Download Size Distribution of Plant Cell Aggregates in

The Chenrical Engineering
Journal,
35 (1987)
B9
B9 - B14
Size Distribution of Plant Cell Aggregates in Batch Culture
F. MAVITUNA
and J. M. PARK
Chemical Engineering
(Received
August
Department,
25,1985;
UMIST, Sackville
in final form
October
M60 IQD (U.K.)
17,1986)-
ABSTRACT
Plant cells in suspension cultures tend to
form aggregates. This can affect the physiology of the cells and hence the production
of
valuable secondary metabolites. Aggregation
is also important from a process engineering
point of view. In this work, the change in the
size distribution of plant cell aggregates in a
batch culture is studied. The growth of suspension cultures of Capsicum frutescens was
found to follow Monod kinetics with a maximum specific growth rate of 0.3125 day-’
and a saturation coefficient of 16.67gglucose
1-l. It was found that the size distribution
became Gaussian after the exponential growth
phase. Using a probability plot, the median
size of cell aggregates and the standard
deviation of the size distribution were determined to be 2.29 mm and 1.21 mm respectively.
1. INTRODUCTION
The size of a single plant cell can vary from
20 pm to 150 E.cm.In suspension cultures,
they mainly exist in aggregates of 2 to 300
cells. After cell division in the early exponential phase of growth, daughter cells do
not usually separate. Also, plant cells tend to
adhere to one other because of their sticky
surfaces. This adhesion is especially strong in
the late exponential growth phase due to
increased excretion of polysaccharides by the
plant cells [ 11.
Since there is considerable interest in the
commercial production of fine chemicals by
plant cell cultures, the aggregation characteristics of plant cells have some significance
from the process engineering point of view.
Although plant cells have high tensile
0300-9467/87/$3.50
Street, P.O. Box 88, Manchester
strength, they have low resistance to shear
[l]. Therefore, in reactors containing plant
cells gentle agitation conditions are required.
This, in turn, results in problems with mixing
because the aggregates tend to sediment or
stick to the reactor surfaces forming extensive
wall growth or crusts. Plant cell aggregates
can also create rheological problems, and they
can block the openings and pipes in a reactor.
Despite these problems, aggregation may be
desirable because of the positive effects on
the cell physiology and biochemistry which
may increase the yields of commercially important fine chemicals [2]. For this reason,
there is growing interest in plant cell immobilization using porous reticulate foam
matrices (biomass support particles [ 31) in
order to create a milieu in which plant cells
can exist in the form of large aggregates but
avoiding some of the problems mentioned
above [4]. In the physical entrapment of
plant cell aggregates in the pores of foam
matrices the initial aggregate size distribution
in relation to the pore size distribution is the
most important factor affecting the efficiency
of immobilization [ 51.
Therefore, it is important to understand
the factors affecting plant cell aggregation.
In this work the changes in aggregate size
distribution of suspension cultures of
C. frutescens have been followed during batch
growth.
2. EXPERIMENTAL
METHODS
AND MATERIALS
The plant cells used in this study are
cultures of C. frutescens (chilli pepper), which
produce the pungent flavour capsaicin.
Suspension cultures initiated from callus
were grown and subcultured in Erlenmeyer
flasks (500 ml) containing 100 ml of modified
0
Elsevier Sequoia/Printed
in The Netherlands
BlO
Schenk and Hildebrandt (SH) medium and
agitated on a New Brunswick psychrotherm
shaker at 150 rev min- ’ and 25 “C.
The ingredients which differed from those
in the original SH medium [6] were as follows: glucose (30 g l-l), KN03 (2.5 g l-l),
KH,PO,, (0.35 g 1-l) and 2,4-dichlorophenoxyacetic acid (0.75 X lop3 g 1-l). The
medium was made up in double distilled
water and the pH was adjusted to 5.8 with
0.1 M KOH and sterilized by autoclaving at
121 “C and 15 lbf in-’ for 20 min. Cells were
subcultured every 20 - 25 days using approximately 20% inoculum (vol.%) from a dense,
early stationary phase culture.
The size of the cell aggregates was measured by a wet sieving technique using stainless steel wire sieves, the aperture sizes of
which were 0.5, 1.0, 1.5, 2.0, 3.0, 4.0 and
5.0 mm. Cells and cell aggregates retained on
each sieve were subjected to dry weight
analysis in order to obtain the aggregate size
distribution in terms of the dry weight
percentages. The fraction of the suspension
culture which passed through the finest sieve
(0.5 mm) was collected on filter paper
(Whatman No. 1).
The dry weights of cell aggregates were
determined by filtering the aggregates through
preweighed filter paper under a vacuum,
washing with distilled water, and drying the
filter paper and the aggregates at 90 “C!for
24 h.
The amount of glucose in the liquid
medium was measured using a glucose
analyzer (Beckman Instrument Co., U.S.A.).
-.’
15 _
1.
t-
.
0
10 -
5-
-3
- 21
- x
~
_I, \,_ _
o0
10
-0
20
Age
of Culture Idayl
Fig. 1. Time course of cell growth and glucose consumption.
30
20
z
8
T-
10
3. RESULTS AND DISCUSSIONS
0
The typical batch growth curve of
C. frutescens is given in Fig. 1 together with
the time course of glucose consumption. As
seen in Fig. 1, the cell growth curve is almost
symmetrical with the curve of glucose consumption. From the double reciprocal plot of
specific growth rate against glucose concentration, the batch growth of the suspension
culture was found to follow Monod kinetics
[7] as shown in Fig. 2. From this
Lineweaver-Burk plot, the maximum specific
growth rate pm and the saturation constant k,
were obtained as 0.3125 day-’ and 16.67
g 1-l, respectively. The biomass yield Y, per
I
I
I
I
I
0.1
0.2
0.3
0.L
0.5
115 II/g1
Fig. 2!. Lineweaver-Burk plot.
unit of glucose was also determined graphically as 0.6 (g-dry weight)(g-glucose))‘.
The size distributions of the cell aggregates
during batch culture are given in Figs. 3 - 9 as
frequency polygons on semi-logarithmic
scales.
In Figs. 7 - 9 the size distribution curves of
the stationary phase culture resemble the
Gaussian distribution. The equation for the
Gaussian (normal probability) curve is given
as
Bll
50
& 50
"~
40
o
~ 3c
~3o
{2o
0.5
1.0
S*ze (mm)
2'0
10
3"0 4-0 5-0
lo
0.5
Fig. 3. Size d i s t r i b u t i o n o f t h e i n o c u l u m .
1.0
Size (mr'n)
2.0
3"0
5.0
Fig. 7. Size d i s t r i b u t i o n o f s u s p e n s i o n c u l t u r e o n t h e
1 8 t h day.
§ so
,o
~5o!
3O
§ .c
30
~2o
{20
o
0.5
1.0
2"0
3.0
5.0
10
Size (ram]
lO
o
Fig. 4. Size d i s t r i b u t i o n o f s u s p e n s i o n c u l t u r e o n t h e
5 t h day.
0"5
1.0
Size [rnm}
2.0
3.0
5'0
Fig. 8. Size d i s t r i b u t i o n o f s u s p e n s i o n c u l t u r e o n t h e
21st day.
~so
~o
~ 30
so
~ 20!
~o
0
0
0.5
1-0
Size [ rnrn)
2"0 3.0
10
5-0
Fig. 5. Size d i s t r i b u t i o n o f s u s p e n s i o n c u l t u r e o n t h e
8 t h day.
~_~2o
~o
00.1
0.5
1-0
Size (turn}
2.0
3"0
5"0
10
Fig. 9. Size d i s t r i b u t i o n of s u s p e n s i o n c u l t u r e o n t h e
2 5 t h day.
~3o
2o
~' ~o
0.5
1.0
Size (mini
2.0
3.0
10
5-0
Fig. 6. Size d i s t r i b u t i o n o f s u s p e n s i o n c u l t u r e o n t h e
1 3 t h day.
f(a)-
o(2~)1/2
exp
202
(1)
If t h e area u n d e r t h e n o r m a l c u r v e b e t w e e n
sieve a p e r t u r e size a = 0 a n d a = oo is t a k e n as
u n i t y , t h e a r e a b e t w e e n a = 0 a n d a = 5 + a,
w h e r e d is t h e m e d i a n size 0 . 8 4 1 3 . This
v a l u e is o b t a i n e d f r o m t a b l e s o f t h e n o r m a l
probability function. Therefore, the area
b e t w e e n a = ~ + o a n d a = ~ is 1 - - 0 . 8 4 1 3 =
0 . 1 5 8 7 . In o r d e r t o f i n d t h e v a l u e s o f 5 a n d
a, an a r i t h m e t i c p r o b a b i l i t y p l o t has t o be
p e r f o r m e d . In t h i s p l o t , o n e axis is l i n e a r a n d
is u s e d f o r t h e sieve a p e r t u r e size; t h e o t h e r
axis is m a r k e d o f f a c c o r d i n g t o t h e p r o b a b i l i t y i n t e g r a l a n d it is u s e d f o r t h e c u m u l a t i v e
undersize or oversize data.
T h e v a l u e o f t h e s t a n d a r d d e v i a t i o n o can
be o b t a i n e d f r o m t h e a r i t h m e t i c p r o b a b i l i t y
p l o t using t h e v a l u e o f a at 8 4 . 1 3 % a n d sub-
B12
tracting the value of 6. Alternatively, the
value of a at 15.87% can be subtracted from
a, i.e.
-ti=ti-ai6S
0 =a84%
(2)
These two values of u may not coincide
precisely, so a mean value is taken as
a84% -16%
o=
(3)
2
The coefficient of variation (CV), as a percentage, is given by
CV=
1000
_
a
1000
= a5m
(4)
or
2QW0
(5)
In order to check whether the size distribution of cell aggregates of C. frutescens is
Gaussian or not, a method proposed by
Powers [8] was used. This method provides
a simple means of recording the size distribution in terms of two parameters.only, the
mean particle size and a statistical quantity,
the coefficient of variation CV, expressed as a
percentage.
If a size distribution is Gaussian the following two requirements should be satisfied:
(a) in the arithmetic probability plot mentioned above the data between about 10% and
90% probability should lie on a straight line
and (b) the median size E corresponding to
50% probability should coincide with the size
at the peak of the distribution curve.
As can be seen in Fig. 10, these two requirements for a Gaussian distribution are
satisfied, which implies that the size distribution of the stationary phase culture is
Gaussian. From Fig. 10
3.5 - 1.08
(T= -____
= 1.21 mm
2
(6)
and
cv
I.
OD 0.5
Fig. 10.
analysis.
1.0
1.5
. I . . . . .
2.0 2.5 3.0 3.5 4.0 4.5
Sieve aperture
(nun)
Probability
plot
5.0
for cell aggregate
size
course of a batch culture (time used as a
parameter). In this figure, the highest peaks
during the stationary phase of growth (after
the 18th day) correspond to the median size
6 (2.29 mm). Multipeaks in this plot do not
smooth out with time but move to larger
sizes.
The cell mass in each size range at various
times can be seen in Fig. 12. Up to the
stationary phase, the change in cell mass of a
certain size range is closely related to the
changes in the adjacent size ranges. For
example, the agglomeration of cell aggregates
of 1.5 - 2.0 mm range, the cell mass of which
is reduced despite the exponential growth
phase, could be deduced from the sharp
increase in cell mass in the 2.0 - 3.0 mm
range.
4. CONCLUSIONS
=
3w.21)
2.29
= 52.84%
(7)
Figure 11 shows the size distribution, in terms
of dry weight concentration, during the time
Because of the biological nature of cell
aggregation it is difficult to devise a simple
theory or indeed to use an existing theory,
e.g. one based upon crystallization, to predict
B13
6-
i
.F
i
f
3
x
b
1
2
3
Meon
Fig. 11.
tration
day; X,
0, 25th
L
5
10
Size distribution
in terms of biomass concenwith time as a parameter:
a, inoculum;
n, 5th
8th day; 0,13th
day; v, 13th day; 0, 21st day;
day.
the size distribution
of plant cell cultures.
Plant cells are cultured normally in batch
systems, which makes analysis difficult and
aggregate size distribution
can be affected
by biomass growth, agglomeration
and
break-up. Growth depends upon the concentration of nutrients and ceases when a limiting
nutrient is exhausted. Also, if the cell physiology is affected by aggregate size then aggregates with various sizes will grow at different
rates, which implies that the growth distribution has to be taken into account.
The stickiness of the cell surface, excretion
of polysaccharides
and high cell concentrations towards the end of batch growth will
promote the agglomeration of cell aggregates.
Depending on the shear rates in the culture,
the aggregates may be broken up and even
lose their viability due to their low tolerance
of shear.
20
10
Size imml
Age
of
Culture Iday)
Fig. 12. The change of cell mass in each size range
with time: 0, 0 - 0.5 mm; 0, 0.5 - 1.0 mm; A, 1.0
1.5 mm; 0, 1.5 - 2.0 mm; A, 2.0 - 3.0 mm; ., 3.0
4.0 mm;O, 4.0 - 5.0 mm;X,
5.0 - 10.0 mm.
-
Following exponential
growth, lysis and
necrosis may affect the cell mass during
batch culture. If large aggregates become
hollow due to autolysis, then the cell dry
weight may decrease even though the size of
the aggregates remains the same.
REFERENCES
M. W. Fowler, in S. H. Mantel1 and H. Smith
(eds.), Plant Biotechnology,
Cambridge University
Press, Cambridge, 1984, p. 3.
K. Lindsey and M. M. Yeoman, in S. H. Mantel1
and H. Smith (eds.), Plant Biotechnology,
1984,
p. 39.
B. Atkinson, G. M. Black, P. J. S. Lewis and A.
Pinches, Biotechnol
Bioeng., 21 (1979) 143.
K. Lindsey, M. M. Yeoman,
G. M. Black and
F. Mavituna, FEBS Lett., 155 (1983)
143.
J. M. Park and F. Mavituna, in Proc. I. Chem. E.
Symp. Process Engineering Aspects of Zmmobilised Cell Systems, UMIST, I. Chem. E.
Publ.,
Rugby,
1986,
pp. 295
- 303.
B14
6 R. U. Schenk and A. C. Hildebrant,
Can. J. Bot.,
50 (1972) 199.
7 J. Monod, Recherches sur la Croissance des
Cultures Bactbriennes, Hermann, Paris, 2nd edn.,
1942.
8 H. E. C., Powers, Znt. Sug. J., 50 (1948) 149.
APPENDIX
a
a
A: NOMENCLATURE
sieve aperture size (mm)
median size of cell aggregates (mm)
cv
k,
S
coefficient of variation (5%)
saturation constant for glucose (g 1-l)
glucose concentration
(g 1-l)
Greek symbols
specific growth rate (day-‘)
maximum
specific growth rate (day-‘)
Pn
0
standard deviation (mm)
c1