Objective: To investigate the effect of using different carbon source on the growth of yeast cells.
NAME: FARAH NADZIRAH ROSLI
CLASS: ALM 7 M 13
Title: Using Different Carbon and Source for Growth
Objective: To investigate the effect of using different carbon source on the growth
of yeast cells.
Background summary:
Growth depends upon both the type of the nutrients available and their concentration. Cells are largely made up of the four elements: carbon, hydrogen, oxygen and nitrogen with smaller, but significant, quantities of phosphorus and sulphur. Accounting as they do for 90% of the cell's dry mass, all six elements are essential for growth. Hence, like all living organisms, microorganisms require an energy source, a carbon source and a range of nutrients for metabolic and cell growth.
Microorganisms are small, easily dispersed and quick to multiply given a suitable environment. They grow on a wide diversity of substrates making them ideal subjects for commercial application. The microorganisms need organic carbon source, such as carbohydrates, as most of them are heterotrophic. Carbohydrates act as a respiratory substrate of cells. Microorganisms also need a nitrogen source for synthesis of DNA, RNA, ATP, coenzymes and chlorophylls. Apart from that, each species has its own optimum conditions within which it grows best.
Respiration involves a series of metabolic pathway, which is a series of enzyme-controlled chemical reaction, where the product of one reaction becomes the substrate for the following reaction. It consist of two different type: aerobic respiration and anaerobic respiration. Aerobic respiration involves glycolysis, Krebs cycle and oxidative phosphorylation while anaerobic respiration only involves glycolysis. The ultimate purpose of respiration is to produce ATP, the free energy for cells.
Yeast only consumes carbohydrate in the form of simple sugars such as monosaccharide and disaccharides for respiration. Therefore, in this experiment, we will be investigating the ability of yeast to utilize different carbon sources as a substrate in anaerobic respiration. A range of different carbohydrates are used as carbon sources, which are glucose and sucrose, and relative rate of respiration will be determined by measuring the production of acid by the yeast cells: the faster the rate of respiration, the faster the rate of acid production.
Materials:
2 % sugar solutions of glucose and sucrose, 500 ml flask with cotton wool plugs, ammonium phosphate, ammonium sulphate, burette and stand, 0.1 M of sodium hydroxide, phenolphthalein indicator solution.
Method:
. 200 ml of 2% of different sugar solutions are added to separate, labelled flask. One flask containing 200 ml of distilled water is included to act as a control.
2. 2 g dried yeast and 1 g of culture nutrients are added to each flask. The culture nutrients are a mixture of equal masses of ammonium phosphate and ammonium sulphate. The flask is swirled or the content is stirred thoroughly with a glass rod to ensure that the nutrients dissolved and that the yeast is resuspended.
3. Each flask is plugged with cotton ...
This is a preview of the whole essay
Method:
. 200 ml of 2% of different sugar solutions are added to separate, labelled flask. One flask containing 200 ml of distilled water is included to act as a control.
2. 2 g dried yeast and 1 g of culture nutrients are added to each flask. The culture nutrients are a mixture of equal masses of ammonium phosphate and ammonium sulphate. The flask is swirled or the content is stirred thoroughly with a glass rod to ensure that the nutrients dissolved and that the yeast is resuspended.
3. Each flask is plugged with cotton wool and incubated overnight at 25ºC.
4. A burette containing 0.1 mol dm-3 sodium hydroxide solution is set up.
5. After incubation, each flask is swirled thoroughly to mix the contents and 25 ml sample of each culture is removed.
6. The sample then is place into separate, labelled conical flask. 2 or 3 drops of phenolphthalein indicator solution is added to each flask.
7. Then, the sample is titrated against the sodium hydroxide to find the volume of alkali required to neutralize the acid produced by the yeast.
Results:
Solution
Glucose
Sucrose
Distilled water
Titration
2
2
2
Burette final reading/ cm3
4.80
27.20
41.30
3.70
24.45
36.90
Burette initial reading/ cm3
0.00
4.80
27.20
0.40
3.70
24.45
Titre value/ cm3
4.80
2.40
4.10
3.30
0.75
2.45
Average titre value/ cm3
3.60
3.70
1.60
Bar chart showing the volume of sodium hydroxide required to neutralised the acid produced by yeast in each sugar solution.
Discussions:
Yeasts are unicellular, saprotrophic fungi and are heterotrophic. They require an organic source of carbon such as carbohydrates, lipids and proteins. When carbohydrates are available, they are used first in respiration by the yeast cells before lipids and proteins are used. However, the carbohydrates must be in the form of simple sugar such as monosaccharide and disaccharides before they enter the respiratory pathway. Therefore, respiration is actually an oxidation process of an organic compound, the respiratory substrate to simple inorganic compound with the release of energy.
Yeast can undergo both aerobic and anaerobic respiration. In this experiment, yeast is grown in an anaerobic condition that is without oxygen. The product of anaerobic sugar respiration by yeast should be ethanol and carbon dioxide. Therefore, the equation is as followed:
a) Respiration of glucose
C6H12O6 2C2H5OH + 2CO2 + heat energy
b) Respiration of sucrose
C12H22O11 + H2O 4C2H5OH + 4CO2 + heat energy
From the equation, anaerobic respiration of glucose and sucrose by yeast produced carbon dioxide gas. The gas dissolves in water to produce carbonic acid:
CO2 + H2O H2CO3
Carbonic acid in water dissociates producing H+ and CO32- ions:
H2CO3 2H+ + CO32-
H+ ion produced make the solution become acidic. Thus, sodium hydroxide of known concentration is used to neutralise the acid contained in a 25 ml of each sample:
H+ + OH- H2O
The higher the volume of sodium hydroxide needed to neutralise the solution, the higher the concentration of acid present in the solution. The higher the production of acid by yeast, the faster the rate of respiration. It is essential to note that yeast cells in distilled water produced little CO2 because they do not have a source of sugar. Apart from that, distilled water is also acidic in nature causing it to be able to undergo neutralization by sodium hydroxide.
The relative rates of respiration are linked with the growth of the yeast. When the yeasts are able to utilise a certain carbon source very well, it is said to be in a suitable growing condition. The yeast cells will grow faster by multiplying its number over time. The increase number of yeasts results in more respiration to occur in the culture medium. This experiment can be further extended to find the relative growth rate of yeast in different sugars by removing a sample from the culture at regular intervals. These samples are filtered, dried and then weighed. The mass of each sample can then be plotted against time to give a graph of growth rate.
Glucose should yield the highest amount of carbon dioxide than sucrose or other sugars. This is because glucose is a monosaccharide, the simplest form of carbohydrate, and thus, can be utilised directly by the yeasts cells. Besides that, glucose can undergo phosphorylation directly in the first stage of glycolysis as it is the main substrate to produce pyruvate. The enzyme involved in this reaction is hexokinase that is present in the yeast cells. It is thought that the structure and shape of the glucose molecule fit very well with the active site of the enzyme. Therefore, the more similar the shape of a sugar molecule to glucose, the more likely it will fit to the active site of the enzyme.
In contrast to glucose, sucrose is disaccharides, which consists of glucose and fructose, and thus should give lower yield of carbon dioxide compared to glucose. Despite the high amount of carbon dioxide it can produce during respiration, sucrose is expected to produce CO2 at a slower rate because it is a disaccharide and must first be converted to glucose by the cell. Sucrose is transformed into glucose and fructose by a yeast enzyme, the invertase. The glucose produced can go into the glycolysis process immediately as mentioned above. The fructose, a glucose isomers, produced however, must be rearranged first by the action of isomerase enzyme before they can go into the glycolysis process.
However, in this experiment, the amount of sodium hydroxide used to neutralize the acid in the mixture containing sucrose solution is slightly higher than glucose with difference of 0.1 cm3. Such result is obtained because there might be an error occurred during the experiment causing the titre value to vary slightly from the first and the second reading. The reason is that the pale pink colour of the neutral solution cannot be seen clearly in the cloudy yeast suspension. This caused excess sodium hydroxide to be used in order to justify the pale pink colour in the suspension.
Besides that, the titre value taken is not concordance for the first and the second reading causing an error to the burette reading. There is a lot of difference between the two readings taken for each sample, which made the average titre value to become inaccurate. This is because the titration done is in a group and each individual takes turn to do the titration. Therefore, there is a different justification on the pale pink colour of the yeast suspension from one individual to another.
To reduce the error, some safety precautions could be taken. One of them is to take a titrated sample where the pale pink colour can be used as a standard for the next titration. For example, by taking the first sample of the titrated sample and compare the colour with the next titrations. Therefore, we must not throw away the sample titrated as it can be used as a guidance for determining the right pale pink colour for further titration samples.
In addition to that, when reading the burette, the eyes must be parallel to the reading observed to avoid parallax error. It is essential to take a concordance value between the first and second titration for calculating the accurate average titre value. The first and the second titre must not differ from 0.20cm3. To ensure that this is achieve, the titration must be done carefully with the conical flask containing the sample is swirled continuously during the titration. This is to ensure that the sodium hydroxide solution and the acid in the sample react evenly.
In this experiment, the other variables are kept constant. For example, the amount of nitrogen, phosphorus and sulphur sources, which are ammonium phosphate and ammonium sulphate, are the same for every sample. The samples are kept in an incubator with the same temperature, at 25ºC, for an overnight. Each flask is plugged with cotton wool during the incubation so that no carbon dioxide can escape from the conical flask to the atmosphere.
Besides that, the volume and the percentage of the sugar solutions used are the same for sucrose and glucose, as well as the concentration of sodium hydroxide used to titrate the sample. These are to ensure that the effect observed is only caused by the different carbon source. The solutions only have a minimal risk to us but a safety precaution must also be taken. Take care to rinse any yeast spillage from skin in case of possible irritation.
Conclusions:
The respiration rate of yeasts is faster when glucose is used as the carbon source compared to that of sucrose because yeasts can utilise glucose better than sucrose for respiration.
References:
. John Adds, Erica Larkcom, and Ruth Miller. Nelson Advanced Science: Respiration and Coordination (revised edition). 2001. Nelson Thornes Ltd. Cheltenham.
2. D.J, Taylor, N.P.O. Green, and G.W. Stout. Third Edition: Biological Science. 1998. Cambridge University Press. Cambridge.
3. Glenn and Susan Toole. New Undestanding Biology For Advanced Level Fourth Edition. 1999. Stanley Thornes Ltd. Cheltenham.
4. Phil Bradfield, John Dodds, Judy Dodds and Norma Taylor. A2 Level Biology. 2002. Pearson Education. Essex.
5. UA007544 - Edexcel AS/Advanced GCE Biology and Human Biology Coursework Guide.