3/10/2014
A New Era For Fermentation Ecology— Routine tracking of all microbes in all places
Department of Viticulture and Enology
Department of Viticulture and Enology Terroir of Wine (regionality)
Source: Wine Business Monthly
1 3/10/2014
Department of Viticulture and Enology
Can Regionality Be Observed (Scientifically) by Chemical/Sensory Analyses?
Department of Viticulture and Enology
Department of Viticulture and Enology
What about the microbes in each environment?
Is there a “Microbial Terroir”
2 3/10/2014
Department of Viticulture and Enology We know the major microbial players
Department of Viticulture and Enology Where do the wine microbes come from?
Department of Viticulture and Enology
Methods Quality Filtration Developed Pick Operational Taxonomic Units (OTUs)
Nick Bokulich Assign Taxonomy
3 3/10/2014
Department of Viticulture and Enology Microbial surveillance: Next Generation Sequencing
Extract DNA
>300 Samples
PCR
Quantify ALL fungal and bacterial populations in ALL samples simultaneously Sequence: Illumina Platform
Department of Viticulture and Enology Microbial surveillance: Next Generation Sequencing
Department of Viticulture and Enology
Example large data set: Bacterial Profile
1 2 3 4 5 6 7 8 Winery Differences Across 300 Samplings
4 3/10/2014
Department of Viticulture and Enology
Microbial surveillance process 1. Compute UniFrac distance (phylogenetic distance) between samples 2. Principal coordinate analysis to compress dimensionality of data 3. Categorize by metadata 4. Clusters represent samples of similar phylogenetic similarity
Department of Viticulture and Enology Can you define a region by the grape microbiota at harvest?
Department of Viticulture and Enology Early Fermentation Microbiota
• 240 musts (2010) • 4 regions: Napa, Sonoma, Northern San Joaquin Valley (NSJV) Central Coast •8 Wineries • Ran both fungal (yeast) and bacterial NGS analysis
5 3/10/2014
Department of Viticulture and Enology Regional Variation — total Bacteria Fungi PC1 (9.45%) PC1 (10.74%)
P = 0.001 P = 0.001
PC1 (16.19%) Sonoma PC1 (13.8%) Napa Central Coast PNAS 2013 NSJV
Department of Viticulture and Enology Regional Variation — Napa vs. NSJV Bacteria Fungi PC2 (10.96%) PC2 (10.93%)
P = 0.001 P = 0.001
PC1 (26.32%) PC1 (14.37%) NSJV Napa
Department of Viticulture and Enology Regional Variation — total
PNAS 2013 Chardonnay Linear Discriminate Analysis Effect Size (Huttenhower)
6 3/10/2014
Cabernet Savingnon and Chardonnay Fungal Regionality Department of Viticulture and Enology
Varietal Variation
Linear Discriminate Analysis Effect Size (Huttenhower) PNAS 2013
Department of Viticulture and Enology
Regional Variation: Coast Sonoma Taxonomic differences NSJV Napa
0.35
P = 0.001 0.3
0.25
0.2 Napa Sonoma P = 5.85E-7 0.15 NSJV P = 2.05E-6 Central P = 1.77E-8 coast Relative Abundance Relative 0.1 P = 0.042
0.05
0 Botryotinia fuckeliana Cladosporium Aspergillus sp H4381 Penicillium thomii Alternaria alternata cladosporioides
PNAS 2013
Department of Viticulture and Enology
Varietal Variation?
7 3/10/2014
Department of Viticulture and Enology
Regional Variation — Taxonomical differences
P = 0.001
P = 5.85E-7 P = 2.05E-6 P = 1.77E-8
P = 0.042
PNAS 2013
Department of Viticulture and Enology
Varietal Variation — Cab Sauvignon vs Zinfandel
Bacteria Fungi PC2 PC2 PC2 (12.63%) (15.19%)
P = P = 0.001 0.001
PC1 PC1 (27.18%) Cabernet (24.72%) Zinfandel
Department of Viticulture and Enology Can we determine how microbes move through a winery?
• Can we track microbial transitions during grape harvest? • What processes drive microbial community patterns? • What reservoirs exist for spoilage microbes?
8 3/10/2014
Department of Viticulture and Enology Can we determine how microbes move through a winery?
Surface swabbing Moe Ohta
Department of Viticulture and Enology Seasonal Surveillance: UC Davis Winery
PLoS One (2013)
Department of Viticulture and Enology
Bacterial
Fungal
PLoS One (2013)
9 3/10/2014
Department of Viticulture and Enology
PLoS One (2013)
Department of Viticulture and Enology D. bruxellensis
Department of Viticulture and Enology What about other food/beverage fermentations?
10 3/10/2014
Department of Viticulture and Enology Artisan Cheese Facility (AEM 2013)
Bacteria Fungi
Department of Viticulture and Enology Sake Facility
AEM (submitted)
Department of Viticulture and Enology
11 3/10/2014
Department of Viticulture and Enology
Where could one using this?
• Vineyard effects • Quality prediction: Vineyard acquisition, problem blocks? • Environmental Mapping • Barrel surveys, winery vectors, regional mapping •Treatments •Vineyard: sprays • Winery: Barrel cleaning, must
additions, SO2
Department of Viticulture and Enology Wine Quality Prediction? How do we currently assess grape quality before winemaking?
Does this tell you anything about the history of the grape?
Department of Viticulture and Enology What is the future?
Is the aggregate grape microbiota witnessed in the press pan indicative of the quality of the ensuing wine?
12 3/10/2014
Department of Viticulture and Enology
Microbiology of the Built Environment Program
Department of Viticulture and Enology Should we be routinely monitoring the microbiota of all food and beverage production?
Department of Viticulture and Enology
Summary • Next generation sequencing tools enable complete mapping of fungal and bacterial microbiota within the grape—wine continuum • Regional, subregional and even vineyard specific differences in grape must microbiota can be robustly defined • Varietal level differences in grape must microbiota can be robustly defined • In some cases, regional differences can be witnessed throughout the winemaking process • In some cases, winery influence on wine microbiota can be observed • Examining winery microbiome can track microbial flow through winery
13 3/10/2014
Department of Viticulture and Enology
The most important thing we do is train people!
Department of Viticulture and Enology
Acknowledgements Financial/In-Kind Support
Mills Lab: • American Vineyard Foundation Nick Bokulich, Moe Ohta, • Oregon Wine Board Mariya Ryazantseva, Chad •MicroTrekInc. Masarweh, Mariko Zelin •Constellation Brands MicroTrek Inc. • Far Niente/Dolce/Nickel & Nickel Paul Richardson •Terraspase Constellation Brands John Thorngate, Jim Orvis, Financial support for Nick Bokulich: Oren Kaye, Chris Dannon Probiotics Fellow Program Cunningham Wine Spectator Scholarship
Far Niente/Dolce/Nickel & American Wine Society Educational Nickel Wineries Foundation Greg Allen and team American Society for Brewing Chemists Foundation
Department of Viticulture and Enology
Thanks
14