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Powder Technology 325 (2018) 476–489

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Powder Technology

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

Encapsulation of Saccharomyces cerevisiae in hydrogel particles based


gellan ionically cross-linked with zinc acetate
Camelia-Elena Iurciuc (Tincu) a, Alexandru Savin a, Leonard Ionut Atanase d,e, Maricel Danu a,
Patrick Martin b, Marcel Popa a,c,d,e,⁎
a
“Gheorghe Asachi” Technical University, Faculty of Chemical Engineering and Protection of the Environment, Department of Natural and Synthetic Polymers, 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, Iasi, Romania
e
Research Institute “Academician Ioan Haulica”, 3, str. Muzicii, Iasi, Romania

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

Article history: The purpose of this study is to obtain ionically cross-linked gellan particles containing immobilized yeast cells.
Received 9 December 2016 The zinc acetate was chosen as a cross-linking agent due to its beneficial role in the growth and nutrition of
Received in revised form 25 July 2017 yeast cells and also due to the advantages of its use in fermentation processes. The physicochemical and morpho-
Accepted 7 November 2017
logical properties as well as the fermentative activity of the obtained particles were studied. The zinc acetate con-
Available online 21 November 2017
centration has an influence on particles stability in water, on the mechanical stability and also on the swelling
Keywords:
degree. The morphology of the obtained particles was analyzed by SEM and proves that the yeast cells are
Yeast immobilized in large numbers in the polymeric matrix. The cell viability is maintained at high values after several
Immobilization fermentation cycles. These obtained micro-bioreactors were tested in terms of fermentative activity and the fer-
Zinc acetate mentation process was optimized by studying the influence of several factors. The gellan gum particles can be
Gellan easily recovered by filtration and they can be reused in repeated fermentation cycles. The results obtained in
Fermentative processes the presence of these cross-linked particles are comparable to those achieved in the free yeast fermentation.
Ionically cross-linked © 2017 Elsevier B.V. All rights reserved.

1. Introduction Moreover, these cells are chemoorganotrophs and they use, as an


energy source, sugars such as hexoses and disaccharides. In the pres-
Immobilization of yeast cells in various macromolecular carriers, in ence of oxygen, they are aerobic microorganisms but they can be anaer-
order to obtain ethyl alcohol is a research field that has expanded due obic when are subjected to fermentation for the production of ethanol.
to its attractive technique and to its economic benefits. Continuous pro- Optimal conditions for the proliferation of yeast cells are represented
cesses are preferred as they are characterized by a substantially im- by a temperature between 30 and 33 °C [4] and a pH range of 4 to 6
proved efficiency, a higher productivity and lower operating costs [1,2]. [5–7].
Saccharomyces cerevisiae are unicellular microorganisms, eukaryotic, The techniques involved in the encapsulation of the microbial cells
classified as fungus and they are also known as baker's yeast or beer in the polymeric matrix are emulsification [8,9], extrusion [10], complex
yeast. Yeast cells reproduce asexually by a process called budding at coacervation [11], spray drying [12], gel entrapment [13] and polymer-
every 90 min and their diameter is usually between 3 and 4 μm. When ization by radiation [14]. Each of these techniques has different advan-
the yeast cells are stored under adverse conditions, such as for example tages and disadvantages. The optimal technique depends on the type
lack of nutrients in the medium or high temperatures, they do not die of polymer used for encapsulation, on the microorganism and on the de-
but are undergoing a process called sporulation. Yeast spores can with- sired properties of the immobilized product. For microbial cells, the ex-
stand long periods without nutrients, at the low and high temperatures, trusion is the most often used method due to the mild processing
until the conditions are appropriate for reproduction and then they start conditions that enable optimal encapsulation and does not affect the vi-
to sprout all over again [3]. ability of microorganisms. This method is usually used in the case of al-
ginates and carrageenan, and involves the extrusion in small droplets of
the biopolymer solution containing the yeast cell suspension in the
⁎ Corresponding author at: “Gheorghe Asachi” Technical University, Faculty of Chemical
Engineering and Protection of the Environment, Department of Natural and Synthetic
cross-linking agent solution [15].
Polymers, Prof.dr.docent Dimitrie Mangeron Str., nr. 73, 700050, Iasi, Romania. The obtained particles with immobilized yeast cells are larger than
E-mail address: marpopa@ch.tuiasi.ro (M. Popa). the free cells and can be recovered easily from the reaction medium

https://doi.org/10.1016/j.powtec.2017.11.017
0032-5910/© 2017 Elsevier B.V. All rights reserved.
C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489 477

by filtration. These particles must be stable under various conditions with that of the free yeast. Micro-bioreactors cross-linked with zinc
such as pH, osmotic pressure and turbulence. Besides protecting the en- ions and with immobilized yeast cell can be an approach for solving
capsulated cells from unfavorable environment, the polymer matrix the zinc deficiency, problem which faces brewers.
must ensure the efficient exchange of nutrients and metabolites with
the environment. Micro-bio-reactors efficiency depends on their specif- 2. Materials and methods
ic surface area, porosity, swelling capacity and stability [16,17]. The re-
covered particles can be reused in the subsequent fermentation 2.1. Materials
process as shown by Tan et al. [9] for gellan particles.
Polysaccharides have been shown to be effective in immobilizing In this study was used deacetylated Gellan Kelkogel characterized by
microbial cells because they are biodegradable, non-toxic and preserve an Mw = 2.351 × 105 g/mol, determined at 25 °C by using a method de-
the cell viability over extended periods of time [18]. Gellan is a water scribed by Reed et al. [33]. Zinc acetate and Glucose were procured from
soluble microbial exopolysaccharide, produced by the bacterium Sigma Aldrich and used as received.
Sphingomonas paucimobilis, with a molecular weight varying between
1–2 × 106 g·mol−1 for the high-acyl gellan and 2–3 × 105 g·mol− 1 2.1.1. Cell yeast type
for low-acyl gellan [19,20]. Is a linear anionic biopolymer having For the immobilization, was used a commercial bakery yeast
tetrasaccharide repeating sequences and consisting of two residues of strain (Pakmaya, Romania) which is found in a compressed form
β-D-glucose, one of β-D-glucuronic acid and another of α-L-rhamnose and has a water content of 70%. Saccharomyces cerivisiae cell charac-
in a ratio of 2:1:1 [21,22]. teristics are: diameter: 2–8 μm; volume: 0.8–2 × 10− 3 cm3; weight:
Gellan is a biocompatible, biodegradable and non-toxic polysaccha- 0.2–0,4 × 10− 10 g; number of cells/g: 8–14 × 109; specific surface:
ride with high stability at pH values ranging from 2 to 10 [23]; it is 8–14 × 109 (m2/g).
resistant to enzymes such as pectinase, amylase, cellulase, lipase and This type of yeast was chosen as it represents a type of yeast cell bio-
papain [20,24], but it is significantly degraded in the presence of mass of the species (top fermenting yeast) composed of living cells ca-
galactomannanase [25]. Gellan have been used to immobilize yeast pable of producing sugars fermentation. These yeast cells easily absorb
cells and to obtain a biocatalyst with real perspectives of being used the hexoses (glucose), and then the sucrose and the maltose [34–39].
for the sparkling wine production technology. Spherical particles, with To achieve immobilized microorganisms with high performance assets
immobilized yeast cells, were obtained by extruding the gel containing in the fermentation process is recommended that these be allowed to
a suspension of yeast cells through a capillary in a bath of CaCl2 solution grow directly into the gel [40,41].
(the ionic cross-linking agent) [26]. Tan et al. [9] have immobilized yeast
cells, using gellan gum as a support, with an emulsification method and 2.2. Methods
have demonstrated the possibility of reusing the obtained micro-
bioreactors. The production of ethanol during the first three cycles 2.2.1. Preparation of gellan particles with and without immobilized
was comparable to that of free yeast. Micro-bioreactors are stable and yeast cells
readily recovered from the fermentation medium by filtration and can In order to obtain ionically cross-linked gellan particles with zinc ac-
be reused for at least 10 cycles of fermentation with relatively high etate, 0.25 g gellan was dispersed in 25 mL bi-distilled water and the
yield of ethanol. temperature was fixed at 85 ± 1 °C until all gellan was dissolved.
It is known that the yeast cells require a wide range of metals to their The formed polymer solution was extruded dropwise using a syringe
proliferation and metabolism [27]. In the fermentative processes, espe- and a capillary (injection needle), with the size of 0.6 × 30 mm
cially in the beer industry, the key metal ions are magnesium and zinc as (22 gauges), into a cross-linking bath, with a volume of 125 mL, contain-
they act as cofactors for different metabolic and biosynthetic enzymes, ing zinc acetate solutions with different concentrations (from 0.05 to
including glycolytic enzymes and alcohol dehydrogenase [28,29]. The 0.3%). Because the gellan solution do not form stable particles at the
heavy metals, even at micromolar concentrations, can be toxic to the time of extrusion into the cross-linking bath, 1.25 mL zinc acetate, of dif-
yeast cells [30]. An environment deficient in zinc ions can lead to slow ferent concentrations (from 0.02 to 0.1%), was added to the aqueous
or incomplete fermentation and this issue was observed in the beer in- gellan solution prior to extrusion. The obtained gellan particles were
dustry. The zinc ions play a significant role in the metabolism of the washed with bi-distilled water in order to remove the zinc acetate ex-
yeast cells, not only because it is an enzymatic activator but also because cess and these particles were kept at 4–6 °C in tightly closed containers
it can boost the absorption of maltose and maltotriose in the yeast syn- until further characterization. For yeast cell immobilization in gellan
thesis process thus achieving high fermentation yields [29–31]. These particles was used the same method as described above. Prior to extru-
ions may have also a beneficial role on the stability and dynamics of sion, a suspension of yeast cells, obtained from 1.25 g yeast and 5 mL of
the cell membrane. When yeast is facing changes in the environment, water, was added into the gellan solution at a temperature of 38–40 °C.
such as a nutrient or metal ions deficiency, accumulation of toxic metab- The temperature at which the extrusion of the polymer solution is car-
olites or the temperature variation, the plasma membrane must adapt ried out is 30 °C. The extrusion process takes approximately 15 min and
before internal structure. Maintaining membrane fluidity is an essential the particles obtained are spherical and have a diameter of about 2–
factor in maintaining its functions [32]. Yeast cells have the ability to ab- 2.5 mm. From the total quantity of the yeast only 30% represents yeast
sorb, very efficient, essential minerals and to exclude the non-essential cells. The amount of yeast cells in 1.25 g yeast used as previously
ones. Zinc ions can be absorbed by the cells in the fermentation medium indicated is therefore equal to 0.375 g and the ratio of yeast cells/gellan
in order to perform essential physiological roles [27]. is 1.5:1 (w/w). The obtained particles are left in the cross-linking bath
The aim of this paper is to obtain ionically cross-linked gellan parti- for 2 h, and 12 h, respectively and then were washed with bi-distilled
cles with immobilized yeast cells, by the extrusion method. The origi- water in order to remove the zinc acetate excess and these particles
nality of this work consists in the use of zinc acetate as a cross-linking were kept at 4–6 °C in tightly closed containers until further
agent in order to obtain gellan particles with immobilized yeast cells characterization.
and the study of various factors that can influence the fermentation
process. The zinc acetate was used as cross-linking agent as is not 2.2.2. Particles stability in aqueous medium
toxic, it has a positive effect on the yeast cells metabolism and plays The stability of the gellan particles with and without immobilized
an important role in the fermentation processes. The morphological yeast cells was determined not only in the cross-linking solution (super-
and the physico-chemical characterization of the obtained particles natant) but also in bi-distilled water. It is considered that from the less
was achieved and the fermentative activity was tested and compared stable particles, polymer fragments can be drawn and this causes the
478 C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489

turbidity of the aqueous solutions in which they are kept. Also, the tur- 2.2.4. Structural stability of the particles the average size of the network
bidity of the solution prepared in the presence of gellan particles with meshes (ξ)
immobilized yeast cells may be caused by yeast cells that can spread Structural stability of the particles was assessed, indirectly, by rheo-
from the polymer matrix. The obtained particles were kept into the ex- logical amplitude and frequency sweep test. It was assumed that higher
trusion solution for 24 h, after which they were washed with bi-distilled values of storage modulus (G′) are proving a stronger structure, i.e.
water and stored in water for an additional 48 h. This method for deter- more cross-linked structure, highlighting a better mechanical stability.
mination of the particles stability was used in the previous studies [42]. Rheological tests were made on a Physica MCR 501 modular rheometer
The solution turbidity was evaluated by measuring the transmit- (Anton Paar) equipped with parallel plate geometry and under con-
tance T% at a wavelength of 550 nm and different intervals of time, trolled shear stress and strain. All measured rheological parameters
using a spectrophotometer BOECO S-22, both in extrusion solution were determined under dynamic oscillation [43,44].
and in bi-distilled water. The transmittance measurements were per- The same number of particles from each sample were placed under
formed in triplicate and the standard deviation is equal to ±0.2%. the plates of the device and selected for the same diameter, while the
upper plate was lowered until it touched the particles, ensuring a slight
pressure.
2.2.3. Cell viability and the concentration of yeast cells Amplitude sweep tests were used to determine the linear viscoelas-
Cell viability and the concentration of yeast cells were determined tic range limit (LVE range). In this case, the frequency was kept constant
using an optical microscope and a Marienfeld (Neubauer) counting (ω = 10 rad/s) and the strain (γ) varies in the range 0.01 ÷ 100%. For
room. The concentration of yeast cells (cells/mL) of the supernatant the frequency sweep test, the deformation was maintained constant in
(or in the bi-distilled water) where the particle was suspended, was de- the linear viscoelastic range (γ = 0.05÷0.1) and the frequency was var-
termined after 12 h and 24 h, respectively. The cell viability was deter- ied between 0.1 and 100 rad/s. All measurements were performed at a
mined as a function of the number of live and dead yeast cells that are constant temperature of 30 °C.
found in the supernatant after the above-mentioned periods of time. In order to better understand the correlations between the structural
This method is frequently applied to assess the quality of the com- properties of the resulted gellan-based hydrogel particles, after cross-
pressed yeast, the physiological state and the percentage of autolysed linking with Zn2 + ions, the G′ values were determined. These values
cells (dead cells) but also the influence of environmental factors on were used to calculate the average size of the network mesh (ξ) and
the physiological activity of the yeast cell. This method is based on the the weight average molecular weight between cross-links (Mc) in the
fact that unviable yeast cells, obtained after the autolysis process or by hydrogel.
the action of an external factor that causes irreversible changes in the The average size of the network mesh (the distance between two
structure of proteins, lose their fermentative properties. By suspending cross-links) was calculated on the basis of the theory of elastic rubber
the yeast cells in dilute methylene blue and sodium citrate solution with which can also be applied to hydrogels having an elastic behavior. The
a concentration of 2%, the physiologically inactive cells will be colored in equations for calculating the parameters ξ and Mc are:
blue because, after the inactivation of reductases, the passage of the dye
in the colorless leuco-derivative form does not longer take place, as oc-  0 −1=3
curs in the active living cell. G  NA
ξ¼ ð2Þ
The number of the unviable cells (autolysed cells) can be deter- RT
mined by using the Marienfeld counting room and the following formu-
la was used:  
cρRT
Mc ¼ 0 ð3Þ
G
N living cells
Vc ¼  100 ð1Þ
ðN living cells þ N dead cellsÞ where, G′-storage modulus, T-temperature at which the analysis
was conducted (303 K), NA-Avogadro's number, R-constant for ideal
gases (J·mol−1 × K), c-polymer concentration (w/v), ρ-water density
where: Vc is the cells viability, Nlive cells are the number of living cells, at 30 °C (kg·m−3) [45,46].
Ndead cells are the number of unviable cells (autolysed).
In order to determine the concentration of yeast cells, which are 2.2.5. Particles swelling degree
found in the supernatant after the particles preparation, a calibration The swelling degree was determine in order to study the hydrogel
curve was drawn. This curve correlates the number of yeast cells with behavior of the particles. The particles swelling degree was determined
their weight. Measurements of cell viability were carried out in tripli- gravimetrically and is express as the ratio between the amount of the
cate and the standard deviation is within ±0.3%. retained water and their weight in the dry form. For this determination,
the samples without the yeast cells were dried at 30 °C until their
weight is constant (Mdry). The dried samples were immersed in 5 mL
2.2.3.1. Determination of cell viability and cell amounts from the particles
of bi-distilled water and the water was filtered off at intervals of 1 h.
after each fermentation cycle. An essential condition for the use of
The excess water from the sample surface was removed by using filter
particles in several fermentation cycles is to maintain a high level of
paper. Swollen particles mass (Mswollen particles) was determined by
cell viability during the fermentation process. The following procedure
weighing the samples until their weight remains constant. The retained
was use in order to determine if the yeast cells proliferate in the poly-
water by the particles M water represents the difference between
meric matrix from a fermentation cycle to another: from each studied
Mswollen particles and Mdry. After weighing, the samples were immersed
sample, 2–3 particles were precisely weighed and then were disrupted
in water, and this operation was repeated every hour until equilibrium
mechanically by the means of an Ultraturrax at 2000 rpm in 100 mL of
was reached. The swelling degree was expressed as the ratio between
double distilled water leading to a homogeneous dispersion. By staining
the amount of water retained by the particles and the amount of parti-
this dispersion with methylene blue, using the method described
cles completely dry, at each time interval, and was calculated with the
above, it was determined both the cell concentration and cell viability
relationship:
in the particles. The amount of particles which is found in the cells
after each fermentation cycle (g cell/g particles) was evaluated as a
function of the number of cells/mL (cell concentration) from the calibra- Mwater
Q ð%Þ ¼  100 ð4Þ
tion curve. Mdry sample
C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489 479

where: Mwater – amount of water retained by the particles, M dry sample – temperature of 30 °C (303 K). The volume of gas at 0 °C will be
amount of dry particles. expressed in L/h.
Measurements of swelling degree were carried out in triplicate and
 . 
the standard deviation is within ±0.3%.   vCO2 ml
 273  60 L  
VCO2 0° C ¼ min
; ð6Þ
2.2.6. Scanning electron microscopy 303  1000 h

The particles were characterized by scanning electron microscopy


in order to determine their morphology, porosity and to qualitatively • The flow of ethanol (gethanol/h)is determined from the equation of the
assess the extent to which the yeast cells are found in the polymer glucose fermentation ethanol
matrix. The particles were cut and dried, were metalized with gold
using a sputter deposition device and analyzed using a Vega Tescan
instrument.  
V CO2 0 ° C  2  46
Gv ¼ ; gethanol =h ð7Þ
2  22:4
2.2.7. Testing the fermentative activity of particles with immobilized
yeast cells
Testing the ability of immobilized yeast cells to produce glucose
• The specific productivity (gethanol/h × gyeast) is the ratio between the
fermentation was carried out anaerobically in a fermentation reactor,
ethanol flow and the mass of dry yeast.
in which was introduced a volume solution of 50 mL glucose with
a concentration of 8% and different zinc acetate concentrations. The
fermentation was conducted at 30 °C, monitoring the amount of CO2
emitted. Based on this amount it was possible to determine the amount The amount of compressed yeast used for each sample was 1.25 g.
of glucose fermented as a function of time and thus the effectiveness of The total amount of dry yeast represents 30% of the total compressed
the process. Each fermentation cycle lasting 255 min, and the volume of yeast and is equal to 0.375 g.
CO2 was noted after every 15 min. After each cycle of fermentation,
the particles were recovered, washed with bi-distilled water to remove Gv
PS ¼ ; getanol =h  gyeast ð8Þ
the excess of glucose and magnesium acetate, and kept in refrigerator at 0:375
4–6 °C for 24 h until the next cycle of fermentation [42].
The fermentation yield was calculated from the fermentation equa- The fermentation rate is calculated as the slope of the linear portion
tion of glucose and as a function of the measured amount of CO2. of the curve of the glucose fermentation yield vs time for a period of 0 to
In order to optimize the fermentation process, the influence of 45 min.
several factors, which can increase the fermentation efficiency, was
studied: a) the concentration of zinc acetate in the cross-linking bath; 3. Results and discussion
b) the concentration of zinc acetate added prior to extrusion; c) the par-
ticle storage time in the extrusion bath immediately after extrusion; The preparation of ionically cross-linked gellan particles is based on
d) the pH of the fermentation bath; e) the concentration of zinc acetate the ion exchange reaction between gellan, in his sodium salt form, and
in the fermentation bath. zinc acetate, as illustrated in the following equation:

2.2.8. Specific productivity and the fermentation rate


Theoretically, the amount of ethanol formed can be calculated on the 2 Gelan−COO− Naþ þ ðCH3 COO− Þ2 Zn2þ ⟶ðGelan−COO− Þ2 Zn2þ
basis of the measured volume of CO2, because under the chosen operat- þ 2 CH3 COO− Naþ
ing conditions, the alcoholic fermentation is the only metabolic activity
of the yeast. The volume of the CO2 was determined at T = 30 °C and The yeast cells are trapped in the mesh network formed by ionic
1 atm. For the calculation of the fermentation efficiency all the data cross-linking. Zinc ion, as a transition metal, determines the appearance
concerning the CO2 volume were corrected according to the standard of side complexation reactions – coordinative bonds - which lead to the
conditions. The calculations are performed based on the following formation of a stronger gel network (more cross-linked) as compared to
glucose fermentation reaction: the use of other divalent metals of the group IIA. The network structure
is shown in Fig. 1.
C6 H12 O6 → 2C2 H5 OH þ 2CO2 The yeast cell is an electrically charged colloid, but this electric
charge can be neutralized or changed. Initially is positively charged
and after its introduction in the fermentation bath, the yeast cell chang-
180 g 2  46g 2  22:4 L
es his charge to negative after a period of time in which the budding
occurs. At the end of fermentation, the yeast cell electric charge will
The specific productivity in ethanol was determined as follows:
become positive again [47,48]. The zinc ions, in addition to the physio-
logical roles that they have in the yeast cell metabolism, can adhere to
• The fermentation plot: CO2 measured volume VCO2 (mL) versus time T
negatively charged cell plasma membrane, providing a higher stability
(min)
to stressors that may occur in the fermentative processes.
The optimal concentration of the cross-linking agent used, in both
From the slope of the linear portion is calculated and determine the
bath fermentation and the pre-extruded, for the preparation of stable
amount of CO2 in mL/min. Tan α represents the slope of the linear por-
particles, with and without immobilized yeast cells, was determined.
tion of the curve variation of the VCO2 vs. time.
An efficient exchange of nutrients must take place between particles,
with immobilized yeast cells, and the fermentation media (which is
V2−V1 the solution of glucose) in order to obtain cell proliferation, multiple fer-
tan α ¼ ; tan α ¼ VCO2 ðmL=minÞ ð5Þ
T2−T1 mentation cycles and higher fermentation efficiency. To determine this
optimal concentration, the influence of the zinc acetate concentration
• The volume of gas at 0 °C (273 K) is calculated based on the volume of was investigated in several samples in both pre-extruded and the extru-
CO2 (mL/min), obtained in the previous step, at the collection sion bath. The influence of this parameter on the structural stability of
480 C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489

The transmittance was measured for all the obtained samples, at the
wavelength of 550 nm, as a function of time in both the extrusion solu-
tion and bi-distilled water, and the results for some of the samples with-
out immobilized yeast cells are given in Table 2.
It can be seen from the transmittance values, given in Table 2, that
the stability of the particles in the aqueous medium is slightly influ-
enced by the concentration of the zinc acetate in the pre-extruded and
in the cross-linking bath.
With the exception of sample N5, the transmittance values increases
if the cross-linking agent concentration in the pre-extruded increases.
Moreover, it can be noticed that the transmittance values increases
with increasing the cross-linker concentration in the extrusion bath
for the majority of the studied samples. In this context the turbidity of
the particles storage solution decreases. From this table, it occurs that
the transmittance values increase with increasing the degree of cross-
linking. The transmittance values are almost constant after particles
storage during 24 h in the extrusion solution. However, a slight decrease
of transmittance values was noted after 48 h storage in bi-distilled
water. Obviously these results are the consequence of strong cross-
linked structures formed not only by ionic bonds but also by coordina-
tive ones between the zinc ion and gellan. The turbidity of the aqueous
solution in which the particles with immobilized yeast cells were kept
was determined by measuring the transmittance as a function of storage
Fig. 1. Schematic presentation of the structure of gellan gum spherical particles with time in the liquid medium. In this case, the turbidity of the environment
immobilized yeast cells ionically cross-linked with zinc acetate. can be influenced not only by the polymer fragments which are not
firmly attached to the polymer network but also by yeast cells that
can diffuses from the particles. For the samples containing immobilized
yeast cells the transmittance, the concentration of cells/mL and the cell
the particles and on the fermentation process behavior (yield, number viability was determined at 12 and 24 h. These results are shown in
of cycles performed) for each of the obtained particles was examined. Table 3.
The experimental condition are provided in Table 1. For all analyzed yeast cells particles it is noted that the transmittance
values rise with increased concentration of the zinc acetate solution in
the extrusion bath or in the pre-extruded. Moreover, it can be noted
3.1. Particles stability and cellular viability that all samples have close transmittance values. For A4D and A5D sam-
ples, after 24 h storage in bi-distilled water, is observed that the trans-
The gellan particles stability in water, with and without immobilized mittance value decreases slightly with increasing the cross-linking
yeast cells, was assessed by measuring the transmittance of the solution degree. These results may be a consequence of the high degree of
in which the particles are suspended during various time periods. As cross-linking therefore in the mesh of the polymeric network cannot
previously mentioned, it was assumed that the less stable particles be immobilized a large number of cells because the polymer chains
may cause a higher turbidity in the storage solution due to the detach- form a dense and compact network. Due to the very stable coordinative
ment from their surface of polymer, segments that were not firmly at- bonds formed by the zinc metal ion, the gellan particles with and with-
tached to the network polymer, or, in the case of particles with cells out immobilized yeast cells are very stable and therefore the transmit-
immobilized yeast, to the diffusion of yeast cells from the particles tance values are quite stable in time and their stability generally
that are not well cross-linked and therefore are unstable. increases with increasing the cross-linking degree. The zinc acetate con-
centration does not have a significant impact on the particles stability as
the transmittance values are high, independent of this concentration.
The concentration of cells/mL which is found in the cross-linking
Table 1 bath or the bi-distilled water decreases as the degree of cross-linking in-
Experimental conditions used for the preparation of gellan particles without immobilized creases and the number of cells that diffuses from the polymer matrix is
yeast cells. The same conditions were used for the preparation of gellan particles with
immobilized yeast cells; in this case the letter D was added to the sample name.

Sample Gellan Concentration of Concentration of Table 2


concentration zinc acetate in the zinc acetate in the Transmittance values for the extrusion solution and double distilled water, respectively, in
(%) pre-extrudeda (%) cross-linking bathb (%) which resulted particles were kept for different periods of time.
A2 0.1 0.02 0.1
Samples Transmittance (%)
A3 0.04
A4 0.06 Extrusion solution Bi-distilled water
A5 0.08
12 h 24 h 12 h 24 h 48 h
L2 0.1 0.02 0.08
L3 0.04 A2 99.00 98.80 99.40 99.20 99.00
L4 0.06 A4 99.40 99.20 99.70 99.40 99.10
L5 0.08 A5 99.60 99.40 99.70 99.60 98.70
N2 0.1 0.02 0.05 L2 99.20 98.60 99.40 99.10 98.50
N3 0.04 L4 99.40 99.10 99.60 99.30 99.00
N4 0.06 L5 99.50 99.20 99.70 99.40 99.20
N5 0.08 N2 99.10 98.20 99.20 98.90 97.10
a N4 99.50 98.40 99.90 99.00 97.70
Volume equal to 1.25 mL.
b N5 99.20 98.80 99.20 98.80 97.90
Volume equal to 50 mL.
C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489 481

Table 3
Transmittance, concentration of yeast cells/mL and cell viability in the extrusion solution and bi-distilled water for gellan particles with immobilized yeast cells.

Samples Transmittance (%) Concentration of yeast cells/ mL × 104 in Cell viability (%)
supernatant

Zinc solution Bi-distilled water Zinc acetate solution Bi-distilled water Zinc acetate solution Bi-distilled water

12 h 24 h 12 h 24 h 12 h 24 h 12 h 24 h 12 h 24 h 12 h 24 h

A2D 98.9 98.1 98.9 97.9 7 10 2.8 5.5 100 95.2 84.6 88
A4D 98.1 97.7 97.5 97.2 16 10.3 9.3 18 97.0 95.3 92.5 92.3
A5D 98.8 97.9 98.9 97.5 2 10 2.8 5 98.9 97.6 91.7 87.0
L2D 98.5 97.9 98.7 97.4 7.3 11.3 3.5 6.3 100 95.7 87.5 89.2
L4D 98.6 96.9 97.6 97.4 7.3 9.3 3 4.5 93.5 92.5 92.3 85.7
L5D 97.6 97.5 98.4 97.8 5.3 6.3 2.5 5 91.3 89.3 83.3 83.3
N2D 98.4 97 98.6 96.5 8.5 12.5 4.8 8 100 92.6 95 91,4
N4D 97.4 97.1 98.2 96.8 7.5 9.3 3 5.3 90.9 90.2 92.3 91.3
N5D 98.2 97.9 99 97.5 5 6.3 2.5 4.8 90.9 89.2 83.3 82.6

low. The concentration of cells/mL in the cross-linking bath, is greater hydrogels for various applications. Moreover, the pore size is important
than the concentration of yeast cells measured in bi-distilled water. for nutrient diffusion, cell growth and cell distribution in the matrix. In
One hypothesis is that some of the yeast cells, that were not order to use the gellan particles in repeated fermentation cycles, it was
immobilized in the network (because the network meshes are too very important to determine their structural stability by carrying out
dense), are free in the extrusion solution. This hypothesis could be sup- rheological tests. For this analysis were selected the most relevant 4
ported by the fact that after 24 h of storage in the extrusion solution, the samples (L4, L4D, N4, N4D) with identical zinc acetate concentration
concentration of cells/mL in the supernatant has only slightly increased. in the pre-extruded (0.06%), with the same polymer concentration
After 24 h of storage in the cross-linking bath, the particles are washed (1%) and various concentrations in the cross-linking bath. The selected
with bi-distilled water and kept in this medium for another 24 h (mea- samples were particles with and without immobilized yeast cells. Due
surements are made every 12 h). The concentration of cells which dif- to their relatively large diameter, yeast cells behave like an elastic mate-
fuse from the particles is much smaller when these particles were rial but are more rigid that the gellan matrix. The deformation and also
kept in bi-distilled water and these results might confirm the above hy- partial disintegration of the hydrogel, generated by the growing of yeast
pothesis. A smaller amount of cells diffuses from the particles, as com- cells inside the matrix, is a complex process influenced by interactions
pared to that remained immobilized in the polymeric matrix, which is between solvent, network domains and cells.
a proof of their stability over time. First of all, an amplitude sweep test (AS) was performed in order to
The samples cell viability is over 88% after 24 h storage in bi-distilled determine the linear viscoelastic domain limits (LVE) (see Fig. 2). Figs. 2
water, with one exception of 82.6% for sample N5D. These results indi- and 3 as well as Table 4 indicates rheological characteristics of these ma-
cate that the gellan particles have the ability to preserve the cell viability terials after 48 h. Using the amplitude sweep tests, were determined
over long time periods and the particles with immobilized yeast cells LVE range of the samples as being between 0.05 and 0.1% (Fig. 1).
could be used in the fermentation processes in repeated fermentation Frequency sweep tests allowed the study of the influence of the yeast
cycles. cells on the stability of hydrogels. Thus, the increase of the difference
between the two moduli (G’ and G”) indicates the presence of an
3.1.1. Structural stability of the particles and the average size of the network internal network with high mechanical and structural stability (Fig. 2).
meshes Moreover, the rheological tests have shown, for all the samples, that
In a general manner, the porosity, the chemical composition and the G’N N G” and that the values of the tangent of the loss angle (tan δ) are
mechanical properties are essential parameters in the preparation of inferior to 1, which demonstrates a predominantly elastic behavior of
these hydrogels.

Fig. 2. Amplitude sweep for samples L4 and L4D respectively N4 and N4D at T = 30 °C, γ = Fig. 3. Frequency sweep for samples L4 and L4D respectively N4 and N4D at T = 30 °C and
0.01 ÷ 100% and ω = 10 rad/s-page 19. ω = 0.1 ÷ 100 rad/s.
482 C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489

Table 4 G” moduli values. This could be explained by the fact that the growing
Rheological characteristics (G′, G″ and tan δ), the average size of the network meshes (ξ) cells press to the surrounding and create a new space for further cells
and the average molecular weight between cross-links (Mc) values for gellan-based hy-
drogel particles.
growth inside the matrix leading to matrix structural changes. There-
fore, yeast cells disrupt the continuity of the polymeric network thus af-
Sample G′ (Pa) G″ (Pa) tan δ (G″/G′) ξ (nm) Mc (kg/mol) fecting the number and the strength of the bonds between the particles.
L4 5270 412 0.0782 5.09 4.77 Finally, the stiffness and the elasticity of the hydrogels can be controlled
L4 + D 2720 169 0.0621 6.34 9.23 by varying the concentration of immobilized yeast cells.
N4 4410 298 0.0675 5.40 5.69
N4 + D 1900 111 0.0584 7.2 13.21
3.2. Swelling degree

The particles L4, without immobilized yeast cells, have a higher The behavior in water of spherical gellan particles, with and without
value of tan δ with respect to sample N4 and a similar behavior is also immobilized yeast cells, was evaluated by their swelling degree (Q%).
observed by comparing the samples L4D and N4D, with immobilized This feature is very important because it determines both the diffusion
yeast cells, which indicates that the elastic behavior of the hydrogels in- rate of the substrate to the immobilized cells in the hydrogel matrix
creases with decreasing the cross-linking density. and the diffusion of the resulting products from the particles. For this
Important information about the properties of the gellan-based hy- determination were selected samples with and without immobilized
drogel particles with or without immobilized yeast cells can be obtained yeast cells. The selected samples are A4, L4, N4, A2, L2, N2, A4D, L4D,
by calculating the average size of the network mesh (ξ) and the weight N4D, A2D, L2D, N2D. Fig. 4 presents the variation of the swelling degree
average molecular weight between cross-links (Mc) by using the values as a function of time for all samples.
of the storage modulus G’ in the LVE range [45,46]. If the concentration The obtained results show that the particles have a strong hydrogel
of zinc acetate in the cross-linking bath increases from 0.05% (N4) to behavior as they can absorb large amounts of water. The swelling de-
0.07% (L4), the size of the network meshes decreases from 5.69 nm to gree depends on the cross-linking degree of the particles. Moreover, it
4.77 nm, as shown in Table 4. Moreover, from Table 4 it can be noticed was observed that the swelling degree decreases with increasing the
an increase of the size of the network mesh when yeast cells are cross-linking density. Particles for which was used a higher concentra-
immobilized within the network. The values of the Mc, for L4D and tion of zinc acetate in the pre-extruded, such as: A4, L4, N4, have a
N4D samples containing yeast cells, is higher as yeast cells can form lower swelling degree as compared with the samples A2, L2, N2. The
bonds with the zinc ions due to the negative charges on the surface of same trend was observed for particles containing immobilized yeast.
the periplasmic cell membrane. It must be pointed out that the elastic For the particles containing immobilized yeast cells the swelling degree
properties of the hydrogels are influenced by the presence of yeast has lower values and it varies in the same order as for particles without
cells in the polymer network which leads to a decrease in the G’ and yeast cells. It is well-known that metal ions can adhere to the cell

(a) (b)

1200
Swelling degree, Q %

900

600

A2
300 L2
N2
0
0 2 4 6 8
Time(h )

(c) (d)

Fig. 4. Time variation of the swelling degree for samples characterized by different cross-linking degrees (T = 30 °C): (a) A4, L4, N4, (b) A4D, L4D, N4D, (c) A2, L2, N2 and (d) A2D, L2D,
N2D.
C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489 483

membrane giving it more strength [32,49], therefore it can be assumed 3.3. Particles morphology
that yeast cells participate to an additional cross-linking. These results
are in concordance with those obtained for the rheological tests and The morphology and the structure of gellan particles, with yeast cells
also with the particles stability testing in the aqueous medium by immobilized was determined by scanning electron microscopy. The se-
measurement of transmittance. Taking into account the significance of lected samples for analysis, A2D and L2D, have identical zinc acetate
the swelling degree on the diffusion of both nutrients and reaction concentration in the pre-extruded but different cross-linker concentra-
products to and from the particles, in the following were selected only tion in the extrusion bath (0.1% for sample A2D and 0.07% for sample
samples containing 0.02% of zinc acetate in pre-extruded because their L2D). Fig. 5 presents the photographs taken by scanning electron mi-
swelling degree is higher and also because A2, L2 and N2 samples croscopy for the two samples.
have previously shown an optimum stability, suggesting that they Scanning electron microscopy was performed on cross-section of
could be used in repeated fermentation cycles. dry gellan particles containing immobilized yeast cells. Micrographs

A2D L2D

Fig. 5. Scanning electron microscopy micrographs for samples A2D and L2D with different cross-linking degrees obtained before the fermentation. The magnification degree is indicated on
each micrograph.
484 C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489

reveal that yeast cells are found in large numbers within the particles 70

Yield in fermented glucose, η %


and are firmly immobilized in the polymer matrix in both analyzed 60
particles. It can be noticed that sample A2D, with a highest cross-
linking degree, has a dense structure with respect to sample L2D. 50
40 η I L2D
3.4. The fermentative activity of the gellan particles with immobilized
30 η I L4D
yeast cells
η I L5D
20
The time variation of fermentation yield was determined, at differ- 0
ent zinc acetate concentrations in the glucose solution at pH 5 and 6 ad- 10
justed with a dilute solution of acetic acid and sodium bicarbonate. 0
Several fermentation cycles were performed for each individual sample 0 100 200 300
and the time period between two fermentation cycle was 24 h. In order Time (min)
to optimize the fermentation process in terms of efficiency, the influ-
ence of several factors was studied:
(a)
a) Influence of the cross-linking agent in the pre-extruded
70

Yield in fermented glucose, η %


60
The selected samples have a different zinc acetate concentration
added before extrusion while the rest of the parameters which may in- 50
fluence the fermentation process is kept constant. The analyzed samples 40 η II L2D
were: L2D, L4D and L5D. The zinc acetate concentration in the fermen- η II L4D
tation solution was equal to 0.007%, and the pH was adjusted to 5 as the 30
η II L5D
previous research reports claims that the optimal pH for the fermenta- 20
tion medium is between 4.5 and 5 [50]. Fig. 6 shows the fermentation 0
yield as a function of time for all these three samples. 10
From Fig. 6, it can be observed that after the first fermentation cycle 0
the yields values increases with the decrease of zinc acetate concentra- 0 100 200 300
tion added before extrusion therefore increase when the cross-linking Time (min)
density decreases. For the second and third fermentation cycles, the fer-
mentation efficiency increases for all the analyzed samples. The fermen- (b)
tation efficiency obtained with the particles containing yeast cells
immobilized is higher than the efficiency obtained in the free yeast fer- 80
mentation in all analyzed cases. It is obvious that the cross-linking de-
Yield in fermented glucose, η%

70
gree influences the behavior of the particles in the fermentation
process. As the zinc acetate concentration in the pre-extruded increases, 60
the particles cross-linking degree increases. It was noticed that for the 50
particles with a high cross-linking degree, the fermentation efficiency 40
decreases with increasing the cross-linking degree of the particles
with immobilized yeast cells due to a slower diffusion of the substrate 30 η III L2D
(glucose). Therefore yeast cells can no longer perform their normal 20 η III L4D
physiological functions and their lysis occurs in time. η III L5D
10
Fig. 7 illustrates the fermentation yield values as a function of the fer- 0
0
mentation cycle numbers for each type of analyzed sample. 0 100 200 300
For all analyzed samples, it occurs that the fermentation yield de- Time (min)
creases as a function of the cycle number. The sample with the best fer-
mentation yield, for which obtained several fermentation cycles were
possible, is the sample L2 with a zinc acetate concentration of 0.02% in (c)
the pre-extruded. Therefore, it can be admitted that the optimal behav-
ior is given by the samples with low cross-linking degree. Fig. 6. Evolution in time of the fermentation yield for samples L2D, L4D and L5D. (a) after
the first fermentation cycle (b) after the second fermentation cycle (c) after the third
fermentation cycle (in the fermentation medium the concentration of zinc acetate
b) Influence of the zinc acetate concentration in the extrusion bath
solution = 0.007%, pH 5).

The analyzed samples in this section are A2D, L2D and N2D. The pH
of the solution in the fermentation bath was adjusted to 5 because the yeast cells from the gellan matrix. At the same time, the nutrients from
optimum pH for the proliferation of the yeast cells is between 4 and 6 the fermentation bath diffuse more easily into the particles with
[51]. Fig. 8 shows the variation of the fermentation yield after the first immobilized yeast cells increasing they proliferation and thus the fer-
cycle of fermentation as a function of time for several samples with dif- mentation efficiency.
ferent concentrations in cross-linking agent. A concentration of 0.007%
zinc acetate was used in the glucose solution (8%) in the fermenter. c) Influence of the particles storage time in the extrusion bath
From Fig. 8, it can be seen that by decreasing the concentration of the
cross-linking agent in the extrusion bath, the fermentation efficiency in- To determine the particles optimal storage time in the reticulation
creases. It is obvious that this effect is due to the lower degree of cross- bath after extrusion, the sample L2D was chosen, as a typical example.
linking and to the higher porosity of the particles, resulting in the inten- The sample L2D was chosen as it exhibited a superior stability compared
sification of diffusion of the substrate, represented by the glucose, to the to that of sample N2D (at which the fermentation yield decreases
C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489 485

100 L2D 80
Yield in fermented glucose,

90

Yield in fermented glucose, η%


80 70
70
60 60
η%

50
40 50
30
40
20
10
30
0 A2D
1 2 3 4 5 6 7 8 9 0
Number of fermentation cycles 20 L2D
N2D
10
(a) 0
0
0 50 100 150 200 250 300
L4D
Yield in fermented glucose,

60

50
Time(min)

40 Fig. 8. Evolution in time of the fermentation yield for samples with different cross-linking
η%

degree, A2D, L2D, N2D, compared with the yield obtained in free yeast fermentation
30 (0) (the fermentation medium had pH = 5 and 0.007% the concentration of zinc acetate).

20
The particles storage in the extrusion solution for a longer period of
10
time induces a increase of the cross-linking degree under the influence
0 of zinc acetate. The efficiency of the fermentation increases when the
1 2 3 4 5 6 7 0 degree of cross-linking is low. It is well known that the yeast cells re-
Number of fermentation cycles quire very small amounts of zinc ions, at micromolar concentrations,
in order to fulfill their physiological role in the cell metabolism. Compar-
ing Fig. 9a to 9b, it can be noted that the fermentation yields are higher
(b)
for the samples that were maintained in the cross-linking solution for
2 h.
60
Yield in fermented glucose,

L5D
50 d) Influence of the pH from the fermentation bath.

40 The pH of the fermentation solution is not considered by many re-


η%

searchers as a key factor in the fermentative processes. However, sever-


30 al studies have reported that the pH of the fermentation solution plays
an important role in the uptake of metal ions by the yeast cells during
20 the fermentation processes. Also, some research stated that the pH has
an important role in the binding of metal cations to the absorption
10
sites in the cell wall [50–52]. For study the influence of the pH from
the fermentation bath, the sample L2D was used and the pH of the fer-
0
1 2 3 4 5 0 mentation bath was adjusted to 5 and 6 respectively. It must be recalled
Number of fermentation cycles that the sample L2D is most effective in terms of fermentation yields.
The particles were kept in the cross-linking bath for 2 h after which
(c) they were washed with bi-distilled water and tested in repeated fer-
mentation processes. The fermentation bath contains glucose dissolved
Fig. 7. Glucose fermentation efficiency obtained in succesive fermentation cycles for in a zinc acetate solution of 0.01%. Fermentation cycles were repeated
samples with immobilized yeast cells compared with free yeast (0); (a) L2D, (b) L4D after 24 h. Fermentation diagrams of the L2D sample, taken at different
and (c) L5D. (concentration of zinc acetate in the glucose solution = 0.01%; pH = 5). values of pH in the fermentation bath, are given in Fig. 10.
The results given in Fig. 10 demonstrates that pH is an important fac-
tor for obtaining optimal fermentation yield of glucose and for the use of
sharply after 6 cycles, and the number of cells that diffuses from the par- the particles in repeated fermentation cycles. Decreasing the pH from 6
ticles after each fermentation cycle is very high) and higher yields were to 5 it was observed that the fermentation efficiency increases and that
obtained compared with A2 sample determined of a lower cross-linking these values are comparable to those obtained for the free yeast fermen-
degree. As a result, the swelling degree is high and cause the diffusion of tation. Moreover, it occurs that the number of fermentation cycles with
nutrients to the yeast cells, as mentioned above. optimal efficiency increases. Thefore, it can be admitted from these re-
The determinations were made after 2 and 12 h respectively. After a sults that the yeast cells can proliferate effectively inside the gellan par-
given storage time in the extrusion solution, the samples were washed ticles at a pH value of 5. Due to the fact that yeast cells are immobilized
in bi-distilled water in order to remove the excess of zinc acetate, and in a polymeric matrix, the diffusion of substrate (glucose) to the cells is
they fermentative activity was determined. The concentration of zinc difficult leading to an increase of the cell proliferation time. The optimal
acetate was equal to 0.015% and the pH was adjusted to 5. Fig. 9 illus- pH for the growth and proliferation of the yeast cells is between 4.5 and
trates the variation of the fermentation yield as a function of the 5 and this parameter is essential for the achievement of a higher num-
fermentation cycles. ber of fermentation cycles [52,53].
486 C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489

100 60

Yield in fermented glucose, η %

Yield in fermented glucose, η %


80 50

40
60
30
40
20
20
10

0 0
1 2 3 4 5 0 1 2 3 4 0
Number of fermentation cycles Number of fermentation cycles

(a) (b)

Fig. 9. Glucose fermentation efficiency obtained in successive fermentation cycles obtained for the sample L2D compared with the efficiency obtained in free yeast fermentation (0). The
sample L2D was kept different periods of time in the cross-linking solution and the fermentation medium differ through pH and zinc acetate concentration, namely a) 2 h, pH 5 and 0.015%;
b) 12 h, pH 5 and 0.015%.

The ethanol yield in the fermentation process is affected by the bath. The fermentation cycle number decreases when a lower concen-
pH of the fermentation medium. It is known that the yeast species tration, such as 0.007%, is used in the fermentation. This behavior of is
Saccharomyces cerevisiae have a maximum increase in acidic environ- due to the fact that the zinc ions are a growth factor for yeast cells in
ments. A small productivity in ethanol at a higher pH may be also due certain concentrations. A lower concentration of zinc acetate in the fer-
to other microbes that can exist in the environment because the mentation bath may result in a lower rate of cell proliferation, and
fermentation takes place without sterilization [54]. therefore, the fermentation yield will decrease. The same decrease of
the efficiency in fermentation cycles is observed when a higher zinc ac-
e) Influence of zinc acetate concentration in the fermentation bath. etate concentration is used. This behavior can be explained by the fact
that higher zinc acetate concentration will increase the cross-linking de-
To study this influence, the fermentative activity of the L2D sample gree of the gellan matrix leading to a more difficult diffusion of nutrients
was tested at various zinc acetate concentrations in the fermentation from the fermentation solution into the particles and the cells cannot
bath (the pH of the fermentation bath was 5 for all the measurements). proliferate. In time, autolysis process occurs and the fermentation
Several zinc acetate concentrations were used, such as: 0.015%, 0.010% process stops. From all the previous results, it was observed that higher
and 0.007% respectively. Fig. 11 illustrates the time variation of the fer- fermentation yields after several fermentation cycles are obtained for
mentation yield after several particles fermentation cycles of the parti- the systems that have a zinc acetate concentration of 0.01% and
cles under the above mentioned conditions. 0.007% respectively and the pH of the fermentation solution is equal
From Fig. 11 it was observed that the fermentation efficiency is a to 5. However, it was difficult to establish a clear dependence between
function of the zinc acetate concentration in the fermentation bath. fermentation yields and other parameters involved in this process.
Moreover, it occurs that the highest number of fermentation cycles is Nevertheless, it occurs that the fermentation yield tends to decrease
obtained for a zinc acetate concentration of 0.010% in the fermentation after five cycles. This variation of the fermentation yield as a function

(a) (b)
Fig. 10. Glucose fermentation efficiency obtained in successive fermentation cycles for L2D sample at: (a) pH 6 and (b) pH 5 compared with the efficiency obtained in free yeast
fermentation (0) (concentration of zinc acetate in the fermentation medium was 0.01%).
C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489 487

As above mentioned, in gellan particle solution was added a suspen-


sion obtained from 1.25 g yeast and 5 mL water. The amount of yeast
cells is equal to 30% of the total amount of yeast, i.e., 0.375 g. Before
each fermentation cycle, based on the calibration curve, it was establish
the amount of yeast cells which was immobilized in the gellan particles
as a function of the number of cells which diffuses in the extrusion
solution. The immobilization efficiency for all samples was over 99%.
After each fermentation cycle it was measured the amount of yeast
cells which diffuses in the fermentation bath. It can be observed that
the particles with immobilized yeast cells are stable in the fermentation
medium. It was assumed that from the less stable particles, a large num-
ber of yeast cells can diffuse. Mariam et al. [54] have immobilized yeast
cells in alginate particles using the calcium chloride as cross-linking
agent. 6 fermentation cycles were obtained with a maximum efficiency
for the fourth cycle and then the yield strongly decreases. The explana-
(a) tion was that the particles are deformed during the fermentation and
become unstable [54]. Considering the theoretical amount of
immobilized yeast as 0.375 g and subtracting from it the amount of
yeast fermentation found in the bathroom, it has been estimated the
number and the amount of imobilized particles remaining in the cells
(Table 5).
The fermentative activity study was carried out in two different fer-
mentation solutions which are distinguished by the amount of zinc ac-
etate in which is dissolved the glucose (0.01% and 0.007% zinc acetate
respectively). The pH of the fermentation solution was in all cases ad-
justed to 5. The results given in Table 4 show that the amount of cells
which diffuse from the particles is much smaller than that of cells
which remains immobilized inside the particles. These results demon-
strate that the gellan particles with yeast cells immobilized are stable.
Moreover, it can be observed that the amount of yeast cells which
diffuses from the particles after each fermentation cycle depend upon
the concentration of zinc acetate of the cross-linking bath. For example,
in the sample A2D, which has a higher cross-linking degree, the cell
amount which diffuses from the particles is smaller than that of the
(b)
sample L2D. The zinc acetate concentration in the fermentation bath
has a significant influence on the amount of yeast cells which diffuses
from the gellan particles.
In order to achieve optimum fermentation efficiency it is necessary
that the zinc ions from the fermentation solution to have an optimum
concentration. From the previous results, it can be admitted that 0.01%
may be the optimal zinc acetate concentration in the fermentation.
The diffusion of a higher amount of yeast cells in the fermentation solu-
tion may be a consequence of the fact that the zinc ions are absorbed ef-
fectively by the yeast cells. These yeast cells can accomplish their
essential physiological roles such as growth and nutrition. Moreover,
they can multiply easier at this concentration which could explain the
more important diffusion in the fermentation solution. The results
described above are in full agreement with the literature data. For
example, Stehlik-Tomas et al. [52] have added to the fermentation me-
dium a concentration of 0.1 g/L ZnSO4 and noticed an improvement in
the fermentation efficiency at pH 5 [52]. The fact that the particles
(c)

Fig. 11. Glucose fermentation efficiency obtained in successive fermentation cycles Table 5
obtained for L2D sample at pH 5 and different zinc acetate concentrations in the The amount of yeast cells measured after each fermentation cycle.
fermentation medium: (a) 0.015%, (b) 0.010% and (c) 0.007%. (pH = 5) compared with
the efficiency obtained in free yeast fermentation. Sample Fermentation Zinc acetate The total The total The theoretical
cycle number concentration number of amount of amount of
in the cells/mL × 104 yeast cells yeast cells
fermentation measured (mg) immobilized
of time is likely to be caused by the proliferation of the yeast cells within bath (%) after each in gellan
the gellan particles. Wang and Hattwer [55] affirm that it exist the pos- fermentation particles (g)
sibility that the immobilized cells to grow better in the gel matrix than cycle
the free ones because they are experiencing limited movement. A2D 6 0.01 51.5 1.06 0.37394
It also should be noticed, that the fermentation efficiency in the pres- 6 0.007 40.25 3.3 0.37414
ence of the gellan particles is superior to that obtained in the presence of L2D 13 0.01 184.5 3.67 0.37133
9 0.007 75.75 1.5 0.3735
free yeast.
488 C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489

with immobilized yeast cells can be easily recovered from the fermenter Table 7
and can be used for several fermentation cycles is a proof that the gellan Fermentation rate and specific productivity in ethanol as a function of the fermentation
cycle for samples A2D and L2D respectively.
matrix protects the yeast cells from the toxic metabolites produced by
ethanol. Moreover, the cell viability is maintained and therefore the Sample Fermentation Zinc acetate Fermentation Specific productivity
yeast cells can proliferate within the gellan particles. cycle number concentration in rate, % glucose in ethanol, g
the fermentation conversion/min ethanol/h × g yeast
Using a zinc acetate concentration of 0.01% in the fermentation bath,
bath (%)
it can be obtained several fermentation cycles with yields comparable to
A2D 1 0.01 0.11 0.59
those obtained in the fermentation of the free yeast, unless if in the fer-
2 0.13 0.59
mentation bath are used higher or lower cross-linker concentrations. 3 0.13 0.59
One explanation could be that a medium deficient in zinc ions can 4 0.13 0.59
slow down the fermentation process and the use of higher zinc acetate 5 0.13 0.2
concentrations in the fermentation bath leads to a further cross-linking 6 0.089 0.2
1 0.007 0.13 0.59
of the gellan particles. In this last case, the nutrients from the fermenta-
2 0.13 0.59
tion solution cannot diffuse into the particles, and the fermentation pro- 3 0.089 0.59
cess stops in time because the cells cannot proliferate and cannot 4 0.13 0.39
exercise their necessary metabolic functions. 5 0.089 0.59
6 0.067 0.37
L2D 1 0.01 0.13 0.59
2 0.13 0.59
3.5. Determination of cell viability and cell amount from the particles after 3 0.11 0.39
each fermentation cycle 4 0.089 0.39
5 0.49 0.74
6 0.089 0.59
It should be noted that the polymeric matrix has a protective role
7 0.067 0.59
against the immobilized cells, allowing their proliferation and thus 8 0.84 0.59
higher cell viability (Table 5). The number, amount and cell viability 9 0.13 0.39
were determined after the disintegration of the particles at the end of 10 0.29 0.82
11 0.089 0.39
the fermentation cycle, as summarized in Table 6. In this manner is
12 0.156 0.39
can be explained the possibility of reusing particles with immobilized 13 0.044 0.59
yeast cells over several fermentation cycles with convenient yields, in 1 0.007 0.13 0.59
most cases higher to that achieved with free yeast. 2 0.13 0.67
3 0.22 0.99
4 0.51 0.99
5 0.22 1.03
3.6. Specific productivity and the fermentation rate 6 0.13 0.59
7 0.13 0.59
The linear section of the kinetic curves of the fermentation process 8 0.13 0.32
9 0.18 0.59
allows the calculation of the fermentation rate. Based on these results
it was possible to determine the specific productivity in ethanol and
the results obtained for each type of particles, compared with free
yeast, as a function of the fermentation cycles are presented in Table 7. 4. Conclusions
For the free yeast, the fermentation rate is 0.067% glucose conver-
sion/min and the specific productivity is 0.51 g ethanol/g glucose x g Gellan particles with immobilized yeast cells were obtained by ionic
yeast. It can be observed a direct correlation between the fermentation cross-linking using zinc acetate as cross-linking agent. The swelling de-
rate and the fermentation yield for all the samples. In general, the fer- gree increases when the cross-linking degree decreases and the obtain-
mentation rate for most fermentative cycles performed in the presence ed results are in concordance with those obtained from the rheological
of gellan particles is higher to that obtained in the presence of free yeast. tests. The gellan particles having the highest cross-linking degree are
In most fermentation cycles, the fermentation rates obtained in the characterized by the highest structural stability. Scanning electron mi-
presence of L2D sample was superior to those obtained for the sample croscopy showed that yeast cells are found in large numbers inside
A2D. Regarding the specific ethanol productivity, it was noticed that the gellan particles and the samples with a higher cross-linking degree
for the most fermentation cycles, it was obtained a higher or equal spe- have a more dense structure. The fermentative activity strongly de-
cific productivity than that of the free yeast. pends on the zinc acetate concentration used in the pre-extruded, in
the cross-linking bath and in the fermentation medium. Other factors
that may affect the fermentative activity of the particles are the pH of
Table 6 the fermentation solution and the particles storage time in the cross-
The number, the amount and the viability of yeast cells immobilized within the gellan linking solution. With the obtained results from this study, it was possi-
matrix cross-linked with Zn2+ ions as a function of the fermentation cycles (L2D). ble to establish the optimal conditions for the preparation of gellan par-
Number of Number of cells × Amount of cells/g Cell ticles with immobilized yeast cells: the gellan solution concentration
fermentation cycles 108/g particles particles, mg viability, % was equal to 1%, the zinc acetate concentration in the pre-extruded
0 4.84 17.93 95.43
should be equal to 0.02%, 0.07% in the reticulation bath and 0.01%
1 4.94 18.3 91.48 in the fermentation medium. Moreover, the optimal pH of the
2 6.75 25 92.69 fermentation solution should be kept at 5 and the optimal particles
3 8.08 29.94 92.63 storage time in the cross-linking bath was 2 h. Gellan particles with
4 7.44 27.57 92.82
immobilized yeast cells can be easily recovered by filtration from the
5 6.6 24.45 92.75
6 6.62 24.53 92.46 fermentation bath and may be used in several fermentation cycles.
7 7.93 29.38 92.50 The specific ethanol productivity obtained in the presence of gellan par-
8 5.51 20.41 91.90 ticles with immobilized yeast cells was similar (in some fermentation
9 6.36 23.56 88.79 cycles even higher) to the specific productivity obtained in the presence
10 6.32 23.45 88.20
of free yeast. The gellan matrix maintains cell viability at high values
C.-E. Iurciuc (Tincu) et al. / Powder Technology 325 (2018) 476–489 489

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