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Different Environmental Conditions On Growth Of Fungi Biology Essay

To determine the effect of different environmental conditions on the growth of fungi and enzyme activity analysis Fungi are eukaryotic organisms in which many of them are dimorphic and the regulation between the unicellular and multicellular configurations are affected by nutritional and physical factors and these factors also affect their growth. The structure of a fungus consists of a thallus (body) and this consists of a mycelium. The mycelium is implanted in; living tissues, soil or organic matter and it consists of a cluster of slender, loosely organised hyphae. Most fungi cells are protected in a wall chitin whereas the rest have cellulose cell walls.

Fungi can either be parasites or saprocytes (use dead organic matter as a source of nutrients), and they grow best in environments that are damp and dark (Prescott et al., 2008). Although fungi require moist areas to grow they will not grow if they are flooded in water as they are aerobic and need oxygen to survive (Madigan., et al 2008). The fungi are cultivated best in media that is rich in carbohydrates such as potato dextrose agar and Starch agar. Another requirement for optimal fungal growth is that the pH of the media has to be in the range of 5 to 6 (F. Wolf and F. Wolf 1947). Carbohydrates are decomposed by heat therefore if the media is cultivated at too high a temperature or conditions too acidic or alkaline they will breakdown and hinder the growth of the fungi.

The majority of fungi are saprophytes therefore in order for the fungal cells to absorb the dead matters nutrients, their mycelial cells release hydrolytic exoenzymes that breakdown the substratum and complex molecules like; proteins and polysaccharides into monomers then these monomers are absorbed by the hyphae via osmotrophy for immediate use or the nutrients can be stored by the older hyphal areas then broken down by autolysis when they are required. Osmotrophy is activated by the mitochondria of the fungal cell which generates a proton motive force which drives the uptake of nutrients. Fungi have no chloroplasts therefore they acquire their energy via respiration by the use of mitochondria and they store the polysaccharides required for this process as glycogen (Prescott et al., 2008).

The absorption of nutrients by fungi is not completely understood as the fungal cell wall does not have pore big enough to allow enzymes to escape. Enzymes are proteins that have a size ranging from 30-50 kDa (Dames., 2011).

The three fungi being studied are Trichoderma, Penicillium and Aspergillus. We are aiming to investigate the optimal conditions for the growth of Trichoderma as it is important in our day to day lives, for example; Trichoderma is a soil fungi and is used to control the diseases in plants and to hasten the growth through quickened root growth (N. Ranasingh et al., 2006). These investigations would allow us to have a mass production of this fungi for the benefit on human and plants.

Penicillium is another important fungus as it aids us in the production of the antibiotic penicillin. The investigation allows us to be able to choose methods to cultivate it, for example the digestion of cellulose tells us that we can cultivate the fungus on trees and we also learn about how they help animals such as cows digest cellulose as they are present in the Rumen. Aspergillus is used in the manufacture of citric acid with is an additive in food (Prescott et al., 2008).

The main objective of the experiment is to study the anatomy and physiology of the fungi; the conditions they grow in optimally such as temperature or pH of the environment. The temperature that is to be used to cultivate the fungi Trichoderma are; 8oC, 25oC, 30oC and 37oC. The pH being used in the study of Trichoderma is; 3, 4, 5, 6, 7, 8 and 9. We also aim to study the effect of the extra cellular enzymes on polysaccharides such as starch and cellulose, and these are all kept at a constant temperature of 25oC. I hypothesize that the growth of the fungi will be directly proportional to temperature up to a certain point which is the optimum temperature then it will decrease after this point like in a normal distribution. The optimum growth at a specific pH range will also follow a normal distribution.

Materials and Method

Experiment 1

Aim: To investigate how temperature affects fungal growth

Materials:

Trichoderma fungal culture PDA plates

12x PDA plates

Cork borer

Plastic for incubating culture plates at 30oC and 37oC

Method:

Two perpendicular lines that intersect at the centre of the PDA plates were drawn underneath them. The plates were then labelled such that there were 3 plates each to inoculate the fungi at the temperatures of; 8oC, 25oC, 30oC and 37oC. The cork borer was then dipped ion ethanol and put into a Bunsen flame to sterilize it. The cork borer was then used to punch as many holes as possible on the outer edges of the fungi.

The tip of a glass Pasteur pipette was then dipped in ethanol and then inserted into a Bunsen flame to sterilize it. The pipette was then use to inoculate each of the 12 plates centrally with the diameter of each inoculum being measured along the lines and then the average calculated. The plates at 30oC and 37oC were placed in a plastic bag so that they would not dry up and lose moisture. All the Plates were then placed in the appropriate incubators and the average changes in diameter were measured every 24 hours for a period of 7 days.

Experiment 2

Aim: To investigate the effect of pH on fungal growth

Materials:

7x 50ml Malt extract agar (MEA) double strength bottles

7x 50ml Buffers of pH; 3,4,5,6,7,8,9 each

21x Petri dishes

Method:

Two perpendicular lines that intersect at the centre of the petri dishes were drawn and the dishes were all labelled such that there were 3 petri dishes for each pH value. The MEA and the appropriate pH buffer were removed from the water baths and placed near a burner (These have to be kept warm or else they will solidify). The MEA and the buffer were then poured together and swirled on the bench before being poured into all the appropriate petri dishes which had the buffer pH noted. This was done for all the pHs.

The MEA and buffer mixture was left to solidify and upon solidification each of the dishes was inoculated centrally with the Trichoderma plugs that were left over from experiment 1. The mean diameter along the lines for each plug was noted then the petri dishes were then all incubated at 25oC. The mean changes in diameter were measured every 24 hours for 1 week.

Experiment 3

Aim: To discover the effect of exoenzymes on the breakdown of starch

Materials:

4x Starch agar plates

Two extra fungal cultures apart from Trichoderma

Pasteur pipette

Method:

The 4 starch agar plates were labelled with the name of two extra fungi (Penicillium and Aspergillus) on one plate each and Trichoderma on the third one. The last plate was left empty so that it would function as a control for the experiment. The plates then had two perpendicular lines intersecting at the centre of them drawn on the back of the plates. A Pasteur pipette was dipped in ethanol and placed in a Bunsen flame to sterilize it. The pipette was used to inoculate the petri dishes centrally with one plug of each fungal culture and the control was empty.

The petri dishes were then left to stand in an incubator at 25oC for 6 days.

Experiment 4

Aim: To show fungal ability to use complex substrates such as cellulose

Materials:

4x Carboxymethycellulose plates (CMC)

Two extra fungal cultures apart from Trichoderma

Pasteur pipette

Method:

The 4 CMC plates had 2 perpendicular lines drawn on each of them that intersected in the centre. The plates were then labelled with the name of two extra fungi (Penicillium and Aspergillus) on one plate each and Trichoderma on the third one. The last plate was left empty so that it would function as a control for the experiment. A Pasteur pipette was dipped in ethanol and placed in a Bunsen flame to sterilize it. The pipette was used to inoculate the petri dishes centrally with one plug of each fungal culture and the control was empty.

The petri dishes were then left to stand in an incubator at 25oC for 6 days.

Results

Table 1a: Showing the growth of Trichoderma in mm at 8oC over 7 days

Day

1

2

3

4

5

6

7

Growth 1

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Growth 2

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Growth 3

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Average

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Table 1b: Showing the growth of Trichoderma in mm at 25oC over 7 days

Day

1

2

3

4

5

6

7

Growth 1

0.00

2+2

12+14

15+13

15+17

45+23

44+45

Growth 2

0.00

1+1

12+13

11+14

16+15

47+28

51+28

Growth 3

0.00

3+3

10+7

-

-

-

-

Average

0.00

1.50

11.33

13.25

15.75

35.75

42

Table 1c: Showing the growth of Trichoderma in mm at 30oC over 7 days

Day

1

2

3

4

5

6

7

Growth 1

0.00

1.5+1.5

10+9

19+12

17+14

21+19

43+25

Growth 2

0.00

2+2

12+12

15+12

15+17

26+17

31+24

Growth 3

0.00

1.5+1

10+14

16+12

17+17

17+17

19+18

Average

0.00

1.58

5.58

14.33

16.17

19.50

26.67

Table 1d: Showing the growth of Trichoderma in mm at 37oC over 7 days

Day

1

2

3

4

5

6

7

Growth 1

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Growth 2

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Growth 3

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Average

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Graph 1: Graph showing the growth of Trichoderma in mm at 8oC for 7 days

Graph 2: Graph showing the growth of Trichoderma in mm at 25oC for 7 days

Graph 3: Graph showing the growth of Trichoderma in mm at 30oC for 7 days

Graph 4: Graph showing the growth of Trichoderma in mm at 37oC for 7 days

Graph 5: Graph showing the growth rate of Trichoderma in mm/day over the four temperatures

mm/day

In Trichoderma growth will not occur if the temperature is below 8oC as indicated by graph 5. Graph 5 also indicates that the maximum temperature for fungal growth is 37oC. In this experiment the optimal temperature for growing fungi in order to have the fastest growth rate possible is 25oC as it has the highest growth rate in Graph 5.

Table 2a: Showing the growth of Trichoderma in mm at a specific pH value at 25oC

pH

3

4

5

6

7

8

9

Day 1

0.00

0.00

0.00

0.00

0.00

0.00

0.00

Day 2

2.83

2.83

2.67

2.67

3.67

1.75

6.67

Day 3

6.83

9.00

8.83

8.25

10.83

5.25

8.5

Day 4

14.80

15.60

26.00

13.67

22.80

8.25

8.33

Day 5

21.00

17.03

21.67

21.00

26.33

9.75

7.50

Day 6

20.88

28.6

27.00

22.50

28.83

11.25

8.33

Day 7

20.50

30.00

29.00

18.75

24.75

17.50

10.83

Table 2b: Showing the growth rate in mm/day of Trichoderma in mm at a specific pH value at 25oC

pH

3

4

5

6

7

8

9

Growth rate

2.93

4.29

4.14

2.68

3.54

2.5

1.55

Graph 6: Showing the growth rate in mm/day of Trichoderma in mm at a specific pH value at 25oC

The growth of the fungi is more at the acidic pH values as compared to the more basic pH values.

Table 3: showing amount of starch and cellulose degraded by fungal exoenzymes in mm at 25oC

Fungi

Distance of starch

degraded/ mm

Average Starch degraded/ mm

Distance of starch

degraded/ mm

Average Starch degraded/ mm

Trichoderma

7+8+6+5+6

6.4

0

0

Aspergillus

3+1+2+2+1

1.8

0

0

Penicillium

8+8+8+5+8

7.4

0

0

Control

None degraded

None degraded

-

Trichoderma, Aspergillus and Penicillium all contain amylase that degrades starch. Penicillium degrades starch the most followed by Trichoderma then Aspergillus.

The table also indicates that none of the fungi have exoenzymes with the ability to degrade cellulose as no cellulose was consumed inside the petri dish.

Discussion

The results of the experiment are as expected which proves my hypothesis correct. Most living systems make use of the normal distribution curve and as shown in graph 5 my results follow this. The fungus grew exponentially up to the optimal temperature which was 25oC. At this temperature this is where growth is fastest because the kinetic energy is highest here meaning that the enzyme activity at the point is the highest. Enzymes are proteins that are biological catalysts and like all proteins their activity and conformation is affected by temperature and pH. Below 8oC there is no growth at this temperature because here, the enzymes present in the fungus lack kinetic energy rendering them inactive. Past 25oC enzymes are starting to be denatured by the high temperatures and therefore at 35oC all the enzymes are denatured and therefore there is no growth.

Graph 6 also shows a normal distribution whereby the fungi grows best at the acidic pH values then as pH approaches the more basic values the growth of the fungi is retarded. The pH affects growth because the enzymes of specific fungi have optimum activity at a specific pH, and above or below that there is minimal activity or the enzymes could be denatured. The pH of the environment also affects growth because it aids them in the hydrolysis of proteins and carbohydrates (F. A Wolf and F. T Wolf., 1947)

The best temperatures to cultivate Trichoderma are 25oC and 30oC as supported by the Graphs; 2, 3 and 5, however the best temperature to grow it is 25oC. It also grows very well in acidic to neutral conditions as opposed to basic conditions (Wolf and Wolf 1947). The growth of Trichoderma at these pH values is expected as it grows in the soil as it lives in symbioses with plants. These pH values help the plants to grow and support nitrogen fixation and the plant may supply the fungi with nutrients. However a more accurate result could have been obtained if petri dish 3 in the 25oC cultivation had not been spoiled by spores spreading all over the container. Trichoderma produces spores in abundance and some spores got released by the conidia of the fungus contaminating the whole container and making measurements difficult to obtain (F. Wolf and F. Wolf 1947) as in petri dish 3 in experiment 1 that was at 25oC. This release of spores can be triggered when the fungi is about to die and it therefore releases spores to neighbouring surroundings to ensure that its survival carries on.

There is an anomalous result at pH 6 and this is because of the petri dish being dropped. Many of the petri dishes had error in them as many dishes were put into one incubator and upon people collecting their cultures they may have dropped the dish thereby displacing the inoculum that was at the centre of the petri dishes.

The enzyme amylase is responsible for the break-down of starch in the fungi. Starch is a big polysaccharide and amylase acts on it to produce oligosaccharides the glucose monomers which can then be absorbed by the hyphae. Trichoderma also contains cellulases but as shown in table 3 it did not degrade cellulose as clearly visible as did the amylase. This could as a result of the fungi releasing amylases to move on ahead and digest the starch before the hyphae arrive, but in the CMC experiment the fungi may only have been using cellulases to digest the substratum, and it may not have released enzymes to digest cellulose on ahead. The choice to secrete enzymes to go and digest complex molecule could be based on the amount of nutrients available at that time. Cellulose contains more glucose units than starch and therefore it would provide more nourishment to the fungi such that it would not need to digest cellulose before the hyphae arrive.

The non-visible digestion of cellulose can also be explained by the reason that cellulose is much bigger and stronger than starch therefore when it comes down to enzyme activity acting on it, it is more difficult to break down and therefore that is why there was a clear ring between the fungi and the starch in experiment 3 and there was no visible ring in experiment 4 which investigated the effect of cellulases. When starch and cellulose are broken down to glucose, this glucose is then used in respiration in the mitochondria. The glucose can either be converted to pyruvate via glycolysis then pyruvate kinase then uses the pyruvate to convert ADP to ATP. The pyruvate could then also enter the Krebs cycle which occurs in the mitochondria to produce more ATP.

Table 3 could not be completed due to the control experiment being contaminated with a fungus which was most probably Aspergillus niger. A spore entered the petri dish and it grew rapidly in the cellulose and proposed by F.A Wolf and F.T Wolf (1947) that fungi need a rich supply of carbohydrates to grow.

A source of error in the experiment was that there were a lot of contaminations in the petri dishes and this is due to the fungus Aspergillus niger which produces spores in abundance (F. Wolf and F. Wolf 1947). Dames (2011) suggests that these spores have been present in the air around the laboratory since last year, therefore if the petri dishes were not close enough to the burner they would probably get contaminated by the fungus as it is very difficult to get rid of.

There was also a decrease in the length of fungi measured for some of the dishes. This error can be down to the measurement being taken, some of the cultures were measured along the lines whereas the others had their longest extension measured. Therefore when each dish was measured by either me or my partner we had different standards or points of measurement thereby some measurements being longer than others.

Another reason for the hyphae shrinking in size could be that the fungus was dying, and this resulted in the hyphae getting smaller. As observed in “table 2” the shrinks were the last results recorded therefore the fungi may have started dying around day six of the experiment. The shrink in the hyphal length is due to the collapse of the cell wall, thereby causing the hypha to have a tubular deflated shape (E.M Miguelez., et al 1999). This is the same principle that is applied to antifungals such as nystatin and nikkomycins that inhibit cell wall synthesis and hyphal growth (Dames., 2011).

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