EthanolRateDetermination_BrandonSkinner

Determination of the Rate of Ethanol Production by Yeast

in WinePresented by

Brandon SkinnerBarton College

School of Sciences

Background• Wine was made by Barton College Biology professor, Dr. Mark

Basinger.• 39-day process• 4 stages• Stage 1 – 7 days• Stage 2 – 10 days• Stage 3 – 8 days• Stage 4 – 14 days

Background• Trial 1• Chilean Pinot Noir• October 24, 2014 – December 1, 2014

• Trial 2• Australian Shiraz• January 30, 2015 – March 9, 2015

• Purpose:• What is the rate at which yeast cells ferment sugars into alcohol?

Fermentation Process• Yeast cells (Saccharomyces cerevisiae) ferment glucose into ethanol• Fermentation is…

• An anaerobic process• Takes place without oxygen

• Used to produce ATP• Adenosine Triphosphate• “Energy” molecule

• Used to regenerate NAD+• Nicotinamide adenine dinucleotide• Involved in redox reactions during cellular metabolism• Converted to NADH during glycolysis• Normally regenerated using aerobic processes

Fermentation Process• Fermentation produces waste byproducts.• When yeast cells undergo fermentation, they produce ethanol and

carbon dioxide as byproducts.

C6H12O6 (aq) + yeast 2 CO2 (g) + 2 CH3CH2OH (aq)

Stage 1: Primary Fermentation• Preparation of Wine• Grape juice• Water• Yeast• Wood chips

• Trial 1: Oak• Trial 2: Roasted Oak

• Clay (Bentonite)

• 7 Days

Stage 2: Secondary Fermentation • Transferred into a sterile plastic carboy• 10 days

Stage 3: Clarifying and Stabilizing• Materials added• Potassium metabisulfite• Potassium sorbate• Chitosan

• Kill remaining microorganisms• Purify• 8 days

Stage 4: Racking• Transferred to another plastic jug• Separate “clean” wine from “dirty” wine• 14 days

Sampling Procedure

Chemical Analysis: Sampling Procedure• 10-15 mL sample taken at the same time every day• Mixed with 1 gram of sodium bisulfite• Quench reaction

• Filtered through 1 micro filters• Transferred into 5-mL vials• Stored in a cool area (refrigerator) for later Gas Chromatograph (GC)

analysis during trial 1.• Immediately analyzed after collection during trial 2.

Chemical Analysis: Preparation of Samples for GC Analysis• Trial 1:• One spiked sample and one non-spiked sample prepared for analysis.

• Trial 2:• Three spiked samples and three non-spiked samples prepared for analysis.

• Spiked sample• 0.5 mL wine sample• 0.5 mL standard solution (10% each of methanol, ethanol, propanol, butanol, and

acetic acid)• Non-spiked sample• 1 mL wine sample

Chemical Analysis: Gas Chromatograph Instrument

Instrument: Clarus 400 GC (PerkinElmer)Detector: FIDColumn types: Capillary Column

Chemical Analysis: Gas Chromatographic Instrument Conditions• Column - ZBWaxplus (from Phenomenex)

with the following specifications:• Type: Capillary• Composition: 100% Polyethylene Glycol

• 100% aqueous stable with high retention for alcohols and chlorinated compounds due to its polarity

• Min/Max Temperature: 20ᵒ – 250ᵒ C• Column dimensions

• Length (L): 30 m• Inner Diameter (ID): 0.32 mm• Film thickness (df): 0.52 μm

Chemical Analysis: Gas Chromatographic Instrument Conditions• Used PerkinElmer Clarus 400 GC• Capillary column GC with flame ionization detector (FID)• Syringe Capacity: 5.0 μL• Oven/Column Temperature:

• Initial: 75 ᵒC, Hold: 2.00 mins.• Ramp #1: 10.0 ᵒC/min 80 ᵒC, Hold: 2.50 mins.• Ramp #2: 10.0 ᵒC/min 145 ᵒC, Hold: 3.50 mins.

• Total runtime: 15.0 mins• Injector Temperature: 250 ᵒC• Carrier Gas: N2 Flow-rate: 14.0 mL/min at 80 psi• Detector: FID Temperature: 245 ᵒC• Injection mode: Split (50 mL/min); Split ratio: 20:1• Injection volume: 2.0 μL

Chemical Analysis: Gas Chromatographs• The data from the gas

chromatography instrument is a plot of current (mV) vs. time.• The time variable is very important

as it used to identify each component.• Time at which a component emerges

from the column and gets detected is the retention time which is noted for each peak detected.• Area under the ethanol peak for each

sample was recorded.10

20

30

40

50

60

70

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0

M ethanol

E thanol

Propanol

B utanol

Acetic Acid

Time/min

mV

mV

Time/min

Data Analysis – Calculating Alcohol Concentration[A]u = Concentration of Ethanol

Iu = Area Under Peak of Sample

[A]u+s = Concentration of Ethanol + Spike

Iu+s = Area Under Peak of Spiked Sample

[A]u / [A]u+s = Iu / Iu+s

(0.5 / 1.0) [A]u + (0.5 / 1.0)(0.10) v/v = [A]u+s

[A]u / (0.5 [A]u+s + 0.05) = Iu / Iu+s

[A]u * Iu+s = 0.5[A]u * Iu + 0.05 Iu

[A]u = (0.05 * Iu) / (Iu+s – 0.50 Iu)

Samples Iu Iu+s [A]u/(v/v) Time (h)S1D1 753.59 40730.49 0.0009 0S1D2 9786.94 44448.16 0.0124 24S1D3 50949.13 67630.99 0.0604 48S1D4 76375.12 83954.75 0.0834 72S1D5 94335.79 90329.21 0.1093 96S1D6 117382.47 102932.22 0.1327 120S1D7 124653.17 107621.75 0.1376 144S1D8 125230.17 102345.79 0.1576 168S2D1 128240.46 108587.54 0.1442 168S2D2 134071.19 111069 0.1522 192S2D3 123013.59 100337.85 0.1584 216S2D4 133856.25 110855.11 0.1524 240S2D5 137256.91 110524.95 0.1638 264S2D6 134115.43 112050.07 0.1490 288S2D7 124604.08 102678.07 0.1543 312S2D8 131542.22 107139.35 0.1590 336S2D9 135386.58 107929.27 0.1682 360S2D10 124069.48 101690.38 0.1564 384S2D11 125055.73 102520.52 0.1563 408

Results – Plots

0 72 144 216 288 360 432 504 576 648 720 792 864 9360

0.05

0.1

0.15

0.2

0.25

Fig. 1. Fraction of Ethanol vs. Time/h for Trial 1Time (h)

Frac

tion

of A

lcoh

ol

Results – Plots

0 72 144 216 288 360 432 504 576 648 720 792 864 9360.00

0.05

0.10

0.15

0.20

0.25

f(x) = 0.000178762544554338 x + 0.0622202799081353R² = 0.823220331092918

Fig. 2. Rate of Ethanol Production for Trial 1

Δt (h)

Δ[A]

u

Results – Plots

0 48 96 144 192 240 288 336 384 432 480 528 576 624 672 720 768 816 864 9120.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Fig. 3. Fraction of Ethanol vs. Time/h for Trial 2Time (h)

Frac

tion

of E

than

ol

Results - Plots

0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384 408 432 456 480 504 528 552 576 6000.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Fig. 4. Fraction of Ethanol vs. Time/h for Trial 2 (First 25 days)Time (h)

Frac

tion

of E

than

ol

Results - Plots

24 48 72 96 120 144 1680.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16f(x) = 0.000891433411015661 x + 0.0106288732162663R² = 0.916629065264641

Fig. 5. Rate of Ethanol Production During Stage 1 of Trial 2

Δt (h)

Δ[A]

u

168 192 216 240 264 288 312 336 360 384 4080.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

f(x) = 4.1981390197825E-05 x + 0.142826197605232R² = 0.251797400150832


Δt (h)

Δ[A]

u

Results - Plots

408 432 456 480 504 528 552 576 6000.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

f(x) = 2.41564595920227E-05 x + 0.147084208974037R² = 0.0840139097218359


Δt (h)

Δ[A]

u

600 624 648 672 696 720 744 768 792 816 840 864 888 9120.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

f(x) = 2.17570240704224E-05 x + 0.118743420658278R² = 0.0665780690892348


Δt (h)

Δ[A]

u

Conclusions and Future Work• Conclusions

• The ethanol production rises quickly during the first few days and then begins to slow as expected.

• During trial 1, the ethanol concentration continues to rise during stage 4 for unknown reasons.• Could be due to contamination, oxidation, or degradation of the standard solution.• It is essential to analyze the samples as soon after they are collected as possible.

• During trial 2, the ethanol concentration stopped increasing at 16%, but fell to 14% during stage 4 when water was added.

• The rate at which ethanol was produced was 0.09% per hour during stage 1, with little to no production in stages 2, 3, and 4.

• Future work• Determine whether yeast or sugar concentration is the determining factor in the reaction.

Acknowledgements

• Dr. John Dogbe• Dr. Mark Basinger • Barton College Science Department

EthanolRateDetermination_BrandonSkinner

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