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The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

The Stress Effect On Already Isolated Yeast Using Different Environmental Samples – Introduction:

The Stress Effect On Already Isolated Yeast Using Different Environmental Samples explains that Yeasts are eukaryotic microorganisms classified in the kingdom Fungi, with 1,500 species currently described (estimated to be 1% of all fungal species). Yeasts are unicellular, although some species with yeast forms may become multicellular through the formation of strings of connected budding cells known as pseudohyphae, or false hyphae, as seen in most molds.Yeast size can vary greatly depending on the species, typically measuring 3–4 µm in diameter, although some yeasts can reach over 40 µm. Most yeasts reproduce asexually by mitosis, and many do so by an asymmetric division process called budding.

The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

History:

he word “yeast” comes from Old English gist, gyst, and from the Indo-European root yes-, meaning “boil”, “foam”, or “bubble”‘Yeast microbes are probably one of the earliest domesticated organisms. People have used yeast for fermentation and baking throughout history. Archaeologists digging in Egyptian ruins found early grinding stones and baking chambers for yeast-raised bread, as well as drawings of 4,000-year-old bakeries and breweries .In 1680, Dutch naturalist Anton van Leeuwenhoek first microscopically observed yeast, but at the time did not consider them to be living organisms, but rather globular structures. In 1857, French microbiologist Louis Pasteur proved in the paper “Mémoire sur la fermentation alcoolique” that alcoholic fermentation was conducted by living yeasts and not by a chemical catalyst. Pasteur showed that by bubbling oxygen into the yeast broth, cell growth could be increased, but fermentation was inhibited – an observation later called the “Pasteur effect”.

By the late 18th century, two yeast strains used in brewing had been identified: Saccharomyces cerevisiae (top-fermenting yeast) and S. carlsbergensis (bottom-fermenting yeast). S. cerevisiae has been sold commercially by the Dutch for bread-making since 1780; while, around 1800, the Germans started producing S. cerevisiae in the form of cream. In 1825, a method was developed to remove the liquid so the yeast could be prepared as solid blocks. The industrial production of yeast blocks was enhanced by the introduction of the filter press in 1867. In 1872, Baron Max de Springer developed a manufacturing process to create granulated yeast, a technique that was used until the first World War. In the United States, naturally occurring airborne yeasts were used almost exclusively until commercial yeast was marketed at the Centennial Exposition in 1876 in Philadelphia, where Charles L. Fleischmann exhibited the product and a process to use it, as well as serving the resultant baked bread.

Nutrition and Growth:

Yeasts are chemoorganotrophs, as they use organic compounds as a source of energy and do not require sunlight to grow. Carbon is obtained mostly from hexose sugars, such as glucose and fructose, or disaccharides such as sucrose and maltose. Some species can metabolize pentose sugars such as ribose.alcohols, and organic acids. Yeast species either require oxygen for aerobic cellular respiration (obligate aerobes) or are anaerobic, but also have aerobic methods of energy production (facultative anaerobes). Unlike bacteria, no known yeast species grow only anaerobically (obligate anaerobes). Yeasts grow best in a neutral or slightly acidic pH environment.

Yeasts vary in what temperature range they grow best. For example, Leucosporidium frigidum grows at −2 to 20 °C (28 to 68 °F), Saccharomyces telluris at 5 to 35 °C (41 to 95 °F), and Candida slooffi at 28 to 45 °C (82 to 113 °F).[18] The cells can survive freezing under certain conditions, with viability decreasing over time.

Structure of Yeast Cell:

Several yeasts, in particular S. cerevisiae, have been widely used in genetics and cell biology, largely because S. cerevisiae is a simple eukaryotic cell, serving as a model for all eukaryotes, including humans, for the study of fundamental cellular processes such as the cell cycle, DNA replication, recombination, cell division, and metabolism. Also, yeasts are easily manipulated and cultured in the laboratory, which has allowed for the development of powerful standard techniques, such as yeast two-hybrid, synthetic genetic array analysis, and tetrad analysis. Many proteins important in human biology were first discovered by studying their homologues in yeast; these proteins include cell cycle proteins, signaling proteins, and protein-processing enzymes.

On 24 April 1996, S. cerevisiae was announced to be the first eukaryote to have its genome, consisting of 12 million base pairs, fully sequenced as part of the Genome Project. At the time, it was the most complex organism to have its full genome sequenced, and took seven years and the involvement of more than 100 laboratories to accomplish. The second yeast species to have its genome sequenced was Schizosaccharomyces pombe, which was completed in 2002It was the sixth eukaryotic genome sequenced and consists of 13.8 million base pairs. As of 2012, over 30 yeast species have had their genomes sequenced and published.

The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

Uses of Yeast:

  • Alcoholic beverages
  • Wines
  • Beer
  • Nutritional supplements
  • Bio-remediation
  • Non alcoholic beverages
  • Aquarium hobby

Objectives:

The main objective of study was to check the effect of stress condition on already isolated yeast from different environmental samples.

Materials:

  • Petri plates
  • Test tubes
  • Glass pipette
  • Micropipette
  • V illuminatorC
  • Test tubes
  • Aluminium foil

Methods:


Activation of cultures:

The isolated yeast strains were activated by inoculating then in YPD broth and incubating them at 37 ͦC for 24 hours.
Preparation for the stress conditions:

For the preparation of stress conditions, 6 sets of autoclaved test tubes, having YPD broth, were prepared in such a way that each set was labelled with U,V, high temperature, osmotic stress, high pH, less glucose concentration and controls.

Inoculation of test tubes and Exposure to the stress conditions:

  1. In order to give the U.V stress the inoculated test tubes were exposed to U.V for 15 minutes and then wrapped in an aluminium foil and incubated at 30 ͦ C for 24 hours.
  2. For the stress by pH, YPD broth was prepared at high pH (10), dispensed in test tubes and respective strains were inoculated, and incubated at 30 ͦ C for 24 hours.
  3. For the stress by osmotic pressure 2M NaCl was added in YPD broh and inoculated with respective strain after dispensing in test tubes. Incubation was given at 30 ͦ C for 24 hours.
  4. For the stress by glucose depletion , only 1% of glucose was added rather than 2% in the broth and was inoculates with respective strain after dispensing in the test tubes. Incubation was given at 30 ͦ C for 24 hours.
  5. For the stress by high temperature, test tubes were inoculated after dispensing broth I them and were exposed to 40 ͦ C for 24 hours.
    For the observation of colony morphology:
  6. For stress (a),50 µl of each strain was spread on its respective plate and paltes were then wrapped in aluminium foil and were incubated at 30 ͦ C for 24 hours
  7. For stress (b), plates were prepared with YPD at high pH and 50 µl of each strain was spreaded on respective plate. And incubated at 30 ͦ C for 24 hours.
  8. For stress (c), plates of YPD were prepared at high salt concentration and 50 µl of each strain was spreaded on respective plate and incubated at 30 ͦ C for 24 hours
  9. For the stress (d), plates of YPD agar were prepared in such a way that 1% of glucose was used rather than 2% and 50 µl each strain was spreaded on respective plate and incubated at 30 ͦ C for 24 hours.
  10. For the stress (e), each strain was spreaded on respective plate and was incubated at 30 ͦ C for 24 hours.

Inspection of colony morphology:

After 24 hours plates were inspected and colony morphology was checked and noted in tables and compared   with controls.

Results:

  1. Colony morphology of controls yeast strains:
Sr. no.CharacteristicsControl

Strain1

Control

Strain2

Control Strain3Control Strain4Control Strain5Control Strain6
1Sizemediummediummediummediummediummedium
2Shaperoundroundroundroundroundround
3Edgessmoothsmoothirregularirregularirregularsmooth
4Elevationconvexconvexconvexconvexconvexconvex
5ColorOff-whiteOff-whiteOff-whiteOff-whiteOff-whiteOff-white
6appearanceglossyglossyshinnyshinnyshinnyshinny
7CFU/ml506045355055
  1. Colony morphology of all stains under stress of high temperature:
Sr. no.CharacteristicsStrain1Strain2Strain3Strain4Strain5Strain6
1Sizesmallmediumsmallsmallsmallsmall
2Shaperoundirregularirregularovalroundround
3EdgesSmoothAmeboidbranchedbranchedsmoothAmeboid
4ElevationConvexConcaveconvexflatflatFlat
5ColorOff-whiteoff-whitewhitishoff-whiteOff-whitewhite
6appearancedrydrydrydrydrydry
7CFU/ml30352050603
  1. Effect of U.V on colony morphology of yeast strains:
Sr. no.CharacteristicsStrain1Strain2Strain3Strain4Strain5Strain6
1SizesmallVery smalllargeVery smallmediumno
2Shapeirregularroundroundroundroundno
3EdgessmoothsmoothroughameboidwavyNo
4ElevationconvexconvexconcaveconvexflatNo
5Coloroff-whiteoff-whiteoff-whiteoff-whiteoff-whiteNo
6appearanceglossyglossypowderyshinnyshinnyno
7CFU/ml1021340no
  1. Effect of high salt concentration on colony morphology of yeast strains:
Sr. no.CharacteristicsStrain1Strain2Strain3Strain4Strain5Strain6
1SizeToo smallsmallsmallsmalllargeMedium
2Shaperoundroundroundroundroundround
3Edgessmoothsmoothsmoothsmoothsmoothsmooth
4Elevationconvexconvexconvexconvexconcaveflat
5Colourwhitewhitewhiteoff-whiteoff-whitewhite
6Appearanceshinnyshinnyglossyglossyglossyshinny
7CFU/ml420391535
  1. Effect of low glucose level on colony morphology of yeast strains:
Sr. no.characteristicsStrain1Strain2Strain3Strain4Strain5Strain6
1SizesmallSmallsmallsmallsmallsmall
2Shaperoundroundroundroundroundround
3Edgessmoothwavysmoothwavywavywavy
4Elevationconvexflatflatflatconvexconvex
5Colorcreamyoff-whiteoff-whiteoff-whiteoff-whiteoff-white
6appearanceglossyShinnyshinnyshinnyglossyglossy
7CFU/ml404650303820
  1. Effect of high pH on colony morphology of yeasts strains:
Sr. no.characteristicsStrain1Strain2Strain3Strain4Strain5Strain6
1SizeNo colony was found on high pH
2Shape
3Edges
4Elevation
5Color
6Appearance
7CFU/ml
  1. Cell morphology of control strains:

                  The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                 The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

         Control strain 1                                        Control strain 2

                                          The Stress Effect On Already Isolated Yeast Using Different Environmental Samples              The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                                     Control strain 3                                      Control strain 4

                                          The Stress Effect On Already Isolated Yeast Using Different Environmental Samples               The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                                    Control strain 5                                        Control strain 6

  1. Cell morphology of yeast under high temperature:

                                        The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                  The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                  Strain 1                                               Strain 2

                                        The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                  Strain 3                                                Strain 4

                                       The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                 The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                       Strain 5                                               Strain 6

  1. Effect of U.V on cell morphology of yeasts:

                                     The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                       The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                                        Strain 1                                                         Strain 2

                                     The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                    The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                                          Strain 3                                                       Strain 4

                                     The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                       The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                                         Strain 5                                                       Strain 6

4. Effect of high salt concentration on yeasts cells morphology:

                                    The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                    The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                                       Strain 1                                                   Strain 2

                                   The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                     The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                                   Strain 3                                                         Strain 4

                                   The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                  The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                                            Strain 5                                               Strain 6

5.  Effect of  less glucose in media on cell morphology of yeasts:

                                         The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                 The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                                            Strain 1                                                      Strain 2

                                        The Stress Effect On Already Isolated Yeast Using Different Environmental Samples                The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                                          Strain 3                                                       Strain 4

                                        The Stress Effect On Already Isolated Yeast Using Different Environmental Samples               The Stress Effect On Already Isolated Yeast Using Different Environmental Samples

                                                          Strain 5                                                      Strain 6

6.  Effect of  high pH on cell morphology of yeasts:

No colonies were found to grow under pH stress.

Discussion:

Yeasts are eukaryotic microorganisms classified in the kingdom Fungi, with 1,500 species currently described (estimated to be 1% of all fungal species). Yeasts are unicellular, although some species with yeast forms may become multi-cellular through the formation of strings of connected budding cells known as pseudohyphae, or false hyphae, as seen in most molds.Yeast size can vary greatly depending on the species, typically measuring 3–4 µm in diameter, although some yeasts can reach over 40 µm. Most yeasts reproduce asexually by mitosis, and many do so by an asymmetric division process called budding.

Yeast is very important in every days life because it has many industrial application and as well as other application. The basic topic or objective of my project was to check the effect of different stresses on yeasts strains already isolated serially from different environmental sample. 6 strains were taken and control samples were also used in order to compare the effect of different stresses on the cell as well as colonial morphology of yeasts. The different stresses that were used: U.V stress, Glucose depletion stress, Osmotic pressure stress, High temperature stress and high pH stress.

First of all, strains were isolated serially and then used for the basic project. When colony morphology for strain 1 was studied it was observed it was seen that it had a medium sized colony in control where as the colony shape was changed in different stresses eg,small in U.V, high temperature, high salt concentration and all other stresses but no colony was observed in the case of high pH. It was because that strains adapt themselves in stress conditions if the are continuously grown in the stress conditions but at fist when they are exposed to the stress conditions the genes that make them adaptable to the stress conditions are not fully activated.

Similarly in the case of cell morphology. Cells shape was altered due to the mutation in the genes and as well as the cfu/ml was decreased, in the case of temperature the CFU/ml also decreased but the cell shape was not that much altered, in the case of high osmotic stress or salt concentration the cells were decreased in size due to the loss of water and cfu/ml was also decreased and in the case of glucose depletion the cell morphology was not altered but the cfu/ml was altered.

In the case of strain 2, the control colony showed a medium size under normal or favorable conditions but different changes were observed in the case of growth under different stresses mentioned in the tables. The reason is same that high pH cause the denaturation of enzymes as a result metabolic activity is altered and colony shape and morphology is altered along with the cell shape and morphology. That is all mentioned in the tables and figures.

In the case of strain 3, the control colony showed a medium size, round shape, smooth edges, convex edges, shinny appearance and a healthy cfu/ml. but under stress conditions the shape is altered from round to irregular, and under stress of high temperature it was seen that colonies had turned to powdery in appearance it was due to the evaporation of water at high temperatures. Usually yeasts grow well at optimum temperature of 30 ͦ c but when the temperature is increased its appearance is altered and give a powdery appearance. So strain 3 also showed altered morphology under stress conditions.

In the case of strain 4, the colony was medium sized , round, smooth and shinny with a normal cfu/ml but under stress conditions the colony morphology was altered but also the cell morphology.it was also due to the fact that strains do not adapt themselves so quickly under stress conditions and show altered growth and cell morphology.

In the case of strain 5 and 6 the colonies were round, smooth and glossy, having medium size under favorable conditions but as soon as they had exposed to the stress conditions their cell as well as colony morphology was altered.

Under the conditions of U.V stress the colonies showed reduced growth it was due to the fact that U.V creates thymidine dimers in the strands f DNA as a result the truncated proteins are formed and cells are not be able to grow properly under these conditions. Only those cells form colonies who have escaped from U.V either light repair mechanism or by some other method.

Under the conditions of high temperature, cells and colonies showed altered morphology because of the inactivation of enzymes. We know that enzymes are denatured and as a result metabolic activities are stopped under these conditions

Under the stress produced by high pH. The cell wall and cell membranes are disrupted due to the imbalance of ions or charge. As a result cells and colony morphology is altered.or in most of the cases cells don’t grow. Easts grow best at low (acidic) or neutral pH.

In the case of high osmotic pressure cells are shrinked due to the movement of water outside the cell and cell shows reduced growth.

Under the stress given by depletion of glucose, cells normally grow well until the glucose is present in the media after that they show reduced growth.

Actually cells activate their MAP kinase pathway in order to activate the silent genes under stress conditions so that they can survive well under stress conditions.

depletion of glucose, cells normally grow well until the glucose is present in the media after that they show reduced growth.

Actually cells activate their MAP kinase pathway in order to activate the silent genes under stress conditions so that they can survive well under stress conditions.

Conclusions:

Yeast grow well under normal conditions but as soon as conditions become unfavorable the MAP kinase pathway is activated and cell adapt themselves to survive but this adaptation needs atleast 72 hours. But we have only exposed the cells to 48 hours as a result cells show stressed growth of cells and as well as altered colony morphology.

References: