New Technoly of Fermernting

J. Insi. Brew., January-February 1998, Vol. 104, pp.
19-31
FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR CONTINUOUS

BEER FERMENTATION: A REVIEW
By P. H. Pilkington1-2, A. Margaritis1*, N. A. Mensour1-2, and I. Russell2

(lDepartment of Chemical & Biochemical Engineering, University of Western Ontario, London, Ontario, Canada N6A5B9,
^Technology Development Department, Labatt Brewing Company Limited, London, Ontario, Canada N6A 4M3)
Received 9 July 1997
Recent fundamental research conducted on immobilised cells with a focus on continuous primary beer
fermentation is presented in this review. The knowledge of whole-cell immobilisation, continuous
fermentation, yeast biochemistry associated with beer flavour production, and bioreactor engineering
design is required to apply immobilised yeast cells for industrial scale beer production. Understanding
how immobilisation and continuous bioreactor operation affect yeast cell metabolism and viability will
provide the groundwork for optimising beer quality. The latest studies on immobilised cell carriers,
viability, vitality, mass transfer characteristics and bioreactor design indicate that an industrial scale
immobilised cell system for primary beer fermentation may soon be a reality in the modern brewery.
Key Words: Immobilised cell carriers, beer production,

continuous fermentation, viability, mass transfer, bioreactor
design, flavour production.
5.
for the production of consistent quality beer using im

mobilised yeast cells.
Introduction
Traditional beer fermentation technology uses freely suspended

yeast cells in batch bioreactors to ferment wort using a variety
of yeast strains to make different beer products. The sugars and
other nutrients present in the liquid phase must diffuse into the
yeast cell to be metabolised by different enzymes into cthanol
and other flavour compounds which must diffuse out of the
yeast cell into the liquid phase. The functional integrity of the
cell membrane plays a key role in determining the viability
characteristics of the yeast cells and their metabolic activity.
Over the last IS years or so, a new development in ferment
ation technology, using immobilised yeast cells for the
continuous production of beer, has gradually emerged world
wide. Earlier research by Margaritis et al.75-16'17--19 an(j otner
researchers8-25-3*65-911 iwa.i3i.i35.i38 has established the superior
characteristics and advantages of using immobilised yeast cells

to continuously produce ethanol in fixed bed and fluidised
bioreactor systems. The large number of publications on yeast
cell immobilisation highlights the importance and interest in
this new technology as we enter the twenty-first century.
Continuous beer fermentation using immobilised cells will out
perform existing mainstream brewing technology by reducing
the time to produce a finished beer, reducing inventory,
reducing floor space, and reducing product variation.
A systematic critical review of the literature on beer pro
duction using immobilised yeast cells revealed the following
problems that need to be solved through more research:
1.
2.
3.
4.
matrix and the flavour and aroma compounds that diffuse

out; and
an integrated approach is needed to incorporate all of the
above information to develop bioreactor control strategies
there is no reliable collection of data on the mixing and

mass transfer of real three-phase fluidised bioreactors;
there is a need to develop an accurate non-invasive method
to measure immobilised cell viability;

much work remains to be done on the effects of dissolved
oxygen concentration and free amino acid concentration on
immobilised yeast cell metabolism and the production of
aroma and flavour compounds which significantly influence
the quality and consumer acceptance of the beer product.
For example, one of the main problems reported is the high
diacetyl concentration in beer produced by immobilised
cells which is detrimental to the beer taste;
more research is needed to measure the diffusivitics of
nutrient compounds that diffuse into the immobilisation
Corresponding author: Dr. A. Margaritis.
In this review, the fundamental aspects of immobilised yeast cell

research needed to expedite the commercialisation of this emerging
technology for continuous beer production are summarised.
Practical importance of cell immobilisation
Producing an alcoholic beverage such as beer using a
continuous fermentation system offers several important
advantages over the commonly used batch systems:
a more uniform product

less process supervision
very high bioreactor volumetric productivity (product
weight/unit time/unit bioreactor volume).
Immobilisation may be used as a tool to confine intact cells

to an inert carrier within a bioreactor. This "tool" will further
increase the efficiency of a continuous fermentation system by
providing5-55-90:
high cell densities per unit bioreactor volume which result

in very high fermentation rates
the reuse of the same biocatalysts (yeast cells) for extended

periods of time due to constant cell regeneration
a continuous process which may be operated beyond the

nominal washout rate
a discrete phase in which cells may be manipulated

easy separation of biocatalyst from the liquid phase where
the desired products are present thus minimising separation
costs
higher cell densities combined with operation at high

dilution rates, decreasing the risk of reactor shutdown due
to contamination
improved tolerance or protection of cells from product
inhibition
smaller bioreactor volumes which may lower capital costs.
Continuous fermentation using immobilised cells also allows

for efficient plant utilisation during peak sales periods. Con
tinuous smaller-scale high rate fermentation systems can be
stepped up to meet peak output when required. This is an
improvement over the current technology utilising large batch
fermenters which are useful during peak production times but
are under-utilised during off-times83. It was also observed that

immobilised cells are less susceptible than free cells to the
This document is provided compliments of the Institute of Brewing and Distilling

www.ibd.org.uk
Copyright - Journal of the Institute of Brewing
20
FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR BEER CONTINUOUS FERMENTATION
(J. Inst. Brew.
Continuous
fermentation using
immobilized
yeast cells
Changes in
cellular
activity
---
-^
Changes due to
Changes due to a
continuous mode
combination of
of reactor operation
yeast cell
immobilization and
immobilization
Changes due to
continuous mode of
reactor operation
111,
Optimization
of reactor
Optimization
of immobilization
design
Optimization
of fermentation
method
conditions
Selection
of appropriate
biocatalvst support
J
Beer of
acceptable flavour
tr
and
quality produced
Fie. I. Considerations for the implementation of a continuous fermentation system using immobilised yeast cells.
effects of certain inhibitory compounds, pH variations, and

nutrient depletion18-77101.
The process advantages offered by continuous fermentation
will have a pronounced impact on the brewing industry. The
traditional beer production process is operated in batch mode
using free cells and generally takes 5-7 days to complete
primary fermentation. Initial studies show that a continuous
immobilised cell system can reduce the time required for
primary fermentation to as little as one day89. Maturation using
immobilised cells coupled with a heat treatment process is
shortened from 5-21 days to 2 hours27.
Many obstacles must be overcome to bring this continuous
process to industrial fruition. Currently, the flavour of beer
produced using immobilised yeast cell technology does not
match that of beer produced using batch systems. Flavourmatching may not be critical for commercial success, but there
may be need for improvement in the flavour of beer produced
using immobilised yeast before this technology becomes viable
on an industrial scale51-96. Two important factors affecting the

formation of these flavour compounds are further described
below.
The first factor involves reactor design. Beer fermentation using
free cells is normally carried out using a batch reactor, in which the
wort is added at the beginning of the fermentation67. Immobilised
cell reactors are operated continuously with fresh wort added to

metabolised wort. This implies that the fermentation process will
need to shift from a constantly changing batch to a steady state
continuous operation. For example, during batch free cell
fermentation, the bioreactor is operated aerobically for the first
segment to allow yeast to utilise the oxygen for cell growth.
Dissolved oxygen levels are subsequently depleted and the reactor

operates anaerobically for the remainder of the fermentation. In a
continuous system, dissolved oxygen will reach a constant, steadystate concentration.
How, then, can the conditions found within the batch reactor
be mimicked in a continuous immobilised cell reactor for the
production of a well-balanced beer? A thorough understanding
of yeast batch growth kinetics is required. It is postulated that
by controlling dissolved oxygen and other substrate levels, the
same relative amount of yeast growth per unit of wort processed
can be obtained in both the batch and continuous systems
The second factor involves the effect of immobilisation on
mass transfer and yeast metabolism. Internal mass transfer
involves the transfer of substrates and products within the
carrier to the yeast cell, whereas external mass transfer refers to
the transfer of nutrients from the bulk medium to the carrier
surface. Alteration of internal and external mass transfer due to
immobilisation may affect the concentration of metabolites in
the immediate vicinity of the immobilised cells and may thus
change the yeast cell's metabolic state and ultimately the flavour
of the beer produced.
The implementation of a continuous fermentation system on
an industrial scale using immobilised yeast will require a team
of applied researchers with expertise in biochemical engineer
ing, microbiology, chemistry, and biochemistry. Project
considerations are outlined in Figure I.
Properties of immobilised cell systems

Whole-cell immobilisation is defined as the localisation of
intact cells to a defined region of space with the preservation of

www.ibd.org.uk
Vol. 104, 1998]
21
(b) Attachment or adsorption
(a) Entrapment within a matrix
to a preformed carrier
(d) Cells contained behind
(c) Self aggregation of
a barrier
cells (flocculation)
o
oo
o
o
o
oo
0
oo
oo
o
00
oo
o
Fig. 2. Basic methods of cell immobilisation.
catalytic activity55. An immobilised cell system is described by

Abbott1 to be any system in which microbial cells are confined
within a bioreactor, thus permitting their reuse. Methods of
immobilisation include physical entrapment within a porous
matrix, attachment or adsorption to a pre-formed carrier, self
aggregation by flocculation or crosslinking agents, and cells
contained behind a barrier. All of these methods have a similar
purpose: to retain high cell concentrations within the bioreactor, leading to increased volumetric productivity of the
system and lowered fermentation costs. Figure 2 illustrates
basic immobilisation techniques.
An immobilised cell system should have the following
properties for large scale industrial application90:
the carrier material must be nontoxic, readily available and

affordable;
the system should be efficient, easy to operate and give good
yields;
the carrier material should allow for high cell loading and
physical strength;
the cells should have a prolonged viability in the support.
bilising yeast cells using entrapment is a relatively simple

method and a high biomass concentration is facilitated84.
Table I lists some characteristics of alginate117 and carrageenan

gels'".
Some brewers have moved away from entrapment matrices
and are currently focusing on adsorption techniques for several
reasons. At present, gel entrapment matrices are not produced
economically on an industrial scale. Diffusion limitations due
to the gel matrix and high biomass loadings cause metabolite
concentration gradients within the polymer beads. This is a
TABLE I.
has been studied extensively. Polymeric beads are usually

spherical with diameters ranging from 0.3 to 3.0 mm. Immo
Aa/)/o-Carrjgecnan
Calcium Alginate
polymer from seaweeds

from the Phaeophycetie class
of algae
polymers from red seaweeds

such as Chondrus crispus
and Eucheuma cottonii
moderate concentrations
thermorcversible gel where

gelling temperature depends
on potassium ion
concentration
method for pilot scale

production of carrageenan
of calcium dictating agents
such as phosphates. 12DTA.
Physical entrapment within a porous matrix

The entrapment of immobilised cells within a porous
polymeric matrix such as calcium alginate1 >.".s5.97.<mu<m.i u.i im3o
or fcap/ra-carrageenan89-90101'27, along with some others44103,
Characteristics of calcium alginate and

kappa-carragtenixn gels
Mg:\ and K* ions will

disrupt alginatc gels
lack of methods for industrial

scale production of alginate
beads

www.ibd.org.uk
beads is currently available8'
22
EUNDAMENTALS Ol IMMOBILISED YEAST CELLS K)R BEER CONTINUOUS FERMENTATION
concern because the flavour components in beer are affected

when mass transfer limitations alter substrate availability or
create a buildup of inhibitory products such as ethanol and
carbon dioxide in the immobilisation microenvironment. How
ever, understanding mass transfer phenomena within entrap
ment matrices may allow one to simultaneously provide
differing conditions at the gel surface and in the bead centre.
One therefore could potentially mimic the aerobic and
anaerobic stages of yeast metabolism during a batch fer
mentation using this entrapped cell system in continuous
fermentation.
This concept of utilising the different microenvironments

within a gel entrapment matrix is being studied for wastewater
treatment systems31 by Dos Santos and colleagues who refer to
the "magic bead concept" in which the nitrifying bacterium
Nitrosomonas europaea and the denitrifier Paracocctis denitri-
ficans are co-immobilised in double-layer gel beads. It was

found that oxygen264761132, due to limitation of its uptake and
diffusion, rarely penetrates greater than a few hundred micro
meters into the gel bead when it is the limiting substrate. Thus,
in a single-stage continuous air lift reactor, the aerobic nitri
fication step was carried out in the outer layer while the
anaerobic denitrification took place in the bead core31. This
result may prove to be useful for achieving aerobic and
anaerobic stages during beer fermentation.
Another limitation of gel entrapment includes the loss of gel
mechanical integrity, by dissolution or by breakdown due to
abrasion, compression, or internal gas accumulation44-84.
Researchers have treated alginate gel beads with stabilising
agents such as sodium meta-periodate and glutaraldehyde13 or
Al'* "2 to improve gel mechanical strength.
Attachment or adsorption to a preformed carrier

Adsorption involves the reversible attachment of biomass to
a solid support mainly by electrostatic, ionic and hydrogen
bonding interactions. Because it is known that yeast cells have
a net negative surface charge, a positively charged support will
be most appropriate for immobilization12. There are actually
two types of whole-cell adsorptive immobilisation carriers: (a)
carriers that allow adsorption only onto external surfaces,
because pore sizes are too small to allow microorganisms to
penetrate inside, and (b) carriers with large enough pores to
allow adsorption onto internal surfaces102. Table II lists some
examples of preformed carrier materials that have been

suggested for immobilisation of yeast.
Biomass loading is generally lower in adsorbed cell systems
than those obtainable in gel entrapment matrices, but mass
transfer may be less limiting. Adsorptive matrices do not have
the additional gel diffusion barrier between the cells and the
bulk fermentation medium. Another advantage to using
adsorption matrices is the regenerability of the support.
The strength of cell attachment to an adsorption carrier
depends on both cell and matrix type. Since there is no barrier
between cells and the surrounding medium, these immo
bilisation matrices may have significant cell leakage. This is not
appropriate for processes requiring a cell-free effluent39.
Changes in environmental ionic strength, pH, temperature,
along with physical stresses such as agitation and abrasion can
induce cell desorption. Another limitation of adsorption celt
TABLE II.
Some carrier types used recently for immobilisation by

attachment or adsorption
Carrier Material
Reference
DEAE cellulose
porous glass
silicon carbide
volcanic rock
59,107
14.59.136.137
sponge
diatomaceous earth
wood blocks
gluten pellets
86.122.123
4,53
115
99
46
10
[J. Inst. Brew.
carriers is the possibility of nonspecific binding of charged
materials within the fermentation medium12.
The thickness of the biofilm attached to the surface of these
carriers ranges from a monolayer of cells to a layer of cells one

millimetre thick55. Flow rate of feed has the biggest effect on
the depth of the biofilm because it affects the turbulence of the
liquid flowing past the adsorbed biolayer. The kinetics of
biofilm formation are complex68 and therefore difficult to
control.
DEAE-cellulose (diethylaminoethyl) support uses ionic

attraction to immobilise yeast cells. It is an inert, nondissolving
cellulose matrix which has a nonuniform granular shape that
provides a large binding area for cell colonisation. Sinebrychoff
Brewery and Cultor Ltd. have worked with DEAE-cellulose to
immobilise yeast cells for use in continuous maturation or
secondary fermentation processes. They found that the
cellulose did not bind most common yeast contaminants and is
washable, reusable and regenerable with a long service life74.
Linko and Kronlof71 conducted a study in which they

compared Spezyme*, a DEAE-cellulose carrier obtained from
Cultor Oy, Finland, with two types of commercially available
porous silica beads used as immobilised cell carriers for the
continuous primary fermentation of beer. Tubular 1.6 L or

conical 5 L packed bed columns were used for the fer
mentations in this study. The silica beads compared were
Bioceramics" from Kirin Brewery in Japan, and Siran8
developed by Schott GmBH, Germany. The researchers
observed that when DEAE-cellulose particles were used, yeast
attached to the surface of the particles only, whereas glass
beads showed yeast growth partially on the surface and
partially inside the pores. The lowest amount of yeast growth
was observed using the glass Siran8 carriers. A beer flavour

profile most similar to traditionally brewed beers was obtained
using these immobilised yeast for primary fermentation. The
only difference found from traditionally brewed beer was
slightly elevated ester and diacetyl levels. To overcome these
limitations, genetically modified yeast with the gene encoding
ct-acetolactate decarboxylase could be used to reduce diacetyl
levels, while ester levels could be controlled by optimising
oxygen levels in the immobilised cell reactor. Of the two glass
carriers, Siran* was found to be superior over Bioceramics1

because of higher mechanical strength and density.
Cashin16 recently compared five different porous carriers in
terms of minimum fiuidisation velocity, biomass loading, and
durability. The carriers compared were the DEAE-cellulose
carrier Spezyme8, three sizes of Siran81 sintered glass beads, and

Hypermics8, a porous ceramic carrier from Kirin Breweries in
Japan. Hypermics8 was found to give the highest biomass
loading, but tended to float and was mechanically weak. The
DEAE-cellulose carrier, Spezyme, had the lowest minimum
fluidisation velocity, but it had to be chemically sterilised which
was more time consuming than the steam sterilisation that was
used for the silica-based carriers. This study found Siran8 to be
the preferred carrier for a fluidised bed system because it gave
the second lowest minimum fluidisation velocity and the second
highest biomass loading, while exhibiting none of the practical
limitations found with the other carriers16.
Meura-Delta, a Belgium-based brewery equipment supply
company, in collaboration with a research team at CERIA, a
Belgian university, integrated a novel immobilisation approach
for the continuous production of beer. They designed a twostage configuration for continuous primary fermentation using
an immobilised cell reactor in the first stage followed by a free
cell system. In the first reactor, multichannel porous silicon
carbide immobilisation rods were arranged in a loop con
figuration123. Open pores in the sintered silicon carbide rods
range in size from 40-60 um, which allow for rapid yeast
colonisation. Silicon carbide is inert, durable, re-usable, steam
sterilisable, and is suitable for CIP cleaning86. Rods with inter
nal channels rather than beads were used for immobilisation in

order to maximise mass transfer between the yeast cells and the
liquid wort flowing through the channels.

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Vol. 104, 1998]
Diatomaceous earth, trade named Celite", is traditionally

used in the brewing industry as a filter aid but it also possesses
desirable qualities for use as an immobilised cell carrier. Celite8

has a uniform composition, high mechanical and chemical
stability, and does not swell during fermentation33. However,
both yeast cells and Celite have negatively charged surfaces

under typical fermentation conditions, causing a relatively
weak interaction between biomass and immobilisation
support. Anderson el al.3 reported that a reduction of

cell leakage rate from Celite is needed for long-term,
large scale fermentations. One group successfully decreased
cell leakage by encasing the Celite8 in a layer of calcium

alginate33.
Zeta potential plays an important role in determining the
extent of cell adhesion to a carrier such as Celite8. When

brought into contact with a polar medium (e.g. fermentation
broth), most substances acquire a surface electric charge63. Ions
of opposing charge will migrate to this surface. The zeta
potential2 is defined as the electrical potential required to shear

off this opposingly charged liquid layer in contact with the
charged solid surface. Factors such as wort composition, yeast
strain, fermentation stage, fermentation conditions, and
medium pH will affect the zeta potential of the Celite8 and the
yeast cells. For example, one would want to select a pH that
would minimise the forces of repulsion between the carrier and
the cells to maximise biomass loading. However, pH also
affects yeast viability, so a compromise between maintaining
yeast viability and productivity while maximising cell loading is
required.
23
with very high flocculation activity may result in low concen
trations of active yeast cells due to diffusion limitation of

substrate to the cells inside the floes60.
Dominion Breweries Limited in New Zealand"-32 has been
successfully using a flocculent lager yeast strain for the con
tinuous fermentation of beer for almost 40 years. This brewery
uses a hold-up vessel followed by two stirred tank fermenters
for the continuous primary fermentation. After primary fer

mentation, the flocculent yeast are separated from the green
beer by gravity in a conical settler. Yeast is then recycled from
the first stirred tank fermenter and the conical settler back into
the hold-up vessel to achieve more precise control of the rate of
fermentation. Labatt Brewing Company Limited in Canada
was also active in earlier work on continuous primary
fermentation of beer37.
Researchers at VTT Biotechnology and Food Research in

Finland13 compared the behaviour of strongly and weakly
flocculent yeast cells immobilised on two different carriers,
Siran (sintered glass) and Spezyme8 (DEAE cellulose) for the
primary fermentation of beer. They found that the strongly
flocculent yeast cells accumulated on the immobilisation
carriers faster and consequently the desired wort attenuation
was reached more quickly than the weakly flocculent yeast
strain. In addition, they noted that a genetically modified
superflocculent yeast strain that was disregarded because it
flocculated too early for use in conventional batch ferment
ations, may prove to be very useful in continuous
fermentations.
Mulchandani el al.93 studied the effect of zeta potential on
the adsorption efficiency of cells onto solid supports including
Celite8. They showed that when the yeast-like fungus Aureo-
basidium pullulans was grown in different pH conditions,

differences in the charge distribution on the cell walls were
found. A. pullulans cells exhibited a net negative charge in
solutions above pH 2.S-3.0. This was thought to be due to
differences in cell wall composition caused by the differing
media conditions. They also found that Celite8 had a negative
zeta potential with maximum negativity at pH 6 and minimum
at pH 2. Since A. pullulans did not synthesise pullulan at pH 2,
they cultured the cells in contact with Celite8 at pH 2 for 72
hours to maximise cell loading and minimise electrostatic
repulsion, and then transferred the immobilised cells to pH 5.5
production medium for optimal fermentation conditions.
Nguyen and Shieh" used Celite8 to immobilise yeast for the
continuous production of ethanol in a fluidised bed reactor.

They obtained only 71% of the theoretical ethanol yield and
this was thought to be due to extended mean cell residence time,
inhibitory effect of high dissolved carbon dioxide concen
trations, and intermittent pH variations in the reactor.
Cells contained behind a barrier

In this type of immobilisation, cells may be retained by the
membrane in soluble form or the cells may be attached to the
surface and/or entrapped within the membrane matrix38. A

barrier formed by the liquid-liquid interface between two
immiscible fluids can also be used for immobilization55. Cell

retention behind a membrane barrier has not been widely used
to immobilise yeast cells for the continuous production of beer,

but there are several groups who have investigated their use for
continuous ethanol production50628081*4. Kyung and

Gerhardt62 investigated continuous ethanol production using
Saccharomyces cerevisiae immobilised in a membranecontained fermenter. Microporous dialysis membranes

provided a barrier which retained the yeast cells in the
fermenter and simultaneously allowed inhibitory fermentation
products such as ethanol to be continuously removed in order
to boost reactor productivity. The problem of membrane
plugging must be overcome for this immobilisation mode to
become a practical industrial-scale method for continuous
ethanol production in the future.
Self aggregation of cells
The formation of cell aggregates by flocculation, shown in

Figure 2(c), is the most simple and least expensive immo
bilisation method, but the least predictable. Stewart118 defines
flocculation as "the formation of an open agglomeration that
relies upon molecules acting as bridges between separate
particles". The natural flocculating ability of yeast cells may be
exploited106 or crosslinkers may be added to bolster the process
of aggregation for cells that do not do so naturally. The control

of cell aggregation is important to maximise bioreactor effi
ciency. Factors which influence the natural flocculation
characteristics of brewer's yeast strains include the genetic
make-up of the strain, the cell wall structure and surface
charge, the growth phase, incubation temperature, medium pH,
cation composition of the medium, and other wort com
ponents"8.
Weak flocculation activity will result in slow cell sediment
ation rates which may cause cells to be "washed out" of the
bioreactor with the fermentation medium. Consequently, a low
cell concentration is maintained in the reactor resulting in
insufficient fermentation rates. On the other hand, larger floes
Immobilised Cell Viability and Vitality
Yeast viability is defined as the percentage of live cells in a

sample, and yeast vitality is a measure of yeast activity or
fermentation performance7-66121. Lentini66, in his compre
hensive 1993 review, specifically describes yeast vitality as "a
function of the total cell viability and the physiological state of
the viable cell population". In order to efficiently and con
sistently produce quality beer using an immobilised cell system,
one must understand the impact of such a system on yeast
physiology and growth.
Many criteria are used to assess yeast cell viability and
vitality. Consequently, the perceived viability of a yeast sample
may vary depending on the criteria selected. It is often
beneficial to monitor a combination of parameters to gain a
more complete understanding of a yeast's physiological state. In
the section below a number of methods of studying yeast cell
viability and vitality are summarised while in later sections
methods for examining the patterns of yeast cell growth within
immobilisation matrices are described.

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24
FUNDAMENTALS Ol: IMMOBILISED YEAST CELLS FOR BEER CONTINUOUS FERMENTATION
Methods for measurement of immobilised cell viability and vitality

Use ofspecific dyesfor assessing cell viability and vitality
Methylene blue is the dye most commonly used for yeast cell
viability staining. Viable cells are able to reduce this stain
making it colourless, whereas non-viable cells are unable to
reduce the stain rendering them a deep blue-purple shade66. A
viable yeast cell count may be completed using a hemocytometer and a light microscope in less than ten minutes.
When buffered and supplemented adequately, methylene blue
has no effect on yeast cell viability64. Methylene blue staining is
considered to be an accurate method only when yeast cell
viability is greater than 90%88. Some examples of methylene
blue solutions include Fink-Kuhles buffered methylene blue
(ASBC international method)120, methylene blue aqueous
solution120, and standard methylene blue solution64. Methylene
blue staining is a good method for assessing the viability of

highly concentrated yeast such as that found in immobilisation
matrices64. Other brightneld stains which have been used to
monitor yeast cell viability include Aniline Blue88 and Crystal
Violet35.
There are also many fluorescent stains designed to assess
yeast cell viability and vitality. When fluorescent stains are used
in conjunction with confocal microscopy9"110 or flow cytometry28.2.4o.48.73 valuable information may be obtained on yeast
cell growth and metabolic state. Kasten56 gives a comprehensive

review of stains available and fluorescence microscopy
techniques.
Capacitance
The principle of this method is that the application of a radio

frequency to a viable cell results in a charge buildup within the
membrane, and a capacitance is generated66. Nonviable cells
are unable to generate this capacitance. A linear correlation has
been demonstrated between capacitance and viable yeast
biomass6.
Kronlof58 recently showed that capacitance probes are
suitable for monitoring viable yeast biomass in both freely

suspended and immobilised cell systems. Yeast cells were
immobilised on porous glass carriers (SiranB) and DEAE-
cellulose (Spezyme8). Kronlof pointed out that although this
method correlates well with methods such as staining for the

detection of viable cells, it does not provide any additional
information about the yeast cell's metabolic state. Therefore
this method is best used in conjunction with other methods for
measuring yeast vitality such as nicotinamide adenine
dinucleotide (NADH) or adenosine triphosphate (ATP).
The power of reproduction as a viability indicator

Standard plate count measures the ability of yeast cells to
proliferate and form colonies on nutrient agar. It generally
takes three days for visible colonies to form and viability is
assessed by counting the number of colony forming units
(CFU). Care must be taken when using this method on very
flocculent yeast66.
Yeast viability by slide culture is also based on the ability of
yeast cells to proliferate. A drop of yeast culture is placed on a
film of nutrient agar and after approximately 18 hours of
incubation the formation of microcolonies is observed under

the microscope. Cells which have given rise to microcolonies are
considered viable whereas single cells that have not formed
microcolonies are considered non-viable120. It is relatively less
time-consuming than standard plate counts but still much
slower than the staining techniques. An advantage of the slide
culture method is that it is accurate at relatively low yeast cell
viabilities.
Viability and vitality methods based on cell metabolic state
Adenosine Triphosphate (ATP)
ATP (adenosine 5' triphosphate) is a good indicator of cell
viability since it is present in all living cells and is degraded
when cells die. ATP allows for the detection of viable cells in a
(J. Inst. Brew.
short amount of time (1015 minutes) when compared with

traditional plating techniques. Since the quantity of ATP per
cell does not vary significantly for a given strain (but varies
between strains), it can be inferred that the amount of ATP
present in a biomass sample is proportional to the number of
viable cells present of that cell type. Another advantage of using

ATP as a viability indicator is that the amount of ATP present
in a cell is roughly independent of the growth rate. Therefore a
correlation between ATP concentration and the amount of

viable cell mass can be made41.
The "firefly assay" is used to determine the quantity of ATP
present in a biomass sample. This invasive method involves
extracting the ATP from the cells and reacting it with firefly
luciferin in a two-step reaction which is catalysed by the enzyme
firefly luciferase. Light is one of the products of this reaction
and a stoichiometric relationship exists between the amount of
light produced and the quantity of ATP in the biomass sample.
Extractants used to release intracellular ATP include boiling in
buffers such as tris-EDTA, cationic detergents, acids, and
organic compounds such as acetone and ethanol. The reactions
taking place are summarised below:
Luciferin+Luciferase+ATP+Mg2* -> (Luciferin-LuciferaseAMP) + Pyrophosphate
(Luciferin-Luciferase-AMP)+O: -Oxyluciferin+Luciferase
+CO2+AMP+Light
ATP concentrations as low as 10"12 g in 100 u.1 volume may be

detected using the firefly method41.
Gikas and Livingston41 used ATP ioluminescence to charac
terise biomass viability in freely suspended and immobilised
cell bioreactors. They concluded that the kinetic data obtained
for freely suspended cells could not adequately describe
immobilised biomass kinetics because immobilised cells may be
in significantly different metabolic states than freely suspended
cells. They questioned whether differences in ATP levels
between freely suspended and immobilised cells reflect differ
ences in the biomass viable fraction or differences in metabolic
state.
NADH Fluorosensor
NADH has successfully been used as a noninvasive, on-line
method of monitoring freely suspended and immobilised yeast
cell metabolism30-69.Viable cells contain nicotinamide adenine
dinucleotide (NAD) coenzyme whereas non-viable cells or

spores normally lose their NAD17. The oxidised form, NAD*
is used by dehydrogenases to accept electrons from their
substrates. For example, in the enzymatic conversion of malate
to oxaioacetate in the presence of oxygen, malate dehydrogenase (MDE) first binds to NAD+ to form a complex of
MDE-NAD*. This complex then combines with malate to
form a ternary complex MDE-NAD+-malate. From here,
NADH, H+ ion, and oxaioacetate are released:
malate+NAD* <- oxaloacetate+NADH+H+
(oxidised)
(reduced)
The reduced form, NADH, fluoresces while the oxidised
form, NAD*, does not. NADH is strongly fluorescent with an

emission maximum at 460 nm wavelength. The total NAD is
the sum of NADH and NAD*. The reducing state is denned as
the ratio of the reduced form to the total amount of NAD:
R=[NADH] / ([NAD*]+[NADH])
Cell metabolic state determines the reducing state which will
remain constant unless there is a shift in metabolism. Thus, the
influence of substrates such as oxygen on the reducing state
may be predicted. When oxygen is in excess, the reducing state
approaches zero because NADH is easily oxidised to form
NAD* and H;O, and when there is a lack of oxygen available
to the cells, R approaches one. The concentration of [NADH]
as well as the intensity of the fluorescent signal are influenced
by the number of viable cells, the reducing state of the cells and
environmental effects. Measuring NADH has an advantage

www.ibd.org.uk
Vol. 104, 1998]
over monitoring dissolved oxygen or pH because it directly

measures, in real time, events occurring within the cell rather
than changes outside the cell environment.
Specific Oxygen Uptake Rate (BRF Yeast Vitality Test)
Researchers at Brewing Research International (BRI)
developed a method to determine the vitality of pitching yeast
by measuring its specific oxygen uptake rate24. Various

groups24-54-82 have shown a correlation between oxygen uptake
rate of yeast and fermentation performance if yeast viability is
less than 90%. The method involves the pitching of yeast into
aerated media and the measurement of the oxygen uptake rate
for one hour M.
A reduced oxygen uptake rate parallels other yeast changes

such as the reduction in yeast lipids, glycogen, acidification
power test value, and yeast viability49. Under these conditions,
oxygen uptake rate correlates well with yeast fermentation
performance. However, Wheatcroft et a/.129 found that oxygen
uptake rate did not correlate well with fermentation per
formance when yeast had been previously acid-washed. Even
though acid-washed yeast showed decreased specific oxygen
uptake rates, they actually showed better fermentation per
formance than non-acid-washed yeast.
The biocatalytic activity of the nitrogen-fixing bacterium,
Vibrio natriegens (Baneckea natriegens), immobilised on
Celite8, was studied by Gikas and Livingston42 using specific

oxygen uptake rate [mg (O2) g~' (dry biomass) h"1]. Specific
oxygen uptake rates for immobilised and free cells in a three
phase air lift reactor were compared and it was found that the
uptake rates were significantly lower for the immobilised cells,
implying a lower biomass activity in the immobilised cell
system.
Acidification Power
The acidification power test developed by Opekarova and
Siglcr104 measures the drop in extracellular pH of a suspension

of yeast cells after the addition of glucose. This method is useful
for detecting large differences in yeast metabolic activity. The
yeast acidification power test requires extensive yeast washing
and multiple sample points which makes it impossible to use

this test on immobilised cells in situ.
Intracellular pH (ICP) Method
The ICP method uses a pH-sensitive fluorescent reagent to
measure the intracellular pH of individual cells and cell mass.
It was found that the intracellular pH of more active yeast cells
does not decrease, even if the extracellular pH is low, whereas

the intracellular pH of less active cells actually decreases under
low extracellular pH conditions49. This test may be able to
detect more subtle changes in yeast cell vitality than acidi
fication power test49.
Measurement of Yeast Vitality by Stress Response
As stated earlier, vitality may be considered a measure of
yeast activity or fermentation performance. It has also been
defined as the ability of cells to endure or overcome stress7.
Therefore one could relate vitality to the response of yeast cells
to stresses such as ethanol, heat shock, and high salt concen
trations. Methylene blue, fluorescent dyes7, and standard plate
counts101 may be used to assess the ability of cells to remain

viable after being subjected to a given stress.
Norton et al.im assessed the ethanol and heat tolerance of
brewers' yeast entrapped in K<arrageenan gel. Free and immo
bilised Saccharomyces cerevisiae cells were exposed to ethanol

concentrations of 0, 18%, and 19.4% (v/v) for a given length of
time and viability was then measured using standard plate
counts. Heat tolerance was measured by incubating yeast cells
at 48C for various lengths of time with periodic shaking. Cell
viablity following heat shock was also measured using standard
plate counts. It was found that there was a significant increase
in yeast survival against ethanol for immobilised cells as com
pared to free cells, but no distinct difference in heat resistance
25
was noted. The study concluded that the carrageenan gel matrix
provided protection to the entrapped yeast cells against ethanol.
When entrapped cells were released from the carrageenan
matrix, their ethanol tolerance returned to that of free cells.
Magnesium Release Test (MRT)
The Magnesium Release Test92 is based on the observation
that low molecular weight species such as magnesium, potas

sium, and phosphate ions are released by yeast immediately
following inoculation into glucose containing medium. Trials
performed on Saccharomyces cerevisiae showed that cells which
released greater quantities of magnesium immediately after
inoculation into high gravity (16P) wort had higher vitality and
fermentation performance than yeast which released lower

amounts of magnesium. Subsequent fermentations performed
using the more vital yeast had shorter lag phases, higher cell
counts, higher end ethanol concentrations, and lower diacetyl
levels. The magnesium release test (MRT) takes less than fifteen
minutes to perform and it uses a commercially available
magnesium test kit (Sigma) which allows the quantitative
colourimetric measurement of magnesium in wort before and
immediately after yeast inoculation.
Methods for determining cell distribution and growth patterns
within immobilised cell matrices
Method ofsuccessive dissolution of layers from gel beads

When calcium alginate is used to entrap yeast cells, calcium
chelators such as phosphates and citrates may be used to
dissolve "shells" of alginate and biomass from the beads18-43"3.

By measuring the concentration and viability of cells inside
each shell, information may be obtained about the spatial
distribution of viable cells within the entrapment matrix.
Usually shells obtained using this method have a thickness

greater than SO um. When biomass profiles are steep within the
beads, greater resolution is required to obtain an accurate
picture of yeast cell distribution. Released biomass from each

shell may be assessed for viability by using methylene blue
staining, fluorescent dyes or by plating. Problems with this
method include uniformity of dissolution of matrix, finding a
suitable nontoxic solution for dissolving the entrapment
matrix, and overall accuracy limitations.
Recently, Parascandola and Alteriis"0 studied the spatial
growth patterns of Saccharomyces cerevisiae cells entrapped in

starch-hardened gelatin discs. They used a multi-step digestion
method to collect cells belonging to five different layers of the
discs. Diluted trypsin was used to release the cells from the
gelatin and yeast viability from each of the layers was then
measured by staining the cells with fluorescent stain

Rhodamine 123 and examining them under a confocal
microscope.
Physical sectioning of immobilised cell matrix

A biomass distribution may be obtained by slicing matrices
containing yeast cells. To avoid matrix compression and to
obtain an accurate distribution, the sample is often dried and
embedded into a resin, or frozen. Shrinkage has been observed
during this preparation134. Beads may then be sliced using a
microtome or razor blade and cell concentration within each

section is obtained using image analysis and the appropriate
viability or vitality stains. However, this invasive slicing method
may cause alterations in cell distribution and other aber
rations9.
NMR imaging
Nuclear magnetic resonance imaging (NMRI) is a noninvasive technique to study spatially chemical and physical
properties of small samples68. Lewandowski et a/.68 used

NMRI to better understand the dynamics of biofilm growth
and external liquid film flow velocity distribution. These studies

are of importance since the extent of biofilm growth is in
fluenced by the local chemical environment and fluid flow

www.ibd.org.uk
26
[J. Inst. Brew.
conditions. Substrates must also be transferred through the

biofilm-bulk medium interface to reach the cells. In addition,
researchers were able to gather information on velocity distri
bution using NMRI near microbially colonised surfaces with

an imaging time of 8-16 minutes.
The use ofconfocal microscopyfor the determination of viable

cell distribution within porous microcarriers
Confocal microscopy is a valuable tool for studying viability
of immobilised cells because of its unique ability to take optical

sections of three dimensional objects in a noninvasive manner.
This depth discrimination property allows only the region of
the object that lies close to the focal plane to be imaged
efficiently87. Using confocal microscopy, one could potentially
DULKUGUtO
PHASE
obtain three-dimensional images of yeast cell growth and
viability distribution within porous microcarriers without
actually disrupting the carrier matrix.
In an elegant study by Lim et al.10, the spatial distribution of

mammalian cells grown on modified macroporous gelatin
microcarriers was studied using confocal microscopy. Optical
sectioning employing confocal microscopy and ethidium
bromide for cell staining allowed the researchers to visualise the
distribution of cells at different stages of growth within the
Fig. 3. Schematic of the transfer of substrate from the bulk medium to

an entrapped yeast cell.
macroporous microcarriers. They found that cells initially only
attach to the external surface or within the external cavities of

the macroporous microcarriers. Subsequently, there was a slow
migration of cells toward the interior of the beads. Confocal
microscopy revealed that penetration and growth of cells to
within the outer half of the radius of the microcarriers
accounted for 87% of surface utilisation efficiency whereas the
central core, comprising the inner half radius of the spherical
particles, contributed only 13% of the total available void
fraction.
Bancel and Hu9 used confocal laser scanning microscopy to
examine the distribution of viable hybridoma cells within
macroporous microcarriers. Confocal microscopy was used to
avoid the potential artifacts that may be generated using
physical sectioning. A cationic fluorescent vital stain (dialkyl

indocarbocyanine) was used for the hybridoma cells and a
second stain (fluorescein isothiocyanate) with a different
emission wavelength was used to render the microcarriers
uniformly fluorescent. The confocal microscope allowed the
researchers to take serial optical sections of the immobilised
cells and the carrier in a noninvasive manner with the
maintenance of cell viability. Their results showed that the
initial spatial distribution of cells within the carrier affects the
kinetics of cell growth and the maximum biomass loading
possible. These factors will have a significant effect on
immobilised cell bioreactor performance.
Mass Transfer Characteristics of Immobilised Cells
Changes in the kinetic behaviour of yeast cells upon

immobilisation may be attributed to either alteration of
physiological/metabolic properties of the cells, or alteration of
the local microenvironment in the immediate vicinity of the
immobilised cells. In the second case, the microenvironment
may be described in terms of solute partitioning effects between
the bulk liquid phase and the solid immobilisation matrix,
external film mass transfer resistance and internal mass transfer
resistance91. These terms may be quantified by the solute parti
tioning coefficient (Kp), the liquid film mass transfer coefficient
(Kl), and the effective solute diffusivity within the immobilisa
tion matrix (De) respectively. The above parameters need to be
quantified in order to determine overall effectiveness factors.
Figure 3 is a schematic of the transfer of substrate from the

bulk medium to an entrapped yeast cell. Substrate must travel
through the bulk medium by diffusion and convection, through
the external liquid film surrounding the bead containing
immobilised cells, through the liquid-solid interface, through
the liquid within the solid gel phase by diffusion, through
resistance caused by microcolony formation, and finally into
the yeast cell where the reactions take place. Mass transfer
occurs by diffusion only within and close to the solid matrix
phase whereas, in the bulk liquid phase, transfer occurs by both
diffusion and convection, leading to concentration gradients
between the two phases.
"Effectiveness factor" is defined as the ratio between the

observed reaction rate and the rate of reaction in the absence of
mass transfer resistance. If the effectiveness factor is less than
one, substrate concentration on the gel surface or within the
matrix is lower than the concentration in the bulk phase. The
Thiele Modulus may be defined as the ratio between the
reaction rate and the internal diffusion rate or, the rate that
substrate is consumed relative to the rate at which it is supplied
by the diffusion process. In other words, a high Thiele Modulus
indicates that the system is mass transfer limited whereas a
lower number indicates that the system is kinetically controlled.
A Thiele Modulus of less than ten is desirable to minimise
substrate concentration gradients. For more detailed dis
cussions of mass transfer in immobilised cell systems, com
prehensive reviews are presented by Karel et al.i$, and more
recently by Westrin and Axelsson128.
The study of the transfer of oxygen in beer fermentation
using immobilised yeast cells is especially important, since a
lack of oxygen due to mass transfer limitations may force yeast
cells to switch from aerobic to anaerobic metabolism, signi
ficantly altering the production of beer flavour compounds.
Internal mass transfer characteristics
Internal mass transfer involves the transfer of substrates and
products within the carrier or solid phase100, which is especially
relevant for entrapped cell systems. The effective solute
diffusivity, Dc, is used to quantify internal mass transfer rates.
Researchers have found that the cell mass is mainly confined
to a biolayer at the periphery of entrapment matrices due to a
lack of substrate at the centre of the beads. This occurs because
substrate concentration gradients are formed, where each point
of a concentration profile reflects a local equilibrium between
supply and consumption68. Internal mass transfer can be
optimised by adjusting the immobilisation matrix size, texture

and porosity. Decreasing bead diameter is a good way to
maximise internal mass transfer. A balance must be made so
that beads are large enough for easy separation from the bulk
phase and small enough to maximise mass transfer. Oxygen is
an essential substrate for yeast cell growth, yet it is only
required in relatively small quantities in brewery fermentations.
Therefore oxygen availability is often limited by mass transfer
restrictions caused not only by the matrix itself, but also by

yeast cells themselves in highly colonised beads.

www.ibd.org.uk
Vol. 104, 1998]
27
Unsteady State Technique for Measuring

Solute Diffusivities
= f(r,t)
CL = f(t)
Merchant et a/.91 developed an unsteady state technique for
measuring effective solute diffusivities within an entrapment

matrix used for whole-cell immobilisation. This method has
advantages over other methods in that it does not rely on the
mechanical stability or the geometry of the entrapment matrix,
and it may be carried out relatively quickly91.
The following assumptions were made in order to derive the

equations needed to determine solute diffusivities:
dr
(1) diffusion of solute occurs radially outward and there is no

concentration variation with angular position;
(2) the physical properties of the sphere are constant;
(3) there is negligible external film mass transfer resistance;
BULK
LIQUID
PHASE
and
(4) at time, t=0, the solute is uniformly distributed throughout
the sphere.
Using the spherical coordinate system shown in Figure 4, a

mass balance for unsteady-state diffusion of solute is described
by the following equation:
Fig. 4. Spherical coordinate system Tor diffusion and reaction of a

solute for a sphere immersed in a finite liquid volume.
A spherical coordinate system used to describe diffusion and

reaction in a spherical shell of thickness "dr" for a single
spherical carrier particle is depicted in Figure 4. Effective
diffusivity, Dc, is defined by Fick's law:
J=-DcaCs/dr
where J is the flux per unit of matrix area, Cs is the amount of
solute per unit liquid volume in the gel phase, and r is the radial
coordinate in the sphere105.
Methods for detection of internal mass transfer characteristics
Use of Microelectrodes
Microelectrodes allow for the measurement of concen
aCs / dt=De [(a2Cs / dr2)+(23Cs / rar)]

To solve this equation, initial and boundary conditions are
needed. Therefore, for a sphere containing solute immersed in
a finite liquid volume initially free of solute, initial and
boundary conditions are listed below.
t=0,
t=o,
t>0,
t>0,
0<r<R,
r>R,
r=0,
r=R,
Cs=constant
CL=0
dCs / (r=0
vL(acL/at)=
KPAsDe(aCs/ar)|r=R
I.C.I
I.C. 2
B.C. 1
B.C. 2
By monitoring the change in concentration of solute in the

liquid phase as solute diffuses out of the sphere, and by solving
the above equation using the aforementioned conditions, one
may evaluate D. Radiotracer techniques may be used to follow
the change in solute concentration since the method requires
very small liquid volumes.
Other researchers34 have measured effective solute diffusi
trations directly inside biofilms. Being able to do this is

important because diffusion gradients within biofilms will
influence their internal structure. For yeast cells entrapped
within calcium alginate or carrageenan gels, the measurement
of substrate concentration profiles using microelectrodes
provides a direct method for characterising the microenvironment of the cells inside the matrix. Microelectrodes have
been developed for pH, oxygen, capacitance, ammonia, and
vities using a similar method except that gel beads are placed in
a buffered aqueous solution in a well-stirred tank. The
diffusion of a given solute into or out of the gel beads is
measured by monitoring the concentration of the bulk liquid
phase. High levels of agititation are required to avoid external
mass transfer resistance at the bead surface. This method is a
relatively simple way to gather data for the calculation of
effective solute diffusivities in immobilised cell systems.
of diffusion properties of components within immobilisation

matrices, the microelectrode tip should be less than 10 urn in
diameter so that it penetrates the biocatalyst without physically
late effective diffusivities of selected simple sugars and organic

acids in calcium alginate gels with and without entrapped
Lactobacillus helveticus bacteria. The mathematical model used
allowed for the calculation of effective diffusivities and
accounted for solution sampling and external film resistance.
Their experiments showed that lower pH (4.5 vs. 5.5 and 6.5),
higher alginate concentration, and lower temperatures each
resulted in lower diffusion rates for both lactose and lactic acid.
Some difficulties are encountered when using micro

electrodes since measurements may be influenced by calibration
conditions, other metabolites, mechanical resistance and
properties of the matrix, and puncture technique. The latter
point refers to the method of insertion of the microelectrode
into the biocatalyst bead. Signal artifacts may occur due to the
sudden breakthrough of the electrode through compressed
material such as the immobilisation matrix95. Flow conditions
in the reactor and in the flow chamber where measurements are
performed must be kept the same for accurate readings. It is
also difficult to determine the exact position of the microelectrode visually and thus spatial resolution is insufficient,
especially since immobilised cell matrices tend to have large
substrate concentration gradients95.
Thin Disk Method for Determining Effective

This method utilises a two compartment
glucose among others21-45-58-95121125. To be useful for the study
changing its configuration68. Response time of the electrode is

also important, especially in dynamic processes using im
mobilised or flocculating cells. A response time of 0.1 seconds
is desirable for these types of systems but an electrode of this
type is extremely fragile and has a short useful life and therefore
is not suitable for routine use15.
Oyaas et al.m also used the multiple bead method to calcu
diaphragm
diffusion cell, where each equal-volume chamber contains a
well-stirred solution placed in contact with either surface of a
thin gel. Diffusion occurs from higher to lower concentration
or from "source" to "sink"52. Concentration-time measure

ments are used to determine the effective diffusivity.
Korgel et a!.51 measured effective diffusivities of galactose in
calcium alginate gel occupied by living Zymomonas mobilis

bacterial cells using this method. The unmetabolizable
galactose was chosen because it allowed for effective diffusivity
measurements independent of consumption in a gel system
containing immobilised living bacterial cells. A thin disc of

www.ibd.org.uk
28
FUNDAMENTALS OF IMMOBILISED YEAST CELLS I OR BEER CONTINUOUS FERMENTATION
TABLK III.
[J. Inst. Brew.
Some advantages and disadvantages of reactors commonly used with immobilised cell systems
Reactor Type
Packed bed reactors
Fluidised bed reactors
Advantages
Disadvantages
simple design
low energy requirements
poor liquid mixing
accumulation of gas pockets

difficulty with scale up
plugging
larger bead sizes needed to maintain
pressure drop
compaction of carrier material limits
reactor height
low shear
foaming
more complex scale up than packed bed
industrial scale systems

arc currently available
benefits from economies

of scale
optimal liquid mixing
heat and mass transfer
easy separation of products
Gas lift draft tube

reactors
lower shear than fluidised beds

benefits from economies
of scale
foaming
more complex scale up than packed bed
industrial scale systems are

currently available
optimal liquid mixing,
heat and mass transfer
reduced pumping costs
compared with fluidised
bed reactor
Loop reactors
easy separation of products
large surface area/volume

easy scale up due to
modular design
industrial scale systems in
developmental stage
fabric-supported calcium alginate gel was placed in a diffusion

cell to measure the rate of diffusion of galactose from source to
sink. Z. mobilis cell concentrations ranged from 0 to ISO g dry
wt/L of gel. Effective diffusivities were found to decline with
increasing entrapped biomass concentration. They also con
cluded that the random pore model of Wakao and Smith126 is
a good predictor of sugar effective diffusivities in gel
entrapment systems.
Cylinder Method of Determining Effective

The cylindrical-shaped gel is initially free of solute, and the

concentrations of source and sink are maintained at C\ and C:
respectively. In this method, the gel is much thicker than the gel
used in the thin disk diffusion-cell. The solute diffuses through
the gel from source to sink in a given time, t. The gel is then
immediately sliced into thin disk-shaped slices. The solute
concentration in each slice is measured, giving concentrationdistance data to be collected within the gel cylinder. Effective
diffusivity, Dc, may then be calculated using these data30.
Colony-expansion Model
When the dynamics of the growth of immobilised cells were
initially studied qualitatively, growth of biomass was described
as expanding at similar rates throughout the beads following
start-up. With time, biomass eventually becomes confined
mainly to areas near the gel surface due to diffusion limitations.
Using a conventional pseudo-homogeneous growth model to
predict cell growth within gel entrapment matrices led to an
over-estimation of macroscopic substrate consumption rates.
This is because diffusion limitations occurring over larger
micro-colonies are not accounted for in this model.
Wijffels el a/.13' theorise that in order to incorporate
diffusion limitation across micro-colonies in a dynamic growth

model, it is necessary to consider biomass growth as expanding
complex design
plugging
lacks economies of scale
micro-colonies, rather than as a homogeneous increase of

biomass in spherical "shells" within the gel beads.When
diffusion limitation over micro-colonies is incorporated into
the dynamics of the immobilised cell system, the colonyexpansion model predicts that inoculum size will affect
substrate consumption rates. At high initial biomass concen
trations, numerous small micro-colonies appear, causing minor
diffusion limitations. However, at low initial biomass concen
trations, fewer but bigger colonies form due to significant
diffusion limitations. This resulted in increased macroscopic
substrate consumption rates in the system containing beads
with smaller microcolonies. These results were confirmed
experimentally in a continuous air lift loop reactor"1.
External mass transfer characteristics

External mass transfer refers to the transfer of nutrients from
the bulk medium to the immobilised yeast cell carrier and is
quantified using the liquid film mass transfer coefficient, KL.
Reactor design factors such as reactor type, size and operating
conditions will influence external mass transfer in immobilised
cell systems. Surface contact between the biocatalyst (immo
bilised yeast cell) and the growth medium (wort) should be
maximised by optimising liquid mixing. Sufficient agitation is
needed so that the thickness of the liquid film surrounding each
carrier particle and consequently external mass transfer resist
ance is minimised, thus facilitating the transfer of species from
the bulk medium to the carrier. The rate of shear on the
particles in a reactor will increase as agitation is increased.
However, for a given agitation rate, different reactor designs
may differ in rate of shear. If shear rates are too high, biomass
may be lost from adsorption matrices, immobilised cell
particles may break, and cell aggregates may be disrupted. It
therefore becomes important to design a reactor which provides
an adequate amount of agitation at a reasonable shear rate.

www.ibd.org.uk
Vol. 104, 1998]
FUNDAMENTALS Op IMMOBILISED YEAST CELLS FOR BEER CONTINUOUS FERMENTATION
The reactors most commonly used for the continuous

production of beer with immobilised cells include packed bed,
fluidised bed, gas lift draft tube, and loop reactors. Packed bed
reactors are the simplest type of stationary reactor. The fer
mentation medium is passed either upward or downward
through the reactor which is packed with immobilised yeast.
When designing and operating a packed bed reactor, one must
ensure that there is no channelling of flow and no reactor
plugging. Even if these pitfalls are successfully avoided, it is
difficult to ensure that nutrients are distributed evenly to all
immobilised cells in the bed due to the lack of mixing and mass
transfer limitations inherent to this type of design109.

In fluidised bed reactors, beads with immobilised cells are
suspended in the upward flow of liquid medium or the

combined liquid and air flow. Good mixing and mass transfer
properties are attractive features of fluidised bed systems.
However, it is important to consider the density of immobilised
cell beads when designing a fluidised bed system because even
if pumping costs are relatively low, a low density difference
between the solid beads and the liquid medium will not result
in high mass transfer rates23.
Gas lift draft tube systems use gas to circulate fermenter

contents internally using a draft tube concentrically located
inside a columnar reactor. These reactors provide good mixing
and aeration, low power consumption, and are of simple con
struction. This makes gas lift systems very attractive for large
scale industrial operations.
The loop bioreactor design123 refers specifically to the
immobilised cell system used for the primary fermentation of

beer developed by Meura-Delta86. Yeast cells are immobilised
on porous rod matrices containing numerous internal channels.
In this design, the fermentation medium flows in a loop from the
bottom of the fermenter, through both the internal channels and
around the matrices for contact with the immobilised yeast, to
the top of the reactor with external recycle. Particle abrasion is
minimised and scale up is simple with this type of design. Table
III summarises some of the advantages and disadvantages of
packed bed, fluidised bed, gas lift draft tube, and loop reactors.
The superior mixing and surface exposure for mass transfer
found in fluidised bed and gas lift draft tube systems make these
types of reactors the most promising for use with industrial
scale immobilised yeast cell fermentation systems55-84.
Conclusions
Immobilised yeast cell technology is an innovation that may
revolutionise the way breweries operate. Using this technology
beer is produced continuously, leading to significant reductions
in production time, inventory, floor space, and product
variation. Over the last ten years or so, publications on this
topic have been on the rise, indicating that immobilised cell
technology is an area of great interest and potential. This flurry

of research activity helps bring together the different
knowledge bases that must converge to develop immobilised
cell technology for industrial primary beer fermentation. There
are already maturation27109 and low-alcohol124 immobilised
29
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3. Anderson, W.A., Bay, P., Legge, R.L. & Moo-Young, M., Journal
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5. Atkinson. B., in Process Engineering Aspects of Immobilized Cell
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J. Insi. Brew., January-February 1998, Vol. 104, pp.

FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR CONTINUOUS

By P. H. Pilkington1-2, A. Margaritis1*, N. A. Mensour1-2, and I. Russell2

Key Words: Immobilised cell carriers, beer production,

for the production of consistent quality beer using im

Traditional beer fermentation technology uses freely suspended

characteristics and advantages of using immobilised yeast cells

matrix and the flavour and aroma compounds that diffuse

there is no reliable collection of data on the mixing and

to measure immobilised cell viability;

Corresponding author: Dr. A. Margaritis.

In this review, the fundamental aspects of immobilised yeast cell

a more uniform product

Immobilisation may be used as a tool to confine intact cells

high cell densities per unit bioreactor volume which result

the reuse of the same biocatalysts (yeast cells) for extended

a continuous process which may be operated beyond the

a discrete phase in which cells may be manipulated

higher cell densities combined with operation at high

Continuous fermentation using immobilised cells also allows

are under-utilised during off-times83. It was also observed that

This document is provided compliments of the Institute of Brewing and Distilling

FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR BEER CONTINUOUS FERMENTATION

(J. Inst. Brew.

effects of certain inhibitory compounds, pH variations, and

on an industrial scale51-96. Two important factors affecting the

cell reactors are operated continuously with fresh wort added to

Dissolved oxygen levels are subsequently depleted and the reactor

Properties of immobilised cell systems

This document is provided compliments of the Institute of Brewing and Distilling

Vol. 104, 1998]

FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR BEER CONTINUOUS FERMENTATION

(b) Attachment or adsorption

(a) Entrapment within a matrix

(d) Cells contained behind

(c) Self aggregation of

Fig. 2. Basic methods of cell immobilisation.

catalytic activity55. An immobilised cell system is described by

the carrier material must be nontoxic, readily available and

bilising yeast cells using entrapment is a relatively simple

Table I lists some characteristics of alginate117 and carrageenan

has been studied extensively. Polymeric beads are usually

polymer from seaweeds

polymers from red seaweeds

thermorcversible gel where

method for pilot scale

of calcium dictating agents

such as phosphates. 12DTA.

Physical entrapment within a porous matrix

or fcap/ra-carrageenan89-90101'27, along with some others44103,

Characteristics of calcium alginate and

Mg:\ and K* ions will

lack of methods for industrial

This document is provided compliments of the Institute of Brewing and Distilling

beads is currently available8'

EUNDAMENTALS Ol IMMOBILISED YEAST CELLS K)R BEER CONTINUOUS FERMENTATION

concern because the flavour components in beer are affected

This concept of utilising the different microenvironments

ficans are co-immobilised in double-layer gel beads. It was

Al'* "2 to improve gel mechanical strength.

Attachment or adsorption to a preformed carrier

allow adsorption onto internal surfaces102. Table II lists some

examples of preformed carrier materials that have been