review primary beer fermentation by immobilised yeast – a...

Journal of Chemical Technology and Biotechnology J Chem Technol Biotechnol 81:1353–1367 (2006)

ReviewPrimary beer fermentation by immobilisedyeast – a review on flavour formation andcontrol strategies†

Ronnie Willaert1∗ and Viktor A Nedovic2

1Department of Ultrastucture, Vrije Universiteit Brussel, Flanders Interuniversity Institute for Biotechnology, Pleinlaan 2, B-1050 Brussels,Belgium2Department of Food Technology and Biochemistry, University of Belgrade, Nemanjina 6, PO Box 127, 11081 Belgrade-Zemun, Serbia andMontenegro

Abstract: Immobilised cells are increasingly being used in bio-industries and may also have benefits for thebrewing industry. The major challenge to applying this technology successfully in breweries is focused on themain fermentation in combination with the secondary fermentation. In particular, the control and fine-tuningof the flavour profile during the main fermentation require further investigation. In this review, the influenceof immobilised cell technology on the production of the flavour-active compounds (i.e. higher alcohols, estersand vicinal diketones) is discussed. Control strategies that are based on the manipulation of parameters duringfermentation such as temperature, feed volume, wort gravity, wort composition and aeration are explained.Finally, bioreactor configurations that may facilitate immobilised cells in performing the primary fermentationare evaluated. 2006 Society of Chemical Industry

Keywords: primary beer fermentation; immobilised cell systems; flavour-active compounds; control strategies;bioreactor configurations

INTRODUCTIONHigh volumetric cell densities of yeasts can be obtainedby packing the microorganisms in a small definedvolume by entrapment or adsorption on the surfaceof a carrier matrix. This leads to higher volumetricproductivities and, in consequence, to smaller biore-actor sizes, with resulting decreased capital costs andshorter residence times. Using immobilised cells, theproduction of beer in a continuous fermentation pro-cess becomes very financially and technically feasible.Additional benefits associated with continuous fer-mentation are ease of biomass separation and recovery(the flocculation properties of brewing yeast strainsused become inconsequential), simplification of pro-cess design, lower risk of microbial contamination ofthe pitching yeast population and potential savings.On the other hand, a number of technical challengesrequire resolution:1 removal of excess yeast, removal ofcarbon dioxide, sustaining yeast viability, optimisationof oxygen (air) feed, prevention of microbial contam-ination and prevention of clogging or channelling ofthe reactors. Regeneration of large amounts of carriermay be rendered unnecessary by the use of a cheap,replaceable carrier, such as wood chips.

Immobilised cell technology (ICT) processes havebeen designed for different stages in the beer fermen-tation process: wort acidification, bioflavouring duringthe secondary fermentation, primary fermentation andfermentations for the production of alcohol-free orlow-alcohol beers (for recent reviews, see Branyiket al.2 and Nedovic et al.3). The most challenging andcomplex application is to the combined main andsecondary fermentation.

Traditional beer fermentation technology uses freelysuspended yeast cells to ferment wort in an unstirredbatch reactor, a time-consuming process. The tra-ditional primary fermentation for lager beer takesapproximately 7 days with a subsequent secondaryfermentation (maturation) of several weeks. How-ever, the resulting beer has a well-balanced flavourprofile, which is well accepted by the consumer. Nowa-days, large breweries use an accelerated fermentationscheme, which is based on using a higher fermenta-tion temperature and a selected specific yeast strain.This allows the production of finished lager beer in12–15 days.

ICT is able to produce lager beer in a much shortertime period (usually 1–3 days). A major difficulty is to

∗ Correspondence to: Ronnie Willaert, Department of Ultrastucture, Vrije Universiteit Brussel, Flanders Interuniversity Institute for Biotechnology, Pleinlaan 2,B-1050 Brussels, BelgiumE-mail: [email protected]†Presented in part at the COST 840 Steering Committee Meeting and Expert’s Conference on ‘Applications of Immobilization/Bioencapsulation in Medicine,Pharmacy, Food Technology and Biotechnology’.(Received 2 December 2005; revised version received 1 March 2006; accepted 1 March 2006)Published online 21 June 2006; DOI: 10.1002/jctb.1582

2006 Society of Chemical Industry. J Chem Technol Biotechnol 0268–2575/2006/$30.00

R Willaert, VA Nedovic

achieve the correct balance of sensory compounds tocreate an acceptable flavour profile in such a short timeframe. ICT for beer production can only be introducedsuccessfully on an industrial scale if the flavour profilecan be controlled and fine-tuned.

CONTROL OF FLAVOUR-ACTIVE COMPOUNDSIN IMMOBILISED-CELL SYSTEMSSome of the most important flavour-active compoundsin beer are higher alcohols, esters and vicinal diketones.The mechanisms of the formation of these compoundsin beer fermentation by freely suspended cell systemsand changes that usually occur when immobilisedsystems are used are explained in the followingsections.

Influence of ICT on higher alcohol productionDuring primary beer fermentation, higher alcohols(also called ‘fusel alcohols’) are produced by yeastcells as by-products and represent the major frac-tion of the volatile compounds. Higher alcoholscan be classified into aliphatic [n-propanol, isobu-tanol, 2-methylbutanol (or active amyl alcohol) and3-methyl butanol (or isoamyl alcohol)] and aromatic(2-phenylethanol, tyrosol and tryptophol) higher alco-hols. Aliphatic higher alcohols contribute to the ‘alco-holic’ or ‘solvent’ aroma of beer and produce a warmmouthfeel. The aromatic alcohol 2-phenylethanol hasa sweet aroma and makes a positive contribution to thebeer aroma, whereas the aromas of tyrosol and tryp-tophol are undesirable. Higher alcohols are synthesisedby yeast during fermentation via the catabolic (Ehrlich)and anabolic pathways (amino acid metabolism).4–7

In the catabolic pathway, the yeast cells use amino

acids from the wort to produce the corresponding α-keto acids via a transamination reaction. The excessoxo acids are subsequently decarboxylated to aldehy-des and further reduced (by alcohol dehydrogenase) tohigher alcohols.8 In the anabolic pathway, the higheralcohols are synthesised from α-keto acids duringthe synthesis of amino acids from the carbohydratesource.6 The pathway choice depends on the indi-vidual higher alcohol and on the level of availableamino acids. The importance of the anabolic path-way decreases as the number of carbon atoms in thealcohol increases5 and increases in the later stage of aconventional batch fermentation as wort amino acidsare depleted.9

Yeast strain, fermentation conditions and wortcomposition all have significant effects on thecombination and levels of higher alcohols that areformed.9,10 Conditions which promote yeast cellgrowth – such as high levels of nutrients (aminoacids, oxygen, lipids, zinc), increased temperatureand agitation – stimulate the production of higheralcohols.11 The synthesis of aromatic alcohols isespecially sensitive to temperature changes. Onthe other hand, conditions which restrict yeastgrowth – such as lower temperature and higher(CO2) pressure – reduce the extent of higher alcoholproduction.11,12 The amino acid composition has amajor effect on higher alcohol formation: growthmedium supplemented with valine, isoleucine andleucine induced the formation of isobutanol, amylalcohol and isoamyl alcohol, respectively.13,14

Decreased higher alcohol production by immo-bilised as opposed to free-cell fermentation has beenreported (Table 1).25,27,32,34,35 This decrease has beenattributed to limited cellular growth in immobilised

Table 1. Higher alcohol production in immobilised-cell and free-cell systems (without reference to a footnote, the data correspond to the analysis of

green beer)

Compound (mg L−1) (flavour threshold) Carrier Immobilised cells Free cells Ref.

Propanol (600,15 80016) Porous glass beads 15.5a/17.2b – 2012.7 – 21

Diatomaceous earth 10.9 – 21DEAE-cellulose beads 11.6 10.4 22

31.8c/31.2d 23.0 23Ca-alginate beads 14.1 12.4 24

9.8e/7.5f 8.0 2519.8 14.0 26

Ca-pectate beads 6.4 6.1 27Silicon carbide rod 20.0 – 28Aspen wood chips 15.7–50.5g (24.9h) 10.0 29Beech wood chips 14.8i/20.4j 10.2 30Spent grains 15 17.5 31κ-Carrageenan 32.5j 9.9j 32

Isobutanol (100,15 80–100,17 20016) Porous glass beads 9.4a/12.1b – 20DEAE-cellulose beads 11.0 9.7 22

10.5k/18.9l/21.3m 10.4k/19.6l/20.6m 2529.0c/32.3d 24.0 23

Ca-alginate beads 9.9 10.6 247.5e/5.8f 16.0 25

(continued overleaf )

1354 J Chem Technol Biotechnol 81:1353–1367 (2006)DOI: 10.1002/jctb

Primary beer fermentation by immobilised yeast

Table 1. Continued


20.6 13.8 26Ca-pectate beads 13.4 19.6 27Silicon carbide rod 14.3 – 28Poly(vinyl alcohol) 31.7n/29.5o 30.1 33Lentikats

Aspen wood chips 7.5–10.8g (8.4h) 8.2 29Beech wood chips 8.0i/9.2j 6.5 30κ-Carrageenan 11.1j 7.8j 32Spent grains 10.1 12.5 31

Isoamyl alcohol (50,15 50–60,17 6516) Porous glass beads 35.8a/42.2b – 2033.9 – 21

Diatomaceous earth 32.8 – 21DEAE-cellulose 35.7 36.5 22


31.0e/38.0f 62.0 25Silicon carbide rod 51.2 – 28Aspen wood chips 30.0–59.5g (47.0h) 51.0 29Beech wood chips 32.5i/29.3j 29.7 30κ-Carrageenan 47.4j 46.7j 32

Amyl alcohol (50,15 50–60,17 7016) Porous glass beads 13.7a/15.8b – 20Beech wood chips 12.1c/12.6j 11.5 30DEAE-cellulose beads 16.2c/16.1d 12.1 23

Isoamyl alcohol + amyl alcohol Ca-alginate beads 70.2 62.2 26Poly(vinyl alcohol) 57.8n/57.2o 55.6 33Lentikats

Ca-pectate beads 56.8 93.4 27Spent grains 60.1 70 31

2-Phenyl ethanol (5,18 40,15 45–50,17 75,19 12516) Poly(vinyl alcohol) 4.1n/4.2o 4.1 33Lentikats

Ca-pectate beads 10.1 14.6 27

a Average over day 0 to 138.b Average over day 378–442.c Day 8.d Day 10.e Packed-bed reactor.f Fluidised-bed reactor.g Range.h Average.i Maturation with immobilised cells.j Conventional maturation.k 8 ◦C.l 15 ◦C.m 20 ◦C.n Recycled CO2.o Gas bottle CO2.

cell systems, leading to poor nitrogen removal. In con-trast, rapid yeast growth leads to enhanced anabolicproduction of amino acid precursors with concomi-tant overflow of higher alcohols, oxo acids, organicacids and vicinal diketones.36 Yeast growth controlby aeration has been employed to increase the levelof higher alcohols (see later). The Kirin Brewery(Japan) developed a two-stage immobilised fermenta-tion system.37,38 The first reactor was a stirred aeratedreactor with suspended yeast cells simulating the yeastgrowth in the beginning of a conventional batch fer-mentation. Most of the nitrogenous compounds wereremoved during this aerobic phase and a sufficientamount of higher alcohols was formed. The second

reactor was the immobilised cell reactor to perform ananaerobic fermentation where ethanol and esters werestill synthesised but not higher alcohols.

In the system of Pajunen et al.,29 where theyeast growth is very limited, it was found thathigher alcohol levels were mostly comparable tofree-cell batch fermentation and regarding some of thealcohols even at a higher level. This was especiallytrue for propanol. The high propanol productionwas linked to the relative high 2,3-pentanedioneconcentration, which was also measured in this system,and to α-ketobutyrate-mediated processes. It washypothesised that the overproduction was the resultof a more active α-ketobutyrate pathway. A high

J Chem Technol Biotechnol 81:1353–1367 (2006) 1355DOI: 10.1002/jctb


propanol production has also been reported for otherimmobilised systems.27,35,39

It has been demonstrated that mass (i.e. aminoacids) transfer limitations also have an influence onhigher alcohol synthesis. In a fluidised-bed reactor,it was found that the free amino nitrogen (FAN)uptake by entrapped yeast cells increased linearly withthe superficial velocity of the wort in the reactor.35,40

This relationship has also been found in a gas-liftbioreactor.41 The rate of amino acid uptake in thefluidised-bed reactor was very similar to that of afree-cell system.35 Amino acids of group I and IIwere removed at a rate equal to that of free cells.It was only group III amino acids (e.g. tryptophan)that were absorbed more slowly by immobilised yeastcells. Aivaisidis et al.42 also found that for yeast cells inalginate, a fluidised-bed reactor produced lower FANlevels than a packed-bed reactor; and higher apparentfluid velocities through the reactors resulted in lowerFAN levels.

Table 1 gives an overview of reported higher alcohollevels in immobilised systems compared with free-cellbatch fermentation. Tables 1–3 also demonstrate thatflavour formation is dependent on bioreactor systemand carrier material. Since fermentation conditionsand yeast strains differ for the data reported inTable 1, it is not possible to evaluate the influenceof the carrier material rigorously. Some researchershave evaluated the effect of the carrier material onthe formation of flavour products. Smogrovicovaand co-workers compared PVA Lentikats with Ca-alginate carrriers,33 and DEAE-cellulose, Ca-pectateand κ-carrageenan beads27 have been studied in a gas-lift bioreactor (Table 1). Higher ethanol evolution wascomparable for yeast cells entrapped in PVA Lentikatsand Ca-alginate carriers. This was also the casefor cells entrapped in Ca-pectate and κ-carrageenanbeads. However, the behaviour of cells adsorbed onDEAE-cellulose was similar to that of free cells, butsignificantly different from that of entrapped cells.Virkajarvi43 found that the carrier material (i.e. porousglass beads, Celite, DEAE-cellulose-based carrier) hadan effect on the higher alcohol concentration, butthe effect varied with the yeast strain used. It washypothesised that the immobilisation method (surfaceadsorption or adsorption on a porous material)and direct effects of the carrier (e.g. differences inadsorption of wort components on the carrier) couldexplain some of the observed effects.

Influence of ICT on ester productionEsters are very important flavour compounds in beer,since they can have very low flavour thresholdsand a major impact on the overall flavour. Theyhave an effect on the fruity/flowery aromas offermented alcoholic beverages. The major esterscan be subdivided into acetate esters and C6 –C10

medium-chain fatty acid ethyl esters. The majoresters in beer are ethyl acetate (aroma: fruity,solvent-like), isoamyl acetate (banana), ethyl caproate

(apple-like with note of aniseed), ethyl caprylate(apple-like) and 2-phenylethyl acetate (roses, honey,apple, sweetish).44,45 They are desirable componentsof beer when present in appropriate quantities andproportions, but can become unpleasant when theyare present in excess.

Esters are produced by yeast cells during the growthphase (60%) and the stationary phase (40%).46 Theyare formed by the intracellular reaction between a fattyacyl-coenzyme A and an alcohol. This reaction is catal-ysed by an alcohol acyltransferase (or ester synthetase).The regulation and control of ester synthesis are not yetfully understood. Ester formation is highly dependenton the yeast strain used45,47 and on certain fermen-tation parameters such as temperature,14,48,49 specificgrowth rate,49 pitching rate49–51 and top pressure.46

Additionally, the concentrations of assimilable nitro-gen compounds,14,52,53 carbon sources,54–57 dissolvedoxygen14,58–60 and fatty acids61,62 can influence theester production rate. The carbohydrate source alsohas an influence on the ester synthesis. Maltose pro-duces less esters than glucose and fructose.56 It hasbeen shown that the main factor controlling esterbiosynthesis is the expression level of the ATF1 gene,which codes for alcohol acetyl transferase I.63,64 ATF1gene expression is repressed by oxygen and unsatu-rated fatty acids.65,66

It has been suggested that the low ester concen-trations that were obtained in some immobilisedprocesses (see also Table 2) are related to the low cellu-lar metabolic activities in these systems.34,68 It has alsobeen reported that for some systems, ester synthesisis increased upon cell immobilisation (Table 2). Thishas been explained as follows: because of mass transferlimitations, oxygen concentrations in the immobilisa-tion matrix are low, causing reduced cellular growth,so that the cellular acetyl-CoA pool can be more avail-able for ester synthesis instead of channelling for fattyacid biosynthesis.68 A reduction in cellular total fattyacid content upon yeast immobilisation on stainless-steel fibre cloth has been measured and supports thisexplanation.69 Ester synthesis during the productionof alcohol-free beer in a packed-bed reactor with sur-face attached cells on DEAE-cellulose beads has beenstudied in detail.70 During fermentation, a simultane-ous increase in the activity of alcohol acetyl transferaseand formation of ethyl acetate and isoamyl acetatewere observed. Additionally, the amount of unsatu-rated fatty acids in wort decreased significantly. Itwas concluded that the anaerobic conditions and theabsence of substantial levels of unsaturated fatty acidslimit cell growth during production and stimulate theformation of acetate esters. In a recent study, theobserved 22% increase in ester concentration uponcell immobilisation on stainless-steel fibre cloth wasinvestigated by measuring the transcription rates ofseveral genes.71,72 The expression level of AFT1 wassignificantly raised in the immobilised cells, resultingin a twofold increase in isoamyl acetate formation.The activity of AFT1 is regulated by the protein



Table 2. Ester production in immobilised-cell and free-cell systems (without reference to a footnote, the data correspond to the analysis of green

beer)


Ethyl acetate (20–30,67 3044) Porous glass beads 23.4a/15.6b – 2024.4 – 21



11.0 e/8.5f 19.0 2518.8 15.2 26

Ca-pectate beads 11.6 16.8 27Silicon carbide rod 31.6 – 28Poly(vinyl alcohol) 10.1g/5.4h 5.2 33Lentikats

Aspen wood chips 20.1–39.8i (26.4j) 21.5 29Beech wood chips 21.4k/24.5l 17.2 30Spent grains 17.9 17.2 31κ-Carrageenan 11.3l 26.4l 32

Isoamyl acetate (0.6–1.2,67 1.244) Porous glass beads 0.7a/0.6b – 201.2 – 21



0.06e/0.05f 2.0 251.25 0.85 26

Ca-pectate beads 3.19 2.11 27Silicon carbide rod 1.3 – 28Poly(vinyl alcohol) 2.09g/0.98h 1.06 33Lentikats

Aspen wood chips 0.3–1.9i (1.0j) 1.5 29Beech wood chips 1.0k/0.6l 1.0 30κ-Carrageenan <0.01l 0.08l 32

Ethyl caproate (0.17–0.21,67 0.2144) Porous glass beads 0.1a/0.1b – 20Poly(vinyl alcohol) 0.54g/0.13h 0.11 33Lentikats

DEAE-cellulose beads 0.08c/0.01d 0.14 23Ca-pectate beads 1.89 1.39 27Aspen wood chips 0.1–0.5i (0.2j) 0.4 29Beech wood chips 0.1k/0.1l 0.1 30

Ethyl caprylate (0.3–0.9,67 0.944) Poly(vinyl alcohol) 0.04g/0.01h 0.01 33Lentikats

Ca-pectate beads 0.21 0.59 27Aspen wood chips 0.2–1.2i (0.6j) 1.4 29

a Average over days 0 to 138.b Average over days 378–442.c Day 8.d Day 10.e Packed-bed reactor.f Fluidised-bed reactor.g Recycled CO2.h Gas bottle CO2.i Range.j Average.k Maturation with immobilised cells.l Conventional maturation.

kinases Sch9p and PKA, involved in stress responsesand nutrient-sensing signalling pathways. The resultsof the expression profile of the stress-related genesHSP12 and SSA3 revealed that the immobilised cellswere under less stressful conditions than free cells.

It was concluded that the particular microenviron-ment created by cell immobilisation possibly activatesthe cAMP/PKA/Sch9 pathway, resulting in an induc-tion of ATF1 expression, leading to enhanced esterconcentrations in the final fermentation product.



Table 3. Vicinal diketone production in immobilised-cell and free-cell systems (without reference to a footnote, the data correspond to the analysis

of green beer)


Diacetyl (0.1–0.1577) Porous glass bead 0.68 – 21Diatomaceous earth 0.79 – 21DEAE-cellulose 0.70 0.40 22

0.40a/0.33b 0.12 23Ca-alginate bead 0.04 0.04 24

0.28c/0.08d 0.20–0.32 26,78Ca-pectate beads 0.09 0.19 27Silicon carbide rod 0.48 – 28

0.45e, 0.36f – 79Poly(vinyl alcohol) 0.11g/0.08h 0.09 33Lentikats

Aspen wood chips 0.1–0.4i 29Beech wood chips 0.02j/0.02k 0.01 30κ-Carrageenan 0.015k <0.02k 32Gluten pellets 0.09l/0.19m/0.18n 0.20p 80Spent grains 0.3 – 31

2,3-Pentanedione (1–1.577) Aspen wood chips 0.1–1.2i – 29Beech wood chips 0.01j/0.01k 0.00 30DEAE-cellulose 0.061a/0.068b 0.02 23

a Day 8.b Day 10.c Batch process.d Continuous process.e Day 2.f Day 30.g Recycled CO2.h Gas bottle CO2.i Range.j Maturation with immobilised cells.k Conventional maturation.l 15 ◦C.m 10 ◦C.n 5 ◦C.

These conflicting results and the data in Table 2indicate that ester synthesis can be inhibited orstimulated when yeast cells are immobilised. Atpresent, it is not clear how ester synthesis inimmobilised systems is regulated and further researchis needed to clarify the observations.

Influence of ICT on production of vicinaldiketones (VDK)VDK are produced as by-products of the synthesispathway of isoleucine, leucine and valine (ILV path-way) during fermentation. Two of these compounds,diacetyl (2,3-butanedione) and 2,3-pentanedione, areimportant in beer. They are characterised by a butteryand sweetish aroma. Diacetyl is sensorily more impor-tant than 2,3-pentanedione. It has a taste thresholdaround 0.10–0.15 mg L−1 in lager beer, approximately10 times lower than that of pentanedione.73

The formation, subsequent re-assimilation bythe yeast and degradation of diacetyl are shownschematically in Fig. 1. Yeast cells possess thenecessary enzymes (reductases) to reduce diacetylto acetoin and further to 2,3-butanediol and 2,3-pentanedione to 2,3-pentanediol. These reducedcompounds have much higher flavour thresholds

and have no impact on the beer flavour.74,75 Thereduction reactions are yeast strain dependent. Thereduction occurs at the end of the conventional mainfermentation and during the maturation. Sufficientyeast cells in suspension are necessary to obtainan efficient reduction. Fermentation parameters thatstimulate yeast cell growth may increase the levelof α-acetolactate (which is a precursor of diacetyl)and consequently also the diacetyl concentration. Thecontent of branched-chain amino acids in group II(valine, leucine and isoleucine are classified in thisgroup)76 also has a significant influence on diacetylformation, owing to the link between the ILV pathwayand VDK synthesis.10

Table 3 shows that for some cases the productionof diacetyl by immobilised cells is much higher thanfor free cells. This was also noticed in early studies onprimary fermentations with immobilised cells.25,81 Ithas been demonstrated that the production of vicinaldiketones can be controlled by the initial yeast cellconcentration in Ca-alginate beads.41,82 This has beenexplained by an increased expression of acetohydroxyacid synthetase gene during growth of the yeast cellsin the carrier.82 The pentanedione pathway seems tobe more active in immobilised yeast systems in a very



Diacetyl

Slow chemical oxidativedecarboxylation

Fermenting wort

Pyruvate

Yeast cell

Extract

α-acetolactate

Diacetyl

Acetoin

2,3-butanediol

Valine

Acetohydroxy acidsynthetase

diacetylreductase

butanedioldehydrogenase

Glucose

EMP pathway

α-acetolactate

Figure 1. Schematic presentation of diacetyl formation, reassimilation and removal.

limited or no growth situation with excess availableamino acids and continuous sugar feed, as was thecase for a recirculation system under pressure.29 Inthis system, the concentration of 2,3-pentanedionewas 2–4 times larger than the diacetyl concentration.When the wort feed was closed but recirculationwas continued, it was possible to reduce both VDKconcentrations below the flavour threshold values.This indicates that the oxidative decarboxylation ofthe VDK precursors was not complete in this system.

In an air-lift reactor with spent grains as immobilisa-tion matrix, high levels of diacetyl (Table 3) were mea-sured in the entire dilution rate range (0.04–0.12 L−1)as a result of the intense biomass growth.31,83 Aftera maturation period of 10 days at 4 ◦C, the con-centration of diacetyl was reduced below its flavourthreshold.31 Low concentrations of diacetyl and a highvolumetric productivity were obtained at high valuesof the total biomass concentration after optimisationof operational parameters in a gas-lift bioreactor withalginate microbeads as yeast carriers.78,84 It has alsobeen reported that diacetyl synthesis decreased as afunction of time during continuous fermentation withimmobilised cells.23,79 This can be explained by areduced cell growth after start-up and increased masstransfer limitations due to the high biomass density.

Control strategiesTemperatureThe influence of the temperature (5–20 ◦C) onthe synthesis of volatile flavour-active by-productshas been investigated for bottom-fermented yeastentrapped in calcium pectate, κ-carrageenan andadsorbed on DEAE-cellulose during batch fermenta-tions.27 Gel-immobilised yeast cells produced lower

amounts of diacetyl and higher alcohols for alltemperatures studied. Ester formation was lowerat temperatures from 5 to 15 ◦C and acetaldehydeformation from 5 to 12 ◦C. Gel immobilisation alsoresulted in a lower amino acid utilisation. The aromacharacter of beers produced by yeast adsorbed onDEAE-cellulose at different temperatures was similarto that of beers produced by free yeast cells. As isthe case for free cells, the concentration of diacetyl,acetaldehyde, higher alcohols and esters in beersfermented with yeast cells immobilised on DEAE-cellulose increased with increasing temperature. Thesame trend has been noted in the repeated batchfermentations of wort at various temperatures (in therange 0–15 ◦C) with freeze-dried immobilised yeastcells on gluten pellets.85 Production rates of ethylacetate and isoamyl acetate were approximately two- tofourfold higher at 12 than 2 ◦C during the productionof alcohol-free beer.70 Pajunen et al.29 noted that evenat high temperatures (16–24 ◦C), low-flavour beerscan be produced with the right process conditions. Incontrast, in a two-stage immobilised cell system usingporous silicon carbide rods, operation at 20 instead of15 ◦C resulted in both reactors in a 60% increase involumetric productivity, but the organoleptic qualityof the beer was converted to an unacceptable level.86

Wort gravity and feed volumeBy controlling the feed volume (and recirculation),the desired attenuation and ethanol production canbe obtained within narrow limits.29 This parametercan be controlled on-line. It has been shown thatwort gravity and alcohol content also have a flavourimpact and can be used to control the flavour.29



In a study to assess the influence of the originalgravity (15, 18 and 21 ◦P) on the productivity ofimmobilised (porous glass beads) yeast reactors,acetaldehyde concentration differed notably from acommercial reference beer.87 However, there was areasonable match for the other measured compounds(propanol, 2-methylpropanol, 3-methylbutyl acetate,2-methy butanol, 3-methylbutanol, ethyl acetate,ethyl caproate). Acetaldehyde production was highestat the highest gravity, which was attributed toincomplete attenuation. For ethyl acetate, propanol, 2-methylpropanol and 3-methylbutanol, the productionwas highest at 24 ◦P. The production of thesecompounds was lowest at 18 ◦C.

When the flow-rate was increased from 500 to750 mL h−1 in a continuous system composed oftwo packed-bed column reactors with porous glassbeads, the concentrations of 3-methylbutanol, 2-methylbutanol, 2-methylpropanol, propanol and ethylacetate were decreased.20 It was mentioned that thechanges may be a result of the shock (sudden increaseof the dilution rate) to the yeast.

Wort compositionImmobilised yeast systems, which are characterisedby a very limited or no cell growth, have a lowconsumption level of FAN. This has an effect onthe beer flavour and gives a higher pH, which impartsthe beer freshness and gives a higher vulnerabilityto microbial contamination. This problem can beapproached by various strategies using low-FANmalt, FAN dilution by adjuncts, pH adjustments orcombination of these strategies.29 Another approachwas to grow freely suspended cells in the wort beforepassing through the column with immobilised cells,37

or to use gas-lift bioreactors,41,83,88 or with inducedliquid circulation.29

AerationVDK and α-amino nitrogen levels have been con-trolled by using a stirred aerated fermenter as the firstreactor in a continuous ICT process.24,89,90 Higheralcohol formation in immobilised cell systems couldbe controlled by aeration.39,91 The effect of aera-tion on the aroma compounds have been investigatedusing mathematical models.20,43 The oxygen transferin the used packed-bed column reactor resulted froma complex function of (among other things) the con-centration of oxygen in the fermenting wort and thesuperficial velocity of gas in the column.

Caution should be exercised in sparging withair, since excess oxygen will lead to low esterproduction but excessive, diacetyl, acetaldehyde andfusel alcohol formation.92–95 The optimisation ofoxygen availability appears critical for the control offlavour-active compound formation in immobilisedsystems.96 Kronlof and Linko97 successfully ensureda constant dissolved oxygen concentration in theirimmobilised yeast reactor by sparging with a mixtureof air and nitrogen as an inert gas. In a gas-lift

bioreactor system with yeast cells immobilised inalginate microbeads and on spent grains, an optimumflavour and aroma profile of the continuous producedgreen beer was obtained at zero airflow in the gas feed(mixing induced by pure N2 and CO2, respectively)and temperatures between 13 and 17 ◦C.31,78,88

TOWARDS ICT APPLICATIONS IN PRIMARYBEER FERMENTATIONThere has been interest in the application ofimmobilised cell technology since its development inthe 1970s. Narziss and Hellich98 developed one of thefirst well-described ICT processes for beer production.Their yeast cell immobilisation method was based ona method for the immobilisation of enzymes.99 Yeastcells were immobilised in kieselguhr (which is widelyused in the brewing industry as a filter aid) and akieselguhr filter was employed as bioreactor (called the‘bio-brew bioreactor’). This process was characterisedby a very low residence time of 2.5 h, but requiredthe addition of viable yeast and a 7-day maturationperiod to reduce the high concentration of VDK inthe green beer. Although this result looks very good,the bio-brew bioreactor overall gave no satisfactoryresults. The most serious problem was the highamount of α-acetolactate in the green beer.100 Theamino nitrogen consumption was reduced (probablyowing to the limited yeast growth), resulting in a lowconcentration of higher alcohols and esters and a highpH. Furthermore, the yeast viability at the reactoroutlet was decreased, the system had a short lifetimeof 7–10 days before clogging occurred and the foamstability of the final beer was decreased.101 Twentyyears later, this process has been further optimised.102

An aerobic reactor was installed immediately upstreamof the bio-brew reactor, the beer flow through the filterwas optimised and a cooling plate was installed in thefilter reactor to control the temperature. This resultedin an increased yeast viability in the reactor and animproved sensory quality of the beer. However, theconcentration of the low molecular weight nitrogenoussubstances in the beer remained too high.

Baker and Kirsop103 were the first to use heattreatment of green beer to accelerate considerablythe chemical conversion of α-acetolactate to diacetyl.They designed a two-step continuous process. Thefirst reactor is a packed-bed reactor containing alsokieselguhr with immobilised yeast cells to performthe primary fermentation. The green beer was heatedusing a heating coil to accelerate the α-acetolactateconversion. It was next cooled before it entered asmaller packed-bed reactor to perform the secondaryfermentation. Problems associated with this processwere gradual blocking of the packed-bed and achanged beer flavour.

In 1973, the first industrial-scale process usinggel (polyacrylamide, later replaced by κ-carrageenan)immobilised non-living Escherichia coli cells (contain-ing aspartase activity) to produce L-aspartic acid from



fumaric acid was implemented by Tanebe Seiyaku inJapan.104 Since the introduction of alginate gel as anentrapment matrix for living yeast cells for the pro-duction of beer by White and Portno in 1979,55 therehas been increased research activity in the develop-ment of ICT processes for the production of beer. Ina laboratory-scale tower fermenter, these researcherswere able to produce beer with a flavour that wascomparable to that of the control (batch fermentationwith free cells). This process was operational during7 months. It was found that gel entrapment gave pro-tection against contaminating bacteria. Furthermore,the concentration of ethyl acetate decreased duringlong-term operation.

Investigations on beer fermentation by yeast cellsimmobilised in alginate beads continued throughthe 1980s and 1990s until today.24,26,34,41,82,84,105–107

In addition, some new porous materials wereintroduced, such as κ-carrageenan, pectate gels andpoly(vinyl alcohol) (PVA).27,78,108–110 It was provedthat the main advantage of the cell entrapmentmethod is the attainment of extremely high cellloadings, consequently providing high fermentationrates. However, in some cases cell proliferation andactivity can be limited by low mass transfer rates withinthe matrices. The reduced cell growth in immobilisedconditions can result in an insufficient free aminonitrogen consumption and as a consequence anunbalanced flavour profile of the final beer.34,111

This was particularly the case for immobilised cellsin packed-bed fermenters where high mass transferrestrictions, accumulation of carbon dioxide, non-uniform temperature profiles, flow channelling andstagnant zones were observed during fermentation.Therefore, different approaches for the adaptation ofimmobilised systems were investigated in order tocorrect the final beer quality. The crucial elementswere cell carriers and bioreactor design.

One of the concepts was to improve the processdesign incorporating packed-bed fermenters. TheJapanese Kirin Brewery developed a multistage systemparticularly aimed at increasing nitrogen consumption

and improving beer flavour. The first stage in thissystem is utilised for an aerobic fermentation, whichfavours adequate yeast cell growth with desirablefree amino nitrogen consumption, while the followingstages are used for ordinary anaerobic fermentations.In the three-stage reactor system of Kirin (Fig. 2), thefirst stage is carried out in a chemostat, followed bya series of packed-bed fermenters.37,89,112 Ca-alginatebeads were initially selected as carrier material toimmobilise the yeast cells; they were later replaced byceramic beads (Bioceramic). Beer could be producedin this process within 3–5 days (see also Tables 1–3).This pilot-scale (100 hL) system was operational for2 years in a restaurant brewery on Saipan Island(Northern Mariana Islands). The production scalewas 5 hL per day and beer of acceptable quality wasproduced.

Hartwell Lahti and VTT Research Institute (Fin-land) developed a primary two-stage fermentationsystem (packed-bed bioreactors) using ICT on a pilotscale of 600 L per day.22,113 This system was laterextended to include a continuous secondary fermen-tation unit with the same capacity.114 Wood chipswere used as carrier material, which reduced the totalinvestment cost by one-third compared with other,more expensive carriers. The results showed that inonly 40 h beer composition and flavour were very sim-ilar to those of beer produced by the traditional batchprocess.

Synebrychoff Brewery (Finland) in collaborationwith Guinness, GEA Liquid Processing Scandinaviaand Cultor Corporation of Finland developed a newICT process in which the concentration of carbondioxide is controlled in a fixed-bed reactor.23 In thisway, forced circulation of fermenting beer is estab-lished, channelling and carbon dioxide accumulationare avoided and mass and heat transfer are enhanced.The CO2 formed is kept dissolved and removed fromthe beer without foaming problems. DEAE-celluloseat the beginning and wood chips later were used ascarrier materials.29,115 Good-quality beer and a con-stant flavour profile were achieved at a production

Yeast

Wort

Greenbeer

BeerAir

Heat exchanger

Off gas

Off gas

Off gasOff gas

First stageStirred tank fermenter

Second stagePacked bed fermenters

Third stagePacked bed maturation

column

Centrifuge

Coolingjackets

Coolingpipes

Figure 2. Kirin’s three stage fermenter system for continuous fermentation (adapted from Inoue, 1995).37



time of 20–30 hours (see also Tables 1–3). In a pilotplant (reactor volume 10 hL, 12 hL per day), thefermentation process and flavour formation could becontrolled by using the feed rate, recirculation rateand fermentation temperature as control variables.29

Fluidised-bed fermenters have been used to improvemass transfer further in immobilised cell systems.The upward flow-rate of feed medium in thesefermenters is high enough to provide fluidisation of cellcarriers, resulting in enhanced mixing properties andmore uniform medium distribution as compared withpacked-bed fermenters. The same type of solid porouscarriers as in packed-bed systems are used.116–118

However, drawbacks of fluidised systems are possiblecarrier abrasion and damage. In addition, fluidisationof glass and ceramic carriers may require high mediumflow–rates, which could result in high pumping costsand cell leakage. On the other hand, in the case of low-density carriers, fluidisation may require low flow-ratesat which the mass transfer rates could be too low forpractical applications.

A silicon carbide cartridge loop bioreactor systemwas developed by the Belgian company Meura in anattempt to overcome problems inherent to packed andfluidised-bed fermenters.28,79,86,95,119–121 It consists ofan immobilised cell reactor where partial attenuationand yeast growth occur, followed by a free-cellstirred-tank reactor for complete attenuation, esterformation and flavour maturation. The immobilisedcell bioreactor contains silicon carbide multichannelporous rods (60% void volume) seeded with yeastcells and perfused in parallel with recirculating feedmedium (Fig. 3). The stirred tank in this system iscontinuously inoculated by free cells, which escapefrom the immobilised cell reactor. The system ischaracterised by a simple design, which can be easilyscaled up. Disadvantages are the relatively high costof silicon carbide matrices and lower cell growthand specific productivity in this system as comparedwith free cells. The immobilised reactor system hasbeen found to be stable over a period of more than6 months, producing ale and lager beer of excellentquality (Tables 1–3).119

CO2 CO2

WortVessel

BeerVessel

Heatexchanger

Immobilised YeastFermenter

Suspended YeastFermenter

Siliconcarbide

rod

Figure 3. Silicon carbide cartridge loop fermenter (adapted fromAndries et al., 2000).119

Figure 4. Gas-lift bioreactor system (adopted from Nedovic et al.,2005).3

A gas-lift bioreactor system, which was intro-duced in beer fermentation experimental studiesby the research group at Belgrade University,105 isanother promising concept for main beer fermenta-tion (Fig. 4). In this system, mixing is establishedby the circulation of liquid and solid phases, pro-viding high liquid recirculation rates, low shear envi-ronment and good mass transfer properties.122–126

Other important characteristics of gas-lift fermentersare high loading of solids, simple construction, lowrisk of contamination, easy adjustment and controlof the operational parameters and simple capac-ity enlargement.26,107,127 Low-density alginate,106,128

carrageenan,27,109,110 pectate27,129 and PVA33,78,84

particles are typically used in three-phase gas-lift fer-menters as carriers of yeast cells.26,27,88,105,108 In spiteof the fact that gel carriers such as alginate, car-rageenan and pectate demonstrated limited mechani-cal stability and resistance when applied in packed-bedreactors, these porous matrices represent a good solu-tion for yeast immobilisation in conjunction withgas-lift bioreactors. They also provide higher cell con-centrations and better cell retention in comparisonwith preformed firm carriers. In addition, several newtechniques (such as jet cutter technology and vibrationtechnology)130,131 for cell immobilisation in porousmatrices allow for large (industrial)-scale productionof gel particles. Laboratory- and semi-pilot-scale biore-actor systems were developed with alginate microbeads(0.8 mm in diameter) and lens-shaped PVA particles(LentiKats) as carriers for yeast cells.28,78,84 Fullbeer attenuation in these studies was reached within7.5–20 h depending on solid loading (10–40%). Thefinal beers had the desired sensory and analytical pro-files.



A combined continuous immobilised system for pri-mary fermentation and maturation has been developedand characterised.33 The primary fermentation wascarried out in a gas-lift bioreactor and the maturationin a packed-bed reactor. The green beer was heattreated (to accelerate the conversion of α-acetolactateto diacetyl) before it was fed to the maturation reac-tor. Yeast cells were entrapped in lens-shaped PVAparticles and used to perform both the primary fer-mentation and the maturation. The system was stablefor 2 months at a residence time of 24–36 h. Thebeer produced was characterised by a compositionand flavour profile which was similar to that of beerproduced by a classical batch fermentation (see alsoTables 1–3).

A similar system using yeast cells entrapped in small(1 mm) κ-carrageenan gel beads in a gas-lift reactorwas developed by Labatt Breweries (InBev, Canada)in collaboration with the Department. of Chemicaland Biochemical Engineering at the University ofWestern Ontario.32,108,109 Pilot-scale (50 L) researchshowed that in this system full attenuation was reachedin 20–24 h compared with 5–7 days required in thetraditional batch fermentation. Although the flavourprofiles of the beer produced using ICT and the batch-fermented beer differed somewhat (see Tables 1–3),a taste panel judged the immobilised cell product tobe acceptable and, overall, similar to the conventionalcontrol fermentation product.

As an alternative, preformed support material basedon spent grains, a brewing by-product with a consid-erable cellulose content, has been proposed recentlyfor yeast cell immobilisation for beer fermentation inan air-lift bioreactor.31,83,132,133 This is an interestingcarrier for cell immobilisation since spent grains area waste by-product from the brewing process. Threemechanisms of yeast immobilisation were described:cell–carrier adhesion, cell–cell attachment and celladsorption (accumulation) inside natural shelters onthe carrier’s surface.134 Initial experiments demon-strated the feasibility of the system.83 Immobilised cellsshowed an approximately 40 ± 20% lower specificsugar consumption rate compared with suspendedcells and contributed 45–75% to the total fermen-tation in the system. The productivity of the systemin terms of ethanol concentration in green beer wassatisfactory, although the diacetyl concentration washigh. The operational conditions in terms of vol-umetric productivity and organoleptic quality werefurther optimised.31 An optimum higher alcohols-to-esters ratio in green beer was found at approximately2 mg L−1 of oxygen dissolved in wort, mixing inducedby pure CO2 and temperatures of 13–16 ◦C. At hightotal biomass concentration, the diacetyl formationwas low and the volumetric productivity of the sys-tem was high. In a comparison between the aromaprofiles of two lager beers produced by a continu-ous primary fermentation with immobilised cells andcombined with a batch maturation and an industrialbatch process with free cells, no significant differences

were found, except for the slightly lower content ofhigher alcohols.31 Despite the significant mechanicalstability of the spent grains, it was noticed that the car-rier material gradually disintegrated and was washedout during a continuous fermentation run of 55 days;this was solved by repeatedly replacing the carriermaterial during reactor operation.54 Another disad-vantage is that a rather complex and time-, energy-and chemical-consuming process is necessary to treatthe spent grains before they can be used as immobili-sation matrix. The results obtained indicate a high riskof cell detachment from the carrier surface since theimmobilised cell system was sensitive towards shearstress.

CONCLUSIONThe use of immobilised yeast cell systems in industryhas been extensively reported in the literature andthe technology has the potential to revolutionisethe brewing industry by replacing traditional batchbrewing operations by continuous ones. Earlierattempts to use continuous (free-cell) fermentationor even totally continuous breweries in the 1960sand 1970s were generally not successful, althoughthe continuous process from Morton Coutts inNew-Zealand (DB Breweries) has been operationalsince 1959. For the ‘conservative’ brewer to considerchanging, the benefits of the new technology mustbe large compared with the classical process andboth scientifically and technologically well established.Industrial-scale systems utilising immobilised yeastcells have already been used for the production oflow alcohol beers and for the maturation of beer.

A major challenge to the successful applicationof ICT on an industrial scale is the control andfine-tuning of the flavour profile during a combinedprimary and secondary fermentation, since manyparameters can influence flavour formation. Variouscontrol strategies have been tested at both laboratoryand pilot scales and which have resulted in satisfactoryflavour profiles in several instances. However, furtherresearch is needed, especially to provide a betterunderstanding of the physiological behaviour ofimmobilised yeast during fermentation. Observeddifferences in flavour profile could then be betterexplained and a more focused metabolic optimisationperformed.

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