Journal of Biotechnology 236 (2016) 45–56

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Yeast cells immobilized in spherical gellan particles cross-linked with

magnesium acetate
Camelia Elena Iurciuc (Tincu) a , Liana Alupei a , Alexandru Savin a , Constanţa Ibănescu a ,
Patrick Martin b , Marcel Popa a,c,d,e,∗
a
“Gheorghe Asachi” Technical University, Faculty of Chemical Engineering and Protection of the Environment, Department of Natural and Synthetic
Polymers, 73 Prof.dr.docent Dimitrie Mangeron Street, 700050 Iasi, Romania
b
Université d’Artois, IUT Béthune, Département Chimie, CS20819, 62408 Béthune, France
c
Academy of Romanian Scientists, Splaiul Independentei Str. 54, 050094 Bucuresti, Romania
d
Faculty of Dental Medicine, University “Apollonia”, 3, Str. Muzicii, Iasi, Romania
e
Research Institute “Academician Ioan Haulica”, 3, Str. Muzicii, Iasi, Romania

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

Article history: In this paper we report on the production of microbioreactors using ionically cross-linked gellan con-
Received 13 February 2016 taining immobilized yeast cells with potential application in glucose fermentation. Cross-linking was
Received in revised form 2 August 2016 achieved through a novel extrusion process in capillary by ionotropic gelation under the action of mag-
Accepted 3 August 2016
nesium acetate. Compared to commonly used methods, this provides a host of practical advantages.
Available online 4 August 2016
The particles were physico-chemically and morphologically characterized as their mechanical stability,
behavior in aqueous media, and bio-catalytic activity are influenced by the amount of cross-linker used.
Keywords:
This demonstrated their ability to be reused in a large number of fermentation cycles without losing
Gellan
Magnesium acetate their bio-catalytic activity. Our results are wholly comparable with the behavior of free yeast. We show
Yeast that fermentation cycles can succeed either immediately or at variable intervals, ensuring high yields of
immobilization glucose transformation, comparable-if not superior-to results currently obtained using free yeast.
Glucose fermentation © 2016 Elsevier B.V. All rights reserved.

1. Introduction
Yeast cell immobilization on different supports for obtaining
ethylic alcohol through fermentation has been studied for over four
decades, and continues to attract research interest worldwide. The
advantages of using yeast cells immobilized over their traditional
method – in free form – are numerous. A short list includes: (i)
cell protection from destruction via mechanical application, envi-
ronmental factors, or toxic metabolites such as those produced by
ethylic alcohol, which preserves viability and activity for long time
periods (Kandylis et al., 2012; Torresi et al., 2011); (ii) increased
productivity of fermentation equipment by increasing the popula-
tion density of the cells and the use of high substrate flow rates
in the production process; (iii) reusing the biological catalyst (Tan
et al., 2011); (iv) reducing contamination danger of the fermenta-
∗ Corresponding author at: “Gheorghe Asachi” Technical University, Faculty of
Chemical Engineering and Protection of the Environment, Department of Natu-
ral Polymers, 73 Prof.dr. docent Dimitrie Mageron Street, 700050, Iasi, Romania;
Academy of Romanian Scientists, Splaiul Independentei Str. 54, 050094 Bucuresti,
Romania.
E-mail addresses: marpopa2001@yahoo.fr, marpopa@ch.tuiasi.ro (M. Popa).
tion product by separating the biocatalyst at the end of the process
through simple filtration; (v) the possibility of moving to con-
tinuous processes. This last method is preferred, due to its high
efficiency, productivity, and lower operating costs (Kourkoutas
et al., 2005).
Yeast cell in the food immobilization remains much explored
industry as it is a key driver for wine and beer production which rely
upon continuous fermentative processes. The techniques involved
in microbial cells encapsulation – especially in the polymer sup-
ports – are: emulsification (Heng et al., 2003; Tan et al., 2011),
extrusion (Wan et al., 1994), complex coacervation (John et al.,
2011), spray drying (Luna-Solano et al., 2005) and gel entrap-
ment. Each of these techniques provides benefits and drawbacks.
Choosing the optimal working technique depends upon the encap-
sulation support used, the immobilized microorganism, and the
immobilized product properties. For microbial cells, inclusion in
spherical polymeric particles with hydrogel character using the
extrusion technique is the most often chosen due to mild processing
conditions which allow optimal encapsulation while maintaining
microorganisms viability (Park and Chang, 2000; Nedovic et al.,
2005; Koyama and Seki, 2004).

http://dx.doi.org/10.1016/j.jbiotec.2016.08.002
0168-1656/© 2016 Elsevier B.V. All rights reserved.
46 C.E. Iurciuc (Tincu) et al. / Journal of Biotechnology 236 (2016) 45–56
The most commonly used materials for immobilization are algi-
nates (Fumi et al., 1987; Yoo et al., 1996; Corton et al., 2000; Idris
and Wahidin, 2006), carrageenan (Rathore et al., 2013), ceramic or
glass carriers (supports) (Goncalves et al., 1992), cellulosic materi-
als (Bardi and Kountinas, 2004), ␥-alumina (Loukatos et al., 2000),
silica sol-gel films (Inama et al., 1993), polyacrylamide gels (Aykut
et al., 1988) and combinations of various materials (Peinado et al.,
2006).
The alginate gels are commonly used but show certain disadvan-
tages such as low mechanical stability and reduced stability in the
presence of chemical compounds, which can determine the diffu-
sion of a large number of yeast cells along with their proliferation
(Gerbsch and Buchholz, 1995; Maicas 2001; Callone et al., 2008;
Brownlee et al., 2009).
Another approach is based on the construction of covalent poly-
mer networks around biomolecules or cells which permanently
remain trapped in the matrix (Gill and Ballesteros, 1998; Gill and
Ballesteros, 2000-part 2; Gill and Ballesteros, 2000-part 1). Silica-
based materials display some physical and chemical properties
such as Si O bond strength (425 J mol-1), which provides material
stability. It is posible to obtain porous materials with suitable poros-
ity for mass transport, the cells being well caught in the matrix.
Sol-gel immobilization requires several steps and the Si-O bond
formation around the yeast cells or cell aggregates could be harm-
ful due to the potential interactions between silica and biological
agents. This could represent a negative aspect in the food indus-
try. To prevent this drawback hydrogel particles of alginates with
immobilized yeast cells have been prepared by ionic crosslinking,
using various cations such as calcium and barium, subsequently
coated with a layer of silica gel. Thus, the yeast cells are protected
through the alginate matrix, and the layer of silicon on the sur-
face of the particles provides stability and their ability to be used
in continuous fermentation processes (Callone et al., 2008).
The low structural stability of the alginate particles with immo-
bilized yeast cells motivated the research for using synthetic
polymers as matrices for cell immobilization. Călinescu et al. (2012)
prepared two supports for the immobilization of yeast cells using
alginates and polyacrylamide. The polyacrylamide gel was widely
used to immobilize several types of microbial cells (Dinu et al.,
2007). The matrices with immobilized yeast cells prepared were
tested in terms of fermentative activity and it was shown that poly-
acrylamide gel has a fermentation activity comparable with the
alginates particles, but the polyacrylamide matrix has exhibited a
better stability (Călinescu et al., 2012).
Aside from the large amount of biomass that needs to be incor-
porated, the supports chosen for the yeast cells immobilization
must have other important features, such as mechanical and chem-
ical stability, should be non-toxic and biocompatible with the cells,
and have a high diffusion coefficient for both substrates (Baptista
et al., 2006).
The polysaccharides remain the main materials used in micro-
bial cell immobilization due to their biocompatibility and lack of
toxicity. Gellan is the most recent polysaccharide to find broad
use as a gelification agent in the food industry (Morris et al.,
2012; Hasheminya and Dehghannya, 2013). It is a linear anionic
biopolymer with repeating tetrasaccharide sequences which are
made of two ␤-d-glucose residues: one of ␤-d-glucuronic acid and
one of ␣-l-rhamnose in the ratio 2:1:1 (O’Neill et al., 1983). It
is a biodegradable polymer-free of toxicity-and has a stable pH
value ranging from 2 to 10 (Moslemy et al., 2003). There are,
however, few studies dedicated to obtaining hydrogels based on
ionically cross-linked gellan, designed to include yeast cells. Tan
et al. (2011) obtained CaCl2 cross-linked gellan microparticles con-
taining encapsulated yeast cells (microbioreactors) through an
emulsifying method and demonstrated the possibility of using
the microbioreactos in multiple fermentative cycles. The micro-
bioreactors proved to be stable, easy to recover by filtration from
the fermentation medium, and can be reutilized for at least 10
fermentation cycles with a relatively high ethanol yield. Ethanol
production in the first three fermentation cycles was comparable
to that obtained with a single free yeast fermentation (Tan et al.,
2011). This biocatalyst holds genuine promise for use in sparkling
wine production technology; it is composed of gellan spherical par-
ticles in which yeast cells have been immobilized, (as proposed
by Mantaluta et al., 2012). The particles were obtained by extrud-
ing the gel formed through a capillary in a cross-linking bath that
contained CaCl2 solution with a concentration of 2% (used as ionic
cross-linking agent).
The yeast, especially Saccharomyces species, is usually selected
due to its features: production of ethanol, high capacity of fermen-
tation, tolerance to ethanol and other inhibitors, ability of rapid
growth under anaerobic conditions which are characteristic to the
fermentation equipments widely used in the food industry (Ivanova
et al., 2011).
The yeast cells require a wide range of metals for their growth
and metabolism (Walker, 1994; Youatt, 1993; Udeh et al., 2014).
The bioavailability of metal ions in the fermentation medium is
an important factor that affects yeast cell physiology and ethanol
production in fermentation processes (Udeh et al., 2014).
In the present work, we obtain hydrogel – like spherical parti-
cles containing yeast cells, based on ionically cross-linked gellan –
ionotropic gelation – through an extrusion process using magne-
sium acetate as the cross-linking agent. As far as we can tell, the
scientific literature makes no mention of obtaining such particles
with immobilized yeast cells. We have chosen this specific ionic
cross-linker due to the advantages Mg(CH3 COO)2 has over CaCl2 .
CH3 COO− ion, as opposed to the Cl− ion, promotes a shift in equi-
librium of the ionic chain-reaction towards forming the network,
which leads to more mechanically stable structures.
For concentrations under 0.7% (mass) the acetate ion, does not
affect the cell’s viability, in direct contrast to Cl− ion’s behavior.
The benefits of using magnesium ions for cell growth and
fermentation processes are manifold. The magnesium ion, most
abundant in the yeast cells, is an important divalent metal ion
that is involved in a wide range of metabolic processes (Elin,
1994; Hartwig, 2001; Cyert and Philpott, 2013). This ion consti-
tutes about 0.3% of the entire quantity of dry yeast and activates
over 300 enzymes (Rees and Stewart, 1997; Walker et al., 2006;
Bose et al., 2011). Maintaining the cell integrity, which includes
a structural stabilization of the nucleic acids, polynucleotides,
chromosomes, polysaccharides and lipids, was attributed to the
magnesium ion (Birch and Walker, 2000). Magnesium is actively
absorbed by the yeast cells from the fermentation medium to
perform physiologically essential roles (Udeh et al., 2014). It is
required for the activation of some glycolytic enzymes such as glu-
cokinase, glucose-6-phosphate dehydrogenase, phosphoglycerate
kinase and enolase, and its role in cell growth and cell protec-
tion during fermentation processes is well documented (Birch
and Walker, 2000). Magnesium ion deficiencies lead to a sluggish
metabolism in the yeast cells and a may slow or stop the fermen-
tation processes (Udeh and Kgatla, 2013; Udeh et al., 2014).
Finally, we note a key positive attribute: the cross-linker lacks
toxicity.
The micro-bioreactors obtained preserve the cell viability for a
long period of time (over 87.93% after 48 h of storage in double
distilled water) and protects yeast cells from the toxic metabolites
produced by ethanol (the cell viability is over 88% after a period
of 84 h of storage in an alcoholic solution with a concentration of
100 g ethanol/l).
The particles we obtained, characterized from the physico-
chemical and morphological points of view, are mechanically stable
(form, size), and possess the capacity for being reutilized in mul-
 C.E. Iurciuc (Tincu) et al. / Journal of Biotechnology 236 (2016) 45–56
tiple fermentative cycles without losing their biocatalytic activity
(up to 40 fermentation cycles with a break of 4 h between cycles).
Their productivity remains high—a result comparable to those
obtained in free yeast fermentation. The results are superior to
those obtained by Tan et al. (2011). In his study they obtained 10
fermentation cycles using microparticles with immobilized yeast
cells prepared through emulsion method.
2. Materials and methods
2.1. Materials
Kelco Biopolymers supplied Gellan of the Kelkogel food grade
type having a molecular weight between 2 and 3 × 105 Da for
deacetylated gellan.
Magnesium acetate and glucose – procured from Sigma Aldrich.
Yeast was purchased from Pakmaya, Romania with a concentra-
tion of 30% cells (w/w).
2.2. Methods
2.2.1. Obtaining the gelan particles with and without yeast
The gellan quantity (0.25 g) dissolves in 25 mL double distilled
water, under stirring, at 80–90 ◦ C. Since the gellan solution is quite
fluid and does not form stable particles when it is added drop-
wise into a bath containing our ionic cross-linker, we proceeded
with its cross-linking before the extrusion, by introducing 1.25 mL
magnesium acetate solution having various concentrations, thus
obtaining a viscous fluid, ready to be extruded. After the solution
cools down at 30–35 ◦ C, it is extruded using a syringe, through nee-
dles of various diameters, in 125 mL magnesium acetate solution of
different concentrations (1–4%). The extrusion process of the entire
polymer solution lasts about 10 min. The gellan spherical particles
were obtained instantaneously, were maintained in the extrusion
solution for 2 h, and were then washed with double distilled water
to remove the magnesium acetate excess. Finally, particles were
separated by filtration and kept refrigerated at 4–6 ◦ C until per-
forming further characterizations.
The gellan spherical particles with immobilized yeast cells were
obtained in a similar manner. First we added the gellan solution,
then a yeast suspension (1.25 g yeast in 5 mL water), thus ensuring
a yeast/gellan mass ratio of 5/1. Since the cells percentage in yeast
is approximately 30%, it follows that their mass is 0.375 g, and the
yeast cells/gellan ratio is 1.5:1. The yeast cell particles were kept at
4–6 ◦ C until subsequent testing.
We used for immobilization a commercial yeast strain,
employed in bakery, in compressed form, with 70% water content.
Cell characteristics are as follows:
- dimension (size) of a cell: 2–8 ␮m
- volume: 0.8–2 × 10−3 cm3
- weight of a yeast cell: 0.2–0.4 × 10−10 g
- number of yeast cells/g: 8–14 × 109
- surface area m2 /g: 8–14 × 109
We chose this type of yeast because it represents a biomass
of cells of the Saccharomyces cerevisiae species (superior ferment-
ing yeast), composed of living cells, which absorb easiest hexoses
(glucose), then sucrose and maltose (Dobbs et al., 1982; Reed and
Nagodawithana, 1991; Potthoff et al., 2012; Randez-Gil et al., 2013;
Klis et al., 2014)
2.2.2. Stability in water and alcohol solution
Maintaining the particles in aqueous or alcoholic mediums can
produce detachment of some fragments from the polymer matrix,
47
as well as yeast cell migration. This produces an increase in tur-
bidity and a corresponding decrease in transmittance values of
the liquid suspension. Here, a lower transmittance value correlates
with reduced structural stability of the particles. Transmittance was
measured with a BOECO S22 UV–vis spectrophotometer at a wave-
length of 550 nm. It was evaluated and determined for all samples
in both the supernatant’s obtained from the extrusion process, and
from the double distilled water, at various durations. The samples
were stored in a refrigerator at 4–6 ◦ C after each transmittance
determination.
Stability in an alcoholic medium is an important metric because
toxic metabolites found in methanol tend to protect yeast cells
immobilized in a polymer matrix. For this determination we ana-
lyzed samples containing yeast cells that were maintained in the
extrusion solution for 12 h and then separated by filtration and
washed with double distilled water to remove the magnesium
acetate traces. These were then suspended in a volume of 50 mL
alcohol solution containing 100 g/L ethanol and the transmittance
and cell viability at different time intervals were determined.
Before determining cell viability and transmittance, samples
were kept in an alcoholic solution in an oven at 30 ◦ C (the specific
temperature for glucose fermentation) for 255 min (the duration
of one fermentation cycle). After each determination samples con-
taining yeast cells were kept in a refrigerator at 4–6 ◦ C until further
measurments. The transmittance and cell viability measurements
were performed in triplicate and in the Tables 2, 3 and 4 we present
the average values. Standard deviations were limited to ± 0.3%.
2.2.3. Swelling degree of the particles without yeast
The particles obtained have an obvious hydrogel character;
therefore their behavior in water is important. Two series of deter-
minations were performed, and we selected four particle types for
the study. The first series of determinations consisted of monitoring
the particles’ behavior during controlled drying, while the second
series observed swelling behavior in double distilled water.
Using the first method, a precisely measured quantity of
swollen particles (maintained 24 h in double distilled water) were
controlled dried at T = 30 ◦ C (the temperature for glucose fer-
mentation). At 1-h intervals, the particles were weighed and the
operations were repeated until they reached a constant weight
which signified the complete loss of water (or the weight of the
particles in a perfectly dried state).
The swelling degree represented the ratio between the amount
of water still present in the particles at every time interval and the
quantity of particles which were completely dry.
The dried samples (a well-known quantity) were then re-
suspended in water at the same temperature (30 ◦ C), and
periodically (1 h intervals) the quantity of retained water was
established (gravimetrically, after dabbing the particles with filter
paper to absorb surface water). The swelling process was stud-
ied until equilibrium (defined as occurring when the quantity of
retained water remained constant).
In both cases, the swelling degree computations were obtained
via:
Q % = Mwater ⁄Mdrysample × 100 (1)
where: Mwater – quantity of water remained/absorbed in samples;
Mdrysample – quantity of dry particles.
The swelling degree measurements were performed in tripli-
cate and in Figs. 2 and 3 we present the average values. Standard
deviations were limited to ±3%.
2.2.4. Mechanical stability of the particles
The structural stability of the particles was evaluated, indirectly,
through rheological tests in amplitude and frequency sweeps,
respectively. We started from the assumption that higher values
48 C.E. Iurciuc (Tincu) et al. / Journal of Biotechnology 236 (2016) 45–56
of the accumulation module (G’) were determined by a stronger,
more cross-linked structure, showing better mechanical stability.
The tests were made on a modular rheometer Physica MCR 501
(Anton Paar, Austria), using the Peltier system for temperature reg-
ulation and parallel plate geometry. All the measured rheological
parameters were determined in the dynamical oscillatory regime.
The same number of particles from each sample were placed under
the plates of the device and selected for the same diameter, while
the upper plate was lowered until it touched the particles, ensur-
ing a slight pressure. The amplitude sweep test was applied in
order to determine the limit of the linear viscoelastic domain (LVE
range), which allowed us to establish the maximum deformation
tolerated by the sample before destruction of its internal structure.
Frequency was maintained constant (␻ = 10 rad/s) while the defor-
mation (␥) varied in the range 0.01 ÷ 100%. For the frequency sweep
test, the deformation was kept constant in the linear viscoelastic
domain (␥ = 0.05 ÷ 0,1) and the frequency varied between 0.1 and
100 rad/s. All measurements were made at a constant temperature
of 30 ◦ C, while monitoring the variation in time of the accumulation
and loss modules.
2.2.5. Cell viability and yeast cell quantity from the particles
The cell viability and the concentration of cells were determi-
nated based on the number of cells that diffuse from the particles
in the solution in which they were preserved.
In order to determine the quantity of yeast cells remaining in
the supernatant we used a special technique (employed in bio-
chemistry) for determining the number of living cells in a given
suspension volume.
The cell viability and quantity (number) of yeast cells from the
mediums in which particles have been suspended, were deter-
mined using an optical microscope and a counting chamber. This
method is applied frequently to evaluate the quality of compressed
yeast, the percentage determination of the autolyzed cells, and
environmental factors that influence the physiological activity of
yeast cells. It relies on the fact that the nonviable yeast cells, as
a result of either the autolysis process or an exterior factor that
produces irreversible modifications in their protein structure, lose
their fermentative properties. By suspending the yeast cells in a
methylene blue diluted solution in sodium citrate 2%, the phys-
iological inactive cells become blue, because after the reductase
inactivation the pigment does not pass through in its colorless
leuco derivative form, as it does in the active (living) cells. Using
the counting chamber (Marienfeld Neubauer) we distinguished the
nonviable cells (autolyzed) from the total cells and thereby assessed
their percentage expression (number or weight, the latter being
determined from the calibration curve).
In order to evaluate structural stability of the gellan particles, the
yeast cell concentration and cell viability (number of cells/mL) from
the aqueous medium in which the particles with immobilized yeast
cells were suspended (50 mL) was determined. The yeast particles
were washed and re-suspended in double distilled water and the
cell concentrations were assessed after 12 h and 24 h. Based on the
number of cells which were alive versus lysed in the cross-linking
solution after the aforementioned time periods, cell viability (Vc )
was determined; this represents the percentage of living cells found
in the total number of supernatant cells.
Nlivecells
Vc = × 100
Nlivecells + Nlysedcells
In order to determine whether the gellan particles with imo-
bilized yeast cells are stable in the fermentation medium, we
established the yeast cell quantity that diffuses from the particles
after each fermentation cycle. Based on the results obtained and
knowing the initial weight of the cells that have been immobilized
(representing 30% from the amount of yeast used, namely 0.375 g)
it was estimated the amount of cells caught in the polymer matrix
(we did not consider the amount of cells that could proliferate in
time in the gelan particles).
As a preliminary step, we plotted a calibration curve that cor-
relates the number of existing yeast cells with their weight using
optical microscopy.
The precision of this method was verified by determining
the weight of a cell, found to be 3.7 × 10−11 g/cell; this is in
excellent agreement with data found in literature (e.g., Haddad
and Lindegren, 1953 specifies values between 2.4 × 10−11 and
12 × 10−11 g/cell).
To determine the number of yeast cells remaining in particles
after each fermentation cycle, the number of existing yeast cells
in the fermentation medium (50 mL) was measured by the same
method and converted to the yeast cell quantity based on a calibra-
tion curve. The resulting amount was subtracted from the initial
amount of cells (0.375 g) to obtain the quantity left in the particle’s
with immobilized yeast cells after every fermentation cycle.
The cell viability measurements were performed in triplicate
and in the Tables 3 and 4 we present the average values. The stan-
dard deviations were placed in the limits of ± 0.3%.
2.2.6. Scanning electron microscopy
To help understand the morphology of the particles contain-
ing encapsulated yeast cells, and the way they are influenced by
the extrusion conditions, SEM photos of the particle cross-sections
were performed. Here, a HITACHI SU 1510 scanning electron micro-
scope was used.
Samples with immobilized yeast cells were cross-sectioned and
dried. The particles thus prepared were placed in a microscope
device and then coated with gold using a sputter device (108 Cress-
ington auto).
2.2.7. Testing the fermentative activity of particles containing
yeast
In a fermenter, we tested particles with immobilized yeast cells
to determine their capacity to produce glucose fermentation. Here,
a volume of 50 mL glucose solution (c = 8%) was added, containing
a magnesium acetate solution concentration (c = 0.5%). The fer-
mentation process was carried out at 30◦ C, while monitoring the
volume of CO2 released. Here, the fermented glucose quantity over
time was established, along with the efficiency of the process, given
that the pH of the fermentation medium was 6.5. Each fermenta-
tion cycle lasts 255 min and the CO2 volume was read and recorded
every 15 min. After each fermentation cycle, the particles were
recovered from the fermenter, washed with double distilled water
(to remove glucose and magnesium acetate excess), and the parti-
cles were either reused immediately (0.5 h was the time between
fermentative cycles) or kept in the fridge at 4–6 ◦ C for 4 h until the
next available cycle.
3. Results and discussions
3.1. Obtaining the gellan particles with and without yeast
Obtaining yeast cells immobilized in a gellan matrix is based
upon the fixing of yeast cells into meshes formed through the
(2) polysaccharide cross-linking, following the ionic bond of the mag-
nesium cations to the gellan carboxylate anions. The main reaction
is:
− 
Mg 2+ OCOCH3 + 2Gelan − COO− Na+ → Gelan −
2
COO− Mg 2+ − OCO − Gelan + 2CH3 COO− Na+
 C.E. Iurciuc (Tincu) et al. / Journal of Biotechnology 236 (2016) 45–56 49

Table 1
Types of gellan particles obtained by ionotropic gelation using different concentrations of magnesium acetate added before extrusion and in the cross-linking bath: the
extrusion process parameters and their average diameter.

Samples Capillary characteristics Concentration of the Concentration of the Concentration of the Average diameter of
(diameter × length) (mm/mm) gelan solution% Mg(OCOCH3)2 solution Mg(OCOCH3)2 solution particle (mm)
(g/100 mL) in preextrudate% in extrusion bath%
(g/100 mL) (g/100 mL)

P1 0.6 × 30 0.8 0.4 2 2.5

P2 0.6 × 30 1.15 0.4 2 2.6
P3 0.6 × 30 1.3 0.4 2 3
P4 0.6 × 30 1 0.2 2 3.3
P5 0.6 × 30 1 0.4 2 2.8
P6 0.6 × 30 1 0.6 2 2.7
P7 0.6 × 30 1 0.8 2 2.5
P8 0.6 × 30 1 0.4 1 3.1
P9 0.6 × 30 1 0.4 3 2.6
P10 0.6 × 30 1 0.4 4 2.5
P11 0.8 × 40 1 0.4 1 3.7
P12 0.8 × 40 1 0.4 2 3
P13 0.8 × 40 1 0.8 2 2.6

Table 2
Transmittance values for the extrusion solution and double distilled water, respectively, in which resulted particles were kept for different periods of time.

Samples Concentration of the Concentration of the Concentration of the Transmittance, T (%)

gelan solution % Mg(OCOCH3)2 solution Mg(OCOCH3)2 solution
(g/100 mL) in preextrudate % in extrusion bath %
(g/100 mL) (g/100 mL)

in extrusion solution in double distilled water

30 min 12 h 3h 12 h 24 h

P4 1 0.2 2 99.6 98.8 98.5 97 95.6

P5 1 0.4 2 99.8 99 99 98.2 96.7
P9 1 0.4 3 99.2 99.1 99.1 99 97.9
P10 1 0.4 4 99.8 99.3 99.1 99 98.2

In order to determine optimal conditions for obtaining the
spherical gellan particles through ionotropic gelation, several
parameters were varied, in steps, during the production process:
- the gellan solution concentration: 0.8%, 1%, 1.15%, 1.3% (w/v);
- the Mg(OCOCH3 )2 solution concentration added before extrusion
(v = 0.5 mL): 0.2%, 0.4%, 0.6%, 0.8% (w/v);
- the Mg(OCOCH3 )2 solution concentration in the extrusion bath
(v = 50 mL): 1%, 2%, 3%, 4% (w/v).
(samples P8, P5, P9–P12). The geometrical characteristics of the
capillary influence the particles’ diameter, in the sense that it
increases with the increasing of the capillary diameter (here length
does not vary significantly) (samples P8, P11).
For the particles’ subsequent characterization, we only consid-
ered those obtained by capillary extrusion with the characteristics
diameter/length = 0.6/30 mm.
3.2. The particles’ morphology

We obtained most of our particles through extrusion in capil-
lary (using a syringe needle) with interior diameter of 600 ␮m and
length of 30 mm. Trying to use a capillary with 800 ␮m diameter
at a length of 40 mm, leads to larger spheres, less stable in aque-
ous medium (lower transmittance values). In all cases, the resulting
particles had spherical shapes with sizes ranging between 2.5 and
3.7 mm depending on the process’ parameter values and the capil-
lary diameter through which the gellan solution had been extruded.
Table 1 presents some of the samples obtained, with their
codification—the values of the parameters listed above and the
diameter of the resulted particles.
An increase in the gellan solution concentration determines a
slight increase in the sphere’s diameter (samples P1–P3); this is a
natural effect, considering that the cross-linking, which is done on
account of the same cross-linker quantity added before extrusion
and in the extrusion bath, becomes less intense. The most intense
effect, however, is produced by the cross-linking agent solution
concentration that is added before extrusion (samples P4–P7; P11,
P13), when the diameter decreases from 3.3 to 2.5 mm. The cross-
linking agent concentration from the extrusion bath contributes
to additional cross-linking of the gellan particle, especially on its
surface, which gives it mechanical stability; the particle’s diameter
decreases with the increase in cross-linking agent concentration
The previously observed influence of the cross-linking agent
quantity on the size of the particles with immobilized yeast cells
required us to analyze their morphology by SEM. There was an
obvious influence of the cross-linker concentration used on this
characteristic. Samples P9Y and P10Y were chosen for illustrative
purposes; they differ by the cross-linking agent concentration used
in the coagulation bath (Fig. 1).
It is obvious from the photos, irrespective of resolution, that
sample P10 has a more compact structure due to using a greater
quantity of cross-linker. The figures also reveal the presence of
the gellan cross-linking matrix that anchors yeast cells with the
more porous cross-sectional surface as seen in the P9 sample.
According to the morphological appearance of the particle’s cross-
sectional surface, there are some physicochemical properties, such
as structural stability, swelling behavior in aqueous medium and
mechanical stability.
3.3. Stability in water and cell viability
In principle, high transmittance values suggest increased sta-
bility of the particles leading to fewer polymer fragments detached
from the gellan matrix. In the case of the particles with immobilized
yeast cells, transmittance can also be affected by the presence in the
50 C.E. Iurciuc (Tincu) et al. / Journal of Biotechnology 236 (2016) 45–56

Table 3
Transmittance values and cell viability in the extrusion solution and double distilled water, respectively, containing encapsulated yeast cells.

Samples Transmittance, T% Concentration of yeast cells/mL × 104 Cell viability, %

in supernatant

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

4h 12 h 4h 12 h 24 h 12 h 12 h 24 h 12 h 12 h 24 h

P4Y 93.7 86.5 94 85.3 86.4 16.5 38 58.5 96 95.73 94.15

P5Y 96.3 94.8 96.8 90.2 88 10.75 11 23.5 93 93.18 93.49
P9Y 97.7 96.4 97.4 96.4 95 6.25 9.25 18.5 92 89.19 88.24
P10Y 99.8 98.8 98.3 96.7 95.5 2.5 6 14.5 90 88.07 87.93

Table 4
The transmittance values and cell viability in alcoholic solution (100 g/L), in which the samples with yeast cells P5Y, P9Y and P10Y were suspended.

Samples Transmittance, T% Yeast cells concentration/mL × 104 Cellular viability, %

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

P5Y 99.2 98.3 98 97.6 3 3.5 4.5 5.5 100 93.3 90 88

P9Y 99.4 99.1 98.3 97.8 2.75 3.5 4 5.25 100 93.3 94.11 91.3
P10Y 99.7 99.4 99 98.1 2 2.5 3.5 4 100 90.9 93.33 88.88

Fig. 1. SEM images performed on the cross section of the dry particles, metalized with gold, samples P9Y (a) and P10Y (b) (three resolutions each).
 C.E. Iurciuc (Tincu) et al. / Journal of Biotechnology 236 (2016) 45–56
Fig. 2. Time variation of the swelling degree in water when particles are dried until constant weight. (a) Particles without yeast; (b) particles with yeast.
Fig. 3. Time variation of the swelling degree in water for the particles, which were previously dried out (a) without yeast and (b) with yeast.
aqueous medium of yeast cells that are not fixed in the polymer
network.
The high transmittance values prove that the particles are stable
in time, both in the extrusion solution and in double distilled water;
some results are presented in Table 2.
The transmittance values in the extrusion solution is over 99%
after 30 min, albeit slightly decreasing after 12 h, suggesting cap-
ture in the network of the entire quantity of gellan, but also
suggesting the fact that, practically, the entire yeast cell’s quantity
have been immobilized. Keeping the samples in double distilled
water after separation from the supernatant (the solution from the
extrusion bath) shows a gradual decrease in transmittance values
with storage time (between 3 and 24 h, its value is still maintained
at very high levels). The highest transmittance values are regis-
tered for particles with a higher cross-linking degree, respectively
those with more cross-linking agent added either before extrusion
(P5) or in the extrusion bath (P10). The effect is natural since gel-
lan macromolecules are much better fixed in the network as the
Mg(OCOCH3 )2 concentration increases.
The transmittance values of the particles with encapsulated
yeast are lower than the ones without yeast, but are still quite high,
which indicates that the yeast has been immobilized efficiently
within the particles (Table 3).
When we kept the particles in the magnesium acetate solution
from the extrusion bath, the transmittance values ranged from 86.5
to 99.8 (after 4 h), slightly decreasing in time. Transmittance values
increase with an increase of the cross-linking agent concentration
added either before extrusion (samples P4, P5) or in the extrusion
bath (samples P9, P10), keeping the particles both in the extru-
sion solution and also in the double distilled water, regardless of
the storage duration. Naturally, the lowest transmittance values
51
are registered for longer periods of particle storage in water (24 h).
Transmittance is well correlated with yeast cell concentrations that
diffuse from the particles, and with the gellan matrix cross-linking
degree. We noticed that maintaining the particles both in the extru-
sion solution that contains cross-linking agent and in the double
distilled water, the free yeast cells concentration decreases when
the cross-linking degree increases, and increases with the stor-
age duration in these liquids (transmittance values vary in inverse
proportion).
Although the number of cells that diffuse from the polymeric
matrix decreases with the increase of its cross-linking degree, the
number of autolyzed cells from the total number of free yeast cells
slightly increases (cell viability decreases both in the extrusion
solution and in the double-distilled water). The effect is logi-
cal: increasing cross-linking degree allows more difficult diffusion
of the nutrients to the yeast cells immobilized in the polymeric
matrix; therefore a higher proportion of cells become nonviable
and the autolysis appears to a greater number of cells and are found
in the cells that diffuse from the matrix in the extrusion solution in
a higher proportion (cell viability decreases).
3.4. Stability in alcoholic solutions and cell viability
One of the encapsulation effects of yeast cells in gellan particles,
as well as in other polymers, is protecting the bio-catalyst from the
mediums in which they work that can sometimes be aggressive.
This effect allows the reuse of the particles with yeast cells immo-
bilized in successive fermentative cycles. It is required, however,
for the particles with immobilized yeast cells to be stable in alco-
holic solutions (formed after fermentation) with a concentration
of at least 80 g/L. For this reason, a few particles types (previously
52 C.E. Iurciuc (Tincu) et al. / Journal of Biotechnology 236 (2016) 45–56

discussed) were analyzed from the point of view of their stability in

an alcoholic solution of 100 g/L, this property being evaluated, as in
the previous case, by monitoring the alcoholic medium transmit-
tance values in which the gellan spheres with encapsulated yeast
were suspended, at different time intervals (between 12 and 84 h).
The results appear in Table 4.
We found that the polymer matrix protects the immobilized
yeast cells from toxic metabolites found in ethanol, and keeps their
cell viability for a longer time, at high values.

3.5. Swelling characteristics in water

For this determination we used samples (P4, P5, P9, P10), the
most stable ones (with higher transmittance values) and with
various magnesium acetate concentrations added either before
extrusion or in the extrusion bath. The swelling degree was estab-
lished in two ways as it was described in Methods.
1. The swelling degree variation in time determined in the first Fig. 4. Amplitude sweep test for the samples P5, P5Y, P10 and P10Y,
(T = 30 ◦ C,␥ = 0,01 ÷ 100%, ␻ = 10 rad/s).
case is illustrated in Fig. 2 (for gellan spheres without yeast – a, and
with yeast – b, respectively).
Note that the sample with the highest cross-linking degree (P10)
has the lowest value for swelling degree, the effect being a result
of their higher network density. Similar evolutions of this property
are observed in the other three types of particles (Fig. 2a). In the
case of identical cross-linking degree, particles with immobilized
yeast cells have lower swelling degree values. This effect supports,
indirectly, the hypothesis that between the polymer and protein
functional groups that enter in the yeast composition, there are
strong interactions; one consequence is an increase in the polymer
network density (Fig. 2b). In the case of sample P10, this effect is
less evident; its behavior is similar to three other samples, proba-
bly because the maximum network density is ensured by the large
quantity of the ionic cross-linker, while the interactions between
yeast and gellan do not contribute significantly to the cross-linking
density.
2. The second version consisted in immersing the completely
dried particles (Md ) in double-distilled water, at 30 ◦ C, and period-
ically determining the amount of retained water until equilibrium.
Fig. 5. Frequency sweep test for samples P5, P5Y, P10 and P10Y (T = 30 ◦ C,
The processes of losing and retaining water are not reversible.
␻ = 0,1 ÷ 100 rad/s).
Drying out the samples leads to the formation of strong hydrogen
bonds between the gellan chains, increasing the cross-linking den-
sity of the matrix. The swelling degree obtained through immersing quantities used. We assumed they could offer conclusions that
particles in water is inferior to that obtained with the previous might later be generalized to the other samples which, from the
method over the entire process (Fig. 3). Naturally, the swelling viewpoint of their cross-linking degree (indirectly evaluated by the
degree decreases with the increase of the cross-linking density swelling degree in water) range between the two chosen samples.
(samples P5, P9, P10). These are P5 and P10, with and without yeast.
The swelling degree for particles containing yeast cells depends, The first set of tests was the amplitude sweep (AS). Here, we
as well, on the cross-linking degree. For identical cross-linking were helped by the linear viscoelastic domain limits previously
degree, the particles with immobilized yeast cells have lower measured as 0.1% for samples P5 and P5Y and 0.05% for P10 and
swelling degree values compared to the ones without yeast, via P10Y (Fig. 4).
the possible participation of the bio-catalyst in the polysaccharide The second set of tests, performed on the same samples, was
cross-linking. done using frequency sweep (FS-frequency sweep), the results are
shown in Fig. 5.
3.6. Mechanical stability of particles evaluated through The frequency sweep tests demonstrated the influence of the
rheological tests cross-linking agent and yeast addition, respectively, on the stability
of the particles. The higher values of accumulation module G’ and
Using the particles with immobilized yeast cells in repeated fer- the increase of the difference between the two dynamic modules
mentation cycles requires high mechanical stability. Preliminary denote the presence of a more stable internal network; in short,
information was obtained by evaluating the stability in aqueous better mechanical and structural stability.
mediums, as previously discussed. We also found it useful to per- Note that the G’ values for the two samples are close to each
form rheological tests into this property. other, illustrating the fact that their stability and structural resis-
The oscillatory tests are extremely useful for describing the tance are close, in accordance, also, with the transmittance values
structure of viscoelastic materials, and provide data about the of the solutions from the supernatant. The sample with a higher
structure and elasticity of a material. cross-linking degree (4% concentration in the extrusion bath, as
Two samples have been chosen for the study, which differ in opposed to 2% concentration in the extrusion bath for the other
cross-linking degree as determined by the different cross-linker sample) presents a slightly superior stability, explained by the
 C.E. Iurciuc (Tincu) et al. / Journal of Biotechnology 236 (2016) 45–56 53

higher cross-linking degree (indeed it had the maximum concen-

tration of the ionic cross-linker). These results are in accordance
as well with the swelling characteristics of the samples: a lower
maximum swelling degree for the more cross-linked sample is
determined by its superior cross-linking density, which also confers
higher structural resistance (hence increased rigidity).
The rheological tests also highlight the fact that the stability
of particles with immobilized yeast is higher than it is for parti-
cles without immobilized yeast cells, i.e., higher values of stability
correspond to particles with a higher cross-linking degree. In this
case as well, there is complete agreement with the previous results
regarding the particles behavior when swollen, enforcing the con-
clusion that the yeast itself contributes to the strengthening of the
network’s structure.

3.7. Testing fermentative activity of the particles with yeast cells

immobilized

Testing the fermentative activity was accomplished using the

fermentation apparatus. The fermentation process temperature
was adjusted by circulating heated water through the fermentor
shell.
All samples of particles with immobilized yeast cells were tested
in fermentative processes and we noted that a high fermentation
yield was obtained, comparable with measurements done using
free yeast. Here, three types of particles were selected (P5Y, P9Y,
P10Y), as they were used in a large number of repeated fermenta-
tion cycles.
Fig. 6 presents fermentation curves (representing variations in
time of the yield in fermented glucose) for the three types of par-
ticles compared to the free yeast, for a few fermentation cycles
(cycles 1–4). Note that the fermentation cycles were performed at
time intervals up to 15 min apart–time intervals used to wash out
the particles after the previous fermentation cycle and to set the
thermostat for the substrate solution to 30 ◦ C. Only the curves for
the first four fermentation cycles have been represented, to avoid
overloading the manuscript (if interested, please contact the author
for full details and data files). For comparison, on each figure we also
present the fermentation curve for free yeast (the same quantity as
the one encapsulated in the gellan particles) – P0.
We noticed that the highest fermentation efficiency was
reached with particle P5Y, characterized by a lower cross-linking
degree (with the exception of cycle 2, which is more efficient for
sample P9Y). One explanation lies in the reduced cross-linking den-
sity for the hydrogel network, that facilitates an easier diffusion
of the voluminous substrate molecules (glucose) to yeast cells. In
further support of this hypothesis, note fermentation in the pres-
ence of sample P10: here, the yields are the lowest we encountered,
probably due to it having the highest cross-linking degree, in corre-
lation also with the swelling curves (Figs. 2, 3). Twenty fermentative
cycles were achieved, all with high fermentation yields, some even
superior to the first cycle.
For further studies, sample P5Y was analyzed. Fig. 7 presents the
fermentation curves for the first 5 cycles, compared to a quantity of
free yeast equivalent to the one immobilized in the spherical gellan
matrices.
The P5Y sample has a concentration of 2% magnesium acetate
in the crosslinking bath. Călinescu et al. (2012) prepared alginate
particles with yeast cells immobilized with calcium chloride used
as cross-linking agent, and found that the best efficiency in fer-
mentation was obtained for particles that were obtained using
Fig. 6. Evolution in time of the glucose fermentation efficiency for the samples P5Y,
a concentration of 2% CaCl2 in the crosslinking bath. A lower P9Y and P10Y in different fermentation cycles: (a) cycle 1; (b) cycle 2; (c) cycle 3;
crosslinking degree leads to a higher mass transfer due to diffusion (d) cycle 4. (T = 30◦ C, Time requiered for a fermentation cycle: 255 min).
of the substrate to the yeast cells within the matrix. The limita-
tion of the mass transfer is a problem in the inclusion method used
for immobilization because the cells could maintained a high cell
54 C.E. Iurciuc (Tincu) et al. / Journal of Biotechnology 236 (2016) 45–56
Fig. 7. Variation of the glucose fermentation efficiency in time in the presence of the
particles with yeast cells immobilized P5Y, in 5 succesive fermentation cycles, com-
pared with free yeast. (T = 30◦ C, Time requiered for a fermentation cycle: 255 min).
viability and it could yield a higher ethanol quantity only if there
is an efficient exchange between nutrients from the fermentation
medium and the particles with yeast cells immobilized (Wang and
Hettwer, 1982).
The high fermentation yield values obtained even after 5 cycles,
suggested further use of particles with immobilized yeast cells up
until 20 cycles. In Fig. 8 we illustrate the maximum yield obtained
in fermented glucose for a select subset of the 20 cycles. We wish
to draw attention to fermentation efficiency, irrespective of the
fermentative cycle.
Indeed, choose any particular cycle—it will be superior to the
corresponding one attained in the presence of free yeast. This is
confirmed by other researchers in the field and results published
in the literature (Tan et al., 2011).
The surprising fact is that the lowest fermentation efficiency is
reported at the first fermentation cycle, even it is superior to the
one obtained with the free yeast cells. A significant increase occurs
between fermentation cycles 2 and 4.
Mariam et al. (2009) have obtained micro-bioreactors pre-
pared from sodium alginate with yeast cells immobilized, ionically
crosslinked, using calcium chloride as cross-linking agent. We
tested them from the point of view of their fermentative activ-
ity and the maximum increase of the fermentation yield was after
the 4th fermentation cycle, while the fermentation yield in the 5th
and 6th fermentation cycle decreased sharply. The maximum num-
ber of fermentation cycles performed with these micro-bioreactors
was 6. The explanation of this behavior was that micro-bioreactors
Fig. 8. The maximum yield in fermented glucose obtained in the presence of the sample P5Ycompared with the yield obtained in the presence of free yeast (0), for some of
the 20 fermentation cycles performed at 30◦ C, 255 min for each fermentation cycle (within 15 min between fermentation cycles).
become unstable after the 4th cycle, when a change in shape takes
place.
Another possible explanation would be that after the first cycle a
more pronounced swelling of the particles takes place and this facil-
itates substrate diffusion of the immobilized cells. After the sixth
fermentation cycle, the maximum yield value slightly decreases,
maintaining relatively constant values up to cycle 20. The gradual
decreases of the fermentation efficiency in the next cycles may be
due, in principle, to the reduction in the number of viable yeast
cells from the gellan matrix. This finding is in accordance with the
study of Wang and Hettwer (1982), who assert the possibility of
immobilized yeast cells to proliferate better than the free ones, due
to the limited motions of the cells within the particles.
To evaluate whether the particles are stable in the fermentation
medium after each fermentation cycle, we determined, based on
the calibration curve, the number of yeast cells from the fermen-
tation bath and their weight. As such, we noticed that the cells are
firmly trapped in the polymer network and do not diffuse in large
numbers in the fermentation medium even after many cycles of
fermentation.
We determined that after each fermentation cycle, a quantity of
cells diffuses from the gellan matrix that is very low in compari-
son with the ones from the particles with yeast cells immobilized
(0.006323 g). This explains the slight decrease of the fermentation
efficiency starting from the sixth cycle, but it also explains mainte-
nance at very high values.
It is obvious that fermentation takes place in the spherical gellan
matrix in which glucose diffuses. The only explanation for obtaining
high efficiency rates even after 20 cycles is that the gellan matrix
protects the yeast cells from inactivation and preserves their viabil-
ity; it also protects the magnesium ion from the magnesium acetate,
at a concentration of 0.5% in the fermenter. Moreover, it not only
preserves the cellular viability but it is possible by its diffusion in
the polymer matrix to contribute to the proliferation of the cells
immobilized in it.
As previously mentioned, the fermentation efficiency also
depends on the type of particles in which the yeast has been
immobilized, and on the conditions in which these were obtained.
Therefore, the efficiency depends on the magnesium acetate solu-
tion concentration from the coagulation bath, and on the particle
cross-linking degree. As the cross-linking density increases, the fer-
mentation efficiency decreases – an effect that was observed well
past (long after) the first cycle. The explanation may be the follow-
ing: on the one hand, with the increase of the cross-linking degree
responsible for the decrease of the particle swelling degree in the
aqueous medium, a slower diffusion of the substrate (glucose solu-
 C.E. Iurciuc (Tincu) et al. / Journal of Biotechnology 236 (2016) 45–56
Fig. 9. Glucose fermentation efficiency attained after 255 min, in successive fermentation cycles for the sample P5Y,compared with the efficiency obtained in the free yeast
fermentation. T = 30◦ C. (Random presentation of some cycles from the total of 40 accomplished).
tion) is accomplished inside the polymer matrix, and to the yeast
cells. On the other hand, the dense network, whose meshes become
probably comparable with the yeast cell’s size, prevents their pro-
liferation, due to the slower diffusion of the magnesium acetate
(that stimulates proliferation).
We also noted that the yeast cell numbers in the fermentation
bath after each fermentation cycle become smaller – they dimin-
ish; this signifies that they are strongly immobilized in the polymer
matrix through which they cannot diffuse anymore (these results
of ours are only mentioned in passing and not presented in detail
because of space constraints). Note, too, that the number of fer-
mentation cycles that can be obtained with immobilized particles
decreases for the three particles types, from 20 (sample P5), to 9
(sample P9), then to 6 (sample P10).
The lack of nutrients (glucose) in yeast cells can lead to their lysis
(death); that is why the nutrient exchange between the fermenta-
tion medium and the particles that contain yeast must be optimal.
Therefore, the gellan particles containing yeast cells should not
be too cross-linked and must have good porosity to allow this
exchange.
The excellent results obtained from sample P5 encouraged us
to try to perform as many fermentation cycles as possible. Here,
we increased the time interval between fermentation cycles to 4 h
in order to obtain indirect information on this crucial parameter
and on the performance of particles with immobilized yeast cells.
To-date, 40 cycles have been accomplished and it is highly prob-
able that the particles could be used further—up until the total
loss of their bio-catalytic activity. In Fig. 9 we present only the
fermentation efficiency achieved after 255 min, for a few cycles
randomly considered. Please note that in all cases we obtained
superior fermentation efficiency to the one attained in the pres-
ence of a quantity of free yeast, quantity which is equivalent to the
one immobilized.
On the other hand, the yield reached at various fermentation
cycles is practically constant (at least until the 20th cycle), a little
less than the yield from cycle one. An unexpected increase in yield
occurs in the last 15 cycles of fermentation. One explanation might
be that yeast cells proliferated in the polymer matrix, thus increas-
ing their bio-catalytic activity. We also observed that increases of
the time interval between cycles lead to slight decreases in effi-
ciency, in comparison with the previous case, when the cycles
succeeded each other with a time interval of only 15 min.
55
4. Conclusions
Magnesium acetate is a suitable cross-linking agent for obtain-
ing gellan particles for yeast cells immobilization leading to
mechanically stable structures without affecting cell viability.
Increasing the cross-linker concentration creates a more compact
structure of the spherical gellan matrix by increasing the cross-
linking degree, increasing the structural stability of the particles
with yeast cells immobilized, and reducing the maximum swelling
degree in aqueous media. By using the particles that contained
immobilized yeast cells, glucose fermentation ability increased
with the reduction of the cross-linker concentration used, because
this facilitates the substrate diffusion to the yeast cells from the
polymer particles. Gellan particles containing immobilized yeast
cells can be reused a large number of fermentation cycles (at least
40 cycles) without any significant loss of their bio-catalytic activ-
ity, and provide yield values in fermented glucose which are higher
than the yield obtained in free yeast fermentation. The bio-reactors
obtained have potential applications in fermentative processes in
the food industry-a prospect made even brighter by the nontoxic
properties of the cross-linker used.
Acknowledgement
This work was supported by the strategic grant POS-
DRU/159/1.5/S/133652, co-financed by the European Social Fund
within the Sectorial Operational Program Human Resources Devel-
opment 2007–2013.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.jbiotec.2016.08.
002.
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