The Chapman & Hall Enology Library

Principles and Practices of by Roger B. Boulton, Vernon L. Singleton, Linda F. Bisson, and Ralph E. Kunkee

Wine Microbiology by Kenneth C. Fugelsang

Winery Utilities Planning, Design and Operation by David R. Storm

Wine Analysis and Production by Bruce W. Zoecklein, Kenneth C. Fugelsang, Barry H. Gump, and Fred S. Nury

Forthcoming Titles Winemaking From Growing to Marketplace by Richard P. Vine, Bruce Bordelon, Ellen M. Harkness, Theresa Browning and Cheri Wagner WINE MICROBIOLOGY Kenneth C. Fugelsang California State University at Fresno

Springer-Science+Business Media, B.V Cover design: Sald Sayrafiezadeh, emDASH inc. Art Direction: Andrea Meyer

Copyright © 1997 by Springer Science+Business Media Dordrecht Originally published by Chapman & HaU in 1997 Softcover reprint of the hardcover 1st edition 1997

ISBN 978-1-4757-6972-2 ISBN 978-1-4757-6970-8 (eBook) DOI 10.1007/978-1-4757-6970-8

AU rights reserved. No part of this book covered by the copyright hereon may be reproduced or used in any form or by any means-graphic, electronic, or mechanical, including photocopying, recording, taping, or information storage and retrieval systems-without the written permission of the publisher. 2 3 4 5 6 7 8 9 10 XXX 01 00 99 98 97 Library of Congress Cataloging-in-Publication Data Fugelsang, K. C. Wine microbiology / Kenneth C. Fugelsang. p. em. -- (The Chapman & Hali enology library) Jncludes bibliographical referenees and index. ISBN 978-1-4757-6972-2 1. Wine and wine making--Microbiology. L Title. II. Series. QR151.F676 1996 663' .203--de20 96-9521 CIP British Library Cataloguing in Publication Data available CONTENTS

Preface Xlll Acknowledgments XVI Introduction XIX

Chapter 1: The Lactic Acid Bacteria 3 1.1 INTRODUCTION 3 1.1.1 Heterofermentative and Homofermentative Metabolism 4 1.1.2 Ecology and Population Dynamics of LAB 7 Presence of LAB in the 7 Growth of Native LAB in Must, , and Wine 10 1.2 UTILIZATION OF COMMERCIAL "STARTERS" FOR MLF 11 1.2.1 Starter Culture Preparation 12 Rehydration, Growth, and Expansion Media Composition 12 Nitrogen requirements 13 Acid Adjustment 14 Sterilization 14 1.2.2 Pure and Coculture Methods 15 1.2.3 Monitoring Population Changes 16 1.2.4 Timing of LAB Addition 17 1.2.5 Use of Ongoing MLF or ML- as Inoculum 18

v vi Contents

1.3 GROWTH OF LAB IN WINE (THE MALOLACTIC ) 18 1.3.1 Biochemistry and 19 1.3.2 Changes in Wine 20 1.3.3 Environmental Conditions and Winemaking Decisions 21 Impacting MLF 21 pH 21 Levels 22 Nutritional Status 22 Cellar Temperature 23 Dioxide 23 Microbial Antaganism 25 26 Lactic Acid Bacteria 27 Bacteriophage 28 1.4 LAB SPOILAGE 29 1.4.1 Formation 29 1.4.2 Refermentation 29 1.4.3 Geranium Tone 31 1.4.4 Mousiness 32 1.4.5 (Citric Acid Utilization) 33 1.4.6 Tartaric Acid Utilization 34 1.4.7 Mannitol Formation, Ropiness, and Polysaccaride Formation 36 1.4.8 Metabolites of (Acrolein) 37 1.5 LABORATORY PROCEDURES (IDENTIFICATION OF BACTERIAL ISOLATES) 37 1.5.1 Gram stain 37 1.5.2 Catalase Test 38 1.5.3 General Growth Medium for LAB 39 1.5.4 Heterofermentative Versus Homofermentative Utilization of Sugar 40 1.5.5 Mannitol Salt Formation 41 1.5.6 Production from 42 1.5.7 Monitoring MLF (Paper Chromatography) 44

Chapter 2: Acetic Acid Bacteria 48 2.1 INTRODUCTION 48 2.1.2 Growth on Carbohydrates 49 2.1.3 Growth of 51 2.1.4 Ecology of Acetic Acid Bacteria 52 Vineyard 53 Primary Processing and Fermentation 54 Post-Fermentation 55 2.1.5 Control of Acetic Acid Bacteria 57 2.1.6 Reduction in Volatile Acidity 58 Contents vii

2.1.7 Sensory Implications 59 Acetic Acid 59 59 60 60 60 2.2 OTHER WINE-ASSOCIATED BACTERIA 60 2.2.1 Bacillus 61 2.2.2 Other Bacterial Involvement 61 2.3 LABORATORY PROCEDURES FOR IDENTIFICATION OF ACETIC ACID BACTERIA 62 2.3.1 Gram Staining 63 2.3.2 Catalase Test 63 2.3.3 Ethanol Oxidation: Separation of Acetobacter from Gluconobacter 64 Carr Medium 64 Carbonate-Ethanol Medium (Frateur's Medium) 65 Calcium Lactate Extract Agar 65 -Yeast Extract-Carbonate Medium 66 2.3.4 Glycerol Medium (Test for Ketogenesis) 66

Chapter 3: Yeasts and Molds 68 3.1 YEAST CLASSIFICATION AND ECOLOGY 68 3.1.1 Yeasts in the Vineyard 69 3.1.2 Yeasts in the 70 3.2 NATIVE FLORA AND FERMENTATION 71 3.3 SELECTED NATIVE YEASTS 72 3.3.1 /Dekkera 72 73 Morphology 73 Distribution and Ecology 74 Monitoring 77 Sensory Properties 79 3.3.2 80 Taxonomy 80 Morphological Properties 81 Habitat 82 3.3.3 Hansenula anomala 83 3.3.4 Kloeckera apiculata (Hanseniaspora uvarum) 84 3.3.5 84 3.3.6 85 3.3.7 Other Spoilage Yeasts 85 3.4 YEAST IDENTIFICATION 86 3.4.1 Cell Morphology 86 3.4.2 Colony Appearance (Color) 87 viii Contents

3.4.3 Asexual (Vegetative) Reproduction 87 Multilateral Budding 88 Bipolar Budding 89 "Fission" 89 Pseudomycelium Formation 89 3.4.4 Sexual Reproduction (Ascospore Formation) 89 3.4.5 Oxidative Requirements for Nitrogen and Carbon 90 3.4.6 Taxonomic Key 91 3.5 LABORATORY PROCEDURES (FOR IDENTIFICATION OF YEASTS) 96 3.5.1 Isolation 96 3.5.2 Demonstration of Ascospores 98 3.5.3 Carbon and Nitrogen Assimilation Tests 101 3.5.4 Fermentation Broths 105 3.5.5 Diagnostic and "Differential" Media 106 WL-Nutritional and WL-Differential Media 107 Brettanomyces/ Dekkera-Selective Medium 107 Zygosaccharomyces-Selective Medium 108 Slide Culture 109 3.6 MOLDS OF IMPORTANCE IN WINEMAKING 110 3.6.1 Identification 111 Mold Life Cycle III 3.6.2 (Gray Mold) 112 3.6.3 Other Molds 113 Penicillum sp. (Blue-Green Molds) 114 Aspergillus (Black Mold) 114 Mucor and Rhizopus (Pin Molds) 114 3.7 LABORATORY PROCEDURES (FOR OBSERVATION AND IDENTIFICATION OF MOLDS) 114

Chapter 4: Prefermentation Processing 117 4.1 INTRODUCTION 117 4.2 AND TRANSPORT 118 4.3 FRUIT QUALI1Y ASSESSMENT 118 4.4 PREFERMENTATION PROCESSING 119 4.4.1 120 4.4.2 Initial Fermentable Sugar Levels 121 4.4.3 Hydrogen Ion Concentration (pH) 121 4.4.4 Suspended Solids 122 4.4.5 Potential () 123 4.4.6 Available Nitrogen 124 4.4.7 Inert Gassing 127 4.5 JUICE (MUTE) STORAGE 127 4.6 PROCESSING MODIFICATIONS FOR MICROBIALLY DETERIORATED FRUIT 129 4.6.1 Processing Botrytis-Infected Fruit 129 Contents ix

Chapter 5: Fermentation and Post-Fermentation Processing 132 5.1 INTRODUCTION 132 5.2 YEAST STARTERS (PREPARATION AND PROPAGATION) 133 5.3 FERMENTATION TEMPERATURE 136 5.4 NATIVE FLORA AND FERMENTATION 136 5.5 137 5.5.1 Temperature and Alcohol-Related Problems 138 5.5.2 Nitrogen Supplementation 139 5.5.3 Revitalizing 140 5.6 POST-FERMENTATION GROWTH 141

Chapter 6: Bottling 143 6.1 INTRODUCTION 143 6.2 FILTRATION 144 6.3 PRESERVATIVES AND STERILANTS 146 6.3.1 Sulfur Dioxide 147 6.3.2 Sorbic Acid 148 6.3.3 Fumaric Acid 151 6.3.4 Benzoic Acid 151 6.3.5 Dimethyldicarbonate 152 6.3.6 OTHER ANTIMICROBIAL AGENTS 153 Lysozyme 153 Nisin 154 Carbon Monoxide 154 6.4 LABORATORY PROCEDURES 155 6.4.1 Bottling Line Sampling 155 6.4.2 Sampling Low-Density Populations 155

Chapter 7: Wmery Sanitation 159 7.1 INTRODUCTION 159 7.2 PRELIMINARY CLEANING 160 7.3 WATER QUALI1Y 160 7.4 DETERGENTS 161 7.4.1 Alkalies 161 7.4.2 Sequestering Agents 162 7.4.3 Surfactants 162 7.4.4 Acids 162 7.5 SANITIZERS 162 7.5.1 -Based Sanitizers 164 7.5.2 Iodine 165 7.5.3 Quaternary Compounds 165 7.5.4 Detergent-Sanitizer Formulations 165 7.5.5 Sulfur Dioxide 166 7.5.6 Physical Sterilants 166 x Contents

7.6 SANITATION MONITORING 166 7.6.1 Swab Tests 167 7.6.2 Direct-Contact Tests 167

Appendix A: Basic Microscopy and Laboratory Setup 169 Al MICROSCOPE 169 Magnification 170 Resolution 170 Contrast 170 A2 CENTRIFUGE/FILTERS 171 A3 AUTOCLAVE 172 A.4 INCUBATOR 172 A.5 WATERBATHS 173 A.6 DISPOSABLE VERSUS REUSABLE PIPETfES 173 A.7 MEDIA 174 A.8 MISCELLANEOUS SUPPLIES 174 A.9 ALTERNATIVES 175

Appendix B: Media Preparation and Transfer Techniques 176 B.1 INTRODUCTION 176 B.2 PHYSICAL/CHEMICAL REQUIREMENTS FOR MEDIA 177 B.2.1 Utilizable Source of Carbon and Nitrogen 178 B.2.2 Oxygen Requirements 178 B.2.3 Hydrogen Ion Concentration (pH) 179 B.2.4 Moisture and Water Activity (Aw) 179 B.2.5 Incubation Temperature 180 B.3 STERILIZATION OF LABORATORY MEDIA AND SUPPLIES 180 B.3.1 Steam Sterilization 181 B.3.2 Boiling Water 182 B.3.3 Dry Heat 182 B.3.4 Sterile Filtration 183 B.3.5 Chemical Sterilization 183 Ethylene Oxide 183 Dimethyldicarbonate 183 Ethanol 184 B.4 MEDIA PREPARATION 184 B.4.1 Fermentation Broths 185 B.4.2 Solidified Media 186 Plates 186 Slants (Slopes) 187 Agar Deeps 188 B.5 STERILE TRANSFER TECHNIQUES 188 B.5.1 Transfers from Plate to Plate ("Streaking for Isolation") 188 B.5.2 Transfer from Slant to Slant 189 B.5.3 Transfer Between Agar "Deeps" 190 B.5.4 Transfer from Plates to Liquids 190 B.5.5 Liquid-to-Liquid and Liquid-to-Solid Transfers (Using Pipettes) 191 Contents xi

Appendix C: Estimation of Population Density 193 C.1 INTRODUCTION 193 C.2 PREPARATION OF DILUTIONS 194 C.3 POPULATION MEASUREMENTS (PLATING OR INDIRECT METHODS) 197 C.3.1 Pour-Plate Technique 197 C.3.2 Spread-Plate Technique 198 C.3.3 Membrane Filtration 199 C.4 MICROSCOPIC (DIRECT) EXAMINATION USING VIABILI1Y STAINS 201 C.4.1 Methylene blue 201 C.4.2 Ponceau-S 202 C.4.3 Walford's stain 202 C.4.4 Viable Cell Counting 203 C.4.5 Epifluorescence Methods 205 C.4.6 Bioluminescence 206 C.5 RECENT TECHNOLOGY 207 C.5.1 Immunochemical Techniques 207 -linked Immunosorbent Assay (ELISA) 207 Immunochemical Fluorescence Microscopy 208 C.5.2 Identification Methods 208 Nucleic Acid Hybridization Probes 209 Polymerase Chain Reaction 209 Characterization ("Fingerprinting") 209 Appendix D: Chemical/Physical Instabilities 210 D.1 INTRODUCTION 210 D.2 CRYSTALLINE DEPOSITS 211 D.2.1 Bitartrate and Calcium Tartrate 211 pH Test for Potassium Bitartrate and Calcium Tartrate 212 Test for Calcium 213 D.2.2 "Crystallike" Particulates ( Dust) 213 D.2.3 Diatomaceous Earth 215 D.3 FIBROUS MATERIALS (CELLULOSE AND ASBESTOS) 216 D.4 AMORPHOUS PRECIPITATES 217 D.4.1 Protein/Phenolics 217 D.4.2 Alternative Test for Protein (Tannic Acid Precipitation) 217 D.4.3 Phenolics 218 D.4.4 Alternative Procedure for Phenolics 219 D.5 , GLUCANS, AND OTHER 219 D.5.1 Glucan Instability 219 D.5.2 Instability 220 D.5.3 Starch Instability 220 D.6.1 METAL INSTABILITIES 220 D.6.2 Potential Metal Instabilities (Prebottling) 222 Bibliography 223 Index 241 PREFACE

The partnership between industrial microbiologists, the industries they serve, and the microbes upon which they rely is (or should be) a close one. Brewers, bakers, and those involved in fermented dairy products come to mind as being leaders in this regard. By comparison, the wine industry, with the notable exception of a few technologically astute world• wide, has historically given relatively little attention to this crucial step in processing. In the last two decades, this pattern has reversed itself and the rank and file of winemaking personnel have become more intensely inter• ested in the subject of wine microbiology. Several reasons come to mind that, in part, account for this. First, prior to the late seventies, much of the research in the area of wine microbiology (particularly, the ) had yet to crystalize to yield a cohesive enough picture to be of value to the winemaker. During this period and into the early eighties, the U.S. wine industry began to utilize lower levels of sulfur dioxide than did their predecessors-the goal being to reduce input of chemicals and to minimize concerns from an increasingly health conscious population of consumers. Unfortunately, the practice had a significant downside; micro• organisms that had previously been controlled suddenly reemerged as sig• nificant threats to wine production. In this regard, the yeast Brettanomyces

xiii xiv Preface

received (and continues to receive) considerable attention in the wine• making community-some regarding it as clearly a spoilage agent and others as contributing to complexity. The history and current status of this yeast is considered in Chapter 3. At about the same time, the ancillary juice and concentrate industry began to proliferate from a few sites in the San Joaquin Valley of California to become a thriving worldwide business. Along with the unique environment of concentrate came another difficult (in the opinion of some, impossible) to control yeast, Zygosaccharomyces. In addition to its unique habitat in the high sugar environment of concen• trate, the organism is seemingly refractile to most methods of control. Further, it grew extraordinarily well in sweetened with contaminated concentrate and, thus, the problem spread from concentrate producers to wineries using the product for blending. Further consideration of Zygosac• charomyces is also found in Chapter 3. By the late eighties, a handful of U.S. winemakers began to experiment with the use of native yeast and bacteria for fermentation as their forefa• thers had done 100 years before. Undaunted by the criticism of many, these individuals continued to ply their art in the belief that the unique contributions of native flora conferred upon the process a dimension of complexity not seen in parallel fermentations using commercial yeast start• ers. Although conventional wisdom continues to argue against this view• point, it is clear that those using native fermentations require a thorough knowledge of the cast of characters, good as well as bad. During the last 20 years, the technical information network has blos• somed. Winemakers now have the opportunity to attend numerous na• tional and international symposia, conferences, and workshops as well re• gional meetings. Many of these now deal, in part or completely, with specialized topics relative to the microbiology of wine. Beyond this, local winemakers groups meet regularly (often monthly) for exchange of ideas and research findings. In terms of getting the information to the end user, the wine industry has been fortunate to have several well-written trade journals which have specialized in the distribution of information in a relatively user-friendly, applied mode. In the years to come, electronic network communication will most certainly become important. Several pioneering efforts in this regard are ongoing. In the spirit of information transfer, this text utilizes an applied approach to the subject as well. The first three chapters deal with origins, development, and identification of bacteria and yeasts that impact the winemaking process. Chapters 4, 5, and 6 address issues of primary processing of , must, juice, the fermen• tation process, and bottling. Special concerns such as storage of juice, processing microbially impaired fruit, reactivating stuck fermentation, and bottling-line sampling are addressed in this section of the text. Preface xv

Microbiological problems in the winery may be cumulative or, seem• ingly, spontaneous and isolated in occurrence. Fundamental to the control of microbiological spoilage is a well-functioning sanitation program. Chap• ter 7 deals with the basics of sanitation as well as monitoring success. Finally, four appendices are included. It is recommended that aspiring wine microbiologists review each of these before beginning. Even more experienced laboratory supervisors may find the included procedures use• ful in training new personnel. Appendix A deals with the issue of costs involved in establishing a "modest" laboratory within the setting of an already established analytical environment. Current costs of basic equip• ment and supplies are discussed as well as potential cost-saving measures. As noted in the concluding remarks in the appendices, some may find it less costly to send out work to already established commercial laboratories. Appendix B outlines the basics of laboratory media preparation and skills involved in handling without contamination. For those who need to brush up on technique, the fundamentals of routine aseptic transfer are covered in detail. Appendix C addresses monitoring population density. Indirect as well as direct methods are covered as well as a discussion of recent and developing technology for rapid detection/enumeration. Finally, even trained personnel occasionally confuse microorganisms with debris that may originate from chemical/physical instabilities in the wine. Appendix D is included to facilitate separation of abiotic from mi• crobiological haze/precipitate. ACKNOWLEDGMENTS

The author would like to thank the following individuals and organizations for their assistance in preparation of this manuscript: The California Agricultural and Technology Institute, C.S.U. Fresno, for partial funding support Dr. C. Prahl, Christian Hansen Laboratory, Horsholm, Denmark, for photomicrographs Ms. Ann Dumont, Lallemand, Inc. Montreal, Canada, for photo• micrographs Ms. Angelica Malagon, Research Associate, C.S.U. Fresno, for editorial assistance. Drs. CJ. Muller, California State University, Fresno and B.W. Zoecklein, Virginia Polytechnic Institute, for technical review of portions of the manuscript

xvii INTRODUCTION

Considering the diversity of microorganisms that may be found in other environments, the "cast of characters" that emerge during winemaking is limited. The explanation generally offered for this centers on the interac• tive and cumulative effects of ethanol and pH combined with nutrient depletion and the use of preservatives in processing. Two groups of bacteria play prominent roles in winemaking. The acetic acid bacteria represented by Gluconobacter and Acetobacter may play an early role in grape quality and, secondarily, in the case of Acetobacter, in stored• wine stability. Growth in this case is clearly undesirable. The second major group of bacteria, the lactic acid bacteria (lAB), is represented by the three genera Lactobacillus, Leuconostoc, and Pediococcus sp. Whether their growth is viewed as positive or negative depends on winemaking philoso• phy, , and the organism involved. On occasion, other bac• teria may become important. Actinomycetes in/on packaging materials, such as corks, boxes, and pallets, under certain conditions may produce sensorially powerful metabolites which have been collectively referred to as "cork taint." Bacillus sp. also has a sporadic history of isolation from wine. Recent reports identify the organism, again, in bottled imported wines.

xix xx Introduction

Several yeasts appear during the course of winemaking. These include the oxidative species, Pichia and Candida, that either do not ferment or are very weakly fermentative. Although capable of growth in musts and juice, they are important members of the film yeast community in stored wine. The weakly fermentative species Hansenula anomala and Kloeckera apiculata/ Hanseniaspora uvarum are seen early in the course of fermentation and are capable of producing 2-4% ethanol and, in the process, objectionally high levels of acetic acid and ethyl acetate. Fermentative species including Bret• tanomyces/Dekkera, Schizosaccharomyces pombe, , and Zygosaccharo• myces, are capable of complete, albeit in some cases, slow fermentation. Compared with already mentioned yeasts these species grow in sugar and alcohol rich environments and their documented presence in the vineyard is rare. deserves special mention among the yeasts associated with fermentation. On the one hand, Saccharomyces plays a partnership role with the winemaker in transformation of sugar to alcohol during fermentation. But, on the other, it becomes a significant adversary where oxidative conditions permit its growth during cellar aging. In this latter role, Saccharomyces play an important role in the film yeast commu• nity. Further, Saccharomyces is still the most likely culprit in post-bottling fermentation. Although some would argue that Zygosaccharomyces is more important in this regard, the latter appears to be a winery-specific problem whereas the farmer may be an industry-wide problem.

SPECIES OR STRAIN

The degree of physical (morphological) and physiological similarity be• tween microorganisms serves as the basis for establishing identity and re• latedness to other organisms. Modern-day classification increasingly relies on similarities at the molecular level, including protein and nucleic acid comparisons. Development of sophisticated techniques for identification based on unique differences in the genetic code will undoubtedly continue to clarify the relationship of one organism to another. Unfortunately, these still require highly trained personnel in appropriately equipped labs. Until such time as these techniques can be reduced to a form usable by workers in the field, most of us must rely on more traditional methods of identifi• cation which include physical and physiological properties. Biologists utilize a system of classification based on the degree of simi• larity between organisms. This orderly classification of living things relative to others is called taxonomy. Taxonomists use a classification hierarchy that begins with the most general criteria and become progressively more restrictive (kingdom, phylum or division, class order, family, genus, species, Introduction xxi

and strain and, in the case of some yeasts, variety of race). Procedurally, one begins the task of the identification of an unknown organism (plant, animal, bacteria) in the broadest sense by establishing to what biological kingdom it belongs. This might seem a trival matter in the case of everyday things (redwood trees, doberman pincers, etc.) but becomes more difficult as we deal with organisms, such as bacteria, algae, and protozoa, nearer the base of the evolutionary ladder. Even among biologists, establishing the relationship in the broadest taxon (plant, animal, bacterial) is chal• lenging and, in some cases, the affinities of some members have been a source of debate for decades. For example, the Cyanobacteria (currently claimed by the bacteriologists) were historically classified among the blue• green algae and claimed by botanists. This also points out that the business of biological classification (taxonomy) is not static but continually chang• ing. As it relates to winemaking, species of the Saccharomyces serve as an example. Over the years, individual industrially important species such as S. baynus, S. oviformis, and S. carlsbergensis among others, have been consol• idated into the current species S. cerevisiae. Because the differences be• tween these yeasts were to minute to retain their identity as individual species, but yet not similar enough to consider them as one-and-the-same, taxonomists classified these as races of Saccharomyces cerevisiae. Aside from those already noted, cerevisiae, capensis, and chevalieri can also be included. This "special purpose" toxonomy was created (or retained) for purposes of industrial microbiologists which require recognition of unique physio• logical properties and may not reflect current thought relative to the spe• cies as a whole. The fundamental unit (taxon) of biological classification is the species. Classically, general biology texts describe the species as being a reproduc• tively isolated population; that is, it is different from all other similar or• ganisms to the extent that it can no longer interbreed. Whereas this defi• nition is adequate for organisms with an established and regular sexual phase in the life cycle, it is less clear as to how it may be applied in the case of those that normally reproduce by asexual means. Thus, the concept of species among bacteria and to a lesser extent many yeasts is difficult to interpret. Although bacteriologists retain use of the concept of species, it has been extended to encompass (potentially) many very similar strains. In theory, a strain consists of progeny of a single cell-a concept that appears functionally equivalent to the clone among viticulturalists. Thus, the use of strain includes a collection of similar-appearing organisms that differ only in terms of a few minor physiological properties. These "minor physiolog• ical properties" then give rise to strains within the species. For example, Leuconostoc oenos may have nearly 100 different strains (including the fa• miliar ML-34 and PSU-I). Although differences may be minor to the tax- xxii Introduction

onomist, they may have major impact on brewers and vintners as well as others that rely on these organisms for some type of useful conversion. As already noted, with the case of Saccharomyces cerevisiae, the classic definition of species may similarly complicate yeast classification. Brettano• myces is another example. Reviews have shown widely varying opinions regarding the importance of the yeast in winemaking. One winery reports extraordinary success, whereas another complains of disasterous results due to Brett-contaminated cooperage. Some even refer to "benign strains" that convert substrate without significantly impacting the wine's sensory properties. Are these different strains, or the same organism growing un• der different environmental conditions? At present, the answer is un• known. In this last example, and others, such observations point to the importance of complete identification of microorganisms.