SWISS GERMAN UNIVERSITY

CELL BIOLOGY LABORATORY REPORT

Subject
Lecturer
Instructor
Faculty/Class
Date of Experiment
Date of Lab. Report
Semester
Time of Experiment

Experiment:

: Cell Biology Laboratory

: Dr.rer.nat. Maruli Pandjaitan
: Ms. Nani Pasaribu, M. Si; Ms. Sylvia Yusri, S. Si
: Life Science/LS 1 A
: 25 September 2013
: 9 October 2013
:1
: 14-00 16.00 pm

Fermentation

NAME

Group

Campus BSD City

Bumi Serpong Damai
Tangerang 15321 Indonesia

: Felicia Melissa, Kristania

Hadhiwaluyo, Chita Sakina Putri,
Jonathan Winarno,
Nabila Putri
: 4

Tel.
Fax.

+62 21 537 6221

+62 21 537 6201

[email protected]
www.sgu.ac.id

I.

Objectives

To determine which fermentation of sugar, alcoholic fermentation using lactose or

starch, that produces more products in the form of carbon dioxide gas, CO2(g).

II.

Theoretical Background
Carbohydrates
First and foremost, carbohydrates are large biological molecules or macromolecules that
plays a significant role in living things. They play an important role as an energy source
through the process of oxidation, energy storage, precursor to other important biological
molecules, and dietary fiber in the form of cellulose. Carbohydrates also form part of the
structures of some cells and tissues. In terms of its structure, this macromolecules is also
known as saccharides (saccharo , Greek for sugar) that contain large quantities of
hydroxyl groups along with aldehydes or ketones functional group. Theoretically speaking,
it can be divided into varieties of categories such as monosaccharide (as its monomer),
disaccharides, oligosaccharides, and polysaccharides.

Monosaccharide
Primarily monosaccharide is the monomer of this macromolecules, it plays an important role
as chemical energy storehouse & durable building material for biological construction, it
exists with the general formula (CH2O)n . In terms of its structure, it is composed of carbon
atom connected with single bonds as its backbone, one carbonyl group (C=O), and at least
two hydroxyl (-OH) groups. It is colorless, crystalline, and water soluble due to the presence
of polar hydroxyl group that is able to form hydrogen bond with adjacent water molecules.
Additionally due to the presence of a chiral carbon atom (4 different groups surrounding the
central carbon atom), carbohydrates exist with a stereoisomer properties which enable them
to form 2 identical molecules of carbohydrates with different arrangement due to the
difference in their positioning in space, even though they are made of the same atoms,
connected in the same sequence or in other words enantiomers. On the other hand, the cyclic
form of carbohydrates also can exist as isomer with difference in their structural
arrangement of atoms, raising many different types of monosaccharaides shown below.

If hydroxyl group of carbon 1 is below plane of

If -OH above plane of ring pyranose or

ring -pyranose or -glucose

glucose

Four isomers are cyclic, two of them with a pyranose (six-membered) ring, two with a
furanose (five-membered) ring. Galactofuranose occurs in bacteria, fungi and protozoa.

The

monosaccharide

itself

is

actually divided into 2 big groups

depending

on

the

type

of

functional group that it contains

and the number of carbon atoms
that are present in its backbone. In
terms of number of carbon atoms
in its backbone, monosaccharide
can be defined as triose (3
carbons), tetrose (4 carbons),
pentose (5 carbons), hexose (6 carbons), or heptose (7 carbons) sugar. However in terms of
functional group, usually they are classified into either aldose or ketose sugars. Ketose
sugars is primarily a sugar containing a ketone functional group. It is also known as fruit
sugar or fructose. Pure, dry fructose is a very sweet, white, odorless, crystalline solid and is
the most water-soluble of all the sugars, it is found in honey, tree and vine fruits, flowers,
berries, and most root vegetables. Whereas aldose sugars is primarily a type of sugar
containing aldehyde functional group. Two main examples of aldose sugars are glucose, an
important carbohydrate in animal (including human) physiology, diet and nutrition, and
galactose, which present in mammals milk and exist as epimer of glucose.

Disaccharide
The second category of carbohydrates is called disaccharides. It is formed when two
monosaccharides join together to make larger molecuels through condensation reaction
shown in the picture below to form glycosidic linkage or bond between carbon-oxygen-andcarbon between the two monosaccharides residues. They serve primarily as readily available
energy stores and different monosaccharides join together to form different disaccharides,
for example maltose, lactose and sucrose, shown in the table below.

MALTOSE
It is the least common disaccharide in
nature. Through condensation reaction, it is
connected by an -1-4 glycosdic linkage.

LACTOSE
Is a disaccharide sugar derived from
galactose and glucose. It is known as 4-O -D-galactosylpyranosyl-a-Dglucopyranoside. Lactose is only found in
milk. It is digested by the enzyme lactase.
Pure lactose is found in whey, the watery
byproduct of cheese production. And
similar to maltose it is also connected by a
1-4 glycosidic linkage.

SUCROSE or table sugar

Also known as table sugar. Both cyclic
carbons of glucose and fructose are tied
together in -1-2 glycosdic linkage.

Oligosaccharide
This third category of carbohydrates has a similar structural features as polysaccharide, it is
made up monomers of monosaccharide that joined together to form a polymer however with
smaller units (3 to 10 monosaccharide units) compared to polysaccharide. This type of
carbohydrates may be found in foods such as in beans and peas. It is usually undigested until

it reaches the large intestines triggering the release of hydrogen, carbon dioxide, and
methane gas. However oligosaccharide also plays an important role as it usually attached to
lipids and proteins converting them into glycolipids and glycoprotein that is particularly
important on plasma membrane from which they project.
Polysaccharide

Lastly the polysaccharides, this class of carbohydrates is made up of many, many sugars
(monossacharides as its monomers) hooked together to form a very large molecule.
Primarily there are three main types of polysachharides, they are glycogen, starch, and
cellulose. Glycogen is a polymer made up of -glucose which are joined by (1>4) &
(1>6) glycosidic linkage bonds, the presence of an (1>6) glycosidic linkage bonds
resulted as a branched part of the molecule. Glycogen plays an important role as a chemical
energy storehouse in most animals.

Starch, similar to glycogen is a polymer made up of -glucose, it exists as an energy storage

house in plants, and stored as densely packed granules (starch grain) found in membranebound plastids within plant cells. Like lactose, an enzyme called amylase is required to
break down starch. In terms of its structure, it is composed of two different polymers of
amylose and amylopectin. Amylose is an unbranched, helical molecule of sugars joined by
(1>4) glycosidic linkage whereas amylopectin is a branched polymer of -glucose with
similar structure as glycogen but with an irregular branching pattern.

Whereas cellulose, different from the starch and glycogen which is made up of polymer of
-glucose, this carbohydrates is made up of polymer of -glucose, which are joined by
(1>4) glycosidic linkage bonds. It is a major component of the plants cell wall and it is
exist as a long, unbranched polymer arranged in such a way to form molecular cables that
resist pulling (tensile) forces. Cellulose also plays an important role as a dietary fiber, which
is necessary to be consume by human as it aids digestion by absorbing water and pushing
food along the digestive tract, making bowel movements easier. However it is proven that
even though cellulose present as the most abundant organic material on Earth, most
multicellular organisms lack enzyme to degrade it, however this enzyme which is called as
cellulase is found at unicellular organisms such as fungi, bacteria, and protists.

Fermentation
Fermentation is primarily a process in which microorganisms such as yeast, in the case of
this investigation, breaks down organic substance like sugar into simpler substances. It
occurs in an anaerobic condition (oxygen is not present) and triggers the breakdown of sugar
molecule into ethanol and CO2. In the real life, fermentation has been playing an important
implication particularly in the food industry where fermentation using the action of
microorganisms is considered desirable and necessary to produce alcoholic beverages such
as wine, beer, and cider. The uses of this process is also important in the pastry where yeast
is used in the process of leavening of the bread, and for preservation techniques to create
lactic acid in sour foods such as sauerkraut, dry sausages, kimchi and yogurt, or vinegar
(acetic acid) for use in pickling foods (Wilder, B n.d.).

In the context of cellular respiration

The actual process where fermentation happen is actually found in the process of cellular
respiration. The first step in the cellular respiration is called glycolysis, where glucose is
broken down into pyruvate through series of steps and with the help of various kinds of
enzyme without the presence of oxygen. Once glycolysis has finished thus producing
pyruvate, then with the presence of the oxygen pyruvate can undergoes an aerobic
respiration through the TCA or Kerbs cycle producing a bigger amount of energy in the
form of ATP. However, if oxygen is still not present during the process, pyruvate can
undergoes an anaerobic respiration through fermentation pathway either forming lactate or
ethanol as its product.

Anaerobic respiration is an important process of respiration despite the fact that it only
produces lesser amount of ATP than aerobic respiration, because it is the sole source of ATP
for many anaerobic bacteria, and used by many eukaryotic cells when their oxygen supply is
low. Additionally the product of anaerobic respiration which is the coenzyme NAD+ from
NADH is considered crucial as it helps to replenish the limited supply of NAD+ during
glycolysis to proceed with its net yield of 2 ATP molecules per glucose and would cease
when its supply was exhausted, resulting in cell death. The anaerobic respiration itself is
divided into two kinds of fermentation pathway, they are lactic acid fermentation in
contracting muscle and alcoholic fermentation which is found in plants or microorganism,
for example fermentation that will be conducted by the help of yeast in this investigation.

Lactic Acid Fermentation

Lactic acid fermentation occurs by converting pyruvate into lactate using the help of
an enzyme called Lactate dehydrogenase. In this process the pyruvate accept
electrons and hydrogen from NADH thus producing NAD+ which is used to
replenish the supply NAD+ in the glycolysis process. It is responsible of the sour
taste in some food such as sauerkraut and yogurt and as a temporary energy source
for human muscle cells, however this process also triggers the buildup of lactate in
the muscle, which can damage the muscle tissue.

Alcoholic fermentation
Different from lactic acid fermentation, alcoholic fermentation is a two-step process.
Firstly, pyruvate is converted into acetaldehyde with the help of the enzyme called
Pyruvate decarboxylase and water releasing CO2. Then, after acetaldehyde has
formed, this compound accept electrons and hydrogen from the coenzyme NADH
which regenerates NAD+ which is used to replenish the supply NAD+ in the
glycolysis process, forming ethanol with the help of the enzyme Alcohol
dehydrogenase.

Yeast
Yeast (Saccharomyces cerevisiae) is a microscopic, unicellular organism that belongs to the
group of organisms called fungi. There are various kinds of yeast and some of them has
been playing an important role to humans. Some of the known uses of yeast are in the
production of bread, beer, cheese, wine, and whiskey, production of diet supplement as they
are rich in B vitamins. They are also used in genetic engineering to produce large quantities
of certain hormones and enzymes, which are used for such medical purposes as healing
wounds and reducing inflammation. Some types of yeast, however, cause disease
(HowStuffWorks 2013) such as candidiasis.

In terms of its structure, yeast cell is relatively bigger in size compared to the cell of a
bacterium however smaller than a plant cell. It is oval or round in shape and has a thin
membrane. Their cell walls consist of an outer layer of mannoprotein which are associated

with glucanes, an inner layer of glucannes associated with chitin, and a cytoplasmic
membrane with a high protein complex content. Yeast are eukaryotic organisms, therefore
its cell contains similar organelles with those eukaryotic cells in general. It contains nucleus
with 16 linear chromosomes, mitochondria as its powerhouse to generate ATP, ribosomes
(with the same size as plant and animal cells), however without the presence of chloroplasts.

Yeasts are known for its ability to undergoes fermentation process (an anaerobic respiration)
in which they secrete enzymes that break down food in the form of organic matter,
particularly sugars, into nutrients they can absorb, producing carbon dioxide and ethanol.
Those organic matters that can be fermented are fructose, glucose, and other
monosaccharide (simple sugars), which are found in most fruits. The enzymes found in the
yeast chemically break down these simple sugars into products that the cell can use,
however there are other species of yeast found in nature that can also chemically break
down disaccharides (double sugars) into its monossacharide units. The application of this
process is commonly used to turn fruit juices into wine and helps turn wort (diluted grain
mash) into beer or whiskey. The carbon dioxide produced by fermentation makes the
bubbles in beer and some kinds of wine, and causes bread to rise. As bread bakes, the
alcohol produced by fermentation evaporates (HowStuffWorks 2013).

Primarily in the process of fermentation, yeast uses various kinds of enzymes to help them
in breaking down the sugars. Biologically speaking, enzymes are substances that are used to
increase the rate of reaction without itself undergoing a chemical change by lowering the
overall activation energy of the reaction. Theenzymes that may be found in the yeast are
sucrase (invertase), zymase, maltase (glucase), lactase, hexosephosphatase, reductase,
carboxylase, melibiase, and endo-tryptase, as well as proteolytic enzymes. However it is
proven that not all kinds of yeast containing the same enzymes, instead each type of yeast
behave differently towards the various kinds of sugar. For example, most yeasts can invert,
and then ferment, cane-sugar, because the enzyme sucrase (invertase) is of common
occurrence in the yeasts. On the other hand, the enzyme lactase is absent from the majority
of yeasts, and hence these are incapable of fermenting milk-sugar.

Primarily in this investigation there are two different kinds of sugars that is used in the
fermentation process, firstly is starch (a polysaccharide sugar) and lactose (a disaccharide).
In this investigation, these two different sugars are expected to produce different amount of
product in the form of CO2 (carbon dioxide gas) during the process.

YEAST AND STARCH

Theoretically, yeast cannot effectively break down the starch even though it is composed of
units of sugar molecules which is simply polymer of a of -glucose. Therefore in the
production of fermented beverages like beer, the starch that is found in the raw material
must be broken down into its monosaccharide or disaccharides sugar units through series of
steps by the help of enzymes as yeast is unable to digest it unless it is broken down into its
glucose units. In terms of its structure, the starch itself exist as a mixture of two different
polymer, an unbranched chain form called amylose and a branched form called amylopectin,
and for yeast to ferment a starch, these two polymers must be broken down by a specific
enzyme called amylase or alpha and beta-amylase to be specific.
Amylase enzyme must be present in order to break down starch into its monomer. Alphaamylase catalyze the breakdown of amylopectin in starch into smaller chain of various
lengths, the smaller chain main contain a monosaccharide unit (glucose), a disaccharide
(maltose), or a larger molecules which contain many glucose units. Whereas beta-amylase
help catalyze the breakdown of amylose in starch into separate molecules of maltose, a
disaccharide sugar. Finally once the starch has been decomposed containing only
monosaccharide and disaccharide, the yeast produce the enzyme maltase to break the
maltose, formed by the breakdown formed with the help of beta-amylase, into simple sugars
(glucose) molecules that it can ferment producing alcohol and carbon dioxide.

YEAST AND LACTOSE

Primarily, lactose is unable to be fermented by yeast in fermentation process. It is due to the
fact that the enzyme used in the breakdown of lactose into its component, galactose and
glucose, is not common in yeast and absent in major species of yeast. Thus are only able to
breakdown lactose into its component when they are genetically engineered to produce
lactase. Primarily, lactase is an enzyme that has an ability to split lactose into its sugar
monomers. It is found in the intestine of young mammals, in plants, fungi, yeasts and bacteria.
Although this enzyme is commonly absent from the yeast, through the advance of geneticrelated technologies, a species of yeast has been genetically modified and biotechnology
created in such a way that they will have the ability to break down lactose which is called
Kluyveromyces lactis.
Kluyveromyces lactis or diary yeast is a species of a scientifically and biotechnologically nonSaccharomyces important yeast that may be found in milk-derived products.

Since the 1950s, K. lactis has been used as a source of lactase (beta-galactosidase), an
enzyme that degrades milk suga(lactose) and is necessary for production of lactose-free
dairy products. And these days, it is commonly used for genetic studies and in the food
industry due to is ability to break down lactose into its constituent sugar components.
Therefore yeast is unable to decompose lactose into simple sugar as the enzyme is
commonly absent in major species of yeast found commercially, whereas yeast still will be
able to break down starch into simple sugars even though it is not effective as breaking
monosaccharide (simple sugars) or disaccharide sugars (except lactose) it is due to the fact
that the polymer of starch which are amylose and amylopectin must be broken down into
shorter chain by various types of enzymes before it can undergoes fermentation by yeast.

III.

IV.
V.

Apparatus and Materials

Apparatus

Materials

Procedure
Data

VI.

Discussion

VII.

Conclusion

VIII.

References
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8
Cell Biology Laboratory Module