Process Biochemistry 37 (2001) 513– 519

www.elsevier.com/locate/procbio

Immobilization system of Kluy6eromyces marxianus cells in barium

alginate for inulin hydrolysis
Esteban Barranco-Florido a, Mariano Garcı́a-Garibay b, Lorena Gómez-Ruiz b,
Alejandro Azaola a,*
a
Departamento de Sistemas Biológicos, Uni6ersidad Autónoma Metropolitana, Xochimilco, A.P. 23 -181, México D.F. 16000, Mexico
b
Departamento de Biotecnologia, Uni6ersidad Autónoma Metropolitana, Iztapalapa, A.P. 55 -535, México D.F. 09000, Mexico

Received 9 October 2000; received in revised form 14 May 2001; accepted 12 June 2001

Abstract

A suspension of Kluy6eromyces marxianus cells (256 mg ml − 1) with inulinase activity were immobilized in barium alginate
treated with glutaraldehyde. After five runs of inulin hydrolysis, the immobilized cells system had up to 85% residual activity.
When beads were heat treated at 65 °C for 5 min, the hydrolysis rate of inulin was improved without affecting the stability of
the system in five hydrolysis batches. Immobilized enzyme was thermostable at temperatures between 45 and 65 °C. The system
efficiency (p) was highly dependent on bead diameter, reaching values up to 0.79 with a bead diameter of 1.43 mm. Inulin diffused
into alginate matrix with a diffusion coefficient (DE) of 2.5× 10 − 6 cm2 s − 1, resulting in apparent kinetic values of the
immobilized enzyme of K%m =0.522 mM and V%max =113.7 mmol min − 1; while values for free enzyme were 0.15 mM and 250 mmol
min − 1. Thiele module values (b) were close to 1, indicating that the limitations of the system were due to diffusional resistance
of substrate. © 2001 Elsevier Science Ltd. All rights reserved.

Keywords: Kluy6eromyces marxianus; Immobilized cells; Barium alginate; Inulinase; Diffusion coefficient; Thiele module

1. Introduction Fructose is used as sweetener in food and pharma-

Cell immobilization in alginate gels produces systems
with high levels of bioconversions, but their main limi-
tation is the diffusional restriction of substrates and
products through the matrix [1]. These systems offer
high biocatalytic activity and durability [2], and they
are thermally stable allowing use at elevated tempera-
tures [3]. Alginate beads are affected by cations such as
Na+, Mg2 + , and K+, as well as by anions such as
ethylenediamine tetraacetic acid (EDTA), citrates, and
phosphates, which cause the loss of Ca2 + [4]. The use
of barium has been proposed as a gel-forming agent to
improve the mechanical resistance of the alginate ma-
trix [5].
* Corresponding author.
E-mail address: azaola@cueyatl.uam.mx (A. Azaola).
ceutical industries. It has better organoleptic properties
being sweeter than sucrose and glucose, and it has also
some physiological advantages that allow its utilization
as a sugar substitute in food and pharmaceutical prepa-
rations [6]. An interesting alternative is to produce
fructose syrups starting from inulin hydrolysis. Inulin, a
polymer of vegetable origin, serves as reserve in plants
of the genus Compositae and Gramineae. It consists of
polymers of 35 residues of D-fructose on average, with
b-2,1 bonds, ending in a D-glucose residue [7].
Inulinase is a non-specific b-fructosidase (EC 3.2.1.7)
that releases fructose molecules from polyfructans as
inulin. It may be secreted to the culture medium or
remain associated with the cell wall. Inulin acts as an
inducer of inulinases [8].
The purpose of this study was to design and to
characterize a system of Kluy6eromyces marxianus cells
with inulinase activity immobilized in a barium alginate
matrix.

0032-9592/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 2 - 9 5 9 2 ( 0 1 ) 0 0 2 3 5 - 7
514 E. Barranco-Florido et al. / Process Biochemistry 37 (2001) 513–519
2. Material and methods
2.1. Microorganism and culture conditions
K. marxianus CDBB-L-278 was grown in a medium
containing 1% inulin (Sigma Chemicals Co., St. Louis,
MO, USA), and 0.5% yeast extract (Bioxon, Mexico
City) (pH 4.5). A 500 ml Erlenmeyer flask with 100 ml
medium was inoculated with 10 ml containing a cell
concentration of 0.3 mg ml − 1 and incubated in a
rotatory shaker (200 r.p.m.) at 30 °C for 24 h.
Growth was determined by turbidity at 550 nm,
converting to biomass dry weight (mg ml − 1) by means
of a standard curve.
2.2. Immobilization technique
A solution of 1.5% sodium alginate (w/v) kept at
65 °C was added to a cell suspension (100 mg dry
biomass/ml alginate solution). The suspension was ex-
truded dropping through a hypodermic needle using a
peristaltic pump to obtain a uniform particle size, col-
lected in a flask containing 0.125M BaCl2 and agitated
overnight. To harden the beads, they were suspended in
0.1, 0.3 or 0.6 M glutaraldehyde and gently shaken for
30 min. Subsequently, they were washed with distilled
water and stored in 0.125 M BaCl2. Cell concentration
within the beads was determined by dissolving 1 ml
beads in 50 ml of 0.1 M EDTA solution at 65 °C for 15
min, and after that 0.2 g K2CO3 were added to com-
plete dissolution. One millilitre of cell suspension was
taken and cell concentration measured by turbidity.
2.3. Enzymic acti6ity determination in free and
immobilized cells
To determine enzymic activity, a solution of 6%
inulin in 0.3 M acetate buffer, pH 5.0, was used, mixed
with 0.5 g dry cells. The reaction was performed at
50 °C, shaking at 200 r.p.m. for 15 min. Samples of 0.1
ml were taken at fixed times and reducing sugars were
determined as described by Miller [9]. For the immobi-
lized system, the same conditions were used as for free
cells, taking as many beads as necessary to produce 0.5
g biomass on a dry weight basis. A unit of inulinase
activity (uI) was defined as the amount of enzyme
necessary to produce 1 mmol reducing sugars per min at
50 °C.
Apparent kinetic parameters of K%m and V%max of the
free and immobilized cell system were determined by
Lineweaver–Burk plot.
All experiments were carried out in triplicate. When
appropriate, analysis of variance was performed using
the SAS software pack (SAS Institute, Cary, NC,
USA).
2.4. Determination of diffusion in barium alginate
beads.
A volume of 15 ml beads with inactivated cells at
90 °C was placed in a glass container and diffusion
determined as reported by Chresand et al. [10]. Aliquots
were taken at different times. Inulin concentration was
determined by a variant of the phenol–sulphuric acid
technique [11]. The diffusion coefficient (DE) was calcu-
lated using the TURBO PASCAL program, with Eqs.
(1)–(3) that are presented in Appendix A.
3. Results and discussion
3.1. Inulin hydrolysis by free and immobilized cells
In a preliminary experiment, inulin hydrolysis was
performed by either free or immobilized cells. In both
cases, approximately 80% of inulin hydrolysis was ob-
tained. During the first 60 min a greater reaction rate
was present in free cells than in immobilized cells.
Later, the hydrolysis rate levels were similar. When the
immobilized system without glutaraldehyde treatment
was re-used, productivity fell by 25% for the second
and 62% for the third batch. In the present study,
barium alginate beads without glutaraldehyde increased
their diameter from 2.68 to 3.4 mm with loss of me-
chanical stability and, hence, of enzymic activity due to
the loss of cells from the immobilization system. When
beads with immobilized cells were stored in BaCl2 they
recovered their original diameter. Beads were therefore
stored in 0.125 M BaCl2 at 4 °C between experiments.
3.2. Effect of glutaraldehyde on barium alginate bead
stability
Table 1 shows enzymic activity of K. marxianus cells,
immobilized with barium alginate after five runs. Im-
mobilized cells were treated with three glutaraldehyde
concentrations (0.1, 0.3 and 0.6 M) with a cellular
concentration of 260 mg ml − 1 and diameter 2.1 cm.
Inulinase activity for each batch was determined run-
Table 1
Enzymic activity of beads treated with different concentrations of
glutaraldehyde after five runs (uI)
Glutaraldehyde Run 1 Run 5
Control 0.482 0.190a
0.1 M 0.452 0.358b
0.3 M 0.446 0.380b
0.6 M 0.460 0.384b
Beads characteristics: cellular concentration, 260 mg ml−1; diameter,
2.1 cm. Figures in rows with the same superscripts are not signifi-
cantly different according to analysis of variance (PB0.05).
 E. Barranco-Florido et al. / Process Biochemistry 37 (2001) 513–519
Table 2
Enzymic activity obtained with three bead diameters and three concentrations of cell mass in the barium alginate immobilization system with 0.3
M glutaraldehyde (uI)
Cellular concentration (mg cell ml−1)
181
256
348
ning one activity determination every 15 days, for a
total of five runs (total 75 days). In the first run, beads
without glutaraldehyde (control) had the highest en-
zymic activity; however, after five runs, it decreased to
39% of the initial activity. Meanwhile, for immobilized
cells treated with glutaraldehyde, residual activity was
79, 85 and 83% after five cycles for 0.1, 0.3 and 0.6 M
glutaraldehyde concentrations, respectively. No signifi-
cant differences (P B0.05) in enzymic activity were
observed between the immobilized systems with differ-
ent glutaraldehyde concentrations. Bajpai and Margari-
tis [12] found that the treatment with glutaraldehyde of
cells immobilized in calcium alginate showed an activity
decrease, as was found here in cells immobilized with
barium alginate.
3.3. Influence of the cellular concentration and bead
diameter on the efficiency of the immobilization system
To determine the optimal operation conditions of the
barium alginate system with glutaraldehyde, the effect
of the cellular concentration and diameter of the bead
was studied. Table 2 presents the effect of different
cellular concentrations and bead diameter on enzymic
activity. No difference in enzymic activity was observed
among the three diameters for each concentration of
immobilized cells. The beads with a cellular concentra-
tion of 181 mg ml − 1 displayed the highest activity, and
beads with a cellular concentration of 348 mg ml − 1
showed the lowest enzymic activity for the three tested
diameters. This effect was probab1y due to cellular
agglomeration caused by glutaraldehyde, which very
likely affected substrate diffusion through the beads.
To select the optimal system, another criterion was
used, termed efficiency, which was defined as the en-
zymic activity of immobilized cells with respect to en-
zymic activity of free cells determined under the same
conditions, as defined by Jamuna and Ramakrishna
[13]. Results are presented in Table 3. The most effi-
cient combinations were cellular concentrations of 181
and 256 mg ml − 1 and diameters of 1.8 and 2.1 mm.
Considering the highest mean value, a diameter of 2.1
mm and a cellular concentration of 256 mg ml − 1 were
selected as the most efficient conditions.
515
Diameter (mm)
1.8 2.1 2.8
0.46 90.047 0.452 9 0.039 0.450 9 0.016
0.336 9 0.002 0.334 9 0.015 0.310 90.017
0.242 9 0.022 0.238 9 0.011 0.200 90.026
3.4. Increase in hydrolysis rate of inulin by heat
treatment of immobilized cells
Fig. 1 shows the profile of hydrolysis rate of inulin
for the immobilized system with previous thermal treat-
ment at 65 °C for 5 min compared with the immobi-
lized system without treatment used as control. As
shown, thermal treatment applied to immobilized cells
significantly increased reaction rate for the first 20 min;
at 10 min the rate was doubled, and at 20 min it was
25% higher than the control. However, after 30 min the
two profiles were similar. Workman and Day [14] tried
to increase the permeability of the cellular wall using
ethyl acetate, but their results did not show an increase
of inulin diffusion across the wall. Thonart et al. [15]
tested thermal treatment of cells before immobilization,
but it did not improve enzymic performance. In the
present work, efficiency was improved with thermal
treatment as it increased to 0.65–0.73 with respect to
previous efficiency results that were 0.62–0.65 (see
Table 3).
To study the stability of the immobilization system
after thermal treatment along several batches, five runs
were performed with an interval of 7 days between; as
part of the stability assessment of the beads, runs were
combined with this relative long storage period. Fig. 2
shows productivity of immobilized cells exposed to
thermal treatment based on reducing sugar production
of every batch. The control consisted of immobilized
cells without previous thermal treatment. Results
showed that thermal treatment did not affect the stabil-
ity of the system, since productivity remained constant
during the five runs, revealing no significant differences
(PB 0.05) among each other.
3.5. Determination of the kinetic parameters
Apparent Km and Vmax values for immobilized and
free cells were determined using the Lineweaver–Burk
plot (not shown). The values obtained were K%m =
0.5229 0.06 mM and V%max = 113.898.1 mmol min − 1
for immobilized cells while values for free cells were
0.159 0.01 mM, and 2509 19 mmol min − 1, suggesting
some diffusion barriers through immobilized yeast cells
and even using non-immobilized cells. Other authors
516 E. Barranco-Florido et al. / Process Biochemistry 37 (2001) 513–519
Table 3
Efficiency (p) of cells immobilized in alginate with glutaraldehyde in the three bead diameters and three cell mass concentrations
Cellular concentration (mg cell ml−1)
181
256
348
have reported different Km values for inulinase from K.
marxianus on inulin. Vandame and Derycke [7] re-
ported a value of 7.4 mM, while Cruz-Guerrero et al.
[21] reported 3.04 mM, the latter for the enzyme from
the same strain reported here. According to Vandame
and Derycke [7], the Km of inulinase depends on the
molecular weight of the polymer.
3.6. Inulin diffusion through the barium alginate beads
Results of inulin diffusion through barium alginate
beads are shown in Fig. 3. An initial concentration of
inulin of 11 mg ml − 1 diminished diffusing in the beads
until it reached the equilibrium concentration after
approximately 20 min.
The diffusion coefficient of inulin was determined
according to the model proposed by Cranck [16]. The
value was DE = 2.5× 10 − 6 cm2 s − 1. Diffusional limita-
tion is one of the reasons for reduction of system
effectiveness. The diffusivity values for small molecules
(such as glucose) in calcium alginate gels have been
published elsewhere [17], but no such data exist for
molecules like inulin. Diffusion in porous polymers is
influenced by the characteristics of the matrix, and the
molecular form and volume of the solutes. Longo et al.
[18] determined diffusivity coefficients of small
molecules and of proteins with higher molecular
weights than inulin in alginate beads. They proposed a
linear plot of molecular weight versus diffusion coeffi-
cient that allows one to determine intermediate values
of diffusion. The value of the diffusion coefficient cal-
culated in the present study is similar to the expected
value interpolating in this plot.
Doherty et al. [19] reported that, to estimate the
diffusion coefficient, it is important to take into ac-
count the size of the beads and their microscopic obser-
vation, since their dimensions are reduced when they
lose humidity and this affects diffusion.
3.7. Effect of temperature on the immobilization system
To determine the effect of temperature on the reac-
tion rate of hydrolysis by immobilized cells (Table 4),
various runs were performed at different temperatures
between 45 and 75 °C. The total reaction time was 60
Diameter (mm)
1.8 2.1 2.8
0.646 9 0.046 0.452 9 0.039 0.450 90.016
0.654 9 0.005 0.334 9 0.015 0.310 90.017
0.620 9 0.012 0.238 90.011 0.200 9 0.026
min. The hydrolysis rate at 55 and 65 °C increased
approximately by 40% compared with activity obtained
at 45 °C. Finally, at 75 °C, activity showed a drastic
fall.
This effect was observed by Bajpai and Margaritis
[20] in a system in which the optimal temperature was
60 °C, and inulinase activity decreased by approxi-
mately 15% after 60 min exposure to 70 °C. The bar-
ium alginate immobilization system could protect the
enzyme of thermal effects allowing activity at 65 °C
and this is convenient since high temperatures would
increase productivity and diminish microbial contami-
nation. In addition, it has been demonstrated elsewhere
that inulinase from this strain is thermostable [21].
3.8. Determination of the efficiency (p) of the
immobilization system
Fig. 4 shows the relationship between the immobi-
lization system efficiency and the diameter of the bead,
considering six different sizes. Cellular concentration in
alginate was 260 mg ml − 1. Maximal efficiency reached
was 0.79 when the minimal bead diameter (1.43 mm)
was obtained. With a bead diameter of 4.2 mm, the
value for p was 0.35, less than one-half of the efficiency
obtained with 1.43 mm. It seems obvious that the
efficiency of the immobilization system depends on the
Fig. 1. Effect of thermal treatment (65 °C for 5 min) on immobilized
cells (256 mg ml − 1; 2.1 mm bead diameter; treated with 0.3 M
glutaraldehyde). (") Control, immobilized cells without thermal
treatment; (2) immobilized cells with thermal treatment.
 E. Barranco-Florido et al. / Process Biochemistry 37 (2001) 513–519 517

Table 4
Hydrolysis rate of the yeast in barium alginate immobilization system
with glutaraldehyde at different incubation temperatures and times
(mmol min−1)

T (°C) Time (min)

10 20 30 45 60

45 0.128 0.180 0.236 0.260 0.265

55 0.240 0.298 0.360 0.370 0.400
65 0.214 0.304 0.335 0.376 0.451
75 0.060 0.036 0.026 0.0161 0.019

3.9. Analysis of the immobilization system through the

Fig. 2. Effect of the thermal treatment (65 °C for 5 min) on immobi-
lized cells in runs with 7 day intervals, compared with immobilized
cell control, with no thermal treatment. Both with 256 mg ml − 1; 2.1
mm bead diameter; treated with glutaraldehyde 0.3 M.
size of the bead, since the immobilization system tends
to equal the conditions of the free cell system while
bead size diminished.
Diffusion depends on the properties of matrices, as
indicated by Slininger et al. [22]. These authors pro-
posed that the penetration of substrate molecules
within the immobilization beads of calcium alginate
depends on the diffusion velocity. They obtained the
effectiveness factor by calculating the ratio between the
specific speed of production of ethanol by immobilized
yeast cells and the specific speed of production of
ethanol by free cells; the value was p= 0.4 due to
substrate diffusion problems in the alginate beads. In
the current work, with a similar system, a higher effi-
ciency was obtained.
Thiele module (b)
To predict the behaviour of the immobilization sys-
tem with respect to diffusion and enzyme activity, the
system has been considered as a special case of a
chemical reaction limited by diffusion, applied to whole
cell immobilization system as reported previously [23].
The adimensional parameter Thiele module (b) was
used for this purpose. It was assumed that: (a) the
system presents a homogeneous reaction with constant
diffusion and partition coefficient; (b) the system dis-
plays spherical geometry and enzymic activity is found
in a stationary state with a substrate that diffuses
according to Fick’s law and is simultaneously hy-
drolyzed by the enzyme; (c) the influence of other
solutes is ignored and only inulin is considered; (d) the
kinetics of enzyme activity follows the Michaelis–
Menten model. Kinetic parameters already reported
and the diffusion coefficient of the immobilized system
were used to calculated Thiele module values (b),
which were 9, 11, 13, 17 and 21 for diameter sphere of
1.43, 1.81, 2.1, 2.8 and 3.4 mm, respectively. When
b1, the problems of diffusion are minimal and the
only limitation is the enzyme reaction; when bB1,
limitations are due to diffusion; and when b\ 1, prob-

Fig. 3. Profile of inulin concentration decrease within the liquid to

determine inulin diffusion coefficient through beads of cells immobi-
lized in barium alginate with 0.3 M glutaraldehyde (256 mg ml − 1; 2.1 Fig. 4. Efficiency of the immobilization system related to free cells in
mm bead diameter). different barium alginate bead diameters.
518 E. Barranco-Florido et al. / Process Biochemistry 37 (2001) 513–519

lems are due to diffusion and reaction limitations.
Therefore, results indicate that limitations of the system
were due to diffusion and reaction limitations. Low
diffusion is a characteristic shown by alginate immobi-
lization systems; however, the alternative is interesting
since the immobilization process is simple and cheap,
and it is thermoresistant and adequately stable, with
durable enzymic activity. Barium alginate was satisfac-
tory since it had greater resistance and was innocuous
to the inulinase activity.
each set of experimental values of t and CL. Then a
value of CL is obtained and compared with experimen-
tal values. Depending on the difference between the two
values of CL for each DE value, the process is repeated
until the differences between calculated and experimen-
tal values are relatively small. The process turns out
several diffusivity values, and that which adjusts better
to experimental CL values is selected.
A.1. Internal diffusional restrictions

Internal diffusion restrictions are caused by the small

4. Conclusions size and tortuosity of support porous, while high loads
of immobilized cells can cause also internal diffusion
The use of barium alginate was adequate to immobi- restrictions. The significant variables that affect internal
lize K. marxianus cells with inulinase activity. The use diffusion restrictions are quantified by Thiele modulus
of glutaraldehyde as a hardening agent offered greater (b) according to Eq. (4).
mechanical resistance and durability, and did not affect
enzymic activity substantially. Temperature, an impor- F= Lu =L
' V%max
(4)
tant factor for any enzymic activity, had a permeabi- K%mDE
lization effect on yeast cells and did not affect the
enzyme activity within a large interval in the immobi-
Appendix B. Nomenclature
lized system.
The cellular concentration affected the speed of hy-
drolysis, immobilized cell efficiency and gel resistance.
The size of the beads, and therefore the surface of
CL concentration at a given time (mg ml−1)
contact, influenced system efficiency.
C oL initial concentration (mg ml−1)
The parameters of diffusion coefficient, efficiency and
DE diffusion coefficient (cm2 s−1)
Thiele module (b) were applied to characterize the
K%m apparent Michaelis–Menten constant (mM)
system and to explain the diffusion-reaction phe-
L sphere radius (cm)
nomenon. Experimental values occurred within the
qn nonzero roots of tan (qn )
module region close to 1, which implies diffusion prob-
R bead radius (cm)
lems. The size of the substrate molecule was the main
t time of sampling (s)
cause of diffusion difficulties.
VL liquid volume (ml)
VS bead volume (cm3)
V%max apparent maximum rate (mmol min−1)
Appendix A. Diffusion coefficient calculation
p efficiency
 equilibrium concentration (mg ml−1)
The measurement of diffusion of a solute through an
immobilization system is based on the derivation pro-
posed by Cranck [16]. Diffusion is given by Eq. (1),
which allows the prediction of solute concentration in
the liquid phase, CL, as a function of the time of References
contact t, when the values for R, h, C oL and DE are
known. The value for qn is determined by a false-true [1] SivaRaman H, SeetaramaRao B, Pundle AV, SivaRaman C.


solution of the Eq. (3). Continuos ethanol production by yeast cells iumobilized in open

CL =C o
h
+ 6h %

q2nt/R2
e − DE n (1)
pore gelatin matrix. Biotechnol Lett 1982;4:359 – 64.
[2] Dervakos GA, Webb C. On the merits of viable-cell immobiliza-
1+h
L 2
n = 19(1 +h) +h qn tion. Biotechnol Adv 1991;9:559 – 612.
[3] Guiseley KB. Chemical and physical properties of algal polysac-
VL charides used for cell immobilization. Enzyme Microb Technol
h= (2)
VS 1989;11:706 – 16.
[4] Cheetham PSJ, Blunt KW, Bucke Ch. Physical studies on cell
3qn immobilization using calcium alginate gels. Biotechnol Bioeng
tan qn = (3)
3+hq 2n 1979;21:2155 – 68.
[5] Tanaka H, Irie S. Preparation of stable alginate beads in elec-
A value is established for DE within a predetermined trolyte solutions using Ba2 + and Sr2 + . Biotechnol Tech
range of values (for example 10 − 4 to 10 − 8 cm2 s − 1) for 1988;2:115 – 20.
 E. Barranco-Florido et al. / Process Biochemistry 37 (2001) 513–519
[6] Gelardi RC. Alternative Sweeteners. New York: Lyn O’Brien
Nabors and Marcel Dekker, 1991:219 –46.
[7] Vandamme EJ, Derycke DG. Microbial inulinases: fermentation
process, properties, and applications. Adv Appl Microbiol
1983;29:139 – 76.
[8] Parekh S, Margaritis A. Inulinase (b-fructofuranosidase) produc-
tion by Kluy6eromyces marxianus in batch culture. Appl Micro-
biol Biotechnol 1985;22:446 –8.
[9] Miller L. Use of dinitrosalicylic acid reagent for determination of
reducing sugars. Anal Chem 1959;31:426 – 8.
[10] Chresand TJ, Dale BE, Hanson SL. A stirred bath technique for
diffusivity measurements in cell matrices. Biotechnol Bioeng
1988;32:1029 – 36.
[11] Rao P, Pattabiraman TN. Reevaluation of the phenol –sulphuric
acid reaction for the estimation of hexoses and pentoses. Anal
Biochem 1989;181:18 –22.
[12] Bajpai P, Margaritis A. Improvement of inulinase stability of
calcium alginate inmobilized Kluy6eromyces marxianus cells by
treatment with hardening agents. Enzyme Microb Technol
1985;7:34– 6.
[13] Jamuna R, Ramakrishna SV. Continuous synthesis of ther-
mostable a-amylase by Bacillus cells immobilized in calcium
alginate. Enzyme Microb Technol 1992;14:36 – 41.
[14] Workman WB, Day DF. Enzymatic hydrolysis of inulin to
fructose by glutaraldehyde fixed yeast cells. Biotechnol Bioeng
1984;26:905 – 10.
519
[15] Thonart P, Roblain D, Rikir R. Improvement of inulin hydroli-
sis yeast cell reactor by mutants selection. Appl Biochem Bio-
technol 1988;17:193 – 202.
[16] Cranck J. The Mathematics of Diffusion, 2nd ed. London:
Oxford University Press, 1975:93 – 6.
[17] Estapé D, Gòdia F, Solà C. Determination of glucose and
ethanol effective diffusion coefficients in Ca-alginate gel. Enzyme
Microb Technol 1992;14:396 – 401.
[18] Longo MA, Novella IS, Garcı́a LA, Dı́az M. Diffusion of
proteases in calcium alginate beads. Enzyme Microb Technol
1992;14:586 – 90.
[19] Doherty Speirs B, Halling PJ, Mcneil B. The importance of bead
size measurement in mass-transfer modelling with immobilised
cellls. Appl Microbiol Biotechnol 1995;43:440 – 4.
[20] Bajpai P, Margaritis A. Immobilization of Kluy6eromyces marxi-
anus cell containing inulinase activity in open gelatin matrix: 1.
Preparation and enzymatic properties. Enzyme Microb Technol
1985;7:373 – 6.
[21] Cruz-Guerrero A, Garcı́a-Peña I, Barzana B, Garcı́a-Garibay M,
Ruiz L. Kluy6eromyces marxianus CDBB-L-278: a hyperproduc-
ing strain. J Ferment Bioeng 1995;80:159 – 63.
[22] Sliniger PJ, Bothast RJ, Black LT, McGhee JB. Continuos
conversion of D-xylose to ethanol by immobilized Pachysolen
tannophilus. Biotechnol Bioeng 1982;24:2241 – 51.
[23] Karel SF, Libicki SB, Robertson CR. The immobilization of
whole cells: engineering principles. Chem Eng Sci 1985;40:1321 –
54.