The Effect of Temperature on the Growth of Aspergillus Oryzae: An Experiment

A2 Practical Investigations
Title: The effect of Temperature on the growth of Aspergillus oryzae
Develop a Hypothesis
This particular investigation is to discover how a range of temperatures effects the growth rate of the fungi Aspergillus oryzae. Most fungi’s tend to survive within the temperature range of 5-35oC, with the optimum depending on their normal environmental temperature.
The fungi Aspergillus oryzae is heterotrophic which means they taken in their food from dead organic matter and cannot produce food for themselves and use the organic carbon in dead organic matter to grow. Temperature can influence shape of the enzyme within the fungi, which effects growth. Enzymes tend to have an optimum temperature for enzyme activity; however it structure can be effected if not within optimum temperature. Fungi’s secretes enzymes on to its food substrate and absorbs the soluble products of the extra-cellular digestion, on which the fungus grows. Therefore optimum temperature will increase growth. The diffusion rate of these dead organic matters is affected by temperature. According to Kinetic theory higher temperatures increase the energy and therefore the movement of the molecules, increasing the rate of diffusion, therefore increasing growth. Lower temperatures decrease the energy of the molecules, thus decreasing the rate of diffusion. The optimum diffusion rate varies with different fungi’s.
The growth of Aspergillus oryzae requires favourable conditions in terms of temperature (around 25oC) which affects components such enzyme activity and diffusion rate which ultimately influence the growth of these particular fungi.

Hypothesis: My hypothesis is that Aspergillus oryzae will grow at a slower rate when the temperature increases or decreases from 25oC, as it increases in temperature it will reach a temperature where growth will stop.
Prediction of Results
I predict that that the Growth of the fungi Aspergillus oryzae at lower temperatures will be smaller as the temperature decreases from 250C. However I also predict that the in higher temperatures the Growth of the Aspergillus oryzae will be a greater surface rea, until it reaches a temperature around 40oC were growth will decreases rapid and stop completely as the enzyme becomes denatured. My prediction for the optimum temperature for Growth of Fungi is 25oC and any temperature above or below this value will have less growth.
Plan a procedure
Equipment and Materials
Bunsen burner
White try
Methylated spirit
Cotton wool
5x Petri dish
Bottle of starch agar
Water-bath
Inoculation loop
Aspergillus oryzae
Incubator
Range of independent variables
The chosen independent variable for the growth of the fungus (Aspergillus Oryzae) is Temperature. The temperature range of the Aspergillus oryzae varies from 20-40oC increasing in from every 5oC 20oC, 25oC, 30oC, 35oC and 40oC. I used these range of temperatures as gives an indication of the growth of fungi either side of it optimum temperature (25oC) and how growth rate is affected in high and low temperatures. For example higher temperature could cause the enzyme within Aspergillus oryzae to become denatured which would rapidly decrease the growth rate and cause the fungi to die. Having a consistent spread of temperatures increasing gradually ensures the result are valid and can be used to compare how growth changes as it move towards it optimum temperature and then when temperature exceeds the fungi optimum temperature.
Method Plan
Growth of Fungus (Aspergillus oryzae) on an Agar Plate over a range of Temperatures.
White tray Preparation
1. Light Bunsen burner (yellow tipped flame)
2. Using methylated spirits dipped in cotton wool swab the white tray (around 1m x 0.5m)
Agar Plate Preparation
1. Place a Petri dish on the white tray you prepared earlier.
2. Place a bottle of starch agar in water-bath until in a molten state, ensuring that it not to hot
3. Remove the stopper with your little finger (a) from the bottle of agar. Be careful not to place the stopper on bench as this would cause contamination. Also the bottle must not be held vertically as they will once again cause contamination.
4. Careful flame the neck of the agar bottle, ensuring not to burn the fungi
5. With your other hand lift the lid of the petri dish (b). When lifting the lid, simply raise the lid, to provide enough space for the medium to be poured to a depth of approximately 5 millimetres. Do not lift the lid to the side.

6. After replacing the lid, gently route the Petri dish to ensure the medium is homozygous.
7. Leave undisturbed for 20 minutes, to allow the medium to set.
8. After the agar is set, invert the Petri dish and label with your initials and the date. Also mark an inoculation target such a small dot in the middle of the dish.
9. Repeat the process in five different Petri dishes
Inoculation of Plate
1. Place the plate, lid side facing you, on the white tray.
2. Using a match light the Bunsen burner.
3. Holding the inoculating loop in your right hand, draw in back and forth through the flame until it gives a red hot glow, this is the sterilisation process.

4. Loosen the screw cap of the Aspergillus culture
5. Holding the bottle containing the slope culture of Aspergillus your left hand. Remove the top with your little finger of your right hand. Flame the neck of the bottle with yellow flame.
6. Carefully bring together the loop into the bottle, allowing the end of the loop to come into contact with an uncolonised region of the ager slope. This ensures it has been fully cooled.
7. Remove some of the Aspergillus oryzae culture, then flame the neck of the bottle and replace the top.
8. Lift the Petri dish at one side and dap the loop containing the Aspergillus oryzae on the centre of the agar. Quickly close lid of Petri dish and seal using tape.
9. Repeat steps 6-8 for each of the five Petri dishes.
10. Invert the dish and place in incubator. Each dish has to colonise in a range of temperatures with each in a different temperature incubator. The temperature have to be set at 5oC 20oC, 25oC, 30oC, 35oC and 40oC for five different dishes.

Controlled Variables
All other variable within the experiment have to be controlled to ensure that the independent variable (temperature) is the only factor which can affect the growth of Aspergillus oryzae. The controlled variables include pH and oxygen levels (aerobic conditions), light and same agar culture. Variables are physical, chemical or biological quantities or characteristics that will affect growth of fungi.
The pH of the medium is important for it influences mineral availability, enzyme activity and membrane function. Generally speaking, fungi can tolerate a wide range of pH, though most media used to culture fungi are acidic. The agar provides a constant pH for all the cultures which ensures a fair test as on temperature is affecting growth of fungi.
The aerobic conditions can be controlled as all dishes will remain in the same volume of air within the same classroom. This will limit the effects that oxygen will have on the growth of each culture.
Light is controlled through holding the Petri dishes within the same environment. Although light does not play a major part in metabolism and growth of fungi, controlling this variable will ensure, that a fair test is carried out and the results can be seen as reliable.
Lastly using same agar culture from the same bottle will ensure that that the components within is the same for all the cultures. If the volume of the agar plates where different then this may have encouraged or limited growth depending on the plates. The volume of the agar plate has to be constant for all five cultures. This ensures a fair test as only the independent variable (temperature) has been allowed to affect the dependent variable (growth of Aspergillus oryzae).
Planning for Analysis
Units used
The Growth of the dependent variable the fungi Aspergillus oryzae is measured in mm2. The degree of accuracy will be to two decimal points which will reduce measurement errors. Whereas the independent variable (temperature) is degrees/oC. In order to calculate the surface area of the fungi, I plan to draw around the fungus growth and using squared paper to calculate the growth in mm2.
Format to present Results
The Table of result will include the Title: The effect of Temperature on the surface area of growth of Aspergillus oryzae. It will also illustrate the independent variable, Temperature/ oC with the five temperatures used 5oC 20oC, 25oC, 30oC, 35oC. It will also include the dependent variable, surface area of Growth of Fungi (Aspergillus oryzae) /mm2.
The Graph will also include the Title: The effect of Temperature on the growth of Aspergillus oryzae. The independent variable is always on the horizontal x-axis, and the dependent variable is always on the vertical y-axis. This means the independent variable, Temperature/ oC is on the x-axis and the dependent variable, Growth of Fungi (Aspergillus oryzae)/mm2 is on the y-axis. The graph will be a standard line graph.
Methods of Analysis
The method of analysis which is the most appropriate for continues data include student’s t-test or confidence limits. Continuous variation is variation that has no limit on the value that can occur within a population. A line graph is used to represent continuous variation. The type of statistical test for discontinuous data is Chi squared test. This statistical test isn’t suitable as the results do not have distinct groups for organisms to belong to. As temperature and surface area of growth of fungi is continuous data (no limit), could use either student’s t-test or confidence limit. However due to time restraints confidence limit is acceptable as it requires the least replicates of each treatment. Also I choose to use Confidence limit, as t-test only used to compare to different treatments (temperatures), whereas 95% Confidence limits has no restrictions on sample means.
Successful Analysis
In order for the results for confidence limits to be reliable, required a certain number of replicates which would help to reduce error. In Confidence limits the standard deviation of the mean indicates how close the sample surface area is to the population mean. By attaching Confidence limit to surface area of fungi you can make it more reliable. Confidence limit is 95% sure that the population mean will within these limits. If there were no restrictions, the recommended number of replications would be 10 or more at each temperature range. However this isn’t feasible to limiting factors such as time and equipment. As we only have a relatively short period of time to carry out these replications, the minimum number of replicates which can be used and still be reliable are 3 at each treatment (temperature). I will have to reduce the number of replicates because of lack of availability of equipment within school. This can be justified by using the class’s results to include in the replications for each treatment. Another limiting factor which has contributing to using a smaller number of replication is time restrictions. There would not be enough time to do 10 replications for all five temperatures. Despite these factors three replications for each treatment will still provide adequate analysis of the how temperature affects fungi growth.
The effects that a range of Temperatures has on the surface area of growth of Aspergillus oryzae within an agar plate of Starch agar.
Temperature/oC
Replicate
Surface area of growth of Aspergillus oryzae/ mm2
Average surface area of growth of Aspergillus oryzae/ mm2
15

20

25

30

Surface area of Aspergillus oryzae/ mm2

Temperature/oC
Replicate
15
20
25
30
1
0
22
299
475
2
0
30
112
374
3
0
68
321
533
4
0
61
269
484
5
0
54
519
493
6
0
54
506
421
7
0
77
391
399
8
0
75
417
429
9
0
79
458
618
10
0
70
566
556
11
0
40
346
764
12
0
54
198
456
13
0
103
434
893
14
0
109
445
745
15
0
118
696
862
16
0
108
738
788
17
0
132
436
829
18
0
65
660
848
19
0
24
142
501
20
0
14
157
397
Average Surface area of Aspergillus oryzae/ mm2
0
67.85
405.5
593.25
Degree of freedom
(n-1)
20-1=19
20-1=19
20-1=19
20-1=19
Students t value
2.093
2.093
2.093
2.093
Standard Deviation
0
33.40
180.04
181.02
Standard Deviation (error) of the mean
0
7.47
40.26
40.48
Upper Confidence Limit
0
83.48
489.76
677.97
Lower Confidence Limit
0
52.22
321.24
508.53 A Table showing the effect that a range of Temperatures has on the Class results for the Growth of Aspergillus oryzae within an Ager plate of Starch aga
Interpretation

Temperatures/ oC

15
20
25
30
My results for Average surface area of growth of Aspergillus oryzae/ mm2
0
26
205.5
424.5 Class results Mean/mm2
0
67.85
405.5
593.25
Upper Confidence Limit
0
83.48
489.76
677.97
Lower Confidence Limit
0
52.22
321.24
508.53
Width of Confidence Limit
0
31.26
168.52
169.44

Assessment of the reliability of the data using statistical evidence
I assessed the reliability of the data using statistical evidence. Through plotting 95% confidence limits on a graph I was able to assess the reliability of the sample mean (growth of Aspergillus oryzae) in a range of different temperatures. The sample size we used was 20 replicates at each temperature, two of which was my results.
As you can see from the table 15oC had no effect on the growth of Aspergillus oryzae. For 20oC the 95% confidence limits was relatively narrow with a width of 31.26mm2 from 83.28mm2 upper and 52.22mm2 lower confidence limit. My result for surface area of growth of Aspergillus oryzae in 15oC was 26mm2 which smaller than the class mean (67.85mm2) and is outside the lower confidence limit (52.22mm2). This could have affected the reliable of the result for class mean as my result was considerable below the sample mean for 20oC.
In terms of 25oC the 95% confidence limits is a lot wider, which results in poor reliability for the class mean for the growth of Aspergillus oryzae. In relation to 405.5mm2 mean, the upper confidence limit was 489.76mm2 and the lower 95% confidence limit for the sample size of Aspergillus oryzae was 321.24mm2. This is a width of the 95% confidence limits is 168.52mm2 in relation the mean. This is a relatively wide confidence limit which would suggest that the reliability is poor. My results for surface area for the growth of Aspergillus oryzae was 205.5mm2, which again was greatly lower than the class mean with a difference of 200mm2, which is also smaller than the lower confidence limit. This may have affected the reliability of the data obtained for class mean.
The 95% confidence limits for 30oC was also wide and may have affected the reliability of the data for surface area growth of Aspergillus oryzae obtained. The class results mean for growth of the fungi (Aspergillus oryzae) was 593.35mm2. The width of the confidence limits was large at with the upper 95% confidence limit at 677.97mm2 and lower 508.53mm2. The width of 95% confidence limits for 30oC is 169.44mm2, which has poor reliability. My results for growth at 30oC feel outside the 95% confidence limits (see table), which is a large variation in compared to the class mean which can have an effect on the reliability of the mean surface area.
For both 25oC and 30oC, the confidence limits where relatively wide, a width of 168.52mm2 and 169.44mm2 respectively. This reliability could have been improved through an increase in the number of replicates, which would help to make 95% confidence limits narrower and therefore improve reliability for the growth (surface area) of the fungi (Aspergillus oryzae) at these two temperatures. However for 15oC the 95% confidence limits was fairly narrow (31.26-52.22mm2) and therefore reliable. To further increase the width of the 95% confidence limits I could increase the number of replicates further, would help to achieve more precise results and fewer variation in growth. Trends identified using Confidence limit and Explanation of trends
From my results you can clearly see that temperature has a direct effect on the growth of the fungi Aspergillus oryzae. In order to use the sample mean for surface area of Aspergillus oryzae to be reliable, we need calculated 95% confidence. The confidence limited ensured that 95% probability that the mean surface area growth (of Aspergillus oryzae) for each temperature lies within that range.
As you can see from the table above, that the surface area of growth of Aspergillus oryzae did increase as the temperature increased. Therefore the most growth was at 30oC with the class mean at 593.25mm2 and the lowest with no growth at the lowest temperature 15oC. Consequently 25 oC had the second highest growth at 405.5mm2 and thirdly was 20oC with 67.85. At 15oC for both my results and the class results there was no growth. These results generally agreed with my hypothesis,
“Aspergillus oryzae will grow at a slower rate when the temperature increases or decreases from 25oC, as it increases in temperature it will reach a temperature where growth will stop.”
However from the means from our class results we established that for the optimum temperature for growth Aspergillus oryzae was 30oC compared to 25oC as stated in my hypothesis. Also the growth medium starch agar was an appropriate culture to grow fungi and wasn’t affected by temperature. This ensured reliability at these higher temperatures. As you can see from the graph and table the 95% confidence limits of the samples (Aspergillus oryzae) from the different temperatures (15 oC, 20 oC, 25 oC and 30 oC) did not overlap, hence this strongly suggests (95% level of probability) that the four samples of Aspergillus oryzae is significantly different. For example the upper confidence limit for 20oC is 83.48mm2, with the lower confidence limit for 25oC at 321.24mm2 and difference of 237.76mm2 from overlapping. There also a significant difference between 30oC and 25oC, 95% confidence limits with 25oC at 32.24-489.76mm2 and 30oC lower and upper confidence limits at 508.53-677.97mm2.
In order to get a better understanding of how temperature affected Aspergillus oryzae we could have used a wider range of temperatures. This would help to confirm my hypothesis that the enzyme would become denatured at around 40oC. Aspergillus oryzae did not growing at 15oC and this could have been due to slow or non-existent diffusion rate and also at lower temperature the enzyme within Aspergillus will less readily fit in the substrate.
As I explained 30oC had the most significant growth. This could be due the enzymes within Aspergillus working better (more complementary shape) at this temperature as it closer to its optimum. More growth would have occurred at 30 oC compared to 25 oC and 20 oC as the fungi will secretes enzymes on to its food substrate at a faster rate and absorbs the soluble products, which Aspergillus oryzae uses grows. Therefore optimum temperature will increase growth.
The particle theory could also have had an impact on the mean growth being greater at 30oC. At higher temperatures, it increases energy and therefore the movement of the molecules, increasing the rate of diffusion and therefore growth. Lastly the metabolism of Aspergillus oryzae works better at higher temperatures, therefore increasing growth.

Evaluation of the Practical Procedure
When carrying out a practical procedure it is important the independent variable is appropriate in showing a comprehensive impact that the independent variable (temperature) has on Growth of Aspergillus oryzae. In terms of the range of temperatures we used 15oC, 20 oC, 25 oC and 30oC. All the temperatures except for 15 oC were appropriate as Aspergillus oryzae grew at these temperatures. However at 15 oC there was no growth meant that the range of the independent variable (temperature) was appropriate, as it helped to illustrate what temperatures the fungi will starts to grow at. Despite this, a wider range with shorter intervals would provide more detailed results and would demonstrate how Aspergillus oryzae is affected from when it starts to grow, to when growth stops through the enzyme becoming denatured. However we were unable to heat the fungi above 30oC within school as it is too close to body temperature and there is a risk that the fungi (Aspergillus oryzae) will become pathogenic, which could cause illness
Procedures to prevent variation of factors not under investigation
To avoid other factors (variables) affecting the investigation you can adapt your procedure to ensure that temperature was the only factor effecting the growth of Aspergillus oryzae.
Same culture bottle
Through using the same culture bottle ensures that for all four temperatures have the same sample of Aspergillus oryzae. This helped to ensure that each sample was the same specific form of fungi. However for the mean class results, some students used another culture bottle which may have caused variations in the growth of fungi. In order to prevent this variation in the future we need to ensure to only use the same culture bottle for all samples.
Light
The agar plates containing the Aspergillus oryzae for 20oC, 25oC and 30oC were kept within the same light conditions. This was controlled within the incubators which were dark throughout the growth period. However for the 15oC samples the Aspergillus was within a different light condition as they were held within a growth chamber, where light was present. This may have affected the growth of the spores as many types of fungi grow better in a particular light condition. This variable in the experiment could have been improved by holding all four temperatures within an incubator.
Growth medium (pH, nutrients)
For all four temperatures the same growth medium starch agar was used to culture the Aspergillus oryzae within the agar plates. This starch agar helped to control the pH of Aspergillus oryzae, which ensure the temperature was the only variable that had an effect on the growth of Aspergillus oryzae. Also using the same growth medium ensured that each sample had the same nutrients available and ultimately constant conditions for the spores to culture. However to improve this procedure; I could have measured out equal volumes of the starch agar.
Time
All the agar plates (containing the Aspergillus oryzae) were left in the incubator at a constant temperature for a week. We did this carefully for all the agar plates to prevent any sample having a longer time to culture. This ensured that the results where reliable as every sample had the same amount of time to grow.
Appropriateness of the observations/ measurements
In order to measure surface area of growth of Aspergillus oryzae, we drew around the growth area with transparent paper and then counted the surface area on mm graph paper. I felted that using the small squares to count surface area would produce accurate and specific results. However this was both a long and difficult process. Also at times counting half squares could have produced varied results which could have effected precision of the data. Another method of measuring the surface area may have been more appropriate and accurate.
The use of the agar plate containing starch agar as the growth medium helped with producing a consistent measurement of the growth. It ensures you could see the growth of the fungi as the plates were transparent. This was relevant with the measurements as the surface area of the growth could be drew around in order to get a quantitative data for surface area of Aspergillus oryzae.
Validity of experimental design and Procedures
Errors
There were a number of errors within this practical procedure which may have caused results for surface area of growth of Aspergillus oryzae which is outside the true value. This is the accurate value which would be found if the surface area could be measured without any errors. Data are only valid if the measurements that have been made are affected by a single independent variable (temperature) only. They are not valid if the investigation is flawed and control variables have been allowed to change. Conclusions are only valid if they are supported by valid and reliable data measured to an appropriate level of accuracy.
The main error in the practical is the temperature of the 15oC samples were not controlled accurately. Due to lack of resources there were not any available incubators to control the temperature. Hence the 15oC, samples were kept in a growth chamber where the temperature wasn’t regulated. Therefore the conditions would have varied and as this independent variable was not kept constant. This would have affected the validity of my results; however we didn’t expect growth at this temperature and so it did not affect the outcome of the experiment.
Another error which could have affected the analysis of the class results, was that some samples may have incurred hot spots within the incubator. This may have produced some anomalous results, which would have affected the class mean for surface area growth of Aspergillus oryzae. This may have been explained by replicate 13 growths at 30oC which was 893mm2, which is 299.75mm2, above the average (593.35). However this variation could not have been corrected and would have affected the validity of the 95% confidence limits.
When carrying out the practical each person may have used different Aseptic techniques during the practical procedure. For example when transferring the spores from the culture bottle to the starch agar (within the agar plate), the spores may have dropped off. As the spores are not visible you are not aware if the spores have been lost during transfer.
Also we could not fully control the mass of spores placed within the agar dish. This also may have produced anomalous results which would produce a less precise class mean.
Lastly the actual fungi Aspergillus oryzae has validity issues as it is a biology material which can not be controlled. The growth of the Aspergillus of the two different bottles used may not have been the same as each other. As the class used two different culture bottles, then two loops would have extract different cultures.
Despite these potential errors I feel they didn’t have a conclusive impact on the results I obtained.
Practical outline of how light affects the growth of fungi (Aspergillus oryzae)
Another independent variable which has an effect on growth of fungi is light conditions. In order to manipulate the light conditions the agar plates containing the Aspergillus oryzae is placed within three different light intensities. The first set of samples will be in a dark cupboard (no light present). The second set samples of Aspergillus oryzae are placed in normal lighting and the third condition will be with a lamping shining directly on the agar plates. To ensure that it is a fair test, the temperature of the agar plates is at kept constant through all the samples being cultured at room temperature as well as all samples being placed in the same mass of starch agar. Then after seven days, compare the growth (through measuring surface area) of Aspergillus oryzae in different light intensities.