wine_yeasts_for_the_future_7294.pdf

M I N I R E V I E W

Wineyeasts for the futureGraham H. Fleet

Food Science, School of Chemical Sciences and Engineering, University of New South Wales, Sydney, NSW, Australia

Correspondence: Graham H. Fleet, Food

Science, School of Chemical Sciences and

Engineering, The University of New South

Wales, Sydney, NSW, Australia, 2052. Tel.:

161 2 9 3855564; fax: 161 2 93855931;

e-mail: [email protected]

Received 9 June 2008; revised 11 July 2008;

accepted 13 July 2008.

First published online 12 September 2008.

DOI:10.1111/j.1567-1364.2008.00427.x

Editor: Patrizia Romano

Keywords

wine yeasts; Saccharomyces ; non-

Saccharomyces ; selection; fermentation.

Abstract

International competition within the wine market, consumer demands for newer

styles of wines and increasing concerns about the environmental sustainability of

wine production are providing new challenges for innovation in wine fermenta-

tion. Within the total production chain, the alcoholic fermentation of grape juice

by yeasts is a key process where winemakers can creatively engineer wine character

and value through better yeast management and, thereby, strategically tailor wines

to a changing market. This review considers the importance of yeast ecology and

yeast metabolic reactions in determining wine quality, and then discusses new

directions for exploiting yeasts in wine fermentation. It covers criteria for selecting

and developing new commercial strains, the possibilities of using yeasts other than

those in the genus of Saccharomyces, the prospects for mixed culture fermentations

and explores the possibilities for high cell density, continuous fermentations.

Introduction

International competition within the wine market, consumer

demands for newer styles of wines and increasing concerns

about the environmental consequences of wine production

are providing new challenges for innovation in wine fermen-

tation technology (Bisson et al., 2002; Pretorius & Hoj, 2005).

Until about the 1980s, the contribution of yeasts to wine

production was seen as a relatively simplistic concept. Essen-

tially, grape juice underwent a natural or a spontaneous

alcoholic fermentation that, almost invariably, was dominated

by strains of the yeast, Saccharomyces cerevisiae. Consequently,

pure cultures of this yeast were isolated and developed as

starter cultures for conducting wine fermentations. Many

other species of yeasts were known to occur in grape juice

and contribute to the first stages of fermentation but,

generally, they were considered to be of secondary significance

or undesirable to the process. By appropriate inoculation of

starter cultures and addition of sulphur dioxide to the juice,

winemakers aimed to eliminate or minimize the influence of

yeasts other than the strain of S. cerevisiae they inoculated.

With this technology, winemakers had established reasonably

good control of the process (Benda, 1982; Lafon-Lafourcade,

1983; Reed & Nagodawithana, 1988).

During the last 25 years, major advances have occurred in

understanding the ecology, biochemistry, physiology and

molecular biology of the yeasts involved in wine production,

and how these yeasts impact on wine chemistry, wine

sensory properties and appeal of the final product (see

reviews of Pretorius, 2000; Fleet, 2003, 2007; Swiegers et al.,

2005). Now, the yeast ecology of the fermentation has been

found to be much more complex than assumed dominance

of the inoculated strain of S. cerevisiae, and the metabolic

impact of yeasts on wine character is much more diverse

than simple fermentation of grape juice sugars. With this

greater knowledge and understanding, alcoholic fermenta-

tion is now seen as a key process where winemakers can

creatively engineer wine character and value through better

yeast management and can strategically tailor wines to a

changing market.

This article discusses new directions for exploiting yeasts

in wine fermentation. It covers criteria for selecting and

developing new commercial strains, the possibilities of using

yeasts other than those in the genus of Saccharomyces, the

prospects for mixed culture fermentations and explores the

possibilities for high cell density, continuous fermentations.

The yeast ecology of fermentation

A sound knowledge of the yeast species that conduct the

alcoholic fermentation and a sound knowledge of the

kinetics of their growth throughout this fermentation are

FEMS Yeast Res 8 (2008) 979–995 c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

essential first steps in understanding how yeasts impact on

wine quality and in developing new directions. It has been

known for a long time that freshly crushed grape juice

harbours a diversity of yeast species, principally within the

genera Hanseniaspora (anamorph Kloeckera), Pichia, Candi-

da, Metschnikowia, Kluyveromyces and Saccharomyces. Occa-

sionally, species in other genera such as Zygosaccharomyces,

Saccharomycodes, Torulaspora, Dekkera and Schizosaccharo-

myces may be present (reviewed in Fleet & Heard, 1993;

Fleet, 2003). These yeasts originate from the microbial

communities of the grape berry and the microbial commu-

nities of the winery environment. It is also well known that

many of these non-Saccharomyces species (especially species

of Hanseniaspora, Candida, Pichia and Metschnikowia)

initiate spontaneous alcoholic fermentation of the juice,

but are very soon overtaken by the growth of S. cerevisiae

that dominates the mid to final stages of the process – most

often being the only species found in the fermenting juice at

these times (Fleet & Heard, 1993; Fleet, 2003). Based on

these early ecological studies, S. cerevisiae and the related

species Saccharomyces bayanus were considered to be the

yeasts of main relevance to the process and, logically, they

became the species around which starter culture technology

was developed (Reed & Nagodawithana, 1988; Degre, 1993).

It remained until the 1980s before a sound understanding

was obtained about the quantitative growth of individual

yeast species throughout juice fermentation (Fleet et al.,

1984; Heard & Fleet, 1986). These studies showed that the

non-Saccharomyces species usually achieved maximum po-

pulations of 107 CFU mL�1 or more in the early stages of

fermentation before they died off. It was concluded that this

amount of biomass was sufficient to impact on the chemical

composition of the wine and that the contribution of these

yeasts to overall wine character was much more significant

than thought previously. Under certain circumstances, such

as fermentation at lower temperatures, some non-Sacchar-

omyces species did not die off and remained at high

populations in conjunction with S. cerevisiae until the end

of fermentation (Heard & Fleet, 1988). Moreover, it was

shown that these indigenous non-Saccharomyces yeasts also

grew in grape juice fermentations to which starter cultures

of S. cerevisiae had been inoculated (Heard & Fleet, 1985).

Consequently, broadly accepted assumptions that starter

cultures would overwhelm the growth of non-Saccharomyces

yeasts and prevent their contribution to wine character were

not necessarily valid. Many studies in various wine regions

of the world have now confirmed the important contribu-

tion that non-Saccharomyces species make to the overall

kinetics of yeast growth during both spontaneous and S.

cerevisiae-inoculated wine fermentations (Mora et al., 1988;

Schutz & Gafner, 1994; Lema et al., 1996; Egli et al., 1998;

Granchi et al., 1999; Pramateftaki et al., 2000; Povhe-Jemec

et al., 2001; Jolly et al., 2003a; Combina et al., 2005a, b; Zott

et al., 2008). This conclusion is also supported by more

recent ecological studies using culture-independent mole-

cular methods for yeast analyses (Cocolin et al., 2000; Mills

et al., 2002; Xufre et al., 2006; Nisiotou et al., 2007).

Consequently, the impact of non-Saccharomyces yeasts on

wine fermentations cannot be ignored. They introduce into

the process an element of ecological diversity that goes

beyond Saccharomyces species and they require specific

research and understanding to prevent any unwanted con-

sequences they might cause or to exploit their beneficial

contributions (Ciani & Picciotti, 1995; Jolly et al., 2003b).

Using molecular techniques that enable differentiation of

strains within a species, further ecological sophistication of

the fermentation has been discovered. Within each species,

there is an underlying growth of a succession of different

strains. As many as 10 or more genetically distinct strains of

S. cerevisiae have been found to contribute to the one

fermentation (Sabate et al., 1998; Pramateftaki et al., 2000;

Cocolin et al., 2004; Ganga & Martinez, 2004; Sipiczki et al.,

2004; Santamaria et al., 2005). In some cases, strains of S.

cerevisiae inoculated as starter cultures were not able to

compete successfully with indigenous strains and, therefore,

did not dominate the fermentation as expected (Querol

et al., 1992; Schutz & Gafner, 1994; Constanti et al., 1997;

Egli et al., 1998; Gutierrez et al., 1999; Ganga & Martinez,

2004; Santamaria et al., 2005). Such failures have important

practical consequences, and reasons for their occurrence

need to be understood. The evolution of strain diversity

throughout fermentation has also been reported within the

non-Saccharomyces species (Schutz & Gafner, 1994; Povhe-

Jemec et al., 2001).

It is now accepted that wine fermentations, whether

spontaneous or inoculated, are ecologically complex and

not only involve the growth of a succession of non-Sacchar-

omyces and Saccharomyces species but also involve the

successional development of strains within each species.

Such complexity presents a challenge to conducting con-

trolled fermentations with particular yeast cultures designed

to impose a special character or style on the final product. In

such cases, predictable, dominant growth of the inoculated

strain or a mixture of strains would be required. Many

factors such as grape juice composition, pesticide residues,

sulphur dioxide addition, concentration of dissolved oxy-

gen, ethanol accumulation and temperature affect the

kinetics of yeast growth during wine fermentations, but

little is known regarding how these factors might affect the

dominance and succession of individual species and strains

within the total population (Fleet & Heard, 1993; Bisson,

1999; Fleet, 2003; Zott et al., 2008). It is generally considered

that the successional evolution of strains and species

throughout fermentation is largely determined by their

different susceptibilities to the increasing concentration of

ethanol – the non-Saccharomyces species dying off earlier in

FEMS Yeast Res 8 (2008) 979–995c� 2008 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

980 G.H. Fleet

the process because they are more sensitive to ethanol than

S. cerevisiae. However, there are increasing reports of wine

isolates of Hanseniaspora, Candida and Kluyveromyces spe-

cies with ethanol tolerances similar to those of S. cerevisiae

(Mills et al., 2002; Pina et al., 2004; Xufre et al., 2006;

Nisiotou et al., 2007). In addition to ethanol, other phe-

nomena such as temperature of fermentation, dissolved

oxygen content, killer factors, quorum-sensing molecules

and spatial density influences are known to affect the

competitive interaction between yeast species and strains in

wine fermentations (Yap et al., 2000; Fleet, 2003; Nissen

et al., 2003; Hogan, 2006; Perez-Nevado et al., 2006), but

more research is needed to understand the diversity and

mechanisms of such reactions.

Yeasts affect wine character by variousmechanisms

Understanding how yeasts influence the key properties of

wine aroma, flavour and colour provides the basic platform

for selection of strains for development as starter cultures

and management of the alcoholic fermentation. Such me-

chanisms now extend beyond the simple, glycolytic meta-

bolism of grape juice sugars and, broadly, can be considered

as follows:

(1) Metabolism of grape juice sugar and nitrogen com-

ponents.

(2) Enzymatic hydrolysis of grape components to affect

wine aroma, flavour, colour and clarity.

(3) Autolysis.

(4) Bioadsorption.

The metabolism of grape juice sugars and amino acids

into ethanol and a vast array of other flavour-impacting

substances such as organic acids, glycerol, higher alcohols,

esters, aldehydes, ketones, amines and sulphur volatiles is

well known as the main reaction by which yeasts influence

wine character (Lambrechts & Pretorius, 2000; Swiegers

et al., 2005). Many studies have reported the qualitative and

quantitative profiles of these metabolites for various strains

of Saccharomyces and non-Saccharomyces yeasts (reviewed

in Fleet, 1992; Heard, 1999; Lambrechts & Pretorius, 2000;

Romano et al., 2003; Swiegers et al., 2005). These profiles

vary significantly between yeast species and strains, so that

extensive strain screening is necessary to select for those

with positive attributes (e.g. enhanced glycerol production,

enhanced ester formation) and reject those with distinct

negative impacts (e.g. overproduction of acetic acid or

hydrogen sulphide). On this basis, species within Hanse-

niaspora, Candida, Pichia, Zygosaccharomyces, Kluyvero-

myces and other genera have the potential to contribute

additional flavour diversity and complexity to wine produc-

tion (Ciani & Maccarelli, 1998; Heard, 1999; Romano et al.,

2003; Jolly et al., 2003b).

The biochemical transformation of flavour-inactive grape

juice constituents into flavour-active components has

emerged, in recent years, as an important, additional

mechanism whereby yeasts substantially impact on wine

aroma and flavour and facilitate greater expression of grape

varietal character. Knowledge about the diversity and com-

plexity of these reactions, however, is still in the discovery

phase (Swiegers et al., 2005; Hernandez-Orte et al., 2008).

Of these reactions, the liberation of terpenes is most studied.

Monoterpene alcohols such as citronellol, geraniol, linalool

and nerol occur naturally in grapes, especially Muscat,

Riesling and other white varieties, giving characteristic

fruity, estery, spicy and vegetative aromas. However, a good

proportion of these grape terpenes are covalently linked to

glucose or disaccharides of glucose and other sugars, where

the bound form has no flavour impact. Glycosidases pro-

duced by yeasts break down this linkage to release the

volatile terpene that significantly impacts on wine character.

The production of glycosidases by yeasts varies with species

and strain, but there are increasing data suggesting that non-

Saccharomyces yeasts such as species of Hanseniaspora,

Debaryomyces and Dekkera are stronger producers of such

enzymes than S. cerevisiae and, therefore, probably have a

greater role in the release of terpene aromas (Fia et al., 2005;

Maicas & Mateo, 2005; Swiegers et al., 2005; Villena et al.,

2007). In a similar way, yeasts can significantly enhance the

wine concentration of volatile thiols such as 4-mercapto-

4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol

(3MH) and 3-mercaptohexyl acetate (3MHA). These thiols

give desirable passionfruit, grapefruit, citrus and other

aroma characters that are most important in Sauvignon

Blanc wines, but can also be relevant in other varieties such

as Riesling, Semillon, Merlot, and Cabernet Sauvignon.

These thiols occur in grapes as nonvolatile forms that are

conjugated to cysteine. During fermentation, yeasts (S.

cerevisiae, S. bayanus) produce a cysteine lyase that decon-

jugates these thiols into their volatile form. The ability to

release these volatile thiols varies with the Saccharomyces

strain (Swiegers et al., 2005; Dubourdieu et al., 2006;

Swiegers & Pretorius, 2007). As yet, little is known about

their production by other wine yeasts. Thus, yeasts with

good properties of terpene and volatile thiol liberation can

be particularly significant in the development of varietal

flavours for some types of grapes.

Yeasts produce many other enzymes such as esterases,

decarboxylases, sulphite reductases, proteases and pectinases

that, in various ways, could impact on wine flavour

and other properties (Charoenchai et al., 1997; Fernandez

et al., 2000; Strauss et al., 2001), but further studies of these

possibilities are needed. The decarboxylation of hydr-

oxycinnamic acids in grape juice to form undesirable

amounts of volatile phenols such as 4-ethyl phenol and

4-ethylguaiacol is well known for Dekkera species but may


981Wine yeasts for the future

also occur in other wine yeasts (Dias et al., 2003; Suarez

et al., 2007).

The roles of yeast autolysis and bioadsorption in affecting

wine flavour have not been given detailed consideration in

the past and, possibly, this reflects their minor impact.

Nevertheless, such influences may be underestimated and

could be of value in future developments. It is well docu-

mented, however, that yeast autolysis contributes a unique

character and value to champagne and other sparkling wines

(Alexandre & Guilloux-Benatier, 2006), and autolytic po-

tential is considered to be a significant property in selecting

strains of S. cerevisiae for the secondary fermentation of

these wines (Martinez-Rodriguez et al., 2001). During alco-

holic fermentation, yeasts grow to produce significant levels

of cellular biomass (108–109 CFU mL�1) that consists of a

mixture of dead and living cells of different species and

strains. These cells, which comprise Saccharomyces and non-

Saccharomyces yeasts, sediment towards the end of fermen-

tation, to become a major part of the lees. Such biomass is

not inert. Dead yeast cells autolyse, which is characterized by

the enzymatic degradation of cell proteins, nucleic acids and

lipids to end products such as amino acids, peptides, fatty

acids and nucleotides, and the release of soluble mannopro-

teins from the cell wall (Hernawan & Fleet, 1995; Zhao &

Fleet, 2005; Alexandre & Guilloux-Benatier, 2006). Most of

these products have flavour impact or flavour-enhancing

potential (Charpentier et al., 2004, 2005; Comuzzo et al.,

2006), but their specific contributions to wine character

require more focused research, and will depend on the

extent to which the wine is exposed to the lees (Perez-

Serradilla & Luque de Castro, 2008). Moreover, there is

evidence that peptides released during yeast autolysis could

have antioxidant and other bioactive properties (Alcaide-

Hidalgo et al., 2007). Yeast mannoproteins may modulate

the perception of flavour metabolites and tannin character

(Feuillat, 2003; Caridi, 2007; Chalier et al., 2007). While

significant information is known about the autolysis of S.

cerevisiae, little is understood about autolysis in other yeast

species such as the non-Saccharomyces species associated

with wine fermentation. The autolytic behaviour of Kloeck-

era apiculata is somewhat different from that of S. cerevisiae

and Candida stellata (Hernawan & Fleet, 1995).

Yeast cells are enveloped by a cell wall that can represent

up to 30% of the cell dry weight. The wall is composed

mostly of glucan polysaccharides and mannoproteins (Klis

et al., 2006) that have the potential to modulate wine flavours

by bioadsorption of grape components, including mycotox-

ins, and microbial metabolites by mechanisms that, as yet,

are relatively unknown (Feuillat, 2003; Moruno et al., 2005;

Chalier et al., 2007). The bioadsorption properties of man-

noproteins appear to be particularly significant and contri-

bute to the colloidal stability of wines by interaction with

grape tartrates and proteins (Caridi, 2007; Perez-Serradilla &

Luque de Castro, 2008). The fine structure of mannoproteins

varies with the yeast species (Klis et al., 2006) and could

introduce diversity into their bioadsorption properties.

Yeasts impact on the colour and pigment profiles of

wines, especially red wines. Such influences vary with the

strain of S. cerevisiae and need to be considered in the

portfolio of properties when selecting yeasts for starter

culture development (Medina et al., 2005; Hayasaka et al.,

2007; Caridi et al., 2007). The impact of non-Saccharomyces

yeasts on wine colour is relatively unexplored. Yeasts affect

the colour of red wines by several mechanisms. They

produce ethanol and other metabolites that extract the

anthocyanin pigments from the grape skins. Some yeasts

produce extracellular pectinases, and these enzymes could

facilitate breakdown of grape tissue and extraction of

pigments. After extraction, the anthocyanin–phenolic con-

stituents responsible for colour must remain stable, but

there is increasing evidence that yeasts can affect this

property. Some of the anthocyanins are glycosylated, and

glycosidase production by yeasts may break this linkage and

decrease colour (Manzanares et al., 2000). Yeast cell wall

polysaccharides can adsorb anthocyanins and, in this way,

remove colour from the wine (Caridi, 2007; Caridi et al.,

2007). Finally, yeasts may indirectly contribute to the

stabilization of wine colour during the maturation stage by

production of acetaldehyde and pyruvic acid that facilitate

the chemical formation of pyranthocyanins (Morata et al.,

2006; Monagas et al., 2007).

Propensity to undergo autolysis and parietal absorption

activity are relatively new properties to consider in the

selection and development of new wine yeasts (Caridi,

2007; Giovani & Rosi, 2007).

Fermentation options

Microbial fermentations can be conducted as either batch

processes or continuous processes. Almost all wines are

produced by batch fermentation, which means that the juice

is placed in a vessel and the entire batch is kept there until

fermentation is completed, usually after 5–10 days (Divies,

1993). Continuous fermentations enable much faster, more

efficient processing, and present new directions for the wine

industry that will be discussed in a later section.

With batch fermentations, there are two options in wine

production: spontaneous or natural fermentation or starter

culture fermentation (Pretorius, 2000). The ecological con-

cepts of these two options have already been mentioned.

Spontaneous fermentations can give high-quality wines

with a unique regional character that provides differentia-

tion and added commercial value in a very competitive

market. Unfortunately, reliance on ‘nature’ brings dimin-

ished predictability of the process, such as stuck or slow

fermentations, and inconsistencies in wine quality.


982 G.H. Fleet

Nevertheless, a good proportion of wine is commercially

produced by this process, especially in European countries

(Rainieri & Pretorius, 2000; Mannazzu et al., 2002). Gen-

erally, a combination of artisanal and technological expertise

is required for success with these fermentations. Starter

culture fermentations, in contrast, offer the advantages of a

more predictable and rapid process, giving wines with

greater consistency in quality. They are well suited for

producing mass market wines, and their acceptance by the

wine industry has been enhanced by the commercial avail-

ability of dried concentrates of selected yeast strains that can

be conveniently reconstituted for inoculation into grape

juice (Degre, 1993; Manzano et al., 2006). Numerous S.

cerevisiae and S. bayanus strains are available as commercial

preparations, and these yeasts have been selected on the

basis of various criteria (see later), including their ability to

tailor wine character (Pretorius, 2000; Bisson, 2004). Never-

theless, there has been increasing recognition that starter

culture wines may be lacking in flavour complexity and are

too standardized and ordinary in character (Rainieri &

Pretorius, 2000; Mannazzu et al., 2002). However, such

criticisms are providing new challenges to enhance the

appeal and value of wine produced by this fermentation

technology. This is being achieved by selecting and develop-

ing novel strains of starter culture yeasts and conducting

fermentations with controlled mixtures of yeast species and

strains. Such initiatives now have a greater chance of a

successful outcome because there is a clearer understanding

of the yeast ecology of wine fermentations, and how this

ecology can be managed under the practical conditions of

the winery. Greater understanding of yeast biochemistry and

physiology is enabling the selection and development of

yeast strains that have defined specific influences on process

efficiency and wine quality.

Criteria for selecting and developing newstrains of wine yeasts

Criteria for the selection and development of yeasts for

wine fermentation have evolved over many years and are

discussed in Degre (1993), Rainieri & Pretorius (2000),

Mannazzu et al. (2002), Pretorius & Bauer (2002), Bisson

(2004) and Schuller & Casal (2005). Basically, these criteria

can be considered under three categories: (1) properties that

affect the performance of the fermentation process, (2)

properties that determine wine quality and character and

(3) properties associated with the commercial production of

wine yeasts. Within each category, there are properties of

varying degrees of significance and importance, some being

essential and some being desirable.

Fast, vigorous and complete fermentation of grape juice

sugars to high ethanol concentrations (48% v/v) are essen-

tial requirements of wine yeasts. The yeast should be tolerant

of the concentrations of sulphur dioxide added to the juice as

an antioxidant and antimicrobial, exhibit uniform dispersion

and mixing throughout the fermenting juice, produce mini-

mal foam and sediment quickly from the wine at the end of

fermentation. These processing properties should be well

expressed at low temperatures (e.g. 15 1C) for white wine

fermentations and at higher temperatures (e.g. 25 1C) for red

wine fermentations. It is most important that the yeast does

not give slow, sluggish or stuck fermentations (Bisson, 1999).

With respect to wine quality and character, it is essential

that any yeast produces a balanced array of flavour metabo-

lites, without undesirable excesses of volatiles such as acetic

acid, ethyl acetate, hydrogen sulphide and sulphur dioxide.

It should not give undesirable autolytic flavours after

fermentation. It should not adversely affect wine colour or

its tannic characteristics. In essence, the yeast must give a

wine with a good clean flavour, free of sensory faults, and

allow the grape varietal character to be perceived by the

consumer (Lambrechts & Pretorius, 2000; Bisson, 2004;

Swiegers et al., 2005).

Companies that produce yeasts for the wine industry also

have basic needs that must be built into the selection and

development process (Degre, 1993). The cost of production

needs to be contained so that the final product is affordable

to the wine industry. Consequently, the yeast must be

amenable to large-scale cultivation on relatively inexpensive

substrates such as molasses. Subsequently, it needs to be

tolerant of the stresses of drying, packaging, storage and,

finally, rehydration and reactivation by the winemaker

(Soubeyrand et al., 2006). These requirements need to be

achieved without loss of the essential and desirable wine-

making properties.

Although technologies are well established for the selec-

tion, development and production of wine yeasts with good

basic oenological criteria, there are increasing environmen-

tal pressures for a wine industry that is more efficient and

more sustainable, and there are increasing demands from

consumers for wines with more distinctive and specific

styles, including those with a healthier appeal (e.g. less

ethanol, increased antioxidant levels; Bisson et al., 2002;

Bisson, 2004). These directions require a more strategic

approach to the development of wine yeasts than in the

past. For this purpose, a desired wine attribute is identified,

and the property to give that quality is designed into the

yeast selection and development process, without compro-

mising the essential oenological criteria already mentioned.

Pretorius and colleagues (Pretorius & Bauer, 2002; Pretorius

& Hoj, 2005; Verstrepen et al., 2006) and Bisson (2004)

broadly describe these developmental targets under five

categories. These are, with some examples, as follows:

(1) Improved fermentation performance (e.g. yeasts with

greater efficiency in sugar and nitrogen utilization, in-

creased ethanol tolerance, decreased foam production).



(2) Improved process efficiency (e.g. yeasts with greater

production of extracellular enzymes such as proteases,

glucanases and pectinases to facilitate wine clarification;

yeasts with altered surface properties to enhance cell

sedimentation, floatation and flor formation, as needed;

and yeasts that conduct combined alcoholic-malolactic

fermentations).

(3) Improved control of wine spoilage microorganisms (e.g.

yeasts producing lysozyme, bacteriocins and sulphur

dioxide that restrict spoilage bacteria).

(4) Improved wine wholesomeness (e.g. yeasts that give less

ethanol, decreased formation of ethyl carbamate and

biogenic amines, increased production of resveratrol

and antioxidants).

(5) Improved wine sensory quality (e.g. yeasts that give

increased release of grape terpenoids and volatile thiols,

increased glycerol and desirable esters, increased or de-

creased acidity and optimized impact on grape phenolics).

Sources of new wine yeasts

During the past 50–75 years, wine production has been

transformed into a modern, industrialized process, largely

based around the activities of only two yeast species: S.

cerevisiae and S bayanus. Future developments will continue

to be based on innovation with these species, but opportu-

nities for innovation using other species of yeasts cannot be

overlooked. As mentioned already, various species of Han-

seniaspora, Candida, Kluyveromyces and Pichia play signifi-

cant roles in the early stages of most wine fermentations,

and there is increasing interest in more strategic exploitation

of these species as novel starter cultures (Ciani & Maccarelli,

1998; Heard, 1999; Ciani et al., 2002; Jolly et al., 2003b).

Their limitations with regard to ethanol tolerance may not

be a hurdle in the production of wines with lower, final

ethanol contents. Various species of Zygosaccharomyces,

Saccharomycodes and Schizosaccharomyces are strong fer-

menters and are ethanol tolerant. Although they are gen-

erally considered as spoilage yeasts, there is no reason to

doubt that a good programme of selection and evaluation

within these yeasts would not discover strains with desirable

winemaking properties (Romano & Suzzi, 1993; Fleet,

2000a). It needs to be recalled that not all strains of S.

cerevisiae produce acceptable wines, and that a systematic

process of selection and evaluation was needed to obtain

desirable strains (Degre, 1993). Consequently, in searching

for and developing new yeasts, the wine industry of the

future must look beyond Saccharomyces species. In addition,

it must look beyond grapes and give broader consideration

to other fruits as the starting raw material. With such vision,

many new yeasts and wine products await discovery.

Essentially, there are two strategies for obtaining new

strains of wine yeasts for development as commercial starter

cultures: (1) isolation from natural sources and (2) genetic

improvement of natural isolates. Once a prospective isolate

has been obtained, it is screened in laboratory trials for

essential oenological criteria as mentioned already. Isolates

meeting acceptable criteria are then used in micro-scale

wine fermentations and the resulting wines are then sub-

jected to sensory evaluation. Strains giving good fermenta-

tion criteria and acceptable-quality wines under these

conditions are then selected for further development as

starter culture preparations. Mannazzu et al. (2002) have

outlined an experimental strategy for the selection and

evaluation of yeast strains for starter culture development,

and some applications of this approach can be found in

Regodon et al. (1997), Perez-Coello et al. (1999), Esteve-

Zarzoso et al. (2000), Cappello et al. (2004), Cocolin et al.

(2004) and Nikolaou et al. (2006).

Natural sources

Generally, wine yeasts for starter culture development have

been sourced from two ecological habitats, namely, the

vineyard (primarily the grapes) and spontaneous or natural

fermentations that have given wines of acceptable or unique

quality.

As mentioned already, yeasts are part of the natural

microbial communities of grapes (Fleet et al., 2002). Under-

standably, therefore, grapes are always considered a potential

source of new wine yeasts. Moreover, there is an attraction

that unique strains of yeasts will be associated with parti-

cular grape varieties in specific geographical locations and,

through this association, they could introduce significant

diversity and regional character or ‘terroir’ into the wine-

making process (Vezinhet et al., 1992; Pretorius et al., 1999;

Jolly et al., 2003a; Martinez et al., 2007; Raspor et al., 2006;

Valero et al., 2007). Thus, in the interests of preserving

biodiversity and regional influence on wine character, grapes

of the region would represent an important source of yeasts

for starter culture development.

Yeasts associated with grape berries have been studied for

over 100 years (reviewed in Fleet et al., 2002), although more

detailed studies are needed to obtain a clearer understanding of

their ecology and factors that affect this ecology. The yeast

species and populations evolve as the grape berry matures on

the vine and are influenced by climatic conditions such as

temperature and rainfall, application of agrichemicals and

physical damage by wind, hail and attack by insects, birds and

animals. The predominant semi-fermentative and fermentative

yeasts isolated from grapes at the time of maturity for wine-

making are mostly species of Hanseniaspora (Kloeckera),

Candida, Metschnikowia, Pichia and Kluyveromyces, although

the data are not always consistent (Martini et al., 1996; Jolly

et al., 2003a; Prakitchaiwattana et al., 2004; Combina et al.,

2005a, b; Renouf et al., 2005, 2007; Raspor et al., 2006; Barata


984 G.H. Fleet

et al., 2008). If the berries are over-ripe, become damaged or

are infected with filamentous fungi (mould), the yeast popula-

tions tend to be higher and include a greater incidence of

fermentative species such as those of Saccharomyces, Zygosac-

charomyces, Saccharomycodes and Zygoascus (Barata et al.,

2008; Nisiotou et al., 2007). Consequently, grapes will be a

very good source of non-Saccharomyces yeasts, should there be

a future direction for using such species as novel starters in

wine fermentations.

It is difficult to isolate Saccharomyces species from ma-

ture, undamaged grapes by direct culture on agar media, but

they are frequently found by enrichment culture methods,

suggesting their presence in very low numbers. Grape berries

that are aseptically harvested from vines and crushed will

eventually ferment and strains of S. cerevisiae and S. bayanus

are easily isolated from the fully fermented must (Vezinhet

et al., 1992; Khan et al., 2000; van der Westhuizen et al.,

2000; Demuyter et al., 2004; Schuller et al., 2005; Mercado

et al., 2007; Valero et al., 2007). Strains of Saccharomyces

paradoxus, capable of producing wine, have also been

isolated from grapes (Redzepovic et al., 2002). However,

recovery of Saccharomyces species from such ferments is not

always consistent and can be determined by many factors

that are likely to affect the occurrence and survival of yeasts

on the grape surface, such as amount of rainfall, tempera-

ture and applications of agrichemicals. We have observed

that the frequency of isolation of Saccharomyces species from

aseptically harvested and crushed grapes can be significantly

increased by removing the skins from the macerate and

allowing the juice without skins to ferment (S.S. Bae & G.H.

Fleet, unpublished data). Possibly, such modification gives

low initial numbers of Saccharomyces a better chance to

compete with the higher populations of other species. As

mentioned already, damaged grape berries are more likely to

yield Saccharomyces species than nondamaged grapes. Based

on molecular analyses using pulsed field gel electrophoresis

and restriction fragment length polymorphism of mtDNA,

grape isolates of S. cerevisiae exhibit substantial genomic

diversity, because many different strains have been obtained

from grapes within the one vineyard or geographical region.

In some cases, particular strains have been unique to one

location, leading to the notion of a yeast ‘terroir’ (Vezinhet

et al., 1992; Khan et al., 2000; van der Westhuizen et al.,

2000; Raspor et al., 2006). In other cases, strain diversity

within the one location and over consecutive years has been

too extensive and inconsistent to support a ‘terroir’ concept

(Versavaud et al., 1995; Schuller et al., 2005; Valero et al.,

2007). However, further research at the biochemical, phy-

siological and oenological level is required to determine

whether S. cerevisiae strains have adapted to grapes in

certain geographical locations.

Wines that have undergone a successful natural fermenta-

tion have been a main source of yeasts for development as

starter cultures. In the past, the focus has been to isolate

suitable strains of S. cerevisiae and S. bayanus, but these

wines will also be a good reservoir of non-Saccharomyces

species. The precise origin of the indigenous strains of

Saccharomyces in these wine fermentations has been a

question of much debate and controversy (Martini et al.,

1996; Mortimer & Polsinelli, 1999). Clearly, the grape itself

is a primary source of the yeasts that occur in the juice and it

is logical to conclude that any Saccharomyces strains from

this source would be prominent in the final fermentation.

However, processing of the juice and its transfer to fermen-

tation tanks contributes added microbial communities.

These microbial communities originate as contamination

from the surfaces of winery equipment and are widely

considered to be ‘residential’ flora that have built up in the

winery over time, through a process of adaptation and

selection, despite cleaning and sanitation operations. These

flora are dominated by fermenting ethanol-tolerant yeast

species such as S. cerevisiae and S. bayanus because of the

selective conditions presented by the properties of ferment-

ing grape juice. Many researchers consider winery flora to be

the main source of the diversity of strains of Saccharomyces

involved in grape juice fermentations and, in recent years,

this has been demonstrated using molecular techniques to

track strain origin and development. Strains isolated from

winery equipment, in addition to any inoculated strain, are

found in the fermenting wine, and similar strains can be

found in the one winery in consecutive years, suggesting a

carry-over from 1 year to the next (Constanti et al., 1997;

Gutierrez et al., 1999; Cocolin et al., 2004; Santamaria et al.,

2005; Mercado et al., 2007). Although data are still very little,

it seems that S. cerevisiae strains on the grape may have only

minor contributions to the overall fermentation (Mercado

et al., 2007). However, this could depend on their initial

populations and how these are influenced by grape-crushing

procedures, cold-maceration processes and grape maturity

(Hierro et al., 2006; Sturm et al., 2006). Presumably, the

Saccharomyces flora in the winery originally came from

grapes and evolved with time. The source of Saccharomyces

yeasts on the grapes is still a mystery, but contamination

from insects in the vineyard is thought to be a likely

possibility (Mortimer & Polsinelli, 1999; Fleet et al., 2002).

Genetic improvement of isolates

Through genetic improvement and metabolic engineering

technologies, it is now possible to develop wine yeasts with a

vast array of specific functionalities as mentioned already in

the section on essential and desirable criteria for selecting

wine yeasts. Basically, the desired property is defined (e.g.

strain with enhanced glycerol production; strain with bac-

teriocin production), and then the appropriate genetic tools

are applied to construct the strain with that property.



Usually, it is necessary to start with a proven wine yeast at

the outset, and to ensure that any genetic manipulation does

not adversely affect its basic winemaking properties. The

principles of these technologies and their application to

wine yeasts have been reviewed by Barre et al. (1993),

Pretorius (2000), Pretorius & Bauer (2002), Bisson (2004),

Giudici et al. (2005), Schuller & Casal (2005) and Verstrepen

et al. (2006) and include:

(1) Mutagenesis.

(2) Spheroplast fusion.

(3) Intraspecific and interspecific hybridization.

(4) Transformation and recombinant DNA techniques.

(5) Adaptive evolution.

(6) Systems biology and functional genomics.

Although early studies (reviewed in Barre et al., 1993)

showed that yeast mating and hybridization methods could

be used to develop strains of S. cerevisiae with improved

properties (e.g. flocculation, less hydrogen sulphide produc-

tion), these classical approaches were overtaken by more

precise and convenient recombinant DNA techniques that

have now yielded a plethora of strains with well-defined

oenological traits as listed in criteria already described (see

also, tables in Rainieri & Pretorius, 2000; Pretorius & Bauer,

2002; Bisson, 2004; Schuller & Casal, 2005). Recent reports

on the metabolic engineering of wine strains of S. cerevisiae

that give enhanced release of volatile thiols (Swiegers et al.,

2007) and decreased ethyl carbamate production (Coulon

et al., 2006) are further examples that illustrate the ‘targeted’

application of this technology. However, consumer and

government concerns about the public health and environ-

mental safety of microbial strains engineered by recombi-

nant DNA technologies remain a hurdle to the commercial

use of these yeasts (Akada, 2002; Schuller & Casal, 2005;

Verstrepen et al., 2006). So far, only one recombinant strain

of wine yeast has received approval for commercial use (in

North America), and this is a strain of S. cerevisiae con-

structed to contain a malate-permease gene from the yeast,

Schizosaccharomyces pombe, and the malolactic gene from

the bacterium, Oenococcus oeni. This strain offers the

advantage of improved process efficiency by eliminating the

need for bacterial malolactic fermentation that is usually

conducted after alcoholic fermentation (Husnik et al., 2007;

Main et al., 2007).

Hybridization (Giudici et al., 2005; Belloch et al., 2008),

adaptive evolution (Barrio et al., 2006; McBryde et al., 2006)

and systems biology (Bisson et al., 2007; Borneman et al.,

2007; Pizarro et al., 2007) are strategies that do not

compromise consumer and government sensitivities with

respect to safety and environmental risks, and now attract

significant focus for the development of a new generation of

wine yeasts. Inter- and intraspecies hybrids within strains

of Saccharomyces (e.g. S. cerevisiae� S. bayanus and

S. cerevisiae� S. kudriazevii) have been isolated from spon-

taneous fermentations and similar hybrids, now commer-

cially available, have been produced by mating yeasts under

laboratory conditions (Gonzalez et al., 2006, 2007; Belloch

et al., 2008). Hybrids between S. cerevisiae and other species

within Saccharomyces are also available (e.g S. cerevisiae� S.

cariocanus, S. cerevisiae� S. paradoxus and S. cerevisiae� S.

mikatae). A list of commercially available strains of wine

yeasts, including newly developed hybrid strains, has been

compiled by Henschke (2007). Hybridization expands the

tolerance of some strains to the stresses of winemaking such

as temperature of fermentation and ethanol concentration

and increases the pool of strains available to enhance

diversity in wine flavour (Belloch et al., 2008; Bellon et al.,

2008).

Adaptive evolution is another concept for selecting strains

with oenological performance and flavour profiles matched

to a particular winemaking need. In this case, yeasts are

continuously and repeatedly cultured under a defined

combination of conditions from which strains that have

specifically adapted to these conditions can be isolated

(McBryde et al., 2006). In essence, it is a laboratory

approach for speeding up what occurs naturally, over time,

in winery habitats (Barrio et al., 2006). Systems biology

exploits knowledge of the total genome and bioinformatic

methods to select and develop new strains of wine yeasts

with very specific functionalities and criteria, as determined

by production, consumer and environmental demands

Borneman et al., 2007; Pizarro et al., 2007). Because

genomic information about wine yeasts is still very limited,

this approach is at a conceptual stage of development and

practical outcomes are yet to be realized.

Controlled fermentations with mixed strains orspecies of yeasts

The realization that yeasts other than Saccharomyces species

are ecologically and metabolically significant in wine fer-

mentation has laid the platform for more creative and

controlled exploitation of their use in wine production.

The potential of using them as stand-alone, single starter

cultures in the development of new styles of fermented

beverages has already been mentioned, but not seriously

explored to date. However, some of these species are limited

in their ability to fully ferment the grape juice sugars and in

their ability to produce sufficient concentrations of ethanol.

Some may grow too slowly in comparison with other

indigenous yeasts, and not establish themselves. Neverthe-

less, they have other properties of oenological relevance that

would be worth exploiting. For example, some Hansenias-

pora/Kloeckera species may produce more appealing mix-

tures of flavour volatiles, and higher amounts of glycosidases

and proteases than Saccharomyces species (Zironi et al.,

1993; Capece et al., 2005; Mendoza et al., 2007; Moreira


986 G.H. Fleet

et al., 2008), C. stellata gives increased levels of glycerol

(Ciani & Comitini, 2006), Kluyveromyces thermotolerans

gives increased levels of lactic acid (Mora et al., 1990),

Torulaspora delbrueckii produces less acetic acid (Lafon-

Lafourcade et al., 1981; Ciani et al., 2006; Bely et al., 2008)

and Schizosaccharomyces species decrease wine acidity

through malic acid metabolism (Gao & Fleet, 1995; Taillan-

dier et al., 1995). Some wine strains of C. stellata have

recently been reclassified as Candida zemplinina (Csoma &

Sipiczki, 2008). Conducting wine fermentations by con-

trolled inoculation of mixtures of different yeast starter

cultures is one strategy to harness the unique activity of

such yeasts. This concept is not new and early studies have

been reviewed by Heard (1999) and Ciani et al. (2002).

However, it now attracts greater interest because of its

potential to introduce specific characteristics into wine and

also because winemakers have a more thorough knowledge

of the ecology and biochemistry of wine fermentation and

how to manage this process (Jolly et al., 2003b). This area of

wine technology is very likely to grow in future applications;

consequently, it is useful to collate significant studies to date

(Table 1).

Most studies (Table 1) have aimed to understand how

non-Saccharomyces species grow interactively with S. cerevi-

siae in comparison with monocultures of the respective

yeasts. Growth profiles are generally reported, along with

glucose and fructose utilization, and the production of key

metabolites such as ethanol, acetic acid, glycerol, ethyl

acetate and, in some cases, various higher alcohols, higher

acids and other esters (e.g. see Jolly et al., 2003b). Essentially,

these studies confirm that non-Saccharomyces yeasts grow in

sequential patterns similar to those observed for sponta-

neous wine fermentations, but conditions such as tempera-

ture, sulphur dioxide addition, inoculum levels and time of

inoculation can be manipulated to enhance the extent of

their survival and contribution to the overall fermentation.

Inoculating ethanol-sensitive or slower-growing non-Sac-

charomyces yeasts into the grape juice several days before

inoculating S. cerevisiae (sequential inoculation) is one

strategy for enhancing their contribution to the fermenta-

tion. Data are not always consistent on how interactive

growth affects the production of flavour metabolites, but

several studies show that the unacceptably high volatile acid

and ester production by some Hanseniaspora/Kloeckera

species does not occur when these yeasts are grown in mixed

culture with S. cerevisiae (Herraiz et al., 1990; Zironi et al.,

1993; Zohre & Erten, 2002; Mendoza et al., 2007; Moreira

et al., 2008). In some cases, overall ethanol production is

decreased in mixed culture fermentations, but in other cases

this is not observed. Using different Saccharomyces species

and strains, Howell et al. (2006) concluded that mixed

culture impacts on the metabolic performance of individual

strains within the mixture. Wines made with mixed cultures

of the yeasts gave a combination of volatile aroma

Table 1. Studies of wine fermentation inoculated with defined mixtures of yeast species

References Yeast species inoculated Objective of study

Heard & Fleet (1988) S. cerevisiae, K. apiculata, Candida spp. Ecological interactions

Nissen et al. (2003) S. cerevisiae, T. delbrueckii, K. thermotolerans Ecological interactions

Ciani et al. (2006) S. cerevisiae, H. uvarum, K. thermotolerans, T. delbrueckii Ecological interactions

Perez-Nevado et al. (2006) S. cerevisiae, Hanseniaspora spp. Ecological interactions

Mendoza et al. (2007) S. cerevisiae, K. apiculata Ecological interactions

Herraiz et al. (1990) S. cerevisiae, K. apiculata, T. delbrueckii Flavour modulation

Moreno et al. (1991) S. cerevisiae, T. delbrueckii Flavour modulation

Zironi et al. (1993) S.cerevisiae, H. guilliermondii, K.apiculata Flavour modulation

Gil et al. (1996) S.cerevisiae, H. uvarum, K. apiculata Flavour modulation

Soden et al. (2000) S. cerevisiae, C. stellata Flavour modulation

Garcia et al. (2002) S. cerevisiae, D. vanriji Flavour modulation

Zohre & Erten (2002) S. cerevisiae, K. apiculata, C. pulcherrima Flavour modulation

Clemente-Jimenez et al. (2005) S. cerevisiae, P. fermentans Flavour modulation

Kurita (2008) S. cerevisiae, P. anomala Flavour modulation

Moriera et al. (2008) S. cerevisiae, Hanseniaspora spp. Flavour modulation

Mora et al. (1990) S. cerevisiae, K. thermotolerans Acid (lactic) enhancement

Silva et al. (2003) S. cerevisiae, S. pombe Deacidification (malic acid)

Toro & Vazquez (2002) S. cerevisiae, C. cantarellii Glycerol enhancement

Ciani & Comitini (2006) S. cerevisiae, C. stellata Glycerol enhancement

Bely et al. (2008) S. cerevisiae, T. delbrueckii Decrease volatile acidity

Howell et al. (2006) S. cerevisiae, S. bayanus (three strain mixtures) Metabolic and flavour interactions

Jolly et al. (2003b) S. cerevisiae, C. colliculosa

S. cerevisiae, C. stellata

S. cerevisiae, K. apiculata

S. cerevisiae, C. pulcherrima

Ecological interactions

Flavour modulation



metabolites different from that obtained by blending to-

gether monoculture wines made with the same component

yeast strains. Thus, with respect to production of flavour

volatiles in wine, the metabolic interactions of yeasts during

mixed culture could be quite complex and difficult to

predict. Consequently, the ultimate evaluation of such

fermentations should be based on sensory testing but,

unfortunately, few studies in Table 1 report such data. In

this context, the study of Soden et al. (2000) is significant

and reports the chemo-sensory outcome in a Chardonnay

wine produced by fermentation with a mixed culture of

C. stellata and S. cerevisiae. The sensory impact of C. stellata,

as well as its production of increased levels of glycerol, were

only evident when the yeast was used in a sequential

inoculation protocol (i.e. inoculated 15 days before

S. cerevisiae), as compared with coinoculation of the two

species at day zero. Jolly et al. (2003b) conducted small-scale

wine fermentations with S. cerevisiae mixed with either

Candida colliculosa, C. stellata, Kloeckera apiculata or Can-

dida pulcherrima. Wines with enhanced sensory appeal were

obtained with many of the mixed yeast fermentations, but

this depended on the grape variety and the length of time

the wine had been aged.

The impact of non-Saccharomyces yeasts in mixed culture

with S. cerevisiae can be more definitive when specific wine

properties are targeted, such as decreasing malic acid con-

centrations using Schizosaccharomyces species or using

Torulaspora delbrueckii to prevent volatile acidity produc-

tion in sweet wine fermentations. Sequential inoculation of

S. pombe before S. cerevisiae appears to be necessary for a

successful deacidification but, unfortunately, this yeast can

give off-flavours to the wine (Snow & Gallander, 1979; Silva

et al., 2003). Possibly, a programme of selection for this yeast

could avoid these problems. In the case of T. delbrueckii,

coinoculation with S. cerevisiae was necessary because it

causes stuck fermentations if inoculated before S. cerevisiae

(Bely et al., 2008).

The studies listed in Table 1 provide an excellent platform

for future development of wine fermentation technology

based on the controlled use of mixed cultures. Further

research is required to understand the biological mechan-

isms of how yeasts interact ecologically and metabolically

when grown in coculture and sequential culture under

winemaking conditions. These fundamental studies must

be linked with well-controlled sensory evaluations of wine

flavour and colour so that useful practical outcomes are

achieved.

More efficient wine fermentation

Alcoholic fermentation is conducted as a batch process,

where the grape juice is placed in tanks or barrels, and kept

there until the fermentation is completed. In most modern

wineries, large stainless-steel, cylindro-conical vessels are

used. Good understanding of the growth and biochemical

activities of yeasts in these batch systems, as well as ability to

control variables such as temperature, agitation and aera-

tion, have ensured the widespread successful use of this

technology (Moresi, 1989; Divies, 1993). Nevertheless, batch

fermentations have inherent inefficiencies. The process is

relatively slow because the duration of fermentation de-

pends on the growth of yeast cells to final populations of at

least 108 cells mL�1. Most wine fermentations, therefore,

require 4–7 days or longer. The process is discontinuous

because a new batch of wine cannot be fermented until the

previous batch is finished and removed from the vessel.

Consequently, large fermenter capacity and associated capi-

tal investment are required. At the end of the process,

microbial cells need to be separated from the product,

thereby adding to inefficiency and product losses. From

time to time, winemakers experience frustration with these

limitations, especially during vintage when there are peak

demands on volume capacity, or when there are unforeseen

delays due to stuck or sluggish fermentations. Consequently,

the question arises – can wine fermentations be conducted

more efficiently, and what are the alternatives?

During the last 20–30 years, technologies have been

developed to immobilize microbial cells and apply them to

commercial fermentations. The basic concept is that very

high concentrations of microbial cells can be entrapped

within or attached to a solid matrix without destroying their

viability or biochemical activity. Immobilized or entrapped

populations of 109–1010 cells mL�1 can be achieved, com-

pared with the levels of 107–108 cells mL�1 usually obtained

in batch cultures. These high cell densities are very reactive,

and can conduct biochemical reactions, such as sugar-

alcohol fermentations, at rates 10–100 times faster than

those that occur during batch culture (reviewed in Pilking-

ton et al., 1998; Verbelen et al., 2006; Bai et al., 2008). Several

strategies have been used for the capture and immobiliza-

tion of high densities of microbial cells and these include:

(1) Physical entrapment within a porous matrix (e.g. gels

of calcium alginate or k-carrageenan, usually in the form

of beads about 2mm in diameter).

(2) Attachment or adsorption to the external surface of

an inert solid matrix such as cellulose particles, porous

glass, volcanic rock, diatomaceous earth, particles of

gluten and fruits.

(3) Confinement within a defined space by membrane

filters (e.g. membrane bioreactors).

(4) Self-aggregation of highly flocculent cells.

Immobilized cell reactors enable faster, more efficient

fermentations, which can be conducted on a batch or a

continuous basis. Usually, the immobilized cell matrix is

packed into columns, and the material for fermentation is

then passed through the column, giving the transformed


988 G.H. Fleet

product on exit. Their application to wine production has

been discussed in several reviews (Divies, 1993, 1994; Fleet,

2000b; Ciani et al., 2002; Kourkoutas et al., 2004; Strehaiano

et al., 2006). Most focus has been on the use of bacterial

reactors for malo-lactic fermentation, and the use of yeasts

entrapped in alginate gels for the secondary fermentation in

sparkling wines. Schizosaccharomyces pombe metabolizes

malic acid, and several studies have described the experi-

mental use of alginate-immobilized cells of this yeast to

deacidify wines (reviewed in Strehaiano et al., 2006). Silva

et al. (2003) successfully used S. pombe entrapped within

beads of alginate in a batch process to deacidify grape juice

before fermentation with S. cerevisiae, and suggest that this

process is at a stage for commercialization.

Application of new bioreactor technologies to the alco-

holic fermentation grape juice is still at beginning stages of

development. Some early laboratory studies showed that the

length of batch fermentations could be decreased using

concentrates of yeast cells, freely suspended or immobilized

on alginate, recycled from previous fermentations (Rosini,

1986; Suzzi et al., 1996). Wine strains of S. cerevisiae

immobilized on a wide range of supports such as volcanic

rock, alumina, calcium alginate, cellulose, gluten pellets and

particles of apple, quince, grape skin, watermelon and raisin

berries have been used to conduct batch fermentations. The

immobilized yeasts could be easily recovered from each

fermentation and reused many times, reportedly giving

fermentations two to three times faster than control fermen-

tations (Kourkoutas et al., 2004). Even faster fermentations

could be achieved when the grape juice was continuously

passed through columns packed with yeast cells immobilized

on these matrices (Fleet, 2000b; Kourkoutas et al., 2004;

Tsakris et al., 2004; Reddy et al., 2008). Candida stellata,

immobilized on calcium alginate, has been used in conjunc-

tion with S. cerevisiae to enhance glycerol production in both

batch and continuous wine fermentations (Ciani & Ferraro,

1996; Ferraro et al., 2000; Ciani et al., 2002).

Membrane bioreactors operated on a continuous, cell-

recycle basis offer greater efficiencies and faster reactions

compared with other reactor technologies because much

higher cell densities can be achieved, and the yeast cells are

in direct contact with the grape juice. Their application to

wine production has been briefly mentioned by Strehaiano

et al. (2006) and Divies (1993, 1994). We have shown that a

laboratory-scale, 1-L membrane bioreactor charged with

109–1010 cells mL�1 of S. cerevisiae could produce 1 L of fully

fermented wine per hour. The reactor performed continu-

ously for 10 days without loss of fermentative activity

(Charoenchai, 1995). Similar experiences with these reactors

have been described by Takaya et al. (2002). Crapisi et al.

(1996) reported an interesting use of a hollow-fibre mem-

brane reactor, charged with cells of Kloeckera apiculata/

Hanseniaspora uvarum to produce a low-alcohol wine.

There is little doubt that new bioreactor technologies

have the potential to significantly enhance the efficiency of

wine fermentation and facilitate innovations with yeasts

other than Saccharomyces species. However, their scale-up

to commercially successful operations presents a number of

challenges that require further research. Some of these issues

are discussed in Fleet (2000b), Kourkoutas et al. (2004) and

Bai et al. (2008). Any particulate material in the juice or

wine will, in due course, physically clogs column reactors or

fouls membranes in membrane reactors, eventually stopping

their performance. Similarly, indigenous bacteria and yeasts

in the juice or wine are likely to accumulate in the reactor

and compromise its performance. Prior clarification and

pasteurization of the juice or wine may be necessary, with

consequences on processing economy and product quality.

Long-term stability and reactivity of yeast cells in the

reactors are essential to maintaining processing costs. Yeast

cells are exposed to many new physical, chemical and

biological stresses in reactors that need to be understood

and optimized to enhance their long-term performance.

Large amounts of carbon dioxide are produced, whose

consequences need to be managed. Finally, wines produced

by new reactor technologies must have acceptable sensory

qualities. The metabolic behaviour of yeast cells operating

under reactor conditions may be significantly different from

that for cells growing in batch culture, thereby giving a

different profile of flavour volatiles in the final product. A

programme of strain selection and development will be

required to obtain wine yeasts that perform under contin-

uous, high cell density conditions.

Conclusion

The wine industry is faced with an exciting but challenging

future. Continuing advances in yeast biology and fermenta-

tion technology provide many opportunities for innovation

and adaptation to a changing market. These will enable

further refinements of existing technologies and products

but also the development of new products based on the

exploitation of new strains of Saccharomyces yeasts, non-

Saccharomyces yeasts, novel bioreactor technologies and the

use of fruits other than grapes. Environmental issues present

new challenges. Current agrichemical use in the vineyard

will not be sustainable, and climate change is likely to have a

cascading effect on grape cultivation, grape juice composi-

tion, the yeast ecology of wine production, fermentation

kinetics and wine character.

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