Pamantasan ng Lungsod ng Maynila

(University of the City of Manila)

Intramuros, Manila
College of Engineering and Technology
Department of Chemical Engineering

Introduction to Biotechnology:

Fermentation of Apple Juice for

the Determination of Wine and
Ethanol Concentrations

Prepared by:
AMADO, Rosenn B.
DANDAMUN, Benbellah Ali Y.
EBREO, Mencyn Michelle Kellie
GONZALES, Belinda Jean A
IGAYA, Deanne May G.
JUAGCO, Cris-Anne III
LOPEZ, Charmaine A.
TORRES, Angela Jane R.

BSCHE-IV

Submitted to:
Engr. Ronald Allan Co
 ACKNOWLEDGEMENT

We acknowledge the presence of Almighty God on

our side that guide and enlighten our minds in coping up
with such great pressure that has been put on our shoulders.

And also, the thorough help of Engr. Ronald Allan S. Co

for the review of our study. We consider his advises as our
prior objective to fulfill.

Lastly, to our parents whom unconditionally understand

what we were going through and for the financial support.

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 2
 Chapter I: INTRODUCTION

BACKGROUND OF THE STUDY

Apple juice is a fruit juice made by the maceration and pressing of

apples. The resulting expelled juice may be further treated by enzymatic and
centrifugal clarification to remove the starch and pectin, which holds fine
particulate in suspension, and then pasteurized for packaging in glass, metal or
aseptic processing system containers, or further treated by dehydration
processes to a concentrate. In the study, apple juice was used as our primary
material for the fermentation. Fermentation typically refers to the conversion of
sugar to alcohol using yeast. In its strictest sense, fermentation is the anaerobic
metabolic breakdown of a nutrient molecule, such as glucose, without net
oxidation. Fermentation is also used much more broadly to refer to the bulk
growth of microorganisms on a growth medium.

Alcohol is formed when yeast feeds on sugar. Alcohol is ethanol or ethyl

alcohol. There are many informal names for alcohol (e.g. booze, bevvy, drink),
and there are many different types of alcoholic drink (e.g. beer, lager, wine,
spirits, cider). But they all contain ethanol. Ethanol is made from the sugar in
grain or fruit and it is a colorless, clear liquid. Yeast is a one-cell fungus which
reproduces by budding off new cells. This tiny organism grows and multiplies by
feeding on sugar in foods such as grain and fruits. As the yeast feeds on the
sugar, carbon dioxide and alcohol are produced. They are the waste products
of the yeast’s anaerobic respiration – that’s respiration without oxygen.

Sugar energy + alcohol + carbon dioxide

C6H12O6 energy + 2C2H5OH + 2CO2

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 3
 When yeast respires anaerobically, only a small amount of energy is
released from the sugar and so most of the sugar stays in the alcohol. When the
alcohol concentration reaches about 15% of alcohol by volume, the yeast cells
die. Ethanol concentration can be based by determining the density
measurement of the ethanol.

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 4
 OBJECTIVES

The objective of this study is to determine the wine and ethanol concentrations
using bio-chemical reaction and stabilization process.

Specific Objectives:

¸ It aims to determine ethanol concentration based by the density

measurement of ethanol

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 5
 Chapter II: Methodology

Materials Needed

∑ (28) 500 ml PET bottles

∑ (14) bending straws
∑ (2) 1L TIPCO Apple Juice
∑ 1 gallon distilled water
∑ 10 g yeast (Not all will be consumed)
∑ Funnel
∑ Analytical Balance
∑ Thermometer
∑ (2) beakers
∑ Vials or any glass bottles for weighing sample

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 6
 Procedure
Preparation:

1. Bottles obtained were of the same size and brand. Labels were removed.
2. Bottles and covers were cleaned with soap and water.
3. After cleaning, it was sterilized with hot water.
4. Bottles and covers dried by wiping the excess water and turned the bottle
down on a clean cloth for the condensate will run down and be
absorbed by the cloth.
5. Bottles were paired, making up 14 pairs. 7 for the first batch. Another 7 for
the second batch.
6. While it is still drying, covers were being bore with the use of cutter.
7. 2 straws were connected together and locked by scotch tape. And to be
inserted on the bottle caps.
8. One end of the straw must be cut to make it shorter (for the Wine Mixture
bottle). While, the other end of the straw must be long to have contact
with the water.
9. Bottles were labeled from 1 to 7, on both batches.

Preparation for Actual Set-up:

1. For basis, beaker was put on the analytical balance and set into zero.
2. 300 ml water were poured, and weighed. Record the data (mass, volume,
density, average mass, average volume, average density)
3. For the next basis: beaker was put on the analytical balance and set into
zero.

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 7
 4. 150 ml apple juice were poured, plus one pinch of yeast and weighed.
Record the data (mass, volume, density, average mass, average volume,
average density)
5. After the preparation, bottles has to be placed in a dark room and shake
free place.

Actual Set-up:

1. Everyday, take a 3-trial of obtaining twenty (20 ml) for each bottle.
(Everyday there should be four bottles to take samples from. 2 from the
first batch (Wine mixture and Ethanol), and another 2 from the second
batch (Wine mixture and Ethanol)
2. For every trial, It should be put on a dry vial, or any glass bottle to weigh
the sample.
3. Record the data. (mass, volume, density, average mass, average volume,
average density).

Solving for Concentration:

% EtOH Concentrated ( theo) = 100%

( .)− ( ) - % wine mixture
=
ℎ − ( )
%
Where:
= (100%)
( )
X= fractional concentration
% ( )
CEtOH = xρEtOH
% ( ℎ )− % ( )
= (100%)
% ( ℎ )
Cwine = (1-x) ρwine(initial)

( ) Where:
%

= CEtOH = Concentration of Ethanol

(g/ml)

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 8
Cwine = Concentration of wine (g/ml) ρEtOH= density of pure ethanol
(0.8059 g/ml)ρwine = density of wine
@t=0, initial value

Chapter III. Data and Results

Table of Data
DATA AND RESULTS: Apple Wine

Basis:

Volume (ml) Mass (g) Density (g/ml)

Water 300 300 1
Wine Mixture 150 180 1.2

1st batch (Ethanol)

Day 1 Volume (ml) Mass (g) Density (g/ml)
Trial 1 20 21.2 1.06
Trial 2 20 20.8 1.04
Trial 3 20 21.2 1.06
Average 20 21.0667 1.0533

Day 2 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21 1.05
Trial 2 20 20.6 1.03
Trial 3 20 20.6 1.03
Average 20 20.7333 1.0367

Day 3 Volume (ml) Mass (g) Density (g/ml)

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 9
 Trial 1 20 20.9 1.045
Trial 2 20 20.9 1.045
Trial 3 20 20.6 1.03
Average 20 20.8 1.04

Day 4 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 20.7 1.035
Trial 2 20 20.7 1.035
Trial 3 20 20.5 1.025
Average 20 20.6333 1.0317

Day 5 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 20. 6 1.03
Trial 2 20 20.6 1.03
Trial 3 20 20.8 1.04
Average 20 20.6667 1.0333

Day 6 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 20. 6 1.03
Trial 2 20 20.5 1.025
Trial 3 20 20.7 1.035
Average 20 20.6 1.03

Day 7 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 20.7 1.035
Trial 2 20 21 1.05
Trial 3 20 21 1.05
Average 20 20.9 1.045

1st batch (Wine Mixture)

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 10
 Day 1 Volume (ml) Mass (g) Density (g/ml)
Trial 1 20 21.4 1.07
Trial 2 20 21.9 1.095
Trial 3 20 21.9 1.095
Average 20 21.7333 1.0867

Day 2 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 20.7 1.035
Trial 2 20 20.6 1.03
Trial 3 20 20.9 1.045
Average 20 20.7333 1.0367

Day 3 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 20.8 1.04
Trial 2 20 20.9 1.045
Trial 3 20 20.5 1.025
Average 20 20.7333 1.0367

Day 4 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21 1.05
Trial 2 20 21.6 1.08
Trial 3 20 21.8 1.09
Average 20 21.4667 1.0733

Day 5 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21.5 1.075
Trial 2 20 21.1 1.055
Trial 3 20 21.6 1.08
Average 20 21.4 1.07

Day 6 Volume (ml) Mass (g) Density (g/ml)

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 11
 Trial 1 20 21.2 1.06
Trial 2 20 20.9 1.045
Trial 3 20 21 1.04
Average 20 21.0333 1.0517

Day 7 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21.9 1.095
Trial 2 20 20.8 1.04
Trial 3 20 20.8 1.04
Average 20 21.1667 1.0583

2nd Batch (Ethanol)

Day 1 Volume (ml) Mass (g) Density (g/ml)
Trial 1 20 21.4 1.07
Trial 2 20 21.6 1.08
Trial 3 20 20.5 1.025
Average 20 21.1667 1.0583

Day 2 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21 1.05
Trial 2 20 20.8 1.04
Trial 3 20 21 1.05
Average 20 20.9333 1.0467

Day 3 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 20.9 1.045
Trial 2 20 20.5 1.025
Trial 3 20 20.3 1.015
Average 20 20.5667 1.0283

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 12
 Day 4 Volume (ml) Mass (g) Density (g/ml)
Trial 1 20 20.6 1.03
Trial 2 20 20.6 1.03
Trial 3 20 20.4 1.02
Average 20 20.5333 1.0267

Day 5 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21 1.05
Trial 2 20 20.9 1.045
Trial 3 20 21 1.05
Average 20 20.9667 1.0483

Day 6 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21.1 1.055
Trial 2 20 21.2 1.06
Trial 3 20 20.8 1.04
Average 20 21.0333 1.0517

Day 7 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21.3 1.065
Trial 2 20 21.2 1.06
Trial 3 20 21.1 1.055
Average 20 21.2 1.06

2nd Batch (Wine Mixture)

Day 1 Volume (ml) Mass (g) Density (g/ml)
Trial 1 20 21.7 1.085
Trial 2 20 21.8 1.09
Trial 3 20 21.7 1.085

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 13
 Average 20 21.7333 1.0867

Day 2 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 20.2 1.01
Trial 2 20 20.6 1.03
Trial 3 20 20.9 1.045
Average 20 20.5667 1.0283

Day 3 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21.1 1.055
Trial 2 20 20.7 1.035
Trial 3 20 20.9 1.045
Average 20 20.9 1.045

Day 4 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21.5 1.075
Trial 2 20 21.2 1.06
Trial 3 20 21.1 1.055
Average 20 21.2667 1.0633

Day 5 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21.9 1.095
Trial 2 20 21.2 1.06
Trial 3 20 21.2 1.06
Average 20 21.4333 1.0717

Day 6 Volume (ml) Mass (g) Density (g/ml)

Trial 1 20 21 1.05
Trial 2 20 21 1.05
Trial 3 20 21.5 1.075
Average 20 21.1667 1.0583
Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 14
 Day 7 Volume (ml) Mass (g) Density (g/ml)
Trial 1 20 21.6 1.08
Trial 2 20 21.3 1.065
Trial 3 20 21.4 1.07
Average 20 21.4333 1.0717

Concentration Data
Batch Time ρ(EtOH) ρ x C EtOH C Wine
(Wine)
1 0 1 1.2 0 0 1.2
1 Day 1 1.0533 1.0867 0.28749 0.23169 0.855011
1 Day 2 1.0367 1.0367 0.41436 0.33393 0.702766
1 Day 3 1.04 1.0733 0.32149 0.25909 0.81421
1 Day 4 1.0317 1.0367 0.41436 0.33393 0.702766
1 Day 5 1.0333 1.07 0.32987 0.26584 0.804161
1 Day 6 1.03 1.0517 0.3763 0.30326 0.748439
1 Day 7 1.045 1.0583 0.35955 0.28976 0.768536

% EtOH (actual) %EtOH (theo) % Wine % Error

(mixture)
0 0 100 ---
27.09771783 28.74904846 71.25095154 5.74395
47.51713966 41.43618371 58.56381629 14.6755
31.82109511 32.14920071 67.85079929 1.02057
47.51713966 41.43618371 58.56381629 14.6755
33.05786949 32.98655164 67.01344836 0.2162
40.51904326 37.63004314 62.36995686 7.67738
37.70339078 35.95534128 64.04465872 4.86172

Batch Time ρ(EtOH) ρ x C EtOH C Wine

(Wine)

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 15
 2 Day 0 1 1.2 0 0 1.2
2 Day 1 1.0583 1.0867 0.28749 0.23169 0.8550114
2 Day 2 1.0467 1.0283 0.43568 0.35111 0.6771885
2 Day 3 1.0283 1.045 0.3933 0.31696 0.7280386
2 Day 4 1.0267 1.0633 0.34687 0.27954 0.7837605
2 Day 5 1.0483 1.0717 0.32555 0.26236 0.8093377
2 Day 6 1.0517 1.0583 0.35955 0.28976 0.7685359
2 Day 7 1.06 1.0717 0.32555 0.26236 0.8093377

% EtOH (actual) %EtOH (theo) % Wine % Error

(mixture)
0 0 100 ---
27.09771783 28.74904846 71.25095154 5.74395
51.84840752 43.56762243 56.43237757 -19.007
43.5363516 39.33011926 60.66988074 -10.695
35.66644975 34.68662776 65.31337224 -2.8248
32.41690808 32.55518904 67.44481096 0.42476
37.70339078 35.95534128 64.04465872 -4.8617
32.41690808 32.55518904 67.44481096 0.42476

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 16
 1.4

1.2

1
Figure 1.1 Line graph
0.8
C EtOH obtained from data
0.6 on Batch 1 (Ethanol
C Wine
and Wine
0.4 Concentration)

0.2

0
0 2 4 6 8 10

1.4

1.2

0.8
C EtOH Figure 1.2 Line
0.6 C Wine graph obtained from
data on Batch 2
0.4

Fermentation
0.2 of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 17

0
0 2 4 6 8 10
 (Ethanol and Wine Concentration)

Time Xa
Day 0 0
Day 1 0.28749
Day 2 0.414362
Day 3 0.321492
Day 4 0.414362
Day 5 0.329866
Day 6 0.3763
Day 7 0.359553
Zero Order Reaction (1)

0.5
y = 0.0333x + 0.1629
0.45 R² = 0.3713
0.4
0.35
0.3
0.25 Series1

0.2 Linear (Series1)

0.15
0.1
0.05
0
0 2 4 6 8 10

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 18
 Time Xa 1/Xa2
Day 0 0 0
First Order Reaction (1) Day 1 0.28749 12.0991
Day 2 0.414362 5.824257
0 Day 3 0.321492 9.675193
0 2 4 6 8 Day 4 0.414362 5.824257
-0.2 y = 0.018x - 1.1081 Day 5 0.329866 9.190225
R² = 0.0817 Day 6 0.3763 7.062046
-0.4
Day 7 0.359553 7.735229
-0.6 Series1

-0.8 Linear (Series1)

-1

-1.2

-1.4
Time Xa 1/Xa
Day 0 0 0
Day 1 0.28749 3.478376
Day 2 0.414362 2.41335
Day 3 0.321492 3.110497
Day 4 0.414362 2.41335
Day 5 0.329866 3.031538
Day 6 0.3763 2.657451
Second Order Reaction (1)
Day 7 0.359553 2.781228

3.5 y = -0.0601x + 3.0811

R² = 0.1101
3

2.5

2 Series1
Linear (Series1)
1.5

0.5
Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 19
0
0 2 4 6 8 10
 Third Order Reaction (1)

14
y = -0.3965x + 9.7873
12 R² = 0.1412

10

8
Series1
6 Linear (Series1)

0
0 2 4 6 8 10

Time Xa 1/Xa^2
Day 0 0 0
Day 1 0.28749 -1.24657
Day 2 0.414362 -0.88102
Day 3 0.321492 -1.13478
Day 4 0.414362 -0.88102
Day 5 0.329866 -1.10907
Day 6 0.3763 -0.97737
Day 7 0.359553 -1.02289

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 20
 Zero Order Reaction (2)

0.5
y = 0.0269x + 0.1881
0.45 R² = 0.2464
0.4 Time Xa
0.35 Day 0 0
0.3 Day 1 0.28749
0.25 Series1 Day 2 0.435676
0.2 Linear (Series1) Day 3 0.393301
0.15 Day 4 0.346866
Day 5 0.325552
0.1
Day 6 0.359553
0.05
Day 7 0.325552
0
0 2 4 6 8 10

First Order Reaction (2)

0
Time Xa LN Xa
0 2 4 6 8 10 y = -0.0071x - 1.0195
-0.2 R² = 0.0128 Day 0 0 0
Day 1 0.28749 -1.24657
-0.4 Day 2 0.435676 -0.83086
Day 3 0.393301 -0.93318
-0.6 Series1 Day 4 0.346866 -0.10582
-0.8 Linear (Series1) Day 5 0.325552 -1.12223
Day 6 0.359553 -1.10289
-1 Day 7 0.325552 -1.12223

-1.2

-1.4

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 21
 Second Order Reaction (2)

4
y = 0.01x + 2.8347
3.5 R² = 0.0031
Time Xa 1/Xa
3
Day 0 0 0
2.5 Day 1 0.28749 3.478376
2 Series1 Day 2 0.435676 2.295282
Linear (Series1) Day 3 0.393301 2.542581
1.5
Day 4 0.346866 2.882955
1 Day 5 0.325552 3.071707
Day 6 0.359553 2.781228
0.5
Day 7 0.325552 3.071707
0
0 2 4 6 8 10

Third Order Reaction (2)

14
y = -0.0031x + 8.4052
12 R² = 9E-06 Time Xa 1/Xa^2
Day 0 0 0
10 Day 1 0.28749 12.0991
8
Day 2 0.435676 5.268332
Series1 Day 3 0.393301 6.464716
6 Linear (Series1) Day 4 0.346866 8.311432
Day 5 0.325552 9.435384
4
Day 6 0.359553 7.735229
2 Day 7 0.325552 9.435384

0
0 2 4 6 8 10

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 22
 DOCUMENTATION

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 23
Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 24
 INTERPRETATION OF RESULTS
The results of the experiment depicts how an ideal fermentation process
would occur wherein the growing cells are consuming the substrate, and
producing more cells as time passes. In this case, the concentrations of ethanol
and wine increases and decreases respectively with time.

The stages of yeast growth are noticeable in the graphs. From Day 0 to
Day 2, the yeast undergoes an exponential growth. From Figure 1.1, the ethanol
concentration increases from 0 to 0.333934 and the wine concentration
decreases from 1.2 to 0.702766. From Figure 1.2, the concentration of ethanol
increases from 0 to 0.351111 and the concentration of the wine decreases from
1.2 to 0.677189. Then from Day 2 to Day 5, the deceleration phase is seen. From
Figure 1.1, the concentration of ethanol begins to decrease from Day 2,
0.333934, to Day 3, 0.25909 while the concentration of wine increases from
0.702766 to 0.81421. From Figure 1.2, the concentration of ethanol begins to
decrease from Day 2, 0.351111, to Day 5, .262362 while the concentration of the
wine increases from .728039 to 0.809338. The concentrations of wine and
ethanol begin to become constant as the yeast approaches the stationary
phase.

As for the order of reaction, Zero Order Reaction yields an R2 closer to 1 (In
the first batch R2 is equal to .3713 and in the second batch it’s value is .2464). For
batch 1, the R2 that is farther to 1 is from the First Order Reaction which yields an
R2 of .0817 while for batch 2; the R2 from the Second Order Reaction is farther to
1 which is equal to .0031.

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 25
 Chapter IV: CONCLUSION
The fermentation of apple juice is relatively fast because it only takes 1-2
weeks for the juice to ferment.

The lag phase (wherein the yeast matures and acclimates to the
environment and no growth occurs) was not visibly seen in the graph. The cause
of which may have been the variation of time (Day to Day). The rate of reaction
in the exponential growth of the yeast follows first order kinetics. Parameters like
the substrate and waste concentrations have an effect on the kinetics of yeast
growth as seen in the deceleration phase of the yeast. The stationary phase
(wherein no growth occurs) was only partially seen in the graph. Other factors
that affected the fermentation process may have been the stabilization of the
set-up, the thermal conditions, and the lighting in the area. The application of
other kinds of yeast would also vary the fermentation process.

The order that is best fit for the fermentation of apple juice is the Zero
Order Reaction. The zero order reaction yielded a value of R2 that is closer to 1
than the first, second, and third order reactions.

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 26
 Chapter V: Recommendation

The researchers recommend to have a better proportion of amount of

yeast to the concentration of apple Juice. Enormous amount of yeast may lead
the apple juice to ferment easily and flow out of the bottle. On the other hand,
there are many fruits in the market that can be fermented, too. During obtaining
of the sample, if using syringe, avoid making too much holes from the bottle so
that air wont pass through. Make sure that bottles were sealed enough by
packaging tapes. Use analytical balance to have an accurate data.

Fermentation of Apple Juice for the Determination of Wine and Ethanol Concentrations Page 27