Article

Journal of
Copyright © 2017 American Scientific Publishers
Nanoscience and Nanotechnology
All rights reserved Vol. 17, 4827–4836, 2017
Printed in the United States of America www.aspbs.com/jnn

Yeast Cells Immobilized in Ionic Crosslinked Hydrogel

Particles Based on Gellan and Gellan/Carboxymethyl
Cellulose—Comparative Study
Camelia Iurciuc (Tincu)1
2
∗ , Alexandru Savin1 , Patrick Martin2 ,
Catalina Anisoara Peptu1 , and Marcel Popa1
3
1
“Gheorghe Asachi” Technical University, Faculty of Chemical Engineering and Protection of the Environment,
Department of Natural and Synthetic Polymers, 700050 Iasi, Romania
2
Département Chimie, IUT Béthune, Université d’Artois, CS20819, 62408 Béthune, France
3
Academy of Romanian Scientists, Splaiul Independentei Str., nr. 54, sector 5, 050094 Bucuresti, Romania

The present work deals with the study of obtaining the spherical particles containing brewer yeast
cells immobilized in a hydrogel matrix based on gellan and gellan/carboxymethyl cellulose (sodium
salt). The influence of some factors of the process for obtaining the particles, by the ionic gelation
with magnesium acetate used as crosslinking agent, by an extrusion process through capillary, on
the particles properties such as structural stability, on morphological characteristics and on behavior
in aqueous Delivered bymedium
and alcoholic Ingentaofto:theState University
particles with or of New yeast
without York cells
at Binghamton
immobilized was stud-
ied. The particles wereIP:obtained
185.89.100.229 On: Tue,
from the mixtures 18 two
of the Apr polysaccharides
2017 13:00:49 and theirs properties
were compared to those based Copyright:
only onAmerican
gellan. OneScientific Publishers
can observe a certain influence of the CMCNa
amount on the above mentioned properties essentially determined by their higher crosslinking den-
sity of the polymers mixture compared to those prepared only with gellan. Biocatalytic properties of
the particles with yeast cells immobilized depends also on the crosslinking density; the bioreactors
with a higher concentration of CMCNa present a slightly lower biocatalytic activity. The particles
with immobilized yeast cells can be used for a higher number of hydrolitic cycles; the number of
cycles decreases when the CMCNa content increases in the particles composition due to the higher
crosslinking degree leading to the inhibition of the yeast cells proliferation.
Keywords: Gellan, Carboxymethyl Cellulose, Immobilized Yeast Cells, Hydrogel Particles.

1. INTRODUCTION are immobilized protects them from the mechanical and

Yeast cells (Saccharomyces cerevisiae) immobilisation, for
obtaining ethanol by sugars fermentation is a research field
that has been intensively developed due to its attractive
technique and economic benefits. In the last years con-
tinuous processes are preferred since an improved fer-
mentation efficiency and a higher productivity with lower
operating costs are obtained.1
By immobilization, usually in polymer matrices, yeast
cells are protected by the metabolites toxicity produced by
ethanol. The viability of the immobilized cells is main-
tained and consequently a higher efficiency of the biorector
can be obtained. Polymer matrix in which yeast cells
∗
Author to whom correspondence should be addressed.
environmental factors.2
3 Particles with immobilized yeast
cells can be considered real bioreactors, whose dimensions
higher than of the free cells allow their facile recovery
from the reaction medium by simple filtration. The recov-
ered bioreactors can be reused in subsequent fermenta-
tion processes.4 The techniques involved in the microbial
cells encapsulation are: emulsification, extrusion, complex
coacervation, spray drying, gel entrapment and polymer-
ization by radiation. The extrusion method is most often
used due to the mild processing conditions that allow a
optimal encapsulation and the viability of the cells is not
affected. This method was often used for alginates and
carrageenan and involves the extrusion of small droplets
from the biopolymer solution in which a yeast cells were

J. Nanosci. Nanotechnol. 2017, Vol. 17, No. 7 1533-4880/2017/17/4827/010 doi:10.1166/jnn.2017.14324 4827

Yeast Cells Immobilized in Ionic Crosslinked Hydrogel Particles Based on GLL and GLL/CMC
suspended through capillaries in a bath that contains dif-
ferent concentrations of the crosslinking agent.5
The particles with immobilized yeast cells (microbiore-
actors obtained) should remain stable in various conditions
such as pH, osmotic pressure and turbulence.
Besides protecting the encapsulated cells from the unfa-
vorable environment, polymer matrix should ensure the
efficient exchange of nutrients and metabolites with the
environment. The microbioreactors efficiency is depend-
ing on their specific surface area, porosity, the swelling
capacity and stability.6
7
The polysaccharides fulfill the conditions required for
the immobilization of a biologically active ingredient:
they are non-toxic, pharmacologically inert, do not con-
tain any impurities, additives or residual components of
the crosslinking processes. Also, they present a chemical
structure clearly defined and their ability to form physical
gels even at very low concentrations is one of their most
important functional properties. The ability of formation
of three-dimensional networks provides a way to increase
chemical and mechanical stability of the system. 8 to the initial values after cooling.20 The CMCNa is an
Mixtures of polysaccharides naturally obtained and
binary gels can be used as models for a complex cellular
structure.9
Due to their qualities, the polysaccharide hydrogels are
currently used in the biomaterials field for artificial tissues,
controlled release systems of the biologically
Delivered by Ingentaactive princi-
to: State University of New York at Binghamton
IP:certain
185.89.100.229
On: Tue, 18 Aprpaper
ples and encapsulation systems for food principles.
Biologically active principles are fixedCopyright: American
in gel matrix either Scientific Publishers
by similar processes to absorption, either by the chemical
processes leading to the formation of the complexes. In the
case of the polysaccharides, as gellan and carboxymethyl
cellulose, hydroxyl and carbonyl groups can be directly
bonded to the active principle.10
12
Gellan is one of the newest biopolymer used as gelling
agent available on the market of food industry. It is
an anionic polymer, linear, with repeating tetrasaccharide
units which consist of two residues of -D-glucose, one
of -D-glucuronic acid and the other of -L-rhamnose in
the ratio is 2:1:1.13
14 Gellan is a biodegradable and non-
toxic polymer; it is very stable at pH, the value ranging
between 2 and 1015 and it is resistant to the action of
enzymes such as pectinase, amylase, cellulase, papain and
16
lipase however a significant gellan degradation occurs in
galactomanase presence.17
Previous studies showed that gellan is suitable for the
yeast cells immobilization. Therefore, the literature rep-
ports that a biocatalyst with real prospects of being used
in sparkling wine production technology was obtained in
the form of spherical gellan particles in which the yeast
cells were immobilized. The particles were developed by
extruding the gel formed through a capillary in a bath of
2% CaCl2 solution (used as ionic cross-linking agent).18
Tan et al.,6 investigated the feasibility for yeast cells
encapsulation using as support the gellan gum, obtaining
4828
Iurciuc (Tincu) et al.
microbioreactors through an emulsification method with
the possibility to reuse them. Ethanol production during
the first three cycles using microbioreactors was compara-
ble to that of using free yeast. Microbioreactors are stable
and were easily recovered from the fermentation medium
by filtration. Therefore, they can be reused for at least 10
fermentation cycles with a relatively high yield of ethanol.6
Carboxymethyl cellulose (CMC) is a cellulose ether
containing the grouping of –CH2 –COOH (Na) bound by
the ether linkage to the hydroxyl group from C-6 of the
cellulose. The degree of substitution ranges from 0.4 to
2.2 and it is usually presented as sodium salt (CMCNa)
having a high solubility in water.19
The CMCNa dissolution in water leads to the formation
of colloidal solutions, that contain at low concentrations
threadlike macromolecules and gels at high concentrations.
CMCNa solutions are pseudoplastic and the gels are
thixotropic. The CMCNa solutions at acidic pH are vis-
cous (the viscosity increases with decreasing the pH val-
ues). The viscosity decreases with temperature but returns
anionic biocompatible polymer, that has applications in the
biomedical field,21 but according to our information it has
not been used to immobilize yeast cells. To the best of our
knowledge, in literature does not exist information on the
preparation of the spherical particles based on gellan and
CMC ionic crosslinked for yeast cells encapsulation.
This
2017presents
13:00:49a comparative study on the prepa-
ration, physical—chemical characterization of yeast cells
immobilized in gellan and in the matrices based on
gellan/CMCNa (in various concentrations ratios) both
with hydrogel characteristics and their application in glu-
cose fermentation processes. The polymer particles were
obtained by the ionotropic gelation of a polymer solution
that contains a suspension of yeast cells in the presence
of magnesium acetate. The shape of particles was ensured
using the extrusion method.
2. EXPERIMENTAL PART
2.1. Materials
Kelco Biopolymers supplied Gellan of the Kelkogel food
grade type having a molecular weight between and 2–3 ×
105 Da for deacetylated gellan.
Carboxymethyl cellulose was purchase from Sigma
Aldrich.
Magnesium acetate was purchase from Sigma Aldrich
and was used as solution, in double distilled water, in a
concentration of 2% (w\w).
Commercially available Pakmaya yeast, containing 30%
yeast cells (w/w) was used.
2.2. Preparation and Physical Chemical
Characterization of the Particles
In principle, the particle preparation it is based on the ionic
gelation of the two polysaccharides, in the presence of
J. Nanosci. Nanotechnol. 17, 4827–4836, 2017
Iurciuc (Tincu) et al. Yeast Cells Immobilized in Ionic Crosslinked Hydrogel Particles Based on GLL and GLL/CMC
Mg2+ ions from magnesium acetate used as crosslinking
agent. The particles characterization has included the eval-
uation of their stability in aqueous media in which they
have been formed and stored, the particles morphology, the
swelling characteristics determination in water, the analy-
sis of the biocatalysis capacity of the glucose fermentation
process in respect with the free yeast, and especially after
repeated fermentation cycles.
2.3. Obtaining Gellan and Gellan/CMCNa Particles
With and Without Brewers Yeast
0.25 g gellan (GLL) or mixture GLL/CMCNa in various
weight ratios was dissolved in 25 ml double distilled water
(the polymer concentration was 1% w/v) under stirring, at
80–90  C. Due to the low viscosity of the polymer solu-
tion does not result stable particles when it is added in the
bath containing the ionic crosslinker, a crosslinking before
extrusion was performed using 1.25 ml of magnesium
acetate solution with a concentration of 0.4% until a vis-
cous fluid is obtained which can be further extruded. The
solution was cooled to 30 –35  C, and then was extruded
using a syringe through a needle having a diameter of
600 m (22 Gauge diameter) and a length of 30 mm, in a
bath with 125 ml of 2% magnesium acetate solution.
The extrusion process of the polymer solution which
lasts about 10 minutes is performed under a constant pres-
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IP: 185.89.100.229 On: Tue,
diameter. Gellan and gellan/CMCNa spherical particles
Copyright: are Scientific
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formed instantly and they are maintained in the crosslinker
solution for 2 h followed by washing with double distilled
water to remove the excess of magnesium acetate. The
obtained particles were filtrated and kept at 4–6  C before
further characterizations.
The particles containing the brewers yeast were simi-
larly prepared and it can be mentioned that prior to the
extrusion in the gellan solution was added a suspension of
yeast prepared from an amount of yeast of 1.25 g in 5 ml
water, thus providing a mass ratio of 5/1 (yeast/polymer).
After preparation, the immobilized particles are stored at
4–6  C until further tests.
2.4. Stability in Aqueous Media
In aqueous or alcoholic medium the particles can suffer the
detachment of some fragments from the polymer matrix
and also yeast cells can come out from the particles both
causing the increase of the solution turbidity in which the
particles were suspended. A lower value of the transmit-
tance it is correlated with a lower structural stability of
the particles. Transmittance has been determined using a
BOECO S-22 UV-Vis spectrophotometer at 550 nm wave-
length and it was evaluated for all samples obtained both in
the supernatant after the extrusion process, and in the dou-
ble distilled water after different time periods. The trans-
mittance measurements were performed in triplicate and in
J. Nanosci. Nanotechnol. 17, 4827–4836, 2017
the Tables II–IV were presented the average values. The
standard deviations were placed in the limits of ±0.3%.
2.5. The Swelling Degree of the Particles
The obtained particles have a hydrogel character therefore
their behavior in water is important.
Five types of particles with and without immobilized
yeast cells have been selected for this study and the
swelling behavior in double distilled water was monitored.
Prior to this experiment the samples were dried at 40  C
in a vacuum oven until all the water from particles it was
eliminated and their weight remained constant.
A certain amount of dried sample were suspended in
15 ml water at 30  C (the temperature to which the glucose
fermentation takes place) and periodically (after every 1 h)
the amount of water retained was determined gravimetri-
cally. The swelling process was studied until equilibrium
(the amount of water retained remained constant).
The swelling degree was calculated using the equation:
Mwater
SD% = × 100 (1)
Mdry sample
Where: Mwater = Mswollen sample −Mdry sample —amount of dried
particles. —the water amount absorbed in the sample.
2.6. The Cell Viability and the Amount of
Yeast York
Cells at Binghamton
Immobilized
18 Apr 2017 13:00:49
For analysing the particles behavior during the fermenta-
Publishers
tion process it is very important to know the exact amount
of immobilized yeast cells. In this sense, the amount of
yeast cells from the supernatant after the particles prepara-
tion was necessary to know. The amount of cells from the
particles is represented by the difference between the ini-
tial amount of cells and the cell amount in the supernatant.
The amount of remaining cells in the supernatant was
determined by an indirect way using a specific technique.
Prior to this experiments, the calibration curve which cor-
related the cells number determined by optical microscopy
with the cells weight was plotted.
In all types of gellan or gellan/CMCNa particles it was
encapsulated the same amount of yeast, 1.25 g respec-
tively. The total amount of yeast cells (30% of the total
weight of the yeast), it is 0.375 g.
In order to determine cell viability, particles with immo-
bilized cells were maintained in 50 ml of an aqueous solu-
tion of magnesium acetate having a concentration of 2%
(extrusion solution) for 24 hours, then washed with bidis-
tilled water to remove the excess of magnesium acetate
and kept for another 48 hours in 50 ml of bidistilled water.
After each determination the particles were stored in a
refrigerator to a temperature of 4–6  C in extrusion solu-
tion or in bidistilled water, according to the environment
where this characteristic was evaluated.
In order to determine cell viability in alcoholic environ-
ments in which the particles are suspended, after washing
4829
Yeast Cells Immobilized in Ionic Crosslinked Hydrogel Particles Based on GLL and GLL/CMC Iurciuc (Tincu) et al.

250 Table I. Synthesized particles coding and their composition.

The number of yeast cells/ml×104
y = 26,993x Samples code
200 2
R = 0,9991 Without yeast With yeast GLL (%, w) CMCNa (%, w)

150
P0 PoD 1 0
P1 P1D 0,80 0,20
P2 P2D 0,65 0,35
100 P3 P3D 0,50 0,50
P4 P4D 0,35 0,65
50

0 made the counting for nonviable cells (autolysates) from

0 2 4 6 8 the total cells and their expression in percentage (num-
Yeast cells weight (mg/ml ×10–2) ber or weight, the latter determined from the calibration
curve—Fig. 1).
Figure 1. The calibration curve for the correlation of the yeast cells
number with their weight.
The yeast cells concentration (number of yeast cells/ml)
from the extrusion solution was determined after 12 and
they were suspended in a volume of 50 ml ethanolic solu- 24 h then the yeast particles were washed and resuspended
tion (100 g/l) and the transmittance and cell viability were in bidistilled water; concentration of the cells was deter-
determined at different time intervals. mined after 12 h, 24 h, 36 h and 48 h. Depending on the
Before determination the samples were maintained in number of living and lysed yeast cells that are found in the
alcohol solution at 30  C for 255 min. These parameters crosslinking solution after the aforementioned time peri-
were taken into account because the glucose fermentation ods to determine the cell viability (CV,%) the following
takes place at these temperature and each fermentation equation has been applied;
cycle lasts 255 min. Nliving cells
Specifically, cell viability and the yeast cells quantity CV % = × 100 (2)
Nliving cells − Nlysed cells
from the environment Delivered
in which by
particles with immobi-
Ingenta to: State University of New York at Binghamton
lized yeast cells were suspended, IP:
were determined usingOn:
185.89.100.229 an Tue, 18
ByApr 2017the
making 13:00:49
difference with the initial amount of
optical microscope (Optika Model B-330) Copyright: American Scientific
and a counting Publishers
yeast cells the remaining yeast cells immobilized in parti-
chamber. The method is often used for quality assessment cles has been determined.
of compressed yeast, determining the percentage of cells The cell viability measurements were performed in trip-
autolysates and the evaluation of the influence of some licate and in the Tables III and IV were presented the
environmental factors on the physiological activity of the average values. The deviations were placed in the limits
yeast cell. With Marienfeld counting chamber it can be of ±0.3%.

Table II. The transmittance value of the aqueous environments

in which the particles with and without encapsulated yeast were
suspended∗ .

Transmittance, T %

In extrusion
solution In double distilled water

Sample 12 h 24 h. 12 h 24 h. 36 h. 48 h

P0 99.8 99.0 99.0 98.2 96.7

P1 99.3 99.1 99.8 99.5 98.9 98.6
P2 99.3 98.8 99.5 99.4 98.8 98.5
P3 99.0 98.4 99.5 99.1 98.7 98.3
P4 98.4 98.2 99.3 98.7 98.3 98.1
P0D 96.3 94.8 96.8 90.2 88.0
P1D 98.4 98.0 99.1 98.7 98.3 98.0
P2D 98.4 97.4 99.1 98.6 98.1 97.8
P3D 97.9 97.4 99.2 98.2 97.5 97.0
P4D 97.3 96.7 99.0 98.2 97.4 96.9

Figure 2. Schematic structure of ionic interpenetrated network between Note: ∗ Concentration of the Mg(OCOCH3 2 before extrusion = 04%. Concentra-
GLL and CMCNa formed by ionic crosslinking with the Mg2+ . tion of the Mg(OCOCH3 2 in the extrusion solution = 2%.

4830 J. Nanosci. Nanotechnol. 17, 4827–4836, 2017

Iurciuc (Tincu) et al. Yeast Cells Immobilized in Ionic Crosslinked Hydrogel Particles Based on GLL and GLL/CMC

2.7. Scanning Electron Microscopy
The particle morphology was analysed using an electron
microscope HITACHI SU 1510.
The samples without yeast cells were cross sectioned
and analyzed on ice.
The samples with yeast cells were sectioned, dried and
covered with gold (environ 7 nm) (108-Cressington auto)
and examined at microscope.
2.8. Fermentative Activity Assay
Testing the glucose fermentation capacity of the particles
with yeast cells immobilized was performed in a fermenter
in which a volume of 50 ml glucose solution (c = 8%) in
a solution of magnesium acetate (0.5%) was introduced.

Fermentation was done at 30 C, monitoring the volume of
CO2 resulted from the reaction which was direct propor-
tional with the amount of glucose fermented in time and
the process efficiency. The time required for one fermen-
tation cycle is 255 min and the amount of CO2 released
was measured at every 15 min.
After each fermentation cycle the particles were recov-
ered from the fermenter, washed with double distilled
water for removing the excess of glucose and magnesium
acetate and then kept in a refrigerator to a temperature of

4–6 C until the next fermentation cycle.
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3. RESULTS AND DISCUSSION
IP: 185.89.100.229 On: Tue, 18 Apr 2017 13:00:49
Polysaccharides selected for the studyCopyright:
are polyanions that Scientific
American
varies by molecular mass value (CMCNa presents a lower
mass) and by the carboxylate anion density along the poly-
mer chain: an anion for every four glucose units from
GLL (structural unit) and 0.75 anions respectively on
each glucose unit in CMCNa. Consequently, the CMCNa
although it is characterized by a lower molecular weight,
can generate denser networks with gellan by the ionotropic
crosslinking with divalent cations. The magnesium acetate
was selected as crosslinking agent due to its advantages:
nontoxicity, CH3 COO− has a slight bazic behavior helping
the formation of the matrix due to the shifting of the reac- (P0-P4 compared P0D-P4D). The slightly lower transmit-
tion equilibrium and resulting a stable mecanichal struc-
ture, this anion does not affect the cells viability if the
concentration is 0,7% or lower and magnesium ions help
the brewer yeast cells proliferation.
The preparation of the particles with immobilized yeast
cells is based on the fixing of the yeast cells in the net-
work meshes formed by ionic crosslinking of the polymer
chains, due to the ionic bonds between magnesium cations
and the carboxylate anions from GLL and CMCNa. Pre-
liminary tests have proved that while GLL can form parti-
cles alone the CMCNa doesn’t form alone stable particles
probably due to lower molecular weight.
But by using these two polysaccharides in mixture, an
stable interpenetrating/interconnected network is formed.
Schematic structure of such network is presented in
Figure 2. The yeast cells, with negative electric charge in
the surface are reinforcing the polymer network structure
due to the bonds formed with the Mg2+ ions.
Based on preliminary results, in order to perform the
comparative study 5 types of particles were prepared, with
and without brewers yeast whose notation and composition
of the starting polysaccharides mixture are presented in
Table I.
In all the cases, the obtained particles are spherical with
a diameter of about 2.5 mm in the swelling state, as it were
obtained from the extrusion and crosslinking process. For
determination of the particles stability in time, that it is
important for their use in many fermentation cycles, it was
proceeded
of New to the
York determination
at Binghamtonof the solution turbidity in
which the particles
were suspended. Starting from the idea
Publishers
that less stable particles can release polymer fragments and
yeast cells in the environment in which are suspended, the
transmittance of the supernatant may present lower values.
This characteristic was determined at different time
intervals both in the crosslinking solution and in double
distilled water. Table II presents results obtained after mea-
suring the turbidity of the two aqueous environments in
which the particles were suspended.
One can observe that the particles without cells have
high transmittance values both in the crosslinking solution
after 24 h, as well as in double distilled water after 48 h
tance value in double distilled water was observed in the
case of plain gellan particles. It can therefore affirm that

Table III. The results obtained by determining of the cell viability and the cell concentration in the medium in which they were
kept.

Concentration of the yeast cells/ml × 104 in supernatant Cell viability, %

In solution of In solution of
2% (Ac) 2 Mg In double distilled water 2% (Ac) 2 Mg In double distilled water

Samples 12 h 24 h 12 h 24 h 36 h 48 h 12 h 24 h 12 h 24 h 36 h 48 h

P0D 165 38 585 − − 96 9573 94.15 − −

P1D 25 45 15 25 275 45 100 9474 100 90.91 9166 90
P2D 275 6 2 3 375 5 100 96 8889 92.31 9375 909
P3D 725 9 275 7 925 12 100 9473 100 94.11 9487 9411
P4D 775 95 575 8 95 14 100 95 9444 93.33 95 9492

J. Nanosci. Nanotechnol. 17, 4827–4836, 2017 4831

Yeast Cells Immobilized in Ionic Crosslinked Hydrogel Particles Based on GLL and GLL/CMC Iurciuc (Tincu) et al.

Table IV. The transmittance and the cell viability values in alcoholic environments.

Concentration of the
yeast cells/ml × 104 in
Transmittance, T% crosslinking solution Cell viability, %

Sample 12 h 36 h 60 h 84 h 12 h 36 h 60 h 84 h 12 h 36 h 60 h 84 h

P0D 99.2 98.3 98 97.6 3 3.5 4.5 5.5 100 93.3 90 88

P2D 98.9 98.4 98.2 97.8 1.75 2.75 4 5 87.5 91.66 94.11 90.9
P4D 98.9 98.8 98.6 97.9 3.75 5 6 7.25 93.75 90.91 92.3 90.62

hydrogel particles formed from mixtures gellan/CMCNa It is found that for the samples that have a lower trans-
are slightly more stable compared with gellan particles mittance value, the cell concentration in supernatant is
obtained under the same conditions, obviously due to higher which means that the supernatant turbidity is given
higher network density. For all the particles based on not only by the polymer fragments separated from the net-
gellan/CMCNa mixture the transmittance values in both work but also by the cells that are not encapsulated in
aqueous media are slightly higher than for the particles the polymer matrix. By increasing the CMCNa concen-
obtained only from gelan. In addition, it is found that tration in the polymer mixture, an increase in the cells
increasing the amount of CMCNa in the particles a slight concentration can be observed. These results are in good
decrease of the transmittance values can be observed. correlation with those obtained from transmittance mea-
The only possible explanation is that due to the CMCNa surements. Increasing the polymer crosslinking degree, the
macromolecules that have much smaller dimension com- network meshes become smaller, the network is denser,
pared to those from gellan, the ones located on the sur- and a higher cells number is not caught in the network
face of the particles, not being bonded to the network, and remains free in the medium in which the particles are
they could be detached in high number when the poly- crosslinked, being adsorbed only on the surface.
mer ratio CMCNa to GLL increases. It must be mentioned In the case of the P0D sample one can be observed that
that CMCNa alone doesn’t form stable particles. Also, the cells number that diffuses from the particles is much
for particles containingDelivered
yeast cells,by
increasing
Ingenta the CMCNa
to: State University ofcompared
higher New Yorktoat theBinghamton
cells number that diffuses from the
content, the transmittance values IP: decrease
185.89.100.229 but Tue, 18 Apr 2017 13:00:49The samples containing CMCNa
slightly, On: other analyzed samples.
still remain higher compared with the sample contain- Scientific
Copyright: American Publishers
forms a denser interpenetrating network than the ones con-
ing only gellan. In the case of all the particles, with and taining only gellan (P0D), due to the higher number of
without brewers yeast encapsulated, the stability slightly carboxylic groups participating in ionic crosslinking. Con-
decreases in time, but it is still high (minimum transmit- sequently, the yeast cells are more strongly anchored in
tance value is 96.7%). For the use of the particles con- the hydrogel matrix and diffuses less from the particles
taining yeast cells in the repeated fermentation cycles, the in supernatant. It is noted that cell viability presents high
biocatalytic efficiency depends on the cells viability and values for all the samples analyzed after various periods
of their proliferation ability. Table III presents the results of time. These high values of the cell viability confirm
obtained after the particle stability evaluation in aqueous the fact that the polymer matrix in which yeast cells are
medium.
800

2000 700
P1 P2
1800
Swelling degree, SD%

P3 P4 600
Swelling degree, SD %

1600
P0
1400 500
1200
400
1000
800 300

600 200
400
100 PD1 PD2 PD3
200
PD4 PD0
0 0
0 5 10 15 20 25 0 10 20 30 40
Time (h) Time (h)

Figure 3. Swelling kinetics for samples P0, P1, P2, P3, P4 without Figure 4. Swelling process kinetics for particles containing immobi-
yeast. lized yeast cells.

4832 J. Nanosci. Nanotechnol. 17, 4827–4836, 2017

Iurciuc (Tincu) et al. Yeast Cells Immobilized in Ionic Crosslinked Hydrogel Particles Based on GLL and GLL/CMC

(a) (b) (c)

Figure 5. Scanning electron microscopy for particles without yeast cells performed cross section on frozen state (photos with different degrees of
magnification) for the samples: (a) P0, (b) P2, (c) P4.

immobilized protects cells against some degradative fac- for the P2D and P4D samples compared with those of the
tors and therefore their viability is maintained for a longer P0D sample.
time. For all the samples the transmittance has high values
The stability of the Delivered by Ingenta to: State Universitymeans
particles with immobilized yeast that of New that the at
York particles stability is higher in alcohol.
Binghamton
cells in alcoholic environments was evaluated in order to The results show
IP: 185.89.100.229 On: Tue, 18 Apr 2017 13:00:49 that the transmittance value increases
highlight the fact that the polymer matrix protect
Copyright: with increasing
them Scientific
American of the CMCNa content. Cell viability has
Publishers
from the toxic metabolites from ethanol. In the Table IV high values even after 84 h of storage in alcoholic media
are presented the transmittance and the cell viability values (over 90%), for particles with immobilized yeast cells that

(a) (b) (c)

Figure 6. Scanning electron microscopy for particles with immobilized yeast cells, performed in cross-sectional surface, in dry state, through met-
allisation for samples: P0D (a), P2D (b), P4D (c) (photos with different degrees of magnification).

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Yeast Cells Immobilized in Ionic Crosslinked Hydrogel Particles Based on GLL and GLL/CMC Iurciuc (Tincu) et al.

contain CMCNa (samples P2D and P4D), being slightly
lower than for the gellan particles (sample P0D has the
cell viability value of 88%).
Based on the presented results it can be concluded that
the polymer matrix protects the immobilized yeast cells by
the toxic metabolites from ethanol and that gellan/CMCNa
particles offers improved protection for immobilized yeast
cells as compared with the particles that contain only
gellan.
3.1. Swelling Characteristics in Water
As all the particles show a strong hydrogel character was
of interest the determination of their swelling abilities in
water by analysing the variation of the swelling degree in
a time interval of 20 hours (Fig. 3).
For particles containing CMCNa lower values of the
maximum swelling degree were obtained in comparison
with P0 sample, a consequence of the higher crosslinking
density of the particles in accordance with the transmit-
tance values discussed above.
The maximum swelling degree decreases with the
increasing of CMCNa concentration in the particles.
For all particles containing immobilized yeast cells it
was found that the maximum swelling degree for all sam-
ples is lower compared with the maximum swelling degree
of the particles without yeast cells (Fig. 4). (see for exam-
ple sample P4 from Fig. 3, for which SD = 684% com-
pared with P4D from Fig. 4 SD = 323%).
This effect proves that yeast cells, with negative electric
charges on the surface, practically participate to crosslink-
ing reinforcing the structure and thus remaining strongly
anchored within the polymeric network. Again in this
case the value of the maximum swelling degree decreased
with increasing of CMCNa concentrations for the reason
already discussed.
3.2. The Morphology of the Particles
The cross-sectional morphology of the particles in equilib-
rium state water/ice and in the dry state was evidentiated
by scanning electron microscopy. Figure 5 presents
microphotographs of the particles without yeast cells.

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Figure 7. The evolution of the fermented glucose yield in time, for 9 successive fermentation cycles in the presence of the gellan and gellan/CMC
particles that contain yeast cells. Numeric index is the number of the fermentation cycle.

4834 J. Nanosci. Nanotechnol. 17, 4827–4836, 2017

Iurciuc (Tincu) et al. Yeast Cells Immobilized in Ionic Crosslinked Hydrogel Particles Based on GLL and GLL/CMC

It is found that for the sample P0, the cross sectional The fermentation efficiency after 255 min for 9 fermen-
surface is not uniform and present a higher porosity. tation cycles, for all 5 types of particles is presented in the
Increasing the CMCNa concentration, the particles diagrams in Figure 8.
cross-sectional surface has became more uniform and From the fermentation curves and diagrams it can be
the porosity decreases. In Figure 6 are presented scan- seen that for the P0D sample process efficiency is higher
ning electron microscopy photographs performed in cross- compared with the other samples analyzed, starting with
section for dry particles with immobilized yeast cells. the fourth fermentation cycle. The explanation can be cor-
related with higher porosity of the particles respectively
3.3. The Evaluation of Glucose Fermentation with the lower crosslinking density, which facilitates the
Ability in Presence of the Particles with substrate diffusion (glucose) to the immobilized yeast cells.
Immobilized Yeast Cells The gellan/CMCNa particles with immobilized yeast
Testing the fermentative activity of the particles containing cells manifest a slightly higher efficiency compared with
yeast cells it was performed in a fermenter that allows the the P0D sample in the first three fermentation cycles.
measurement of the CO2 volume released in time. This can be determined by the fact that the lower
Based on the volume of CO2 released it was deter- crosslinking degree allows the diffusion of a larger num-
mined the glucose fermentation yield, consequently the ber of yeast cells from the polymer matrix, demonstrated
process efficiency. Fermentative activity was assessed for by the results obtained for cell viability (Table III).
all particles with immobilized yeast cells obtained at 30  C Gellan/CMCNa particles present a denser and more sta-
and pH = 5. After each fermentation cycle the particles ble interpenetrated network due to their higher crosslink-
were recovered from the fermenter by filtration, washed ing degree and as a consequence from the polymer matrix
with double distilled water and refrigerated at 4–6  C a lower number of cells are coming out and the ones
until the next fermentation cycle. The time required for an that remain in the particle determine a higher fermentation
fermentation cycle was 255 mn and the interval between efficiency.
cycles was 24 h. Is important to mention that the particles with immo-
Several fermentation curves obtained are shown in bilized yeast cells, regardless of the matrix type, can be
Figure 7. reused in many fermentative cycles. In the present study
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(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

Figure 8. The maximum yield in fermented glucose after several fermentation cycles for particles with immobilized yeast cells compared to the free
yeast.

J. Nanosci. Nanotechnol. 17, 4827–4836, 2017 4835

Yeast Cells Immobilized in Ionic Crosslinked Hydrogel Particles Based on GLL and GLL/CMC Iurciuc (Tincu) et al.

Table V. The productivity of the bioreactors for the 5 types of parti- density depends on one hand by the amount of crosslinker
cles containing immobilized yeast cells for several fermentation cycles
used but also by the composition of the initial polymers
(random choice).
mixture that increase with the amount of CMCNa in the
Number of
Productivity particles. This property is reflected in all the characteris-
[(g ethanol/h × g yeast])/particles type tics of the particles obtained, starting from the structural
the fermentation
cycles Free yeast P0D P1D P2D P3D P4D stability, morphology, behavior in aqueous media and fer-
I 0.18 0.21 0.19 0.18 0.18 0.18 mentation ability.
II 0.18 0.24 0.24 0.18 0.18 The bioreactors obtained from the mixture of the two
III 0.55 0.18 0.18 0.24 0.18 polysaccharides can be used in a large number of fermen-
IV 0.39 0.18 0.24 0.18 0.18
tative cycles, without showing a significant loss of their
V 0.52 0.18 0.18 0.18 0.18
VI 0.6 0.18 0.18 0.18 0.18 biocatalytic activity and can be readily separated from the
VII 0.18 0.18 0.24 0.18 0.18 fermentation media of glucose by simple filtration.
VIII 0.3 0.18 0.18 0.18 0.18
IX 0.47 0.18 0.18 0.18 0.18
Acknowledgments: This work was supported by the
strategic grant POSDRU/159/1.5/S/133652, co-financed by
were performed up to 9 fermentation cycles without an
the European Social Fund within the Sectorial Operational
important loss of the biocatalytic activity. Therefore, the
Program Human Resources Development 2007–2013.
polymer matrix maintains the cells viability and protect
the cells by the toxic metabolites produced by ethanol and
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IP: 185.89.100.229
The particles are easily recovered On: Tue, 18J. Apr
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Received: 17 November 2015. Accepted: 14 December 2015.

4836 J. Nanosci. Nanotechnol. 17, 4827–4836, 2017