Biochemical Engineering Journal 41 (2008) 79–86

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Biochemical Engineering Journal

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Cyclodextrin production by Bacillus firmus strain 37 immobilized

on inorganic matrices and alginate gel
Cassiana Mazzer, Lı́via Rosas Ferreira, Júlia Regina Tedesco Rodella,
Cristiane Moriwaki, Graciette Matioli ∗
Pharmacy and Pharmacology Department, State University of Maringá (UEM), Av. Colombo 5790, CEP 87020-900, Maringá-PR, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Production of ␤-cyclodextrin (␤-CD) by Bacillus firmus strain 37 cells, immobilized by adsorption on
Received 21 December 2007 silica–titania (SiO2 /TiO2 ) and silica–manganese dioxide (SiO2 /MnO2 ) matrices, was optimized for tem-
Received in revised form 20 March 2008 perature, substrate concentration and initial biomass. The immobilization process was most efficient at
Accepted 22 March 2008
60 ◦ C with 10% maltodextrin and 1.0 g of cells, resulting, after a 5-day assay, in a ␤-CD production of
11.7 ± 0.1 mM for cells immobilized on SiO2 /TiO2 and 11.2 ± 0.1 mM in SiO2 /MnO2 . Entrapment in alginate
Keywords:
gel resulted in a maximum ␤-CD production of 4.1 ± 0.1 mM, which was maintained constantly until the
Bacillus firmus
end of a 10-day assay. During this same period, free cells produced 8.3 ± 0.2 mM, and cells immobilized
Cyclodextrins
Immobilized cells
on SiO2 /TiO2 and SiO2 /MnO2 , 16.7 ± 0.4 and 17.3 ± 0.5 mM, respectively. ␤-CD production by cells immo-
Adsorption bilized in calcium alginate in four repetitive cycles of 5 days each, showed an increase up to the third
Batch processing cycle, reaching 4.8 ± 0.2 mM, while production by free cells started falling from the second cycle. In this
Enzymes same assay, cells immobilized on SiO2 /TiO2 and SiO2 /MnO2 , showed the best ␤-CD production results at
the end of the first cycle, with a gradual fall occurring due to the desorption of cells thereafter.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
Cyclodextrin glycosyltransferase (CGTase) is the enzyme that
converts, through a process of cyclization, starch into cyclodex-
trins (CDs) [1,2]. CDs have the ability to encapsulate a wide range
of organic and inorganic molecules, altering the stability, reactivity
and solubility of the inclusion complexes formed with them [3,4].
These characteristics make CDs, and their derivatives, suitable for
applications in biotechnology [5], agriculture [6], pharmaceutical
[3,7], food [8] and cosmetics [6] industries.
The majority of bacterial CGTases produce mainly ␣-CD, ␤-CD,
and a small quantity of ␥-CD, consisting of six, seven or eight units
of glucose, respectively [1]. CGTase is an extracellular enzyme, and
is a member of the amylolytic glycosylases family [9]. These are
produced by Thermoanaerobacter [10] and by some species of Bacil-
lus, such as Bacillus circulans [4], Bacillus macerans [11], Bacillus
stearothermophilus [12], Bacillus agaradhaerens [13], and Bacillus
firmus [14]. Alkalophilic bacilli have attracted much attention for
industrial applications because of their high level of activity in wide
pH and temperature ranges. These bacilli have come to be regarded
as the most promising microorganisms for the production of CDs,
as they mainly form ␤-CD without an accumulation of ␣-CD and,
∗ Corresponding author. Tel.: +55 44 3261 4301; fax: +55 44 3261 4119.
E-mail addresses: gmatioli@uem.br, gramatioli@yahoo.com.br (G. Matioli).
furthermore, the purification of the ␤-CD from the reaction mixture
is made easier by using the difference in solubility between ␤-CD
and ␥-CD. These factors have lead to ␤-CD having a more accessible
price compared to other CDs [15].
CGTases have been immobilized in innumerable matrices with
the aim of enabling the continuous production of CDs [11,16–21].
However, there are very few studies that report on the immobi-
lization of cells with the aim of producing these enzymes (Bacillus
amyloliquefaciens [22], B. circulans ATCC 21783 [23,24], B. firmus
[25], Bacillus licheniformis [26], and B. agaradhaerens [27]).
The technique of cell immobilization for the production of
extracellular enzymes has various advantages over the process
of conventional fermentation using free cells. These include the
ability to separate the cell mass from the fermentation medium,
the ease of carrying out continuous operations for long periods
of time, an increase in productivity in the reactor, the prolonged
and repeated use of cells, reduced risks of contamination, continu-
ous fermentation using less sophisticated reactors and elimination
of the necessity to purify the enzymes [25,28,29]. For this reason,
various methods of immobilization, including adsorption, covalent
binding and entrapment in gel, have been used to obtain cells with
high levels of activity and stability [30]. The most commonly used
method of immobilization is entrapment in gel [31]. This is mainly
due to the compatibility of the gel, the simplicity of the immobi-
lization technique and the high viability and productivity of the
immobilized cells [26]. The easiest and oldest method is that of

1369-703X/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
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80 C. Mazzer et al. / Biochemical Engineering Journal 41 (2008) 79–86
adsorption, in which many organic and inorganic materials have
been used [32].
The sol–gel method, first reported 150 years ago, is a method
used for the production of inorganic materials at room tempera-
ture. The process provides a suitable model for the incorporation,
immobilization, entrapment, and encapsulation of a wide vari-
ety of materials, including organic and inorganic compounds,
biomolecules, microorganisms, tissues, and indicators [33]. The
sol–gel process has aroused interest mainly because it is carried out
at room temperature and in conditions that are not severe enough
to cause the denaturation of the encapsulated biomolecules [34].
Previous studies on the production of ␤-CD through the immo-
bilization of B. firmus strain 37 in inorganic matrices by the sol–gel
method, without the need to purify the CGTase, have been carried
out by this research group, and important results were obtained
[25]. Therefore, the aim of this study was to optimize the condi-
tions of ␤-CD production by B. firmus strain 37 cells adsorbed onto
an inorganic matrix, and to compare it to that obtained through
immobilization by entrapment in gel.
2. Materials and methods
2.1. Microorganism and culture conditions
B. firmus strain 37, isolated from the soil of a cassava plantation
by Matioli et al. [35], was cultivated in culture plates containing
solid culture medium composed of (%, w/v): 1.0 soluble starch, 0.5
polypeptone, 0.5 yeast extract, 0.1 K2 HPO4 , 0.02 MgSO4 ·7H2 O, 0.01
Congo red dye, 1.0 Na2 CO3 , and 1.5 agar. The plates were incubated
at 37 ◦ C for 48 h and the colonies formed in the culture medium
were transferred to 2 l Erlenmeyer flasks containing 1 l of liquid
culture medium with the same composition to that of the solid
medium, except for the agar and dye. The flasks were incubated at
37 ◦ C in an incubator under orbital shaking (120 rpm) for 48 h. The
cells were then removed from the culture medium by centrifuga-
tion (2000 × g, 10 min, 4 ◦ C) and washed. The cells were diluted to a
proportion of 1:2 in liquid medium containing 10% glycerol in order
to obtain 107 colony-forming units (CFU) per milliliter. This cell sus-
pension was kept at −80 ◦ C and later used in the immobilization
procedures and in the assays with free cells.
2.2. Microorganism reactivation procedure
Three milliliters of the bacterial suspension containing
107 CFU/ml, which corresponds to 1 g of cells (7.2% dry-cell weight),
were transferred to 250 ml Erlenmeyer flasks containing 40 ml
of liquid culture medium. The flasks were incubated at 37◦ C in
an incubator under orbital shaking (120 rpm) for 24 h. The cells
were then removed from the culture medium by centrifugation
(2000 × g, 10 min, 4 ◦ C) and washed.
2.3. Preparation of the inorganic matrices
The preparation of the inorganic matrices was carried out
according to the method described by Moriwaki et al. [25].
The silica–titania (SiO2 /TiO2 ) and silica–manganese dioxide
(SiO2 /MnO2 ) matrices had specific surface areas of 503 m2 /g and
454 m2 /g, mean pore volumes of 0.35 and 0.16 ml/g, and mean pore
diameters of 2.8 × 10−3 and 1.4 × 10−3 ␮m, respectively.
2.4. Immobilization procedures
For immobilization on inorganic matrices by the adsorption
method, the cells, after reactivation, were suspended in 50 ml of
sterile distilled water and transferred to a 250 ml Erlenmeyer flask
containing 1.0 g of sterilized matrix. The suspension was main-
tained at 37 ◦ C in an incubator under orbital shaking at 120 rpm
for 24 h.
For the gel-entrapment technique, the cells were immobilized
using sodium alginate by the ionotropic method [36]. After reac-
tivation, the cells were added to 17 ml of sodium alginate solution
(1.5%, w/v) and the mixture was extruded into 30 ml of calcium
chloride solution (0.5%, w/v), resulting in the formation of spheri-
cal beads, which were washed with sterile distilled water. The mean
diameter of the particles was 3.91 mm.
2.5. Influence of temperature, substrate concentration and initial
biomass on the production of cyclodextrin by cells immobilized on
inorganic matrices
The production of CDs by cells immobilized on SiO2 /TiO2 and
SiO2 /MnO2 and by free cells was evaluated at different tempera-
tures (37, 50, 60, and 70 ◦ C), at different substrate concentrations
(1.0, 5.0, 10, and 15% of maltodextrin) and at different concen-
trations of initial biomass (1.5, 3.0, 4.5, and 6.0 ml of bacterial
suspension). Each assay was carried out for 5 days and the ␤-CD
concentration and CGTase activity were then determined.
The tests were carried out in 250 ml Erlenmeyer flasks contain-
ing reaction medium, which consisted of 50 ml of maltodextrin in
50 mM Tris–HCl buffer, pH 8.0, and 5 mM CaCl2 . Maltodextrin was
chosen for this study due to the standardization of its use in the pre-
vious studies carried out by this research group [14,25,35,37–39].
The assay was carried out in an incubator under shaking at 120 rpm.
Aliquots of 1 ml were periodically collected (0, 12, 24, 36, 48, 72, 96
and 120 h) and diluted in 1 ml of distilled water for later determi-
nation of the ␤-CD produced and the enzymatic activity.
2.6. Cyclodextrin production by the adsorption on inorganic
matrix and entrapment in gel methods
The production of CDs by B. firmus strain 37 cells immobilized
on SiO2 /TiO2 , SiO2 /MnO2 , and in calcium alginate, as well as by
free cells, under the optimized conditions (temperature of 60 ◦ C,
10% (w/v) maltodextrin and 3.0 ml of cell suspension) was evalu-
ated in this study. The assay lasted for 10 days, without changing
the substrate or carrying out any other modification. Samples were
collected in duplicate every 24 h.
The method used in this study was described by Matioli et al.
[35] and adapted by Moriwaki et al. [25]. The tests were carried out
in 250 ml Erlenmeyer flasks containing 50 ml of reaction medium.
For each CD production cycle, the reaction medium was composed
of 50 ml of 10% (w/v) maltodextrin in 50 mM Tris–HCl buffer, pH 8.0,
and 5 mM CaCl2 . The assay was carried out at 60 ◦ C and at 120 rpm.
CD production in repetitive cycles was also carried out with free
cells, and cells immobilized on SiO2 /TiO2 , SiO2 /MnO2 , and in cal-
cium alginate. Four cycles of 120 h were carried out and at the end of
each cycle, the cells immobilized in calcium alginate were filtered
and washed, and the free cells and those immobilized on SiO2 /TiO2
and SiO2 /MnO2 were removed from the production medium by
centrifugation (800 × g, 3 min, 4 ◦ C), washed with sterile distilled
water and added again to the reaction medium. For each cycle, a
new CD production medium was prepared. All the experiments
were carried out in duplicate. Aliquots of 1 ml were collected peri-
odically (0, 24, 36, 48, 72, 96 and 120 h), diluted in 1 ml of distilled
water and boiled for later quantification of the ␤-CD produced. The
CGTase activity in the reaction medium was determined at the end
of each cycle.
To check whether cell desorption occurred, the number of CFUs
present in the supernatant was evaluated for recently immobilized
samples and after the end of the first CD production cycle.
 C. Mazzer et al. / Biochemical Engineering Journal 41 (2008) 79–86 81

2.7. Scanning electron microscopy (SEM)
Samples collected after 24 h of immobilization were evaluated
using scanning electron microscopy. The observations were carried
out according to the method described by Moriwaki et al. [25], using
a scanning electron microscope (Shimadzu, model SS 550) with an
acceleration voltage of 10 kV.
2.8. Analytical methods
2.8.1. ˇ-Cyclodextrin determination
␤-CD concentration was determined by the method described
by Tardioli et al. [16], which is based on the dye-extinction of phe-
nolphthalein at 550 nm, which occurs after its complexation with
␤-CD. The assay was performed by mixing 0.5 ml of sample contain-
ing ␤-CD with 2.5 ml of 0.06 mM phenolphthalein working solution
containing 0.12 M carbonate-bicarbonate buffer, pH 10.5, and the
absorbance was read at 550 nm. For the blank, the sample was sub-
stituted by distilled water. The phenolphthalein working solution
was prepared at the time of dosage from a stock solution of 3 mM
phenolphthalein in 95% ethanol (2 ml of phenolphthalein stock
solution and 20 ml of 0.6 M carbonate-bicarbonate buffer, pH 10.5,
with the volume being made up to 100 ml with distilled water).
The ␤-CD concentration was calculated using Eq. (1), which was
obtained through the determination of the equilibrium constant
(K␤-CD ) for the formation of the inclusion complex of ␤-CD with
phenolphthalein. This constant was determined by a non-linear
correction of Eq. (2) for a series of absorbance data in function of
the standard concentrations of ␤-CD (0–1 × 10−3 M) prepared in
distilled water.
  
A550 A0/550
C␤-CD = 0.3 1− 1 + 1.0813
A0/550 A550
where C␤-CD is the concentration of ␤-CD (mM), A550 and A0/550 are
the absorbances of the samples and the blank, respectively.
   and SiO2 /MnO2 , respectively, were observed. The specific activity
A550 A0/550
C␤-CD = a 1− 1+
A0/550 Kˇ−CD aA550
where a is the total concentration of phenolphthalein in the assay
cuvette (5 × 10−5 M).
The equilibrium constant (K␤-CD ) was calculated using the
Quasi-Newton method, resulting in 21627.10 ± 85.61 M−1 for the
confidence interval of 95%. Eq. (1) was obtained by the substitution
of K␤-CD and the a value in Eq. (2).
tion time of 0 min, for which the CGTase was firstly inactivated and
the substrate was then added [14]. To obtain the specific activity,
the enzyme was purified, as described by Matioli et al. [35], and
protein contents were determined by the method of Bradford [40],
using bovine serum albumin as standard, giving 0.52 mg protein/ml
of solution.
2.9. Statistical analysis
The results were submitted to analysis of variance (ANOVA) and
Tukey means tests, with a 5.0% level of probability, using the soft-
ware Statistica 6.0/2001 (Stat Soft, Inc. Tulsa, OK, EUA).
3. Results and discussion
3.1. Effect of temperature on ˇ-cyclodextrin production by
immobilized B. firmus strain 37 cells
The CGTase enzyme obtained from B. firmus strain 37, isolated
from the soil of a cassava plantation, was characterized by Matioli et
al. [37]. They reported that the greatest stability occurred at 60 ◦ C
and the highest specific activity was obtained for the enzyme at
65 ◦ C. In the present study, due to the fact that the enzyme was
not used directly, it was deemed appropriate to determine the best
temperature for the production of ␤-CD from the immobilized cells.
The tests were carried out with a reaction medium contain-
ing 10% (w/v) maltodextrin in 50 mM Tris–HCl buffer, pH 8.0,
5 mM CaCl2 and 3.0 ml of microbial cell suspension, and a tem-
perature range of 37–70 ◦ C. There were significant differences in
␤-CD production among all the temperatures studied for the cells
immobilized on SiO2 /TiO2 and SiO2 /MnO2 (Fig. 1). There were
(1) increases in ␤-CD production for the immobilized cells as the
temperature increased, reaching a maximum at 60 ◦ C. At this tem-
perature, ␤-CD productions of 6.0 ± 0.1 mM for the free cells, and
11.6 ± 0.2 and 11.1 ± 0.5 mM for the cells immobilized on SiO2 /TiO2
(2) of the CGTase was 75 U/mg for the free cells and 146 U/mg and
140 U/mg for the cells immobilized on SiO2 /TiO2 and SiO2 /MnO2 ,
respectively. However, an increase in temperature to 70 ◦ C resulted
in a reduction in ␤-CD production of the order of 92% for the free
cells, and of 87 and 88% for the cells immobilized on SiO2 /TiO2 and
SiO2 /MnO2 , respectively.
Similar results to these were obtained by Nemati and Webb [41],
who also showed that the influence of temperature on microor-
ganism activity is increased when the cells are immobilized. The

2.8.2. Determination of the enzymatic activity of cyclodextrin

glycosyltransferase
CGTase activity was determined as a function of the initial ␤-CD
production rate using dextrin as a substrate. The ␤-CD concen-
tration in the samples was measured by the colorimetric method,
described in Section 2.8.1. The enzymatic activity of the soluble and
immobilized enzymes was calculated from the inclination of the ␤-
CD concentration curve versus time. One unit (U) was defined as the
quantity of enzyme that produces 1 ␮mol of ␤-CD/min under the
assay conditions. The assay conditions consisted of a substrate solu-
tion containing 1.0% (w/v) maltodextrin (D.E. 10) in 50 mM Tris–HCl
buffer, pH 8.0, and 5 mM CaCl2 at 50 ◦ C. One milliliter of the sub-
strate was placed in each of six tubes and then 1 ml of the enzymatic
solution was added to each of them. The tubes were shaken and
incubated for up to 30 min at 50 ◦ C, with one tube being removed
every 5 min. The CGTase was inactivated by being placed in a boil-
Fig. 1. Effect of temperature on ␤-cyclodextrin (␤-CD) production. () Free cells,
ing water bath for 10 min. The reaction time and the dilution of
( ) cells immobilized on SiO2 /TiO2 matrix, () cells immobilized on SiO2 /MnO2
the enzyme were selected from the linear relation between ␤-CD matrix. Assay conditions: 10% (w/v) maltodextrin in 50 mM Tris–HCl buffer, pH 8.0,
production versus time. The control was obtained by using a reac- 5 mM CaCl2 and 3.0 ml of microbial cell suspension.
82 C. Mazzer et al. / Biochemical Engineering Journal 41 (2008) 79–86

kinetic study that they developed was carried out in a packed-bed

bioreactor inoculated with Thiobacillus ferrooxidans immobilized on
polyurethane foam support particles. Their experimental results
showed that the kinetics of Fe (II) oxidation by biofilms at 30 ◦ C
were significantly faster than those at 20 ◦ C. According to these
authors, temperature has an important role in enzymatic activity,
as it can influence the reaction even when immobilized cells are
being used. They further stated that temperature could be used as
an effective parameter in the control of the biomass level inside the
reactor of the immobilized cells. In later studies, when Gómez et al.
[42] immobilized the same microorganism on a nickel alloy fiber
support, the use of higher temperatures decreased iron-oxidation.
It can therefore be seen that the best temperature for ␤-CD pro-
duction depends on the microorganism that is being used in the
immobilization process.
Fig. 2. Effect of maltodextrin substrate concentration on ␤-cyclodextrin (␤-CD)
It can also be observed in Fig. 1 that there were significant differ- production. () Free cells, ( ) cells immobilized on SiO2 /TiO2 matrix, () cells
ences in ␤-CD production for the free cells, with the exception of the immobilized on SiO2 /MnO2 matrix. Assay conditions: temperature 60 ◦ C, 50 mM
productions at 37 ◦ C and 50 ◦ C. It was found that the optimal condi- Tris–HCl buffer, pH 8.0, 5 mM CaCl2 , and 3.0 ml of microbial cell suspension.
tions for the production of ␤-CD from immobilized cells were not
the same as those used for free cells, and it was therefore necessary Abdel-Naby et al. [22] found that as well as concentration, the
to optimize all the parameters involved in the process. Furthermore, origin of the substrate can also influence CGTase production. They
it was concluded that immobilized cells showed greater activity evaluated the effects of various sources of carbon (glucose, galac-
at higher temperatures compared to free cells, which were most tose, fructose, maltose, lactose, sucrose, sorbose and xylose) on
active at 37 and 50 ◦ C. The use of higher temperatures also has the CGTase production by B. amyloliquefaciens cells immobilized in cal-
advantage of lowering the risk of contamination. cium alginate. The best results were obtained using glucose at a
The fact that ␤-CD production was different when using immo- concentration of 2.0%.
bilized and free cells can be explained by Gómez et al. [42] and
Jamuna and Ramakrishna [43], who stated that the conditions
3.3. Effect of initial biomass on ˇ-cyclodextrin production by
of stress induced by immobilization may alter the mechanism of
immobilized B. firmus strain 37 cells
enzyme production, although the mechanism and nature of this
change are still not clear. This is probably due to certain physiolog-
The effect of initial biomass on ␤-CD production was investi-
ical alterations in the microorganism during immobilization.
gated by preparing production media containing 0.5–2.0 g of cells,
with 3.0 ml of cell suspension corresponding to 1.0 g of cells. The
3.2. Effect of variation in substrate concentration on
assay conditions were the same as those described above, with mal-
ˇ-cyclodextrin production by immobilized cells
todextrin concentration being 10% (w/v) and the temperature being
60 ◦ C.
The influence of variations in the concentration of maltodex-
The results showed that ␤-CD production for the cells immo-
trin on ␤-CD production using B. firmus strain 37 cells immobilized
bilized on inorganic matrices increased together with the increase
on inorganic matrices was investigated over a period of 120 h.
in initial biomass up to the value of 1.5 g of cells (Fig. 3). The ␤-CD
Maltodextrin concentration varied from 1.0 to 15% (w/v), while
productions obtained were 12.2 ± 0.3 mM for cells immobilized on
temperature (60 ◦ C) and the quantities of cells (3.0 ml of bacterial
SiO2 /TiO2 and 12.0 ± 0.5 mM for cells immobilized on SiO2 /MnO2 ,
suspension) and inorganic matrix (1.0 g) were kept constant.
with CGTase enzymatic activities of 94.8 and 93.2 U/g of cells h,
With the increase in maltodextrin concentration from 1.0 to 15%,
respectively. However, these values were not significantly different
␤-CD production increased from 4.5 ± 0.1 to 7.0 ± 0.3 mM for free
to the values obtained for 1.0 g of cells. For the cells immobilized
cells, 4.6 ± 0.2 to 12.8 ± 0.6 mM for cells immobilized on SiO2 /TiO2 ,
on SiO2 /MnO2 , ␤-CD production was also not significantly differ-
and 4.7 ± 0.2 to 12.4 ± 0.8 mM for cells immobilized on SiO2 /MnO2
(Fig. 2). The increase in maltodextrin concentration from 1.0 to 5.0%
resulted in an increase in ␤-CD production of over 100%, both for
cells immobilized on SiO2 /TiO2 and on SiO2 /MnO2 . A significant
increase in ␤-CD production was also observed when the maltodex-
trin concentration was increased from 5.0 to 10%, however, this did
not occur in the increase from 10 to 15%. Therefore, using a mal-
todextrin concentration of 10% in the reaction medium seems to be
more economically advantageous. The same pattern was seen for
CGTase enzymatic activity.
The results obtained indicate that the concentration of the sub-
strate is an essential factor in ␤-CD production when using the
method of immobilizing cells on inorganic matrices. It has also been
stated in previous literature that the concentration of the substrate
can influence ␤-CD production for cells immobilized by entrap-
ment in gel. Kunamneni et al. [36] examined this relationship when
studying the influence of starch concentration on CGTase produc-
Fig. 3. Effect of initial biomass on ␤-cyclodextrin (␤-CD) production. () Free cells,
tion by Bacillus sp cells immobilized in alginate. In their work, a
( ) cells immobilized on SiO2 /TiO2 matrix, () cells immobilized on SiO2 /MnO2
reduction in starch concentration from 4.0 to 1.0%, resulted in the matrix. Assay conditions: temperature 60 ◦ C, 10% (w/v) maltodextrin in 50 mM
specific CGTase production decreasing from 8.37 to 6.95 U/ml h. Tris–HCl buffer, pH 8.0, and 5 mM CaCl2 .
 C. Mazzer et al. / Biochemical Engineering Journal 41 (2008) 79–86
ent between the initial biomass values of 1.0 and 2.0 g. It is worth
noting that the increase in initial biomass to 2.0 g did not result
in an increase in the production of this cyclic oligosaccharide. For
the free cells, ␤-CD production was significantly different at all ini-
tial biomass concentrations, with the greatest production occurring
with 1.0 g of cells.
Moriwaki et al. [25] also evaluated the influence of initial
biomass on ␤-CD production using B. firmus strain 37 on the same
matrices and with the same reaction medium used in the present
study, with the only differences being temperature (50 ◦ C instead
of 60 ◦ C) and reaction time (144 h instead of 120 h). In their study,
for cells immobilized on SiO2 /TiO2 , an increase in biomass from
1.2 to 3.0 g promoted a 20% increase in ␤-CD production, and for
cells immobilized on SiO2 /MnO2 , an increase in biomass from 1.2
to 1.8 g lead to a 25% increase in ␤-CD production, which remained
constant when 2.4 and 3.0 g of cells were used.
Similar studies were reported by Abdel-Naby et al. [22], in which
maximum CGTase activity was achieved using 1.2 g of Bacillus amy-
loliquefaciens cells, and an increase to 1.4 g resulted in a slight
reduction in activity.
The reason why an increase in ␤-CD production does not occur
when initial biomass is increased has been explained by Moriwaki
et al. [25] as being that the biofilm is produced in layers on the
external surface of the matrix and, therefore, the activity is limited
to just the biocatalyzer located in the outermost layer of the biofilm
that has contact with the substrate.
3.4. ˇ-CD production by the methods of adsorption on inorganic
matrix and entrapment in gel
Various studies have reported the immobilization of cells by
the entrapment in gel technique [22,23,28,29,31,36,44]. This stim-
ulated our research group to carry out assays in the present study
on the immobilization of B. firmus strain 37 in calcium alginate as
well.
␤-CD production by free cells and cells immobilized by the
adsorption on SiO2 /TiO2 and SiO2 /MnO2 methods were compared
to that of cells entrapped in calcium alginate for a period of
10 days without renewal of the maltodextrin substrate, which
had an initial concentration of 10% (w/v). The cells immobi-
lized on SiO2 /MnO2 showed a ␤-CD production of 17.3 ± 0.5 mM,
while the cells immobilized on SiO2 /TiO2 showed a production of
16.7 ± 0.4 mM, with increasing production throughout the whole
assay period. For the cells immobilized in calcium alginate, ␤-CD
production increased gradually until the 3rd day, reaching a maxi-
mum value of 4.1 ± 0.1 mM, which remained constant until the end
of the assay. This value was lower than that obtained for free cells
(8.3 ± 0.2 mM), which showed similar behavior (Fig. 4). CGTase spe-
cific activity also increased during the evolution of the 10-day assay,
reaching values of 211 U/mg for cells immobilized on SiO2 /TiO2 ,
218 U/mg for cells immobilized on SiO2 /MnO2 and 103 U/mg for free
cells. For cells immobilized in calcium alginate, enzymatic activity
reached 53 U/mg.
Kunamneni et al. [36] and Jamuna and Ramakrishna [29] evalu-
ated the production of CGTase and ␣-amylase, respectively, from
Bacillus sp cells immobilized in calcium alginate. The results
obtained by Kunamneni et al. [36] revealed that CGTase production
initially declined for 3 days, although the quantity of enzyme then
remained constant for 18 days (400 U/ml), and thereafter fell. In the
study by Jamuna and Ramakrishna [29], ␣-amylase activity reached
its maximum value within 120 h and then declined gradually until
the end of the assay.
According to Jamuna and Ramakrishna [29], the process of
immobilization leads to modifications in the microenvironment,
and various metabolic and morphological alterations can occur in
83
Fig. 4. ␤-cyclodextrin (␤-CD) production by free cells and cells immobilized by
adsorption and entrapment in gel () Cells immobilized on SiO2 /MnO2 matrix,
() cells immobilized on SiO2 /TiO2 matrix, (䊉) free cells, () cells immobilized in
calcium alginate). Assay conditions: temperature 60 ◦ C, 10% (w/v) maltodextrin in
50 mM Tris–HCl buffer, pH 8.0, 5 mM CaCl2 and 3.0 ml of cell suspension.
the cells. Herrero et al. [44] also observed an environmental alter-
ation for immobilized cells, compared to free cells, which affected
their physiological conditions. Taking this into consideration in the
case of immobilization in calcium alginate in the present study, the
stress induced by the conditions of immobilization to which the
cells were submitted, might have compromised CGTase synthesis
and ␤-CD production.
Another possibility is that the granules of calcium alginate func-
tion as a barrier against the access of the enzyme to the substrate.
According to Laca et al. [45], the gel must have appropriate diffu-
sivity values to enable interaction between the molecules. In the
present study, this possibility was investigated by SEM, carried out
with samples collected after 24 h of immobilization. In Fig. 5B, a
transversal section, it can be seen that the cells are trapped inside
the granule. However, as Fig. 5A shows, there are no microorgan-
isms on the surface of the gel. It was also observed in the present
study that the alginate beads were still intact after 10 days of assays.
Herrero et al. [44] evaluated malolactic fermentation using Oeno-
coccus oeni immobilized in alginate beads, and also observed, by
optical microscopy, that the appearance of the beads did not alter
during the 17 days of fermentation, that is, that they did not rupture.
However, for SiO2 /MnO2 and SiO2 /TiO2 , the SEM results showed
that the cells were adsorbed on the surfaces of these inor-
ganic matrices (Fig. 5C and D, respectively). The SiO2 /TiO2 matrix
shows good characteristics of adsorption, although the quantity of
adsorbed cells is greater on the SiO2 /MnO2 matrix. These results
are in agreement with those of the studies by Moriwaki et al. [25],
in which good adsorption of B. firmus strain 37 was observed on
SiO2 /MnO2 and SiO2 /TiO2 matrices.
3.5. ˇ-Cyclodextrin production in repetitive cycles (operational
stability)
The possibility of the repeated use of immobilized B. firmus
strain 37 cells for the production of ␤-CD was evaluated using four
cycles of 5 days each (Fig. 6).
For the cells immobilized in calcium alginate, the best results
were achieved in the third cycle, obtaining 4.8 ± 0.2 mM of ␤-CD,
which corresponds to a 20% increase in production in relation to the
first cycle. A significant reduction in ␤-CD production was observed
at the end of the fourth cycle (480 h of assay), in which the cells
84 C. Mazzer et al. / Biochemical Engineering Journal 41 (2008) 79–86
Fig. 5. Scanning electron microscopy of immobilized Bacillus firmus strain 37 cells. (A) Absence of microorganisms on the external surface of a calcium alginate gel granule and
(B) presence of microorganisms inside a calcium alginate gel granule (transversal section). (C) Cells immobilized on SiO2 /MnO2 matrix. (D) Cells immobilized on SiO2 /TiO2
matrix.
only achieved 54% of the production obtained in the third cycle.
This reduction may have been a function of cell death, consid-
ering that there was no process of cell desorption. According to
Laca et al. [45], there is a very low concentration of oxygen inside
the granule, and this can help to cause cell death. This fact was
confirmed by determining the number of CFUs present in the super-
natant before and after the first production cycle. The number of
CFUs in the supernatant deriving from the separation of the cells
recently immobilized in calcium alginate was less than 10 ml−1 ,
and there was no cell growth at the end of the cycle. The specific
enzymatic activity obtained in the third cycle for the CGTase of the
cells immobilized in calcium alginate was 62 U/mg. The free cells
showed a decrease in production from the second cycle, falling
from 6.4 ± 0.2 to 1.1 ± 0.1 mM of ␤-CD. At the end of the fourth
cycle, the ␤-CD production by the free cells was around 10 times
Fig. 6. ␤-cyclodextrin (␤-CD) production in four repetitive cycles from Bacillus fir-
mus strain 37 cells. () Cells immobilized on SiO2 /TiO2 matrix, () free cells, (䊉) cells
immobilized in calcium alginate, () cells immobilized on SiO2 /MnO2 matrix). Assay
conditions: temperature 60 ◦ C, 10% (w/v) maltodextrin in 50 mM Tris–HCl buffer, pH
8.0, 5 mM CaCl2 and 3.0 ml of cell suspension.
lower than the value obtained for the cells immobilized in calcium
alginate.
Mamo and Gessesse [28] also observed that repetitive produc-
tion of amylase using Bacillus sp cells immobilized in alginate,
agar and agarose was stable, and that productivity increased until
the third cycle for all the matrices. They argued that immobiliza-
tion may have reduced autolysis and that cell growth may have
occurred.
For the B. firmus strain 37 cells immobilized on SiO2 /TiO2 and
SiO2 /MnO2 , the best results for ␤-CD production were obtained
at the end of the first cycle, achieving values of 11.0 ± 0.2 and
11.4 ± 0.3 mM of ␤-CD, respectively. At the end of the second cycle
(240 h of assay), the cells immobilized on SiO2 /TiO2 and SiO2 /MnO2
maintained 55 and 61% of the production obtained in the first cycle,
respectively. Thereafter, there was a gradual fall in ␤-CD produc-
tion until the fourth cycle, due to the weak bonds between the
cells and their inorganic matrices, resulting in their desorption.
The process of desorption was confirmed by determining the num-
ber of CFUs/ml in the supernatant at the end of the first cycle,
which produced values of 2.3 × 103 and 4.0 × 102 for SiO2 /TiO2 and
SiO2 /MnO2 , respectively.
The low activity of the cells entrapped in calcium alginate
gel occurred due to the diffusional limitations of the substrate
molecules in penetrating the interior of the gel. However, due to
the entrapment of the cells, they did not desorb from their support,
as occurred with the cells immobilized on the inorganic matrices.
Moriwaki et al. [25] observed similar behavior to that seen in
the present study when using the same microorganism, inorganic
matrices and assay conditions as employed in the present study, but
with different cell cultivation conditions. In their study, cell revital-
ization was carried out for every cycle in order to obtain a new lot
of cells, while in the present study, a single lot was prepared and
stored at −80 ◦ C. The best results were also obtained in the first
 C. Mazzer et al. / Biochemical Engineering Journal 41 (2008) 79–86
cycle, with there being a decline starting from the second cycle, in
which both of the matrices maintained around 60% of the produc-
tion obtained in the first cycle. Moriwaki et al. [25] also evaluated
the possibility of cell desorption by determining the protein content
of the inorganic matrices containing the adsorbed cells. Desorption
was confirmed due to a reduction in the protein content at the end
of the production cycle.
4. Conclusion
The best conditions for the production of ␤-CD from B. firmus
strain 37 cells immobilized on SiO2 /TiO2 and SiO2 /MnO2 inorganic
matrices were determined by using different temperatures, sub-
strate concentrations and biomasses. The optimized conditions
were: 1.0 g of cells, a temperature of 60 ◦ C, and a reaction medium
containing 10% (w/v) maltodextrin, which resulted in ␤-CD pro-
ductions of 11.7 ± 0.1 and 11.2 ± 0.1 mM by cells immobilized on
SiO2 /TiO2 and SiO2 /MnO2 , respectively, after 120 h of assay. There
was no significant difference in ␤-CD production by the cells immo-
bilized on the two matrices, and the use of these matrices resulted
in superior production to that obtained by free cells.
On comparing ␤-CD production by cells immobilized by adsorp-
tion and by entrapment in gel, it can be observed that the
values obtained using the inorganic SiO2 /TiO2 (16.7 ± 0.4 mM)
and SiO2 /MnO2 (17.3 ± 0.5 mM) matrices were superior to those
obtained by the use of calcium alginate (4.1 ± 0.1 mM) and free cells
(8.3 ± 0.2 mM). It was also observed that the cells immobilized on
the inorganic matrices maintained their activity throughout the
whole period of assay (10 days) and, further, showed a constant
increase in ␤-CD production. The fact that the cells immobilized in
calcium alginate achieved lower production to that of the free cells
is due to the stress induced by the conditions of immobilization to
which they were submitted, which compromised ␤-CD production.
The repetitive-cycle assays demonstrated that there was an
increase in ␤-CD production from cells immobilized in calcium
alginate up to the third cycle, which was around 10 times higher
than that obtained by the free cells at the end of the fourth cycle.
The cells immobilized on the SiO2 /TiO2 and SiO2 /MnO2 inorganic
matrices maintained good ␤-CD production up to the end of the
second cycle (240 h of assay), after which there was a gradual
decline in the production of this cyclic oligosaccharide up to the
fourth cycle, as a function of the process of cell desorption from the
matrices.
It was concluded that the choice of immobilization method
depends on the production system used, as immobilization on inor-
ganic matrices and in calcium alginate have different characteristics
and advantages. The results of this study show that when the inter-
est is the continuous use of the immobilized cells, the method of
adsorption is better, but when repetitive cycles are being used, the
method of entrapment in gel was observed to be superior.
The use of cells immobilized on SiO2 /TiO2 and SiO2 /MnO2 is a
promising method for the industrial production of ␤-CD, while also
having the added benefit of being able to recover the cells from the
reaction medium. Another advantage of this method is the possi-
bility of the use of alkaline conditions, which are required for the
growth of B. firmus and which can reduce the risk of contamination
by other microorganisms. A method that enables selectivity in the
production of cyclodextrins is of industrial interest, and, according
to Martins et al. [27] and Tardioli et al. [46], immobilization offers
this possibility.
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