 Biology SL Sample Internal Assessment

Table of content
Rationale
Research question
Background information
Procedure
Scientific justification
Statistical analysis
Test of hypothesis
Conclusion
Evaluation
References

# Effect of temperature on the rate of respiration of yeast in aerobic conditions

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## Rationale

Since my childhood, I have been extremely fond of baked foods. The appetite for breads, cakes and other bakery foods has intrigued me to learn a little bit of the ways to prepare them. During my baking classes from my mother, who is an expert in this I was introduced to the fact that a certain microorganism- yeast is an unavoidable ingredient for this and my mistakes while baking a cake taught me that the temperature at which baking is done and amount of yeast added plays a major role in controlling the volume of the cake as well as its flavor. Further knowledge during my Biology classes at IB Diploma Program especially in the sub-topic enzymes, my concept in this arena became a little clearer. This exploration and a childhood interest coupled with my hobby of baking cakes has finally led me to select the topic of effect of temperature on rate of respiration of yeast in aerobic conditions as my topic of investigation for my Biology Internal Assessment.

## Research question

How does the rate of aerobic respiration in yeast depends on the temperature at which it is carried out, determined using carbon dioxide gas sensor?

## Background information

### Respiration in yeast

Yeast can respire in both aerobic (in presence of oxygen) and anaerobic (absence of oxygen) pathways. figure 1 - Respiration In Yeast Figure 2 - The overall equations for aerobic and anaerobic respiration are

### Enzymes involved in aerobic respiration

Aerobic respiration is an enzyme controlled process. It occurs in two parts- Glycolysis and Kreb’s cycle and each step in this process are controlled by enzymes. Figure 3 - Schematic Representation Of Kreb’s Cycle And Glycolysis

The enzymes used in this experiment is a multi enzyme system. The process is divided into two separate cycles- glycolysis and Kreb’s cycle. All steps in each of the cycle are catalysed by specific enzymes. Glycolysis converts glucose into pyruvate and then into Acetyl CoA using the enzyme pyruvate dehydrogenase.   The aerobic respiration occurs in several steps; catalysed by specific enzyme from the system in each step. The functioning of this enzyme depends on physical conditions like pH, temperature. Beyond a temperature (optimum temperature) the enzyme gets denatured and loses its shape. Hence as per the Lock and Key mechanism, it cannot function any more. The production of carbon dioxide reaches a constant volume and the rate of respiration becomes a limiting factor.

### Hypothesis Figure 4 - Literature Curve For The Variation Of Rate Of Respiration With Temperature

Null hypothesis: The variables rate of respiration in aerobic mode and the temperature has no correlation.

Alternate hypothesis: Both the variables rate of aerobic respiration and temperature have significant level of correlation.

### Variables

Type of variable
Variable
Method of measure
Apparatus used
Independent
Temperature at which ferementation is carried out – 0°C to 40°C at an interval of 10°C.
The suspension of yeast and sucrose was kept in an ice bath or heated in water bath to vary the temperature.
Thermometer.
Dependent
Quantity of carbon dioxide produced

A CO2 gas sensor was introduced in the yeast suspension.

CO2 gas sensor

Figure 5 - Table On Variables

### Controlled variables

Variable
Why is it controlled?
How is it controlled?
Apparatus used
Ratio of yeast solution and sucrose solution
The quantity of microorganism added per unit mass of the substrate (sucrose) will affect the volume of carbon dioxide produced.
The ratio was maintained at 1:1 for all trials.
Measuring cylinder.
Concentration of sucrose solution
Higher the concentration of sucrose solution, greater the volume of carbon dioxide produced.
Same sucrose solution was used for all trials.
Time of immersion of gas sensor
The volume of carbon dioxide as measured by the gas sensor will depend on the time of contact between the probe and the reaction mixture.
For all trials, the gas sensor was immersed for 3 minutes.
Stop-watch
Apparatus used
Since there are differences in calibration use of different apparatus for the same purpose introduces random error.
Same apparatus was used wherever possible.
Figure 6 - Table On Controlled Variables

### Materials and apparatus required

Materials/ Apparatus
Quantity
Least count
Uncertainty
Sucrose
10 g
NA
NA
Yeast solution

50 cm3

NA
NA

CO2 gas sensor

1
0.01
±0.01 ppm
Digital mass balance
1
0.001
±0.001 g
Stop-watch
1
0.01
± 0.01 s
Thermometer
1
0.1
±0.05 °C
Reaction vessel ( A jar with a single opening in the lid to insert the gas sensor)
1
NA
NA
Measuring cylinder
1
0.1

±0.05 cm3

100 cm3 beaker

1
40

±0.20 cm3

Figure 7 - Table On Materials And Apparatus Required

NA= Not Applicable

## Procedure

### Preparation of 100 cm3 of 0.1 moldm-3 sucrose solution

• Weigh 3.42 g ( 0.01 moles) of  crystals of sucrose using a watch glass and digital mass balance.
• The weighed crystal was transferred into a 100 cm3 volumetric flask.
• Distilled water was added up to the mark.
• The sucrose was allowed to dissolve.

### Primary procedure

• 20 cm3  of yeast solution, was taken using the measuring cylinder from a beaker containing it  and transfer it to the jar.
• 20 cm3 of the sucrose solution was taken using a measuring cylinder, and transferred to the same jar, thus creating the ratio 1:1
• The jar was kept in the freezer to set the temperature of the solution at O°C.
• The gas sensor probe was inserted into the solution and timed for 3 minutes using a stop-watch.
• The same procedure was repeated for other temperatures created using the Bunsen burner.

### Safety Precautions

• Protective clothing like gloves, lab coats were used.
• None of the chemicals were ingested or exposed to skin.
• The yeast solution was handled carefully.

### Environmental concerns

• All waste liquids were disposed off safely into the waste bin so that the environment is not harmed.
• Emission of carbon dioxide was within a closed vessel which did not escape into the atmosphere and thus do not harm it.

### Ethical considerations

• Minimum amount of chemicals were used.
• Minimum amount of microorganism yeast was used.

## Data collection Figure 8 - Table On Change in levels of CO2 at various temperature Figure 9 - Table on Calculation of mean, median, mode and standard deviation

Sample calculations:

Calculation of mean value = (24.66 + 81.66 + 142.00 + 172.66 + 183.33) /5 = 120.86

Median value = Value in the middle position = 120.86

### Graph Figure 10

### Discussion of the graph

The graph plots the rate of aerobic respiration along y axes as it is the dependent variable and temperature in degree Celsius along x axes as it is the independent variable.

The graph obeys a polynomial trend line obeying the equation:

y = -0.262 x2 + 22.37 x + 65.17

y = rate of respiration.

x = temperature at which respiration is carried out.

At the maximum value of  y, $$\frac{dy}{dx}$$ = 0

y  =  - 0.262 x2  +  22.37  x  +  65.17

$$\frac{dy}{dx}$$ =  -  (2 X 0.262)x + 22.37 = 0

-0.524 x  +  22.37 = 0

x  = $$\frac{22.37}{0.524}$$  =  42.69

It means that at a temperature of 42.69 °C, the rate of respiration of yeast in the sucrose solution attains maximum value. Hence this temperature (42.69°C) may be considered as the optimum temperature of the enzyme controlled reaction involved. As quite evident from the graph and the discussion done above, the increase in the value of the level of CO2 from 0°C to 30°C is uniform and linear. The graph reaches maxima at 42.69°C and levels out beyond it.

The correlation factor of the graph plotted is found to be 0.995 which clearly indicates a strong positive correlation between the rate of respiration and the temperature at which fermentation is carried out.

## Scientific justification

Initially, the rate of respiration increases, the level of CO2 produced increases with the increase of temperature. With the rise of temperature, the average kinetic energy of the reactant molecules increases. More number of substrate molecules collide with the enzyme and leads to the formation of products. At the maxima (42.690C) , the activity of the enzyme reaches the maximum value. Figure 11

Beyond this temperature, the structure of the enzyme gets disrupted. The tertiary structure of protein gets distorted due to breaking of the intermolecular hydrogen bonds in them. The enzyme is no longer able to bind to the substrate. Fermentation is absolutely an enzyme catalysed reaction. As the enzyme does not bind with the substrate, enzyme –substrate complex are not formed and as a result the products are also not generated. The temperature becomes a limiting factor and hence the rate of respiration and thus the level of carbon dioxide levels out; turning out the graph as parallel to x axis. The change in shape of the active sites makes the substrate and enzyme non complementary and inhibit them for binding each other.

## Statistical analysis

### Chi-square test Figure 12 - Table On Chi-Square Test

## Test of hypothesis

As there are 5 variables selected,

the number of degrees of freedom = Number of variables – 1 = (5-1) = 4

Value of p = 93.36 % (as calculated from the chi square distribution table using the chi square statistic)

This high value of p clearly indicates that the null hypothesis cannot be rejected.

### T Test

The value of t as calculated using an online calculator is 0 while the p value is 1. As the p value is > 0.5, the results are considered to be significant.

Significance value= 0.05

Hypothesis : Two tailed Figure 13 - Table On T Test

### Calculation of different scores

Parameter
Treatment 1
Parameter
Treatment 2

N1

5

N2

5

Df1

5-1 = 4

Df2

5-1 = 4

M1

362.6

M2

362.6

SS1

0

SS2

160407.2

S21

0

S22

40101.8
Figure 14 - Calculation of different scores

T-value calculation:

s2p = (( df1 / (df1 +df2 ) ) – s21 ) + ( ( df2 /(df2 + df2 ) –s22 )

= ( ( 4/8) -0 ) + ( (4/8 ) – 40101.8 ) = 20050.9

s2M1 = s2p/N1 = 20050.9 /5 = 4010.18

s2M2 = s2p/N2 = 20050.9/5 = 4010.18

T= ( M1-M2 ) / ( s2M1 + s2 M2 )1/2  = 0 /  ( 8020.36 ) ½ = 0

### Annova test Figure 15 - Table On Annova Test

The f ratio value is 0.04644

The p value is 0.995592

Hence the result is not significant as p < 0.05

## Conclusion

The main aim of the investigation was to answer the research question:

How do the rate of respiration of yeast at aerobic conditions depends on the temperature at which it is carried out?

As clearly supported and evident from the data collected, the rate of aerobic respiration in yeast increases as an enzyme increases with the increase of temperature until 49.62 °C. Beyond this, the enzyme gets denatured, the changes in tertiary structure of the enzyme deforms the active site of the enzyme. The enzyme and the active site becomes non complementary to each other. The enzyme is no longer able to bind with the substrate. The reaction is inhibited, production of carbon dioxide ceases down, rate of respiration becomes the limiting factor.  The curve becomes limiting.

The discussion above is also supported coherently by the data collected. With the rise of temperature from O°C to 30°C the level of carbon dioxide increases rapidly from 74 ppm to 426 ppm but beyond a temperature of 300C, the level becomes constant in the range of 510-560 ppm.As already determined from the equation of polynomial trend line, the optimum temperature for the enzyme to work is 49.62°C. The activity of the enzyme is deformed or lost above this temperature.

As indicated from the results of the chi square test, the null hypothesis cannot be neglected. So the correlation between the temperature and the level of carbon dioxide produced by fermentation cannot be stated very significantly and evidently. Huge percentage error might have attributed to these high value of chi square statistic.

As indicated by the t test result as per two trail hypothesis method and taking significance level as 0.5, the results obtained are significant and indicates the acceptance of alternate hypothesis.

Overall, based on the statistical analysis and evaluation of the graph as my processed data, I conclude a weak positive correlation between the level of carbon dioxide produced and temperature at which sucrose is fermented.

## Evaluation

### Strength

• Sufficient data has been collected and repeated readings have been taken to reduce random errors.
• The range of temperature selected were in the normal range (OOC to 400C). As reported in literature reports, the optimum temperature for this process is 37.2 °C
• Statistical analysis has been done extensively to derive a conclusion about which hypothesis to be accepted

### Weakness

Weakness
How does it affect?
Suggestion of improvements
Inaccuracy of the apparatus
Contributes to random error of the experimentally collected data and makes it less precise.
Use of apparatus with higher precision. The glass apparatus used must be properly calibrated. More accurate debvices like burette can be used instead of measuring cylinder for the same purpose.
pH of the yeast-sucrose suspension
The activity of enzyme depends on pH of the medium. Enzyme shows maximum activity at an optimum pH.
The pH of the yeast-suspension mixture must be adjusted to the optimum pH by using a standard buffer solution.
Production of ethanol
Ethanol is a by-product of the fermentation process. As the concentration of ethanol increases beyond a limit, it becomes toxic for the yeast and they cannot survive any more. So the metabolic functions in yeast leading to production of zymase stops which hinders the process.
The substrate concentration must be selected in such a way so that the amount of ethanol secreted from fermentation does not become toxic and inhibits the metabolism in the cells of yeast.
Lack of oxygen
As the experiment is conducted in an anaerobic environment, there is no supply of oxygen which interferes with the growth of the yeast. The basic metabolism inside the cells of the microorganism is disturbed which finally limits the secretion of the enzyme zymase.
As fermentation can also occur in presence of limited supply of oxygen, it is not essential to ensure that the environment is completely anaerobic.
Figure 16 - Table On Weakness

### Limitations

• Although fermentation is ideally performed in an anaerobic condition, this experiment was not done in that way.
• Measure to control or adjust the pH of the medium to the optimum level for the enzyme to work best was not done.
• The gas sensor used to determine the level of carbon dioxide has a zero error and issues with calibration as well.

### Determination of percentage error

Fermentation is ideally carried out at a temperature of 32-35°C3. Considering 34°C as an

optimum temperature in this case, the percentage error is calculated.

Optimum temperature as calculated from the graphical method = 49.62°C

Percentage error = [( Literature value Experimental value ) / Literature value ] X 100

= [( 49.62 – 34) /34]  X 100 = 45.94

The percentage error of the experiment was found to be 45.94 %

### Further extension

Sugar molecules other than sucrose especially starch may be used.

Amount of ethanol may be determined as this reaction is mostly used in wine making process.

Variation of pH can be done to study the effect of pH on the rate of the reaction and derive the optimum pH.

The reaction can be done in an absolutely anaerobic condition to yield better and more accurate results.