food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123

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Food and Bioproducts Processing

journal homepage: www.elsevier.com/locate/fbp

Physico-chemical characteristics and fermentative

activity of the hydrogel particles based on
polysaccharides mixture with yeast cells
immobilized, obtained by ionotropic gelation

Camelia-Elena Iurciuc (Tincu) a , Alexandru Savin a ,

Leonard Ionut Atanase d,e , 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, Prof. Dr. docent Dimitrie Mangeron Str., nr. 73, 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, Iaşi, 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: The paper reports the preparation of particles, having a hydrogel behavior, with immo-
Received 18 February 2017 bilized yeast cells from a mixture of polysaccharide based on gellan, i-carrageenan, and
Received in revised form 4 May 2017 carboxymethyl cellulose sodium salt. These particles were ionically cross-linked, using the
Accepted 16 May 2017 extrusion method, with magnesium acetate, calcium acetate and zinc acetate and were
Available online 24 May 2017 characterized, by FTIR and SEM, in terms of some physicochemical properties, morphol-
ogy, stability in water, and water retention capacity. These properties are influenced by the
Keywords: composition of polysaccharide mixture, which determines the number of cross-linkable
Gellan functional groups, and also by the type and the concentration of the cross-linking agent.
i-Carrageenan Analysis, by scanning electron microscopy, reveals that the yeast cells are well immobilized
Carboxymethyl cellulose and in high amounts in the polysaccharides matrix. This matrix maintains the cell viability
Yeast cells at high levels in aqueous media. High fermentation yields were obtained and the specific pro-
Immobilization ductivity in ethanol was equal to 0.79 g ethanol/h × g yeast cells. Highest ethanol yield was
Hydrogel particles obtained for the sample cross-linked with Mg2+ , and the lowest for the sample cross-linked
with Zn2+ . Polysaccharides matrix provides structural stability to yeast cells and maintains
the cells viability, at values higher than 82% even after 10 fermentation cycles, as well as their
ability to proliferate. Moreover, the obtained particles are stable, can be readily recovered
by filtration from the fermentation medium and can be reused for at least 10 fermentation
cycles. The obtained bio-reactors could represent a new approach to solving the problem of
producers in the alcoholic beverages industry concerning the deficit of mineral nutrients.
© 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1. Introduction for its use as biofuel. Production costs, environmental issues, fossil fuel
depletion and rising gas prices have been identified as major factors
Currently, there is a general interest regarding the increase of the that led to intensified research in order to find effective methods of
ethanol production efficiency for alcoholic beverage industry but also obtaining ethanol by fermentation processes (Bai et al., 2004; Hill et al.
2006; Blieck et al., 2007; Bai et al., 2008; Silva et al. 2008). In this regard,
the use of immobilized cells is a research area that has expanded due
to an attractive preparation method and to the economic advantages
∗
Corresponding author. that they present. Continuous processes are preferred as they have sub-
E-mail address: marpopa@ch.tuiasi.ro (M. Popa).
http://dx.doi.org/10.1016/j.fbp.2017.05.003
0960-3085/© 2017 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.
 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123
stantially improved efficiency, higher productivity and lower operating
costs (Kourkoutas et al., 2005). Also, production of ethanol, for use as
biofuel, has enjoyed of a great interest in recent years. The production
of fuel from ethanol is different from the beverage production due to the
fact that it is not necessary a slow process for providing a quality final
product; in this case, it is preferably to use a rapid and complete fermen-
tation process in order to ensure a maximum profitability (Westman
and Franzén, 2015).
Yeasts, especially Saccharomyces species, are preferred due to a
large fermentation capacity, to their tolerance to ethanol and to
other inhibitors, and to their ability of rapid growth under anaer-
obic conditions, which are characteristic of fermentative processes
(Groboillot et al., 1994; Ivanova et al., 2011). The advantages of yeast
cells immobilization on various supports are multiple: higher volu-
metric productivity as a result of the high cells density, increased
tolerance to various stressors, such as higher ethanol concentrations,
toxic metabolites produced by fermentation, high temperature and pH,
improved mechanical and chemical stability, low risk of contamination
and inhibition, maintenance of cell viability, lowering the production
costs due to the fact that these micro-bio-reactors can be recovered
easily from the fermentation medium and reused in other processes,
and the possibility of using these systems in continuous fermentative
processes (Brányik et al., 2001; Verbelen et al., 2006; Duarte et al., 2013).
Several techniques have been developed for the cells immobiliza-
tion on a organic or inorganic support matrix by physical (adsorption)
or chemical (covalent) binding, by flocculation (aggregation of the
biomass) and by encapsulation in microcapsules or in more or less
porous polymeric particles (Rathore et al., 2013). These techniques
should be simple, cheap and it is essential that the immobilized cells to
have an adequate fermentative activity, to be stable in the fermentation
medium, and to maintain a high viability (Baptista et al., 2006). Methods
commonly used to obtain systems with immobilized cells having appli-
cations in the food industry 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), gel entrapment
and radiation polymerization (Saraydın et al., 2002). Among them, the
extrusion method is most often used due to the mild processing condi-
tions that enable optimal encapsulation and also due to the fact that the
cellular viability is not affected (Rathore et al., 2013). Some of the sub-
strates used for immobilization are: alginates (Fumi et al., 1987; Corton
et al., 2000; Idris and Wahidin, 2006; Yoo et al., 1996) glass or ceramic
substrates (Goncalves et al., 1992; Santos et al., 2005), cellulosic materi-
als (Credou and Berthelot, 2014), ␥-alumina (Loukatos et al., 2000), silica
sol–gel films (Inama et al., 1993), polyacrylamide gel (Aykut et al., 1988)
and combinations of various materials (Peinado et al., 2006; Câmara
et al., 2016). Choosing the proper media for immobilization is essential
and the required characteristics are: good chemical and mechanical
stability, absence of toxicity, biocompatibility, and high diffusion coef-
ficient for both substrates and fermentation products (Baptista et al.,
2006).
Alginate-based matrices obtained by the ionotropic gelation, even if
frequently used, have some drawbacks: limited diffusion of nutrients,
of metabolites and of oxygen as a consequence of the gel matrix and of
the high density of cells immobilized into the particles (Nedovic et al.,
2005) Moreover, alginate gels have a low mechanical and chemical sta-
bility which can lead to a diffusion of a large number of cells in the
same time with their proliferation (Maicas, 2001; Idris and Wahidin,
2006; Călinescu et al., 2012; Nedović et al., 2015). These properties may
be improved by grafting synthetic polymers on alginate or by the incor-
poration, in gels, of inorganic materials such as silica, sand and alumina
(Rosevear, 1984; Hong et al., 2016). The polyacrylamide gel was widely
used to immobilize several types of microbial cells (Călinescu et al.,
2012; Dinu et al., 2007) and the fermentative activity of these systems
with immobilized yeast cells was shown to be comparable to those
based on alginates, nevertheless having a higher stability (Călinescu
et al., 2012). The systems based on polyacrylamide may be obtained
by polymerization using gamma radiation (Deshpande et al., 1987;
Ahmed, 2015), which, in comparison with the chemically initiated poly-
merization, has the advantage that it can occur at low temperatures,
down to −75 ◦ C. These conditions prevent the inactivation caused by
105
temperature and samples can be frozen in any form: particles, tubes or
membranes. This technique has been also extended to the cell immo-
bilization in gelatin (El-Hadedy et al., 2014).
Hydrogel particles based on alginates were prepared by ionic cross-
linking using Ca2+ , Ba2+ , and then covered with a layer of silica gel.
Thus, the yeast cells are protected by the alginate-based matrix and the
silica layer provides stability and the possibility to use these systems
in continuous fermentation processes. The strength of the Si O bond
(425 J mol−1 ) which provides a higher stability to the material (Callone
et al., 2008).
Besides alginates, the possibility of using other polysaccharides, as
matrices for cell immobilization, was investigated in the literature.
Gellan, an anionic biopolymer (O’Neill et al., 1983; Grasdalen and
Smidsrod, 1987; Morris et al., 2012) allowed the preparation of a bio-
catalyst in the form of spherical particles with real prospects of being
used for sparkling wine production technology. The particles have been
formed by extruding the polysaccharide solution, containing a sus-
pension of yeast cells, through a capillary in a bath with a solution
of 2% CaCl2 , as ionic cross-linking agent (Mantaluta et al., 2012). Tan
et al. (2011), have immobilized yeast cells using gellan as support by
a emulsification method and have stated that the obtained micro-bio-
reactors can be reused. The production of ethanol during the first three
fermentation cycles was comparable to that obtained in the presence
of free yeast. Moreover, these authors have indicated that the micro-
bio-reactors are stable and readily recovered from the fermentation
medium, by filtration, and that they can be reused for at least ten
fermentation cycles with a relatively high yield of ethanol (Tan et al.,
2011).
Carboxymethyl cellulose sodium salt (CMCNa) is a water soluble
derivative of cellulose (Sannino et al., 2009). His mixture with a graft
copolymer based on N-vinyl-2-pyrrolidone (PVP) was used to immobi-
lize the baker’s yeast Saccharomyces cerevisiae, by the extrusion method,
using AlCl3 as a cross-linking agent. The obtained spherical particles
were tested in the fermentation process for 27 h and allowed the prepa-
ration of ethanol at a concentration of 57 g/L. Furthermore, it was
indicated that the yield of ethanol increases when the particles cross-
linking degree decreases (Yiğitoğlu et al., 2007; Gokgoz and Yiğitoğlu,
2011).
Carrageenan is a polygalactan rich in sulfate esters, character-
ized by a low solubilization temperature and low gel strength (Necas
and Bartosikova, 2013). K-carrageenan has been successfully used as
a matrix for the yeast cells immobilization allowing the preparation
of ethanol with a high yield. Hettwer and Wang (1982) have added
hydroxyapatite or tricalcium phosphate to the gel matrix based on
k-carrageenan. Tricalcium phosphate maintains the pH and the cell
viability at an optimal level, increases the gel density and the ethanol
productivity (Wang and Hettwer, 1982). Particles of k-carrageenan with
immobilized yeast cells were obtained, by the extrusion method using
KCl as cross-linking agent, by Martinez et al. (2016) and Godia et al.
(1987). K-carageenan particles were also obtained by the emulsification
technique, which involves the dispersion of the polysaccharide aque-
ous solution in a non-miscible organic phase, followed by the gelation
and the separation of microparticles. Since the k-carrageenan particles
are not very stable it can be coated with a layer of chitosan in order to
reduce the pore size leading thus to an increase of the gel mechanical
and chemical stability (Raymond et al., 2004).
The yeast cells require a wide range of metal ions for their prolif-
eration and metabolism and these ions have a significant impact on
important parameters, such as: rate conversion of sugar into ethanol,
the final yield in ethanol, growing and multiplying yeast, cell viabil-
ity, stressors and yeast flocculation behavior (Walker, 1994; Cyert and
Philpott, 2013). Cations of magnesium and zinc are mostly used as
they act as cofactors for several important glycolytic enzymes and also
as modulators of the stressors for yeast (Rautio et al., 2007; Gibson,
2011), such as: rapid fluctuations in temperature, high osmotic pres-
sure, increased concentration in ethanol and lack of nutrients that
adversely affects the metabolism of the yeast cells leading to an inef-
fective fermentation process (Ding et al., 2009; Zhao and Bai, 2009).
Different studies have pointed out that the yeast cells tolerance to
stressors is dependent on the nutritional composition of the fermenta-
106 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123
tion medium (Wang et al., 2007; Gibson, 2011). Metal ions, especially
magnesium, have an important role in the regulation of the yeast
cell metabolism (Walker et al., 1982; Walker, 1994), in maintaining the
structural integrity and functionality of the intracellular organelles
induced by cell–cell interactions, such as: flocculation, regulation of
gene expression, mechanism of nutrients absorption, activation of
enzymes involved in metabolism (Udeh and Kgatla, 2013; Udeh et al.,
2014). Yeasts have the ability to efficiently absorb essential miner-
als from the fermentation medium and to exclude the non-essential
ones. Yeast mineral nutrition increases the ethanol yield allowing the
production of high quality products and therefore is important for pro-
ducers of beer, wine and also for those who produce bio-ethanol and
distilled beverages (Udeh and Kgatla, 2013).
The aim of this study is related to the yeast cell immobiliza-
tion in matrices obtained from a mixture of three polysaccharides,
such as: gellan, carrageenan and carboxymehyl cellulose (CMCNa), by
ionotropic gelation using the extrusion method in the presence of cal-
cium acetate, magnesium acetate and zinc acetate, as cross-linking
agents. The originality is given, not only, by the mixture of three
polysaccharides for the matrix preparation but also by the use of three
different cross-linkers. The mixture of these three polysaccharides is
justified by the following aspects:
– Particles can be obtained from gellan by ionotropic gelation, but the
porosity of these particles are not adequate for the fermentation
processes.
– CMCNa increases the particles cross-linking degree due to a high
number of carboxylate groups.
– Carrageenan is involved in the cross-linking reaction by means of
the SO3 H groups allowing also the preparation of highly porous
particles which facilitates the diffusion of the substrate and of the
reaction products (Guzman-Villanueva et al., 2013; Varghese et al.,
2014). At the best of our knowledge, this polysaccharide has not
been used until now to immobilize yeast cells. Therefore, in this
study, it was investigated the influence of the ratio between the three
polysaccharides on physical, chemical and morphological character-
istics as well as on the fermentation capacity of the micro-reactors
formed by yeast cell immobilization into spherical matrices based
on the polysaccharides mixture. Based on literature data concern-
ing the different cross-linking capacity of divalent metal cations,
as well as their influence on yeast cell proliferation, it was studied
the influence of three metal cations (Mg2+ , Ca2+ , Zn2+ ) on parti-
cles physicochemical properties and especially on the fermentative
activity of the obtained micro-reactors.
2. Materials and methods
2.1. Materials:
- Kelco Biopolymers supplied Gellan (Gel) of the Kelkogel
food grade type having a molecular weight between 2 × 105
and 3 × 105 Da, for deacetylated gellan, and a COO− /mol
(646 Da) group.
- Carboxymethyl cellulose (CMCNa) was purchased from
Sigma Aldrich having 0.75 anions on each glucose unit and
a COO− /mol (233.7 Da) group.
- I-caragenan (Car), containing two HOSO3 − /mol (464 Da)
groups was also purchased from Sigma Aldrich.
- Zinc acetate (Ac2 Zn), magnesium acetate (Ac2 Mg), calcium
acetate (Ac2 Ca) and glucose were supplied by Sigma Aldrich.
- Yeast was purchase from Pakmaya, Romania with a concen-
tration of 30% cells (w/w).
The type of yeast used is a commercial bakery yeast strain
which is found in a compressed form and has a water con-
tent of 70%. The characteristics of Saccharomyces cerivisiae
cells are: 2–8 ␮m in diameter; a volume of 0.8–2 × 10−3 cm3 ;
weight between 0.2 and 0.4 × 10−10 g; number of cells/g equal
to 8–14 × 109 ; specific surface: 8–14 × 109 (m2 /g). This type of
yeast was selected due to the fact that it represents a cell
biomass of the top fermenting yeast and it is composed of
living cells capable of producing fermentation of sugars with-
out requiring a special growth or culture medium. These cells
absorb the easiest hexoses (glucose), and 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. Methods
2.2.1. Particles preparation by ionotropic gelification
For the preparation of particles based on Gel\CMCNa\Car ion-
ically cross-linked with Ac2 Ca, Ac2 Mg and Ac2 Zn different
polysaccharides concentrations were used, as indicated in
Table 1.
The experimental program which was used is provided in
Table 1.
The particles with immobilized yeast cells have identical
weight ratios between the polysaccharides and were obtained
under identical conditions. To differentiate the particles with-
out yeast cells from those with immobilized yeast cells, the
later will be designated such as: D1Y, D2Y, D3Y and D4Y, where
Y stands for immobilized yeast cells. All the obtained particles
are spherical and have a mean diameter of 2–3 cm.
The total amount of the polysaccharides mixture (0.25 g)
was dissolved in 25 ml double distilled water, under stirring at
a temperature of 70–80 ◦ C, in order to obtain a polysaccharides
solution with a concentration of 1%. As the viscosity of this
solution is low and does not form stable particles by dropping
into the ionic cross-linker solution, it was necessary to pro-
ceed to a pre-cross-linking process, prior to extrusion. This
reaction was realized by mixing the polysaccharides solution
with a volume of 1.25 ml cross-linking agent (Ac2 Ca, Ac2 Mg
and Ac2 Zn) solution, at various concentrations, leading to the
preparation of a viscous fluid, which can be easily extruded.
Then the solution was cooled to 30◦ –35 ◦ C, and extruded by
means of a syringe through a needle with a diameter of 23
Gauge in 125 ml of cross-linking agent solution of various con-
centrations, such as: 2.1 × 10−1 M Ac2 Mg, 2.1 × 10−1 M Ac2 Ca or
1.1 × 10−2 M Ac2 Zn. The extrusion process of the entire solu-
tion takes about 20 min.
Spherical particles of Gel\CMCNa\Car are formed instan-
taneously and they are stored in the extrusion solution for
2 h, and then washed with double distilled water in order to
remove the excess of the cross-linking agent. The particles
are separated by filtration and stored in a refrigerator at 4–6 ◦ C
before their characterization. Spherical particles with immo-
bilized yeast cells were obtained by a similar procedure only
that in this case the polysaccharides solution was mixed with
a yeast suspension (1.25 g yeast in 5 ml of water) at a tempera-
ture of 40 ◦ C, thus providing a yeast/polysaccharides mixture
mass ratio of 5:1. The particles with immobilized yeast cells
are kept at a temperature between 4–6 ◦ C subsequent to their
characterization.
2.2.2. FTIR-ATR spectroscopy
Car, Gel and CMCNa spectra as well as the spectra of sam-
ples D4Ca şi D4Zn, prepared with the mixture of these three
polysaccharides in a mass ratio of 1:1:1, were obtained using
a spectrometer FTIR Cary 630 with ATR at the interface of the
samples (Agilent Technologies, USA) over a frequency range
between 4.000 to 650 cm−1 and a resolution of 4 cm−1 . The soft-
 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123
Table 1 – Experimental program.
Sample Gel % Car % CMCNa % Concentration of cross-linking agent
added before extrusion, Ma
Ac2 Mg Ac2 Ca
D1 40 30 30 2.8 × 10−2 2.8 × 10−2 1.1 × 10−3
D2 40 20 40
D3 20 40 40
D4 33 33 33
a
Cross-linker solution volume in the pre-extruded equal to 1,25 ml.
b
Cross-linker solution volume in the extrusion bath equal to 125 ml.
ware used for data collection was Cary 630 MicroLab PC and
the software Agilent Resolution Pro was used to determine the
position of the peaks.
2.2.3. Particles stability study in different aqueous media
The storage of particles in aqueous media can cause the
detachment of polymer fragments from the polysaccharides
matrix, as well as the diffusion of yeast cells, leading to a
turbidity increase and consequently to a decrease of the stor-
age medium transmittance. A lowered transmittance value
correlates with a lower structural stability of the particles.
A spectrophotometer BOECO-S22, UV–vis, at a wavelength of
550 nm, was used to determine the transmittance, at different
time periods, for all the obtained samples in both the super-
natant resulted after the extrusion process, and the distilled
water in which the particles were stored. The method has been
previously described by Iurciuc (Tincu) et al. (2016a).
Particles without yeast cells (10 g) were kept in 50 ml
cross-linking solution for 36 h and then were washed with
double distilled water. The particles with immobilized yeast
cells were stored for 24 h in identical conditions. Measure-
ment of transmission was performed every 12 h. Between
measurements, yeast cell-free particles were stored at room
temperature whereas the particles with immobilized yeast
cells were stored at 4–6 ◦ C. Measurements of the transmittance
were performed in triplicate and average values are provided
in Tables 2 and 3. The standard deviation is within ± 0.3% lim-
its.
2.2.4. Cell viability and yeast cells concentration
It may be assumed that a higher amount of yeast cells can
diffuse from the least stable particles with immobilized yeast
cells. In order to determine if the cell viability is preserved in
time, it was necessary to calculate the concentration of cells
and the cell viability in the aqueous solution in which they are
stored. As previously indicated, the particles with immobilized
yeast cells were stored in 50 ml of solution containing different
concentrations of cross-linking agent (Ac2 Mg—2.1 × 10−1 M,
Ac2 Ca—2.1 × 10−1 M, Ac2 Zn—1.1 × 10−2 M) for 24 h, then they
were washed with double distilled water in order to remove
the cross-linker excess and stored for another 36 h in 50 ml of
double distilled water. After each determination the particles
were stored in a refrigerator at the temperature of 4–6◦ C in
the extrusion solution or in double distilled water.
The cell viability and the number of yeast cells in media
in which the particles were suspended in a dilute solution of
methylene blue and 2% sodium citrate were determined by
an optical microscope (Optika Model B-330) and a counting
chamber Marienfeld (Neubauer) with which it is possible to
identify the number of non-viable cells, autolysed cells colored
107
Concentration of the cross-linking
agent in the extrusion bath, Mb
Ac2 Zn Ac2 Mg Ac2 Ca Ac2 Zn
2.1 × 10−1 2.1 × 10−1 1.1 × 10−2
in blue, from the total amount of cells. The method has been
previously described by Iurciuc (Tincu) et al. (2016a,b).
Depending on the number of live and lysed yeast cells that
are in the extrusion solution after the above-mentioned peri-
ods of time it was determined the cell viability (Vc ).
N living cells
Vc = × 100 (1)
(N living cells + N dead cells)
Standard deviation is within ±0.3% limits.
Keeping the cell viability during the fermentation process
is a prerequisite for the particles to be used in several fer-
mentation cycles. Samples of type D4Y were use in order to
determine whether this process take place and also to inves-
tigate the influence of metal ions on cell proliferation. The
following procedure was used in order to determine if the
cells proliferate in the polysaccharides matrix between dif-
ferent fermentation cycles. From each sample tested for the
fermentative activity, 2–3 particles were precisely weighted
and then mechanically disrupted by means of an Ultratur-
rax at 2000 rpm in 100 ml of double distilled water in order
to obtain a homogeneous dispersion of the cells. By staining
these cells dispersion with methylene blue, using the method
described by Iurciuc (Tincu) et al. (2016a), both the cell concen-
tration and the cell viability in the particles were determined.
It was possible to evaluate the amount of cells which is found
in the particles after each fermentation cycle (g cell/g particles)
by taking into account the number of cells/ml (cell concentra-
tion) of the drawn calibration curve. This method with some
modifications was used by Tang and Le (2013) and Callone et al.
(2008).
2.2.5. Particles swelling degree
Since the particles obtained in this study have a hydrogel
behavior, it was considered useful to estimate their swelling
degree (Q,%). Q% was determined gravimetrically for both
particles with and without immobilized yeast cells. Sam-
ples were dried at 40◦ C until equilibrium was reached (Mdry )
then were suspended in 5 ml of water, and periodically (every
1 h) removed by filtration. The mass of the swollen sample
(Mswollensample ) was determined by weighing the particles after
dabbing them with filter paper to absorb water from their sur-
faces. The swelling was studied until equilibrium was reached
(when the amount of water retained remained constant).
The mass of water retained (Mwater ) represent the difference
between the mass of the swollen samples Mswollensample and
the mass of the dry samples Mdry . (Iurciuc (Tincu) et al., 2016a;
Ahmed, 2015; Pasqui et al., 2012). The swelling degree was
determined for all the obtained samples, with or without
immobilized yeast cells, cross-linked with the three different
cross-linker agents.
108 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123

Table 2 – Transmittance values for samples without immobilized yeast cells determined after various storage periods in
aqueous media.
Sample Transmittance, T %

In the gelifiation solution In double distilled water

12 h 24 h 12 h 24 h 36 h

D1Mg 99.60 98.70 99.00 98.50 97.10

D2Mg 99.60 98.50 99.10 98.50 97.80
D3Mg 99.60 99.10 98.90 98.00 95.50
D4Mg 99.40 99.20 99.20 98.50 95.30

D1Ca 99.40 98.20 98.90 97.30 96.00

D2Ca 98.90 98.10 99.20 97.90 96.10
D3Ca 99.10 98.40 98.50 97.00 95.90
D4Ca 98.30 98.00 98.80 97.20 96.80

D1Zn 98.70 96.80 99.70 99.00 96.70

D2Zn 98.30 97.10 99.70 99.10 97.50
D3Zn 99.30 97.30 99.70 98.50 97.60
D4Zn 99.50 97.30 99.50 98.70 98.00

Table 3 – Transmittance, concentration of yeast cells/ml and cell viability values for different samples in cross-linking
solution and in double distilled water.
Samples Transmittance, T% Concentration of yeast cells: Cell viability, %
number of cells/ml x 10−5
In the In double In the In double In the In double
cross-linking distilled cross-linking distilled cross-linking distilled
solution water solution water solution water
12 h 24 h 12 h 24 h 12 h 24 h 12 h 24 h 24 h 12 h 24 h

D1YMg 96.60 93.20 99.10 98.30 0.68 0.85 0.28 0.53 94.40 95.50 93.60
D2YMg 96.00 94.00 98.80 98.30 1.48 1.90 0.73 0.95 96.20 95.80 94.60
D3YMg 95.90 94.30 98.20 96.90 1.00 1.48 0.98 1.45 96.70 96.70 96.60
D4YMg 96.50 94.70 98.80 97.50 0.98 1.30 0.53 0.70 96.30 96.60 92.70

D1YCa 98.20 96.50 99.30 98.50 1.30 1.73 0.43 0.75 95.30 95.50 95.20
D2YCa 97.30 95.60 99.30 98.40 2.20 3.00 0.30 0.75 96.70 94.70 93.00
D3YCa 93.50 92.00 98.70 96.80 4.00 5.00 2.50 3.38 95.20 94.30 93.10
D4YCa 96.00 94.70 98.90 96.90 1.30 1.50 0.33 0.95 95.20 96.00 94.30

D1YZn 98.90 96.50 99.70 98.60 1.30 1.73 0.19 0.80 95.80 87.50 86.50
D2YZn 98.50 98.40 99.50 99.00 2.20 3.00 0.30 0.58 95.20 92.30 88.50
D3YZn 95.70 90.50 95.00 86.50 2.50 3.50 1.25 2.00 93.30 94.30 90.90
D4YZn 98.60 98.50 97.90 95.30 1.30 1.50 0.33 1.18 92.30 92.90 92.20

The swelling degree was calculated using the following 2.2.7. Testing the fermentative activity of particles with
equation: immobilized yeast cells
Testing the ability of particles with immobilized yeast cells
to induce the glucose fermentation was carried out in a fer-
Mwater
Q(%) = × 100 (2) menter in which has been introduced a volume of 50 ml
Mdry particles
glucose solution (c = 8%) in 3.5 × 10−3 M calcium chloride,
3.5 × 10−3 M magnesium acetate and 3.8 × 10−5 M zinc acetate.
The fermentation was conducted at 30 ◦ C, in anaerobic con-
Measuring the degree of swelling was made in triplicate
ditions and the amount of CO2 emitted was monitored. Each
and the standard deviation is within ±3% limits.
fermentation cycle is carried out during 255 min and the vol-
ume of CO2 is recorded every 15 min. After each fermentation
cycle, the particles were recovered from the fermenter, washed
2.2.6. Particles morphology
with double distilled water in order to remove the excess
The particles were characterized by scanning electron
of both glucose and cross-linker agent, and then stored in
microscopy in order to determine their morphology, porosity
the refrigerator at 4–6 ◦ C until the next cycle. The duration
and to qualitatively assess the extent to which the yeast cells
between two fermentation cycles was 24 h and the pH of
are found in the polysaccharides matrix. The particles were
the fermentation broth was adjusted to 5 with a 2% acetic
cut and dried, were metalized with gold using a sputter depo-
acid solution and sodium bicarbonate (NaHCO3 ) solution. The
sition device and analyzed using a Vega Tescan instrument.
method has been previously described in the article (Iurciuc
Samples selected to be analyzed by SEM were D4YMg,
(Tincu) et al., 2016a).
D4YCa and D4YZn. The polysaccharide mass ratio was 1:1:1.
For each sample were performed 10 fermentation cycles
The SEM micrographs have been performed before testing the
and the yield obtained after each cycle was compared to that
fermentative activity of the particles with immobilized yeast
obtained from the fermentation in the presence of free yeast.
cells.
 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123
The ethanol yield (fermentation efficiency) was given as
a ratio between the practical amount (Cp) of ethanol deter-
mined experimentally from the measured volume of CO2 and
the theoretical amount (Ct) of ethanol which can be obtained
from a solution of glucose having a concentration of 8%.
Cp
% = × 100
Ct
2.2.8. Specific productivity and fermentation rate
Theoretically, the produced amount of ethanol it can be
deducted from the measured volume of CO2 because, under
the chosen operating conditions, the alcoholic fermentation
is the only metabolic activity of the yeast. The calculations
are performed based on glucose fermentation reaction:
C6 H12 O6 → 2C2 H5 OH + 2CO2
180g 2×46g 2×22,4L
The specific productivity in ethanol was determined as fol-
lows:
The fermentation plot: CO2 measured volume VCO2 (mL)
versus time T (min).
From the slope of the linear portion is calculated and deter-
mine the amount of CO2 in mL/min. Tg␣ represents the slope
of the linear portion of the curve variation of the VCO2 vs. time.
V2 − V1
tg˛ = , tg␣ = vCO2 (mL/min)
T2 − T1
• The volume of gas at 0 ◦ C (273 K) is calculated based on the
volume of CO2 (mL/min), obtained in the previous step, at
the collection temperature of 30 ◦ C (303 K). The volume of
gas at 0 ◦ C will be expressed in L/h.
vCO2 × 273 × 60
L
VCO2 (0◦ C) = , (6)
303 × 1000 h
• The flow of ethanol (g ethanol/h) is determined from the
equation of the glucose fermentation
VCO2 (0 ◦ C) × 2 × 46
Gv = , getanol /h
2 × 22, 4
The specific productivity (g ethanol/h × g yeast) is the ratio
between the ethanol flow and the mass of dry yeast. The total
amount of dry yeast represents 30% of the total compressed
yeast and is equal to 0.375 g.
Gv
PS = ,g /h × gyeastcells
0, 375 etanol
The fermentation rate is calculated as the slope of the lin-
ear portion of the glucose fermentation yield curve vs time for
a period of 0–45 min.
3. Results and discussions
Polysaccharides investigated in this study have a polyanion
behavior, and consequently a ionotropic gelation ability lead-
ing to the formation of hydrogel networks due to their strong
hydrophilicity. Preliminary studies have concluded that the
mixture of Gel with Car, in the presence of divalent cations,
leads to the formation of a interconnected polysaccharides
network with a high porosity. Moreover, it was shown that
yeast cells immobilized in these particles diffuse out of the
matrix in large amount in the storage medium and that the
109
particles become unstable over time. In order to create a
more stable and dense polysaccharides network, in which the
yeast cells will be well immobilized, CMCNa of low molecular
weight was chosen as additional component besides Gel and
Car. Previous studies have shown that CMCNa increases the
cross-linking density of hydrogel particles due to their higher
amount of carboxylate groups in the structural unit (Iurciuc
(3)
(Tincu) et al., 2017).
3.1. Particles preparation
Immobilization of yeast cells in the polysaccharides matrix
is based on fixing them in the mesh network obtained by
the ionic cross-linking of the polysaccharides chains due to
(4)
the binding of metal cations to the carboxylate anions of Gel,
CMCNa and to the acid sulfate groups of the Car. Using mix-
tures of the three polysaccharides, a stable interconnected
matrix is formed with suitable porosity which allows the opti-
mum diffusion of nutrients in the matrix in order to preserve
the cell viability in time. Schematic structure of such a network
is shown in Fig. 1. The yeast cells with negative electric charge
on the surface can bind M2+ ions thus reinforcing polysaccha-
rides network structure.
Yeast cell is an electrically charged colloid and may lose the
electrical charge or change the sign. It is positively charged in
(5)
the fermentation medium but, after a period of time, budding
occurs and it becomes negatively charged. At the end of the
fermentation, the electric charge of the yeast cell will become
positive again (Malm, 1946; Eddy and Rudin, 1958; Kregiel et al.,
2012). Divalent metal ions, in addition to the physiological
roles that they have in the yeast cell metabolism, may attach
to the cells plasma membrane, which is negatively charged,
giving it higher stability to stressors that may occur in the
fermentation processes (Udeh and Kgatla, 2013; Udeh et al.,
2014).
Between the particles with immobilized cells yeast and the
fermentation medium, which contains the glucose solution,
there must be an efficient exchange of nutrients in order that
(7)
the cells to proliferate, to obtain multiple fermentation cycles
and optimum ethanol yield.
Previous systematic studies (Iurciuc (Tincu) et al., 2016a,
2017) have demonstrated that, when using gellan as a matrix,
the optimum concentration of Ac2 Ca and Ac2 Mg solutions in
the cross-linking bath is equal to 2.1 × 10−1 M, thus this con-
centration was used for the preparation of all particle samples.
(8)
Preliminary cross-linking attempts realized in the presence
of Ac2 Zn, at the same concentration, led to particles with
immobilized yeast cells unable practically to trigger the fer-
mentation process. The explanation stems from the fact that
the network density is too high due to participation to cross-
linking of Zn, which is a transition metal, not only by ionic
but also by coordinative bonds. Yields and fermentation rates
comparable to those obtained in the presence of Ca2+ and
Mg2+ ions, which are metals of group II in the Mendeleev table,
were obtained at Zn2+ ions concentrations with an lower order
of magnitude, such as 1.1 × 10−2 M Ac2 Zn, corresponding to
a concentration of 0.2% wt. The concentrations of the three
cations were also different in the polysaccharides solution
before extrusion: 2.8 × 10−2 M for Ca2+ and Mg2+ acetates, and
1.1 × 10−3 M for the Zn2+ salt. The different cations concentra-
tions are justified and from another reason: for their optimal
growth the yeast cells requires a larger amount of magnesium
110 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123
Fig. 1 – Schematic representation of the polymeric matrix with immobilized yeast cells.
and calcium ions, while the zinc ions are needed in very small
quantities (Walker, 2004).
3.2. Particles characterization by FTIR-ATR
spectroscopy
FTIR spectroscopy was used in order to determine the pres-
ence of polysaccharides within the obtained particles. IR
spectra were recorded for the three polysaccharides (Gel, Car,
CMCNa) as well as for the cross-linked particles D4 with Ac2 Ca
and Ac2 Zn and these data are illustrated in Fig. 2.
FTIR spectra from Fig. 2 proves qualitatively the presence
of each polysaccharide in the particles composition. For sam-
ple D4Ca the band at 1052.9 cm−1 is specific for HSO3− group
present in the Car. The bands at 1029.4 cm−1 , specific for the
CH–OH group of the cyclic alcohols, at 1409.4 cm−1 , specific to
OH of carboxylic group, and at 1604 cm−1 , specific to salts of
carboxylic acids, show the presence, in particles, of Gel and of
CMCNa. These two polysaccharides have almost identical FTIR
spectra and thus their differentiation is not possible. Similar
results were obtained from the analysis of the IR spectrum of
particles cross-linked with zinc acetate where the signals cor-
responding to the above mentioned bands are easily shifted.
3.3. Stability study over time of particles without
immobilized yeast cells in different aqueous media
In order to determine if the particles can be used in repeated
fermentation processes it is important that their stability to be
tested in different aqueous media. As mentioned in the “meth-
ods” section, polysaccharides fragments can detach from the
least stable particles into the storage solution leading to an
increase of the turbidity. The turbidity of the aqueous solu-
tions was determined by measuring the transmittance and
the results for the samples without immobilized yeast cells
are shown in Table 2. In order to differentiate the samples,
the following notations will be used: the particles cross-linked
with Ac2 Mg, Ac2 Ca and Ac2 Zn are designated as DiMg, DiCa
and DiZn, respectively (where i represent the sample number).
From this table it can be observed that, after 24 h of stor-
age in the cross-linking solution, the transmittance values
slightly decrease, lowest values being noticed for particles
cross-linked with Ac2 Zn. This behavior may be due to a slightly
lower density of the polysaccharides matrix which induces the
diffusion in the storage medium of shorter macromolecules
from the outer layers of the matrix.
In double distilled water, all particles show a slight reduc-
tion of the transmittance after 36 h. Surprisingly, the particles
cross-linked with Ac2 Zn seem to be more stable, probably due
to the fact that, after the diffusion of the polysaccharides
chains from outer layers, the core matrix has a high cross-
linking degree and therefore the structure is more stable.
Moreover, it can be noticed that the transmittance values
do not vary widely with the polysaccharides ratio. However,
it can be seen that the sample D2, regardless of the cross-
linking agent used, has a transmittance value higher than D1.
For sample D3, characterized by a maximum Car concentra-
tion in the mixture, the transmittance values has a tendency
to decrease when compared to other samples In this case,
higher tensions occurs in the polysaccharides network lead-
ing to the partial detachment of polymer fragments having as
consequence the slight decrease of the transmittance values.
It can be assumed, therefore, that these high transmittance
values are a proof of stability of the particles without yeast
cells in aqueous media.
3.4. Stability study over time of particles with
immobilized yeast cells—evaluation of diffused cells
concentration and of cell viability
In order to determine if particles with immobilized yeast
cells are stable in aqueous media, several characteristics were
determined, such as: transmittance average values, the con-
centration of cells/ml and cell viability in the cross-linking
 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123
Fig. 2 – FTIR spectra for: (a) Gel, (b) Car, (c) CMCNa and for cross-linked particles in the presence of (d) calcium acetate and (e)
zinc acetate.
solution and in distilled water after 12 h and 24 h of particles
storage. The resulting data are shown in Table 3.
From this table, a first general observation is related to
the fact that the transmittance values decrease slightly with
storage time in both aqueous media for all the samples. In
particular, it can be seen that particles cross-linked with Zn2+
have the highest stability even if Ac2 Zn is used in a much
lower amount as compared to other two divalent cations. This
behavior can be considered as a confirmation of the assump-
tion previously discussed, which is that the Zn2+ participates
in the formation of the network not only via ionic binding,
but also by coordinative bonds, leading thus to stable struc-
tures. However, an exception is D3YZn sample, which after
24 h of storage in the cross-linking solution has the highest
turbidity, indicating a more intense diffusion of the cells from
the polysaccharides matrix. The plausible explanation can be
that in the composition of this sample is found the highest
amount of Car, which increases the porosity of the polysac-
charides matrix. This explanation is confirmed also by the
lower transmittance values obtained for other two D3Y sam-
ples, cross-linked in the presence of Ca2+ and Mg2+ . Moreover,
111
it was noticed that particles cross-linked with Mg2+ have the
lowest values of transmittance, probably because this cation
is an electropositive element weaker than Ca2+ (Krieck and
Westerhausen, 2017) and therefore the ionic bonds formed in
the polysaccharides matrix have a weaker strength.
After 24 h storage in double distilled water, samples D4YZn
and D3YZn have the lowest values of transmittance com-
pared to the same samples cross-linked with Ac2 Mg or Ac2 Ca
probably also due to higher porosity induced by Car; identical
behavior was observed for the same samples cross-linked with
Ca2+ and Mg2+ . From these result, it can be assumed that the
transmittance should vary in the following order, if this feature
would be affected only by increasing the particles porosity:
T%D2Y > T%D1Y > T%D4Y > T%D3Y
For most of the analyzed samples, regardless of the cross-
linking agent used, the values of the transmittance vary in this
order.
The concentration of yeast cells in aqueous media in which
the particles are suspended correlates generally with the
transmittance values. The concentration increases with stor-
112 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123
age time and has the highest values for samples which have
higher amounts of Car in composition (D3Y, D4Y).
As expected, the concentration of cells in both aqueous
media increases with time. It is noted, however, that a higher
concentration of cells is found in the cross-linking bath than
in double distilled water.
The values of the transmittance for the particles stored
in double distilled water, even if varies in the same way as
in the case of particles stored in the cross-linking solution,
are however lowered. The effect is explained by the fact that
the storage in water is realized after 24 h of storage in the
cross-linking solution, and during this period of time a high
amount of the weaker cells trapped in the network has already
diffused. It is worth mentioning, however, that the same ten-
dency is observed, for the cells concentration, in both double
distilled water and cross-linking solution.
Cell viability in storage media was investigated in order to
determine if the polysaccharides matrix can maintain cell via-
bility for a longer period of time. High values of cell viability,
ranging between 86.5% and 96.59%, attests that the polysac-
charides matrix can maintain cell viability at optimal values
for fermentation processes.
3.5. Particles swelling capacity
The behavior in water of spherical particles of gellan/i-
carrageenan/CMCNa with and without immobilized yeast
cells was evaluated by the swelling degree (Q%). The swelling
degree is representative of the cross-linking density of the net-
work, and indicates the ability of the polysaccharides matrix
to allow the diffusion of the substrate and of the fermenta-
tion products. In Fig. 3 are shown the time variations of the
swelling degree obtained for all the samples.
From this figure it can be observed, as a general remark,
that the time variation of the swelling degree directly depends
on the amount of Car in the polysaccharides mixture. Thus,
in most cases presented in Fig. 3, the Q% values increase
with increasing the amount of Car. This behavior is due to
the increased porosity of the particles, which can retain large
amounts of water not only in the polysaccharides matrix but
also in the pores. The variation order of Q%, for particles with
and without immobilized yeast cells and regardless of the
cross-linking agent used, is the following:
Q D3 > Q D1 > Q D4 > Q D2
Swelling degree values are directly influenced by the par-
ticles cross-linking degree, respectively by the number of
anionic groups (carboxylate, sulfate) which are involved in the
formation of ionic bonds with the metal. This explains why
the sample D2 shows, regardless of the cross-linker used, the
lower Q% value; this sample contains the maximum amount of
CMCNa (which increases the cross-linking density) and min-
imum Car (which increases the porosity). Sample D1, with
smaller amounts of CMCNa but higher concentration of Car,
have higher values of Q % as a result of two complemen-
tary effects: low cross-linking degree and higher porosity. The
swelling degree depends also on the type of the cross-linker
used, for both particles with and without immobilized yeast
cells, and this feature varies in the following order:
QMg2+ > QZn2+ > QCa2+
It is well known that Ca2+ cations provide the formation
of stronger gels than those obtained with Mg2+ (Morris et al.,
2012; Iurciuc (Tincu) et al., 2016b).
The concentration used for the solution of Ac2 Zn in the
cross-linking bath was much lower (around 10−2 M) but suf-
ficient to provide a cross-linking degree comparable to that
achieved by other two divalent cations, due to the involve-
ment of Zn2+ ion also in complexation reactions. Moreover, it
was stated that a greater proportion of this cross-linking can
become toxic to cells (Walker, 2004).
For samples containing particles with immobilized yeast
cells, the swelling degree value is smaller than for particles
without yeast cells. It is well known that metal ions can adhere
to the cell membrane providing its more resistance (Lipke and
Ovalle, 1998; Learmonth and Gratton, 2002; Foster et al., 2014)
and that an additional particles cross-linking can be induced
by the bonds formed between the metal ions and the yeast
cells.
3.6. Particles morphology
Images taken by scanning electron microscopy reveals the
morphology of particles with immobilized yeast cells. As a
typical example, sample D4Y, which has equal weight ratio
between the three polymers, was selected and in Fig. 4 are
presented images for samples cross-linked in the presence of
the three cations used in this study: Mg2+ , Ca2+ and Zn2+ .
Scanning electron microscopy images were made in cross-
section and points out that the yeast cells are immobilized in
large number inside the particles and firmly attached in the
polysaccharides network. Furthermore, it was noticed that the
metal cations may influence the particles cross-linking degree.
The morphology induced by Ca2+ is thicker which could be a
consequence of a higher cross-linking degree. Particles cross-
linked with Mg2+ have a higher porosity, with a less dense
network as a consequence of a lower cross-linking degree as
compared with samples cross-linked with Ca2+ ions. Cross-
linked particles with Zn2+ ions shows densities comparable to
those obtained in the presence of Ca2+ , even if the concen-
tration of Ac2 Zn is an order of magnitude smaller than in the
other two cases; this behavior was explained above.
3.7. Testing the fermentative activity
Depending on the CO2 volume resulted from the glucose fer-
mentation in the presence of particles with immobilized yeast
cell, it was possible to calculate the ethanol yield (␩%) (Xing
et al., 2016). Although it is not considered a critical factor that
might affect the ethanol yield, the pH plays an important role
in the uptake of the metal cations by the yeast cells during
the fermentation process (Liu et al., 2015a,b). Different studies
have been made regarding the influence of pH on the fermen-
tation processes and it was shown that this parameter has an
essential role in the binding of metal cations in the absorption
sites from the cell membrane. Moreover, previous research
reported that the optimum pH for the fermentation medium is
around 4.5–5 (Rees and Stewart, 1997; Bose et al., 2011; Reddy
and Reddy, 2011). Consequently, the pH of the glucose solu-
tion, dissolved at various cross-linking agent concentrations,
introduced in the fermentation bath was adjusted to 5.
High yields in ethanol were calculated from the testing of
the fermentative activity for all the samples and these values
are comparable to those obtained in the presence of free yeast.
10 fermentation cycles were carried out for each sample, and
 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123 113

1400 700

1200 600
Swelling degree, Q%

Swelling degree, Q%
1000 500

800 400

600 300

400 D1 Mg 200 D1D Mg

D2 Mg D2D Mg
200 D3 Mg 100 D3D Mg
D4 Mg D4D Mg
0 0
0 2 4 6 8 10 12 0 5 10 15 20 25
Time (h) Time (h)
(a) (b)

800 600

Swelling degree, Q%
700
500
Swelling degree, Q%

600
400
500
400 300
D1D Ca
300 D1Ca 200 D2D Ca
200 D2Ca
D3D Ca
D3Ca 100
100 D4D Ca
D4Ca
0 0
0 10 20 0 5 10 15 20
Time (h) Time (h)
(c) (d)

600

1000
500
Swelling degree, Q %

Swelling degree, Q %

800
400

600
300

400
200 D1D Zn
D1 Zn
D2D Zn
200 D2 Zn
100 D3D Zn
D3 Zn
0 D4 Zn D4D Zn
0
0 10 20 30 0 5 10 15 20 25
Time(h) Time (h)

(e) (f)
Fig. 3 – Swelling degree time variations for: (a) samples without yeast cell cross-linked with Ac2 Mg, (b) samples with
immobilized yeast cells cross-linked with Ac2 Mg, (c) samples without yeast cell cross-linked with Ac2 Ca (d) samples
with immobilized yeast cells cross-linked with Ac2 Ca, (e) samples without yeast cell cross-linked with Ac2 Zn, (f) samples
with immobilized yeast cells cross-linked with Ac2 Zn.

after each fermentation cycle, the particles were washed and a function of time for four samples, D1Y, D2Y, D4Y, and D3Y,
kept in a refrigerator at a temperature of 4–6◦ C until the next cross-linked with magnesium acetate.
cycle. Fig. 5 shows the variation of the fermentation yield as It is well known that the Mg2+ ion represents about 0.3%
of the total mass of dry yeast and it activates more than 300
114 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123

Fig. 4 – SEM images for sample D4Y using as cross-linking agents Ac2 Mg, Ac2 Zn, and Ac2 Ca.

enzymes. Moreover, magnesium maintains the integrity of
the cell, which includes a structural stabilization of nucleic
acids, polynucleotides, chromosomes, polysaccharides and
lipids (Walker, 1994; Rees and Stewart, 1997; Reddy and Reddy,
2011; Bose et al., 2011; Birch and Walker, 2000; Barros de Souza
et al., 2016).
It was noted that the fermerntation yields are similar for all
4 samples and that these values are higher than that obtained
in the presence of free yeast. However, the highest values were
obtained for samples D3Y and D4Y which can be explained
by the highest values of Q%. There is thus a direct correla-
tion between the swelling capacity of the particles in aqueous
media and the fermentation capacity, explained by a more
important diffusion of the substrate and of the resulting prod-
ucts through less cross-linked matrices (Poreda et al., 2013).
Furthermore, it was observed that the ethanol yield decreases
 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123 115

80 70
70 60

Ethanol yield, η%

Ethanol yield, η%
60 50
50 40
I I
40 II II
30
30 IV IV
VI 20 VI
20
VIII IX
10 X
10 X 0
0 0
0
0 100 200 300
0 100 200 300
Time(min) Time(min)
(a) (b)
80 70
70 60
60
Ethanol yield, η %

50

Ethanol yield, η %
50
40
40 I I
30
III III
30
V 20 V
20 VI VIII
VIII 10 X
10 X 0
0 0
0 0 100 200 300
0 100 200 300
Time(min) Time(min)

(c) (d)

Fig. 5 – Variation of the ethanol yield as a function of time for several fermentation cycles (noted with I to X) for samples
cross-linked with Ac2 Mg: (a) D1Y, (b) D2Y, (c) D3Y, (d) D4Y. The line noted with O is the yield obtain from the fermentation in
the presence of free.

over time from cycle to cycle but it still has a higher value than
that obtained in the fermentation in the presence of free yeast.
For D4Y and D3Y samples, cross-linked with Ac2 Ca, it was
also possible to carry out 10 fermentation cycles with high
yield values, in most cases comparable to those obtained in
the presence of particles cross-linked with Mg2+ .
On the contrary, for particles cross-linked with Ac2 Zn only
4 fermentation cycles have been achieved for D3Y sample
and 8 fermentation cycles for sample D4Y. The sample D3Y,
with 0.4% carrageenan, 0.2% gellan and 0.4% CMCNa, con-
tains a higher amount of functional cross-linkable groups, in
comparison with the sample D4Y, which leads to a higher
requirement of zinc ions for cross-linking. Previous research
regarding the influence of metal ions on the fermentative pro-
cesses have shown that occasionally the lack of some minerals
in the fermentation medium may negatively affect the fer-
mentation process in the presence of yeast cells (Walker and
Stewart, 2016). Zinc is an important element in the fermen-
tative processes as it acts as an enzyme activator of alcohol
dehydrogenase and a medium deficient in zinc can lead to
slow or incomplete fermentation process (Zhao and Bai, 2012).
Therefore, even if the particles cross-linking degree and their
structural stability are comparable to samples cross-linked
with Ca2+ and Mg2+ , however the amount of Zn2+ is insufficient
to provide repeated fermentation cycles.
Fig. 6 shows a comparison of the fermentation yields for
D4Y and D3Y samples cross-linked with Ac2 Ca, Ac2 Mg and
Ac2 Zn. In Fig. 6a, the fermentation yields, determined for 10
fermentation cycles, are compared for D3Y samples obtained
by cross-linking in the presence of Ac2 Mg and Ac2 Ca. For D4Y
samples, obtained by using the three cross-linking agents, the
fermentation yields are given for eight cycles (Fig. 6(b)).
As a general remark, it can be seen, from this figure, that
the ethanol yield of samples cross-linked with Ac2 Mg is com-
parable to samples cross-linked in the presence of Ac2 Ca.
Ca2+ ions have a important role in protecting yeast cells
against the toxic effects of ethanol as well as Zn2+ and Mg2+
ions. Because the antagonistic effect of magnesium, the pos-
itive effects of cell proliferation can be compromised when
additional Ca2+ ions are added to the medium. Therefore, a
special attention should be taken during the addition of Ca2+
ions as if the calcium ions are added in high concentrations
they may replace the magnesium in a number of bio-chemical
pathways. However, this exchange could damage the cell in
time (Udeh and Kgatla, 2013; Liu et al., 2015a,b).
Moreover, the fermentation yields, for samples cross-
linked with Ac2 Mg, are higher compared to those obtained
for the same samples cross-linked with Ac2 Ca. This may be
due to the fact that yeast cells have a higher requirement for
Mg2+ than for Ca2+ , as indicated by Rees and Stewart (1997). At
the same time, the particles cross-linked with Mg2+ present
greater swelling capacity in water, thus a more pronounced
diffusion of the substrate and of the reaction products through
the polysaccharides matrix. Obtaining comparable fermen-
tation yields, for each fermentation cycle, for samples D4Y
and D3Y cross-linked with Mg2+ and Ca2+ may be a result
116 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123
Fig. 6 – Variation of the ethanol yield in the presence of free yeast (0), of samples D3Y and D4Y with immobilized yeast cells
and cross-linked in the presence of different metalic ions: (a) sample D3Y cross-linked with Ac2 Ca (D3Y Ca) and Ac2 Mg (D3Y
Mg), (b) sample D4Y cross-linked with Ac2 Ca (D4Y Ca), Ac2 Mg (D4Y Mg) and Ac2 Zn (D4Y Zn).
of the efficient exchange of nutrients between the fermen-
tation medium and the immobilized yeast cells. Starting with
the ninth fermentation cycle it appears however a downward
trend in yields. As it will be show later, this tendency is deter-
mined by the decrease of both cells number and viability of
cells which remain in the polysaccharides matrix after each
fermentation cycle.
For sample D4YZn, Fig. 2b shows that at the first fermen-
tation cycle it was obtain a maximum yield that exceeds the
corresponding yield for sample D4YCa and is almost equal to
the yield obtained for sample D4YMg. With the exception of
cycle 4, the fermentation yield decreases progressively from
cycle 2 to cycle 8 after which the fermentation stops. As previ-
ously stated, this behavior may be due to a deficiency in Zn2+
ions. A complete characterization has been carried out for all
types of samples D4 since the previous analysis showed that
they are stable and that the fermentation yields are superior
to other samples.
Fig. 7 presents the evolution of the fermentation yields in
time for samples D4YMg, D4YCa and D4YZn, for the first 8
fermentation cycles by comparing the results with the yield
obtained in the presence of the free yeast fermentation.
From this figure, it can be seen that fermentation yields
are similar for samples D4YCa and D4YMg and that these val-
ues are slightly variable from one cycle to another. On the
contrary, for sample D4YZn the ethanol yield decreases with
the number of cycles, behavior which was explained previ-
ously. From the high values of fermentation yields and from
the fact that they are almost constant for several fermenta-
tion cycles it can be concluded that a efficient exchange is
achieved between the fermentation medium and the immo-
bilized cells. Sample D4Y, containing equal proportions of the
three polysaccharides, is both sufficiently porous, due to the
quite high amount of Car, and structurally stable enough,
due to the high number of carboxyl groups involved in cross-
linking. These features explain the enhanced diffusion of
various molecular species through the polysaccharides matrix
but also the mechanical stability which allows the use of these
particles in repeated fermentation cycles without disintegrat-
ing.
3.8. Assessment of cell viability and cell amount from
the particles after each fermentation cycle
In order to obtain several fermentation cycles and a higher
ethanol yield, it is imperative that yeast cells to proliferate
within the polysaccharides matrix. By using the technical pro-
cedure described in the “Methods”, it was determined the
number of yeast cells in suspension and consequently in the
particles. Cell viability and cell number on one gram of parti-
cles are shown in Table 4.
From Table 4 it can be seen that, for all the samples, the cells
number is higher than that obtained in the presence of free
yeast. Moreover, the number of cells increases with increasing
the fermentation cycles. However, these values have a maxi-
mum at cycle VI for particles cross-linked in the presence of
Ca2+ and Zn2+ . Furthermore, it can be also observed that, for
all samples, the cell viability decreases slightly as a function
of the fermentation cycles, but these values are maintained at
a high level, which indicates that the polysaccharides matrix
protects the cells quite effective. Raymond et al. (2004) have
demonstrated, by determining the cell viability and the cell
number, that yeast cells proliferate within k-carrageenan par-
ticles coated with chitosan. Callone et al. (2008) have reached
at the same conclusion by determining the cells number in
alginate particles coated with a layer of silica.
Fig. 8 presents the evolution of the amount of cells/gram
of particles as a function of the fermentation cycle for sam-
ples D4Y compared to the maximum yield obtained after each
fermentation cycle.
From this figure it is obvious that the yeast cells proliferate
within the polysaccharides matrices. In the case of D4YMg
samples, the amount of yeast cells before fermentation is
equal to 17.2 mg/g and increases steadily from a fermentation
cycle to another until cycle 8, when it reaches 36.6 mg/g parti-
cles. A decrease occurs after this fermentation cycle, followed
by a slight upward trend. The maximum fermentation yield
follows basically the same trend up to cycle 8 (when a maxi-
mum is observed) and these values are in perfect correlation
with the evolution of the amount of cells from the particles.
After eight cycles, however, the maximum yield decreases
slightly, apparently in discordance with the variation of the
amount of cells. By analyzing in detail these results, in con-
nection with the results provided in Table 2, it is obvious that
these low fermentation yields are a direct consequence of the
number of living cells decrease even if the total number of cells
increases after the cycle 8.
A good agreement between the two features can be
observed for D4YCa sample, in which the initial weight of
yeast cells is 18.98 mg/g of particles. The amount of yeast cells
increases up to cycle 6, when it is equal to 31.4 mg cells/g parti-
cles, and decreases after cycle 7. In this case, a similar trend is
 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123 117

70 70

Ethanol yield, η %
60 60

Ethanol yield, η %
50 50
40 40
30 30
I D4YMg
20 I D4YCa 20 II D4YMg
I D4YZn II D4YCa
10 10
0 II D4YZn
0 0
0
0 100 200 300 0 100 200 300
Time(min) Time(min)

(a) (b)
70 70
60 60

Ethanol yield, η %
Ethanol yield, η %

50 50
40 40
30 30
III D4YMg IV D4YMg
20 20
III D4YCa IV D4YCa
10 III D4YZn 10 IV D4YZn
0 0 0
0
0 100 200 300 0 100 200 300
Time(min) Time(min)

(c) (d)
70 80
60 70
Ethanol yield, η %

Ethanol yield, η %

50 60
50
40
40
30
30 VI D4YMg
20 V D4YMg
20 VI D4YCa
V D4YCa
10 V D4YZn 10 VI D4YZn
0 0
0 0
0 50 100 150 200 250 300 0 100 200 300
Time(min) Time(min)

(e) (f)
70 70
60 60
Ethanol yield, η %

Ethanol yield, η %

50 50
40 40
30 30
VII D4YMg
20 20 VIII D4YMg
VII D4YCa
10 VIII D4YCa
VII D4YZn 10 VIII D4YZn
0 0
0 0
0 100 200 300
0 100 200 300
Time(min) Time(min)

(h)
Fig. 7 – Evolution of the ethanol yield in time for the samples D4YMg, D4YCa and D4YZn in different fermentation cycles:
(a)—cycle 1; (b)—cycle 2; (c)—cycle 3; (d)—cycle 4; (e)—cycle 5; (f)—cycle 6; (g)—cycle 7; (h)—cycle 8, compared with the yield
obtained in free yeast fermentation. (T = 30 ◦ C, fermentation cycle duration: 255 min).

noted for the maximum fermentation yield. These results are Sample D4YZn has a slightly different behavior. The
in agreement with previous research which attest that yeast amount of cells increases from one fermentation cycle to
cells immobilized in different supports retain their viability another and becomes almost constant after cycle 7. How-
(Kregiel et al., 2013; Berlowska et al., 2013). ever, the fermentation yields decrease continuously, excepting
118 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123

Table 4 – Cell viability and the cells number/gram of particles.

Fermentation cycle Cells number × 108 /g particles Cell viability, Vc%

D4YMg D4YCa D4YZn D4YMg D4YCa D4YZn

a
0 4.70 5.10 4.40 95.90 96.50 97.10
I 5.20 5.90 5.50 95.90 94.50 96.50
VI 7.80 8.70 9.40 95.00 94.70 92.20
VIII 7.40 7.60 8.80 94.30 94.10 92.00
X 8.20 6.90 7.50 93.00 88.60 87.80

a
Fermentation in the presence of free yeast.

Fig. 8 – Variation of the amount of cells from particles as a function of the fermentation cycles for sample D4Y cross-linked
with: (a) Ac2 Mg, (b) Ac2 Ca and (c) Ac2 Zn.

for cycle 4 in which a maximum is observed. Lower yields,
determined in last fermentation cycles, might be a direct
consequence of the cell viability decrease despite of the pro-
liferation or constant amount of cells. Studies regarding the
influence of the zinc ions on the fermentative processes for
brewing shows that supplementation of wort with zinc ions
leads to higher flocculation, caused probably by its binding
to cell membranes. In this context, Taylor and Orton showed
that the zinc inhibits the flocculation only when it is found at
very high concentrations in the fermentation medium (Taylor
and Orton, 1973, 1975). Consequently, inside the particles from
sample D4YZn, the yeast cells can form aggregates that do not
allow an efficient exchange of nutrients, leading to a decrease
of ethanol productivity. (Tamás et al., 2014; Poreda et al., 2013).
Zinc ions can have two opposite effects: on the one hand they
lead to an increased number of yeast cells inside the polysac-
charides matrix because the zinc acts as an enzyme activator
(this behavior is found in the present study), on the other hand
it forms a complex with both the functional groups of the-
polysaccharides mixture and with the proteins from the cell
membranes (Migockal et al., 2015). In this last case, very strong
coordinative bonds are formed and the ethanol productivity
decreases due to cells aggregation in time and to the limited
nutrients diffusion. Fig. 9 shows the variation of the maximum
fermentation yields as a function of the fermentation number
for samples cross-linking with Ac2 Zn.
From this figure it can be seen that samples D4YZn and
D1YZn have the highest ethanol yield and can be used up
to 8 fermentation cycles. These samples contain the smallest
amount of anionic functional groups, which are involved in the
cross-linking process, and the highest amount of Car, there-
fore, their networks are characterized by lowest cross-linking
 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123 119

Fig. 9 – Maximum glucose fermentation yields in the presence of free yeast (0) as well as in particles with immobilized
yeast cells and cross-linked with zinc acetate: (a) D1Y; (b) D2Y; (c) D3Y and (d) D4Y.

density and higher porosity. These both features have as result
the increase of the diffusion of all molecular species from and
out of the polysaccharides matrix, with positive impact on
the fermentation process. Samples D3YZn and D2YZn have
lower ethanol yields (␩ %) probably due to the higher cross-
linking degree induced by the large number of anionic groups
of the polysaccharides chains. This conclusion is in concor-
dance with the results obtained for the swelling capacity in
aqueous media which varies in the following order:
␩%D3Y > ␩%D2Y > ␩%D4Y > ␩%D1Y
In order to evaluate the stability of the particles with
immobilized yeast cells in the fermentation medium, it was
determined the number and consequently the amount of cells
that are found in this medium after each fermentation cycle.
Mariam et al. (2009) obtained micro-bio-reactors based on
sodium alginate with immobilized yeast cells by ionic cross-
linking using CaCl2 as cross-linking agent. These particles
were tested in terms of their fermentative activity and an
increase in the maximum fermentation yield was observed
after the 4th cycle. This maximum was followed by a sharp
decrease and the maximum number of fermentation cycles
conducted with these micro-bio-reactors was 6. This behav-
ior was explained by the fact that these micro-bio-reactors
become unstable after the 4th fermentation cycle by changing
their morphology and thus the yeast cells can diffuse in large
numbers in the fermentation medium (Mariam et al., 2009).
The present study aims to demonstrate that the yeast cells
are firmly attached to the polysaccharides network and that
they are not diffusing in large numbers in the fermentation
medium even after several fermentation cycles. The obtained
results are given in Table 5.
For this experiment it was determined the number of the
yeast cells that diffuses from the particles in 50 ml solution of
the fermentation medium and based on the calibration curve
it was determined the quantity of yeast cells.
From this table it appears that the total number of cells
which diffuse from the particles cross-linked with Ac2 Mg and
Ac2 Ca increases with increasing the porosity induced by Car.
Moreover, it can be noticed that the amount of cells which
diffuses is higher for samples cross-linked with Ac2 Ca than
for those cross-linked with Ac2 Mg. Therefore, it can be con-
cluded that the particles cross-linked with Ac2 Mg are more
stable than those cross-linked with Ac2 Ca. This behavior can
be explained by the fact that the yeast cells have a higher
affinity for Mg2+ ions than Ca2+ ions and therefore Mg2+ ions
can adhere more easily to the plasma membrane of cells.
This can lead to more stable structures as the bio-catalyst can
practically participate in the cross-linking reaction (Udeh and
Kgatla, 2013; Udeh et al., 2014).
The amount of diffusing cells increases with decreasing
the number of cross-linkable functional groups for all the par-
ticles. However, the number of diffusing cells from particles
cross-linked in the presence of zinc acetate is much lower
than that from particles cross-linked with Ca2+ or Mg2+ . This
behavior may be due to the fact that the Zn2+ ions are absorbed
effectively by the yeast cells and therefore they are stronger
anchored in the polysaccharides matrix.
The results provided in Table 5 demonstrate that a small
number of yeast cells diffuses from the particles tested in
the fermentation process and that this proportion is much
smaller than the number of yeast cells remaining in the parti-
cles. Moreover, these results prove that the fermentation takes
place predominantly in the particles and not in free cells from
supernatant.
120 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123

Table 5 – The amount of yeast cells which diffuses from the particles after each fermentation cycle.
Sample Total number of Total number of yeast cells in the Total amount of yeast cells which
fermentation cycles fermentation medium/ml × 106 diffuse from the particles, mg

D3YMg 10 1.67 2.91

D4YMg 10 1.28 2.46
D3YCa 10 1.70 3.50
D4YCa 10 1.39 2.85
D3YZn 4 0.42 1.18
D4YZn 8 0.56 1.25

Table 6 – Fermentation rate and specific ethanol productivity.

Number of Fermentation rate, % glucose Specific productivity (g etanol/h × g
fermentation cycles conversion/min yeast cells)/particles type
D3Y D4Y D3Y D4Y D3Y D4Y D3Y D4Y D3Y D4Y D3Y D4Y
Mg Mg Ca Ca Zn Zn Mg Mg Ca Ca Zn Zn

I 0.27 0.22 0.24 0.20 0.20 0.20 0.79 0.79 0.79 0.79 0.59 0.79
II 0.27 0.24 0.20 0.27 0.20 0.20 0.70 0.79 0.59 0.79 0.59 0.59
III 0.20 0.20 0.20 0.22 0.16 0.20 0.79 0.77 0.76 0.79 0.40 0.59
IV 0.20 0.27 0.20 0.27 0.20 0.20 0.79 0.79 0.76 0.79 0.40 0.79
V 0.20 0.27 0.22 0.22 0.20 0.79 0.79 0.79 0.79 0.59
VI 0.20 0.20 0.20 0.27 0.20 0.59 0.79 0.59 0.62 0.59
VII 0.27 0.24 0.20 0.20 0.20 0.79 0.72 0.59 0.59 0.59
VIII 0.20 0.24 0.20 0.20 0.13 0.59 0.82 0.59 0.79 0.40
IX 0.29 0.20 0,20 0.22 0.98 0.79 0.79 0.79
X 0.20 0.20 0.22 0.27 0.59 0.63 0.79 0.79

The fact that the particles immobilized with yeast cells
can be easily recovered from the fermenter and can be used
for several fermentation cycles shows that the polysaccha-
rides matrix in which these cells are immobilized protect them
from the toxic metabolites produced by ethanol. Furthermore,
cell viability is maintained and the cells yeast may proliferate
within the obtained particles.
From the linear portion of the kinetic curves which describe
the fermentation process it was possible to evaluate the fer-
mentation rate. The specific productivity in ethanol and the
fermentation rate during 45 min were calculated for all the
samples and these values are given in Table 6.
The fermentation rate in the presence of free yeast and
the specific productivity are equal to 0.067% conversion of glu-
cose/min and 0.51 g ethanol/g glucose × g yeast, respectively.
From Table 6 it can be noted that, for almost all the fermenta-
tion cycles, the fermentation rate is higher than that obtained
in the fermentation of free yeast. A similar trend was observed
for both fermentation rate and fermentation yields. Identical
conclusions can be drawn for the specific ethanol productivity.
Finally, it is worth noting that the highest yield was obtained
with for sample D4Y cross-linked with magnesium acetate.
4. Conclusions
Mixtures based on gellan, carboxymethyl cellulose (sodium
salt) and carrageenan, in different proportions, cross-linked
with divalent metal ions derived from acetates, such as: Ca2+ ,
Mg2+ , and Zn2+ , allow to preparation, by extrusion, of spherical
particles capable of immobilizing yeast cells in the polysac-
charides matrix. The spherical cross-linked matrices with
and without yeast cells, are stable in aqueous media and
the morphology and the physicochemical properties of par-
ticles depend on their composition in polysaccharides and
on the cross-linker nature; the particle porosity increases
with increasing the amount of Car in the mixture. The parti-
cles swelling degree is directly correlated to the cross-linking
degree, which in turn depends on the amount and nature of
the cross-linking agent as well as on the polysaccharides com-
position in the particles. The mixture composition influences
the number of cross-linkable groups and the particles porosity;
higher swelling degree was observed for particles cross-linked
with Mg2+ and also those containing high amounts of car-
rageenan. Particles with immobilized yeast cells exhibit a
higher cross-linking degree due to the fact that the yeast cells
forms ionic bonds with metal ions.
The fermentative activity depends on the ratio between
the polysaccharides of the particles and on the type of cross-
linker; highest ethanol yield was obtained for the sample
D4Y cross-linked with Mg2+ , and the lowest for the sample
cross-linked with Zn2+ . The particles with immobilized yeast
cells can be reused for at least 10 fermentation cycles. The
polysaccharides matrix maintains the cell viability at val-
ues higher than 82% even after 10 fermentation cycles. The
specific ethanol productivity was higher for particles with
immobilized yeast cells than for free yeast. The particles of
gellan/i-carrageenan/CMCNa with immobilized yeast cell can
be used successfully in repeated fermentative processes and
may represent a new approach to solving the problem of metal
ions deficiency facing producers in the alcoholic beverages
industry.
References
Ahmed, E.M., 2015. Hydrogel: preparation, characterization, and
applications: a review. J. Adv. Res. 6, 105–121,
http://dx.doi.org/10.1016/j.jare.2013.07.006.
Aykut, G., Hasirci, V.N., Alaeddinoglu, G., 1988. Immobilization of
yeast cells in acrylamide gel matrix. Biomaterials 9, 168–172,
http://dx.doi.org/10.1016/0142-9612(88)90117-2.
Bai, F.W., Chen, L.J., Zhang, Z., Anderson, W.A., Moo-Young, M.,
2004. Continuous ethanol production and evaluation loss
under very high gravity medium conditions. J. Biotechnol. 110,
287–293, http://dx.doi.org/10.1016/j.jbiotec.2004.01.017.
Bai, F.W., Anderson, W.A., Moo-Young, M., 2008. Ethanol
fermentation technologies from sugar and starch feedstock.
 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123
Biotechnol. Adv. 26, 89–105,
http://dx.doi.org/10.1016/j.biotechadv.2007.09.002.
Baptista, C.M.S.G., Cóias, J.M.A., Oliveira, A.C.M., Oliveira, N.M.C.,
Rocha, J.M.S., Dempsey, M.J., Lannigan, K.C., Benson, P.S., 2006.
Natural immobilisation of microorganisms for continuous
ethanol production. Enzyme Microb. Technol. 40, 127–131,
http://dx.doi.org/10.1016/j.enzmictec.2005.12.025.
Barros de Souza, R., Silva, R.K., Ferreira, D.S., de Sa Leitao Paiva Jr.,
S., de Barros Pita, W., de Morais Jr., M.A., 2016. Magnesium
ions in yeast: setting free the metabolism from glucose
catabolite repression. Metallomics 8, 1193–1203,
http://dx.doi.org/10.1039/C6MT00157B.
Berlowska, J., Kregiel, D., Ambroziak, W., 2013. Physiological tests
for yeast brewery cells immobilized on modified chamotte
carrier. Antonie Van Leeuwenhoek 104, 703–714,
http://dx.doi.org/10.1007/s10482-013-9978-1.
Birch, R.M., Walker, G.M., 2000. Influence of magnesium ions on
heat shock and ethanol stress responses of Saccharomyces
cerevisiae. Enzyme Microb. Technol. 26, 678–687,
http://dx.doi.org/10.1016/S0141-0229(00)00159-9.
Blieck, L., Toye, G., Dumortier, F., Vertrepen, K.J., Delvaux, F.D.,
Thevelein, J.M., Vandijck, P., 2007. Isolation and
characterization of brewer’s yeast variants with improved
fermentation performance under high-gravity conditions.
Appl. Environ. Microbiol. 73, 815–824,
http://dx.doi.org/10.1128/AEM, 02109-06.
Bose, J., Babourina, O., Rengel, Z., 2011. Role of magnesium in
alleviation of aluminium toxicity in plants. J. Exp. Bot. 62,
2251–2264, http://dx.doi.org/10.1093/jxb/erq456.
Brányik, T., Vicente, A.A., Machado Cruz, J.M., Teixeira, J.A., 2001.
Spent grains—a new support for brewing yeast
immobilisation. Biotechnol. Lett. 23, 1073–1078,
http://dx.doi.org/10.1023/A:1010558407475.
Callone, E., Campostrini, R., Carturan, G., Cavazza, A., Guzzon, R.,
2008. Immobilization of yeast and bacteria cells in alginate
microbeads coated with silica membranes: procedures,
physico-chemical features and bioactivity. J. Mater. Chem. 18,
4839–4848, http://dx.doi.org/10.1039/B807301E.
Călinescu, I., Chipurici, P., Trifan, A., Bădoiu, C., 2012.
Immobilisation of Saccharomyces cerevisiae for the production
of bioethanol. U.P.B. Sci. Bull. 74, 33–40
http://www.scientificbulletin.upb.ro/rev docs arhiva/
full2e3 876162.pdf.
Câmara, A., Dupont Beney, S.L., Gervais, P., Rosenthal, A., Correia,
R.T.P., Pedrini, M.R.S., 2016. Fisetin yeast-based bio-capsules
via osmoporation: effects of process variables on the
encapsulation efficiency and internalized fisetin content.
Appl. Microbiol. Biotechnol. 100, 5547–5558,
http://dx.doi.org/10.1007/s00253-016-7425-8.
Corton, E., Piuri, M., Battaglini, F., Ruzal, S.M., 2000.
Characterization of Lactobacillus carbohydrate fermentation
activity using immobilized cells technology. Biotechnol. Prog.
16, 59–63, http://dx.doi.org/10.1021/bp9901217.
Credou, J., Berthelot, T., 2014. Cellulose: from biocompatible to
bioactive material. J. Mater. Chem. B 2, 4767–4788,
http://dx.doi.org/10.1039/C4TB00431K.
Cyert, M.S., Philpott, C.C., 2013. Regulation of cation balance in
Saccharomyces cerevisiae. Genetics 193, 677–713,
http://dx.doi.org/10.1534/genetics.112.147207.
Deshpande, A., D’Souza, S.F., Nadkarni, G.B., 1987.
Coimmobilization of D-amino acid oxidase and catalase by
entrapment of Trigonopsis variabilis in radiation polymerised
Polyacrylamide beads. J. Biosci. 11, 137–144,
http://dx.doi.org/10.1007/BF02704664.
Ding, J., Huang, X., Zhang, L., Zhao, N., Yang, D., Zhang, K., 2009.
Tolerance and stress response to ethanol in the yeast
Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 85,
253–263, http://dx.doi.org/10.1007/s00253-009-2223-1.
Dinu, M.V., Ozmen, M.M., Dragan, E.S., Okay, O., 2007. Freezing as
a path to build macroporous structures: superfast responsive
polyacrylamide hydrogels. Polymer 48, 195–204,
http://dx.doi.org/10.1016/j.polymer.2006.11.022.
121
Dobbs, A.J., Peleg, M., Mudgett, R.E., 1982. Some physical
characteristics of active dry yeast. Powder Technol. 32,
163–169, http://dx.doi.org/10.1016/0032-5910(82)85007-9.
Duarte, J.C., Rodrigues, J.A.R., Moran, P.J.S., Valença, G.P., Nunhez,
J.R., 2013. Effect of immobilized cells in calcium alginate beads
in alcoholic fermentation. AMB Express 3, 31,
http://dx.doi.org/10.1186/2191-0855-3-31.
Eddy, A.A., Rudin, A.D., 1958. The structure of the yeast cell wall.
I. Identification of charged groups at the surface. Proc. R. Soc.
Lond. B Biol. Sci. 148, 419–432
http://rspb.royalsocietypublishing.org/content/
148/932/419.abstract.
El-Hadedy, D.E., Saad, M.M., Hassan, H.M., 2014. Protease
inhibitor expression with immobilized cells of egyptian
Streptomyces lavendulae on different radiated matrices using
gamma radiation. World J. Pharm. Sci. 2, 899–913
http://www.wjpsonline.org/admin/uploads/fJHmxA.pdf.
Foster, A.W., Osman, D., Robinson, N.J., 2014. Metal preferences
and metallation. J Biol. Chem. 289, 28095–28103,
http://dx.doi.org/10.1074/jbc.R114.588145.
Fumi, M.D., Trioli, G., Colagrande, O., 1987. Preliminary
assessment on the use of immobilized yeast cells in sodium
alginate for sparkling wine process. Biotechnol. Lett. 9,
339–342, http://dx.doi.org/10.1007/BF01025800.
Gibson, R.B., 2011. Improvement of higher gravity brewery
fermentation via wort enrichment and supplementation. J.
Inst. Brew. 117, 268–284,
http://dx.doi.org/10.1002/j.2050-0416.2011.tb00472.x.
Godia, F., Casas, C., Castellano, B., Sold, C., 1987. Immobilized
cells: behaviour of carrageenan entrapped yeast during
continuous ethanol fermentation. Appl. Microbiol. Biotechnol.
26, 342–346, http://dx.doi.org/10.1007/BF00256666.
Gokgoz, M., Yiğitoğlu, M., 2011. Immobilization of Saccharomyces
cerevisiae on to modified carboxymethyl cellulose for
production of ethanol. Bioprocess Biosyst Eng. 34, 849–857,
http://dx.doi.org/10.1007/s00449-011-0535-x.
Goncalves, L.M.D., Barreto, M.T.O., Xavier, A.M.B.R., Carrondo,
M.J.T.M., Klein, J., 1992. Inert support for lactic acid
fermentation. A technological assessment. Appl. Microbiol.
Biotechnol. 38, 305–311, http://dx.doi.org/10.1007/bf00170077.
Grasdalen, H., Smidsrod, O., 1987. Gelation of gellan gum.
Carbohydr. Polym. 7, 371,
http://dx.doi.org/10.1016/0144-8617(87)90004-X.
Groboillot, A., Boadi, D.K., Poncelet, D., Neufeld, R.J., 1994.
Immobilization of cells for application in the food industry.
Crit. Rev. Biotechnol. 14, 75–107,
http://dx.doi.org/10.3109/07388559409086963.
Guzman-Villanueva, D., El-Sherbiny, I.M., Herrera-Ruiz, D.,
Smyth, H.D.C., 2013. Design and in vitro evaluation of a new
nano-microparticulate system for enhanced aqueous-phase
solubility of curcumin. BioMed Res. Int. 2013,
http://dx.doi.org/10.1155/2013/724763, Article ID 724763, 9
pages.
Heng, P.W.S., Chan, L.W., Wong, T.W., 2003. Formation of alginate
microspheres produced using emulsification technique. J.
Microencapsul. 20, 401–413,
http://dx.doi.org/10.3109/02652040309178078.
Hill, J., Nelson, E., Tilman, D., Polasky, S., Tiffany, D., 2006.
Environmental, economic, and energetic costs and benefits of
biodiesel and ethanol biofuels. Proc. Natl. Acad. Sci. U.S.A.
103, 11206–11210, http://dx.doi.org/10.1073/pnas.0604600103.
Hong, S.H., Shin, M., Lee, J., Ryu, J.H., Lee, S., Yang, J.W., Kim, W.D.,
Lee, H., 2016. Stable alginate gel prepared by linkage exchange
from ionic to covalent bonds. Adv. Healthcare Mater. 5, 75–79,
http://dx.doi.org/10.1002/adhm.201400833.
Idris, A., Wahidin, S., 2006. Effect of sodium alginate
concentration, bead diameter, initial pH and temperature on
lactic acid production from pineapple waste using
immobilized Lactobacillus delbrueckii. Process Biochem. 41,
1117–1123, http://dx.doi.org/10.1016/j.procbio.2005.12.002.
Inama, L., Dire, S., Carturan, G., Cavazza, A., 1993. Entrapment of
viable microorganisms by SiO2 sol–gel layers on glass
surfaces: trapping, catalytic performance and immobilization
122 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123
durability of Saccharomyces cerevisiae. J. Biotechnol. 30,
197–210, http://dx.doi.org/10.1016/0168-1656(93)90113-2.
Iurciuc (Tincu), C.E., Alupei, L., Savin, A., Ibănescu, C., Martin, P.,
Popa, M., 2016a. Yeast cells immobilized in spherical gellan
particles cross-linked with magnesium acetate. J. Biotechnol.
236, 45–56, http://dx.doi.org/10.1016/j.jbiotec.2016.08.002.
Iurciuc (Tincu), C.E., Savin, A., Lungu, C., Martin, P., Popa, M.,
2016b. Gellan. Food applications. Cellulose Chem. Technol. 13
http://www.cellulosechemtechnol.ro/pdf/CCT1(2016)/p.1-13.pdf.
Iurciuc (Tincu), C.E., Savin, A., Martin, P., Peptu, C.A., Popa, M.,
2017. Yeast cells immobilized in ionic crosslinked hydrogel
particles based on gellan and gellan/ carboxymethyl
cellulose—comparative study. J. Nanosci. Nanotechnol. 17,
4827–4836, http://dx.doi.org/10.1166/jnn.2017.14324.
Ivanova, V., Petrova, P., Hristov, J., 2011. Application in the ethanol
fermentation of immobilized yeast cells in matrix of
alginate/magnetic nanoparticles, on chitosan-magnetite
microparticles and cellulose-coated magnetic nanoparticles.
Int. Rev. Chem. Eng. 3, 289–299
https://arxiv.org/ftp/arxiv/papers/1105/1105.0619.pdf.
John, R.P., Tyagi, R.D., Brar, S.K., Surampalli, R.Y., Prévost, D., 2011.
Bio-encapsulation of microbial cells for targeted agricultural
delivery. Crit. Rev. Biotechnol. 31, 211–226,
http://dx.doi.org/10.3109/07388551.2010.513327.
Klis, F.M., de Koster, C.G., Brul, S., 2014. Cell wall-related
bionumbers and bioestimates of Saccharomyces cerevisiae and
Candida albicans. Eukaryotic Cell. 13, 2–9,
http://dx.doi.org/10.1128/EC.00250-13.
Kourkoutas, Y., Kanellaki, M., Koutinas, A.A., Tzia, C., 2005. Effect
of fermentation conditions and immobilization supports on
the wine making. J. Food Eng. 6, 115–123,
http://dx.doi.org/10.1016/j.jfoodeng.2004.08.003.
Kregiel, D., Berlowska, J., Szubzda, B., 2012. Novel permittivity test
for determination of yeast surface charge and flocculation
abilities. J. Ind. Microbiol. Biotechnol. 39, 1881–1886,
http://dx.doi.org/10.1007/s10295-012-1193-y.
Kregiel, D., Berlowska, J., Ambroziak, W., 2013. Growth and
metabolic activity of conventional and non-conventional
yeasts immobilized in foamed alginate. Enzyme. Microb.
Technol. 53 (4), 229–234,
http://dx.doi.org/10.1016/j.enzmictec.2013.05.010.
Krieck, S., Matthias Westerhausen, M., 2017. Kudos and
renaissance of s-block metal chemistry. Inorganics 5, 17
http://www.mdpi.com/2304-6740/5/1/17.
Learmonth, R.P., Gratton, E., 2002. Assessment of membrane
fluidity in individual yeast cells by laurdan generalised
polarisation and multi-photon scanning fluorescence
microscopy. In: Kraayenhof, R., Visser, A.J.W.G., Gerritsen, H.C.
(Eds.), Fluorescence Spectroscopy, Imaging and Probes—new
Tools in Chemical, Physical and Life Sciences. Springer,
Heidelberg, pp. 241–252 http://citeseerx.
ist.psu.edu/viewdoc/download?doi=10.1.1.528.6834&rep
=rep1&type=pdf.
Liu, S., Hou, Y., Liu, W., Lu, C., Wang, W., Sun, S., 2015a.
Components of the calcium-calcineurin signaling pathway in
fungal cells and their potential as antifungal targets.
Eukaryotic Cell 14, 324–334,
http://dx.doi.org/10.1128/EC.00271-14.
Lipke, P.N., Ovalle, R., 1998. Cell wall architecture in yeast: new
structure and new challenges. J. Bacteriol. 180, 3735–3740
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC107352/.
Liu, X., Jia, B., Sun, X., Ai, J., Wang, L., Wang, C., Zhao, F., Zhan, J.,
Huang, W., 2015b. Effect of initial pH on growth characteristics
and fermentation properties of Saccharomyces cerevisiae. J. Food
Sci. 80, 800–808, http://dx.doi.org/10.1111/1750-3841.12813.
Loukatos, P., Kiaris, M., Ligas, I., Burgos, G., Kannelaki, M.,
Komaitis, M., Koutinas, A., 2000. Continuous wine-making by
g-alumina-supported biocatalyst. Quality of the wine and
distillates. Appl. Biochem. Biotechnol. 89, 1–14,
http://dx.doi.org/10.1385/abab:89:1:1.
Luna-Solano, G., Salgado-Cervantes, M.A., Rodríguez-Jimenes,
G.C., García-Alvarado, M.A., 2005. Optimization of brewer’s
yeast spray drying process. J. Food Eng. 68, 9–18,
http://dx.doi.org/10.1016/j.jfoodeng.2004.05.019.
Maicas, S., 2001. The use of alternative technologies to develop
malolactic fermentation in wine. Appl. Microbiol. Biotechnol.
56, 35–39, http://dx.doi.org/10.1007/s002530100662.
Malm, M., 1946. Changes in the electrical charge of yeast cells
treated with sodium fluoride. Nature 157, 731–732,
http://dx.doi.org/10.1038/157731b0 http://www.nature.com/
nature/journal/v157/n3996/abs/157731b0.html.
Mantaluta, A., Cojocaru, D., Savin, C., Pasa, R., 2012. The testing
of some organic supports for yeasts immobilization
technology used in sparkling wine production. Annnals of the
“Alexandru Ioan Cuza” University. Genet. Biol. Mol. 12, 81–85
http://www.gbm.bio.uaic.ro/index.php/gbm/article/view/933.
Mariam, I., Manzoor, K., Ali, S., Ul-Haq, I., 2009. Enhanced
production of ethanol from free and immobilized
Saccharomyces cerevisiae under stationary culture. Pak. J. Bot.
41, 821–833 http://www.bf.lu.lv/grozs/Mikrobiologijas/
Akt probl/Raksti Journal club/2011 04 15/enhanced
%20production%20of%20ethanol.pdf.
Martinez, A., Vivas, G., Quicazan, M., 2016. Evaluation of alcoholic
fermentation during the production of mead using
immobilized cells in kappa-carrageenan. Chem. Eng. Trans.
49, 19–24, http://dx.doi.org/10.3303/CET1649004
http://www.aidic.it/cet/16/49/004.pdf.
Migockal, M., Kosieradzka, A., Papierniak, A.,
Maciaszczyk-Dziubinska, E., Posyniak, E., Garbiec, A., Filleur,
S., 2015. Two metal-tolerance proteins, MTP1 and MTP4, are
involved in Zn homeostasis and Cd sequestration in
cucumber cells. J. Exp. Bot. 66, 1001–1015,
http://dx.doi.org/10.1093/jxb/eru459.
Morris, E.R., Nishinari, K., Rinaudo, M., 2012. Gelation of gellan—a
review. Food Hydrocoll. 28, 373–411,
http://dx.doi.org/10.1016/j.foodhyd.2012.01.004.
Necas, J., Bartosikova, L., 2013. Carrageenan: a review. Vet. Med.
58, 187–205
http://agriculturejournals.cz/publicFiles/91236.pdf.
Nedovic, V., Willaert, R., Laskošek-Čukalović, I., Obradovic, B.,
Bugarski, B., 2005. Beer production using immobilized cells.
In: Nedovic, V., Willaert, R. (Eds.), Focus on Biotechnology.
Springer, Netherlands, pp. 259–273,
http://dx.doi.org/10.1007/1-4020-3363-X 15.
Nedović, V., Gibson, B., Mantzouridou, T.F., Bugarski, B.,
Djordjević, V., Kalušević, A., Paraskevopoulou, A., Sandell, M.,
Šmogrovičová, D., Yilmaztekin, M., 2015. Aroma formation by
immobilized yeast cells in fermentation processes. Yeast 32,
173–216, http://dx.doi.org/10.1002/yea.3042.
O’Neill, M., Selvendran, R.R., Morris, V.J., 1983. Structure of the
acidic extracellular gelling polysaccharide produced by
Pseudomonas elodea. Carbohydr. Res. 124, 123–133,
http://dx.doi.org/10.1016/0008-6215(83)88360-8.
Pasqui, D., De Cagna, M., Barbucci, R., 2012. Polysaccharide-based
hydrogels: the key role of water in affecting mechanical
properties. Polymers 4, 1517–1534
http://www.mdpi.com/2073-4360/4/3/1517.
Peinado, R., Moreno, J., Villalba, J., Gonzalez-Reyes, J., Ortega, J.,
Mauricio, J., 2006. Yeast biocapsules: a new immobilization
method and their applications. Enzyme Microb. Technol. 40,
79–84, http://dx.doi.org/10.1016/j.enzmictec.2005.10.040.
Poreda, A., Tuszyński, T., Zdaniewicz, M., Sroka, P., Jakubowski,
M., 2013. Support materials for yeast immobilization affect
the concentration of metal ions in the fermentation medium.
J. Inst. Brew. 119, 164–171, http://dx.doi.org/10.1002/jib.77.
Potthoff, E., Guillaume-Gentil, O., Ossola, D., Polesel-Maris, J.,
LeibundGut-Landmann, S., Zambelli, T., Vorholt, J.A., 2012.
Rapid and serial quantification of adhesion forces of yeast
and mammalian cells. PLoS One 7, 0052712,
http://dx.doi.org/10.1371/journal.pone.0052712.
Randez-Gil, F., Córcoles-Sáez, I., Prieto, J.A., 2013. Genetic and
phenotypic characteristics of baker’s yeast: relevance to
baking. Annu. Rev. Food Sci. Technol. 4, 191–214,
http://dx.doi.org/10.1146/annurev-food-030212-182609.
 food and bioproducts processing 1 0 4 ( 2 0 1 7 ) 104–123
Rathore, S., Desai, P.M., Liew, C.V., Chan, L.W., Heng, P.W.S., 2013.
Microencapsulation of microbial cells. J. Food Eng. 116,
369–381, http://dx.doi.org/10.1016/j.jfoodeng.2012.12.022.
Rautio, J.J., Huuskonen, A., Vuokko, H., Vidgren, V.,
Londesborough, J., 2007. Monitoring yeast physiology during
very high gravity wort fermentations by frequent analysis of
gene expression. Yeast 24, 741–760,
http://dx.doi.org/10.1002/yea.1510.
Raymond, M.C., Neufeld, R.J., Poncelet, D., 2004. Encapsulation of
brewers yeast in chitosan coated carrageenan microspheres
by emulsification/thermal gelation. Artif. Cell Blood Sub. 32,
275–291, http://dx.doi.org/10.1081/BIO-120037832.
Reddy, L.V.A., Reddy, O.V.S., 2011. Effect of fermentation
conditions on yeast growth and volatile composition of wine
produced from mango (Mangifera indica L.) fruit juice. Food
Bioprod. Process. 89, 487–491,
http://dx.doi.org/10.1016/j.fbp.2010.11.007.
Reed, G., Nagodawithana, T.W., 1991. Baker’s yeast production. In:
Reed, G. (Ed.), Yeast Technology. Springer, Netherlands, pp.
261–314, http://dx.doi.org/10.1007/978-94-011-9771-7 7.
Rees, E.M.R., Stewart, G.G., 1997. The effects of increased
magnesium and calcium concentrations on yeast
fermentation performance in high gravity wort. J. Inst. Brew.
103, 287–291,
http://dx.doi.org/10.1002/j.2050-0416.1997.tb00958.x.
Rosevear, A., 1984. Immobilised biocatalysts—a critical review. J.
Chem. Technol. Biotechnol. 34, 127–150,
http://dx.doi.org/10.1002/jctb.280340302.
Sannino, A., Demitri, C., Madaghiele, M., 2009. Biodegradable
cellulose-based hydrogels: design and applications. Materials
2, 353–373 http://www.mdpi.com/1996-1944/2/2/353.
Santos, J.C., Mussatto, S.I., Dragone, G., Converti, A., Silva, S.S.,
2005. Evaluation of porous glass and zeolite as cells carriers
for xylitol production from sugarcane bagasse hydrolysate.
Biochem. Eng. J. 23, 1–9,
http://dx.doi.org/10.1016/j.bej.2004.10.001.
Saraydın, D., Öztop, H.N., Karadağ, E., Öztop, A.Y., Işıkver, Y.,
Güven, O., 2002. The use of immobilized Saccharomyces
cerevisiae on radiation crosslinked acrylamide–maleic acid
hydrogel carriers for production of ethyl alcohol. Process
Biochem. 37, 1351–1357,
http://dx.doi.org/10.1016/S0032-9592(01)00337-5.
Silva, D.P., Branyik, T., Dragone, G., Vicent, A.A., Teixeira, J.A.,
Silva, J.B.A., 2008. High gravity batch and continuous
processes for beer production:evaluation of fermentation
performance and beer quality. Chem. Pap. 62, 34–41,
http://dx.doi.org/10.2478/s11696-007-0076-6.
Tamás, M.J., Sharma, S.K., Ibstedt, S., Jacobson, T., Christen, P.,
2014. Heavy metals and metalloids as a cause for protein
misfolding and aggregation. Biomolecules 4, 252–267
http://www.mdpi.com/2218-273X/4/1/252.
Tan, S.M., Heng, P.W.S., Chan, L.W., 2011. Development of
re-usable yeast-gellan gum micro-bioreactors for potential
application in continuous fermentation to produce
bio-ethanol. Pharmaceutics 3, 731–744
http://www.mdpi.com/1999-4923/3/4/731.
Tang, P.D.P., Le, V.V.M., 2013. Fermentation performance of free
and immobilized yeast on cork (Sonneratia caseolaris)
root—application of immobilized yeast to repeated batch
ethanol fermentation. Int. Food Res. J. 20, 1813–1817
http://www.ifrj.upm.edu.my/20%20(04)%202013/42
%20IFRJ%2020%20(04)%202013%20Le%20(461).pdf.
Taylor, N.W., Orton, W.L., 1973. Effect of alkaline-earth metal salts
on flocculence in Saccharomyces cerevisiae. J. Inst. Brew. 79,
294–297, http://dx.doi.org/10.1002/j.2050-0416.1973.tb03543.x.
Taylor, N.W., Orton, W.L., 1975. Calcium in flocculence of
Saccharomyces cerevisiae. J. Inst. Brew. 81, 53–57,
http://dx.doi.org/10.1002/j.2050-0416.1975.tb03661.x.
123
Udeh, H.O., Kgatla, T.E., 2013. Role of magnesium ions on yeast
performance during very high gravity fermentation. J. Brew.
Distilling 4, 19–45, http://dx.doi.org/10.5897/jbd2013.0041.
Udeh, H.O., Kgatla, T.E., Jideani, A.I.O., 2014. Effect of mineral ion
addition on yeast performance during very high gravity wort
fermentation. Int. J. Biol. Biomol. Agric. Food Biotechnol. Eng.
8, 1208–1216 http://waset.org/publications/9999716/effect-of-
mineral-ion-addition-on-yeast-performance-during-very-
high-gravity-wort-fermentation.
Varghese, J.S., Chellappa, N., Fathima, N.N., 2014.
Gelatin–carrageenan hydrogels: role of pore size distribution
on drug delivery process. Colloids Surf. B Biointerfaces 113,
346–351, http://dx.doi.org/10.1016/j.colsurfb.2013.08.049.
Verbelen, P.J., De Schutter, D.P., Delvaux, F., Verstrepen, F.R.,
Delvaux, K.J., 2006. Immobilized yeast cell systems for
continuous fermentation applications. Biotechnol. Lett. 28,
1515–1525, http://dx.doi.org/10.1007/s10529-006-9132-5.
Walker, G.M., Birch-Anderson, A., Hamburger, K., Kramhoft, B.,
1982. Magnesium-induced mitochondrial polymorphism and
changes in respiratory metabolism in the fission yeast,
Schizosaccharomyces pombe. Carlsberg Res. Commun. 47,
205–214.
Walker, G.M., 1994. The roles of magnesium in biotechnology.
Crit. Rev. Biotechnol. 14, 311–354,
http://dx.doi.org/10.3109/07388559409063643.
Walker, G.M., 2004. Metals in yeast fermentation processes. Adv.
Appl. Microbiol. 54, 197–229,
http://dx.doi.org/10.1016/S0065-2164(04)54008-X.
Walker, G.M., Stewart, G.G., 2016. Saccharomyces cerevisiae in the
production of fermented beverages. Beverages 2, 2–12
http://www.mdpi.com/2306-5710/2/4/30.
Wan, L.S.C., Heng, P.W.S., Chan, L.W., 1994. Surfactant effects on
alginate microspheres. Int. J. Pharm. 103, 267–275,
http://dx.doi.org/10.1016/0378-5173(94)90177-5.
Wang, H.Y., Hettwer, D.J., 1982. Cell immobilization in
k-carrageenan with tricalcium phosphate. Biotechnol. Bioeng.
24, 1827–1838, http://dx.doi.org/10.1002/bit.260240809.
Wang, F.Q., Gao, C.J., Yang, C.Y., Xu, P., 2007. Optimization of an
ethanol production medium in very high gravity
fermentation. Biotechnol. Lett. 29, 233–236,
http://dx.doi.org/10.1007/s10529-006-9220-6.
Westman, J.O., Franzén, C.J., 2015. Current progress in high cell
density yeast bioprocesses for bioethanol production.
Biotechnol. J. 10, 1185–1195,
http://dx.doi.org/10.1002/biot.201400581.
Xing, Z., Zhang, Q., Shi, X., Lin, Y., 2016. Saccharomyces cerevisiae
immobilized in alginate for continuous fermentation. J. Clean
Energy Technol. 4, http://www.jocet.org/index.php?m=content
&c=index&a=show&catid=45&id=539.
Yiğitoğlu, M., Işıklan, N., Ozmen, R., 2007. Graft copolymerization
of N-vinyl-2-pyrrolidone onto sodium carboxymethyl
cellulose with azobisisobutyronitrile as the initiator. J. Appl.
Polym. Sci. 104, 936–943, http://dx.doi.org/10.1002/app.25786.
Yoo, I.K., Seong, G.H., Chang, H.N., Park, J.K., 1996. Encapsulation
of Lactobacillus casei cells in liquid core alginate capsules for
lactic acid production. Enzyme Microb. Technol. 19, 428–433,
http://dx.doi.org/10.1016/s0141-0229(96)00016-6.
Zhao, X.Q., Bai, F.W., 2009. Review: mechanisms of yeast stress
tolerance and its manipulation for efficient ethanol
production. J. Biotechnol. 144, 23–30,
http://dx.doi.org/10.1016/j.jbiotec.2009.05.001.
Zhao, X.Q., Bai, F.W., 2012. Zinc and yeast stress tolerance:
micronutrient plays a big role. J. Biotechnol. 158, 176–183,
http://dx.doi.org/10.1016/j.jbiotec.2011.06.038.