ABSTRACT

The study was conducted to determine the effect of the nature of

substrate on the rate of respiration. It was done using the Smith
fermentation tube method. 15 mL of separate solutions of fructose,
glucose, sucrose, lactose, starch and dH2O were placed in six
Smith fermentation tubes. Then, 15 mL of both dH2O and 10%
yeast suspension were added to each of the tube. After thorough
mixing of the solutions on each tube, the openings of the tubes
were plugged with cotton balls. The height of the carbon dioxide
(CO2) bubble formed in each tube was measured every five
minutes for 25 minutes. The volume of CO2 evolved in each tube
was computed to be used in getting the respective the respective
rates of respiration of the setups. The tube containing fructose, a
monosaccharide, as its substrate had the highest computed
respiration rate of . it was followed by the setups containing
glucose (monosaccharide; 0.52 cm3/min.) and sucrose
(disaccharide; 0.25 cm3/min.) as substrates. There was no rate of
respiration computed for the setups with the following solutions as
substrates because no evolution of CO2 gas happened inside the
tube within the given amount of time. Thus, the more complex the
substrate is, the slower the rate of respiration

INTRODUCTION

According to Campbell (1987), metabolism is the overall chemical process of an

organism. It is an emergent property of life that arises from specific interactions between

molecules within the cell. Metabolism is generally concerned with managing the material

and energy resources of the cell. Some pathways of metabolism break down complex

molecules to simpler compounds to release energy, called catabolic pathways. Cellular

respiration is a major passageway of catabolism, in which the sugar glucose and other

organic fuels are broken down to carbon dioxide and water.

 When you go snowboarding, take an aerobics class, or just sit around, ATP allow

the muscles in your body to contract. These ATP molecules are produced during cellular

respiration, a process that requires participation of mitochondria (Mader, 2001). By the

use of enzymes, phosphate groups from ATP are being transferred to other compounds,

which are then said to be phosphorylated.

In many chemical reactions, there happens a transfer of one or more electrons

from one reactant to another. These electron transfers are called oxidation-reduction

reaction, shortly-named redox reactions. During this reaction, the loss of electrons from

one substance is called oxidation, while the addition of electrons to another substance is

known as reduction (Campbell, 1987).

In cellular respiration, the complete breakdown of glucose arises in four stages.

First, 6-carbon glucose is broken in half which forms two molecules of pyruvate with

three carbon atoms each. In the second stage, the two pyruvate molecules each lose a

carbon atom by a release of carbon dioxide. In the citric acid cycle, the bonds of the

remaining 2-carbon molecules are broken and the single carbon atoms combine with

oxygen to form more carbon dioxide. Lastly, during oxidative phosphorylation, the

energy from the broken bonds of carbons in glucose is then transferred to ATP (Tobin

and Dusheck, 1998).

Most of the ATP generated by cellular respiration is the work of oxidative

phosphorylation. Because of this, without oxygen, oxidative phosphorylation ends.

However, fermentation provides a mechanism for cells to oxidize organic fuel and

generate ATP in the absence of oxygen (Campbell, 1987).

 Fermentation consists of glycolysis followed by the reduction of pyruvate by

NADPH to either lactate or alcohol and carbon dioxide (Mader, 2001). In lactate

fermentation, the pyruvate is converted into lactase. On the other hand, in alcohol

fermentation, pyruvate from glycolysis is broken down to acetaldehyde, which accepts

electrons from NADH and thereby becomes ethanol (Starr and Taggart, 1987). Single-

celled fungi, yeasts, are good examples of organisms that use this pathway and generate

alcohol and carbon dioxide. Yeasts are used to leaven bread. It is metabolically flexible

that it allows them to break down organic molecules through fermentation if oxygen is

absent and through aerobic respiration if oxygen is present. In the absence of oxygen,

yeasts ferment the energy sources and release carbon dioxide gas and ethyl alcohol as by

products (Postlethwait and Hopson, 1992).

An initial input of energy is required in most chemical reactions to start. That

energy that must be provided by the molecules in order to react is known as the energy of

activation. In laboratories, heat usually supplies the free energy of activation. However,

many different reactions are simultaneously going on in a cell, and heat would cause

harm to all of these reactions. In order to avoid this problem, cells use enzymes. Enzymes

are protein molecules specialized to serve as catalysts. A catalyst is a substance that

forms a temporary bond with the molecules reacting to lower the activation energy

required for a reaction. Substrate is the molecule on which an enzyme acts. This fits

perfectly on the site of the reactions catalyzed by the enzyme, active site (Raven, et. al.,

2005). Organic compounds such as carbohydrates, fats and proteins can be used as

substrates. However, carbohydrates are essentially used as substrates in yeast (Duka, et.
al., 2009). Carbohydrates are sugars and sugar derivatives and they differ in complexity

(Dickerson and Geis, 1976).

The hypothesis being tested in the study is in the statement: If the type of

substrate will affect the rate of cellular respiration, then, the more complex the substrate

is, the slower the rate of respiration. 15 mL of both distilled H2O and 10% yeast were

placed in six Smith fermentation tubes. Each tube was then added by 15mL of the

respective solutions: 1-fructose, 2-glucose, 3-sucrose, 4-lactose, 5-starch and 6-dH2O.

Within 25 minutes, the height of CO2 gas evolved inside each tube was measured and the

rate of respiration was then computed (See Results and Discussion).

In connection to the hypothesis stated, the study aimed to determine the effect of

the nature of substrate on the rate of respiration. The specific objectives were:

1. to determine which type of substrate will have a higher/lower rate of

respiration; and,

2. to describe the relationship between the nature of substrate and the rate of

respiration.

The study was conducted in the University of the Philippines Los Baños, Institute

of Biological Sciences, Room C-117 last November17, 2014 under the supervision of Dr.

Blesshe Querijero.
 MATERIALS AND METHODS

Smith fermentation tube method was used to determine the effect of the nature of

substrate on the rate of respiration in yeast. Six Smith fermentation tubes were obtained.

Smith fermentation tube is a special tube having a closed vertical arm extending into a

bulbous portion with tapered opening. 15mL of the following solutions were added to the

respective tubes: 1-fructose, 2-glucose, 3-sucrose, 4-lactose, 5-starch and 6-dH2O. All the

solutions were at 10% concentration. Then, 15mL dH2O and 15mL 10% yeast suspension

were added to each tube. It was noted that yeast should be the last to be added to the

tubes. Each tube were gently shaken to make sure that the solutions inside were

thoroughly mixed. Also, trapped bubbles were removed by covering the opening with the

palm of one hand and tilting the tube horizontally.

The openings of the tubes were immediately plugged with cotton balls and then

the tubes were tied together at their vertical arms to keep them upright. The tubes were

then set aside in an area where they will not be disturbed. The height of the area occupied

by the CO2 evolved, which were trapped in the vertical arm, were measured every five

minutes for 25 minutes using a 12-inch commercial ruler.

The volume of the gas evolved, CO2, was computed using the formula:

𝑉𝐶𝑂2 = 𝜋𝑟 2 ℎ

where: r is the radius of the inner opening of the Smith fermentation tube; and

h is the height of CO2 gas formed in every time interval.

 The rate of CO2 gas evolution or the rate of respiration in yeast in every tube was

then determined by the use of the given formula:

𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝐶𝑂2
𝑅𝑎𝑡𝑒 𝑜𝑓 𝑅𝑒𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛 = 𝑡𝑖𝑚𝑒 (min)

RESULTS AND DISCUSSION

Table 1 and Figure 1 show the height of carbon dioxide (CO2) bubble formed in

each of the tube after a given period of time. It was observed that after 25 minutes,

fructose had the highest height of CO2 formed of 7.1 cm. this was followed by glucose

with 6.5 cm, sucrose with 3.1 cm and lactose, starch and dH2O having all equal to 0cm.

The tube with dH2O as substrate served as the controlled setup of the experiment. In this

setup, respiration would not occur since there is no organic substance to be broken down

to generate energy. In the tube with starch as substrate, no respiration took place within

the given period of time. According to Campbell (1993), starch is a storage

polysaccharide of plants. It is a polymer consisting entirely of glucose. Thus, yeast or S.

cerevisiae must carry out the preliminary steps of breaking down the starches to glucose

first, since yeast cannot directly use these complex carbohydrates (Hopson and

Postlethwait, 1992). Lactose is a disaccharide, so as the sucrose. However, respiration did

not occur in the setup with lactose as substrate while respiration occurred on sucrose as

substrate. This is due to the absence of the enzyme lactase from the majority of the yeasts

making them incapable of fermenting milk-sugar (Simmonds, 1919).

Table 1. The height of carbon dioxide bubble formed inside the tubes of different

substrates within 25 minutes

Height of CO2 bubble formed (cm)

Time
Fructose Glucose Sucrose Lactose Starch dH2O
(min)
r=0.75 cm r=0.8 cm r=0.8 cm r=0.9 cm r=0.6 cm r=0.95 cm

0 0 0 0 0 0 0

5 1.5 0.6 0.2 0 0 0

10 2.3 1.6 0.7 0 0 0

15 3.7 2.9 1.4 0 0 0

20 5.2 4.4 2.2 0 0 0

25 7.1 6.5 3.1 0 0 0

In Table 2, the computed volume of CO2 evolved in each setup after 25 minutes

were shown. The tube with glucose as substrate had the highest volume of CO2 evolved

equal to 13.07cm3. It was followed by the tubes with fructose as substrate (12.55cm3) and

with sucrose as substrate (6.233cm3). The tubes with lactose, starch and dH2O as

substrates all have volume of CO2 evolved equal to 0cm3.

Table 2. The volume of CO2 evolved inside the tubes with different substrates after 25

minutes

Type of
Fructose Glucose Sucrose Lactose Starch dH2O
substrate

VCO2
12.55 13.07 6.233 0 0 0
(cm3)

Using the computed volumes of CO2, the rate of respiration of each tube was

determined as shown in Table 3 and figure 2. The tube with glucose as substrate had the

highest respiration rate equal to 0.52cm3/min. The tube with fructose as substrate

(0.51cm3/min) and the tube with sucrose as substrate (0.25cm3/min) have followed.

Table 3. The respiration rate of yeast with different substrates after 25 minutes

Type of
Fructose Glucose Sucrose Lactose Starch dH2O
Substrate

VCO2
12.55 13.07 6.233 0 0 0
3
(cm )

Rate of

Respiration 0.51 0.52 0.25 0 0 0

(cm3/min)
 Height (cm)

Type of Substrate

Figure 1. A bar graph showing the height of carbon dioxide bubble formed inside the

tubes of different substrates within 25 minutes.

 Rate of Respiration (cm3/min)

Type of Substrate

Figure 2. A bar graph showing the respiration rate of yeast with different substrates after

25 minutes.

Fructose and glucose are both monosaccharides that are considered most

important monomers of carbohydrate. They both share the molecular formula C 6H12O6;

however, different properties of these sugars emerge from their different arrangements of

atoms (Hopson and Postlethwait, 1992). Also, fructose and glucose differ in

monosaccharide classifications. Fructose is classified as a ketohexose, containing a

ketone functional group, while glucose is classified as an aldohexose, containing an

aldehyde functional group (Mcmurry, 2012). Based from Zubay, et. al. (1995), there is a

faster fermentation when fructose is used than glucose, though they are both

monosaccharides, because there is direct formation of fructose 6-phosphate in fructose.

Thus, organisms would not need to use phosphoglucose isomerase enzyme in order to

phosphorylate glucose 6-phosphate to fructose 6-phosphate. This would mean that less

time is needed to ferment fructose than glucose; therefore, fructose will have a faster rate

of respiration than glucose. However, the data have shown contradictions to these.

Sucrose is a disaccharide formed in combination of the monosaccharides fructose and

glucose (Hopson and Postlethwait, 1992). Accordingly, sucrose will have a lower rate of

respiration than the aforementioned monosaccharides because it needs to take both of the

monosaccharides’ processes in fermentation.

Based from the stated data above, the mixture with the complex type of substrate

will result to slower rate of respiration and the mixture with the simple type of substrate

will result to faster rate of respiration. This statement leads to verification of the

forenamed hypothesis, “If the type of substrate will affect the rate of cellular respiration,

then, the more complex the substrate is, the slower the rate of respiration.” However,

random errors possibly occurred during the conduct of the experiment that may have

affected the desirable outcomes to be further discussed in the summary and conclusion.
 SUMMARY AND CONCLUSION

The effect of the nature of substrate on the rate of respiration was determined by

the use of the Smith fermentation tube method. Six Smith fermentation tubes were all

placed by 15mL of both dH2O and 10% yeast. Six separate solutions of different types of

substrates were also added to the corresponding tubes: 1-fructose, 2-glucose, 3-sucrose,

4-lactose, 5-starch and 6-dH2O. The height of CO2 bubble formed in each tube within 25

minutes were measured in cm. the gathered data were used to calculate the rate of

respiration of the mixtures in every tube and was presented and analyzed carefully.

Results have shown that the setup with glucose, monosaccharide, as substrate had

the highest rate of respiration (13.07cm3/min) followed by the setup with fructose, a

monosaccharide, as substrate (12.55cm3/min) and setup with sucrose as substrate

(6.233cm3/min). It was also observed that the setups with lactose, starch and dH2O as

substrates have no evolution of CO2 gas within the given period of time. Thus, no rate of

respiration was calculated.

In conclusion, the complexity of the nature of a substrate is inversely related to

the rate of respiration. That means that the higher the complexity of the substrate, the

lower its rate of respiration becomes. However, in the experiment conducted, theoretical

results were not perfectly achieved. In this case, fructose should have a higher respiration

rate than glucose as explained in the results and discussion. The possible error happened

may have been the uncertainties in measurements and usage of devices by the

researchers. Thus, further experimentation is required for validation of the study.

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