Yeast Diversity‐ Native and Immigrant

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C.M. Lucy Joseph Department of Viticulture and Enology UC Davis, [email protected]

What is Microbial Terroir?

• Defining microbial terroir: The use of native fungi for the study of traditional fermentative processes. Daniel Felder, Daniel Burns, David Chang. International Journal of Gastronomy and Food Science 1 (2012) 64–69 • “In the course of developing butabushi, koji and miso, DNA analysis has been performed throughout to understand the impact of our native microbes and to propagate them in controlled environments.“

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Microbial Terroir in Wine Next Generation Sequencing (NGS)

• Microbial biogeography of wine grapes is conditioned by cultivar, vintage, and climate. Nicholas Bokulich, John Thorngate, Paul Richardson, and David Mills. PNAS. Published online November 25, 2013

• “Given that many of the same environmental conditions that govern regional variations in grapevine growth and development also alter microbial communities across space and time, it follows that biogeographical assemblages of grape‐surface microbiota may exist, potentially influencing grapevine health and wine quality. “

NGS Fingerprint

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Microbial Terroir in Wine • Regional microbial signatures positively correlate with differential wine phenotypes: evidence for a microbial aspect to terroir. Sarah Knight, Steffen Klaere, Bruno Fedrizzi & Matthew R Goddard. www.nature.com. 2015 • Vineyard soil bacterial diversity and composition revealed by 16S rRNA genes: Differentiation by geographic features. Kayla N. Burns, Daniel A. Kluepfel, Sarah L. Strauss, Nicholas A. Bokulich , Dario Cantu, Kerri L. Steenwerth. Soil Biology & Biochemistry 91 (2015) 232‐247 • Microbial terroir and food innovation: The case of yeast biodiversity in wine. Vittorio Capozzia, Carmela Garofaloa, Maria Assunta Chiriattib, Francesco Griecob, Giuseppe Spanoa. Microbiological Research 181 (2015) 75–83 •

Microbial Terroir 'Wine ... arguably displays the strongest geographic signatures of all agricultural products and thus is a superb model to evaluate the degree to which there might be a microbial aspect to terroir. However, even for wine, the drivers of terroir remain largely untested.'

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Microbial Fingerprint in Soil

Vineyard soil bacterial diversity and composition revealed by 16S rRNA genes: Differentiation by geographic features Kayla N. Burns, Daniel A. Kluepfel, Sarah L. Strauss, Nicholas A. Bokulich , Dario Cantu, Kerri L. Steenwerth. Soil Biology & Biochemistry 91 (2015) 232‐247

Sampling ‐ NGS

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Processing ‐ NGS or Other Techniques

Improving Data Relevance

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Metabolite Fingerprint (25%)

Regional microbial signatures positively correlate with differential wine phenotypes: evidence for a microbial “However, even for wine, the aspect to terroir. Sarah Knight, Steffen drivers of terroir remain Klaere, Bruno Fedrizzi & Matthew R largely untested." Goddard. www.nature.com. 2015

Yeast in Microbial Terroir

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Recognized Saccharomyces Species

Natural History of Saccharomyces

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Natural History of Saccharomyces

The ecology and evolution of non‐ domesticated Saccharomyces species. P Boynton and D Greig. Yeast 2014; 31: 449–462.

Natural History of Saccharomyces

Genome evolution across 1,011 Saccharomyces cerevisiae isolates. Jackson P, M De chiara, A Friedrich, J Yue, D Pflieger, A Bergström, A Sigwalt, B Barre, K Freel, A Llored, C Cruaud, K Labadie, J Aury, B Istace, K Lebrigand, P Barbry, S Engelen, A Lemainque, P Wincker, G Liti, J Schacherer. 19 APril (2018) V.556, Nature 339.

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Where does Saccharomyces hang out between crushes?

Population Distribution

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Saccharomyces Isolates

Impact of Commercial Strain Use on Saccharomyces cerevisiae Population Structure and Dynamics in Pinot Noir Vineyards and Spontaneous Fermentations of a Canadian Winery. J Martiniuk, B Pacheco, G Russell, S Tong, I Backstrom, V Measday. PLOS ONE August 23, 2016

Seasonal Variation

Exploring the northern limit of the distribution of Saccharomyces cerevisiae and Saccharomyces paradoxus in North America. Guillaume Charron, Jean‐Baptiste Leducq, Chloe Bertin, Alexandre K. Dube & Christian R. Landry. FEMS Yeast Res 14 (2014) 281–288

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Sporulation in Soils as a Survival Mechanism

Sporulation in soil as an overwinter survival strategy in Saccharomyces cerevisiae. Sarah J. Knight, and Matthew R. Goddard. FEMS Yeast Research, 16, 2016

Autochthonous vs Commercial Saccharomyces

The Geographic Distribution of Saccharomyces cerevisiae Isolates within three Italian Neighboring Winemaking Regions Reveals Strong Differences in Yeast Abundance, Genetic Diversity and Industrial Strain Dissemination. A. Viel, J‐L. Legras, C. Nadai, M. Carlot, A. Lombardi, M. Crespan, D. Migliaro, A. Giacomini, and V. Corich. Frontiers in Microbiology, August 2017, Volume 8, Article 1595

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Flavor Affects From Non‐Saccharomyces Yeasts

• Enzymatic release of specific flavor compounds from precursors • Metabolic production of flavor compounds • Synergistic affects with Saccharomyces • Growth affects on Saccharomyces • Growth affects on other microbes • Metabolites affecting other parameters such as acid, alcohol, proteins, polysaccharides

Organism Aroma Compounds Metabolic Compounds Candida cantarellii glycerol Candida zemplinina esters, terpenols, volatile thiols, higher glycerol, mannoproteins, acidity, ethanol (Starmarella bacillaris) alcohols, 2‐PE and PEA Debaryomyces vanriji terpenols Hanseniaspora uvarum higher alcohols, isoamyl acetate, ethyl mannoproteins, ethanol acetate Hanseniaspora vineae 2‐PE and PEA, organic acids, higher alcohols Hanseniaspora guillermondi 2‐PE and PEA, isoamyl acetate, ethyl Yeast acetate Hanseniaspora osmophila ethanol Fermentation Issatchenkia orientalis higher alcohols, acetaldehyde malo‐lactic Lachancea thermotolerans esters, 2‐PE and PEA glycerol, mannoproteins, acidity, ethanol Affects Metschnikowia pulcherrima volatile thiols, higher alcohols, 2‐PE and glycerol, mannoproteins, acidity, ethanol PEA, isoamyl acetate, acetaldehyde Pichia anomala isoamyl acetate Pichia fermentans mannoproteins Pichia kluyveri volatile thiols Saccharomycodes ludwigii higher alcohols mannoproteins Schizosaccharomyces pombe glycerol, mannoproteins, malo‐lactic Starmarella bombicola ethanol Torulaspora delbrueckii terpenols, volatile thiols, higher alcohols, 2‐ mannoproteins, acidity PE and PEA, acetaldehyde, organic acids Williopsis saturnus isoamyl acetate Zygosaccharomyces acetaldehyde, higher alcohols, ethyl glycerol, mannoproteins, ethanol, acidity (Zygotorulaspora) florentinus acetate Zygosaccharomyces spp. ethanol

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Common Yeast Genera on Grapes ‐ Spain

Non‐Saccharomyces in Un‐inoculated Fermentations

non‐Sacch Sacch Brix

8 25

20 6

5 15 CFU/ml 4 Brix Log 10 3

2 5

0 0 0123456789101112131415 Days

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Non‐Saccharomyces in Inoculated Fermentations

Comparison of Fermentation and Wines Produced by Inoculation of Hanseniaspora vineae and Saccharomyces cerevisiae. J Lleixà, V Marn, M del C. Portillo, F Carrau, G Beltran, and A Mas. Frontiers in Microbiology, March 2016, Volume 7 Article 338

FIGURE 5 | Results of triangle (table) and descriptive (graphic) test of Macabeo wine fermented with H. vineae and S. cerevisiae.

Why Define California Native Yeast

• Determine if Saccharomyces in the UC Davis Department of Viticulture and Enology Culture Collection are natives • Preserving diversity • Increasing interest in native yeast in wine fermentations • Role in terroir • Untapped resource • Saccharomyces yeast • Non‐Saccharomyces yeast

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Microsatellite DNA Method • Database Available • 180 Commercial isolates • 100 natural isolates • 30 New Zealand isolates • 30 Chilean isolates • 25 California isolates • Multiplex PCR with 10 primers • Three dyes are used; HEX, FAM, NED • Checked on gel for PCR product • Sized with capillary sequencer • Analyze fragments by color and size to determine pattern

Primers/Genes

Primer Gene Chrom Repeat Label Alleles Size

YOR267C HRK1 XV CAA HEX 42 373‐512

YML091C RPM2 XIII AAT NED 42 212‐362

YOL109W ZEO1 XV TAA,TAG HEX 33 237‐362

YFR028C CDC14 VI GT HEX 32 109‐180

YPL009C XVI CTT FAM 31 387‐497

YDR160W SSY1 IV AAT NED 28 347‐484

YLL049W XII TA FAM 26 260‐332

YBR240C THI2 II TA FAM 19 330‐368

YGL014W PUF4 VII TAA NED 18 115‐168

YGL139W VII CAA FAM 13 95‐135

Control MAT III NA FAM, HEX 2 468, 492

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Hunting Wild Yeast • Known habitats • Natural woodlands • Oak bark • Rotting vegetation • Soil, insects, flowers • Isolated areas • 5 kilometers from commercial sources • Winery, Brewery, Bakery • Geographic barriers • Elevation • Water • Avoid contamination • Clean Sampling • Sterile handling

Autochthonous California Saccharomyces

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Autochthonous California Saccharomyces

Culture Collection Make Up

Cluster Category Native Commercial UCD VEN

#1 Commercial 0 100% 0 #2 Commercial ‐ mixed 3% 39% 58% #3 Native ‐ mixed 52% 3% 45% #4 Commercial 0 47% 53% #5 Commercial 0 81% 19% #6 Commercial 0 100% 0 #7 Commercial 0 88% 12% #8 Native ‐ mixed 31% 15% 54% #9 Commercial 0 100% 0

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California Saccharomyces Isolates

Saccharomyces Species Percentage of Isolates

Saccharomyces cerevisiae 62 Saccharomyces paradoxus 28 Saccharomyces bayanus/pastorianus 5 Saccharomyces kudriavzevii 5

Isolates From New Vineyard in California

Saccharomyces species May 8, 2015 June 16, 2017 Newly Planted Two Years Old

Saccharomyces cerevisiae 100% 15%

Saccharomyces parodoxus 0 85%

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