Process Biochemistry 42 (2007) 1348–1351

www.elsevier.com/locate/procbio

Short communication
Immobilization of yeast cells in PVA particles for beer fermentation
Dejan Bezbradica a, Bojana Obradovic b, Ida Leskosek-Cukalovic c,
Branko Bugarski b, Viktor Nedovic c,*
a
Department for Biochemical Engineering and Biotechnology, Faculty of Technology and Metallurgy, Karnegijeva 4, Belgrade, Serbia
b
Department for Chemical Engineering, Faculty of Technology and Metallurgy, Karnegijeva 4, Belgrade, Serbia
c
Department of Food Technology and Biochemistry, Faculty of Agriculture, Nemanjina 6, Zemun, Serbia
Received 27 November 2006; received in revised form 15 March 2007; accepted 14 April 2007

Abstract
In this study, the potential of application of non-aggressive LentiKat1 technique for brewer’s yeast immobilization on polyvinyl alcohol was
assessed. High cell loads of about 109 cells/ml were achieved by this procedure and immobilization procedure had no adverse effect on cell
viability. The stability and activity of obtained immobilized biocatalyst was tested in the growth studies and fermentations. Immobilized cells
exhibited high fermentation activity in both, laboratory and pilot-scale fermentations. In three successive gas-lift reactor fermentations the apparent
attenuation of around 80% was reached after only 2 days, indicating good potential of immobilized cells for development of continuous primary
beer fermentation. LentiKat1 particles showed high mechanical and fermentative stability, since they endured 30 days of operating time during 6-
month period without significant change of cell activity, particle shape and particle size.
# 2007 Published by Elsevier Ltd.

Keywords: LentiKat1; PVA; Brewer’s yeast; Gas-lift; Immobilization; Fermentation

1. Introduction
The application of systems employing immobilized brewer’s
yeast cells introduced important advantages in brewing
fermentations such as high cell loads, high volumetric
productivities, improved tolerance to inhibitors and reuse of
the same biocatalyst for extended periods of time [1–3].
Immobilized systems have been successfully applied in the
production of alcohol-free beer and in the secondary fermenta-
tion of lager beer [4,5]. Polyvinyl alcohol (PVA) is a promising
support for cell entrapment since it is non-toxic and mechanically
stable. The PVA particles have been produced by multiple
freezing and thawing of PVA solutions, jet cutting technique, and
cross-linking by UV radiation or boric acid [6–12].
A novel non-aggressive method (called LentiKat1) of PVA-
gel preparation at room temperature by means of controlled
partial drying was developed recently [13,14]. Due to the
lenticular shape, LentiKats1 have the beneficial properties of
both, small and large carriers, namely good diffusion properties
as a result of small lens thickness and good mechanical
* Corresponding author.
E-mail address: vnedovic@agrifaculty.bg.ac.yu (V. Nedovic).
properties and simple separation, typical for large carriers [15–
17]. It has been previously reported by Smogrovicova et al. that
beer of good quality was produced using brewer’s yeast
immobilized in LentiKats1 particles [17].
In this work, the viability and growth kinetics of brewer’s
yeast cells immobilized in LentiKats1 were tested in growth
and fermentation studies in shaken flasks and a laboratory scale
gas-lift reactor. The stability of growth kinetics was monitored
in multiple batch fermentations.
2. Materials and methods
Brewing yeast cells (Saccharomyces cerevisiae, kindly provided by Efes
Weifert Brewery, Pancevo, Serbia) were firstly cultivated in sterile growth
medium at 25 8C in shaken flasks. Cells were harvested in the early
exponential phase by centrifugation at 8000 rpm. LentiKat1 Liquid
(80 ml, geniaLab, Germany) was melted at 90–95 8C, cooled down to a
temperature of 30–35 8C and mixed with 20 ml of yeast cell suspension
(108 cells/ml) by a magnetic stirrer. Tip of wires of a LentiKat1 Printer
(geniaLab, Germany) were quickly dipped into the solution, lifted and
pressed on Petri dishes to form droplets. Dishes were then put under the
sterile, laminar airflow at room temperature. Under these conditions, gella-
tion of the droplets occurred in approximately half an hour at a 75% decrease
of the initial mass due to water evaporation. The obtained gel particles in the
form of lenses were stabilized and re-swelled in the stabilizing solution
(geniaLab, Germany) for 2 h. The resulting LentiKats1 lenses were about

1359-5113/$ – see front matter # 2007 Published by Elsevier Ltd.

doi:10.1016/j.procbio.2007.04.009
 D. Bezbradica et al. / Process Biochemistry 42 (2007) 1348–1351
3.5 mm in diameter and 0.3 mm thick with immobilized yeast cells at a
starting concentration of 1  107 cells/ml.
Growth of immobilized yeast cells was investigated in a short-term study by
cultivating LentiKats1 lenses for 85 h in shaken flasks. The flasks, which
contained 200 ml of growth medium and 10 g of carriers or 10 g of yeast
suspension (control sample), were placed on orbital shaker at 115 rpm and
25 8C. The starting overall cell concentration in both experimental groups was
about 0.5  106 cells/ml. The growth medium (pH 5.0) contained: 100 g/l
glucose, 1.5 g/l yeast extract, 2.5 g/l NH4Cl, 5.5 g/l K2HPO4, 0.25 g/l,
MgSO4
7H2O, 1 g/l NaCl, 0.01 g/l CaCl2 and 3 g/l citric acid. LentiKats1
lenses and medium were sampled at timed intervals, and analyzed for cell
viability and concentration. The experiments were carried out in duplicate.
A series of batch fermentations was performed in 500 ml flasks, which
contained 210 ml of sterile malt extract (12%, w/w) and 70 g of LentiKats1
lenses, on orbital shaker at 115 rpm and 17 8C, which lasted 2–4 days, each. The
initial concentration of immobilized yeast cells was about 2  107 cells/ml of
LentiKats1.
Batch fermentations were performed in an internal-loop gas-lift bioreactor
with a working volume of 1 l. Bioreactor consisted of a glass cylinder (80 mm
i.d., 220 mm height) and a coaxially positioned draft tube (50 mm i.d., 100 mm
height). Bioreactor contained 50 g of dry mass of LentiKats1 and 560 ml of
sterile plant wort of 12% extract. Nitrogen was introduced through a glass
sparger at the bottom of the reactor at a flow rate of 200 ml/min. The initial
concentration of immobilized yeast cells was 1  109 cells/ml of LentiKats1.
The size of LentiKats1 lenses with immobilized cells was estimated using a
microscope with an accuracy of 10 mm. Cell concentrations and viabilities were
determined after dissolution of lenses by heating and mixing. Yeast cell
concentration was estimated using a Thoma counting chamber and cell viability
was assessed by means of methylene blue staining technique. Liquid samples
from both, growth and fermentation mediums, were collected aseptically from
the flasks and analyzed for specific gravity, yeast cell counts, and cell viability,
as described previously [18].
3. Results and discussions
Effects of immobilization procedure on viability and
proliferation potential of S. cerevisiae cells were studied in
comparative cultivations of cells immobilized in LentiKats1
carriers and freely suspended cells. Virtually 100% viability of
immobilized cells was assessed immediately after the produc-
tion of LentiKats1 carriers, using the staining technique.
Kinetics of cell concentrations in LentiKats1 carriers and
those released in the medium are presented in Fig. 1A. A lag
phase of 22 h and an exponential growth phase of 18 h with a
Fig. 1. Growth curves for: (A) (*) cells in LentiKat1 particles and (^) cells released in growth medium; (B) (*) cells in LentiKat1 particles + cells released in
growth medium, and (^) free cells.
1349
specific growth rate 0.22 h 1 can be observed on the growth
curve obtained for immobilized cells. Released cells were
detected in the medium only after 20 h of cultivation, which
approximately coincided with the start of intensive prolifera-
tion of immobilized cells (Fig. 1A). The increase of cell
concentration in the medium was exponential with apparent
specific growth rate 0.43 h 1, representing combined effects of
cell proliferation in the medium and cell leakage from the
carriers. The exponential growth phase of immobilized cells
was about double that of released cells (18 h versus 10 h),
which may indicate higher tolerance of immobilized cells to
inhibitor metabolites.
The kinetics of overall cell concentration in the immobilized
system (in carriers and medium) and in free cell suspension, are
compared in Fig. 1B. The immobilized cells exhibited
significantly longer lag and exponential phases than the free
cells (22 and 18 h versus 5 and 10 h, respectively). In addition,
the apparent specific growth rate in the immobilized system was
almost half that of the free cell suspension (0.25 h 1 versus
0.47 h 1, respectively). However, the final overall cell
concentration in the immobilized system was slightly higher
than the concentration achieved in the free cell suspension
(Fig. 1B) due to the prolonged, although slower, growth in the
immobilized system.
The above results indicate that the immobilization procedure
had a negligible effect on cell viability and growth. A prolonged
lag phase of cells entrapped in polymer matrices was also
observed in previous cell immobilization study [19]. However,
the apparent specific growth rate of the cells released in the
medium, which were comparable to that of free cells found in
the present work, implies preserved cell physiology. In
addition, the final cell concentration achieved in LentiKats1
carriers was an order of magnitude higher than the final
concentration of suspended cells (4  108 cells/ml of carrier
versus 3  107 cells/ml) and in the range of previously reported
concentrations of brewer’s yeast cells in Ca-alginate, Ca-
pectate and k-carrageenan [2,4,18].
A series of eight short-term fermentation studies in shaken
flasks was performed in order to assess the activity of
1350 D. Bezbradica et al. / Process Biochemistry 42 (2007) 1348–1351
Fig. 2. Average cell growth in the medium during one fermentation batch in
shaken flasks.
immobilized brewer’s yeast cells. The apparent attenuations of
around 80% were achieved already after 2-day fermentations.
Prolongation of fermentation to 3 or 4 days resulted in only a
small increase of attenuation, not beyond 83%. During repeated
multiple fermentations, cell concentrations within LentiKats1
particles stabilized after three experimental runs at a value of
about 6  108 cells/ml. Cell concentrations measured in the
obtained green beers are presented in Fig. 2. Extension of
fermentation time led to a considerable increase in cell
concentrations in the medium and minor increase of apparent
attenuation, probably due to the intensive cell growth in
peripheral section of particles resulting in the cell release in the
medium. To summarize, Fig. 3 presents cumulative biomass
produced over 4 weeks in a series of batch fermentations. After
the 5 initial days (two experimental runs) a constant rate of
biomass production of about 5.2  10 3 h 1 was determined
indicating good operational stability of the immobilized cells.
Rates of biomass formation in traditional fermentations are
significantly lower than the obtained value. High rates of
biomass formation exhibited by immobilized cells could be
crucial for continuous mode processes providing a stable source
Fig. 3. Cumulative biomass production over eight fermentation batches in
shaken flasks.
of yeast supply and operation at flow rates beyond usual
washout rates. Additionally, the cell activity stayed constant for
about 20 days after the initial adjusting period and LentiKats1
particles remained intact after multiple experimental runs,
confirming chemical and mechanical stability. The good
mechanical properties of LentiKats carriers partly attributed
to cell immobilization, which increased significantly PVA
hydrogel hardness as shown in previous study focused on S.
cerevisiae cells entrapment [20].
Gas-lift reactor was applied since it was previously reported it
provides excellent mass and heat transfer properties due to
intensive mixing of liquid and solid phases and, in the same time,
low shear stresses [21–24]. Three fermentations were performed
in the experimental gas-lift reactor loaded with 8% (w/v) of
LentiKats1 particles, comprising a cell load of
(0.9  0.1)  108 cells/ml of reactor volume. The same batch
of LentiKat1 particles was used in all fermentations. The
progress of fermentation in the three successive experiments is
presented in Fig. 4 as the average apparent attenuation per initial
cell load versus fermentation time. The apparent attenuation of
wort reached 80% after 2 days showing high fermentation
activity of the immobilized cells. In addition, the immobilized
cells continued to grow, so that the overall cell concentration in
the reactor was doubled during the repeated fermentations. Cell
growth was mainly observed as increase of released cells in the
medium due to the already high cell loads in carriers. Cell
‘‘leakage’’ was especially prominent during intensive formation
of carbon dioxide probably due to the creation of pores in the
polymer matrix by arising bubbles. However, it should be noted
that the overall cell concentration in the particles was not affected
by the increased cell release. This result implies that bursts of
CO2, which are inevitable during the brewing process, do not
impair the LentiKats1 particles structure extensively. At the end
of fermentations, the free cells accounted for the 40  5% of the
total cell biomass in the reactor. Although intensive cell
proliferation is not the main purpose of the brewing process,
certain level of cell growth must be achieved since most of the
typical beer flavor compounds are side- or by-products of
metabolism associated with yeast growth. Additionally, it is
Fig. 4. Progress of fermentation as the average apparent attenuation per initial
cell load over time, during fermentations in the gas-lift reactor.
 D. Bezbradica et al. / Process Biochemistry 42 (2007) 1348–1351
plausible that cells growing free in the medium exhibited higher
growth rate since growth medium was readily accessible. Cell
growth and release rates, confirm the potential of LentiKats1
carriers as a permanent source of cells, to allow stable continuous
operation [25]. Achieved attenuations of wort and cell
proliferation imply that the approach based on the use of
LentiKats1 lenses in gas-lift reactors can provide operation with
efficient mass transport without significant internal and external
mass transfer limitations. At the same time, LentiKats1 particles
retained their size and shape over a 6-month storage at 4 8C,
including 30 days of operating time.
4. Conclusion
This study has demonstrated the potential of LentiKats1
particles as carriers of brewing yeast cells for beer fermentation.
The feasibility of the investigated carrier was investigated
through growth studies in batch reactor, while fermentation
activities of the immobilized cells were studied in shaken flasks
and an internal-loop laboratory gas-lift reactor. Results of the
growth studies imply that the immobilization procedure had no
adverse effects on cell viability and proliferation. Although the
growth phases of immobilized cells were prolonged as compared
to freely suspended cells, high final cell concentrations were
achieved of the order of 1  109 cells/ml of LentiKats1.
Furthermore, the immobilized cells retained high fermentation
activity such that the full attenuation in green beer was reached
after 48 h in both shaken flasks and the gas-lift reactor. The
immobilized cells have shown stable fermentation kinetics and
cell growth kinetics (Fig. 4), which is prerequisite for successful
application in industrial continuous beer production. In the
present study, a relatively low solid load (8%, w/v) in the gas-lift
reactor was applied, which implies that even higher fermentation
rates could be achieved at higher solid hold-ups. Finally, a batch
of obtained LentiKats1 particles with immobilized brewer’s
yeast cells endured growth studies, eight batch fermentations in
shaken flasks and three batch fermentations in the gas-lift reactor
comprising 30 days of operating time in a 6-month period
without obvious changes in shape and size.
Acknowledgments
This research was funded by the Ministry of Science and
Environmental Protection, Republic of Serbia (grants 371005
and 142075) and by the support of Efes Weifert Brewery,
Pancevo, Serbia.
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