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

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.

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?

Yeast can respire in both aerobic (in presence of oxygen) and anaerobic (absence of oxygen) pathways.

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.

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.

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.

NA= Not Applicable

• 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.

• 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.

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

• 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.

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

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

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.

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.

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.

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.

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

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

The f ratio value is 0.04644

The p value is 0.995592

Hence the result is not significant as p < 0.05

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.

• 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

• 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.

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 %

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.

Websites:

Effect of Temperature on Fermentation https://westminsterscienceinmotion.com/.pdf Accessed on 18 March,2018 12.35 pm

Pata.” Sucrose Enzyme, 1 Jan. 1970, https://patakaa.blogspot.com///2012/01/sucrose-enzyme.html.Accessed on 17 March,2018 9.05 am

“Welcome to CAB Direct.” CAB Direct, https://www.cabdirect.org/cabdirect/welcome/?target=%2fcabdirect%2fabstract%2f20073018161.Accessed on March18,2018

Journals

²Ppepin, Charles, and Charles Marzzacco. “The Fermentation of Sugars Using Yeast:A Discovery Experiment .” The-Fermentation-of-Sugars-Using-Yeast-A-Discovery-Experiment.pdf,Melbourne FL , https://www.researchgate.net/publication/275031809_The_Fermentation_of_Sugars_Using_Yeast_A_Discovery_Experiment. Accessed on 17 March,2018 9.45 am

Batista, Anderson S., et al. “Sucrose Fermentation by Saccharomyces Cerevisiae Lacking Hexose Transport.” Journal of Molecular Microbiology and Biotechnology, vol. 8, no. 1, 2004,pp. 26–33., doi:10.1159/000082078.

Software used :

https://www.socscistatistics.com//tests/Online Statistical Calculation