Jonathan R. Cave

University of California, Davis

Viticulture and Enology

Oxygen’s Role in Fermentation

Clark Sensor Fluorescence Quenching

Optical Sensor

475 nm Fiber Optic (Blue)

600 nm Fluorescence (Orange)

Sanitizable/Autoclavable

High chemical tolerances

Physically Divided or Direct

Non-Invasive

No consumption

Polarographic (800 mV) Electrode

Traditional Dip-Probe

Developed by L. C. Clark (1956)

O2 Permeable Membrane

Amperometric Ag/AgCl

Movement - Dip, In-Line Flow

Invasive

Consumes O2 – Negligible

Sensors

Requires Flow or Mixing

O2 Diffuses through membrane

2 diffusion processes (Membrane and Solution)

Membrane < 20µm so that equilibration across membrane is time limiting rather than the reaction

10-20 seconds equilibration

Stable in under 1 minute

Clark SensorPolarographic Electrode

Cathode: O2+ 2H2O+ 4𝑒 ̶ @ ՜ 4OH ̶ @ Anode: Ag + Cl ̶ B՜ AgCl(s) + 𝑒 ̶ @

Clark FluorescenceTemperature Sensitive

(External Compensation)

Acetone, Toluene, Chloroform, Methylene Chloride, Chlorine Gas, Organic Vapor

Temperature Technically (Internal Thermistor)

H2 (g) SO2, H2S

Replenish Electrolyte

Interferences

H2S + OH ̶ @ ՜ HS ̶ @ + H2O HS ̶ @ + OH ̶ @ ՜ S2-+ H2O 2Ag++ S2- ՜ Ag2S (Black Precipitate)

475 nm Fiber Optic

Excites Fluorescent DyeFOXY – Hydrophobic Sol-GelPt-porphyrinFluorescent Dye in Polymer Matrix

600 nm Fluorescence

Dynamic Fluorescence Quenching

Collision of O2 with fluorophore causes “non-radiative energy transfer” exciting O2 into triplet state

Fluorescence Quenching5

O2 ∝ 1𝐹𝑙𝑢𝑜𝑟𝑒𝑠𝑐𝑒𝑛𝑐𝑒 𝑅𝑒𝑠𝑝𝑜𝑛𝑠𝑒

Experimental Relevance

0.5cm, Physically Divided (Sight Glass)

Flow Rate Independent

pH, CO2, H2S, SO2, Ionic Species

Chemical Tolerance NaOH, H2O2, HCl

CIP - autoclave, steam

Linear Range 1-1800 ppb Accuracy ± 1 ppb LOD: 1 ppb

Minimal Cross SensitivityYes: Acetone, Chlorine

GasNo: CO2, H2S, SO2

Compatible with Ethanol

PreSens Oxygen Sensor Spots 4

Winery Applicability

GoalComprehensive model of oxygen availability, necessity, benefit, and detriment from vine to glass

Oxygen Management in Winery Operations

Jonathan Cave, Nick Gislason, Andrew Waterhouse

Cap Manipulation

Racking

Crush

Pressing

Barreling Down

Bottling

Aerative PumpoversSplash Racking, Rack and Return,

Delestage-ish

High Anticipated Oxygen Solvation

Desired Oxygen Uptake

Early in Fermentation - Low EtOH/High Sugar

SO2 - Oxygen scavenger and Interaction Inhibitor?

Winery Operations

Observed 29 Pumpovers 23 Aerative 6 Closed Controls

Within first 3 days of fermentation

Pumpovers by experienced cellar staff Well practiced technique Not harvest interns

No alteration by experimenters

No interference in the production process

Required Observational Treatments

Experimental Design

Oxygen Sensor Spots– Paired Values

Drop – Distance from Screen to Wine

Splash – Radius and WallsFlow Rate – From Racking ArmFlow Type – Screen interaction

Parameters

Two ConditionsDrop – Large/Small

10” vs. 4”Splash – Intense/Mild

Spread and ArcingFlow Rate – Fast/SlowFlow Type – Turbulent/Laminar

Range: 70 - 2300 ppb

Closed PO Control – 0 ppb

Drop – Most Relevant STDEV of lower [O2] too high

CV > 75%

Oxygen Solvation/Assimilation Data

Oxygen Assimilation for main observable Treatments

Splash Flow Rate Flow Type Drop

  Intense Mild Fast SlowTurbule

ntLamina

r Large Small

Average (ppb)

1563 573 1102 518 1473 947 1282 205

STDEV 553 500 874 564 536 717 643 183

t-Test: Two-Sample Unequal VariancesLarge Small

Mean 1282 205Variance 412948 33518Observations 93 28df 119t Stat 14.3P(T<=t) one-tail 5.4x10-28

t Critical one-tail 1.66P(T<=t) two-tail 1.1x10-27

t Critical two-tail 1.98

Non-Separable TreatmentsCoincident Treatments

Interdependence of Rate, Type and Splash

Cannot discern combination of effects or sole influence

Drop is the only separable Parameter

This is not to say they are irrelevant – need more data

Data Analysis

Treatment OccurrenceTurbulent with Large Drop

95%

Turbulent with Small Drop

5%

Laminar with Large Drop

77%

Laminar with Small Drop

23%

Total Turbulent 27%Total Laminar 73%

Experimental Variation of Large DropWe should expect no significant difference

Enough variability that operations are unpredictable

Distinct groups within the single treatment

Combination of effects may attribute to variation

Refinement of current technique is necessary

Variability

Large Drop Treatment ANOVA

Df Sum Sq Mean Sq F value Pr(>F)

Experiment

14 29417359 2101240 23.015 < 2.2e-16 ***

Residuals 76 6938609 91297

Experiment

Average (ppb)

Statistical Group

27 343 a16 416 a24 700 ab13 878 ab22 945 ab11 966 ab25 1231 bc8 1248 bc7 1277 bc5 1330 bc

17 1623 cd15 1681 cd6 1826 cde9 2197 de

23 2286 e

Conclusions and Future Work

Nick GislasonAndrew Waterhouse

References

1.) Andreasen, A. A., & Stier, T. J. B. 1953. Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in a defined medium. Journal of Cellular and Comparative Physiology, 41, 23–36

2.) Andreasen, A. A., & Stier, T. J. B. 1954. Anaerobic nutrition of Saccharomyces cerevisiae. II. Unsaturated fatty acid requirement for growth in a defined medium. Journal of Cellular and Comparative Physiology, 43, 71–281

3.) Ough, C.S. and M.A. Amerine. 1988. Methods for analysis of musts and wines, 2nd, Wiley, New York.

4.) Huber, C., T.-A. Nguyen, C. Krause, H. Humele and A. Stangelmayer. 2006. Oxygen ingress measurement into pet bottles using optical-chemical sensor technology. BrewingScience 5-15.

5.) http://www.oceanoptics.com/Products/sensortheory.asp

Acknowledgments

Supplemental Materials