A study of the factors affecting Saccharomyces cerevisiae population growth Essay

Abstraction:

An experiment was designed and conducted to look into the population growing of the barm Saccharomyces cerevisiae under assorted environment conditions such as temperature, pH degrees and glucose concentration. The research inquiries were so arrived as: What is the consequence of differing temperatures on Saccharomyces cerevisiae population growing?

What is the consequence of differing pH degrees on Saccharomyces cerevisiae population growing?

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What is the consequence of differing glucose concentrations on Saccharomyces cerevisiae population growing?

The different temperatures were chosen based on dynamicss and each temperature differing from the other by at least 10oC, so a noteworthy alteration in the yeast population to be observed. Two of the temperatures chosen were below the optimal temperature and two above and one in the optimal temperature.

Based on the optimal pH degrees for the growing of the barm, certain buffers with two pH values above and two below of the optimal pH and one in the optimal pH were prepared and stored.

The glucose concentration that was used in civilizations which tested for the consequence of temperature and pH was chosen in such a manner that would enable the barm population to turn without restriction every bit far as glucose is concerned. One of the options for proving the consequence of glucose over the yeast growing was the absence of glucose from the civilization. The other options were to halve the optimal glucose concentration and the last was higher of the optimal value.

When proving the different temperatures, the consequences showed that there was small growing in comparative low and high temperatures and really high growing in the optimal temperature ( the population about quadrupled ) . In the different pH degrees the barm growing was small in low and high pH degrees but was increased as pH was making the optimal pH. In the instance of different glucose concentrations, the consequences showed that with no glucose in the civilization was a little growing ; in the glucose concentration of halve of the optimal value there was growing but once more less than the optimum ; in the glucose concentration above optimum there was really high growing as there was in the optimal value.

Chapter 1: Introduction

Research Questions:

What is the consequence of differing temperatures on Saccharomyces cerevisiae population growing?

What is the consequence of differing pH degrees on Saccharomyces cerevisiae population growing?

What is the consequence of differing glucose concentrations on Saccharomyces cerevisiae population growing?

The barm:

Saccharomyces cerevisiae is a individual celled fungus that reproduces asexually by budding or division. It is one of the most good studied eucaryotic theoretical account beings in both molecular and cell biological science.

Saccharomyces cerevisiae is possibly the most of import and used fungus in the history of the universe even from ancient times because of its usage in the brewing of beer and in lifting of dough in staff of life. That is the ground why is called beer maker ‘s barm and baker ‘s barm, due to the usage of different strains of Saccharomyces for the alcoholic and sugar agitation.

S. cerevisiae is a really good type of barm for biological surveies owing to the rapid growing ( duplicating clip 1.5-2 hours at 30 A°C ) , the spread cells and the easiness of reproduction planting. Furthermore is a non-pathogenic being, so can be handled dauntlessly with merely small safeguards. Besides big sums of commercial baker ‘s barm are available with consequence being an easy and inexpensive beginning for biochemical surveies.

S. cerevisiae has round to ovoid cells between 3-8I?m in diameter

Figure 1- S. cerevisiae cells

Respiration:

In biological science, respiration is defined as: “ the procedure by which the energy in nutrient molecules is made available for an being to make biological work ” ( Kent, 2000 ; p.100 ) . It is besides called Cellular respiration. This procedure of cellular respiration happens in every life cell as it is the lone manner to obtain energy in a signifier that will be useable for the cell, so it can transport out the maps of motion, growing and reproduction ( ibid ) .

The nutrient in barms must be obtained as they can non bring forth it on their ain. For barms, a really good beginning of energy is sugars. All strains of S. cerevisiae can metabolise glucose ( a hexose sugar ) , maltose and trehalose.

Adenosine Triphosphate ( ATP ) :

Adenosine Triphospate known besides as ATP is the signifier of chemical energy that cells use to transport out biological activities. Without ATP an being ca n’t last. During cell respiration the energy that is found in nutrient molecules is transformed to ATP ( Kent, 2000 ; p.100 ) .

Types of Respiration:

There are two chief types of respiration that take topographic point within a cell: Anaerobic respiration ( without O ) and Aerobic respiration ( with O ) . S. cerevisiae can metabolise sugars in both ways, but in this research the civilizations of barm were exposed to air hence to oxygen, so aerophilic respiration was chiefly the manner that barm cells grew and reproduced.

Aerobic Respiration:

Aerobic respiration is a complex procedure which involves different stairss of reactions and its intent is to metabolise nutrient molecules. As these reactions take topographic point and nutrient is broken down, energy is released which is so used to synthesise ATP from ADP ( Adenosine diphosphate ) and inorganic phosphate ( Kent, 2000 ; p.101 ) . These reactions are carried out by particular enzymes. There are the three major metabolic phases in aerophilic respiration: glycolysis ( which is besides portion of anaerobiotic respiration ) , Krebs rhythm, electron conveyance concatenation and oxidative phosphorylation.

Krebs rhythm: The cardinal stage of the aerophilic respiration and occurs in the mitochondrial matrix. It involves the production of acetylcoenzyme A ( acetyl-CoA ) ( Kent, 2000 ; p.104 ) .

Electron Transport Chain: It involves the highest production of ATP during respiration, intending the 90 % of ATP is produced in this phase. This metabolic phase occurs in the interior mitochondrial membrane ( Greenwood. et Al. 2007 ; p.127 ) .

Glycolysis:

Cell respiration has to make with the production of ATP by the oxidization of sugars, fats or other substrates. In this research as substrate was used glucose. When glucose is the substrate, the first metabolic tract of cell respiration is glycolysis, which is carried out by enzymes in the cytol of the cell. A little sum of ATP is produced in this tract by the oxidization of glucose. Glycolysis consists portion of aerophilic and anaerobiotic respiration because no O is used ( Allot, 2007 ; p.73 ) .

Figure 2- The phases of glycolysis

Enzymes:

Thousands of chemical reactions are carried out within a cell. These reactions most of the times occur in a really slow rate. For that ground populating beings make biological accelerators which are called enzymes and rush up these reactions. “ Enzymes are ball-shaped proteins which act as accelerators of chemical reactions ” ( Allot, 2007 ; p.18 ) . An enzyme can increase to more than a billion of times the rate of a chemical reaction. Besides cells can command which reaction occurs in their cytol by doing some enzymes and non others. Enzymes achieve to increase the rate of a reaction by diminishing the activation energy ( the lower limit sum of energy required for a reaction to happen ) ( Green. Et Al. 2008 ; p.167 ) of the substrate or the substrates, when adhering to the activation site ( “ is the portion of the enzyme ‘s surface into which the substrate is bound and undergoes reaction ” ) ( Greenwood. et Al. 2007 ; p.114 )

Enzymes are sensitive molecules with really specific construction which enables them to transport out specific reactions. This construction including the active site can be damaged by assorted conditions and substrates. This harm is called denaturation and is normally lasting for an enzyme and if denaturation is occurred the enzyme can no longer transport out its map. As a consequence when enzymes are required to catalyse a reaction, is necessary that they have appropriate conditions. It should be remembered that different enzymes have different ideal conditions. The factors that affect the enzyme activity are: the temperature, the pH, the substrate concentration. In a specific point for each of the old factors, enzymes work in the most effectual manner, known as optimal conditions.

The consequence of temperature, pH and substrate concentration upon the enzyme activity which affects the growing of S. cerevisiae barm cells are studied in this research.

Consequence of Temperature:

As the temperature is increased in an enzyme-catalysed reaction, the rate of reaction is increased up to maximum in a specific temperature. This is called optimal temperature. The optimal temperature of Saccharomyces cerevisiae is 30o- 32oC.

In temperatures below of the optimum, when increasing the temperature there is an addition in the kinetic energy of the reactants and there are more frequent hits between the active site and the substrates, so the activity of the enzymes is increased.

The rate still rises as the temperature increases ; till it reaches the highest rate where is the optimal temperature hence the highest enzyme activity.

Above this temperature the rate starts to drop quickly. This is due to the high energy that causes quiver inside the enzyme with consequence the bonds which maintain the construction of enzyme to interrupt. This causes denaturation and the active site can no longer suit the substrate.

Overall, at really low temperatures the enzyme activity therefore the rate is low due to the low kinetic energy of the substrate but there is no denaturation, at the optimal temperature the rate is the highest and degrees off because the addition in kinetic energy of substrate is cancelled out by the denaturation of the enzyme and at high temperatures enzymes are denaturated and the rate falls dramatically because denaturation exceeds the high kinetic energy of the substrates. These are summarized in the undermentioned graph.

Figure 6- Effect of temperature on enzyme activity

Consequence of pH ( hydrogen ion concentration ) :

Most of the enzymes operate efficaciously in a little scope of pH values. Between these pH values there is an optimal pH value in which the enzyme activity is the highest. The optimal pH of Saccharomyces cerevisiae is 5.5. Acids and bases cause denaturation of the construction of the enzyme by interrupting chiefly H and ionic bonds with consequence the substrate ca n’t suit the active site. Furthermore the charges of the amino acids within the active site are affected by pH alterations, so the enzyme is non able to organize an enzyme-substrate composite. Above and below the optimal pH the enzymatic activity therefore the rate is reduced well.

Figure 7- The consequence of pH on enzyme activity

Consequence of Substrate concentration:

In an enzyme-catalysed reaction the rate additions in direct proportion to the substrate concentration. The optimal glucose concentration of Saccharomyces cerevisiae is 2 % . At low substrate concentrations, the rate of enzymatic activity increases aggressively as the substrate additions. This occurs due to the more frequent hits between the substrate molecules and the unoccupied active sites. On the other manus, at high substrate concentrations the biggest portion of the active sites have been occupied with consequence when increasing the substrate concentration there is small consequence on the rate of enzymatic activity.

Figure 8- The consequence of substrate concentration on enzyme activity

Chapter 2: Methodology

Aims of the survey:

To find how the different temperatures affect the growing of population of S. cerevisiae.

To find how the different pH values affect the population growing of S. cerevisiae.

To find how the different glucose concentrations affect the population growing of S. cerevisiae.

Hypothesis:

Hypothesis 1: The population of S. cerevisiae will turn the most at the optimal temperature, intending between 28oC to 32oC, and besides the population growing at temperatures below the optimum will be higher than the population growing at temperatures above the optimum.

Hypothesis 2: In the optimal pH, intending at low acidic conditions of pH 5.5 to pH 6, there will be the highest S. Cerevisiae barm cell population growing. At pH degrees above and below the optimal pH there will be less growing but this growing degree will be comparatively of the same grade for the values of pH above and below.

Hypothesis 3: In the optimal glucose concentration, intending about 2 % glucose, will happen the highest barm growing. In the glucose concentration below of the optimum there will be much lower growing, whereas in the absence of glucose there will be about none yeast growing.

Variables:

When proving the consequence of differing temperatures on S. cerevisiae population growing:

Independent variable: Temperature ( 5o C, 15oC, 30oC, 50oC, 60oC ) .

Dependent variable: Number of S. cerevisiae cells.

Controlled variables: 7mL buffer of pH 6 in every trial tubing, glucose concentration 2mL ( 2 % glucose solution ) in every trial tubing and 1mL barm ( 0.02 % yeast solution ) in every trial tubing.

When proving the consequence of differing pH degrees:

Independent variable: pH ( 3, 4, 6, 8 ) .

Dependent variable: Number of S. cerevisiae cells.

Controlled variables: Temperature ( 30oC ) , glucose concentration 2mL ( 2 % glucose solution ) in every trial tubing, 7mL buffer in every trial tubing, 1mL barm ( 0.02 % yeast solution ) .

When proving the consequence of differing glucose concentrations on S. cerevisiae population growing:

Independent variable: Glucose concentration ( 0mL, 1mL, 2mL, 3mL of 2 % glucose solution each ) .

Dependent variable: The figure of S. cerevisiae cells.

Controlled variables: Temperature ( 30oC ) , 9mL buffer of pH 6 at 0mL glucose, 8mL buffer of pH 6 at 1mL glucose, 7mL buffer of pH 5.5 at 2mL glucose, 6mL buffer of pH 6 at 3mL glucose, 1mL barm ( 0.02 % yeast solution ) in every trial tubing.

Materials/ Apparatus:

Test tubing

Burette

Micropipettes

Pipets

Pipette-fillers

Graduated cylinder of 10mL, 250mL and 1000mL

Volumetric Flasks of 250mL and 1000mL

Funnels

Spatula

Weight boats

Beakers

Plastic wash bottles

Plastic bottles

Cover faux pas

Haemocytometer

Microscope

Digital multi-log

Balance

Waterbath

Magnetic scaremonger

Thermometer

Ethanol 70 %

0.1M Citric acid

0.2M Sodium H phosphate

Distilled H2O

Yeast: Saccharomyces cerevisiae

Beginning of barm: YIOTIS S.A, INDUSTRY OF NUTRITIONAL PRODUCTS, ATHENS, GREECE.

Procedure:

Day 1:

The first measure before the start of the aerophilic agitation of barm was to fix the buffers. For the readying of buffers of different pH, citric acid ( 3-carboxy-3-hydroxypentanedioic acid ) and sodium H phosphate ( Na2HPO4 ) were used. Four plastic bottles, labeled each with one pH value ( 3, 4, 6, 8 severally ) , were required. 100mL of each of the buffers were prepared.

The stock solutions of citric acid and Na2HPO4 foremost prepared.

For the readying of stock solution of citric acid of concentration 0.1M and volume 1L, 19.2g of citric acid and 1L distilled H2O required.

For the readying of stock solution of Na2HPO4 of concentration 0.2M and volume 1L, 28.4g Na2HPO4 and 1L distilled H2O required.

A balance and a weigh boat required for the measurement of multitudes. The solutions were added and stored in two volumetric flasks of 1L severally, which measured the volume of distilled H2O. Citric acid and Na2HPO4 were added into the flasks with the assistance of funnels.

Table of volumes for each buffer of different pH:

pH

0.1M Citric acid

0.2M Na2HPO4

3

79.45mL

20.55mL

4

61.45mL

38.55mL

6

36.85mL

63.15mL

8

2.75mL

97.25mL

The volumes were measured and put into four different plastic bottles by utilizing two burettes of 50mL. The cogency of each pH value checked by utilizing a digital multi-log.

The following measure was to fix the glucose solution. For the readying of glucose one volumetric flask of 500mL used to mensurate the volume of distilled H2O and to hive away the glucose solution. 10g of glucose were weighed by utilizing a balance, a weigh boat and a spatula. Half of a 100mL beaker filled with distilled H2O was used to fade out the 10g of glucose. A magnetic scaremonger used for better disintegration. After glucose was complete dissolved, was added to the 500mL flask utilizing a funnel. The remainder of the flask was filled up to 500mL with distilled H2O.

Then, the yeast solution prepared for the intent of the experiments of that twenty-four hours. Every twenty-four hours a new barm solution was prepared. For the yeast solution 0.10g of dry barm were weighted from sachet with a spatula and placed on the weight boat. The barm was added to a 1000mL volumetric flask filled with 500mL distilled H2O with the assistance of a funnel in order to avoid staking of dry barm in the cylindrical walls of the flask. Afterwards the solution was swirled by smooth shaking.

After everything was ready the experiments for the perusal of the consequence of differing temperatures on S. cerevisiae growing initiated. Three H2O baths were prepared and each one adjusted in three different temperatures 30oC, 50oC and 60oC. Each temperature was tested by utilizing a thermometer and a digital multi-log detector. Two iceboxs were used for the low temperatures and adjusted at 5oC and 15oC. After all temperatures have been reached, the readying of civilizations started. Five trial tubings labelled with one temperature each. The civilizations were prepared with half an hr difference in order to prove the stableness of the temperature and to take a sample from each trial tubing and number the initial population. A pipette of 25mL used to present the glucose to the trial tubing. A 10mL calibrated cylinder used to mensurate the volume of the buffer and so was introduced into the trial tubing besides. Then with another 25mL pipette, 1mL barm was taken and placed besides into the trial tubing. The yeast solution was shaken before taking the sample as barm cells tend to drop to the underside of the flask due to their weight. Afterwards by utilizing a micropipette, a sample was taken from the civilization inside the trial tubing and placed on haemocytometer and so to the microscope to number the initial population ( the cells found in the boundary lines of the Chamberss were counted ) .

The haemocytometer is a specialized microscopical setup used to number cells and other cell organs. A haemocytometer consists of two numbering Chamberss. Each chamber consists of an agreement of squares of different sizes which are used to number easy the cells. These squares of different size signifier different grid layouts. In the Centre of each chamber it is found a grid of squares of 0.2mm 0.2mm 0.1mm dimensions. There is another grid of squares of dimensions 0.25mm 0.25mm 0.1mm, in each of the four corners around the cardinal grid. The grids of squares of 0.25mm 0.25mm 0.1mm dimensions were used for the numeration of the barm cells. A screen faux pas is placed above the Chamberss, so the samples are spread every bit due to capillary action on the numeration country.

Figure 9- A haemocytometer

Figure 10- One of the two Chamberss of a haemocytometer

Figure 11- Grid of 0.25mm 0.25mm 0.1mm squares

The trial tubing was so placed for 24hours in the temperature matching to what was labeled. This process was the same for the remainder four trial tubing. In the terminal of the twenty-four hours the glucose solution 2 % was placed in the icebox, the 1000mL flask with the barm solution, the haemocytometer, the screen glass and all the other setup was cleaned with ethanol 70 % and washed with distilled H2O and left to dry. The usage of 70 % ethyl alcohol for the cleansing of haemocytometer does n’t hold any negative consequence on the barm cells that were topographic point on it to be counted. This happened in the terminal of every twenty-four hours.

Day 2:

The following twenty-four hours each trial tubing was removed with half an hr difference in the order that they were left for agitation. Then a sample was taken with the usage of a micropipette and placed on haemocytometer and once more to microscope to number the barm cells.

After completing with temperature proving the following thing was to analyze the consequence of pH degrees on S. cerevisiae population growing.

A yeast solution was prepared the same manner as Day 1. The glucose solution was removed from the icebox. Clean trial tubings taken and labeled with different pH values 3, 4, 6, 8. A H2O bath adjusted at 30oC. Again, every civilization was prepared the same manner as Day 1 and placed in a trial tubing with half an hr difference. All trial tubings with different pH degrees were placed in the same H2O bath for 24hours. Before each trial tubing was placed in H2O bath, a sample was taken to number the initial population of each.

Day 3:

The civilizations were removed in the order that were left to ferment and samples were taken to number the yeast population from each one. Between each measuring the haemocytometer was cleaned as was mentioned in Day 1.

Finally, the consequence of glucose concentration on yeast population growing was left. New yeast solution was prepared. The H2O was adjusted at 30oC. In clean trial tubes the new civilizations were prepared to prove the glucose concentrations. The trial tubings were labelled each with one concentration value. Samples were taken from each to number the initial population. The civilizations were placed in H2O bath to agitation.

Day 4:

The civilizations were removed from H2O bath and samples taken to number the yeast population.

Failings and Improvements:

Failing

Improvement

In the populations of barms cells that were counted in the microscope, there were both alive and dead cells

or denaturated cells.

A dye such as methylene blue could be used to find in each numbering the unrecorded and the dead or inactive cells. The cells which would stay colorless would bespeak enzyme activity and the dead or denaturated cells would be turned into bluish.

Methylene blue should be used merely after the agitation has finished because it inhibits the barm cells by devouring the H ions that are produced during respiration.

The trial tubing, where the barm civilizations were left for agitation, were somewhat closed on the top with cotton in order to forestall the entryway of other micro-organisms. This cotton stopper prevented the easy flow of fresh air ( incorporating O ) inside the trial tubing. This limited the handiness of O supply that the barms required in order to turn aerobically.

The trial tubing can be placed to ferment aerobically in a closed container such as BioFlo 3000. This sort of bio treating systems provide a broad scope of options that enables the research worker to set a standard air flow which includes different options of certain proportions O ggand air which can react to oxygen-demanding barms or any other micro-organism.

There was absence of some basic component beginnings in every barm civilization that are necessary for better agitation conditions such N and P beginnings. Lack of such beginnings lead to comparatively low cell growing comparing to the growing that could be achieved without the absence of such elements.

Bacto-peptone can be used as an organic N beginning. Yeast extract makes available many bio foods required for the agitation of barm cells. It besides provides indispensable H2O soluble vitamins, aminic acids, peptides and saccharides.

Chapter 3: Datas Collection and Processing

Data Collection:

The consequence of differing temperatures on S. cerevisiae population growing:

Table with the initial population:

Temperature

( oC ) A±0.5

Number of cells ( A±1 )

Chamber 1

Chamber 2

Grid 1

Grid 2

Grid 3

Grid 4

Grid 1

Grid2

Grid 3

Grid 4

5

21

23

30

28

29

33

26

24

15

30

32

28

34

36

39

29

33

30

31

44

38

46

27

35

40

42

50

35

40

42

39

35

42

35

47

60

39

40

37

34

54

32

44

41

Table with the 24 hours fermented population:

Temperature

( oC ) A±0.5

Number of cells ( A±1 )

Chamber 1

Chamber 2

Grid 1

Grid 2

Grid 3

Grid 4

Grid 1

Grid2

Grid 3

Grid 4

5

39

35

27

32

41

33

30

37

15

63

61

69

55

51

49

66

56

30

168

175

155

120

152

175

113

168

50

46

39

45

42

40

48

42

55

60

38

44

46

37

38

46

52

48

The consequence of differing pH degrees on S. cerevisiae population growing:

Table with the initial population:

pH

Number of cells ( A±1 )

Chamber 1

Chamber 2

Grid 1

Grid 2

Grid 3

Grid 4

Grid 1

Grid2

Grid 3

Grid 4

3

38

35

29

42

64

35

25

29

4

40

45

27

31

50

39

56

24

6

30

22

39

40

22

32

37

38

8

37

26

33

29

39

35

29

22

Table with the 24 hours fermented population:

pH

Number of cells ( A±1 )

Chamber 1

Chamber 2

Grid 1

Grid 2

Grid 3

Grid 4

Grid 1

Grid2

Grid 3

Grid 4

3

45

50

41

53

51

44

41

49

4

74

68

77

63

82

76

91

88

6

125

143

133

119

105

111

125

102

8

54

61

52

48

56

54

64

47

The consequence of differing glucose concentrations on S. cerevisiae population growing:

Table with the initial population:

Glucose 2 % concentrations

( milliliter )

Number of cells ( A±1 )

Chamber 1

Chamber 2

Grid 1

Grid 2

Grid 3

Grid 4

Grid 1

Grid2

Grid 3

Grid 4

0

37

56

47

51

39

42

48

43

1

48

46

48

42

35

39

44

42

2

51

44

48

39

49

38

36

44

3

42

44

41

61

28

54

52

36

Table with the 24 hours fermented population:

Glucose 2 % concentrations

( milliliter )

Number of cells ( A±1 )

Chamber 1

Chamber 2

Grid 1

Grid 2

Grid 3

Grid 4

Grid 1

Grid2

Grid 3

Grid 4

0

50

42

59

48

44

53

55

48

1

82

90

76

84

81

73

68

79

2

175

166

181

177

179

176

154

169

3

185

171

178

182

169

172

175

181

Datas Processing:

Average values and Standard divergence

In order to cipher the standard divergence for each of the undermentioned mean values in each tabular array of different factors, digital reckoner “ CASIO, fx-9860 II SD ” was used.

In the undermentioned tabular arraies it is shown the mean values and standard divergences that were calculated by utilizing the information of both chamber 1 and chamber 2, in each different factor ( temperature, pH degrees, glucose concentrations ) .

Tables of average values and standard divergence for differing Temperatures:

Table with the initial population:

Temperatures ( ) A±0.5

Mean value ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

5

26.75

3.99

15

32.63

3.70

30

37.88

6.54

50

39.38

4.31

60

40.13

6.79

Table with the 24 hours fermented population:

Temperatures ( ) A±0.5

Mean value ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

5

34.25

4.68

15

58.75

7.15

30

153.3

24.2

50

44.63

5.18

60

43.63

5.45

Tables of average values and standard divergence for differing pH degrees:

Table with the initial population:

pH

Mean value ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

3

37.13

12.1

4

39.00

11.2

6

32.50

7.33

8

31.25

5.78

Table with the 24 hours fermented population:

pH

Mean value ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

3

46.75

4.62

4

77.38

9.47

6

120.4

14.1

8

54.50

5.86

Tables of average values and standard divergence for differing glucose concentrations:

Table with the initial population:

Glucose 2 % concentrations ( milliliter )

Mean value ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

0

45.38

6.35

1

43.00

4.50

2

43.63

5.53

3

44.75

10.6

Table with the 24 hours fermented population:

Glucose 2 % concentrations ( milliliter )

Mean value ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

0

49.88

5.64

1

79.13

6.81

2

172.1

8.86

3

176.6

5.78

Calculation of cell concentration

In order to cipher the cell concentration for each factor, the comperative mean values, which are displayed above, were used. These average values were applied to the following expression which enables to change over counted cells into cell concentration:

In the above expression, C is the feasible cells/mL, N is the counted cells, D is the dilution factor and 103 is the haemocytometer rectification factor.

An illustration with the application of the expression of cell concentration for the factor of temperature at 5oC and after 24 hours of agitation is shown below:

In the instance of 24 hours of agitation at temperature at 5oC, the feasible counted cells, N=34.25, the dilution factor, D=1. In all experiments, when proving the different factors, the dilution factor is ever one ( D=1 ) .

Representation of calculated informations of cell concentrations

Tables of cell conentration ( cells/mL ) for the differing temperature values:

Table with the initial population:

Temperatures ( ) A±0.5

Cells/mL ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

5

26750

3991.1

15

32630

3700.9

30

37875

6534.2

50

39375

4307.4

60

40125

6791.6

Table with the 24 hours fermented population:

Temperatures ( ) A±0.5

Cells/mL ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

5

34250

4682.8

15

58750

7146.4

30

153250

24235.5

50

44625

5180.7

60

43625

5449.4

Tables of cell conentration ( cells/mL ) for the differing pH degrees:

Table with the initial population:

pH

Cells/mL ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

3

37125

12158.9

4

39000

11212.2

6

32500

7329.0

8

31250

5775.6

Table with the 24 hours fermented population:

pH

Cells/mL ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

3

46750

4621.4

4

77375

9470.8

6

120375

14029.9

8

54500

5855.4

Tables of cell conentration ( cells/mL ) for the differing glucose concentrations:

Table with the initial population:

Glucose 2 % concentrations ( milliliter )

Cells/mL ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

0

45375

6345.7

1

43000

4504.0

2

43625

5527.5

3

44750

10566.1

Table with the 24 hours fermented population:

Glucose 2 % concentrations ( milliliter )

Cells/mL ( Chamber 1, Chamber 2 ) ( counted cells )

Standard Deviation

0

49875

5642.6

1

79125

6812.5

2

172125

8855.0

3

176625

5780.2

Chapter 4: Analysis and Interpretation

4.1 Graphs

The information that is used for the sketching of the graphs is shown in chapter 3, in “ Data Processing ” , “ Representation of calculated informations of cell concentrations ” . The several tabular array values were used for each of the factors.

The package that was used for the sketching of the graphs is, Graph 4.3 ( Ivan Johansen, 2007 ) .

consequence of Temperature on S. cerevisiae population growing

The consequence of pH on S. cerevisiae population growing

The consequence of substrate Glucose concentration on S. cerevisiae population growing

4.2 Interpretation

Testing Hypothesis 1:

Comparing the different temperatures that the S. cerevisiae population left to turn, it can be seen based on both the cell concentration and the graph, that below 30oC the of the population grows quickly as the temperature increases ; the barm population about doubles when temperature increases from 5oC to 15oC and about three-base hits when temperature increases from 15oC to 30oC. Above 30oC the growing of the population is extremely decreased ; yeast population becomes about 3.5 times less when temperature increases from 30oC to 50oC and when temperature increases from 50oC to 60oC the population decreases really somewhat. As a consequence, the highest S. cerevisiae population growing is observed at 30oC. Consequently this should be the optimal temperature. Furthermore, as temperature below the optimal point increases the population increases more from its initial value than it does at temperatures above the optimal point. Overall the hypothesis confirmed.

Testing Hypothesis 2:

Measuring the yeast population growing at the different pH degrees, it can be seen that the addition of population above and below the value of pH 6 is about the same. The fact that at pH 6 it is observed the highest population growing implies that this is the optimal pH degree. The lowest growing is observed at pH 3 and pH 8. In these specific pH degrees the growing is somewhat higher at pH 8 ( population increases about 1.7 times ) than it is at pH 3 ( population increases about 1.3 times ) . The growing is higher in pH 8 as it is closer to the optimal pH. At pH 4 the addition in population is about the same as it is at pH 8. Both pH 4 and pH 8 differ by 2 pH degrees from the optimal degree but the yeast population at pH 4 additions about 1.982 times where at pH 8 the population increases 1.7 times. This shows that S. cerevisiae operates better at acidic conditions. Overall the hypothesis is confirmed.

Testing Hypothesis 3:

Analyzing the growing of S. cerevisiae at different glucose concentrations and for 24 hours of agitation, the consequences obtained show that in the absence of glucose from the civilization the barm population did n’t increase at all. The lone addition that was observed from its initial population was 1.091.1 times, intending that this 0.1 addition may hold occurred due to the capacity of energy within the barm cells. At 1 % glucose concentration it was observed sufficient growing. The barm population about doubled from its initial value ( increased about by 1.8 times ) . In higher glucose concentration the barm cells population respond greater and as a consequence a higher population growing was observed. The initial population increased 3.9 times, significance that about quadrupled. In even higher glucose concentrations the population increased extremely once more but non plenty so to be able to state that at 24 hours of agitation S. cerevisiae requires more energy to make the maximal reproduction capacity. The population increased 3.954.00 times, about the same of that of 2 % concentration. Furthermore, based on the graph plotted for glucose concentrations, it can be seen that after 2 % glucose concentration the yeast population reaches plateau without any farther addition. So the modification growing glucose concentration is at 2 % . Overall the hypothesis is confirmed.

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