Diffusion Characteristics of Substrates
in Ca-Alginate Gel Beads
Hideo Tanaka, Masatoshi Matsumura, and 1. A. Veliky*
Institute of Applied Biochemistry, The University of Tsukuba, Ibaraki, Japan
Accepted for publication July 8, 1983
The diffusion characteristics of several substrates of
varying molecular sizes into and from Ca-alginate gel
beads in well-stirred solutions were investigated. The
values of the diffusion coefficient (D)of substrates such
as glucose, L-tryptophan, and a-lactoalbumin [with mo-
lecular weight (MW) less than 2 x lo4]into and from the
gel beads agreed with those in the water system. Their
substrates could diffuse freely into and from the gel
beads without disturbance by the pores in the gel
beads. The diffusion of their substrates into and from
the gel beads was also not disturbed by increasing the
Ca-alginate concentration in the beads and the CaCI2
concentration used in the gel preparation. In the case of
higher molecular weight substances such as albumin
(MW = 6.9 x lo4),?-globulin (MW = 1.54 x lo5)and fi-
brinogen (MW = 3.41 x lo5),the diffusion behaviors of
the substrates into and from the gel beads were very dif-
ferent. No diffusion of their substrates into the gel
beads from solutions was observed, and only albumin
was partly absorbed on the surface of the gel beads.
The values of D of their substrates from the gel beads
into their solutions were smaller than their values in the
water system, but all their substrates could diffuse from
the gel beads. The diffusion of high molecular weight
substrates was limited more strongly by the increase of
Ca-alginate concentration in the gel beads than by the
increase of the CaCI2 concentration used in the gel
preparation. Using these results, the capacity of Ca-al-
ginate gel as a matrix of immobilization was discussed.
INTRODUCTlON
Natural or synthetic polymers are used as the matrix in
some of the immobilization techniques to entrap pro-
teins, enzymes, whole microbial, plant, and animal cells.
The phsicochemical characteristics of such a matrix in
gel form can have an effect on the reactions of the biologi-
cally active material entrapped in the gel. The pore size of
the gel, reflected by the viscosity of the carrier, due to the
size of the molecule, and/or its concentration can affect
the diffusion of the substrates or products and limit the
reaction rates of the entrapped cells or enzymes. Pore size
is a critical parameter in selecting a matrix for a particu-
lar process. Low-molecular-weight substrates and prod-
*Present address: Division of Biological Sciences, National Research
Council, Ottawa, Ontario, Canada.
Biotechnology and Bioengineering, Vol. XXVI, Pp. 053-058 (1984)
0 1984 John Wiley & Sons, Inc.
ucts can easily diffuse into or from a matrix with large
pores. Large pores can cause problems by allowing the
entrapped enzymes or whole cells to leak out. The major
factors in selecting the most effective system for immobi-
lization of enzymes and whole cells are the pore size de-
fined by the molecular size and the structure of the com-
pound used as the carrier for the active biological
material, as well as the size of the substrate or product
expected to diffuse into or from the matrix.
The diffusion characteristics of the synthetic polyacryl-
amide gels have been studied in detail by White et al.'-3
Proteins and enzymes as well as whole cells, can be immo-
bilized in polyacrylamide gels. The leakage of proteins
and cells is minimal because of the small pore size of the
carrier. On the other hand, the diffusion of low-molecu-
lar-weight substrates, such as KCI and urea, is affected
by the pore size, and their diffusion rates were low. While
the diffusion characteristics of the natural polysaccharide
alginic acid in its Ca-alginate gel form have not been
studied in detail, the average pore size of this gel is esti-
mated to be larger than that of the polyacrylamide gel.
The Ca-alginate gel is therefore used mainly as a carrier
for the immobilization of whole cells.
In this article, the diffusion characteristics of several
substrates of varying molecular sizes into and from the
Ca-alginate gel beads in well-stirred solutions are investi-
gated, and the capacity of the Ca-alginate gel as an im-
mobilization matrix is discussed from the point of view of
diffusion characteristics.
MATERIALS AND METHODS
Materials
Sodium alginate was BDH Chemicals Ltd. and other
materials were Sigma Chemical Co. (St. Louis, MO).
Preparation of Ca-Alginate Gel Beads
The Ca-alginate gel beads were prepared by dropping
sodium alginate (2 or 4%) solutions into calcium chloride
(5OmM or 500mM) solutions (distilled water or pH 7.0,
1M Tris buffer). The beads were cured 2 h in the calcium
CCC 0006-3592/84/010053-06$04.00
chloride solutions and washed with distilled water or the 6(1 + a ) e-D'l:t/a2
buffer. The Ca-alginate gel beads containing one of sev- cp = acLo
(1 + a )
[l +cO3

eral substrates having different molecular weights were
prepared by dropping into the calcium chloride solutions
1M Tris buffer (pH 7.0) containing sodium alginate (2 or
4%) and the substrate (0.570)into the buffer solution
containing calcium chloride and the substrate to prevent
the diffusion of the substrate from the alginate beads.
The beads were cured 2 h in the buffer solution. The con-
centration was equal to that of the solution in the beads
(0.570).All beads used had diameters of 3.3 f 0.2 mm
(average of 20 beads).
Apparatus
A schematic diagram of the reactor is shown in Figure
1. The reactor was a cylindrical grass vessel (40 mm
diameter) with two baffle plates (4 mm width and 15 mm
height). The working volume of liquid was 50 mL. The
liquid was agitated by a Teflon bar (26 mm) on a mag-
netic stirrer. The stirrer speed was 625 rpm. The temper-
ature was kept constant at 30°C.
n=l 9 + 9 a + qia2
a sin(q,r/a)
r sin qn
where a is the diameter of a bead, r is the distance from
the center of a bead, t is time, a is defined by ( V / n )
(47ra3/3), V is the volume of the solution excluding the
space occupied by beads, n is the number of beads, and
the qn terms are the nonzero positive roots of
3q n
tang, =
3 + aqn
If the liquid film resistance around the beads can be ig-
nored, the concentration of substrate just within the sur-
face of a bead, (C,),,,, is the same as that in the solution
(C,)so that, from eq. (l), the substrate concentration in
the solution can be expressed as follows:

Measurements of Molecular Diffusion or

Coefficients of Substrates into and out of
Ca-Alginate Beads CL -
a O3 6(1 + a ) e - D 4 ? t / a 2

The molecular diffusion coefficients (D)of the sub- CLO (1 + a) [ 1 + c n=l 9 + 9a + qia2
strates in the Ca-alginate gel beads were obtained from Similarly, when spherical beads in which the initial con-
the concentration change of the substrates in the well- centration is uniform and equal to C, are suspended in a
stirred solution suspending the beads. It has been postu- substrate-free solution, the substrate is diffused from the
lated that the structure inside the beads was homogene- beads, and the increment of substrate concentration in
ous. When spherical beads which are free of substrate are the solution is given by
suspended in a solution with an initial substrate concen-
tration of CLo,the substrate in the solution is diffused
into the beads, and the substrate concentration, Cp,
within the beads is given by the following e q ~ a t i o n , ~
or

5 6

where CLEis the equilibrium substrate concentration in

the solution and the relationship between CLE and C, is
as follows:

The values for a , n , V, a, CLo,Cpo,CLE,and t were

known, and values for C, and D were calculated on a mi-
crocomputer (Epson Model HC-20).

I
I
0 3 1
I
Analysis
Glucose concentrations were determined by the enzy-
matic-colorimetric method.s The L-tryptophan, cr-lac-
Figure 1. Schematic diagram of the reactor system: (1) glass vessel,
toalbumin, albumin, y-globulin, and fibrinogen concen-
(2) baffle plate, (3) magnetic stirrer, (4) Teflon bar, ( 5 ) thermometer,
( 6 ) pH meter, and (7) water bath.
trations were determined by extinction at 280 nm.

54 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 26, JANUARY 1984

RESULTS

Diffusion of Substrates into Ca-Alginate Gel

Beads from Well-Stirred Solutions
Five-hundred 2% Ca-alginate gel beads prepared in
"
0

5OmM CaClz solution were added to a well-stirred 0.5%

glucose (MW = 180) solution in a glass vessel, and the
glucose concentration in the solution was measured over
a period of time and is shown in Figure 2. Keeping in
mind that the liquid volume increased when the beads
were added to the solution, due to with the volume of the
liquid on the surface of the beads, the values were plot-
ted. The glucose concentrations calculated from eq. (2),
using D of glucose in the pure water system (4.08 X lop4
cm2/min at 30°C)6is shown also in Figure 2. The glucose
concentration decreased rapidly just after the addition of
the beads, and gradually slowed with time, leveling off
after about 30 min. There was good agreement between
the calculated and experimental values.
Next, the glucose diffusion into Ca-alginate gel beads
from solutions of different initial glucose concentration
(0.5, 5, 10, 20, and 30%) was measured and is shown in
Figure 3. All conditions except for initial concentration
were the same as those in experiment using 0.5% glu-
cose. The concentrations calculated from eq. (2) using D 0 10 20 30 40 50
of glucose in the pure water system are shown also in Fig- T i m e (min)
ure 3 . The experimental values for the initial 30% glu-
Figure 3. Diffusion of glucose from solution into Ca-alginate gel
cose solution are scattered in Figure 3, owing to the diffi- beads: (0, 0 , A , A, 0)experimental and (-) calculated values.
culty of mixing the highly viscous solution. However, all
the solutions with the different glucose concentrations
behaved like the solution with the initial glucose concen- bumin (MW = 6.9 X lo4)from their solutions into Ca-
tration of 0.570, and all reached their equilibrium states alginate gel beads were measured in much the same way
after ca. 30 min. All results were in good agreement with as that of glucose; these diffusions are shown in Figure 4,
the calculated values. Therefore, it was found that glu- although CL/CL,rather than CL is used as the ordinate.
cose can diffuse as freely into 2% Ca-alginate gel beads as The initial concentration of the substrates was 0.5% in
in water. 1M Tris buffer (pH 7.0), and the concentration of Ca-
The diffusions of L-tryptophan (MW = 204), a-lac- alginate in the beads, the concentration of the CaC12solu-
toalbumin (MW = 1.56 X lo4), and bovine serum al-

1.00 -

4.0c
v f i ,'
nl

I , , ,
-
2 0 20 40 60
CI) Time ( min )

Figure 2. Diffusion of glucose from solution into Ca-alginate gel Figure 4. Substrate diffusion from solutions into Ca-alginate gel
beads: (0)experimental and (-) calculated values. beads: ( 0 , A, m) experimental and (-) calculated values.

TANAKA, MATSUMURA, AND VELIKY: DIFFUSION CHARACTERISTICS I N ALGINATE GELS

55
tion used in the gel preparation, and the number of beads
added were the same as those in the glucose experiments.
Their diffusions calculated from eq. (2') with D in the
'.OOl
c
P
pure water system at 30°C (~-tryptophan~:4.02 X 10-4;
0.95-
a-la~toalbumin~ :6.10 X and albumin8 :4.27 X
lop5 cm2/min) are shown in Figure 4. The results show -
that there was diffusion of L-tryptophan and a-lac-
-
9
toalbumin from the solutions into Ca-alginate gel beads, 0

'0= O . O O -
and the concentrations of the solutions reached their
equilibrium states after ca. 30 and 120 min, respectively.
The concentration of albumin, however, having a higher
0.85-
molecular weight, decreased for only the first 10 min and
then remained constant. Thus, it can be seen that al-
bumin could not diffuse freely into 2% Ca-alginate gel
beads from the solution.
The effects of the Ca-alginate concentration in the Figure 5. The a-lactoalbumin diffusion into Ca-alginate gel beads
beads and the CaClz concentration used in the gel prepa- made under different conditions: (0) experimental values for beads
composed of 2% Ca-alginate and prepared in 5 O O m M CaCI, solution;
ration on substrate diffusion from solutions into the ( 0 ) experimental values for beads composed of 4% Ca-alginate and
beads were investigated next. The diffusion of a-lac- prepared in 5OmM CaCI2 solution; and (-) calculated values.
toalbumin from the solutions into beads composed of 4%
Ca-alginate and prepared in 5OmM CaC12 solution and
into beads composed of 2% Ca-alginate and prepared in
5OOmM CaC12 solution was measured. The results are
shown in Figure 5. The diffusion of a-lactoalbumin cal-
culated from eq. (2') using D of a-lactoalbumin in the 1.0 -
pure water system at 30°C is shown in Figure 5. As can be
seen from the figure, there was close agreement between 0.8-
the experimental and calculated values. T.herefore, nei-
ther the Ca-alginate concentration in the beads nor the
-
CaC12 concentration used in the gel preparation had any
- 0.8-
w
J
effect on the diffusion of a-lactoalbumin into the beads, 0

and a-lactoalbumin can diffuse as freely into gel beads as

\ 0.4 -
0
in water.

Diffusion of Substrates from Ca-Alginate Gel

Beads into Well-Stirred Solutions
Time l h r l
The diffusion of glucose, a-lactoalbumin, bovine se-
rum albumin, y-globulin (MW = 1.56 X 105)and fibrin- Figure 6. Substrate diffusion into buffer solution from Ca-alginate gel
beads: ( 0 A m) experimental and (-) calculated values.
ogen (MW = 3.41 X 105) from Ca-alginate gel beads
into well-stirred solutions (water for glucose and 1M Tris
buffer, pH 7.0, for the other substrates) was measured.
The results are shown in Figures 6 and 7. The ordinate in
both figures is expressed as C,/CLE in place of C,. The
initial concentration of the substrates contained in the 1.0 -

beads was 0.5% in all experiments, and the concentra-

tion of the Ca-alginate in the beads, the concentration of 0.8 -
-
the CaC12 solution used in the gel preparation and the - 0.6 -
number of beads added were 2%, SOmM, and 500, re- u
2
u
spectively. The diffusions, calculated from eq. (3 '), using \ 0.4 -
D in the pure water system at 30°C y-globulin7:2.7 X 0

lop5 and fibrinogen9 :1.2 X loF5cm2/min) are shown in

the same figures. From both figures, it was found that all
substrates used could diffuse from Ca-alginate gel beads
into solutions. Glucose and a-lactoalbumin were in al-
most perfect agreement with the calculated values; their Figure 7. Substrate diffusion into buffer solution from Ca-alginate gel
substrates were found to diffuse freely from the beads beads: (*) experimental values for y-globulin; (V) experimental values
into solutions. However, higher molecular weight sub- for fibrinogen; and (-) calculated values.

56 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 26, JANUARY 1984

strates, such as albumin, y-globulin, and fibrinogen,
were not in agreement with the calculated values. The lag
in diffusion of the substrates was found to accelerate as
the molecular weight increased. The diffusion of fibrino-
gen was specially irregular in the beginning and the sub-
strate concentration in the solution started to increase af-
ter about one hour.
The effects of the Ca-alginate concentration in the
beads and the concentration of the CaC12 used in the gel
preparation on the substrate diffusion from the beads
into the buffer solutions were investigated. The diffusion
of albumin into the buffer solutions from the beads which
were composed of 2 or 4% Ca-alginate and prepared in
5OmM or 5OOmM CaCI2 solution was measured for 5 h.
The results are shown in Figure 8. The albumin diffusion
calculated from eq. (3’), using D of albumin in the pure
water system, is shown in Figure 8. From Figure 8, it was
apparent that the diffusion of albumin in the beads was
little affected by increasing the CaCI2concentration, but
was disturbed considerably by an increase of the Ca-algi-
nate concentration.
DISCUSSION
There have been no reports of quantitative and system-
atic studies on the diffusion of substrates in Ca-alginate
gels. A very few studieslo-l2 were slightly quantitative,
but almost all of them were studies on substrate diffusion
from the gel into the surrounding solution. The only
study on substrate diffusion into the gel from the sur-
rounding solution was an indirect one which used immo-
bilized whole cells12 and presumed the diffusion of inulin
(MW = 3000-5000) from the solution into the gel beads.
The results presented in this article provide quantita-
tive and systematic information on diffusion of several
substrates into and from Ca-alginate gel beads. The se-
- but all their substrates could diffuse from the gel beads.
1.0
- oa -
-
y 0.6-
0
>OA-
0.2 - ies on the diffusion of substrates in gel beads. In the case
I 1 I 1 1 albumin from the beads, increasing the Ca-alginate con-
1 2 3 4 5 centration in the beads has more effect than increasing
Tirnethrl
Figure 8. Albumin diffusion into buffer solution from Ca-alginate gel
beads made under different conditions: (m) experimental values for
beads composed of 2% Ca-alginate and prepared in 5OmM CaCI, solu-
tion; (0) experimental values for beads composed of 2% Ca-alginate
and prepared in 5OOmM CaCI2 solution; ( 0 )experimental values for
beads composed of 4% Ca-alginate and prepared in 5OmM CaCI, solu-
tion; and (-) calculated values.
TANAKA, MATSUMURA, AND VELIKY: DIFFUSION CHARACTERISTICS IN ALGINATE GELS
lection of the several substrates used in the diffusion ex-
periments was based on chemical, physical and practical
characteristics. All the proteins used here carry slight
negative charges or do not carry charges under the condi-
tions of pH 7. Therefore, the diffusion of the proteins
from the beads could not be affected by the electrostatic
interaction between gel support and protein charges. The
molecular size generally depends on the molecular weight
and the structure, but for convenience, only the molecu-
lar weight is used as an index of the molecular size in
these experiments.
In order to grasp the diffusion characteristics of several
substrates of increasing molecular size into and from Ca-
alginate gel beads, they were calculated from egs. (2’)
and (3’) based on the experimental data, and were com-
pared with D of their substrates in the water system (see
Table I). The D values of substrates such as glucose,
L-tryptophan, and a-lactoalbumin (MW < 2 X lo4)into
and from Ca-alginate gel beads were almost the same.
They were also the same as the D values of the substrates
in the water system. These results suggest that these sub-
strates can diffuse freely into and from Ca-alginate gel
beads, having homogeneous structure, with no distur-
bance from the pore size of the beads. Nor the diffusion
of these substrates is disturbed by increasing the concen-
tration of the Ca-alginate in the beads or the CaCI2 used
in the gel preparation which are the source of the dense
structure and small pore size in the beads. In the case of
higher molecular weight substrates such as albumin,
y-globulin, and fibrinogen (MW > 6.5 X lo4), the two
diffusion behaviors of substrates into and from Ca-algi-
nate gel beads were very different. Their substrates did
not diffuse into the Ca-alginate gel beads from their solu-
tions, and only albumin’s (MW = 6.9 X lo4)concentra-
tion decreased slightly in the solution. The substrate was
assumed to be partly absorbed on the surface of the gel
beads. The D values of substrates from the gel beads into
their solutions are smaller than those in the water system,
Kierstan and Bucke’O reported that glucose oxidase
(MW = 1.54 X 105)and innulinase (MW > lo5), lower
molecular weight substrates than the fibrinogen used by
us, were not diffused from Ca-alginate gel beads. The dif-
ference between their results and ours may be caused by
the differences in the structures of the substrates used.
Accordingly, not only the molecular weight but also the
structures of the substrates should be considered in stud-
of diffusion of higher molecular weight substrates such as
the CaC12concentration used in the gel preparation. The
most noteworthy fact in this experiment is that albumin
can’t diffuse into Ca-alginate, but higher molecular
weight substrates than albumin such as y-globulin and
fibrinogen, can diffuse from Ca-alginate gel beads. Such
a difference of diffusion characteristics may be caused
mainly by the difference of composition of the beads
57
 Table I. Diffusion coefficient of substrates in water and into and from Ca-alginate gel beads at 30°C.

Diffusion coefficients ( X
(cm2/min)
~

Into Ca-alginate gel beads From Ca-alginate gel beads

Substrate in watera 2% Ca-alginate 4% Ca-alginate 2% Ca-alginate 4% Ca-alginate

Glucose 4.08 4.10 - 4.10 -

L-Tryptophan 4.02 4.00 - - -
a-lactoalbumin 0.61 0.61 0.61 0.61 -
Albumin 0.42 no diffusion - 0.21 0.054
y-globulin 0.27 - - 0.12b 0.040b
Fibrinogen 0.12 - - 0.02b -

aReference used are glucose (ref. 6), L-tryptophan (ref. 6), a-lactoalbumin (ref. 7), albumin (ref. 8),
y-globulin (ref. 7), and fibrinogen (ref. 9).
bThe diffusion coefficient was calculated from experimental values 2 h after the start of the experiments.

used. The former beads are composed of only Ca-algi- covered in this article should be useful for judging the ca-
nate, but the latter beads are composed of Ca-alginate pacity of gels as an immobilization matrix and for select-
and a substrate. If the substrate has a small molecular ing the most effective system for immobilizing enzymes
weight, the structure of the Ca-alginate gel may be unaf- and whole cells.
fected by the substrate, but if the substrate has a high
molecular weight, the structure of the Ca-alginate gel
The authors wish to thank Dr. S. M. Martin and Dr. Makoto
may change. Therefore, the diffusion characteristics in Yaguchi of the National Research Council in Canada (Ottawa) for their
the latter beads may change and higher molecular weight helpful suggestions, and Mr. Yasuo Watanabe for technical assistance
substrates may be able to diffuse easily from the beads. on a microcomputer.
From this investigation, it was found that Ca-alginate
gel used as a matrix of immobilization has the following
References
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58 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 26, JANUARY 1984