Effect of Sugars on Rate of Respiration of Baker's Yeast, Lecture notes of Biology

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Effect of Sugars on Rate of Respiration of

Baker’s Yeast

Biology HL Internal Assessment

November 5, 2018

Design

Research Question: What is the effect of different types of sugars (maltose, sucrose, glucose,

fructose) on the rate of anaerobic respiration of Saccharomyces cerevisiae (Baker’s yeast)

obtained by the amount of carbon dioxide (ppm) produced?

Personal Engagement

My interest in this topic was sparked when I helped my mother bake naan , a type of Indian

flatbread. I was fascinated to see how the yeast helped the naan to rise and was curious as to the

scientific explanation behind it. When I learnt more about respiration and its applicability to

real-life (for breadmaking and alcoholic drinks), I was curious about how different sugars may

affect how fast yeast respired.

Background Research

Cellular respiration is the conversion of carbohydrates into usable energy, in the form of

ATP, which is used for metabolism, cell function and maintenance, and growth (Perry &

Burggren, 2007). Respiration is important to study because it is universal; almost every organism

respires (Perry & Burggren, 2007). The formula for cellular respiration is:

C 6 H 12 O 6 (glucose) + 6O 2 → 6CO 2 + 6H 2 O + ATP (energy)

This lab, however, will not be focusing on aerobic respiration which requires oxygen. The

lab investigates the rate of respiration in Saccharomyces Cerevisiae , commonly known as

Baker’s yeast. When it has run out of oxygen, yeast uses anaerobic respiration to break down

glucose molecules and convert them to ATP. After, it starts producing ethanol--this process is

called fermentation. It is important to note that glucose isn’t yeast’s only source of energy, but

the preferred source (Rodrigues, Ludoviko, & Leão, 2006). The first step of anaerobic respiration

is glycolysis. In glycolysis, the starting glucose molecule is rearranged into fructose-6-phosphate

by enzymes, where two phosphate groups attach to it, making it unstable and causing the

molecule to split (Bear & Rintoul, 2013). This uses 2 ATP molecules. The resulting 3-carbon

sugars are converted to pyruvates through several reactions, each 3-carbon sugar producing 2

ATP and 1 NADH (electron carrier) molecule (Bear & Rintoul, 2013). Thus, glycolysis results in

the net production of 2 ATP and 2 molecules of NADH. The second step of anaerobic respiration

is fermentation. The purpose of fermentation is to oxidise NADH so it can be reused as an

electron carrier; fermentation produces ethanol (Bear & Rintoul, 2013). The equation for

fermentation is:

C 6 H 12 O 6 (glucose) → 2CH 3 CH 2 OH (ethanol) + 2CO 2

The metabolism of disaccharides is a bit different from that of monosaccharides. In yeast, an

enzyme called invertase hydrolyses sucrose into fructose and glucose outside of the cell (Godoy

et al., 2014). Maltose is transported into the cell (via active transport) and metabolised by an

enzyme called glucosidases (Godoy et al., 2014). Then the monosaccharides go through

glycolysis. CO 2 is one of the products of respiration. Therefore, the volume of CO 2 produced will

be measured to calculate the rate of respiration. Yeast requires five minutes in warm water to be

activated to carry out respiration (Jaworski). This step will thus be added in the method.

Aim

The yeast S. cerevisiae was used for the experiment because it grows quickly and efficiently

in anaerobic conditions (Rodrigues, Ludoviko, & Leão, 2006). At first glance, the choice of

sugars may seem random. Glucose and fructose are monosaccharides whereas maltose and

sucrose are disaccharides. However, the aim of this lab is to investigate whether sugars are

respiration Type of water Distilled water will be used for all trials. Type of yeast The yeast, S. cerevisiae, will be used for all trials. Supplied by school. Lab environment All trials will be carried out in the same laboratory at 25oC. Weighing scale The same weighing scale will be used for all trials to take all measurements. +/- 0.01g Yeast activation The yeast will have five minutes to activate by being placed in the water bath.

Table 3. Materials

Item Quantity Specifics

Dried yeast 200g +/- 0.01g S. cerevisiae species Distilled water 100ml +/- 0.05 For each solution Sugars 50g per sugar +/- 0.01g The sugars used are maltose, sucrose, glucose, and fructose Weighing scale 1 +/- 0. Beakers 4 +/- 25g Not used for measurement. Graduated cylinder 1 +/- 0.5 ml Stirring rod 4 Water bath 1 Set at 40oC Test tube 5 Timer 1 +/- 0.01s Vernier CO 2 gas sensor 1 +/- 10% ppm (source: Vernier)

Method

1. Prepare a water bath. It should be 40oC at all times.

2. Fill a beaker with 50 ml of water and put 3g of dried S. cerevisiae in it. Use a graduated

cylinder to measure the water (for more accuracy).

3. Prepare five sugar solutions (one for each sugar and one control).

a. Put 10g of each sugar in a separate beaker using a weighing scale to measure the

exact amount.

b. Pour 100 ml of distilled water into each of the beakers using a graduated cylinder

to measure the water.

c. Stir the solutions until the sugar has dissolved completely.

4. Pour 5mL of the yeast solution into a test tube and put it in the water bath for five

minutes to activate the yeast.

5. Pour the yeast solution (5mL) and one of the sugar solutions (100mL) into the carbon

dioxide probe bottle and cap it (with the probe). Make sure to start the timer.

6. Record the amount of CO 2 produced in 1-minute intervals for ten minutes. Take

qualitative observations.

7. Repeat steps 5-6 with the all the sugars (maltose, sucrose, glucose and fructose) and no

sugar added. Do five trials for each sugar.

Note: If the sugar solution or yeast solution are all used up, repeat Steps 2 and 3 to make more as needed keeping the concentration the same.

Table 4. Safety

Safety Categories Description How To Avoid

Physical 1. Glassware can break and

cause injuries.

2. Solutions may spill onto

clothes or into eyes.

1. Handle glassware carefully.

If it breaks, call an adult

immediately.

2. Wear lab coat and goggles.

Ethical 1. Wasting sugar by using it

for a lab and then throwing

it away may be seen as

unethical.

1. Use the minimum amount of

sugar for the experiment.

Environmental 1. Dumping the yeast solution

in the sink will most likely

go down the drain and end

up in the river and pollute

it.

1. Dispose of yeast solution in

a separate container.

Trial 5 0 94 150 188 209 231 244 287 303 325 341 Fructose -- Volume of CO 2 (ppm +/- 10%) Trial 1 0 56 79 98 109 118 149 171 203 214 234 Trial 2 0 59 65 68 75 78 84 84 87 93 100 Trial 3 0 4 35 57 66 78 91 100 113 119 125 Trial 4 0 57 78 100 106 122 135 153 163 181 191 Trial 5 0 94 150 188 209 231 244 287 303 325 341

To obtain accurate data, at minute 0, 0 ppm of CO 2 was assumed. The rest of the values were

subtracted from the starting measurement. For example, if there was 210 ppm at 0 minutes, and

243 ppm at 1 minute, the measurement for 1 minute would be 243-210 = 33 ppm.

Table 6. Qualitative Observations

Sugar Photograph Observations

Control ● There is no reaction at all.

Maltose ● The solution is much hazier than that of

glucose.

● Beaded particles of what I think is

maltose appeared on the sides of the

bottle.

Sucrose ● The solution is lighter in colour than

maltose and glucose.

● A lot of the sucrose particles have sank

to the bottom even though in the sugar

solution, the sucrose had been dissolved.

Glucose ● The solution is a very pale colour and

seemed to get paler over the trial.

● It smelled a bit like rotten eggs.

● The yeast had sort of dissolved in the

sugar solution.

● The solution is very hazy/foggy.

● During one trial, one bubble appeared.

Fructose ● It was harder to dissolve the fructose

into water.

● The solution was yellow-y in colour.

How Data Will Be Processed

The RQ aims to investigate the rate of reaction. Hence, the rate will be calculated by finding the

mean average CO 2 produced and dividing it by time (10 minutes). This is to facilitate comparing

the different rates of reactions. If an instantaneous rate was obtained, it would be difficult to

come to a conclusion as to which sugar is metabolised faster.

Mean average: The mean average CO 2 produced will be calculated using the following formula

to be used for identifying a pattern between rate and amount of CO 2 produced.

m ean average = (^) number of trials total carbon dioxide produced in all trials ( ppm )

Example calculation for glucose:

m ean average = 492+440+512+191+341 5 = 3 95.2 ppm

Rate of reaction: Then, the rate of reaction will be calculated using the following formula so that

the fastest sugar which ferments can be identified.

r ate of reaction = (^) time ( minutes ) mean average amount of carbon dioxide produced ( ppm )

Example calculation for glucose:

r ate of reaction = 10 min 9.52 ppm min

395.2 ppm

−

Standard deviation: Will be done to measure the distribution of data relative to the central

tendency using the following formula.

σ =

√ n

Σ( x − x ) 2

σ = standard deviation, Σ = sum, x = value in data set, x = mean value, n = # of data sets

Table 7. Example calculation for glucose

A bar graph was chosen to represent the mean average rate of reaction of each sugar mixture

because it clearly shows the difference in rate. The data for each sugar is also independent. The

error bars were created by finding the standard deviation of the average rates of reactions for all

the trials of each sugar. Glucose seems to have the fastest mean average rate. However, the rates

of other sugars are pretty close in value. The error bars are overlapping which suggests that the

difference in rates isn’t significantly different.

Discussion

The experiment aimed to investigate the effect of different types of sugars (maltose, sucrose,

glucose, fructose) on the rate of anaerobic respiration of Saccharomyces cerevisiae (Baker’s

yeast). The alternative hypothesis argued that fructose would have the fastest rate of respiration

followed by glucose, sucrose, and maltose because fructose didn’t have to be converted for

respiration to occur, and monosaccharides would ferment faster than disaccharides. The findings

(see Table 7 ) showed that glucose had the highest rate of reaction, 39.5 ppm min-1. This was

followed by maltose (34.6 ppm min-1), sucrose (22.2 ppm min-1) and fructose (19.8 ppm min-1).

The controlled solution with no sugar produced no CO 2. The control was used to ensure that

fermentation by yeast only occurred when sugar was present. Since no CO 2 was produced, it is

safe to assume that this is indeed the case.

The qualitative observations, to a certain extent, supports the quantitative data. After

fermentation, the glucose solution was the most odorous and the solution seemed to get paler

almost immediately after yeast was added. The fructose solution was the most difficult to

dissolve in the water. One trend in the raw data ( Table 4 ) is that the reactions for most trials of

maltose, sucrose and fructose started fast and then gradually slowed down. For glucose,

however, the rate of reaction starts fast, starts slowing down after around five minutes, and

speeds up again. This may be a possible evaluation point. However, it also supports that S.

Cerevisiae is more efficient in fermenting glucose.

The results of this experiment are in accordance with Hopkins (1928) who did a study

investigating the difference in fermentation rates of fructose and glucose by S. Cerevisiae. and

found that glucose fermented faster than fructose. Hopkins (1928) also found that glucose

doesn’t convert into fructose while being fermented by S. Cerevisiae. This is important because

it counters my alternative hypothesis. However, Hopkins (1928) also pointed out that other

species of yeast such as Sauterne yeasts ferment fructose faster than glucose. Therefore, the high

fermentation rate of glucose is attributed to enzymes of S. Cerevisiae being more efficient. This

brings up an evaluation point of this experiment which is that there are many factors which show

the rate of fermentation, not just the production of CO 2. The enzymes used, as pointed out, are a

better indication of fermentation rate. This experiment is reductionist in that sense.

The rate of fermentation of sucrose was slower than maltose contrary to the alternative

hypothesis. Based on the Hopkins (1928) study, this makes sense because maltose is made of

two glucose molecules whereas sucrose--of fructose and glucose. It has been shown that glucose

ferments much faster than fructose as stated above. Furthermore, according to Batista et al., the

metabolic pathway (of fermentation) of sucrose is actually inefficient because it is metabolised

outside the cell which is a bit counter-intuitive because monosaccharides would be easier to

transport into the cell than disaccharides.

Although the fermentation rates of the sugars were different, it is important to know whether

there is a statistically significant difference. Unfortunately, an ANOVA test cannot be done

because the data was continuous. Furthermore, the mean average rates of reaction couldn’t be

used because they were enough data points. Therefore, the study must conclude that there is a

difference between fermentation rates in different sugars by S. Cerevisiae but it isn’t clear

whether the difference is statistically significant or not.

Relating this back to what inspired me to carry out this experiment, my mother uses sucrose,

refined white sugar (“Types of Carbohydrates”), to make naan. My mother and I take two hours

to make naan. However, this experiment suggests that using glucose would be better because the

rate of respiration is faster. Thus, we would take a lesser amount of time to make naan.

Evaluation

Although the data shows that there is a difference in fermentation rates of sugars, there are

several improvements that can be made to this experiment. First is the need for more trials. There

were twenty-five trials in total resulting in twenty-four degrees of freedom. More trials will lead

to better statistical results. Second, the length taken for fermentation was only ten minutes and as

can be seen from Table 4 , a plateau wasn’t reached which suggests that complete fermentation

hadn’t occurred. This is a concern because the data doesn’t represent the full reaction that takes

place and the average rate of fermentation isn’t accurate. An improvement would be to allow the

yeast to respire for a longer period of time. Another improvement concerning time is the

increments in which data was collected. Shorter increments would have led to better data

because the trends and patterns would have been easier to see.

There is also a question of whether the range of independent variables was sufficient or not.

The aim of the experiment was to compare the rate of metabolism of disaccharides to that of

monosaccharides. Considering this aim, the range of independent variables was sufficient

because glucose and fructose are monosaccharides whereas maltose and sucrose are

disaccharides made up of glucose, and glucose and fructose respectively. Thus, monosaccharides

are effectively being compared to disaccharides. Below is a table of more errors and

improvements. A wider variety of sugars could also have been tested to determine the best sugar

for respiration by Baker’s yeast.

Table 9. Sources of Errors

Source of error Significance Improvement There were some clusters of yeast at the bottom of the CO 2 probe’s bottle. Moderate: The yeast may have needed more time to be activated. It was only kept in the water bath for five minutes prior to each trial. No measure was taken of whether the yeast was fully activated or not. This would have affected the rate of fermentation. Put the yeast in the water bath for at least ten minutes. The data wasn’t collected on the same day at the same time. Low: The surrounding environment, although done in the same lab, would have All data should be collected on the same day under highly controlled conditions.

Works Cited

Alba-Lois, Luisa, and Claudia Segal-Kischinevzky. “Yeast Fermentation and the Making of Beer

and Wine.” Nature News , Nature Publishing Group, 2010,

www.nature.com/scitable/topicpage/yeast-fermentation-and-the-making-of-beer-

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.

Bear, Robert, and David Rintoul. “Cellular Respiration.” Principles of Biology , 2013. OpenStax

CNX , cnx.org/contents/24nI-KJ8@24.18:p26rHTib@5/Glycolysis.

“CO2 Gas Sensor.” O2 Gas Sensor | Vernier , Vernier Software & Technology, LLC. ,

www.vernier.com/products/sensors/co2-sensors/co2-bta/.

Godoy, Victor De, et al. “Efficient Maltotriose Fermentation through Hydrolysis Mediated by

the Intracellular Invertase of Saccharomyces Cerevisiae.” BMC Proceedings , vol. 8, no.

Suppl 4, 2014. National Center for Biotechnology Information ,

doi:10.1186/1753-6561-8-s4-p181.

Hopkins, Reginald Haydn. “The Selective Fermentation of Glucose and Fructose by Brewer's

Yeast.” Biochemical Journal , vol. 22, no. 4, 4 July 1928, pp. 1145–1156.,

doi:10.1042/bj0221145.

Jaworski, Stephanie. “Yeast.” Joyofbaking.com , Joyofbaking.com,

www.joyofbaking.com/Yeast.html.

Perry, Steven F., and Warren W. Burggren. “Why Respiratory Biology? The Meaning and

Significance of Respiration and Its Integrative Study.” Integrative and Comparative

Biology , vol. 47, no. 4, 1 Oct. 2007, doi:https://doi.org/10.1093/icb/icm033.

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www.researchgate.net/publication/226259782_Sugar_Metabolism_in_Yeasts_an_Overvi

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