Advances in Bioscience and Biotechnology, 2011, 2, 8-12 ABB
doi:10.4236/abb.2011.21002 Published Online February 2011 (http://www.SciRP.org/journal/abb/).
Published Online February 2011 in SciRes. http://www.scirp.org/journal/ABB
Ca-alginate spheres behavior in presence of some solvents and
water-solvent mixtures
Luis G. To rr es, A ngelica Ve lasquez , Marco A . Brito-Arias
Unidad Profesional Interdisciplinaria de Biotecnología. Instituto Politecnico Nacional. Av. Acueduct o s/n. Colonia Barrio la La guna
Ticoman; delegacion Gustavo A. Madero 07340 D. F., Mexico.
Received 25 June 2010; revised 5 July 2010; accepted 12 July 2011.
ABSTRACT
Immobilization systems more frequently used are
calcium alginate spheres. These biocatalysts have
many potential applications in the immobilization of
enzymes, prokaryotic cells, vegetal and animal cells,
algae, organelles and mixtures of these living compo-
nents. Other applications of immobilized cells imply
the use of non aqueous systems. Some bioconversions
are carried out in the presence of solvents such as
hexane acetone or acetonitrile, or mixtures wa-
ter-solvents. The aim of this work was to investigate
the behaviour of Ca-alginate spheres when put in
contact with different solvents (water, diesel, ethanol,
methanol, acetone, n-hexane, isopropyl alcohol, THF,
acetonitrile, and toluene), or solvent-water mixtures
(i.e., ethanol-water), regarding the resistance of the
alginate spheres after days of contact. Calcium algi-
nate particles suffered different damages, depending
on the solvent t h ey were put in contact. Water did not
damaged the Ca-alginate structure with or without
Ca present. On the other hand different solvents lost
a proportion of volume, i.e., n-hexane (16%), metha-
nol (19%), ethanol (19.5%), toluene (22%), diesel
(34%), acetone (765), isopropyl alcohol (80%), THF
and acetonitrile (total loss, total destruction). Nor the
dielectric constant nor the polarity indexes were ca-
pable of explaining the difference on the volume loss
or total sphere destruction, except for water-ethanol
mixtures.
Keywords: Dielectric Constant; Ca-Alginate;
Immobilization, Solvents; Spheres; Polarity Index
1. INTRODUCTION
Nowadays, there is an increasing interest in producing
high amounts of ethanol as an alternative fuel to fossil
fuels in the whole world. In the USA, the production
demand for 2012 is 7.5 billion gallons per year [1].
Ethanol is a valuable alternative to petroleum-based
transportation fuels. The traditional ethanol manufac-
ture was using sugar and yeast, though there are many
other alternatives such as using wheat, barley, sorghum,
beets, cheese whey, potatoes, and many other feed-
stocks [2]. Over 90% of the ethanol produced in USA is
made of corn. In opposite, in Brazil—the world’s larg-
est producer—most ethanol is made from sugar cane.
Other sugar sources for ethanol production are cellu-
losic feedstocks, i.e. agricultural waste, forestry resi-
dues and even solid municipal waste [2].
Many microorganisms can convert sugars to ethanol,
and they can do it as free cells or into immobilized
systems. These systems have proved to enhance pro-
duction rates based in simple facts as the following: 1)
immobilized systems can achieve high cell loads and
maintain them for longer periods, 2) immobilization
systems have a protector effect over cells, in particular
when products or substrates are toxic to cells [3], and 3)
these systems are flexible and allow the co-immobi-
lization of different microorganisms or microorganisms
and enzymes, or even cofactors.
Immobilization systems more frequently used are
calcium alginate spheres [4]. These biocatalysts have
many potential applications in the immobilization of
enzymes, prokaryotic cells, vegetal and animal cells,
algae, organelles and mixtures of these living compo-
nents. See Chevalier and de la Noue [5] experiments
with co-immobilization of algae and bacteria.
Other applications of immobilized cells imply the
use of non aqueous systems. Some bioconversions are
carried out in the presence of solvents such as hexane
acetone or acetonitrile, or water-solvents mixtures.
In recent years, it has been reported the use of im-
mobilized microorganisms in order to modify the con-
tent of certain molecules present in crudes and fractions,
such as diesel or gasolines. Target molecules are sul-
phur or nitrogen compounds, with linear, branched,
cyclic or even aromatic structures.

L. G. Torres et al. / Advance s i n B io s ci e nce and Biotechnol o gy 2 (2011) 8-12 9
Many other systems have been reported, where Ca-
alginate spheres deals with presence of non aqueous
phases. Hedstrom et al. [6] used this kind of gels to
immobilize Candida antartica lipase in the esterifica-
tion of 2-octanol and hexanoic acid in hexane.
To mention other, Hertzberg et al. [7] worked with
immobilized lipase to catalyze alkyl butanoate forma-
tion, transesterification reactions and hydrolysis of bu-
tyl butanoate. For these reactions, the authors assessed
the stability of calcium alginate beads in ethanol, ace-
tone, pyridine, 2-butanone, hexane, and iso-hexane.
They measured the relative volume change when beads
were transferred from CaCl2 to different solvents.
When calcium alginate spheres are putt in contact
with different solvents and solvents-water mixtures,
different behaviours are depicted. It has been reported
that Ca-alginate spheres as any hydrogel are constituted
mainly by water (typically 96-99%), tend to be dehy-
drated in contact with alcohols, such as ethanol or iso-
propyl alcohol. Other solvents cause other damages in
the Ca-alginate spheres such as shrinking, deformation,
dryness, and even destruction.
The aim of this work was to investigate the behav-
iour of Ca-alginate spheres when put in contact with
different solvents (water, diesel, ethanol, methanol,
acetone, n-hexane, isopropyl alcohol, THF, acetonitrile,
and toluene), or solvent-water mixtures (i.e. ethanol-
water), regarding the resistance of the alginate spheres
after days of contact.
2. MATERIALS AND METHODS
2.1. Ca-Alginate Spheres Preparation.
Ca-alginate spheres were prepared in accord to the pro-
cedure previously reported by Torres et al. [8]. Na-algi-
nate solutions were dropped in 0.1 M CaCl2 solutions
using system in order to control the spheres diameter in
about 3 mm. Spheres were prepared using calcium algi-
nate solutions cont ai ning 3% of sodium alginate.
2.2. Solvents
Double-distilled water was p repared in our laboratory by
ultra filtration. Ethanol, methanol acetonitrile, THF, n-
hexane, acetone, toluene and isopropylic alcohol were
purchased either with J.T. Baker or Aldrich Chemicals.
Diesel was a commercial sample, purchased in a Mexico
City gas station. Some properties of those solvents such
as dielectric constant and polarity index are shown at
Table 1.
2.3. Average Sphere Diameters
Average diameters were determined measuring 20
spheres with a veneer. Standard deviations were calcu-
Table 1. Some solvent physichochemical properties.
Solvent Dielectric constant
DC Polarity Index* PI
Water 80.0 9.0
Ethanol 24.6 5.2
Methanol 32.7 5.1
Acetonitrile 37.5 5.8
THF 4.0 4.0
n-Hexane 1.9 0.0
Acetone 21 5.1
Isopropyl alcohol20.3 3.9
Toluene 2.3 2.4
*From http: //www.chemical-ecology.net/java/solvents.htm
lated using Excel program (Microsoft).
2.4. Spheres Behaviour in Presence of Different
Solvents and Mixtures
200 prepared alginate beads were put in a 250 ml Er-
lenmeyer flask together with 100 ml the selected solvent
or mixture. Flasks were gently shaked in an orbital agi-
tator for 4 days. A sample of 20 spheres was em ployed to
measure initial and final averag e diameters.
3. RESULTS AND DISCUSSION
Table 2 shows the changes suffered by 3% calcium
alginate spheres after 3 days submerged in the different
solvents. In the case of water, a 0.1 MCaCl2 solution was
used to prevent Ca diffusion from the spheres to the wa-
ter. Initial batches of spheres were 3.13 mm in diameter.
Ca-alginate spheres were submerged in the solvents
and observed after 24, 48, 72 and 96 hours. All experi-
ments were carried out at room temperature (about 20 ±
2o C). Spheres submerged in water (added with CaCl2)
did not show any change during the four days, as ex-
pected. At the end of the process the spheres looked just
like at the beginning of the experiment and the average
sphere diameter decreased up to 3.047 ± 0.026 mm, giv-
ing a volume loss of about 2.9%, which is negligible.
That was not the case of the spheres submerged in
n-hexane, where the particles were adhered to the bot-
tom of the flask from the beginning, they turned yellow
and at day four, all spheres seem very soft. After meas-
uring the averaged diameter of 20 particles, the final
diameter was of 2.904 ± 0.177 mm, resulting in a vol-
ume loss of 15.9%, which is an important figure.
When immersing the 200 Ca-alginate spheres on
methanol, no changes were observed during the first 24
hours, but at the second day, some turbidity appeared in
the flask. At third day turbidity was a little higher but no
changes were observed at day 4. Final average diameter

L. G. Torres et al. / Advance s i n B io s ci e nce and Biotechnol o gy 2 (2011) 8-12 10
Table 2. Changes in the Ca-alginate spheres when submerged in different solvents.
Elapsed time (days)
Solvents 0 1 2 3 4
Final diameter
Ave ± std dev Volume loss (%)
CaCl2 in water NCh NCh NCh NCh Some tu rbidity 3.047 ± 0.026 2.9
n-Hexane Spheres stiked to
bottom Yellowi sh sp heresYellow More yellowSoft spheres 2.904 +/- 0.177 15.9
Methanol NCh Some turbidity More turbidityNCh NCh 2.870 ± 0.032 18.8
Ethanol NCh NCh NCh NCh NCh 2.863 ± 0.072 19.5
Toluene Spheres st icke d t o the bottom NCh Some turbidity,
spheres ag-
glomerated NCh Very brilliant
spheres 2.837 ± 0.212 21.6
Diesel Spheres sticked to bottom
Some turbidity,
spheres agglomer-
ated NCh NCh
Very brilliant
spheres 2.676 ± 0.221 34.3
Acetone NCh Yellowish NCh NCh Yellow 2.003 ± 0.154 75.6
Isopropyl alcohol NCh Diameter diminu-
tion NCh NCh Stiff spheres 1.788 ± 0.158 80.4
THF Diameter diminu-
tion Some turbidity NCh NCh More turbidity - -
Acetonitrile Spheres destruc-
tion Lentil form Diameter di- minution NCh Yellow lentils - -
NCh, No changes observed
was 2.80 ± 0.032 mm, with a volume loss of 18.8%. The
behaviour of Ca-alginate spheres in ethanol was very
similar to that observed in methanol, as expected. In this
case, the final sphere diameter was in average 2.863 ±
0.072 mm, and t he vol ume loss was of 19.5 mm.
When Ca-alginate particles were submerged in tolu-
ene, it was observed that particles were adhered to the
bottom from the first day and at the end of the process,
spheres were very brilliant, with an average final d iame-
ter of 2.837 ± 0.212 mm and a volume loss of 21.6%.
In the case of diesel, it was observed that spheres tend
to agglomerate, and at the end of the process the parti-
cles were very brilliant and the volume loss was of 34%.
When Ca-alginate spheres were submerged for 4 days
in acetone, the media turned very yellow and the final
average diameter was of 2.003 ± 0.54 mm, giving a
volume loss of 75.6%. When spheres were put in contact
with iso-propylic alcohol, an important diameter diminu-
tion was observed at the second day, and at the end of
the process very stiff, small particles were produced.
Final average diameter w as of 1.788 ± 0.158 mm and the
volume loss was of 80.4%.
The last two experiments were similar in results.
When immersing the Ca-alginate spheres on acetonitrile
and THF, destruction of the spheres was observed, tur-
bidity appeared from the beginning of the process and it
was impossible to determine for both cases the final
spheres average diameter. It was considered that the
sphere were unre co vered.
It is well known that Ca-alginate spheres are produced
by the interaction of alginic acid, which is a linear poly-
saccharide and the divalent calcium, forming a structure
known as the egg-box model. The destruction of the
Ca-alginate gel should be promoted by the Ca loss, or
either by the solubilisatio n of water in the add ed solvent.
A way to analyze the effect of the characteristics of the
added solvent is the measure of the polarity index and
the dielectric constant of solvents.
The first is the relative measure of the degree of in-
teraction of the solven t with various polar test solutes. In
that way, pentane and 1,1,2-trichlorotrifluoroethane have
a polarity index of 0 (no interaction). Cyclopentane,
heptane, hexane, iso-octane and petroleum ether have
very low polarity indexes (0.1).
On the other hand, dimethyl acetamide (6.5),
n-methylpyrrolidone (6.7) and dimethyl sulfoxide (7.2)
have high polarity indexes. In the case of water, a value
of 9-10 has been reported. In the case of diesel, it is not
possible to give a figure for dielectric constant. Since
this is a mixture of many compounds, and diesel has a
huge variability depending of the source (crude) and
process employed to produce it.
The dielectric constant values are referred to the
measure of extent to which concentrates electrostatic
lines of flux. It is the ratio of the amount of stored elec-
trical energy when a voltage is applied, relative to the
permittivity of a vacuum. The correct name for this pa-
rameter should be relative static permittivity.
With these facts in mind, the loss of volume (%)
measured in every experiments were plotted as a func-
tion of the polarity index in Figure 1. As noted, four of
the points are around 15-20% volume loss, for polarity
indexes between 0 and 5.2. For one of the points (that
corresponding to water added with calcium), volume
loss is negligible (2.9%). Ther e are two points with high
volume losses (acetone and isopropyl alcohol, with po-
larity indexes between 5.1 and 3. That means that polar-
ity index do not explain why some Ca-alginate spheres
shrink in some solvents more than in others.
In a new intent to explain the Ca-alginate volume loss

L. G. Torres et al. / Advance s i n B io s ci e nce and Biotechnol o gy 2 (2011) 8-12 11
Figure 1. Volume loss vs polarity index for differ-
ent solvents.
in the different solvents, Figure 2 shows the plot of the
dielectric constant value for the different solvents, and
the corresponding volume loss (%). Again, most of the
points of dielectric constant between 0 and 35, corre-
sponded to volume loss of around 20%.
A new set of experiments were carried out using water,
ethanol and water-ethanol mixtures. The results of the
experiments after 4 days are those shown in Table 3.
The polarity indexes and dielectric constants for the wa-
ter-ethanol mixtures are also included. It is interesting to
note that volume loss for Ca-alginate particles was of
only 2.7% in the case of water (no CaCl2 was added),
while for pure ethanol the volume loss was of 38.25.
The rest of the volume losses seem to be proportional
to the water-ethanol mixture. If the values of volume
loss for the particles are plotted against the polarity in-
dexes PI for the given water-ethanol mixtures, a straight
line represents the behaviour of the experimental line,
with a R2 value of 0.96 (see Figure 3).
The equation is:
 
573739 Volume loss %..PI (1)
When the volume loss was plotted against the dielec-
tric constant DC a straight line was obtained too (figure
not shown), but the form of the resulting equation is:
 
52 690 595Volume loss %.. DC (2)
With R2 = 0.976.
Solvents with a relative static permittivity greater than
Figure 2. Volume loss vs dielectric constants for dif-
ferent solvents.
Figure 3. Volume loss vs polarity index for
water-ethanol mixtures.
Table 3. Changes in the Ca-alginate spheres when submerged
in water-ethanol mixtures.
Water/ethanol
mix Volume loss (%) Dielectric constant Polarity index
100/0 2.7 80 0
80/20 15.2 67.3 0.88
60/40 20.78 56.6 1.76
40/60 24.4 45.9 2.64
20/80 30.62 35.2 3.52
0/100 38.25 24.6 4.4
From [9].
15 can be further divided into protic and aprotic. Protic
solvents solvate anions (negatively charged solutes)
strongly via hydrogen bonding. Water is a protic solvent.
Aprotic solvents such as acetone or dichloromethane
tend to have large dipole moments (separation of partial
positive and partial negative charges within the same
molecule) and solvate positively charged species via
their negative dipole. Using this new division it is
possible to analyze briefly the situation again. Acetone,
acetonitrile and THF are aprotic solvents, while water
and alcohols are non po lar ones.
4. CONCLUSIONS
Calcium alginate particles suffered different damages,
depending on the solvent they were put in contact. Water
did not damaged the Ca-alginate structure with or with-
out Ca present. On the other hand different solvents lost
a proportion of volume, i.e., n-hexane (16%), methanol
(19%), ethanol (19.5%), toluene (22%), diesel (34%),
acetone (765), isopropyl alcohol (80%), THF and ace-
tonitrile (total loss, total destruction). Nor the dielectric
constant nor the polarity indexes were capable of ex-
plaining the difference on the volume loss or total sphere
destruction, except for the ethanol-water mixtures.
When putting in contact the Ca-alginate spheres with
water, ethanol and water ethanol mixtures, different
volume loss (%) were observed. In this case, both di-
electric constants for mixtures and polarity indexes for
mixtures, explained the volume loss. Two very simple
equations expressing the volume loss as a function of
dielectric constant or polarity indexes were developed,

L. G. Torres et al. / Advance s i n B io s ci e nce and Biotechnol o gy 2 (2011) 8-12 12
which result very interesting in predicting the volume
loss by s for a given water-ethanol mixture. This is par-
ticular important for the case of ethanol production by
immobilized cells.
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