
porosity glass sinter at rates of 1.0, 1.5 and 2.0 1 min
in the absence of antifoam.

Cultures were initiated with a

10% cell inoculum from a 2 1 flask containing 1.2 1 of culture.

The latter had been grown for 7 days on an orbital shaker
(150 r.p.m.)
by Gamborg

at 25C.

The nutrient broth was B5 as described

(4) with supplements

and consequent

cell deposition

as above.

To reduce foaming

around the top of the vessel,

antifoam was used on certain experimental

runs.

The antifoam,

silicone based

(Dow Corning antifoam RD emulsion),

up in medium.

Biomass was measured daily as wet and dry

weight/1-1

(9).

Residual

sucrose and fructose in the spent

m e d i u m were estimated with the anthrone

method

(2).

was made

(total carbohydrate)

Residual broth glucose was estimated with the

Boehringer glucose oxidase/peroxidase

test kit.

were measured with the sulphite oxidation method

172

KLa values
(5).

RESULTS

Increasing
of g r o w t h

aeration
of p l a n t

Fig.

i.

rate
cell

AND

has

DISCUSSION

two o b v i o u s

cultures

(Fig.

effects

on the m o d e

i).

B i o m a s s p l o t s for g r o w t h of c e l l s of
C a t h a r a n t h u s r o s e u s at v a r y i n g a e r a t i o n

rates

C
12

,,

KEY:

= 2.O

First
with

there

i min

is an

a subsequent

-i

Time

(days)

.,

= 1.5

increase

in the

suppression

i0

1 min

length

of the

12

-i

14

.,

of

= 1.0

lag p h a s e

initial

1 min

-i

together

growth

rate.
-i
1 min
to

Secondly,

i n c r e a s e in the a e r a t i o n r a t e f r o m 1.0
-i
1.5 1 m i n
is a c c o m p a n i e d by an i n c r e a s e in the m a x i m u m
-i
g r o w t h rate.
W i t h f u r t h e r i n c r e a s e to 2.0 1 m i n
and beyond,
the m a x i m u m
point

growth

to a p o s i t i v e

growth

of a e r a t i o n

other

culture
Related

cultures
by

have

initial

over

KLa

of p l a n t

values.

systems

shown

a restricted

by K a t o

that
that

range

declines.
between
cell

Similar

and h a v e

studies

and

achieved

interaction

characteristics

range

(i).

rate

the

cultures

(6) w i t h

biomass

the r e l a t i o n s h i p
(KLa v a l u e

173

of

observations
rate

over

have

summarised

et al

final

aeration

effects

been

Both

a restricted

been

noted

by A i b a
tobacco

yield

in

et al.
cell

is a f f e c t e d

is a l i n e a r

f r o m ca.

and

one

5 - 1 0 hr-l).

At a KLa v a l u e of 5.3 hr - K a t o et al f o u n d t h a t g r o w t h
was h a r d l y d e t e c t a b l e

indicating

present work we have used

higher

initial

(6) in an a t t e m p to

i n f o r m a t i o n on the e f f e c t of h i g h

KLa v a l u e s ,

and s e c o n d l y to a s c e r t a i n

the

l i m i t of i n i t i a l KLa v a l u e on final b i o m a s s yield.

A t the l e v e l s of K L a u s e d in o u r s t u d y
data

In the

i n i t i a l KLa v a l u e s m u c h

t h a n t h o s e of K a t o et al

first gain further

upper

limitation.

indicate

( 20 hr -I)

(Table i) t h a t we are a p p r o a c h i n g

at w h i c h the i n i t i a l K L a c e a s e s
on g r o w t h rate,

and indeed,

a point

to h a v e a p o s i t i v e

at l e v e l s

m a y b e g i n to h a v e a n e g a t i v e

the

effect

a b o v e a b o u t 25 hr -I

effect.

TABLE I
GROWTH PARAMETERS FOR CELL CULTURES OF CATHARANTHUS ROSEUS

BIOMASS YIELD

SUCROSE
CONVERSION

AERATION
RATE

GROWTH
RATE

K a
L

gl-1/ days

1 min -1

d -I

hr -!

12.4/8
i1.4/9
9.5/9
9.5/11
7.8/7

56
51
42
42
33

1.O
1.5
1.5
2.0
2.0

0.34
O.41
0.33
0.33
0.38

20.5
25.2
5.1
27.3
6.4

+
+

A = antifoam (+, with) (-, without)

It is also a p p a r e n t
KLa v a l u e s

f r o m the d a t a in T a b l e

at a r o u n d

20 hr -I r e s u l t s

y i e l d and less e f f i c i e n t c o n v e r s i o n

At h i g h a e r a t i o n r a t e s

1 that

increasing

in a r e d u c e d b i o m a s s
of s u c r o s e

into b i o m a s s .

f o a m i n g m a y b e g i n to p r e s e n t p r o b l e m s

so e x p e r i m e n t s w e r e also c a r r i e d out in the p r e s e n c e of a

silicon based antifoam
as a n t i c i p a t e d ,
coefficient,

(Fig. 2).

A d d i t i o n of a n t i f o a m r e s u l t e d ,

in a m a s s i v e r e d u c t i o n

of the m a s s

transfer

b o t h at 1.5 1 m i n -I and 2.0 1 m i n -I a e r a t i o n .

b o t h cases t h e r e was a m a r k e d r e d u c t i o n

in b i o m a s s

the e f f e c t on g r o w t h rate was m i n i m a l .

The less e f f i c i e n t

conversion
resulting

of b i o m a s s

yield while

is p r o b a b l y d u e to p e r m e a b i l i t y b a r r i e r s

f r o m a n t i f o a m action.

by a n t i f o a m ,

In

the r e d u c t i o n

t o g e t h e r w i t h the r e d u c t i o n

The l o w e r i n g

of the KLa v a l u e

in c a r b o n c o n v e r s i o n

into b i o m a s s ,

in m a x i m u m b i o m a s s y i e l d s ,
174

reinforces the view of Kato et al

of low KLa

(6) regarding the limitation

(ie. 5 hr -I) values on biomass yield and carbon

conversion in plant cell cultures.

Fig. 2.

Biomass p!qts for growth of cells of

Catharanthus roseus in the presence of antifoam

12
I

4
n

0
KEY:

= 2.O 1 min

4
-1

6
8
i0
Time (days)
-i
m = 1.5 1 rain

12

It is possible that the lowered growth rates and biomass

yields observed at high aeration rates may result from a
'stripping off' of key volatiles

(eg. C02).

As a preliminary

experiment to gain further information on these possibilities

cultures were run at KLa values similar to those quoted above,
-i
but with a CO 2 'bleed' of 50 ml min
Initial results show
that the addition of such a CO 2 bleed significantly enhances
biomass yield when compared with a control vessel.

Detailed

investigations of this phenomenon are now in progress.

The

narrow range of KLa values over which it appears possible to

operate plant cell bioreactors, without either causing oxygen
limitation or inhibition or CO 2 stripping,

is an important

constraint in the use of air driven reactors for mass cultivation of plant cells.

Against this must be assessed their

obvious low shear characteristics

175

and general physical attributes.

The need to maintain a culture within a narrow range of

KLa values has to be carefully weighed against the
requirements for good mixing, and homogeneous culture
operation.

This is an aspect of mass growth of plant cells

which has perhaps not previously been fully appreciated,

and in view of its obvious importance is one which requires
further detailed study.

ACKNOWLEDGEMENTS

We thank Mr. I. Fletcher for technical support and the

Wolfson Foundation for financial support.

REFERENCES

I.

Aiba, S., Humphrey, A. E. and Millis, N.

Biochemical Engineering.

Academic Press.

(1965)
New York.

Cooper, C. M., Fernstrom, G. A. and Miller, S. A.

(1944)

Ind. Eng. Chem 36. 504 - 506.

.

Fowler, M. W.

(1981) Progress in Industrial Microbiology

17, In press.
.

Gamborg, I., Miller, H. and Ojima, Y.

(1968) Exp. Cell.

Res. 50, 151 - 158.

.

6.

Handel E. van.

Kato, A., Shimizu, Y. and Nagai, S.

Technol.

(1968) Anal. Biochem. 22, 280 - 283.

(1975) J. Ferment

53, 744 - 751.

Turner, E. R. and Quartley, C.

(1956) J. exp. Bot. 7,

362 - 371.
.

Wagner, F. and Vogelmann, H.

(1977) In: Plant Tissue Culture

and its Biotechnological Application.

and M. H. Zenk eds. pp 245 - 252.
.

W. Barz, E. Reinhard

Heidelberg: Springer-Verlag.

Wilson, S. B., King, P. and Street, H. E.

J. exp. Bot. 21, 177 - 207.

176

(1971)