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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

ibd. effects of certain inhibitory compounds. steadystate concentration. Considerations for the implementation of a continuous fermentation system using immobilised yeast cells. The traditional beer production process is operated in batch mode using free cells and generally takes 5-7 days to complete primary fermentation. Two important factors affecting the formation of these flavour compounds are further described below. In a continuous system. during batch free cell fermentation. It is postulated that by controlling dissolved oxygen and other substrate levels. dissolved oxygen will reach a constant. 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 111.20 FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR BEER CONTINUOUS FERMENTATION (J. 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 This document is provided compliments of the Institute of Brewing and Distilling www. Inst. 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. chemistry. the bioreactor is operated aerobically for the first segment to allow yeast to utilise the oxygen for cell growth. The first factor involves reactor design. Brew. microbiology. 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. Dissolved oxygen levels are subsequently depleted and the reactor operates anaerobically for the remainder of the fermentation. Many obstacles must be overcome to bring this continuous process to industrial fruition. pH variations.uk Copyright . Beer fermentation using free cells is normally carried out using a batch reactor. How. whereas external mass transfer refers to the transfer of nutrients from the bulk medium to the carrier surface. and nutrient depletion18-77101. in which the wort is added at the beginning of the fermentation67. The process advantages offered by continuous fermentation will have a pronounced impact on the brewing industry.org. 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. I. Initial studies show that a continuous immobilised cell system can reduce the time required for primary fermentation to as little as one day89. 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. Flavourmatching may not be critical for commercial success. then. 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. the flavour of beer produced using immobilised yeast cell technology does not match that of beer produced using batch systems. Currently. Immobilised cell reactors are operated continuously with fresh wort added to metabolised wort. Maturation using immobilised cells coupled with a heat treatment process is shortened from 5-21 days to 2 hours27. Project considerations are outlined in Figure I.Journal of the Institute of Brewing . 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. For example. and biochemistry. Internal mass transfer involves the transfer of substrates and products within the carrier to the yeast cell. This implies that the fermentation process will need to shift from a constantly changing batch to a steady state continuous operation.

Methods of immobilisation include physical entrapment within a porous matrix. Some brewers have moved away from entrapment matrices and are currently focusing on adsorption techniques for several reasons. Basic methods of cell immobilisation. An immobilised cell system is described by Abbott1 to be any system in which microbial cells are confined within a bioreactor. 12DTA.i im3o or fcap/ra-carrageenan89-90101'27. 104. An immobilised cell system should have the following properties for large scale industrial application90: • • • • the carrier material must be nontoxic. Figure 2 illustrates basic immobilisation techniques. along with some others44103. 2. the carrier material should allow for high cell loading and physical strength.Journal of the Institute of Brewing beads is currently available8' .ibd. Polymeric beads are usually spherical with diameters ranging from 0.<mu<m. readily available and affordable. All of these methods have a similar purpose: to retain high cell concentrations within the bioreactor. At present.org. the cells should have a prolonged viability in the support. 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 This document is provided compliments of the Institute of Brewing and Distilling www. has been studied extensively. This is a TABLE I. attachment or adsorption to a pre-formed carrier.97. the system should be efficient.i u.Vol. bilising yeast cells using entrapment is a relatively simple method and a high biomass concentration is facilitated84.s5.3 to 3. Diffusion limitations due to the gel matrix and high biomass loadings cause metabolite concentration gradients within the polymer beads.". thus permitting their reuse. and cells contained behind a barrier. leading to increased volumetric productivity of the system and lowered fermentation costs. catalytic activity55. Physical entrapment within a porous matrix The entrapment of immobilised cells within a porous polymeric matrix such as calcium alginate1 >. easy to operate and give good yields. self aggregation by flocculation or crosslinking agents. Table I lists some characteristics of alginate117 and carrageenan gels'".0 mm. gel entrapment matrices are not produced economically on an industrial scale. 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.uk Copyright . 1998] 21 FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR BEER CONTINUOUS FERMENTATION (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.

Some carrier types used recently for immobilisation by attachment or adsorption Carrier Material Reference DEAE cellulose porous glass silicon carbide volcanic rock 59. DEAE-cellulose (diethylaminoethyl) support uses ionic attraction to immobilise yeast cells. a porous ceramic carrier from Kirin Breweries in Japan. This document is provided compliments of the Institute of Brewing and Distilling www. 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. To overcome these limitations. It was found that oxygen264761132. Brew. Spezyme®. but mass transfer may be less limiting. and is suitable for CIP cleaning86. Cashin16 recently compared five different porous carriers in terms of minimum fiuidisation velocity.59.136. They designed a twostage configuration for continuous primary fermentation using an immobilised cell reactor in the first stage followed by a free cell system. these immo bilisation matrices may have significant cell leakage.ibd. The researchers observed that when DEAE-cellulose particles were used. had the lowest minimum fluidisation velocity. The lowest amount of yeast growth was observed using the glass Siran8 carriers. or internal gas accumulation44-84. Finland. along with physical stresses such as agitation and abrasion can induce cell desorption. Researchers have treated alginate gel beads with stabilising agents such as sodium meta-periodate and glutaraldehyde13 or Al'* "2 to improve gel mechanical strength. The carriers compared were the DEAE-cellulose carrier Spezyme8. re-usable. Since there is no barrier between cells and the surrounding medium. 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. due to limitation of its uptake and diffusion. They found that the cellulose did not bind most common yeast contaminants and is washable. Adsorptive matrices do not have the additional gel diffusion barrier between the cells and the bulk fermentation medium. genetically modified yeast with the gene encoding ct-acetolactate decarboxylase could be used to reduce diacetyl levels. How ever. multichannel porous silicon carbide immobilisation rods were arranged in a loop con figuration123. and Siran8 developed by Schott GmBH. Tubular 1. by dissolution or by breakdown due to abrasion. The silica beads compared were Bioceramics" from Kirin Brewery in Japan. and (b) carriers with large enough pores to allow adsorption onto internal surfaces102.137 sponge diatomaceous earth wood blocks gluten pellets 86. Silicon carbide is inert. because pore sizes are too small to allow microorganisms to penetrate inside. Hypermics8 was found to give the highest biomass loading. rarely penetrates greater than a few hundred micro meters into the gel bead when it is the limiting substrate. a DEAE-cellulose carrier obtained from Cultor Oy. have worked with DEAE-cellulose to immobilise yeast cells for use in continuous maturation or secondary fermentation processes. Linko and Kronlof71 conducted a study in which they compared Spezyme*. but it had to be chemically sterilised which was more time consuming than the steam sterilisation that was used for the silica-based carriers. 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. 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.Journal of the Institute of Brewing . but tended to float and was mechanically weak. Meura-Delta. Of the two glass carriers. Changes in environmental ionic strength. and Hypermics8. durable. Thus. biomass loading. carriers is the possibility of nonspecific binding of charged materials within the fermentation medium12. ionic and hydrogen bonding interactions. in a single-stage continuous air lift reactor. There are actually two types of whole-cell adsorptive immobilisation carriers: (a) carriers that allow adsorption only onto external surfaces. yeast attached to the surface of the particles only. This is not appropriate for processes requiring a cell-free effluent39.org. Another limitation of gel entrapment includes the loss of gel mechanical integrity. steam sterilisable. integrated a novel immobilisation approach for the continuous production of beer.6 L or conical 5 L packed bed columns were used for the fer mentations in this study. The only difference found from traditionally brewed beer was slightly elevated ester and diacetyl levels.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. Inst.123 4. pH. Because it is known that yeast cells have a net negative surface charge.122.107 14. while exhibiting none of the practical limitations found with the other carriers16. 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. Table II lists some examples of preformed carrier materials that have been suggested for immobilisation of yeast. The DEAE-cellulose carrier. The strength of cell attachment to an adsorption carrier depends on both cell and matrix type. temperature. Sinebrychoff Brewery and Cultor Ltd. three sizes of Siran81 sintered glass beads. 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. compression. which allow for rapid yeast colonisation. a positively charged support will be most appropriate for immobilization12.53 115 99 46 10 [J. a Belgium-based brewery equipment supply company. and durability. Open pores in the sintered silicon carbide rods range in size from 40-60 um. in collaboration with a research team at CERIA. It is an inert. In the first reactor. Germany. Biomass loading is generally lower in adsorbed cell systems than those obtainable in gel entrapment matrices. 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.uk Copyright . with two types of commercially available porous silica beads used as immobilised cell carriers for the continuous primary fermentation of beer. reusable and regenerable with a long service life74. This result may prove to be useful for achieving aerobic and anaerobic stages during beer fermentation. a Belgian university. The kinetics of biofilm formation are complex68 and therefore difficult to control. the aerobic nitri fication step was carried out in the outer layer while the anaerobic denitrification took place in the bead core31. while ester levels could be controlled by optimising oxygen levels in the immobilised cell reactor. whereas glass beads showed yeast growth partially on the surface and partially inside the pores. A beer flavour profile most similar to traditionally brewed beers was obtained using these immobilised yeast for primary fermentation. nondissolving cellulose matrix which has a nonuniform granular shape that provides a large binding area for cell colonisation. Another limitation of adsorption celt TABLE II. Siran* was found to be superior over Bioceramics1 because of higher mechanical strength and density. Attachment or adsorption to a preformed carrier Adsorption involves the reversible attachment of biomass to a solid support mainly by electrostatic. Another advantage to using adsorption matrices is the regenerability of the support.

one must understand the impact of such a system on yeast physiology and growth. causing a relatively weak interaction between biomass and immobilisation support. Cell retention behind a membrane barrier has not been widely used to immobilise yeast cells for the continuous production of beer. Since A. fermentation conditions. 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.0. In order to efficiently and con sistently produce quality beer using an immobilised cell system. they cultured the cells in contact with Celite8 at pH 2 for 72 hours to maximise cell loading and minimise electrostatic repulsion. and yeast vitality is a measure of yeast activity or fermentation performance7-66121. and does not swell during fermentation33. A barrier formed by the liquid-liquid interface between two immiscible fluids can also be used for immobilization55.ibd. 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. may prove to be very useful in continuous fermentations. It is often beneficial to monitor a combination of parameters to gain a more complete understanding of a yeast's physiological state. medium pH. differences in the charge distribution on the cell walls were found. Siran® (sintered glass) and Spezyme8 (DEAE cellulose) for the primary fermentation of beer. Ions of opposing charge will migrate to this surface. On the other hand. 1998] FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR BEER CONTINUOUS FERMENTATION Diatomaceous earth. is the most simple and least expensive immo bilisation method. 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. both yeast cells and Celite® have negatively charged surfaces under typical fermentation conditions. 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.93 studied the effect of zeta potential on the adsorption efficiency of cells onto solid supports including Celite8. 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. Stewart118 defines flocculation as "the formation of an open agglomeration that relies upon molecules acting as bridges between separate particles". Mulchandani el al. However.S-3. incubation temperature. 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. Kyung and Gerhardt62 investigated continuous ethanol production using Saccharomyces cerevisiae immobilised in a membranecontained fermenter. They showed that when the yeast-like fungus Aureo- basidium pullulans was grown in different pH conditions. 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. so a compromise between maintaining yeast viability and productivity while maximising cell loading is required. Lentini66. the growth phase.g. 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. the perceived viability of a yeast sample may vary depending on the criteria selected. Consequently. The control of cell aggregation is important to maximise bioreactor effi ciency. but there are several groups who have investigated their use for continuous ethanol production50628081*4. high mechanical and chemical stability. one would want to select a pH that would minimise the forces of repulsion between the carrier and the cells to maximise biomass loading. cation composition of the medium. fermentation stage. However. This brewery uses a hold-up vessel followed by two stirred tank fermenters for the continuous primary fermentation. Cells contained behind a barrier In this type of immobilisation. A. shown in Figure 2(c). Labatt Brewing Company Limited in Canada was also active in earlier work on continuous primary fermentation of beer37. pullulans did not synthesise pullulan at pH 2. Self aggregation of cells The formation of cell aggregates by flocculation. After primary fer mentation. Consequently.3 reported that a reduction of cell leakage rate from Celite® is needed for long-term. In addition. trade named Celite". fermentation broth). and other wort com ponents"8. and then transferred the immobilised cells to pH 5. Factors which influence the natural flocculation characteristics of brewer's yeast strains include the genetic make-up of the strain.uk Copyright . Zeta potential plays an important role in determining the extent of cell adhesion to a carrier such as Celite8. they noted that a genetically modified superflocculent yeast strain that was disregarded because it flocculated too early for use in conventional batch ferment ations. Factors such as wort composition. specifically describes yeast vitality as "a function of the total cell viability and the physiological state of the viable cell population". 104. 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. For example. This was thought to be due to differences in cell wall composition caused by the differing media conditions.Journal of the Institute of Brewing . Nguyen and Shieh" used Celite8 to immobilise yeast for the continuous production of ethanol in a fluidised bed reactor. yeast strain. in his compre hensive 1993 review. Celite8 has a uniform composition. larger floes Immobilised Cell Viability and Vitality Yeast viability is defined as the percentage of live cells in a sample. large scale fermentations. Many criteria are used to assess yeast cell viability and vitality. This document is provided compliments of the Institute of Brewing and Distilling www. They also found that Celite8 had a negative zeta potential with maximum negativity at pH 6 and minimum at pH 2. and medium pH will affect the zeta potential of the Celite8 and the yeast cells. They obtained only 71% of the theoretical ethanol yield and this was thought to be due to extended mean cell residence time. is traditionally used in the brewing industry as a filter aid but it also possesses desirable qualities for use as an immobilised cell carrier. Anderson el al. and intermittent pH variations in the reactor. 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.org. most substances acquire a surface electric charge63. pullulans cells exhibited a net negative charge in solutions above pH 2. Researchers at VTT Biotechnology and Food Research in Finland13 compared the behaviour of strongly and weakly flocculent yeast cells immobilised on two different carriers. One group successfully decreased cell leakage by encasing the Celite8 in a layer of calcium alginate33.5 production medium for optimal fermentation conditions. the cell wall structure and surface charge. but the least predictable.Vol. pH also affects yeast viability. a low cell concentration is maintained in the reactor resulting in insufficient fermentation rates. When brought into contact with a polar medium (e. inhibitory effect of high dissolved carbon dioxide concen trations. the flocculent yeast are separated from the green beer by gravity in a conical settler. 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.

When buffered and supplemented adequately. A linear correlation has been demonstrated between capacitance and viable yeast biomass6. fluoresces while the oxidised form. it does not provide any additional information about the yeast cell's metabolic state. and when there is a lack of oxygen available to the cells. on-line method of monitoring freely suspended and immobilised yeast cell metabolism30-69. H+ ion. The oxidised form. cationic detergents. Cells which have given rise to microcolonies are considered viable whereas single cells that have not formed microcolonies are considered non-viable120. the influence of substrates such as oxygen on the reducing state may be predicted. 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. 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. NAD* is used by dehydrogenases to accept electrons from their substrates. There are also many fluorescent stains designed to assess yeast cell viability and vitality. acids. 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. malate dehydrogenase (MDE) first binds to NAD+ to form a complex of MDE-NAD*. It is relatively less time-consuming than standard plate counts but still much slower than the staining techniques. the reducing state approaches zero because NADH is easily oxidised to form NAD* and H. ATP allows for the detection of viable cells in a (J. Gikas and Livingston41 used ATP ioluminescence to charac terise biomass viability in freely suspended and immobilised cell bioreactors. NADH Fluorosensor NADH has successfully been used as a noninvasive. in the enzymatic conversion of malate to oxaioacetate in the presence of oxygen. Methylene blue staining is considered to be an accurate method only when yeast cell viability is greater than 90%88.4o. The total NAD is the sum of NADH and NAD*. The concentration of [NADH] as well as the intensity of the fluorescent signal are influenced by the number of viable cells.org. the reducing state of the cells and environmental effects. Yeast cells were immobilised on porous glass carriers (SiranB) and DEAE- cellulose (Spezyme8).Journal of the Institute of Brewing . The "firefly assay" is used to determine the quantity of ATP present in a biomass sample. short amount of time (10—15 minutes) when compared with traditional plating techniques. whereas non-viable cells are unable to reduce the stain rendering them a deep blue-purple shade66. and standard methylene blue solution64. 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. and organic compounds such as acetone and ethanol. NADH. Kasten56 gives a comprehensive review of stains available and fluorescence microscopy techniques. Kronlof pointed out that although this method correlates well with methods such as staining for the detection of viable cells. Brew. Care must be taken when using this method on very flocculent yeast66. When fluorescent stains are used in conjunction with confocal microscopy9"110 or flow cytometry28. 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. does not. Some examples of methylene blue solutions include Fink-Kuhles buffered methylene blue (ASBC international method)120.1 volume may be detected using the firefly method41. 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. 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.73 valuable information may be obtained on yeast cell growth and metabolic state. Other brightneld stains which have been used to monitor yeast cell viability include Aniline Blue88 and Crystal Violet35. and a capacitance is generated66. Inst. Extractants used to release intracellular ATP include boiling in buffers such as tris-EDTA. 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. 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). Thus. Since the quantity of ATP per cell does not vary significantly for a given strain (but varies between strains). and oxaioacetate are released: malate+NAD* <-» oxaloacetate+NADH+H+ (oxidised) (reduced) The reduced form. Viable cells are able to reduce this stain making it colourless. An advantage of the slide culture method is that it is accurate at relatively low yeast cell viabilities. It generally takes three days for visible colonies to form and viability is assessed by counting the number of colony forming units (CFU). NADH.2». 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. Nonviable cells are unable to generate this capacitance. NAD*. Methylene blue staining is a good method for assessing the viability of highly concentrated yeast such as that found in immobilisation matrices64. When oxygen is in excess.48.uk Copyright . NADH is strongly fluorescent with an emission maximum at 460 nm wavelength.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. From here.ibd.O. R approaches one. methylene blue aqueous solution120. A viable yeast cell count may be completed using a hemocytometer and a light microscope in less than ten minutes. 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. Yeast viability by slide culture is also based on the ability of yeast cells to proliferate. This complex then combines with malate to form a ternary complex MDE-NAD+-malate.Viable cells contain nicotinamide adenine dinucleotide (NAD) coenzyme whereas non-viable cells or spores normally lose their NAD17. 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. methylene blue has no effect on yeast cell viability64. Kronlof58 recently showed that capacitance probes are suitable for monitoring viable yeast biomass in both freely suspended and immobilised cell systems. For example. Measuring NADH has an advantage This document is provided compliments of the Institute of Brewing and Distilling www. 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. Therefore a correlation between ATP concentration and the amount of viable cell mass can be made41.

Vibrio natriegens (Baneckea natriegens). However. and yeast viability49. This test may be able to detect more subtle changes in yeast cell vitality than acidi fication power test49. events occurring within the cell rather than changes outside the cell environment. the sample is often dried and embedded into a resin. Parascandola and Alteriis"0 studied the spatial growth patterns of Saccharomyces cerevisiae cells entrapped in starch-hardened gelatin discs. 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. Therefore one could relate vitality to the response of yeast cells to stresses such as ethanol. 1998] FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR BEER CONTINUOUS FERMENTATION over monitoring dissolved oxygen or pH because it directly measures. To avoid matrix compression and to obtain an accurate distribution. glycogen. These studies are of importance since the extent of biofilm growth is in fluenced by the local chemical environment and fluid flow This document is provided compliments of the Institute of Brewing and Distilling www. 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. When entrapped cells were released from the carrageenan matrix. whereas the intracellular pH of less active cells actually decreases under low extracellular pH conditions49. A reduced oxygen uptake rate parallels other yeast changes such as the reduction in yeast lipids. By measuring the concentration and viability of cells inside each shell. Heat tolerance was measured by incubating yeast cells at 48°C for various lengths of time with periodic shaking. immobilised on Celite8. implying a lower biomass activity in the immobilised cell system. Methylene blue. Problems with this method include uniformity of dissolution of matrix. 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. Magnesium Release Test (MRT) The Magnesium Release Test92 is based on the observation that low molecular weight species such as magnesium. Under these conditions.Vol. in real time. 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. fluorescent dyes or by plating. 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 rate24.129 found that oxygen uptake rate did not correlate well with fermentation per formance when yeast had been previously acid-washed. and standard plate counts101 may be used to assess the ability of cells to remain viable after being subjected to a given stress. they actually showed better fermentation per formance than non-acid-washed yeast. and overall accuracy limitations.im assessed the ethanol and heat tolerance of brewers' yeast entrapped in K<arrageenan gel. It was found that there was a significant increase in yeast survival against ethanol for immobilised cells as com pared to free cells. and high salt concen trations. oxygen uptake rate correlates well with yeast fermentation performance. When biomass profiles are steep within the beads. Recently. Norton et al. The biocatalytic activity of the nitrogen-fixing bacterium. Shrinkage has been observed during this preparation134. but no distinct difference in heat resistance 25 was noted. Measurement of Yeast Vitality by Stress Response As stated earlier. vitality may be considered a measure of yeast activity or fermentation performance. higher end ethanol concentrations. fluorescent dyes7. or frozen. finding a suitable nontoxic solution for dissolving the entrapment matrix. Released biomass from each shell may be assessed for viability by using methylene blue staining.68 used NMRI to better understand the dynamics of biofilm growth and external liquid film flow velocity distribution. Usually shells obtained using this method have a thickness greater than SO um. higher cell counts. The method involves the pitching of yeast into aerated media and the measurement of the oxygen uptake rate for one hour M. It has also been defined as the ability of cells to endure or overcome stress7. this invasive slicing method may cause alterations in cell distribution and other aber rations9.uk Copyright . and 19.Journal of the Institute of Brewing . Subsequent fermentations performed using the more vital yeast had shorter lag phases. potas sium.4% (v/v) for a given length of time and viability was then measured using standard plate counts. They used a multi-step digestion method to collect cells belonging to five different layers of the discs. even if the extracellular pH is low. their ethanol tolerance returned to that of free cells. NMR imaging Nuclear magnetic resonance imaging (NMRI) is a noninvasive technique to study spatially chemical and physical properties of small samples68. 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. calcium chelators such as phosphates and citrates may be used to dissolve "shells" of alginate and biomass from the beads18-43"3. 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. Cell viablity following heat shock was also measured using standard plate counts. The study concluded that the carrageenan gel matrix provided protection to the entrapped yeast cells against ethanol. Intracellular pH (ICP) Method The ICP method uses a pH-sensitive fluorescent reagent to measure the intracellular pH of individual cells and cell mass. acidification power test value. and lower diacetyl levels. Various groups24-54-82 have shown a correlation between oxygen uptake rate of yeast and fermentation performance if yeast viability is less than 90%. 18%. Physical sectioning of immobilised cell matrix A biomass distribution may be obtained by slicing matrices containing yeast cells. Even though acid-washed yeast showed decreased specific oxygen uptake rates. information may be obtained about the spatial distribution of viable cells within the entrapment matrix. was studied by Gikas and Livingston42 using specific oxygen uptake rate [mg (O2) g~' (dry biomass) h"1].ibd. This method is useful for detecting large differences in yeast metabolic activity. Lewandowski et a/. 104. and phosphate ions are released by yeast immediately following inoculation into glucose containing medium. However. It was found that the intracellular pH of more active yeast cells does not decrease.org. Wheatcroft et a/. greater resolution is required to obtain an accurate picture of yeast cell distribution. heat shock. Free and immo bilised Saccharomyces cerevisiae cells were exposed to ethanol concentrations of 0. Trials performed on Saccharomyces cerevisiae showed that cells which released greater quantities of magnesium immediately after inoculation into high gravity (16°P) wort had higher vitality and fermentation performance than yeast which released lower amounts of magnesium.

The Thiele Modulus may be defined as the ratio between the reaction rate and the internal diffusion rate or. Confocal microscopy was used to avoid the potential artifacts that may be generated using physical sectioning. and finally into the yeast cell where the reactions take place. 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. substrate concentration on the gel surface or within the matrix is lower than the concentration in the bulk phase. Substrate must travel through the bulk medium by diffusion and convection. Therefore oxygen availability is often limited by mass transfer restrictions caused not only by the matrix itself. Inst. "Effectiveness factor" is defined as the ratio between the observed reaction rate and the rate of reaction in the absence of mass transfer resistance. In an elegant study by Lim et al. in the bulk liquid phase. yet it is only required in relatively small quantities in brewery fermentations. where each point of a concentration profile reflects a local equilibrium between supply and consumption68. through the liquid-solid interface. 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. 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. If the effectiveness factor is less than one. The above parameters need to be quantified in order to determine overall effectiveness factors. and the effective solute diffusivity within the immobilisa tion matrix (De) respectively. They found that cells initially only attach to the external surface or within the external cavities of the macroporous microcarriers. Dc. the rate that substrate is consumed relative to the rate at which it is supplied by the diffusion process. The effective solute diffusivity. external film mass transfer resistance and internal mass transfer resistance91. Substrates must also be transferred through the biofilm-bulk medium interface to reach the cells. This depth discrimination property allows only the region of the object that lies close to the focal plane to be imaged efficiently87. The study of the transfer of oxygen in beer fermentation using immobilised yeast cells is especially important. there was a slow migration of cells toward the interior of the beads. Figure 3 is a schematic of the transfer of substrate from the bulk medium to an entrapped yeast cell.Journal of the Institute of Brewing . leading to concentration gradients between the two phases. For more detailed dis cussions of mass transfer in immobilised cell systems. through the external liquid film surrounding the bead containing immobilised cells. 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.org. These terms may be quantified by the solute parti tioning coefficient (Kp). In other words. A Thiele Modulus of less than ten is desirable to minimise substrate concentration gradients. the spatial distribution of mammalian cells grown on modified macroporous gelatin microcarriers was studied using confocal microscopy. is used to quantify internal mass transfer rates. In the second case. Schematic of the transfer of substrate from the bulk medium to an entrapped yeast cell. the liquid film mass transfer coefficient (Kl). 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. signi ficantly altering the production of beer flavour compounds. Decreasing bead diameter is a good way to maximise internal mass transfer. and more recently by Westrin and Axelsson128. which is especially relevant for entrapped cell systems. but also by yeast cells themselves in highly colonised beads. Mass transfer occurs by diffusion only within and close to the solid matrix phase whereas. 3. the microenvironment may be described in terms of solute partitioning effects between the bulk liquid phase and the solid immobilisation matrix. 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. conditions. 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. Oxygen is an essential substrate for yeast cell growth. a high Thiele Modulus indicates that the system is mass transfer limited whereas a lower number indicates that the system is kinetically controlled. Brew.uk Copyright .10. contributed only 13% of the total available void fraction. macroporous microcarriers. Internal mass transfer characteristics Internal mass transfer involves the transfer of substrates and products within the carrier or solid phase100. These factors will have a significant effect on immobilised cell bioreactor performance. 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. Using confocal microscopy. This occurs because substrate concentration gradients are formed. texture and porosity. transfer occurs by both diffusion and convection. since a lack of oxygen due to mass transfer limitations may force yeast cells to switch from aerobic to anaerobic metabolism. com prehensive reviews are presented by Karel et al. In addition. 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. through resistance caused by microcolony formation. comprising the inner half radius of the spherical particles. 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. researchers were able to gather information on velocity distri bution using NMRI near microbially colonised surfaces with an imaging time of 8-16 minutes. Internal mass transfer can be optimised by adjusting the immobilisation matrix size. through the liquid within the solid gel phase by diffusion. This document is provided compliments of the Institute of Brewing and Distilling www. Subsequently.26 FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR BEER CONTINUOUS FERMENTATION [J. or alteration of the local microenvironment in the immediate vicinity of the immobilised cells.i$.ibd. Bancel and Hu9 used confocal laser scanning microscopy to examine the distribution of viable hybridoma cells within macroporous microcarriers.

C. The latter point refers to the method of insertion of the microelectrode into the biocatalyst bead. higher alginate concentration. t BULK LIQUID PHASE and (4) at time.5 vs. 0<r<R. 1 B. Korgel et a!. A thin disc of This document is provided compliments of the Institute of Brewing and Distilling www. initial and boundary conditions are needed. capacitance. (3) there is negligible external film mass transfer resistance.org. Being able to do this is important because diffusion gradients within biofilms will influence their internal structure. 104. of diffusion properties of components within immobilisation matrices. one may evaluate D«.91 developed an unsteady state technique for measuring effective solute diffusivities within an entrapment matrix used for whole-cell immobilisation. Flow conditions in the reactor and in the flow chamber where measurements are performed must be kept the same for accurate readings. and it may be carried out relatively quickly91. High levels of agititation are required to avoid external mass transfer resistance at the bead surface. For yeast cells entrapped within calcium alginate or carrageenan gels.5). Thin Disk Method for Determining Effective Solute Diffusivities This method utilises a two compartment glucose among others21-45-58-95121125. r=0. Effective diffusivity.m also used the multiple bead method to calcu diaphragm diffusion cell. Some difficulties are encountered when using micro electrodes since measurements may be influenced by calibration conditions. To be useful for the study changing its configuration68.Journal of the Institute of Brewing . and lower temperatures each resulted in lower diffusion rates for both lactose and lactic acid. t>0. Cs=constant CL=0 dCs / (r=0 vL(acL/at)= KPAsDe(aCs/ar)|r=R I.Vol. (2) the physical properties of the sphere are constant. especially since immobilised cell matrices tend to have large substrate concentration gradients95. It is also difficult to determine the exact position of the microelectrode visually and thus spatial resolution is insufficient.51 measured effective diffusivities of galactose in calcium alginate gel occupied by living Zymomonas mobilis bacterial cells using this method. oxygen. Oyaas et al.5 and 6. for a sphere containing solute immersed in a finite liquid volume initially free of solute. 2 B. mechanical resistance and properties of the matrix. The diffusion of a given solute into or out of the gel beads is measured by monitoring the concentration of the bulk liquid phase. is defined by Fick's law: J=-DcaCs/dr where J is the flux per unit of matrix area. other metabolites. Response time of the electrode is also important. 5. 4. the solute is uniformly distributed throughout the sphere. where each equal-volume chamber contains a well-stirred solution placed in contact with either surface of a thin gel. initial and boundary conditions are listed below. This method is a relatively simple way to gather data for the calculation of effective solute diffusivities in immobilised cell systems. and vities using a similar method except that gel beads are placed in a buffered aqueous solution in a well-stirred tank. The unmetabolizable galactose was chosen because it allowed for effective diffusivity measurements independent of consumption in a gel system containing immobilised living bacterial cells.t) CL = f(t) Merchant et a/. Cs is the amount of solute per unit liquid volume in the gel phase. 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. t=0.ibd. Dc. t>0. 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. Concentration-time measure ments are used to determine the effective diffusivity. the measurement of substrate concentration profiles using microelectrodes provides a direct method for characterising the microenvironment of the cells inside the matrix. Other researchers34 have measured effective solute diffusi trations directly inside biofilms. t=0.C. Spherical coordinate system Tor diffusion and reaction of a solute for a sphere immersed in a finite liquid volume. A response time of 0. Signal artifacts may occur due to the sudden breakthrough of the electrode through compressed material such as the immobilisation matrix95. Microelectrodes have been developed for pH. Using the spherical coordinate system shown in Figure 4. Their experiments showed that lower pH (4.C. 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. ammonia. r=R. and r is the radial coordinate in the sphere105. especially in dynamic processes using im mobilised or flocculating cells.C. r>R. 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 puncture technique. 1998] FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR BEER CONTINUOUS FERMENTATION 27 Unsteady State Technique for Measuring Solute Diffusivities = f(r. t=o. 2 By monitoring the change in concentration of solute in the liquid phase as solute diffuses out of the sphere. a mass balance for unsteady-state diffusion of solute is described by the following equation: Fig.I I. and by solving the above equation using the aforementioned conditions. Radiotracer techniques may be used to follow the change in solute concentration since the method requires very small liquid volumes. Therefore.uk Copyright . 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. Diffusion occurs from higher to lower concentration or from "source" to "sink"52. The mathematical model used allowed for the calculation of effective diffusivities and accounted for solution sampling and external film resistance.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.

28 FUNDAMENTALS OF IMMOBILISED YEAST CELLS I OR BEER CONTINUOUS FERMENTATION TABLK III. Wijffels el a/. Cylinder Method of Determining Effective Solute Diffusivities The cylindrical-shaped gel is initially free of solute. KL.When diffusion limitation over micro-colonies is incorporated into the dynamics of the immobilised cell system. However. It therefore becomes important to design a reactor which provides an adequate amount of agitation at a reasonable shear rate. size and operating conditions will influence external mass transfer in immobilised cell systems. immobilised cell particles may break. The rate of shear on the particles in a reactor will increase as agitation is increased. 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. The solute diffuses through the gel from source to sink in a given time. t.org. thus facilitating the transfer of species from the bulk medium to the carrier. In this method. and cell aggregates may be disrupted. However. mobilis cell concentrations ranged from 0 to ISO g dry wt/L of gel. The gel is then immediately sliced into thin disk-shaped slices. numerous small micro-colonies appear. Surface contact between the biocatalyst (immo bilised yeast cell) and the growth medium (wort) should be maximised by optimising liquid mixing. With time. biomass may be lost from adsorption matrices. for a given agitation rate. Z. different reactor designs may differ in rate of shear. 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. If shear rates are too high. fewer but bigger colonies form due to significant diffusion limitations. At high initial biomass concen trations. Brew. growth of biomass was described as expanding at similar rates throughout the beads following start-up. This resulted in increased macroscopic substrate consumption rates in the system containing beads with smaller microcolonies. 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. the gel is much thicker than the gel used in the thin disk diffusion-cell. causing minor diffusion limitations. biomass eventually becomes confined mainly to areas near the gel surface due to diffusion limitations. Reactor design factors such as reactor type. the colonyexpansion model predicts that inoculum size will affect substrate consumption rates. Colony-expansion Model When the dynamics of the growth of immobilised cells were initially studied qualitatively. This is because diffusion limitations occurring over larger micro-colonies are not accounted for in this model. Dc.ibd. These results were confirmed experimentally in a continuous air lift loop reactor"1.13' theorise that in order to incorporate diffusion limitation across micro-colonies in a dynamic growth model. [J. rather than as a homogeneous increase of biomass in spherical "shells" within the gel beads. 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. and the concentrations of source and sink are maintained at C\ and C: respectively. at low initial biomass concen trations. This document is provided compliments of the Institute of Brewing and Distilling www. Inst. The solute concentration in each slice is measured.uk Copyright . Effective diffusivity. Effective diffusivities were found to decline with increasing entrapped biomass concentration. it is necessary to consider biomass growth as expanding complex design plugging lacks economies of scale micro-colonies. 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. giving concentrationdistance data to be collected within the gel cylinder. 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. may then be calculated using these data30.Journal of the Institute of Brewing .

Process Biochemistry. 573. When designing and operating a packed bed reactor.. inventory. M..C. Journal of Chemical Technology ami Biotechnology. J.E. The authors wish to thank their colleagues Robert Stewart and Jadwiga Sobczak for their contribution to the manuscript. 38. Journal of the Institute of Brewing.. Even if these pitfalls are successfully avoided. Pardonova. (C. Nature Biotechnology.D. gas lift draft tube. D. 11. 15. B.S. Bio technology and Bioengineering. Legge.4028. one must ensure that there is no channelling of flow and no reactor plugging. Particle abrasion is minimised and scale up is simple with this type of design. Pilkington and N. Evans. & WijfTels.M. 44. 1996. T. Mattiasson. Journal of Chemical Technology and Biotechnology. M. 1996. P. This document is provided compliments of the Institute of Brewing and Distilling www. 1956.M. M. 94. K. D. 7. to the top of the reactor with external recycle. Biotechnology and Bioengineering. low power consumption. Daoud.E. 79.. fluidised bed.. leading to significant reductions in production time. 824.C. 884..E.. (D.A. 16. P. 8. 1986. Gas lift draft tube systems use gas to circulate fermenter contents internally using a draft tube concentrically located inside a columnar reactor. Biotechnology and Bioengineering.31. Polednikova. fluidised bed. 29. 1978. R. 1990. 27. & Han.. 64. P. Conclusions Immobilised yeast cell technology is an innovation that may revolutionise the way breweries operate.. A. 1970. D. and loop reactors. Casas. 1986. (C. S. G. 1987... 1986. C. 73. These reactors provide good mixing and aeration. Bcjar.M. 1996. Acknowledgements. Ph.-H. Cashin. & Bailey. 85. 14. 10. V.A. 25. 5. Doran.. I.) Manchester: Institute of Chemical Engineers. Choi. G. P. Webb. 892. 1984. C.F. 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. & Kanellaki. & Webb. 17. a low density difference between the solid beads and the liquid medium will not result in high mass transfer rates23. & Mosbach. 93...8. In fluidised bed reactors. Table III summarises some of the advantages and disadvantages of packed bed. M..28.) New York: Academic Press. B. F.P. Gcslrclius. Coults.J. 1990. Bay. Faculteil Landbouwwetenschappen Rijksunirersiteit. & Sola. J. Curin. Kancllaki. There are already maturation27109 and low-alcohol124 immobilised 29 References 1. 1982.S.C. & deary. the fermentation medium flows in a loop from the bottom of the fermenter. 1994. 1988.J. through both the internal channels and around the matrices for contact with the immobilised yeast.. 2289.P.org. V. and product variation. S. 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