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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