MES INDIAN SCHOOL

DOHA,QATAR
CHEMISTRY
PROJECT
(2022-23)
TOPIC : Effect of Various Factors on which
Rate of Fermentation Depends on

Done by: Teacher Guide:

EDWIN SIBY Mrs. Farsana Nanakkal
XII-B Department of Chemistry
 Bonafide
Certificate
This is to certify that Master Edwin Siby of class XII B has
completed his Chemistry Project during the academic year
2022-23 at M.E.S Indian School Doha, Qatar and his project
report is certified and bonafied.

Signature of Teacher In charge: …………………………………………..

Name:………………………………………..
Date:………………………………………….

School Stamp:

Roll Number / Registration Number

Submitted for all Indian senior school certificate practical

examination(Class XII) held at M.E.S Indian School, Doha-Qatar
on ……………………….

Internal Examiner External Examiner

Signature…………………………… Signature………………………………
Name………………………………… Name……………………………………
Designation……………………….. Designation…………………………..
School………………………………….
 ACKNOWLEDGEMENT

Following the successful completion of this project, I am

obliged to give gratitude to many who have helped me
throughout to do so.

To thank, I should start with the Almighty, who gave me

the ability, strength and proper health, to complete this
investigatory project. I am thankful to my teacher, Mrs.
Farsana Nanakkal, who motivated us to do this project. I
would also like to the Head of Department of Chemistry,
and the Chemistry Department along with my classmates
for the help and guidance given to me throughout this
project.

I also take this opportunity to extend my gratitude to our

principal Mrs. Hameeda Khadar and Head Of Section Mr.
Iliyas, as well as the School Governing Board who give the
students excellent service in terms of education.

Last, but not the least I thank my parents without whose

support I would have not been able to complete this
project.
 INDEX
1. Cover page 1

2. Bonafide Certificate 2

3. Acknowledgement 3

4. Index 4

5.Introduction 5

6. Content 9

7. Experiment 19

8. Observations 21

9. Conclusion 23

10. Bibliography 24
 INTRODUCTION
Fermentation is typically the conversion of
carbohydrates to alcohols and carbon dioxide or organic
acids using yeasts, bacteria, or a combination thereof,
under anaerobic conditions (absence of oxygen) by the
action of enzymes. Enzymes are complex organic
compounds, generally proteins. They are highly specific
with regard to their substrates. Fermentation in simple
terms is the chemical conversion of sugars into ethanol.
Ethanol fermentation, also referred to as alcoholic
fermentation is the biological process in which sugars
such as glucose, fructose, and sucrose are converted into
cellular energy and thereby produce ethanol and carbon
dioxide as metabolic waste products. All ethanol
contained in alcoholic beverages is produced by means
of fermentation induced by yeast. Wine is produced by
fermentation of the natural sugars present in grapes and
other kinds of fruit. Ethanol fermentation occurs in the
production of alcoholic beverages and ethanol fuel, and
in the leavening of bread dough. Fermentation is used in
preservation techniques and in production of foods such
as yogurt, cottage cheese (paneer), dhokla, idli,
chocolates, cheese etc. ‘Fermentation’ has been derived
from the Latin word ferver, which means ‘to boil’, as
during fermentation, there is a lot of frothing in the
liquid due to evolution of carbon dioxide. This gives it
the appearance as if it is boiling!
Yeasts are unicellular eukaryotic microorganisms
classified in the kingdom Fungi, Yeast size can vary
greatly depending on the species, typically measuring
3-4 μm in diameter, although some yeasts can reach
over 40 μm. Most yeasts reproduce asexually by
mitosis, and many do so by an asymmetric division
process called budding. Yeasts do not form a single
taxonomic or phylogenetic grouping. The term yeast is
often taken as a synonym for Saccharomyces cerevisiae.
Natural fermentation precedes human history. The
earliest evidence of winemaking dates from eight
thousand years ago, in Georgia, in the Caucasus area.
Seven-thousand-year- old jars containing the remains
of wine have been excavated in the Zagros Mountains
in Iran. There is strong evidence that people were
fermenting beverages in Babylon circa 3000 BC, ancient
Egypt circa 3150 BC, pre-Hispanic Mexico circa 2000
BC, and Sudan circa 1500 BC. Ancient fermented food
processes were developed long before man had any
knowledge of the existence of the microorganisms
involved.
When studying the fermentation of sugar to alcohol by
yeast, Louis Pasteur concluded that the fermentation was
catalyzed by a vital force, called “ferments”, within the
yeast cells. The “ferments” were thought to function only
within the yeast cells. The “ferments” were thought to
function only within living organisms. Nevertheless, it
was known that yeast extracts (Yeast extract is the name
given to processed yeast products made byextracting the
cell contents (removing the cell walls)) can ferment sugar
even in the absence of living yeast cells. While studying
this process in 1897, Eduard Buchner found that sugar
was fermented even when there were no living yeast
cells in the mixture; by a yeast secretion that he termed
zymase, i.e., fermenting activity of yeast is due to active
catalyst of biochemical origin. In 1907 he received the
Nobel Prize in Chemistry for his research and discovery of
“cell-free fermentation.”

Main uses of fermentation

The primary benefit of fermentation is the conversion of
sugars and other carbohydrates, e.g., converting juice
into wine, grains into beer, carbohydrates into carbon
dioxide to leaven bread, and sugars in vegetables into
preservative organic acids.
Food fermentation has been said to serve five main
purposes:
• Enrichment of the diet through development of a
diversity of flavours, aromas, and textures in food
substrates.
• Preservation of substantial amounts of foods through
lactic acid, alcohol, acetic acid, and alkaline
fermentations
• Biological enrichment of food substrates with
protein, essential amino acids, essential fatty acids,
and vitamins
• Elimination of antinutrients
• A decrease in cooking time and fuel requirement
 CONTENT
Objective:
In this project, time taken for fermentation of various fruit /
vegetable juices had to be compared. Fermentation is one of
the oldest methods of processing food into a form that is
suitable for preservation. In fermentation technology, we
stress in understanding the various process in fermentor and
how various intrinsic factors influence the fermentation
process. Fermentation technology being an industrial
microbiology subject are geared in producing maximum
amount of high economical fermentation products. The
objective of this project is to compare the rates of
fermentation of different fruit and vegetable juices. The
information gained from this experiment may be used by
wineries to determine which fruit juice ferments best. But it
is difficult to understand and control the fermentation
process as it involves various components such as effect of
substrates, products inhibition, conditions and complex
microbial interactions. Fermentation is affected by several
factors including the temperature, salt concentration, pH,
oxygen availability and nutrient availability. The rate of
fermentation can be controlled by manipulating any of these
factors.
a) Temperature : Different yeasts tolerate different
temperatures. For Saccharomyces cerevisiae, it is around 35-
400C. A variation of just a few degrees from this temperature
alters the activity of the microbes and affects the quality of
the final product.
b) Nutrients i.e. Sugar content : All bacteria require a
source of nutrients for metabolism. The fermenters require
carbohydrates, in this case sugars glucose and fructose.
The energy requirements of microbes are very high.
Limiting the amount of substrate available can reduce the
rate of fermentation.
c) Effect of oxygen : If oxygen is present, some species of
yeast will oxidize pyruvate completely to carbon dioxide
and water. Thus, these species of yeast will produce
ethanol only in an anaerobic environment. However, many
yeasts such as the baker’s yeast Saccharomyces cerevisiae,
or fission yeast Schizo saccharomyces pombe, prefer
fermentation to respiration. These yeasts will produce
ethanol even under aerobic conditions.
Hence the rate of fermentation varies.
The fermentation process is not only complex but always in
a state of flux. Process, we are therefore in a situation to
always be adaptive and reactive to these changes so that
throughout the fermentation process we are always
sustaining the conditions in a narrow window of optimal
fermentation conditions. In order to help us do this we
need to know fermentation kinetics. When we talk about
fermentation kinetics we are talking about fermentation
models. Kinetics and modellings are very useful to us as
tools to make fermentation predictions and enhancing our
experimental designs to be more focused to the specific
problems such as the rate limiting steps or product
inhibition.
The study of fermentation kinetics helps us by providing
clear quantitative data for us to understand the process
and improve the process accordingly. Peering into
observation ports might be good advertising gimmick for
fermentation technology but do not really help much in
understanding the process or even to control and
predict the fermentation outcome. Subjective
observations will rarely help in producing optimum
fermentation process and thus affect profitability
studies and making decisions. Its numbers that count!
Thus the importance of the study of fermentation
kinetics or models.
The first step in the study of fermentation kinetics is to
understand the various processes involved in the whole
process. Such questions such as inputs and outputs, the
metabolic pathways involved and type of products or
side products formed. The various individual reactions
involved and what factors control the metabolite levels.
Then only after all the relevant data are obtained do we
start formulating the models.
SCOPE :
The scope of this project is as wide as the scope of
process of fermentation. This project aspires to explore
one of the innumerable applications of the biochemical
concept of breakage of highly ordered large molecules
into smaller ones by the action of microorganisms or
enzymes. Some of the applications include:
THE PRODUCTION OF ALCOHOL
Beers, wines and spirits are all produced by fermenting
various carbohydrates. Yeasts do this naturally to sugars;
a property that has been utilized by humans for
thousands of years. Ethanol is also produced industrially
on a large scale for use as a biofuel. This has traditionally
involved a two step fermentation procedure using
aerated tanks containing the yeast Saccharomyces
cerevisciae and substrate carbohydrates.
THE PRODUCTION OF CITRIC ACID
Citric acid is a useful product in both the food and
pharmaceutical industries; it is used in food as a
preservative and to produce an acidic, sour taste in soft
drinks and other beverages. In the pharmaceutical
industry it can be used as buffering agent and to clean
equipment. Citric acid is formed by the fermentation of
a molasses substrate by the fungus Aspergillus Niger.
The biochemical pathway involved includes the
production of pyruvate in glycolysis, followed by its
conversion to citric acid via the condensation of acetyl
co-enzyme A and oxaloaecetate.
ACETIC ACID PRODUCTION
In the presence of the Acetobacter bacterium and
oxygen, fermented carbohydrates, ciders or wines can
be converted to vinegar (acetic acid). The result is
usually is usually a 5 % solution of acetic acid. Acetic
acid is used in diluted form in the food industry as a
condiment and pickling agent. It is also employed in
industry as a solvent and an important reagent in many
organic synthesis reactions.
A VERSATILE REACTION
Fermentation certainly produces a diverse range of
chemicals and is obviously a key reaction in many
industries. The one thing all these processes have in
common is an initial culture containing carbohydrates
and particular species of microorganism.
LIMITATIONS :
One of the limitations of fermentation as a process is
its requirement for multiple reagents. Secondly, in
many cases the time taken is quite long and this
creates a need for catalyst. Without catalysts, the
reaction is extremely slow. The limitation of our project
is the slight error in the result and the project is limited
to the fermentation of the juices with Baker’s yeast and
not under normal conditions i.e. without adding
Baker’s yeast. Owing to the different criterion on which
the rate of fermentation depends, if the experiment is
not carried out in the optimal temperature range, the
rates will turn out to be different than the actual rates
of the juices that have been taken. It is not possible to
get the exact theoretically estimated value due to
impurities in the reagents as well as the compounds.
Another point to be noted is that the rates calculated
from this experiment is just one case and this can’t
actually access the rate of fermentation of the fruit. An
average needs to be taken to access its actual value.
PRINCIPLE/THEORY :
Fermentation is the slow decomposition of complex organic
compounds into simpler compounds by the action of enzymes.
Enzymes are biological molecules that catalyse (i.e, increase
the rates of) chemical reactions. Fruit and vegetable juices
contain sugar such as sucrose, glucose and fructose. The
chemical equations below summarize the fermentation of
sucrose, whose chemical formula isC12 H22 O11. One mole of
sucrose is converted into four moles of ethanol and four moles
of carbon dioxide:
C12H22O11 + H2O + Invertase 2 C6H12O6
Glucose + Fructose
C6H12O6 + Zymase 2 C2H5OH + 2CO2
Glucose + Fructose
Sucrose is hence first converted to glucose and fructose with
the enzyme invertase, while enzyme zymase converts glucose
and fructose to ethyl alcohol.
Invertase
Invertase (systematic name: beta-fructofuranosidase) is an
enzyme that catalyses the hydrolysis (breakdown) of sucrose.
Related to invertases are sucrases. Invertases and sucrases
hydrolyse sucrose to give the same mixture of glucose and
fructose. Invertases cleave the O-C (fructose) bond, whereas
sucrases cleave the O-C (glucose) bond. For industrial use,
invertase is usually derived from yeast. It is also synthesized by
bees, who use it to make honey from nectar. Optimum
temperature at which the rate of reaction is at its greatest is
600 C and an optimum pH of 4.5.
 Invertase
C12H22O11 + H2O C6H12O6 + C6H12O6
Sucrose Glucose Fructose

Zymase
Zymase is an enzyme complex (“mixture”) which
catalyzes the fermentation of sugar into ethanol and
carbon dioxide. They occur naturally in yeasts. Zymase
activity varies among yeast strains.
Zymase
C6H12O6 + C6H12O6 2C2H5OH + 2CO2
Glucose Fructose Ethanol

Chemical test: Fehling’s solution

To test for the presence reducing sugars to the juice, a
small amount of Fehling’s solution is added and boiled
in a water bath. During a water bath, the solution
progresses in the colors of blue (with no glucose
present), green, yellow, orange, red, and then brick red
or brown (with high glucose present). A colour change
would signify and the presence of glucose.
Sucrose (table sugar) contains two sugars (fructose and
glucose) joined by their glycosidic bond in such a way as to
prevent the glucose isomerizing to aldehyde, or the
fructose to alpha-hydroxy-ketone form. Sucrose is thus a
non-reducing sugar which does not react with Fehling’s
solution.(Sucrose indirectly produces a positive result with
Benedict’s reagent if heated with dilute hydrochloric acid
prior to the test, although after this treatment it is no
longer sucrose.) The products of sucrose decomposition
are glucose and fructose, both of which can be detected
by Fehling’s as described above.
By comparing the time required for completion of
fermentation of equal amounts of different substances
containing starch the rates of fermentation can be
compared.
Addition of yeast
In wine making, yeast is normally already present on
grape skins. Fermentation can be done with this
endogenous “wild yeast,” but this procedure gives
unpredictable results, which depend upon the exact types
of yeast species present. For this reason, a pure yeast
culture is usually added, this yeast quickly dominates the
fermentation. Baker’s yeast is the common name for the
strains of yeast commonly used as a leavening agent in
baking bread and bakery products, where it converts the
fermentable sugars present in the dough into carbon
dioxide and ethanol. Baker’s yeast is of the species
Saccharomyces cerevisiae, which is the same species
commonly used in alcoholic fermentation, and so is also
called brewer’s yeast.
Pasteur’s salt
Pasteur’s salt solution is prepared by dissolving ammonium
tartarate, 10.0 g; potassium phosphate, 2.0 g; calcium
phosphate, 0.2 g; and magnesium sulphate, 0.2 g dissolved in
860 ml of water.
The Pasteur’s salts in solution act as a buffer to any acids the
yeast may create. Since yeast only converts sugar (most likely
sucrose or glucose) to ethanol under anaerobic conditions, and
it is unreasonable to assume that there will be no oxygen
present in the laboratory, some acetic acid is created as a
result. The Pasteur salts act as buffers to the acidity so that the
proteins in the yeast do not become denatured.
 EXPERIMENT
Aim:
To compare the rates of fermentation of some fruit/vegetable
juices and determine the substance which has the highest
rate of fermentation amongst the various samples taken.
Requirement:
a. Chemical Requirement
1. Pasteur’s salts
2. Yeast
3. Fehling’s reagent
b. Apparatus Requirement
4. Conical flasks
5. Test tubes
6. Beaker
7. Bunsen burner, tripod stand and watch glass
PROCEDURE
1. 5.0 ml of apple juice was taken in a clean 250 ml conical
flask and diluted with 50 ml of distilled water.
2. 2.0 gram of Baker’s yeast and 5.0 ml of solution of
Pasteur’s salts
were added to the above conical flask.
3. The contents of the flask were shaken well and the
temperature of the
reaction mixture was maintained between 35-400C.
4. After 10 minutes 5 drops of the reaction mixture were
taken from the flask and added to a test tube containing 2 ml
of Fehling reagent. The test tube was placed in a boiling water
bath for about 2 minutes. The Colour of the solution or
precipitate was then noted.
5. Step 4 was repeated after every 10 minutes until the
reaction mixture stopped giving any red colour or
6. This time taken, i.e. time taken for the completion of
fermentation was noted. precipitate.

7. All the above steps were repeated by taking 5 ml each of

grape juice, black grape juice, sweet lime juice, orange juice
and carrot juice.
Precautions:
• All apparatus should be clean and washed properly.
• The flask should not be rinsed with any of the solution.
 OBSERVATIONS
Volume of fruit juice taken = 5.0 ml
Volume of distilled water added = 50.0 ml
Weight of baker’s yeast added = 2.0 g
Volume of solution of Pasteur’s salts = 5.0 ml

Time Colour of reaction mixture on reaction with Fehling solution

( in
minutes )
Apple Sweet Carrot Orange Tomato
Juice lime Juice Juice Juice
Juice

10 Red Red Red Red Red

20 Red Red Red Red Brownish Red

30 Red Red No change Red Brown

40 Red Red No change No change No change

50 Brownish Greenish No change No change No change

Red Brown

60 Brown No No change No change No change

change

70 No change No No change No change No change

change
Graph
 CONCLUSION
• The time taken for fermentation of carrot juice was
well before the rest ofthe juices, it’s recorded time
being 30 minutes.
• This means that carrot juicehas the highest sucrose
content from the various samples taken.
• After 50 minutes orange and tomato juices gave
positive test for fermentation with Fehling’s solution.
For sweet lime juice time taken for fermentation was
60 minutes and for apple juice it was 70 minutes.
 BIBLIOGRAPHY
• Wikipedia - The free encyclopedia -
(http://en.wikipedia.org)
• Comprehensive Practical Chemistry
• Academia.edu