handbook of enology volume 1 the microbiology of wine...

Handbook of EnologyVolume 1

The Microbiology of Wine and Vinifications2nd Edition

Pascal Ribereau-GayonDenis DubourdieuBernard Doneche

Aline Lonvaud

Faculty of EnologyVictor Segalen University of Bordeaux II, Talence, France

Original translation by

Jeffrey M. Branco, Jr.Winemaker

M.S., Faculty of Enology, University of Bordeaux II

Revision translated by

Christine RychlewskiAquitaine Traduction, Bordeaux, France

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Pascal Ribereau-GayonDenis DubourdieuBernard Doneche

Aline Lonvaud

Faculty of EnologyVictor Segalen University of Bordeaux II, Talence, France

Original translation by

Jeffrey M. Branco, Jr.Winemaker

M.S., Faculty of Enology, University of Bordeaux II

Revision translated by

Christine RychlewskiAquitaine Traduction, Bordeaux, France

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Library of Congress Cataloging-in-Publication Data:

Ribereau-Gayon, Pascal.[Traite doenologie. English]Handbook of enology / Pascal Ribereau-Gayon, Denis Dubourdieu, Bernard

Doneche ; original translation by Jeffrey M. Branco, Jr.2nd ed. /translation of updates for 2nd ed. [by] Christine Rychlewski.

v. cm.Rev. ed. of: Handbook of enology / Pascal Ribereau Gayon . . . [et al.].

c2000.Includes bibliographical references and index.Contents: v. 1. The microbiology of wine and vinificationsISBN-13: 978-0-470-01034-1 (v. 1 : acid-free paper)ISBN-10: 0-470-01034-7 (v. 1 : acid-free paper)

1. Wine and wine makingHandbooks, manuals, etc. 2. Wine and winemakingMicrobiologyHandbooks, manuals, etc. 3. Wine and winemakingChemistryHandbooks, manuals, etc. I. Dubourdieu, Denis. II.Doneche, Bernard. III. Traite doenologie. English. IV. Title.

TP548.T7613 2005663.2dc22

2005013973

British Library Cataloguing in Publication Data

A catalogue record for this book is available from the British Library

ISBN-13: 978-0-470-01034-1 (HB)ISBN-10: 0-470-01034-7 (HB)

Typeset in 10/12pt Times by Laserwords Private Limited, Chennai, IndiaPrinted and bound in Great Britain by Antony Rowe Ltd, Chippenham, WiltshireThis book is printed on acid-free paper responsibly manufactured from sustainable forestryin which at least two trees are planted for each one used for paper production.

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ContentsRemarks Concerning the Expression of Certain Parameters of Must and Wine Composition viiPreface to the First Edition ixPreface to the Second Edition xiii

1 Cytology, Taxonomy and Ecology of Grape and Wine Yeasts 1

2 Biochemistry of Alcoholic Fermentation and Metabolic Pathways of Wine Yeasts 53

3 Conditions of Yeast Development 79

4 Lactic Acid Bacteria 115

5 Metabolism of Lactic Acid Bacteria 139

6 Lactic Acid Bacteria Development in Wine 161

7 Acetic Acid Bacteria 183

8 The Use of Sulfur Dioxide in Must and Wine Treatment 193

9 Products and Methods Complementing the Effect of Sulfur Dioxide 223

10 The Grape and its Maturation 241

11 Harvest and Pre-Fermentation Treatments 299

12 Red Winemaking 327

13 White Winemaking 397

14 Other Winemaking Methods 445

Index 481

Remarks Concerning the Expressionof Certain Parameters of Mustand Wine CompositionUNITS

Metric system units of length (m), volume (l) andweight (g) are exclusively used. The conversion ofmetric units into Imperial units (inches, feet, gal-lons, pounds, etc.) can be found in the followingenological work: Principles and practices of wine-making, R.B. Boulton, V.L. Singleton, L.F. Bissonand R.E. Kunkee, 1995, The Chapman & HallEnology Library, New York.

EXPRESSION OF TOTAL ACIDITYAND VOLATILE ACIDITY

Although EC regulations recommend the expres-sion of total acidity in the equivalent weight of tar-taric acid, the French custom is to give this expres-sion in the equivalent weight of sulfuric acid. The

more correct expression in milliequivalents perliter has not been embraced in France. The expres-sion of total and volatile acidity in the equivalentweight of sulfuric acid has been used predomi-nantly throughout these works. In certain cases, thecorresponding weight in tartaric acid, often used inother countries, has been given.

Using the weight of the milliequivalent of thevarious acids, the below table permits the conver-sion from one expression to another.

More particularly, to convert from total acidityexpressed in H2SO4 to its expression in tartaricacid, add half of the value to the original value(4 g/l H2SO4 6 g/l tartaric acid). In the otherdirection a third of the value must be subtracted.

The French also continue to express volatileacidity in equivalent weight of sulfuric acid. Moregenerally, in other countries, volatile acidity is

Desired Expression

Known Expression meq/l g/l g/l g/lH2SO4 tartaric acid acetic acid

meq/l 1.00 0.049 0.075 0.060

g/l H2SO4 20.40 1.00 1.53 1.22

g/l tartaric acid 13.33 0.65 1.00

g/l acetic acid 16.67 0.82 1.00

Multiplier to pass from one expression of total or volatile acidity to another

viii Remarks Concerning the Expression of Certain Parameters of Must and Wine Composition

expressed in acetic acid. It is rarely expressedin milliequivalents per liter. The below table alsoallows simple conversion from one expression toanother.

The expression in acetic acid is approximately20% higher than in sulfuric acid.

EVALUATING THE SUGARCONCENTRATION OF MUSTS

This measurement is important for tracking grapematuration, fermentation kinetic and if necessarydetermining the eventual need for chaptalization.

This measurement is always determined byphysical, densimetric or refractometric analysis.The expression of the results can be given accord-ing to several scales: some are rarely used, i.e.degree Baume and degree Oechsle. Presently, twosystems exist (Section 10.4.3):

1. The potential alcohol content (titre alcoomet-raque potential or TAP, in French) of mustscan be read directly on equipment, which isgraduated using a scale corresponding to 17.5or 17 g/l of sugar for 1% volume of alcohol.Today, the EC recommends using 16.83 g/l asthe conversion factor. The mustimeter is ahydrometer containing two graduated scales:one expresses density and the other gives adirect reading of the TAP. Different methodsvarying in precision exist to calculate the TAPfrom a density reading. These methods take var-ious elements of must composition into account(Boulton et al., 1995).

2. Degree Brix expresses the percentage of sugarin weight. By multiplying degree Brix by 10,the weight of sugar in 1 kg, or slightly lessthan 1 liter, of must is obtained. A conversiontable between degree Brix and TAP exists inSection 10.4.3 of this book. 17 degrees Brixcorrespond to an approximate TAP of 10% and20 degrees Brix correspond to a TAP of about12%. Within the alcohol range most relevant toenology, degree Brix can be multiplied by 10

and then divided by 17 to obtain a fairly goodapproximation of the TAP.

In any case, the determination of the Brix or TAPof a must is approximate. First of all, it is notalways possible to obtain a representative grapeor must sample for analysis. Secondly, althoughphysical, densimetric or refractometric measure-ments are extremely precise and rigorously expressthe sugar concentration of a sugar and water mix-ture, these measurements are affected by other sub-stances released into the sample from the grapeand other sources. Furthermore, the concentrationsof these substances are different for every grapeor grape must sample. Finally, the conversion rateof sugar into alcohol (approximately 17 to 18 g/l)varies and depends on fermentation conditions andyeast properties. The widespread use of selectedyeast strains has lowered the sugar conversion rate.

Measurements Using Visibleand Ultraviolet SpectrometryThe measurement of optic density, absorbance, iswidely used to determine wine color (Volume 2,Section 6.4.5) and total phenolic compounds con-centration (Volume 2, Section 6.4.1). In theseworks, the optic density is noted as OD, OD 420(yellow), OD 520 (red), OD 620 (blue) or OD 280(absorption in ultraviolet spectrum) to indicate theoptic density at the indicated wavelengths.

Wine color intensity is expressed as:

CI = OD 420 + OD 520 + OD 620,Or is sometimes expressed in a more simplifiedform: CI = OD 420 + OD 520.

Tint is expressed as:

T = OD 420OD 520

The total phenolic compound concentration isexpressed by OD 280.

The analysis methods are described in Chapter 6of Handbook of Enology Volume 2, The Chemistryof Wine.

Preface to the First EditionWine has probably inspired more research andpublications than any other beverage or food. Infact, through their passion for wine, great scientistshave not only contributed to the development ofpractical enology but have also made discoveriesin the general field of science.

A forerunner of modern enology, Louis Pasteurdeveloped simplified contagious infection mod-els for humans and animals based on his obser-vations of wine spoilage. The following quoteclearly expresses his theory in his own words:when profound alterations of beer and wine areobserved because these liquids have given refugeto microscopic organisms, introduced invisibly andaccidentally into the medium where they thenproliferate, how can one not be obsessed by thethought that a similar phenomenon can and mustsometimes occur in humans and animals.

Since the 19th century, our understanding ofwine, wine composition and wine transformationshas greatly evolved in function of advances in rel-evant scientific fields i.e. chemistry, biochemistry,microbiology. Each applied development has leadto better control of winemaking and aging con-ditions and of course wine quality. In order tocontinue this approach, researchers and winemak-ers must strive to remain up to date with the latestscientific and technical developments in enology.

For a long time, the Bordeaux school of enologywas largely responsible for the communication ofprogress in enology through the publication ofnumerous works (Beranger Publications and laterDunod Publications):

Wine Analysis U. Gayon and J. Laborde (1912);Treatise on Enology J. Ribereau-Gayon (1949);

Wine Analysis J. Ribereau-Gayon and E. Peynaud(1947 and 1958); Treatise on Enology (2 Volumes)J. Ribereau-Gayon and E. Peynaud (1960 and1961); Wine and Winemaking E. Peynaud (1971and 1981); Wine Science and Technology (4 volu-mes) J. Ribereau-Gayon, E. Peynaud, P. Ribereau-Gayon and P. Sudraud (19751982).

For an understanding of current advances inenology, the authors propose this book Handbookof Enology Volume 1: The Microbiology of Wineand Vinifications and the second volume of theHandbook of Enology Volume 2: The Chemistry ofWine: Stabilization and Treatments.

Although written by researchers, the two vol-umes are not specifically addressed to this group.Young researchers may, however, find these booksuseful to help situate their research within a par-ticular field of enology. Today, the complexity ofmodern enology does not permit a sole researcherto explore the entire field.

These volumes are also of use to students andprofessionals. Theoretical interpretations as wellas solutions are presented to resolve the problemsencountered most often at wineries. The authorshave adapted these solutions to many different sit-uations and winemaking methods. In order to makethe best use of the information contained in theseworks, enologists should have a broad understand-ing of general scientific knowledge. For example,the understanding and application of molecularbiology and genetic engineering have becomeindispensable in the field of wine microbiology.Similarly, structural and quantitative physiochem-ical analysis methods such as chromatography,

x Preface to the First Edition

NMR and mass spectrometry must now bemastered in order to explore wine chemistry.

The goal of these two works was not to createan exhaustive bibliography of each subject. Theauthors strove to choose only the most relevant andsignificant publications to their particular field ofresearch. A large number of references to Frenchenological research has been included in theseworks in order to make this information availableto a larger non-French-speaking audience.

In addition, the authors have tried to conveya French and more particularly a Bordeaux per-spective of enology and the art of winemaking.The objective of this perspective is to maximizethe potential quality of grape crops based on thespecific natural conditions that constitute their ter-roir. The role of enology is to express the char-acteristics of the grape specific not only to varietyand vineyard practices but also maturation condi-tions, which are dictated by soil and climate.

It would, however, be an error to think that theworlds greatest wines are exclusively a result oftradition, established by exceptional natural con-ditions, and that only the most ordinary wines,produced in giant processing facilities, can ben-efit from scientific and technological progress.Certainly, these facilities do benefit the most fromhigh performance installations and automation ofoperations. Yet, history has unequivocally shownthat the most important enological developmentsin wine quality (for example, malolactic fermenta-tion) have been discovered in ultra premium wines.The corresponding techniques were then applied toless prestigious products.

High performance technology is indispensablefor the production of great wines, since a lackof control of winemaking parameters can easilycompromise their quality, which would be less ofa problem with lower quality wines.

The word vinification has been used in thiswork and is part of the technical language ofthe French tradition of winemaking. Vinificationdescribes the first phase of winemaking. It com-prises all technical aspects from grape maturityand harvest to the end of alcoholic and some-times malolactic fermentation. The second phaseof winemaking winematuration, stabilization and

treatments is completed when the wine is bottled.Aging specifically refers to the transformation ofbottled wine.

This distinction of two phases is certainly theresult of commercial practices. Traditionally inFrance, a vine grower farmed the vineyard andtransformed grapes into an unfinished wine. Thewine merchant transferred the bulk wine to his cel-lars, finished the wine and marketed the product,preferentially before bottling. Even though mostwines are now bottled at the winery, these long-standing practices have maintained a distinctionbetween wine grower enology and wine mer-chant enology. In countries with a more recentviticultural history, generally English speaking, thevine grower is responsible for winemaking andwine sales. For this reason, the Anglo-Saxon tradi-tion speaks of winemaking, which covers all oper-ations from harvest reception to bottling.

In these works, the distinction between vinifi-cation and stabilization and treatments has beenmaintained, since the first phase primarily concernsmicrobiology and the second chemistry. In thismanner, the individual operations could be linkedto their particular sciences. There are of course lim-its to this approach. Chemical phenomena occurduring vinification; the stabilization of wines dur-ing storage includes the prevention of microbialcontamination.

Consequently, the description of the differentsteps of enology does not always obey logic asprecise as the titles of these works may leadto believe. For example, microbial contaminationduring aging and storage are covered in Vol-ume 1. The antiseptic properties of SO2 incited thedescription of its use in the same volume. This lineof reasoning lead to the description of the antioxi-dant related chemical properties of this compoundin the same chapter as well as an explanation ofadjuvants to sulfur dioxide: sorbic acid (antisep-tic) and ascorbic acid (antioxidant). In addition,the on lees aging of white wines and the result-ing chemical transformations cannot be separatedfrom vinification and are therefore also coveredin Volume 1. Finally, our understanding of pheno-lic compounds in red wine is based on complexchemistry. All aspects related to the nature of the

Preface to the First Edition xi

corresponding substances, their properties and theirevolution during grape maturation, vinification andaging are therefore covered in Volume 2.

These works only discuss the principles ofequipment used for various enological operationsand their effect on product quality. For example,temperature control systems, destemmers, crushersand presses as well as filters, inverse osmosismachines and ion exchangers are not described indetail. Bottling is not addressed at all. An in-depthdescription of enological equipment would merit adetailed work dedicated to the subject.

Wine tasting, another essential role of thewinemaker, is not addressed in these works.Many related publications are, however, readilyavailable. Finally, wine analysis is an essential toolthat a winemaker should master. It is, however, notcovered in these works except in a few particular

cases i.e. phenolic compounds, whose differentfamilies are often defined by analytical criteria.

The authors thank the following people whohave contributed to the creation of this work:J.F. Casas Lucas, Chapter 14, Sherry; A. Brugi-rard, Chapter 14, Sweet wines; J.N. de Almeida,Chapter 14, Port wines; A. Maujean, Chapter 14,Champagne; C. Poupot for the preparation ofmaterial in Chapters 1, 2 and 13; Miss F. Luye-Tanet for her help with typing.

They also thank Madame B. Masclef in particu-lar for her important part in the typing, preparationand revision of the final manuscript.

Pascal Ribereau-GayonBordeaux

Preface to the Second EditionThe two-volume Enology Handbook was pub-lished simultaneously in Spanish, French, and Ital-ian in 1999 and has been reprinted several times.The Handbook has apparently been popular withstudents as an educational reference book, as wellas with winemakers, as a source of practical solu-tions to their specific technical problems and sci-entific explanations of the phenomena involved.

It was felt appropriate at this stage to preparean updated, reviewed, corrected version, includingthe latest enological knowledge, to reflect the manynew research findings in this very active field. Theoutline and design of both volumes remain thesame. Some chapters have changed relatively littleas the authors decided there had not been any sig-nificant new developments, while others have beenmodified much more extensively, either to clarifyand improve the text, or, more usually, to includenew research findings and their practical applica-tions. Entirely new sections have been inserted insome chapters.

We have made every effort to maintain the sameapproach as we did in the first edition, reflectingthe ethos of enology research in Bordeaux. We useindisputable scientific evidence in microbiology,biochemistry, and chemistry to explain the detailsof mechanisms involved in grape ripening, fermen-tations and other winemaking operations, aging,and stabilization. The aim is to help winemakersachieve greater control over the various stages inwinemaking and choose the solution best suitedto each situation. Quite remarkably, this scientificapproach, most intensively applied in making thefinest wines, has resulted in an enhanced capac-ity to bring out the full quality and character of

individual terroirs. Scientific winemaking has notresulted in standardization or leveling of quality.On the contrary, by making it possible to correctdefects and eliminate technical imperfections, ithas revealed the specific qualities of the grapesharvested in different vineyards, directly related tothe variety and terroir, more than ever before.

Interest in wine in recent decades has gonebeyond considerations of mere quality and takenon a truly cultural dimension. This has led somepeople to promote the use of a variety of tech-niques that do not necessarily represent significantprogress in winemaking. Some of these are sim-ply modified forms of processes that have beenknown for many years. Others do not have a suf-ficiently reliable scientific interpretation, nor aretheir applications clearly defined. In this Hand-book, we have only included rigorously testedtechniques, clearly specifying the optimum con-ditions for their utilization.

As in the previous edition, we deliberatelyomitted three significant aspects of enology: wineanalysis, tasting, and winery engineering. In viewof their importance, these topics will each becovered in separate publications.

The authors would like to take the opportunityof the publication of this new edition of Volume 1to thank all those who have contributed to updatingthis work:

Marina Bely for her work on fermentationkinetics (Section 3.4) and the production ofvolatile acidity (Sections 2.3.4 and 14.2.5)

Isabelle Masneuf for her investigation of theyeasts nitrogen supply (Section 3.4.2)

xiv Preface to the Second Edition

Gilles de Revel for elucidating the chemistryof SO2, particularly, details of combinationreactions (Section 8.4)

Gilles Masson for the section on rose wines(Section 14.1)

Cornelis Van Leeuwen for data on the impactof vineyard water supply on grape ripening(Section 10.4.6)

Andre Brugirard for the section on Frenchfortified winesvins doux naturels (Section14.4.2)

Paulo Barros and Joa Nicolau de Almeida fortheir work on Port (Section 14.4.3)

Justo. F. Casas Lucas for the paragraph onSherry (Section 14.5.2)

Alain Maujean for his in-depth revision of thesection on Champagne (Section 14.3).

March 17, 2005

Professor Pascal RIBEREAU-GAYONCorresponding Member of the InstituteMember of the French Academy of Agriculture

1

Cytology, Taxonomy and Ecologyof Grape and Wine Yeasts

1.1 Introduction 11.2 The cell wall 31.3 The plasmic membrane 71.4 The cytoplasm and its organelles 111.5 The nucleus 141.6 Reproduction and the yeast biological cycle 151.7 The killer phenomenon 191.8 Classification of yeast species 221.9 Identification of wine yeast strains 35

1.10 Ecology of grape and wine yeasts 40

1.1 INTRODUCTION

Man has been making bread and fermented bev-erages since the beginning of recorded history.Yet the role of yeasts in alcoholic fermentation,particularly in the transformation of grapes intowine, was only clearly established in the middleof the nineteenth century. The ancients explainedthe boiling during fermentation (from the Latinfervere, to boil) as a reaction between substances

that come into contact with each other duringcrushing. In 1680, a Dutch cloth merchant, Antonievan Leeuwenhoek, first observed yeasts in beerwort using a microscope that he designed andproduced. He did not, however, establish a rela-tionship between these corpuscles and alcoholicfermentation. It was not until the end of the eigh-teenth century that Lavoisier began the chemicalstudy of alcoholic fermentation. Gay-Lussac con-tinued Lavoisiers research into the next century.

Handbook of Enology Volume 1 The Microbiology of Wine and Vinifications P. Ribereau-Gayon, D. Dubourdieu, B. Doneche and A. Lonvaud 2006 John Wiley & Sons, Ltd

2 Handbook of Enology: The Microbiology of Wine and Vinifications

As early as 1785, Fabroni, an Italian scientist, wasthe first to provide an interpretation of the chem-ical composition of the ferment responsible foralcoholic fermentation, which he described as aplantanimal substance. According to Fabroni, thismaterial, comparable to the gluten in flour, waslocated in special utricles, particularly on grapesand wheat, and alcoholic fermentation occurredwhen it came into contact with sugar in the must. In1837, a French physicist named Charles Cagnardde La Tour proved for the first time that the yeastwas a living organism. According to his findings,it was capable of multiplying and belonged to theplant kingdom; its vital activities were at the baseof the fermentation of sugar-containing liquids.The German naturalist Schwann confirmed his the-ory and demonstrated that heat and certain chem-ical products were capable of stopping alcoholicfermentation. He named the beer yeast zucker-pilz, which means sugar fungusSaccharomycesin Latin. In 1838, Meyen used this nomenclaturefor the first time.

This vitalist or biological viewpoint of the roleof yeasts in alcoholic fermentation, obvious tous today, was not readily supported. Liebig andcertain other organic chemists were convinced thatchemical reactions, not living cellular activity,were responsible for the fermentation of sugar.In his famous studies on wine (1866) and beer(1876), Louis Pasteur gave definitive credibilityto the vitalist viewpoint of alcoholic fermentation.He demonstrated that the yeasts responsible forspontaneous fermentation of grape must or crushedgrapes came from the surface of the grape;he isolated several races and species. He evenconceived the notion that the nature of the yeastcarrying out the alcoholic fermentation couldinfluence the gustatory characteristics of wine. Healso demonstrated the effect of oxygen on theassimilation of sugar by yeasts. Louis Pasteurproved that the yeast produced secondary productssuch as glycerol in addition to alcohol and carbondioxide.

Since Pasteur, yeasts and alcoholic fermen-tation have incited a considerable amount ofresearch, making use of progress in microbiology,

biochemistry and now genetics and molecularbiology.

In taxonomy, scientists define yeasts as unicel-lular fungi that reproduce by budding and binaryfission. Certain pluricellular fungi have a unicellu-lar stage and are also grouped with yeasts. Yeastsform a complex and heterogeneous group foundin three classes of fungi, characterized by theirreproduction mode: the sac fungi (Ascomycetes),the club fungi (Basidiomycetes), and the imper-fect fungi (Deuteromycetes). The yeasts found onthe surface of the grape and in wine belong toAscomycetes and Deuteromycetes. The haploidspores or ascospores of the Ascomycetes class arecontained in the ascus, a type of sac made fromvegetative cells. Asporiferous yeasts, incapable ofsexual reproduction, are classified with the imper-fect fungi.

In this first chapter, the morphology, repro-duction, taxonomy and ecology of grape andwine yeasts will be discussed. Cytology is themorphological and functional study of the struc-tural components of the cell (Rose and Harrison,1991).

Fig. 1.1. A yeast cell (Gaillardin and Heslot, 1987)

Cytology, Taxonomy and Ecology of Grape and Wine Yeasts 3

Yeasts are the most simple of the eucaryotes.The yeast cell contains cellular envelopes, acytoplasm with various organelles, and a nucleussurrounded by a membrane and enclosing thechromosomes. (Figure 1.1). Like all plant cells,the yeast cell has two cellular envelopes: thecell wall and the membrane. The periplasmicspace is the space between the cell wall andthe membrane. The cytoplasm and the membranemake up the protoplasm. The term protoplastor sphaeroplast designates a cell whose cellwall has been artificially removed. Yeast cellularenvelopes play an essential role: they contributeto a successful alcoholic fermentation and releasecertain constituents which add to the resultingwines composition. In order to take advantage ofthese properties, the winemaker or enologist musthave a profound knowledge of these organelles.

1.2 THE CELL WALL

1.2.1 The General Roleof the Cell Wall

During the last 20 years, researchers (Fleet, 1991;Klis, 1994; Stratford, 1999; Klis et al., 2002) havegreatly expanded our knowledge of the yeast cellwall, which represents 1525% of the dry weightof the cell. It essentially consists of polysaccha-rides. It is a rigid envelope, yet endowed with acertain elasticity.

Its first function is to protect the cell. Withoutits wall, the cell would burst under the internalosmotic pressure, determined by the compositionof the cells environment. Protoplasts placed inpure water are immediately lysed in this manner.Cell wall elasticity can be demonstrated by placingyeasts, taken during their log phase, in a hypertonic(NaCl) solution. Their cellular volume decreasesby approximately 50%. The cell wall appearsthicker and is almost in contact with the membrane.The cells regain their initial form after being placedback into an isotonic medium.

Yet the cell wall cannot be considered an inert,semi-rigid armor. On the contrary, it is a dynamicand multifunctional organelle. Its composition andfunctions evolve during the life of the cell, in

response to environmental factors. In addition toits protective role, the cell wall gives the cellits particular shape through its macromolecularorganization. It is also the site of moleculeswhich determine certain cellular interactions suchas sexual union, flocculation, and the killerfactor, which will be examined in detail later inthis chapter (Section 1.7). Finally, a number ofenzymes, generally hydrolases, are connected tothe cell wall or situated in the periplasmic space.Their substrates are nutritive substances of theenvironment and the macromolecules of the cellwall itself, which is constantly reshaped duringcellular morphogenesis.

1.2.2 The Chemical Structureand Function of the ParietalConstituents

The yeast cell wall is made up of two prin-cipal constituents: -glucans and mannoproteins.Chitin represents a minute part of its composi-tion. The most detailed work on the yeast cellwall has been carried out on Saccharomyces cere-visiae the principal yeast responsible for thealcoholic fermentation of grape must.

Glucan represents about 60% of the dry weightof the cell wall of S. cerevisiae. It can bechemically fractionated into three categories:

1. Fibrous -1,3 glucan is insoluble in water,acetic acid and alkali. It has very few branches.The branch points involved are -1,6 linkages.Its degree of polymerization is 1500. Underthe electron microscope, this glucan appearsfibrous. It ensures the shape and the rigidity ofthe cell wall. It is always connected to chitin.

2. Amorphous -1,3 glucan, with about 1500glucose units, is insoluble in water but solublein alkalis. It has very few branches, like thepreceding glucan. In addition to these fewbranches, it is made up of a small number of-1,6 glycosidic linkages. It has an amorphousaspect under the electron microscope. It givesthe cell wall its elasticity and acts as an anchorfor the mannoproteins. It can also constitute anextraprotoplasmic reserve substance.


3. The -1,6 glucan is obtained from alkali-insoluble glucans by extraction in acetic acid.The resulting product is amorphous, water sol-uble, and extensively ramified by -1,3 glyco-sidic linkages. Its degree of polymerization is140. It links the different constituents of thecell wall together. It is also a receptor site forthe killer factor.

The fibrous -1,3 glucan (alkali-insoluble) proba-bly results from the incorporation of chitin on theamorphous -1,3 glucan.

Mannoproteins constitute 2550% of the cellwall of S. cerevisiae. They can be extracted fromthe whole cell or from the isolated cell wallby chemical and enzymatic methods. Chemicalmethods make use of autoclaving in the pres-ence of alkali or a citrate buffer solution atpH 7. The enzymatic method frees the manno-proteins by digesting the glucan. This methoddoes not denature the structure of the mannopro-teins as much as chemical methods. Zymolyase,obtained from the bacterium Arthrobacter luteus,is the enzymatic preparation most often used toextract the parietal mannoproteins of S. cerevisiae.This enzymatic complex is effective primarilybecause of its -1,3 glucanase activity. The actionof protease contaminants in the zymolyase com-bine, with the aforementioned activity to liberatethe mannoproteins. Glucanex, another industrialpreparation of the -glucanase, produced by a fun-gus (Trichoderma harzianum), has been recentlydemonstrated to possess endo- and exo--1,3 andendo--1,6-glucanase activities (Dubourdieu andMoine, 1995). These activities also facilitate theextraction of the cell wall mannoproteins of theS. cerevisiae cell.

The mannoproteins of S. cerevisiae have amolecular weight between 20 and 450 kDa. Theirdegree of glycosylation varies. Certain ones con-taining about 90% mannose and 10% peptides arehypermannosylated.

Four forms of glycosylation are described(Figure 1.2) but do not necessarily exist at thesame time in all of the mannoproteins.

The mannose of the mannoproteins can consti-tute short, linear chains with one to five residues.

They are linked to the peptide chain by O-glycosyllinkages on serine and threonine residues. Theseglycosidic side-chain linkages are -1,2 and -1,3.

The glucidic part of the mannoprotein can alsobe a polysaccharide. It is linked to an asparagineresidue of the peptide chain by an N -glycosyllinkage. This linkage consists of a double unit ofN -acetylglucosamine (chitin) linked in -1,4. Themannan linked in this manner to the asparagineincludes an attachment region made up of a dozenmannose residues and a highly ramified outerchain consisting of 150 to 250 mannose units.The attachment region beyond the chitin residueconsists of a mannose skeleton linked in -1,6with side branches possessing one, two or threemannose residues with -1,2 and/or -1,3 bonds.The outer chain is also made up of a skeleton ofmannose units linked in -1,6. This chain bearsshort side-chains constituted of mannose residueslinked in -1,2 and a terminal mannose in -1,3. Some of these side-chains possess a branchattached by a phosphodiester bond.

A third type of glycosylation was describedmore recently. It can occur in mannoproteins,which make up the cell wall of the yeast. It consistsof a glucomannan chain containing essentiallymannose residues linked in -1,6 and glucoseresidues linked in -1,6. The nature of the glycanpeptide point of attachment is not yet clear, but itmay be an asparaginylglucose bond. This type ofglycosylation characterizes the proteins freed fromthe cell wall by the action of a -1,3 glucanase.Therefore, in vivo, the glucomannan chain mayalso comprise glucose residues linked in -1,3.

The fourth type of glycosylation of yeast manno-proteins is the glycosylphosphatidylinositolanchor (GPI). This attachment between the ter-minal carboxylic group of the peptide chain anda membrane phospholipid permits certain manno-proteins, which cross the cell wall, to anchorthemselves in the plasmic membrane. The regionof attachment is characterized by the followingsequence (Figure 1.2): ethanolamine-phosphate-6-mannose--1,2-mannose--1,6-mannose--1,4-glucosamine--1,6-inositol-phospholipid. A C-phospholipase specific to phosphatidyl inositoland therefore capable of realizing this cleavage


6M[M 6M 6M 6M ]n 6M 6M

2

M

2

M

2

M

2

M2

M

2

M

2

M

3

M

3

M

3

M

P

M

3

M

2

M3

M

3

M

2

M P2

M

3

2 3

M M

MP 6M6

M 4 GNAc 4 GNAc NH Asn

3M 3M 2M 2M O Ser/Thr

(G,M) Xxx

lipid P Ins 6 GN 4 M 6 M 2 M 6 P (CH2)2 NH C O

Fig. 1.2. The four types of glucosylation of parietal yeast mannoproteins (Klis, 1994). M = mannose; G = glucose;GN = glucosamine; GNAc = N-acetylglucosamine; Ins = inositol; Ser = Serine; Thr = threonine; Asn = asparagine;Xxx = the nature of the bond is not known

was demonstrated in the S. cerevisiae (Flick andThorner, 1993). Several GPI-type anchor manno-proteins have been identified in the cell wall ofS. cerevisiae.

Chitin is a linear polymer of N -acetylglucos-amine linked in -1,4 and is not generally found inlarge quantities in yeast cell walls. In S. cerevisiae,chitin constitutes 12% of the cell wall and isfound for the most part (but not exclusively) inbud scar zones. These zones are a type of raisedcrater easily seen on the mother cell under theelectron microscope (Figure 1.3). This chitinic scaris formed essentially to assure cell wall integrityand cell survival. Yeasts treated with D polyoxine,an antibiotic inhibiting the synthesis of chitin, arenot viable; they burst after budding.

The presence of lipids in the cell wall has notbeen clearly demonstrated. It is true that cell walls

Fig. 1.3. Scanning electron microscope photograph ofproliferating S. cerevisiae cells. The budding scars onthe mother cells can be observed


prepared in the laboratory contain some lipids(215% for S. cerevisiae) but it is most likelycontamination by the lipids of the cytoplasmicmembrane, adsorbed by the cell wall during theirisolation. The cell wall can also adsorb lipids fromits external environment, especially the differentfatty acids that activate and inhibit the fermentation(Chapter 3).

Chitin are connected to the cell wall or sit-uated in the periplasmic space. One of themost characteristic enzymes is the invertase (-fructofuranosidase). This enzyme catalyzes thehydrolysis of saccharose into glucose and fruc-tose. It is a thermostable mannoprotein anchoredto a -1,6 glucan of the cell wall. Its molecularweight is 270 000 Da. It contains approximately50% mannose and 50% protein. The periplasmicacid phosphatase is equally a mannoprotein.

Other periplasmic enzymes that have been notedare -glucosidase, -galactosidase, melibiase, tre-halase, aminopeptidase and esterase. Yeast cellwalls also contain endo- and exo--glucanases (-1,3 and -1,6). These enzymes are involved in thereshaping of the cell wall during the growth andbudding of cells. Their activity is at a maximumduring the exponential log phase of the populationand diminishes notably afterwards. Yet cells in thestationary phase and even dead yeasts containedin the lees still retain -glucanases activity intheir cell walls several months after the completionof fermentation. These endogenous enzymes areinvolved in the autolysis of the cell wall during the

ageing of wines on lees. This ageing method willbe covered in the chapter on white winemaking(Chapter 13).

1.2.3 General Organization of the CellWall and Factors Affecting itsComposition

The cell wall of S. cerevisiae is made up of anouter layer of mannoproteins. These mannopro-teins are connected to a matrix of amorphous -1,3glucan which covers an inner layer of fibrous -1,3 glucan. The inner layer is connected to a smallquantity of chitin (Figure 1.4). The -1,6 glucanprobably acts as a cement between the two lay-ers. The rigidity and the shape of the cell wallare due to the internal framework of the -1,3fibrous glucan. Its elasticity is due to the outeramorphous layer. The intermolecular structure ofthe mannoproteins of the outer layer (hydrophobiclinkages and disulfur bonds) equally determinescell wall porosity and impermeability to macro-molecules (molecular weights less than 4500). Thisimpermeability can be affected by treating thecell wall with certain chemical agents, such as-mercaptoethanol. This substance provokes therupture of the disulfur bonds, thus destroying theintermolecular network between the mannoproteinchains.

The composition of the cell wall is stronglyinfluenced by nutritive conditions and cell age.The proportion of glucan in the cell wall increases

Cytoplasm

Cytoplasmic membrane

Mannoproteins and -1,3 amorphous glucan

- 1,3 fibrous glucan

Cell wall

Periplasmic space

External medium

Fig. 1.4. Cellular organization of the cell wall of S. cerevisiae


with respect to the amount of sugar in the cul-ture medium. Certain deficiencies (for example,in mesoinositol) also result in an increase in theproportion of glucan compared with mannopro-teins. The cell walls of older cells are richer inglucans and in chitin and less furnished in manno-proteins. For this reason, they are more resistantto physical and enzymatic agents used to degradethem. Finally, the composition of the cell wall isprofoundly modified by morphogenetic alterations(conjugation and sporulation).

1.3 THE PLASMIC MEMBRANE

1.3.1 Chemical Compositionand Organization

The plasmic membrane is a highly selective barriercontrolling exchanges between the living cell andits external environment. This organelle is essentialto the life of the yeast.

Like all biological membranes, the yeast plasmicmembrane is principally made up of lipids andproteins. The plasmic membrane of S. cerevisiaecontains about 40% lipids and 50% proteins.Glucans and mannans are only present in smallquantities (several per cent).

The lipids of the membrane are essentiallyphospholipids and sterols. They are amphiphilicmolecules, i.e. possessing a hydrophilic and ahydrophobic part.

The three principal phospholipids (Figure 1.5)of the plasmic membrane of yeast are phos-phatidylethanolamine (PE), phosphatidylcholine(PC) and phosphatidylinositol (PI) which repre-sent 7085% of the total. Phosphatidylserine (PS)and diphosphatidylglycerol or cardiolipin (PG) areless prevalent. Free fatty acids and phosphatidicacid are frequently reported in plasmic membraneanalysis. They are probably extraction artifactscaused by the activity of certain lipid degradationenzymes.

The fatty acids of the membrane phospholipidscontain an even number (14 to 24) of carbon atoms.The most abundant are C16 and C18 acids. Theycan be saturated, such as palmitic acid (C16) andstearic acid (C18), or unsaturated, as with oleic

acid (C18, double bond in position 9), linoleic acid(C18, two double bonds in positions 9 and 12) andlinolenic acid (C18, three double bonds in positions9, 12 and 15). All membrane phospholipids sharea common characteristic: they possess a polar orhydrophilic part made up of a phosphorylatedalcohol and a non-polar or hydrophobic partcomprising two more or less parallel fatty acidchains (Figure 1.6). In an aqueous medium, thephospholipids spontaneously form bimolecularfilms or a lipid bilayer because of their amphiphiliccharacteristic (Figure 1.6). The lipid bilayers arecooperative but non-covalent structures. Theyare maintained in place by mutually reinforcedinteractions: hydrophobic interactions, van derWaals attractive forces between the hydrocarbontails, hydrostatic interactions and hydrogen bondsbetween the polar heads and water molecules.The examination of cross-sections of yeastplasmic membrane under the electron microscopereveals a classic lipid bilayer structure with athickness of about 7.5 nm. The membrane surfaceappears sculped with creases, especially duringthe stationary phase. However, the physiologicalmeaning of this anatomic character remainsunknown. The plasmic membrane also has anunderlying depression on the bud scar.

Ergosterol is the primary sterol of the yeast plas-mic membrane. In lesser quantities, 24 (28) dehy-droergosterol and zymosterol also exist (Figure1.7). Sterols are exclusively produced in the mito-chondria during the yeast log phase. As with phos-pholipids, membrane sterols are amphipathic. Thehydrophilic part is made up of hydroxyl groupsin C-3. The rest of the molecule is hydrophobic,especially the flexible hydrocarbon tail.

The plasmic membrane also contains numerousproteins or glycoproteins presenting a wide rangeof molecular weights (from 10 000 to 120 000).The available information indicates that the orga-nization of the plasmic membrane of a yeast cellresembles the fluid mosaic model. This model,proposed for biological membranes by Singer andNicolson (1972), consists of two-dimensional solu-tions of proteins and oriented lipids. Certain pro-teins are embedded in the membrane; they arecalled integral proteins (Figure 1.6). They interact


R' C O

O

CH

H2C O P

O

O

O CH2 CH2 NH3+

Phosphatidyl ethanolamine

R C

O

O

R' C

O

O

CH2

CH

H2C O P

O

O

O CH2 C

H

COO

NH3+

Phosphatidyl serine

OHOH

H H

O

H

OHH

H

HO

OH H

P

O

O

O

CH2

HC

H2C

O

O C

C

O

O

R'

R

Phosphatidyl inositol

R C O

O

CH2

CHOCR'

O H2C O P

O

O

O CH2 CH2 N+(CH3)3

Phosphatidyl choline

R C

O

O CH2

CHOCR'

O H2C O P

O

O

O CH2 C CH2 O P

O

O

O CH2

HC O

H2C O

C

C R

O

R'

O

Diphosphatidyl glycerol (cardiolipin)

R C

O

O CH2

Fig. 1.5. Yeast membrane phospholipids

strongly with the non-polar part of the lipid bilayer.The peripheral proteins are linked to the precedentby hydrogen bonds. Their location is asymmetrical,at either the inner or the outer side of the plasmicmembrane. The molecules of proteins and mem-brane lipids, constantly in lateral movement, arecapable of rapidly diffusing in the membrane.

Some of the yeast membrane proteins have beenstudied in greater depth. These include adenosinetriphosphatase (ATPase), solute (sugars and amino

acids) transport proteins, and enzymes involved inthe production of glucans and chitin of the cellwall.

The yeast possesses three ATPases: in the mito-chondria, the vacuole, and the plasmic membrane.The plasmic membrane ATPase is an integral pro-tein with a molecular weight of around 100 Da. Itcatalyzes the hydrolysis of ATP which furnishesthe necessary energy for the active transport ofsolutes across the membrane. (Note: an active


Polar head: phosphorylated alcohol

Hydrocarbon tails: fatty acid chains

a

b

Fig. 1.6. A membrane lipid bilayer. The integralproteins (a) are strongly associated to the non-polarregion of the bilayer. The peripheral proteins (b) arelinked to the integral proteins

transport moves a compound against the concen-tration gradient.) Simultaneously, the hydrolysis ofATP creates an efflux of protons towards the exte-rior of the cell.

The penetration of amino acids and sugarsinto the yeast activates membrane transport sys-tems called permeases. The general amino acid

permease (GAP) contains three membrane proteinsand ensures the transport of a number of neutralamino acids. The cultivation of yeasts in the pres-ence of an easily assimilated nitrogen-based nutri-ent such as ammonium represses this permease.

The membrane composition in fatty acids andits proportion in sterols control its fluidity. Thehydrocarbon chains of fatty acids of the membranephospholipid bilayer can be in a rigid and orderlystate or in a relatively disorderly and fluid state. Inthe rigid state, some or all of the carbon bondsof the fatty acids are trans. In the fluid state,some of the bonds become cis. The transitionfrom the rigid state to the fluid state takes placewhen the temperature rises beyond the fusiontemperature. This transition temperature dependson the length of the fatty acid chains and theirdegree of unsaturation. The rectilinear hydrocarbonchains of the saturated fatty acids interact strongly.These interactions intensify with their length. Thetransition temperature therefore increases as thefatty acid chains become longer. The doublebonds of the unsaturated fatty acids are generallycis, giving a curvature to the hydrocarbon chain(Figure 1.8). This curvature breaks the orderly

H3C

CH3

CH3

CH3

H3C

HO

H3C

H3C

CH3

CH3

CH2

H3C

HO

H3C

H3C

CH3

CH3

H3C

HO

H3C

H

Ergosterol (24) (28) Dehydroergosterol

Zymosterol

Fig. 1.7. Principal yeast membrane sterols


Stearic acid (C18, saturated)

Oleic acid (C18, unsaturated)

Fig. 1.8. Molecular models representing the three-di-mensional structure of stearic and oleic acid. The cisconfiguration of the double bond of oleic acid producesa curvature of the carbon chain

stacking of the fatty acid chains and lowers thetransition temperature. Like cholesterol in the cellsof mammals, ergosterol is also a fundamentalregulator of the membrane fluidity in yeasts.Ergosterol is inserted in the bilayer perpendicularlyto the membrane. Its hydroxyl group joins, byhydrogen bonds, with the polar head of thephospholipid and its hydrocarbon tail is insertedin the hydrophobic region of the bilayer. Themembrane sterols intercalate themselves betweenthe phospholipids. In this manner, they inhibitthe crystallization of the fatty acid chains at lowtemperatures. Inversely, in reducing the movementof these same chains by steric encumberment, theyregulate an excess of membrane fluidity when thetemperature rises.

1.3.2 Functions of the PlasmicMembrane

The plasmic membrane constitutes a stable,hydrophobic barrier between the cytoplasm andthe environment outside the cell, owing to its

phospholipids and sterols. This barrier presents acertain impermeability to solutes in function ofosmotic properties.

Furthermore, through its system of permeases,the plasmic membrane also controls the exchangesbetween the cell and the medium. The function-ing of these transport proteins is greatly influencedby its lipid composition, which affects membranefluidity. In a defined environmental model, thesupplementing of membrane phospholipids withunsaturated fatty acids (oleic and linoleic) pro-moted the penetration and accumulation of certainamino acids as well as the expression of the gen-eral amino acid permease (GAP), (Henschke andRose, 1991). On the other hand, membrane sterolsseem to have less influence on the transport ofamino acids than the degree of unsaturation ofthe phospholipids. The production of unsaturatedfatty acids is an oxidative process and requires theaeration of the culture medium at the beginningof alcoholic fermentation. In semi-anaerobic wine-making conditions, the amount of unsaturated fattyacids in the grape, or in the grape must, probablyfavor the membrane transport mechanisms of fattyacids.

The transport systems of sugars across the mem-brane are far from being completely elucidated.There exists, however, at least two kinds of trans-port systems: a high affinity and a low affinitysystem (ten times less important) (Bisson, 1991).The low affinity system is essential during the logphase and its activity decreases during the station-ary phase. The high affinity system is, on the con-trary, repressed by high concentrations of glucose,as in the case of grape must (Salmon et al., 1993)(Figure 1.9). The amount of sterols in the mem-brane, especially ergosterol, as well as the degreeof unsaturation of the membrane phospholipidsfavor the penetration of glucose in the cell. Thisis especially true during the stationary and declinephases. This phenomenon explains the determininginfluence of aeration on the successful completionof alcoholic fermentation during the yeast multi-plication phase.

The presence of ethanol, in a culture medium,slows the penetration speed of arginine and glucoseinto the cell and limits the efflux of protons


00

0

0

0

0.0 0.1 0.2 0.3 0.4 0.5 0.60

1

2

3

4

5

6

high affinity transportsystem activity

Length of the fermentation as a decimal of total time

Glu

cose

pen

etra

tion

spee

d (m

mol

/h/g

dry

wei

ght) low affinity

transportsystem activity

Fig. 1.9. Evolution of glucose transport system activityof S. cerevisiae fermenting a medium model (Salmonet al., 1993). LF = Length of the fermentation as adecimal of total time GP = Glucose penetration speed(mmol/h/g of dry weight) 0 = Low affinity transportsystem activity = High affinity transport systemactivity

resulting from membrane ATPase activity (Alexan-dre et al., 1994; Charpentier, 1995). Simulta-neously, the presence of ethanol increases thesynthesis of membrane phospholipids and theirpercentage in unsaturated fatty acids (especiallyoleic). Temperature and ethanol act in synergy toaffect membrane ATPase activity. The amount ofethanol required to slow the proton efflux decreasesas the temperature rises. However, this modifica-tion of membrane ATPase activity by ethanol maynot be the origin of the decrease in plasmic mem-brane permeability in an alcoholic medium. Therole of membrane ATPase in yeast resistance toethanol has not been clearly demonstrated.

The plasmic membrane also produces cellwall glucan and chitin. Two membrane enzymesare involved: -1,3 glucanase and chitin syn-thetase. These two enzymes catalyze the poly-merization of glucose and N -acetyl-glucosamine,derived from their activated forms (uridinediphosphatesUDP). The mannoproteins areessentially produced in the endoplasmic reticulum

(Section 1.4.2). They are then transported by vesi-cles which fuse with the plasmic membraneand deposit their contents at the exterior of themembrane.

Finally, certain membrane proteins act as cel-lular specific receptors. They permit the yeast toreact to various external stimuli such as sexual hor-mones or changes in the concentration of externalnutrients. The activation of these membrane pro-teins triggers the liberation of compounds such ascyclic adenosine monophosphate (cAMP) in thecytoplasm. These compounds serve as secondarymessengers which set off other intercellular reac-tions. The consequences of these cellular mecha-nisms in the alcoholic fermentation process meritfurther study.

1.4 THE CYTOPLASM AND ITSORGANELLES

Between the plasmic membrane and the nuclearmembrane, the cytoplasm contains a basiccytoplasmic substance, or cytosol. The organelles(endoplasmic reticulum, Golgi apparatus, vacuoleand mitochondria) are isolated from the cytosol bymembranes.

1.4.1 Cytosol

The cytosol is a buffered solution, with a pHbetween 5 and 6, containing soluble enzymes,glycogen and ribosomes.

Glycolysis and alcoholic fermentation enzymes(Chapter 2) as well as trehalase (an enzyme cat-alyzing the hydrolysis of trehalose) are present.Trehalose, a reserve disaccharide, also cytoplas-mic, ensures yeast viability during the dehydrationand rehydration phases by maintaining membraneintegrity.

The lag phase precedes the log phase in asugar-containing medium. It is marked by a rapiddegradation of trehalose linked to an increase intrehalase activity. This activity is itself closelyrelated to an increase in the amount of cAMP inthe cytoplasm. This compound is produced by amembrane enzyme, adenylate cyclase, in response


to the stimulation of a membrane receptor by anenvironmental factor.

Glycogen is the principal yeast glucidic reservesubstance. Animal glycogen is similar in structure.It accumulates during the stationary phase in theform of spherical granules of about 40 m indiameter.

When observed under the electron microscope,the yeast cytoplasm appears rich in ribosomes.These tiny granulations, made up of ribonucleicacids and proteins, are the center of proteinsynthesis. Joined to polysomes, several ribosomesmigrate the length of the messenger RNA. Theytranslate it simultaneously so that each oneproduces a complete polypeptide chain.

1.4.2 The Endoplasmic Reticulum,the Golgi Apparatusand the Vacuoles

The endoplasmic reticulum (ER) is a doublemembrane system partitioning the cytoplasm. It islinked to the cytoplasmic membrane and nuclearmembrane. It is, in a way, an extension of thelatter. Although less developed in yeasts than inexocrine cells of higher eucaryotes, the ER hasthe same function. It ensures the addressing ofthe proteins synthesized by the attached ribosomes.As a matter of fact, ribosomes can be either freein the cytosol or bound to the ER. The pro-teins synthesized by free ribosomes remain in thecytosol, as do the enzymes involved in glycolysis.Those produced in the ribosomes bound to the ERhave three possible destinations: the vacuole, theplasmic membrane, and the external environment(secretion). The presence of a signal sequence (aparticular chain of amino acids) at the N -terminalextremity of the newly formed protein determinesthe association of the initially free ribosomes inthe cytosol with the ER. The synthesized proteincrosses the ER membrane by an active transportprocess called translocation. This process requiresthe hydrolysis of an ATP molecule. Having reachedthe inner space of the ER, the proteins undergo cer-tain modifications including the necessary excisingof the signal peptide by the signal peptidase. Inmany cases, they also undergo a glycosylation.

The yeast glycoproteins, in particular the struc-tural, parietal or enzymatic mannoproteins, con-tain glucidic side chains (Section 1.2.2). Some ofthese are linked to asparagine by N -glycosidicbonds. This oligosaccharidic link is constructed inthe interior of the ER by the sequential additionof activated sugars (in the form of UDP deriva-tives) to a hydrophobic, lipidic transporter calleddolicholphosphate. The entire unit is transferred inone piece to an asparagine residue of the polypep-tide chain. The dolicholphosphate is regenerated.

The Golgi apparatus consists of a stack ofmembrane sacs and associated vesicles. It is anextension of the ER. Transfer vesicles transportthe proteins issued from the ER to the sacs of theGolgi apparatus. The Golgi apparatus has a dualfunction. It is responsible for the glycosylationof protein, then sorts so as to direct them viaspecialized vesicles either into the vacuole or intothe plasmic membrane. An N-terminal peptidicsequence determines the directing of proteinstowards the vacuole. This sequence is present inthe precursors of two vacuolar-orientated enzymesin the yeast: Y carboxypeptidase and A proteinase.The vesicles that transport the proteins of theplasmic membrane or the secretion granules, suchas those that transport the periplasmic invertase,are still the default destinations.

The vacuole is a spherical organelle, 0.3 to3 m in diameter, surrounded by a single mem-brane. Depending on the stage of the cellularcycle, yeasts have one or several vacuoles. Beforebudding, a large vacuole splits into small vesi-cles. Some penetrate into the bud. Others gatherat the opposite extremity of the cell and fuseto form one or two large vacuoles. The vacuo-lar membrane or tonoplast has the same generalstructure (fluid mosaic) as the plasmic membranebut it is more elastic and its chemical com-position is somewhat different. It is less richin sterols and contains less protein and glyco-protein but more phospholipids with a higherdegree of unsaturation. The vacuole stocks someof the cell hydrolases, in particular Y carboxypep-tidase, A and B proteases, I aminopeptidase,X-propyl-dipeptidylaminopeptidase and alkalinephosphatase. In this respect, the yeast vacuole can


be compared to an animal cell lysosome. Vacuolarproteases play an essential role in the turn-overof cellular proteins. In addition, the A proteaseis indispensable in the maturation of other vacuo-lar hydrolases. It excises a small peptide sequenceand thus converts precursor forms (proenzymes)into active enzymes. The vacuolar proteases alsoautolyze the cell after its death. Autolysis, whileageing white wine on its lees, can affect wine qual-ity and should concern the winemaker.

Vacuoles also have a second principal function:they stock metabolites before their use. In fact,they contain a quarter of the pool of the aminoacids of the cell, including a lot of arginine as wellas S-adenosyl methionine. In this organelle, thereis also potassium, adenine, isoguanine, uric acidand polyphosphate crystals. These are involvedin the fixation of basic amino acids. Specificpermeases ensure the transport of these metabolitesacross the vacuolar membrane. An ATPase linkedto the tonoplast furnishes the necessary energyfor the movement of stocked compounds againstthe concentration gradient. It is different from theplasmic membrane ATPase, but also produces aproton efflux.

The ER, Golgi apparatus and vacuoles canbe considered as different components of aninternal system of membranes, called the vacuome,participating in the flux of glycoproteins to beexcreted or stocked.

1.4.3 The MitochondriaDistributed in the periphery of the cytoplasm, themitochondria (mt) are spherically or rod-shapedorganelles surrounded by two membranes. Theinner membrane is highly folded to form cristae.The general organization of mitochondria is thesame as in higher plants and animal cells. Themembranes delimit two compartments: the innermembrane space and the matrix. The mitochon-dria are true respiratory organelles for yeasts. Inaerobiosis, the S. cerevisiae cell contains about50 mitochondria. In anaerobiosis, these organellesdegenerate, their inner surface decreases, and thecristae disappear. Ergosterol and unsaturated fattyacids supplemented in culture media limit thedegeneration of mitochondria in anaerobiosis. In

any case, when cells formed in anaerobiosis areplaced in aerobiosis, the mitochondria regain theirnormal appearance. Even in aerated grape must,the high sugar concentration represses the synthe-sis of respiratory enzymes. As a result, the mito-chondria no longer function. This phenomenon,catabolic glucose repression, will be described inChapter 2.

The mitochondrial membranes are rich in phos-pholipidsprincipally phosphatidylcholine, phos-phatidylinositol and phosphatidylethanolamine(Figure 1.5). Cardiolipin (diphosphatidylglycerol),in minority in the plasmic membrane (Figure 1.4),is predominant in the inner mitochondrial mem-brane. The fatty acids of the mitochondrial phos-pholipids are in C16:0, C16:1, C18:0, C18:1.In aerobiosis, the unsaturated residues predomi-nate. When the cells are grown in anaerobiosis,without lipid supplements, the short-chain satu-rated residues become predominant; cardiolipinand phosphatidylethanolamine diminish whereasthe proportion of phosphatidylinositol increases. Inaerobiosis, the temperature during the log phase ofthe cell influences the degree of unsaturation of thephospholipids- more saturated as the temperaturedecreases.

The mitochondrial membranes also containsterols, as well as numerous proteins and enzymes(Guerin, 1991). The two membranes, inner andouter, contain enzymes involved in the synthesis ofphospholipids and sterols. The ability to synthesizesignificant amounts of lipids, characteristic of yeastmitochondria, is not limited by respiratory deficientmutations or catabolic glucose repression.

The outer membrane is permeable to mostsmall metabolites coming from the cytosol since itcontains porine, a 29 kDa transmembrane proteinpossessing a large pore. Porine is present inthe mitochondria of all the eucaryotes as wellas in the outer membrane of bacteria. Theintermembrane space contains adenylate kinase,which ensures interconversion of ATP, ADP andAMP. Oxidative phosphorylation takes place in theinner mitochondrial membrane. The matrix, on theother hand, is the center of the reactions of thetricarboxylic acids cycle and of the oxidation offatty acids.


The majority of mitochondria proteins are codedby the genes of the nucleus and are synthesized bythe free polysomes of the cytoplasm. The mito-chondria, however, also have their own machineryfor protein synthesis. In fact, each mitochon-drion possesses a circular 75 kb (kilobase pairs)molecule of double-stranded AND and ribosomes.The mtDNA is extremely rich in A (adenine) andT (thymine) bases. It contains a few dozen genes,which code in particular for the synthesis of cer-tain pigments and respiratory enzymes, such ascytochrome b, and several sub-units of cytochromeoxidase and of the ATP synthetase complex. Somemutations affecting these genes can result in theyeast becoming resistant to certain mitochondrialspecific inhibitors such as oligomycin. This prop-erty has been applied in the genetic marking ofwine yeast strains. Some mitochondrial mutantsare respiratory deficient and form small colonieson solid agar media. These petit mutants are notused in winemaking because it is impossible toproduce them industrially by respiration.

1.5 THE NUCLEUS

The yeast nucleus is spherical. It has a diameterof 12 mm and is barely visible using a phasecontrast optical microscope. It is located near theprincipal vacuole in non-proliferating cells. Thenuclear envelope is made up of a double membraneattached to the ER. It contains many ephemeralpores, their locations continually changing. Thesepores permit the exchange of small proteinsbetween the nucleus and the cytoplasm. Contraryto what happens in higher eucaryotes, the yeastnuclear envelope is not dispersed during mitosis.In the basophilic part of the nucleus, the crescent-shaped nucleolus can be seen by using a nuclear-specific staining method. As in other eucaryotes, itis responsible for the synthesis of ribosomal RNA.During cellular division, the yeast nucleus alsocontains rudimentary spindle threads composed ofmicrotubules of tubulin, some discontinuous andothers continuous (Figure 1.10). The continuousmicrotubules are stretched between the twospindle pole bodies (SPB). These corpuscles arepermanently included in the nuclear membrane and

Discontinous tubules

Continuoustubules

Nucleolus

Cytoplasmicmicrotubules

Chromatin

Pore

Spindle pole body

Fig. 1.10. The yeast nucleus (Williamson, 1991). SPB =Spindle pole body; NUC = Nucleolus; P = Pore; CHR =Chromatin; CT = Continuous tubules; DCT = Discon-tinuous tubules; CTM = Cytoplasmic microtubules

correspond with the centrioles of higher organisms.The cytoplasmic microtubules depart from thespindle pole bodies towards the cytoplasm.

There is little nuclear DNA in yeasts comparedwith higher eucaryotesabout 14 000 kb in ahaploid strain. It has a genome almost three timeslarger than in Escherichia coli, but its geneticmaterial is organized into true chromosomes. Eachone contains a single molecule of linear double-stranded DNA associated with basic proteinsknown as histones. The histones form chromatinwhich contains repetitive units called nucleosomes.Yeast chromosomes are too small to be observedunder the microscope.

Pulse-field electrophoresis (Carle and Olson,1984; Schwartz and Cantor, 1984) permits the sep-aration of the 16 chromosomes in S. cerevisiae,whose size range from 200 to 2000 kb. Thisspecies has a very large chromosomic polymor-phism. This characteristic has made karyotypeanalysis one of the principal criteria for the iden-tification of S. cerevisiae strains (Section 1.9.3).The scientific community has nearly establishedthe complete sequence of the chromosomic DNAof S. cerevisiae. In the future, this detailed knowl-edge of the yeast genome will constitute a powerfultool, as much for understanding its molecular phys-iology as for selecting and improving winemakingstrains.

The yeast chromosomes contain relatively fewrepeated sequences. Most genes are only present

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