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The Effect of Nature of Substrate on the Rate of Respiration on Yeast

(Saccharomyces cereviciae)¹

Rhoda Ecila Mae Cortes

Group 1 Sec. UV-5L

November 11, 2015

_____________________
¹A scientific paper submitted in partial fulfilment of the requirement in General
Biology I Laboratory under Mr. Jickerson P. Lado, 1st sem., 2015-2016.
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ABSTRACT

The effect of the nature of substrate on the rate of respiration of yeast was
determined using the Smith fermentation tube method. In six fermentation
tubes, 15 mL of both distilled water (H₂O) and 10% yeast solution was
poured. Then, 15 mL separate solution of glucose, fructose, sucrose,
lactose, starch, and distilled H₂O was then poured in tubes labelled 1 to 6,
respectively, and the tube opening was plugged with cotton ball. The
height of the area occupied by the carbon dioxide (CO₂) evolved for each
tube was measured using a 30 cm ruler every three minutes for 30
minutes. The volume of CO₂ evolved in each tube was computed, which
was used for computing the rate of respiration in each tube. Tube 3, with
sucrose substrate had the highest respiration rate of 0.23cm³/min. This was
followed by tube 2, fructose substrate with a respiration rate of
0.20cm³/min and by tube 1, with glucose substrate, with a respiration rate
of 0.17cm³/min. These showed that as time increased, the respiration rate
of yeast also increased until it will go down to zero depending on the
amount of substrate used. There was no respiration rate computed for
tubes 4- 6 with starch, lactose, and distilled H2O, respectively because no
CO₂ had evolved. Thus, the simpler the substrate or sugar, the faster the
rate of cellular respiration in yeast.
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INTRODUCTION

Cellular respiration is the breaking down of organic molecules by the catabolic

pathways of aerobic and anaerobic respiration which produces ATP by the use of an

electron transport chain. These are being done by the transfer of electrons during

chemical reactions or redox reaction, which releases energy stored in organic molecules

for ATP synthesis. (Reece, Urry, Cain, Wasserman, Minorsky and Jackson, 2011).

Anaerobic respiration (fermentation) is the breakdown of sugars by bacteria and

yeast using a method of respiration without oxygen, has two types. The first type is the

lactic acid fermentation in which glucose molecules are converted to lactate catalysed by

lactate dehydrogenase (Campbell and Farrel, 2012). The second type is the alcohol

fermentation in which pyruvate molecules are converted to ethanol in two steps. The first

step involves the release of carbon dioxide by the pyruvate which is converted to

acetaldehyde. At the next step, NADH reduces acetaldehyde to ethanol (Campbell, Reece

and Mitchell, 2011). It involves a culture of yeast and a solution of sugar, producing

ethanol and carbon dioxide with the aid of the enzymes. The alcohol produced has been

used in making wines and beers and the carbon dioxide produced has been used in baking

as it gets trapped in the dough and causes it to rise (Ambrose, 1987).

Some organisms, called obligate anaerobes, carry out only fermentation or

anaerobic respiration. In fact, these organisms cannot survive in the presence of oxygen,
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some forms of which can actually be toxic if protective systems are not present in the

cell. A few cell types, such as cells of the vertebrate brain, can carry out only aerobic

oxidation of pyruvate, not fermentation. Other organisms, including yeasts and many

bacteria, can make enough ATP to survive using either fermentation or respiration. Such

species are called facultative anaerobes (Campbell, Reece and Mitchell, 2011).

Yeast is unicellular fungi characterized by a wide dispersion of natural habitats.

The true yeasts are separated into one main order Saccharomycetales, “saccharomyces”

means `sugar fungi` are made up mostly of protein. Yeast feeds by heterotrophic nutrition

because they lack chlorophyll and therefore are non-photosynthetic. They are widespread

in nature, found in the soil and on plants, commonly found wherever sugar occurs for it

has been used for thousands of years to ferment sugar into ethanol. (Schneiter, 2004)

Yeasts undergo glycolysis in cellular respiration which occurs in the cytosol.

Glycolysis is the breakdown of one molecule of glucose into two molecules of pyruvate,

an organic compound with a backbone of three carbon atoms (Starr and Taggart, 2004).

However factors, such as enzymes which catalyze the reaction and the nature of the

substrate might affect the rate of respiration.

Enzymes are catalysts that speed up reactions; they are made from protein and are

specific as to which substrate they work on (Campbell, Reece and Mitchell, 2011). For

example a zymase-complex enzyme will only bind with a glucose molecule to produce

the ferments carbon dioxide and alcohol. Thus, enzymes must be present for organisms to

respire the specific substrate.

Sugars can be commonly classified into three. The first is monosaccharide (simple

sugar) which is the building block of all carbohydrates. Second is the disaccharide which
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is formed when two monosaccharides are linked by glycosidic bond. The last, being the

polysaccharides, which is formed by the linking together of many monosaccharides

(Campbell, Reece and Mitchell, 2011).

In the method consisting two test tubes: Test tube A and B both contained 4 ml

glucose and 4 ml of 10% yeast using a graduated cylinder. 4 ml distilled water which is

the negative control and a co-factor 4 ml of 0.2M (MgSO4) was placed on Test tube A

and B respectively. This makes them suitable for the application of our hypothesis that

the co-factor or the nature of substrates affects the rate of cellular respiration in yeast.

In method Smith Fermentation Tube Method, a special tube with closed vertical

arm extending towards a bulbous portion with tapered opening. It was the experimental

design regarding the effect of different substrates on the rate of cellular respiration in

yeast. Six fermentation tubes were used, each contained15 ml distilled water and 15 ml of

10% yeast suspension. To test the hypothesis, 5 ml at 10 % solutions different substrates

– glucose, fructose, sucrose, lactose, starch and distilled water which is the negative

control in the set-up were subjected to each tube, 1-6 respectively and shook gently. To

remove the trapped bubbles, the opening was covered with the palm of one head and

tilted horizontally. Openings were plugged with cotton balls as all CO₂ evolved will be

trapped in the vertical arm. Tubes were tied together at the vertical arms to keep them

upright. The height of the area occupied by the CO₂ evolved was measured every three

minutes for thirty minutes.

The hypothesis “If the co-factors are needed to speed up metabolic processes then

the presence of a co-factor will increase the rate of cellular respiration in yeast”, more

CO₂ evolved over time and “if the nature of the substrates affect the rate of cellular
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respiration in yeast then the simpler the substrate, the faster the rate of cellular respiration

in yeast” was tested using Durham tube method and Smith Fermentation Tube Method.

This study aimed to determine the effect of co-factor (MgSO4) and different

nature of substrates used (starch, lactose, sucrose, glucose, and fructose) on the rate of

cellular respiration in yeast. The objectives were:

1. To expound the effect of co-factor and different nature of substrates used on

the rate of cellular respiration in yeast ; and

2. To identify the possible factors that could affect the cellular respiration in

yeast.

The study was conducted at the Institute of Biological Sciences Laboratory,

University of the Philippines Los Baños, Laguna, Philippines on November 4, 2015.

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MATERIALS AND METHODS

In identifying the effect of nature of substrates on the rate of cellular respiration in

yeast, two methods were performed.

I. Durham Tube Method

It was an experimental design regarding the effect of a co-factor on the rate of

cellular respiration. Two test tubes were used: Test tube A and B both contained 4 ml

glucose and 4 ml of 10% yeast using a graduated cylinder. However, to test the 1st

hypothesis, 4 ml distilled water which is the negative control and a cofactor 4 ml of 0.2M

MgSO₄ was placed on Test tube A and B respectively. An inverted Durham Tube was

slide down into each test tube. To remove air bubbles in the inverted Durham Tube, the

opening of the bigger test tube was covered tightly with the palm of one hand and let the

bubbles to escape from the Durham Tube by tilting the bigger tube gently from side to

side. Afterwards, a cotton ball was placed in the opening as shown in Figure 1.
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The height of the area occupied by the gas (in cm) at the bottom of the inverted

Durham Tube was measured at a three-minute interval for thirty minutes.

II. Smith Fermentation Tube Method

In this method, a special tube with closed vertical arm extending towards a

bulbous portion with tapered opening. It was the experimental design regarding the effect

of different substrates on the rate of cellular respiration in yeast. Six fermentation tubes

were used, each contained15 ml distilled water and 15 ml of 10% yeast suspension. To

test the second hypothesis, 5 ml at 10 % solutions different substrates – glucose, fructose,

sucrose, lactose, starch and distilled water which is the negative control in the set-up were

subjected to each tube, 1-6 respectively and shook gently. To remove the trapped

bubbles, the opening was covered with the palm of one head and tilted horizontally.

Openings were plugged with cotton balls as all CO₂ evolved will be trapped in the

vertical arm. Tubes were tied together at the vertical arms to keep them upright as shown

in Figure 2.

Figure 2. Smith Fermentation Tube Method

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The height of the area occupied by the CO₂ evolved was measured every three

minutes for thirty minutes. The volume of CO₂ evolved for both Durham tube method

and Smith fermentation tube method were calculated using the formula:

VCO2=

where: h = height of are occupied by the bubbles.

r = radius of the Durham or Smith Fermentation Tube Method given that

radius in Durham tube method is 0.4 cm and in Smith Fermentation tube method is 0.8

cm.

While the rate of CO₂ production was also computed by the dividing the final

volume of CO₂ evolved by time,

Rate of Respiration = Final Volume of CO2

30 minutes
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RESULTS AND DISCUSSION

The rate at which carbon dioxide is produced can be used to measure the rate of

anaerobic respiration of yeast because carbon dioxide gas accumulates as a waste product

of fermentation in yeast and cellular respiration in many kinds of cells. Fermentation

releases two molecules of the gas from the anaerobic (not requiring molecular oxygen)

degradation of a substrate, usually glucose, as well as two molecules of ethanol plus

usable energy for cell function. More energy is released by cellular respiration than by

fermentation because glucose is completely oxidized in the process. Thus, carbon dioxide

is a waste product of the energy-releasing mechanisms of the cell. Logically, then, carbon

dioxide is an indicator of the rate of substrate degradation in an organism. More carbon

dioxide will be released from an organism as the rate of fermentation or cellular

respiration increases.

In Durham tube method, results have shown that test tube B with a co-factor

containing MgSO4 obtained a larger volume or amount of CO₂ evolved than test tube A

containing the negative control which is distilled water having a final volume of 2.6 cm³

and 0.7 cm³ respectively.

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5
Height of
gas formed 4
(cm) dH₂O
3 MgSO₄

0
3 6 9 12 15 18 21 24 27 30
Time elapsed (minutes)

Figure 1. A line graph showing the height (cm) of the area occupied by the carbon
dioxide (CO₂) that had evolved from each Durham tube every three minutes for 30
minutes which affects the rate of cellular respiration in yeast.

In Table 1 and Figure 2, the height, in cm, of the area occupied by the carbon

dioxide (CO₂) that had evolved from each Smith fermentation tube every three minutes

for 30 minutes. Results revealed that after 30 minutes, tube 3, with sucrose as its

substrate, had the highest height of CO₂ evolved which is 3.5 cm. This was followed by

tube 2, with fructose as its substrate, wherein the CO₂ evolved with a height of 3 cm. The

next was tube 1, with glucose as its substrate, which had a 2.5 cm CO₂ evolved. There

was no CO₂ evolved in tubes 4 and 5 with lactose and starch as its substrate, respectively.

This non-evolution of CO₂ indicated that no respiration occurred in these tubes. Tube 6,

with the distilled H₂O substrate, served as the negative control in the setup of the
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experiment. Since distilled H₂O is not a sugar/substrate, no respiration will occur.

Height of gas formed (cm)

Time
Distilled
elapsed Glucose Fructose Sucrose Lactose Starch
H₂o
(minutes)

3 0 0 0 0 0 0
6 0 0 0 0 0 0
9 0 0 0 0 0 0
12 0.5 0 0 0 0 0
15 0.5 0 0 0 0 0
18 0.8 1 0 0 0 0
21 1.1 2 0 0 0 0
24 1.5 2 0 0 0 0
27 2.1 2 0 0 0 0
30 2.5 3 3.5 0 0 0
Change in
2.5 3 3.5 0 0 0
Height (cm)

Table 1. The height of gas formed on the solution of yeast and distilled water with different
substrates – glucose, fructose, sucrose, lactose, starch, and the negative control
which is water.

Figure 1. Substrates in Smith Fermentation Tube Method

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Using the formula, VCO2= , the computed volume of CO₂ for tubes 1, 2 and 3

(glucose, fructose, sucrose) were 5.02 cm³, 6.03 cm³, and 7.03 cm³, respectively. With

these volumes, the rate of respiration for each tube was computed using the formula

Rate of Respiration = Final Volume of CO2

30 minutes

5
Volume of
CO₂ evolved 4
(cm³)
3

0
Glucose Fructose Sucrose Lactose Starch dH2O
Nature of Substrates

Figure 2. A bar graph showing the volume (cm³) of the area occupied by the carbon
dioxide (CO₂) that had evolved from each Smith fermentation tube which affects the rate
of cellular respiration in yeast.
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As shown in figure 3, the tube with the sucrose substrate, had the highest rate of respiration of

0.23 cm³/min. This was followed by tube 2, with the fructose substrate, having a 0.20 cm³/min.

Then, tube 1, with the glucose substrate, had a 0.17 cm³/min rate of respiration.

3.5

3
Glucose
2.5
Height of Fructose
gas formed 2
(cm) Sucrose
1.5
Lactose
1 Starch

0.5 dH2O

0
3 6 9 12 15 18 21 24 27 30
Time elapsed (minutes)

Figure 3. A line graph showing the height (cm) of the area occupied by the carbon
dioxide (CO₂) that had evolved from each Smith fermentation tube every three minutes
for 30 minutes which affects the rate of cellular respiration in yeast.

Glucose and fructose are both monosaccharides. They have the same number and

kind of atoms but their structure are different. Fructose is a structural isomer of glucose

(Campbell, Reece and Mitchell, 2011). They also differ in their monosaccharide

classifications. Glucose is classified as an aldohexose, which has an aldehyde functional

group, while fructose is classified as a ketohexose, which has a ketone functional group

(Mcmurry, 2012). Thus, the reason behind why fructose obtained the highest rate is that
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fructose had a head start on becoming carbon dioxide over glucose and sucrose.

Theoretically, fructose will be fermented faster by yeast compared to glucose.

Many sugars are fermentable compounds. Monosaccharides combine by a

condensation reaction, and they can be turned back into their single units by hydration.

Reducing sugars such as glucose and fructose are monosaccharides and can be readily

oxidised. When this reaction occurs the sugars lose electrons to another substance, which

is said to be reduced. Some disaccharides are not easily oxidised, notably sucrose- made

up of fructose and glucose, these types of sugars are non-reducing. So in the case of

sucrose, yeast cells produce the enzyme sucrase, it becomes easier to oxidise thus sucrose

is a good substrate for fermentation (Zubay, Parson and Vance, 1995). Thus, sucrose is a

disaccharide composed of 1 equivalent of glucose and 1 equivalent of fructose

(McMurry, 2012). It can be broken down into its simpler components by catalyzing with

the sucrose enzyme (Reece, Urry, Cain, Wasserman, Minorsky and Jackson, 2011). Thus,

the simpler the sugar, the higher the rate of respiration of yeast. Therefore, glucose will

be fermented faster by yeast compared to sucrose.

While fructose and glucose are abundant in plants, Lactose, a dissacharide made

up of glucose and galactose linked together, is found predominantly in milk. That link is

both strong and the enzyme is not in common baker’s yeast. Even some humans lack the

enzyme (lactase) needed to break lactose when they become adults, plus its’ structure is

far too complicated do so for it is a big molecule. (Zubay, Parson and Vance, 1995). And

because the yeast can't break lactose and starch due to its lack of the enzyme to turn them

into digestible pieces and its complex structure, there will be no carbon dioxide produced.
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Among all the substrates used is starch which is a polysaccharide. Unfortunately,

yeast does naturally produce amylase, but it takes for a long time to break down a

quantity of starch. According to the Baking Industry Research Trust, there are many

glucose units joining together in starch that yeast cannot ferment it unless starch is broken

down into its glucose units that involve several enzymes, two of which are the alpha and

beta amylase, which meant that fermentation of starch will be slow.

Based on the data and graphs shown from the experiment done, the information did

not satisfy the hypothesis “If the nature of substrates affect the rate of cellular respiration

in yeast then the simpler the substrate, the faster the rate of cellular respiration yeast.”

The tabulated calculations were inconsistent. The experiment showed a different result

from the expected outcome. This might be due to some human error made by the groups.

1. The cotton balls should be plugged immediately in the openings of Smith

fermentation tube.

2. The holding of tubes should be avoided as the heat generated by the hands can

have an effect on respiration.

3. The experiment should be performed again in a more isolated place and with

higher precautionary measures using upgraded laboratory apparatus.

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SUMMARY AND CONCLUSION

The effect of the nature of substrate on the rate of respiration of yeast was

determined. In six fermentation tubes, solution with 15 mL of both distilled water and

10% yeast was poured. Then, 15 mL of separate solution of glucose, fructose, sucrose,

lactose, starch and distilled water (negative control) were added to their respective tubes.

The height (cm) of the area occupied by the carbon dioxide (CO2) that had evolved in

each tube was measured and recorded every three minutes for 30 minutes.

Results revealed that after 30 minutes, tube 3, with sucrose as its substrate,

had the highest height of CO₂ evolved which is 3.5 cm. This was followed by tube 2, with

fructose as its substrate, wherein the CO₂ evolved with a height of 3 cm. The next was

tube 1, with glucose as its substrate, which had a 2.5 cm CO₂ evolved. There was no CO₂

evolved in tubes 4 and 5 with lactose and starch as its substrate, respectively. This non-

evolution of CO₂ indicated that no respiration occurred in these tubes. Tube 6, with the

distilled H₂O substrate, served as the negative control in the setup of the experiment

done.

The computed volume of CO2 for tubes 1, 2 and 3 were 5.02 cm³, 6.03cm³

and 7.03 cm³, respectively. These computed volumes were used to compute the rate of

respiration in yeast. The tube with the sucrose substrate had the highest rate of

respiration of 0.23 cm³/min. This was followed by tube 2, with the fructose substrate,

having a 0.20 cm³/min. Then, tube 1, with the glucose substrate, had a 0.17 cm³/min rate

of respiration.
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Shown from the experiment done, there will be no conclusions can be derived

from this experiment because results were inconsistent due to some sources of error like

human errors. It is recommended that the experiment should be performed again in a

more isolated place and with higher precautionary measures using upgraded laboratory

apparatus.

Theoretically, yeast with fructose as its substrate will have the highest rate of

respiration, followed by glucose as the substrate, and by sucrose as the substrate. Thus,

the simpler the substrate, the higher the rate of cellular respiration in yeast.
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Accessed November 8,2015.

Campbell, M.K., S.O. Farrell. 2012. Biochemistry. 7th ed. USA: Cengage Brooks/Cole.
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Schneiter, R. 2004. Genetics, Molecular and Cell Biology of Yeast. University of

Freiburg, Schweiz. p. 4, 5.

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Thomson Brooks/Cole. p. 134.

Zubay, G.L., W.W., Parson, D.E. Vance.1995. Principles of Biochemistry. USA: W.C.
Brown, Inc. pp. 315-320
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