Yeast cells are microscopic, one-celled fungi made up mostly of protein which are more important for their ability to ferment carbohydrates in various substances. Yeasts are widespread in nature, found in the soil and on plants.
Uses of Yeast
Yeasts are well known for the making of bread and wine.
Respiration in Yeast
Yeast has to make energy, stored as ATP to carry out all cellular functions. To do this they can respire both aerobically when there is plenty of oxygen, but where oxygen is short, they respire anaerobically; they are therefore called partial anaerobes. This produces less energy, but keeps the yeast alive. The major source for energy production in the yeast, Saccharomyces cerevisiae, is glucose and glycolysis is the general pathway for conversion of glucose to pyruvate, whereby production of energy in the form of ATP is coupled to the generation of intermediates and reducing power in form of NADH for biosynthetic pathways. Fig 4 outlines the two processes.
Aerobic
When the yeast is mixed with sugar or glucose solution, it soon starts to respire. The yeast uses sugar and oxygen dissolved in the water to produce carbon dioxide, water and energy by aerobic respiration. Glucose is phosphorylated, eventually producing two molecules of pyruvate. The presence of oxygen enables, pyruvate to enter the mitochondrial matrix where it is oxidatively decarboxylated to acetyl CoA by the pyruvate dehydrogenase multi enzyme complex. This reaction links glycolysis to the citric acid cycle, in which the acetyl CoA is completely oxidized to give two molecules of CO2 and reductive equivalents in form of NADH and FADH2. these are then used to produce ATP.
Yeast (zymase complex)
Glucose + Oxygen Water + Carbon Dioxide + Energy
C6H12O6 6O2 6H20 6CO2 2880 Kj/mole
In
Anaerobic
When oxygen is not present, hydrogen cannot be disposed of by forming H2O. The electron transport chain stops working and no further ATP is formed by oxidative phosphorylation. Thus pyruvate is decarboxylated to ethanal, then reduced to ethanol.
Yeasts are able to ferment, which means that they metabolise anaerobically in the presence of an organic compound, such as a sugar or an amino acid, and in the absence of an external electron acceptor. During fermentation, the cell obtains energy from the oxidation of organic compounds and excretes one or more products as a way of disposing of the electrons derived from substrate oxidation. Typical examples of fermentation products are CO2 (leavening of bread), ethanol (beer and wine production), lactic acid (formation of dairy products) and propionic acid (in cheese production). In contrast to the process of respiration, in which an external electron acceptor is required and the organic substrates are often fully oxidized to CO2 and H2O, during fermentation the organic compound serves as both, electron donor and, after partial oxidation, as electron acceptor. Fermenting organisms gain fewer ATP molecules from each molecule of food they oxidize than aerobically respiring organisms. They excrete large quantities of only partially oxidized products during growth.
Although yeast can survive during anaerobic respiration, it does not grow and multiply as it would during aerobic respiration. Anaerobic respiration releases much less energy than aerobic respiration, only 210KJ compared to 2880KJ. In anaerobic conditions most energy remains locked in the ethanol. One problem is that alcohol is poisonous in large amounts. If the concentration of alcohol gets more than 14% it kills the yeast and the fermentation stops. Thus, if the experiment was carried out in anaerobic conditions the experiment couldn’t be carried out for too long.
Yeast (zymase complex)
Glucose Ethanol + Carbon Dioxide + Energy
C6H12O6 2C2H5OH CO2 210 Kj/mole
Conclusions
It is important to know how the yeast respire and what conditions are required for this because it clearly makes a difference. Thus when it comes to the experiment, this area needs to be controlled and only one pathway be allowed in order to make it a reliable investigation.
For practical reasons it would be easier for the yeast to respire anaerobically. This is because it would be difficult to maintain constant oxygen concentrations.
Fig 4
Respiration of Substrates Other Than Glucose
Although all yeasts are microorganisms that derive their chemical energy, in the form of ATP, from the breakdown of organic compounds, there is metabolic diversity in how these organisms generate and consume energy from these substrates. It is now well established that most yeasts employ sugars as their main carbon and hence energy source, but there are particular yeasts which can utilize non-conventional carbon sources.
Many sugars are fermentable compounds. While sucrose and glucose are abundant in plants, lactose is found predominantly in milk; hence, the number of organisms that have evolved to ferment lactose is lower than for the other sugars.
Galactose is a 'non-conventional' nutrient for yeast found in sucrose, which can be used as a sole carbon source when glucose is absent from the medium. This is one of the few pathways in yeast, which is regulated in a nearly 'all-or-nothing' mode. Fig 5 outlines what happens
Fig 5
In addition to hexose sugars, yeasts can utilize a number of 'non-conventional' carbon sources. Free glucose is scarce in natural environments or in natural products used to feed yeast cells. For example, disaccharides, such as maltose, sucrose or lactose can easily be accepted as nutrients by the action of corresponding hydrolases which breaks these disaccharides down into their constituent monosaccharides. However, as shown in figure 6, S. cerevisiae cannot hydrolyse lactose.
Fig 6
Variables
In order to make sure that the variable being manipulated is the one making the difference, other possible variables need to be controlled
Temperature affects both the time required to attain maximum activity and the maximum rate of gas production. Generally, the gas production rate doubles over a range of 0-40°C. this is the Q10 rule. Using a thermostatically controlled water bath can control the temperature and part of the trial experiments will be to find a suitable temperature for the yeast to respire at
pH values between 4.0 and 6.0 are optimum for bakers’ yeast that gives a constant and maximum gas production rate within this pH range. Yeast is slowly inactivated at values below pH 4.0 and above pH 6.0. To prevent a drop in gas production, buffering salts such as CaCO3 are added to water brews. In this particular experiment though it is not necessary to use buffers, as the initial rate will be measured.
Ethanol production from sugar has a considerable effect on the rate of gas production. Gas production rate is reduced by about 20 percent at ethanol concentrations of 4 vol %. Since one gram of sugar yields about 0.5 gram of ethanol, the rate of gas production in a water brew will be reduced toward the end of fermentation. The experiment will not be conducted long enough for this variable to have an effect.
The type of sugar has a profound effect on gas production. Glucose, fructose, and sucrose (beet sugar) are fermented rapidly and at similar rates by bakers’ yeast. Sucrose is hydrolysed` (inverted) into glucose and fructose by invertase present on the cell surface of regular bakers’ yeast. Lactose (milk sugar) is not fermented by regular bakers’ yeast. This is the only variable that is being manipulated
Conclusions and Predictions
Having studied the theory on yeast, the substrates, and respiration it is now possible to make some concluding comments and predictions.
Substrates
- It can be predicted that glucose will respire the fastest, sucrose second and it is uncertain whether lactose will be hydrolysed because the yeast may not have the enzymes required to break it down.
Yeast
- For the experiment the yeast will be respired anaerobically. This is because it will be easier to make and maintain anaerobic conditions and oxygen concentrations. Thus a system will be developed whereby no oxygen will be allowed to get in.
-
The rate of respiration will be measured by measuring the volume of CO2 produced. This is the most practical method available
- The yeast will be measured out using a top pan balance.
Initial Method
Apparatus
- Top pan balance
-
250cm3 side arm conical flask (with tubing)
-
500cm3 measuring cylinder
- Stop clock
- Yeast
- 2M glucose, sucrose, lactose
- Trough
- Bung (with two holes in)
- Thermometer
- Syringes
1g of dried yeast was measured out using a top pan balance. This was placed into a 250cm3 side arm conical flask. A bung was then placed over the top and a thermometer inserted through into the flask. 10cm3 of the substrate (either glucose, sucrose or lactose) were measured out using a syringe and placed into the top of the bung. A trough was filled up with water and a 500cm3 measuring cylinder that was also filled with water was placed in so no air bubbles were it. The measuring cylinder was then clamped and the tubing from the conical flask placed into the measuring cylinder. Then the substrate was released in to the conical flask via the syringe and the stop clock started. All this was done at room temperature. Readings were taken every 30 seconds from the measuring cylinder, on the volume of gas that had been produced.
Conclusions From Trials
-
It became apparent that 1g of yeast was not enough and after several trials it was found that 5g of yeast worked well. This meant that more of the substrate was needed and it was found that 30cm3 was enough.
-
The temperature was important in deciding how quickly the yeast would respire and at 30oC the yeast hardly produced any CO2, at 50oC a better yield was produce but it was found that at 50oC the best yield of CO2 was produced. At higher temperatures the yeast started to stop respiring.
- Instead of collecting the gas via a measuring cylinder, a gas syringe was used as an alternative. This was found to be more accurate and practically it was easier to set up.
- It was found that the predictions on the rate at which the substrates were respired were correct. Glucose was the quickest with sucrose and slower but lactose not working at all.
Results from Trials
A table to show the volume of CO2 produced when yeast respired glucose at 30oC
Table1
A table to show volume of CO2 produced when yeast respired glucose at 40oC
Table 2
A table to show volume of CO2 produced when yeast respired glucose at 50oC
Table 3
A table to show volume of CO2 produced when yeast respired sucrose at 50oC
Table 4
There are no results for the trial experiment for lactose because no gas was produced.
Conclusions from trial experiments
- One problem was that when the syringe was pushed down into the conical flask air was pushed out into the measuring cylinder. This was resolved by simply discounting this first wave of air bubbles and as it was a constant for each experiment did not matter.
- An airtight container was achievable but the air that was already in the container could not be remover practically. However, as this would be the same for each experiment it would not make a considerable difference between the results.
-
In the main experiment the reaction will be recorded for 10 minutes. This is because in the sucrose experiment after this time no more CO2 was released.
Main Method
5g of dried yeast was measured out using a top pan balance. This was placed into a 250cm3 side arm conical flask. A bung was then placed over the top and a thermometer inserted through into the flask. 30cm3 of the substrate (either glucose, sucrose or lactose) were measured out using a syringe and placed in a thermostatically controlled water bath at 500C. A gas syringe was attached to the side arm conical flask and clamped horizontally. The side arm conical flask was then placed into the water bath at 500C and the substrate which had been placed in the water bath previously, which was now at 500C was attached to the bung in to conical flask. Then the substrate was released into the conical flask via the syringe and the stop clock started. Readings were taken every 30 seconds from the gas syringe, on the volume of gas that had been produced.