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Candidate: D.

Gustav Christensson
mrmodenait@gmail.com

The Effect of
Temperature on
Fungal Growth
How does the temperature of a nutritive environment affect the
doubling time of Penicillium chrysogenum?

International School of Modena


IB Diploma Programme
Biology Extended Essay
May 2017 Session
3,997 Words
The Effect of Temperature on Fungal Growth

1 ABSTRACT
Penicillium chrysogenum is a fungus in the family Trichocomaceae, often found on food or
in damp indoor habitats. As it reproduces by releasing spores into the environment, it
possesses a species-specific doubling time (the time taken for the population size to
double). This investigation explores how the temperature of the surrounding environment
(studied from 11.3°C to 44.1°C) affects the species’ doubling time. The aim was to find the
optimum and worst temperatures for the species to grow in, and to determine what doubling
time the species would exhibit. To answer the questions, test-tubes containing P.
chrysogenum and Saboraud Dextrose Agar were immersed in a water bath and a
refrigerator to obtain eight regulated temperatures. The population size was measured
before and after immersion using spectrophotometry, by considering the turbidity (tendency
to scatter light) of the sample, which was shielded in a cone of black card. The turbidity was
found by comparing the incident light intensity onto the sample with the transmitted light
intensity, measured with a photometer. From the difference in turbidity, the doubling time
was calculated. The results showed that growth was slowest and that doubling time was the
longest at 35.3°C (141.3 hours), but it was shortest at 44.1°C (20.04 hours). An inverse
fourth-order polynomial regression identified two peaks of maximum doubling time at 16.2°C
and 35.7°C. Additionally, it predicted faster growth in between, with a local minimum
doubling time at 25.3°C. The extrapolation also showed that doubling time tends to zero at
either temperature extreme. Conversely, the large uncertainty and several methodological
issues (such as the cell size interfering with the scattering of light, or the difficulty in
identifying the species) may have compromised the reliability of the data.

Word count: 282 words


D. Gustav Christensson

2 CONTENTS
3 Introduction ................................................................................................................... 1

4 Hypothesis .................................................................................................................... 2

5 Design .......................................................................................................................... 2

5.1 Proposed Set-Up .................................................................................................... 2

5.2 Variables ................................................................................................................. 3

6 Method.......................................................................................................................... 4

6.1 Equipment .............................................................................................................. 4

6.2 Procedure ............................................................................................................... 4

6.3 Diagrams ................................................................................................................ 5

6.4 Risk Assessment .................................................................................................... 7

7 Data Tables .................................................................................................................. 8

7.1 Raw Data ................................................................................................................ 8

7.2 Notes .................................................................................................................... 10

7.2.1 Observations .................................................................................................. 10

7.2.2 Measuring the Spread of Data ....................................................................... 10

7.2.3 Standard Deviation in Elapsed Time .............................................................. 11

7.3 Processed Data .................................................................................................... 11

7.4 Notes .................................................................................................................... 12

7.4.1 Calculation of the Absorbance ....................................................................... 12

7.4.2 Calculation of the Doubling Time ................................................................... 12

7.5 Sample Calculations ............................................................................................. 13

8 Visualised Data ........................................................................................................... 17

9 Results........................................................................................................................ 18

10 Discussion .................................................................................................................. 19

11 Evaluation of Method .................................................................................................. 20

12 Conclusion .................................................................................................................. 21

13 Acknowledgements..................................................................................................... 22
The Effect of Temperature on Fungal Growth

14 References ................................................................................................................. 22

15 Appendices ................................................................................................................. 25

15.1 Agar Composition .............................................................................................. 25

15.2 Calculating Sample Absorbance ....................................................................... 25

15.3 Finding the Doubling Time ................................................................................. 27


D. Gustav Christensson

3 INTRODUCTION
Temperature is a measure of the average kinetic energy of the particles in a system, which
is fundamental in understanding the processes that govern even the simplest forms of life,
such as fungi. It has been called “one of the most influencing factors” affecting fungal growth
(Li, Wadsö, & Larsson, 2009, p. 1494), for these microorganisms cannot regulate internal
cell temperature. Compared to other factors, “Lag phase, growth rate and maximum growth
[are] mainly affected by temperature, whereas the effect of aw [water activity] and pH are
smaller” (Carrillo-Inungaray, et al., 2014, p. 934).

Penicillium chrysogenum, also known as Penicillium notatum, is a fungus in the family


Trichocomaceae, commonly found on mouldy food or in damp, indoor environments.
Although rarely pathogenic, it is significant to understand how temperature affects its growth
when preserving food or controlling room temperatures. However, as Alexander Fleming
discovered in 1928, P. chrysogenum can produce penicillin, a crucial antibiotic against
Streptococcus bacterial infections. Thus, finding a temperature for maximum growth is
equally important for pharmacology.

This experiment will examine the effect of temperature (ranging from 10°C to 45°C) on the
doubling time of P. chrysogenum in order to find the temperatures which yield minimum and
maximum growth. The doubling time of a species is the time taken for the population size to
double (Widdel, 2010), and it is inversely proportional to the doubling rate (which measures
growth per unit time). Hence a lower doubling time indicates faster growth, and vice versa.

To measure the population size of this fungus (a prerequisite for doubling time), this
experiment will exploit a property of all microorganisms known as turbidity. Turbidity occurs
when particles in a fluid scatter incident light in several directions (known as anisotropic
scattering), making the fluid cloudy and less transparent. Turbidity is directly proportional to
the population size of a fungus and can be measured using spectrophotometry by comparing
the incident and transmitted light intensities. Since a spectrophotometer normally calculates
the absorbance of a substance (its ability to absorb light rather than scattering it), this
investigation will use the term “absorbance” to represent “turbidity”. Moreover, the Beer-
Lambert Law governing the calculation (quoted on page 12) specifically employs this term.

1
The Effect of Temperature on Fungal Growth

4 HYPOTHESIS
If the temperature of the nutritive environment increases to 37°C, the doubling time of the
Penicillium chrysogenum sample will decrease since most enzymatic reactions necessary
to sustain life are catalysed best around this temperature. Any temperature on either
extreme should cause a greater doubling time; cold temperatures slow down molecular
movement and hence lower the chance of a successful enzyme-substrate collision, while
high temperatures denature the enzymes.

Normally, for the cell to progress to the subsequent phase in the cell cycle, there must be
enzyme-substrate collisions between proteins called cyclins and enzymes called cyclin-
dependent kinases. These reactions activate other proteins by phosphorylation, making
them carry out the processes of the cell cycle. By impeding cyclin-kinase collisions or cyclin
production itself with unfavourable temperatures, growth of P. chrysogenum should slow
down because more time will be required for sufficient proteins to activate, or for the cyclins
to pass the threshold concentration necessary to progress the cell to the next phase.

5 DESIGN
5.1 PROPOSED SET-UP
Figure 1: Proposed Set-Up of Experiment.
A: Digital Temperature Probe. B: Water Bath. C: Water. D: Test-Tube Rack. E: Test-Tubes.

The fungal samples will be cultivated in test-tubes because they resemble the cuvette in a

2
D. Gustav Christensson

spectrophotometer and because they can be immersed into a water-bath to control the
temperature.
5.2 VARIABLES
The independent variable for this experiment will be the temperature of the water bath. This
will be manipulated by adjusting the device settings, but it will be measured by a digital
temperature probe (sensitivity ±0.1°C) to validate the reading on the dial.

The dependent variable will be the doubling time of the fungal sample. This will be calculated
from the initial population size, the final population size and the time elapsed between the
two measurements. The population size will be estimated by its absorbance, which will be
calculated by Beer-Lambert’s Law knowing the light intensity transmitted by the sample and
the light intensity transmitted by the underlying agar alone. The light intensity (the luminous
flux per unit area) will be measured with a photometer (a photoelectric light intensity meter),
to the nearest 0.1 lux.

The control variables will be:

Table 1: Control Variables in the Experiment

Control Variable Method of Control Significance Effect on Results


Fungi will have the
Utilising test-tubes of the same area to grow on; Prevents test-tube
Test-tube same brand and model light transmitted size from becoming
Diameter (internal diameter equal through the test-tube an uncontrolled
to 21.3mm; standard glass will be refracted limiting factor for cell
deviation of 0.2mm). identically in all test- growth.
tubes.
Reduces chances of
Controls quantity of
Preparing each batch of random error in cell
nutrients given to the
Quantity of Agar cultures with 15ml of growth rate due to
fungi; ensures that the
Used agar, measured with a nutrient quantity; or
agar will absorb the
graduated cylinder to variation in the agar
same light intensity in
±0.5ml. absorbance due to its
all test-tubes.
thickness.
Weighing the fungal
samples to the same Reduces chances of
Starting Mass of Fungi will reproduce
starting mass (0.01g, the variation in growth
Fungal Sample from an identically-
smallest resolvable mass rate due to unequal
sized first generation.
of the available weighing starting size.
scale).
Obtaining the sample Fungi will be from the Reduces chances of
Origin of Fungal from the surface of a same strain; therefore, recording doubling
Sample single slice of white they should reproduce times from other
bread. at the same rate. species.
Preparing each batch of
Type of Agar Fungi will use the same
cultures (10 trials) from Ensures that fungi use
Used organic compounds for
9.75 ± 0.01 g of the same nutrients
reproduction.
Sabouraud Dextrose

3
The Effect of Temperature on Fungal Growth

Agar mixed in 150ml of (casein, animal tissue,


distilled water. glucose) for growth1.
Light intensity striking
Fixing the top of the test- Reduces variation and
the fungal sample will
Arrangement of tube 15cm under the random error in the
not be attenuated
Set-Up lightbulb when absorbance
differently depending
measuring the measurements due to
on the distance from
absorbance. intensity attenuation.
the source.

6 METHOD

6.1 EQUIPMENT
 Photometer (Figure 2).  Spatula.
 Clamp-stand, boss and clamp.  1200-1500ml distilled water.
 Desk lamp with a 100W light-bulb  Heating plate and heat-proof glove.
(Figure 3).  White bread.
 Black card.  Tweezers, or scalpel, or pipette (Figure
 Water bath. 6).
 Temperature probe.  Cutting board.
 Sensor interface and display unit  10 test-tubes, of internal diameter 2cm
(Figure 7).
(Figure 4).
 Weighing scale (resolution ±0.01g) and
 100ml conical flask, with stopper. weighing boats.
 250ml conical flask.  25ml graduated cylinder (resolution
 500ml conical flask. ±0.25ml).
 75-100g Sabouraud Dextrose Agar  100ml graduated cylinder (resolution
(Figure 5). ±0.5ml).

6.2 PROCEDURE
1. Soak a slice of white bread in water inside a 500ml conical flask. Leave it in a warm, dark
place for two weeks.
2. Transfer the bread from the conical flask to the cutting board with tweezers.
3. Separate any turquoise-green fungi from the bread, and isolate them with a stopper in
the 100ml conical flask.
4. Mount the photometer vertically on the clamp-stand, 30cm underneath a desk lamp
(Figure 8).
5. Construct a hollow cone out of thick, black card (Figure 9). The cone should be open on
both sides; the large opening should accommodate the test-tubes’ diameter while the
other should fit the photometer’s width. Rest this cone above the photometer so that the
wider opening faces the desk lamp.

1 See the Appendix (page 25) for a more detailed outline of the substances in the agar.

4
D. Gustav Christensson

6. Surround the upper 3cm of the test-tubes with a roll of black card, and label the tubes 1
to 10 (Figure 10).
7. Weigh up 9.75g of dextrose agar in a weighing boat with the help of a spatula, and
transfer it to the conical flask (Figure 11).
8. Use the 100ml graduated cylinder to measure 150ml of distilled water in two rounds.
Pour the water into the conical flask.
9. Place the conical flask on the heating plate, and set it to 300°C. Agitate the conical flask
regularly with the glove until the agar begins to boil.
10. While the agar heats up, use ten weighing boats and the weighing scale to measure ten
quantities of P. chrysogenum culture from the 100ml conical flask, each with a mass
equal to equal to the smallest resolvable mass of the scale (in this case, 0.01g). Always
zero the scale before adding the fungi (Figure 12).
11. Turn off the heating plate. Use the 25ml graduated cylinder to measure 10 agar volumes,
15ml each, and dispense them into the ten test-tubes on the test-tube rack.
12. Turn on the desk lamp and set the photometer to the 0-600 lux range (Figure 13).
Connect the photometer to the sensor unit (Figure 14). Hold each test-tube vertically in
the black cone so that the opening is 15cm under the lightbulb and measure the
transmitted light intensity.
13. Use tweezers to place one of the ten samples prepared in step 10 into each test-tube.
Measure the transmitted light intensities again.
14. Immerse the test-tubes into the water bath, configured to 20°C (Figure 15). Connect the
temperature probe to the display unit (Figure 14), then immerse its rod into the water and
record the time of day.
15. At the same hour during the following days, record the water temperature.
16. After five days, record the final transmitted light intensity and time of day.
17. Sterilise the test-tubes with boiling water. Expel the agar with tweezers or by shaking
vigorously. Wash with detergent.
18. Repeat steps 7 to 17 with different temperatures in the water bath. Recommended
temperatures are multiples of 5°C between 20°C and 50°C.
19. If possible, use an adjustable refrigerator to grow samples in temperatures under 20°C,
at further multiples of 5°C validated by an alcohol thermometer.

6.3 DIAGRAMS

5
The Effect of Temperature on Fungal Growth

Figure 2: Photometer (Light Figure 3: 116 W Desk-Light Figure 4: Vernier LabPro


intensity meter) Interface

Figure 5: Agar Used in Figure 6: Tweezers (centre), with Figure 7: 2cm Diameter Test-
investigation alternative tools Tube

Figure 8: Photometer mounted Figure 9: Hollow Cone Figure 10: Labelled Test-Tube
vertically constructed out of Black Card for with surrounding Black Card Strip
focusing light.

Figure 11: Transferring Agar to Figure 12: Resetting or "zeroing" Figure 13: Setting the
Weighing Boat the Weighing Scale Photometer to the 0-600 lux
range.

6
D. Gustav Christensson

Figure 14: Connecting the device Figure 15: Controlling Water Bath
to the Display Unit temperature

6.4 RISK ASSESSMENT


The heating plate used to warm the agar mixture to boiling temperature can burn or scald
human tissue. Always wear a heat-proof glove when handling the conical flask containing
the agar.

P. chrysogenum is rarely pathogenic in humans, but airborne spores may cause allergic
reactions in the respiratory system. Wear a surgical mask, a lab-coat, nitrile gloves and
safety goggles when transferring the fungi, and disinfect laboratory surfaces, equipment and
hands afterwards.

The conical flasks are fragile and may shatter if dropped. The subsequent shards may
breach the human epidermis. Do not store the conical flasks close to edges where they may
fall. In the event of broken glass, use a thick cloth to wipe away fragments from all surfaces.

7
The Effect of Temperature on Fungal Growth

7 DATA TABLES
7.1 RAW DATA
Table 2: Comparison of Programmed Water Bath Temperature and Actual Temperatures

Sample Temperatures (± 0.1 °C) Actual Temperature


Target
Temperature (°C) Average Absolute Percentage Standard
Sample I Sample II Sample III Sample IV
(°C) Uncertainty (± °C) Uncertainty (±%) Deviation (°C)
10.0 10.8 11.4 12.8 10.3 11.3 1.3 11.04 1.1
15.0 17.4 16.3 16.3 16.2 16.6 0.6 3.63 0.6
20.0 18.9 18.6 18.5 18.0 18.5 0.4 2.43 0.4
25.0 28.4 28.7 28.6 28.7 28.6 0.2 0.52 0.1
30.0 33.2 33.7 33.5 33.7 33.5 0.3 0.75 0.2
35.0 35.5 35.4 35.3 35.1 35.3 0.2 0.57 0.2
40.0 38.9 40.1 40.6 39.8 39.9 0.9 2.13 0.7
45.0 42.5 43.6 46.1 44.3 44.1 1.8 4.08 1.5

Table 3: Transmitted Light Intensity through Test-Tubes containing Agar only

Target Transmitted Light Intensity (± 0.1 lux) Agar-Only Light Intensity


Temperature Avg. Abs. Uncertain. Perc. Uncertain. St. Dev.
(°C) I II III IV V VI VII VIII IX X
(lux) (± lux) (±%) (lux)
10.0 984.4 830.5 650.1 770.1 692.2 607.2 899.4 710.5 551.7 630.1 732.6 216.4 29.53 130.4
15.0 117.0 215.1 130.2 144.2 138.2 119.2 115.1 173.1 171.1 317.6 164.1 101.3 61.71 59.3
20.0 194.6 152.9 198.8 188.8 280.2 277.1 233.7 141.6 188.4 207.4 206.4 69.3 33.58 43.7
25.0 555.7 630.1 588.8 787.1 535.1 688.0 851.2 844.9 832.6 865.6 717.9 165.3 23.02 126.0
30.0 791.1 956.5 830.1 782.3 590.1 634.2 638.3 592.9 698.7 531.6 704.6 212.5 30.15 125.5
35.0 41.9 40.1 382.6 515.5 332.5 375.5 277.4 419.5 125.6 323.8 283.4 237.7 83.86 154.0
40.0 628.8 417.0 298.5 435.5 401.5 791.3 463.5 558.3 670.3 459.0 512.4 246.4 48.09 140.2
45.0 371.4 519.4 652.9 620.3 562.2 736.7 711.5 383.8 576.3 670.7 580.5 182.7 31.46 119.6

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D. Gustav Christensson

Table 4: Transmitted Light Intensity through Test-Tubes containing Agar and initial generation of P. chrysogenum Sample

Transmitted Light Intensity (± 0.1 lux) Initial Light Intensity


Target
Temperature Abs. Perc. St.
Avg.
(°C) I II III IV V VI VII VIII IX X Uncertain. Uncertain. Dev.
(lux)
(± lux) (±%) (lux)
10.0 470.1 726.8 221.1 701.9 762.1 411.4 702.4 661.6 508.3 192.0 535.8 285.1 53.20 199.2
15.0 35.7 38.9 147.6 42.9 32.5 28.6 22.2 58.7 37.4 70.1 51.5 62.7 121.84 34.7
20.0 135.0 47.0 112.4 73.7 68.8 135.4 127.7 132.6 152.3 105.3 109.0 52.7 48.29 33.0
25.0 207.4 226.8 217.4 300.3 115.7 288.4 558.5 395.2 363.8 730.1a 297.1 221.4 74.54 122.4
30.0 219.8 519.2b 336.6 202.3 112.1 130.3 139.7 112.6 150.2 102.1 167.3 117.3 70.08 71.0
35.0 28.0 25.6 53.4 66.9 56.2 79.5 62.6 32.0 51.5 93.1 54.9 33.8 61.50 21.0
40.0 182.8 377.2 522.2 451.1 377.4 307.7 346.7 201.9 214.2 211.2 319.2 169.7 53.16 110.1
45.0 460.7 555.7 367.8 574.3 770.6 378.1 502.0 448.3 299.6 799.5 515.7 250.0 48.47 156.8
a
This value was excluded as an outlier because it was 2.24 standard deviations from the previous mean (340.3 lux).
b
This value was excluded since it was 2.53 standard deviations from the previous mean (202.5 lux).
Table 5: Transmitted Light Intensity through Test-Tubes containing Agar and final generation of P. chrysogenum Sample

Transmitted Light Intensity (± 0.1 lux) Final Light Intensity


Target
Temperature Abs. Perc.
Avg. St. Dev.
(°C) I II III IV V VI VII VIII IX X Uncertain. Uncertain.
(lux) (lux)
(± lux) (±%)
10.0 72.8 67.9 50.4 75.2 151.6 50.2 64.0 150.2 91.0 29.9 80.3 60.9 75.76 38.6
15.0 9.4 5.6 9.2 7.5 7.9 9.2 9.4 6.0 9.8 8.3 8.2 2.1 25.52 1.4
20.0 14.1 12.6 17.1 15.6 13.2 12.2 16.7 15.4 16.2 13.9 14.7 2.5 16.67 1.6
25.0 10.2 3.6 4.1 7.1 8.4 3.4 3.4 3.2 6.8 2.6 5.3 3.8 71.97 2.5
30.0 5.3 8.1 5.8 9.6 7.1 4.1 6.8 4.9 3.9 5.3 6.1 2.9 46.80 1.7
35.0 6.0 5.3 8.8 5.8 6.8 11.5 6.4 7.3 5.8 9.2 7.3 3.1 42.52 1.9
40.0 94.0 18.4 10.5 20.9 18.2 140.6 87.4 93.1 114.1 36.1 63.3 65.1 102.72 45.1
45.0 30.5 19.9 22.8 12.4 15.8 23.3 22.4 13.7 25.8 23.7 21.0 9.1 43.03 5.4

9
The Effect of Temperature on Fungal Growth

Table 6: Elapsed Time for Different Batches of Fungal Sample

Target Temperature (°C) Elapsed Time (± 5 minutes) Elapsed Time (± 0.08 hours)
10.0 6965 116.08
15.0 9800 163.33
20.0 5605 93.42
25.0 10070 167.83
30.0 8590 143.17
35.0 9810 163.50
40.0 8265 137.75
45.0 5780 96.33

7.2 NOTES
7.2.1 OBSERVATIONS
There was a frequent discrepancy between the water bath's programmed temperature and
the empirical temperature measurements, which is compared in Table 2.

The elapsed time (time allocated for growth) for each batch of samples could not be
controlled as it was dependent on access to the laboratory. It was measured to the nearest
five minutes from the test-tubes’ immersion into the water bath to the moment in which half
the light intensities had been measured. A single standard deviation for the elapsed time
(calculated below) was used for all trials.

7.2.2 MEASURING THE SPREAD OF DATA


The absolute uncertainty for a variable 𝑥 was calculated as:
𝑥Maximum − 𝑥Minimum
∆𝑥 =
2

The percentage uncertainty of mean 𝑥̅ was calculated as:

∆𝑥 𝑥Maximum − 𝑥Minimum
∆%𝑥 = 100% ( ) = 100% ( )
𝑥̅ 2𝑥̅

For the temperature and the elapsed time, a limited number of samples were taken. Hence
the Sample Standard Deviation for the variable 𝑥, tested over 𝑛 trials, was calculated as:

∑(𝑥 − 𝑥̅ )2
𝜎Sample =√
𝑛−1

For the light intensity, the Population Standard Deviation was used because the sample was
larger (ten trials):

10
D. Gustav Christensson

∑(𝑥 − 𝑥̅ )2
𝜎Population = √
𝑛

7.2.3 STANDARD DEVIATION IN ELAPSED TIME


The Standard Deviation for the elapsed time was calculated knowing that the average time
2
between each measurement was 150 hours. If 𝜇 is the average elapsed time, the elapsed
9 7 5 3 1
times of each measurement were approximately 𝜇 − 150, 𝜇 − 150 , 𝜇 − 150, 𝜇 − 150, 𝜇 − 150,
1 3 5 7 9
𝜇 + 150 , 𝜇 + 150 , 𝜇 + 150 , 𝜇 + 150 and 𝜇 + 150 hours. Calculating the Sample Standard

Deviation yields:
2 2 2 2
2 1 9 7 5 3
(𝜎Sample ) = ((𝜇 − − 𝜇) + (𝜇 − − 𝜇) + (𝜇 − − 𝜇) + (𝜇 − − 𝜇)
10 − 1 150 150 150 150
2 2 2 2
1 1 3 5
+ (𝜇 − − 𝜇) + (𝜇 + − 𝜇) + (𝜇 + − 𝜇) + (𝜇 + − 𝜇)
150 150 150 150
2 2
7 9
+ (𝜇 + − 𝜇) + (𝜇 + − 𝜇) )
150 150

2 1 9 2 7 2 5 2 3 2 1 2
(𝜎Sample ) = (2 ( ) + 2( ) + 2( ) + 2( ) + 2( ) )
9 150 150 150 150 150
2 2 81 49 25 9 1 2 165 1 1
(𝜎Sample ) = ( 2
+ 2
+ 2
+ 2
+ 2
)= ( 2
) = ( )( ) (330)
9 150 150 150 150 150 9 150 9 1502
√330
𝜎Sample = ≈ 0.040 hours
450

7.3 PROCESSED DATA


Table 7: Calculation of Initial Absorbance, Final Absorbance and Doubling Time of Fungal Sample

Temperature (°C) Initial Absorbance Final Absorbance Doubling Time (hours)


Standard Average Standard Average Standard Average Standard
Avg.
Deviation (4 s.f.) Deviation (4 s.f.) Deviation (4 s.f.) Deviation
11.3 1.1 0.1359 0.1790 0.9601 0.2227 41.16 28.15
16.6 0.6 0.5036 0.3324 1.300 0.173 119.4 84.8
18.5 0.4 0.2771 0.1604 1.147 0.104 45.57 18.79
28.6 0.1 0.3832 0.1944 2.133 0.219 67.76 20.43
33.5 0.2 0.6244 0.1999 2.063 0.145 83.03 22.76
35.3 0.2 0.7130 0.2884 1.590 0.261 141.3 76.9
39.9 0.7 0.2055 0.1913 0.9080 0.3314 64.26 43.24
44.1 1.5 0.0515 0.1595 1.441 0.142 20.04 18.65

11
The Effect of Temperature on Fungal Growth

7.4 NOTES
7.4.1 CALCULATION OF THE ABSORBANCE
The initial and the final absorbance was calculated using Beer-Lambert’s Law (Clark, The
Beer-Lambert Law, 2013; Clark & Gunawardena, The Beer-Lambert Law). Generally, the
dimensionless absorbance 𝐴 of a substance exposed to incident light intensity 𝐼0 but
transmitting light intensity 𝐼 is:
𝐼0
𝐴 = log ( )
𝐼

Following a derivation in the Appendix2, the formula in this context became:

𝐼Agar
𝐴Sample = log ( )
𝐼Total

Where 𝐴Sample is the sample’s absorbance, 𝐼Agar is the light intensity transmitted by the agar
only (see Table 3) and 𝐼Total is the light intensity transmitted by the agar and the fungal
sample (measured before immersion in Table 4 and after immersion in Table 5). The
absorbance was always given to four significant figures in accordance with most of the light
intensity readings.

The standard deviation was used to represent the spread of data because it takes all of the
measurements into account. The Standard Deviation in the absorbance (𝜎𝐴 ) was calculated
using the formula:

2 2 2 2
𝜕𝐴 𝜕𝐴 1 1
𝜎𝐴 = √((𝜎𝐼0 ) ( )) + ((𝜎𝐼 ) ( )) = √((𝜎𝐼0 ) ( )) + ((𝜎𝐼 ) (− ))
𝜕𝐼0 𝜕𝐼 𝐼0 ln 10 𝐼 ln 10

Where 𝜎𝐼0 is the standard deviation in the incident light intensity, 𝜎𝐼 is the standard deviation
in the transmitted light intensity, 𝜕 represents the partial derivative with respect to a variable
and ln(10) is the natural logarithm of 10.

7.4.2 CALCULATION OF THE DOUBLING TIME


Assuming the fungal sample grew exponentially3, the doubling time 𝑡𝑑 was calculated using
the starting absorbance 𝐴0 , the final absorbance 𝐴 and the time elapsed 𝑡:

2 See page 25.


3 This formula is derived in the Appendix, on page 27.

12
D. Gustav Christensson

𝑡
𝑡𝑑 =
𝐴
log 2 (𝐴 )
0

Where log 2 𝑥 is the logarithm in base 2. The doubling time was given to 4 significant figures
to mirror the precision in the light intensity and the absorbance, and its standard deviation
was calculated as:

2 2 2
𝜕𝑡𝑑 𝜕𝑡𝑑 𝜕𝑡𝑑
𝜎𝑡𝑑 = √((𝜎𝑡 ) ( )) + ((𝜎𝐴 ) ( )) + ((𝜎𝐴0 ) ( ))
𝜕𝑡 𝜕𝐴 𝜕𝐴0

2 2 2

ln 2 𝑡 ln 2 𝑡 ln 2
𝜎𝑡𝑑 = (𝜎𝑡 ) ( ) + (𝜎𝐴 ) (− ) + (𝜎𝐴0 ) ( )
𝐴 𝐴 𝐴
ln (𝐴 ) 𝐴 ln2 (𝐴 ) 𝐴0 ln2 (𝐴 )
0 0 0
√( ) ( ) ( )

7.5 SAMPLE CALCULATIONS


When the temperature was 33.5°C, the following calculations were performed:

Table 8: Sample Data Calculations at 33.5°C

33.2 + 33.7 + 33.5 + 33.7 134.1


Average 𝑇̅ = = ≈ 33.5 °C
4 4
Absolute 33.7 − 33.2 0.5
∆𝑇 = = ≈ ± 0.3 °C
Uncertainty 2 2
Actual Temperature

0.5
Percentage 1000
∆%𝑇 = 100 ( 2 ) = ≈ ± 0.75 %
Uncertainty 134.1 1341
4

134.1 2 134.1 2 134.1 2 134.1 2


√(33.2 − 4
) + (33.7 −
4
) + (33.5 −
4
) + (33.7 −
4
)
Sample 𝜎𝑇 =
4−1
Standard
Deviation 13 2 7 2 1 2 7 2 268
√(− 40
) + ( ) + (− ) + ( )
40 40 40 = √ 1600 = 1 √67 ≈ 0.2 °C
𝜎𝑇 =
3 3 20 3

13
The Effect of Temperature on Fungal Growth

791.1 + 956.5 + 830.1 + 782.3 + 590.1 + 634.2 + 638.3 + 592.9 + 698.7 + 531.6
𝐼̅0 =
Average 10
7045.8
̅
𝐼0 = ≈ 704.6 lux
10
Absolute 956.5 − 531.6 424.9
∆𝐼0 = = ≈ ± 212.5 lux
Uncertainty 2 2
424.9
Percentage 207119
∆%𝐼0 = 100 ( 2 ) =
Agar-Only Light Intensity

≈ ± 30.15%
Uncertainty 7045.8 6869
10
1 7045.8 2 7045.8 2 7045.8 2
𝜎𝐼20 = ((791.1 − ) + (956.5 − ) + (830.1 − )
10 10 10 10
2 2
7045.8 7045.8 7045.8 2
+ (782.3 − ) + (590.1 − ) + (634.2 − )
10 10 10
2 2
7045.8 7045.8 7045.8 2
+ (638.3 − ) + (592.9 − ) + (698.7 − )
10 10 10
2
Population 7045.8
+ (531.6 − ) )
Standard 10
Deviation 1 2163 2 6298 2 3138 2 1943 2 2862 2 3519 2
𝜎𝐼20 = (( ) +( ) +( ) +( ) + (− ) + (− )
10 25 25 25 25 25 50
1657 2 2792 2 147 2 8649 2
+ (− ) + (− ) + (− ) + (− ) )
25 25 25 50
1
𝜎𝐼20 = (157591.6216 … ) = 15759.16216 …
10
𝜎𝐼0 ≈ 125.5 lux
219.8 + 336.6 + 202.3 + 112.1 + 130.3 + 139.7 + 112.6 + 150.2 + 102.1
̅̅̅̅̅̅̅
𝐼Initial =
Average 9
1505.7
̅̅̅̅̅̅̅
𝐼Initial = = 167.3 lux
9
Absolute 336.6 − 102.1 234.5
∆𝐼Initial = = ≈ ± 117.3 lux
Uncertainty 2 2
234.5
Initial Light Intensity

Percentage 16750
∆%𝐼Initial = 100 ( 2 ) = ≈ ± 70.08%
Uncertainty 1505.7 239
9
1 1505.7 2 1505.7 2 1505.7 2
𝜎𝐼2Initial = ((219.8 − ) + (336.6 − ) + (202.3 − )
9 9 9 9
1505.7 2 1505.7 2 1505.7 2
+ (112.1 − ) + (130.3 − ) + (139.7 − )
9 9 9
Population 1505.7 2 1505.7 2 1505.7 2
+ (112.6 − ) + (150.2 − ) + (102.1 − ) )
Standard 9 9 9
Deviation 1
𝜎𝐼2Initial = ((52.5)2 + (169.3)2 + (35.0)2 + (−55.2)2 + (−37.0)2 + (−27.6)2
9
+ (−54.7)2 + (−17.1)2 + (−65.2)2 )
45357.08
𝜎𝐼2Initial =
9
𝜎𝐼Initial ≈ 70.1 lux

14
D. Gustav Christensson

5.3 + 8.1 + 5.8 + 9.6 + 7.1 + 4.1 + 6.8 + 4.9 + 3.9 + 5.3 60.9
Average ̅̅̅̅̅̅
𝐼Final = = ≈ 6.1 lux
10 10
Absolute 9.6 − 3.9 5.7
∆𝐼Final = = ≈ ± 2.9 lux
Uncertainty 2 2
5.7
Percentage 9500
∆%𝐼Final = 100 ( 2 ) = ≈ ± 46.80%
Uncertainty 60.9 203
10
Final Light Intensity

1 60.9 2 60.9 2 60.9 2 60.9 2


𝜎𝐼2Final = ((5.3 − ) + (8.1 − ) + (5.8 − ) + (9.6 − )
10 10 10 10 10
60.9 2 60.9 2 60.9 2 60.9 2
+ (7.1 − ) + (4.1 − ) + (6.8 − ) + (4.9 − )
10 10 10 10
2 2
60.9 60.9
+ (3.9 − ) + (5.3 − ) )
Population 10 10
Standard 1 79 2 201 2 29 2 351 2 101 2 199 2 71 2
Deviation 𝜎𝐼2Final = ((− ) +( ) + (− ) +( ) +( ) + (− ) +( )
10 100 100 100 100 100 100 100
119 2 219 2 79 2
+ (− ) + (− ) + (− ) )
100 100 100
1 293890 293890
𝜎𝐼2Final = ( )=
10 10000 100000
𝜎IFinal ≈ 1.7 lux
Initial Absorbance

704.6
Average 𝐴0 = log ( ) ≈ 0.6244
167.3

2 2
1 1
Standard 𝜎𝐴0 = √((125.5) ( )) + ((70.1) (− ))
704.6 ln 10 167.3 ln 10
Deviation
𝜎𝐴0 = √(0.07735 … )2 + (−0.1820 … )2 = √0.03910 … ≈ 0.1999

15
The Effect of Temperature on Fungal Growth

Final Absorbance
704.7
Average 𝐴 = log ( ) ≈ 2.063
6.1

2 2
1 1
Standard Deviation 𝜎𝐴 = √((125.5) ( )) + ((1.7) (− )) = √(0.07735 … )2 + (0.1210)2 = √0.02063 ≈ 0.1447
704.6 ln 10 6.1 ln 10

143.17 143.17
Average 𝑡𝑑 = = ≈ 83.03 hours
2.063 1.724
log 2 ( )
0.6244
Doubling Time

2 2 2

ln 2 143.17 ln 2 143.17 ln 2
𝜎𝑡𝑑 = √((0.04) ( )) + ((0.1447) (− )) + ((0.1999) ( ))
2.063 2.063 2.063
Standard Deviation ln ( ) 2.063 ln2 ( ) 0.6244 ln2 ( )
0.6244 0.6244 0.6244

0.02798 … 2 14.36 … 2 19.83 … 2


𝜎𝑡𝑑 = √( ) + (− ) +( ) = √(0.02341 … )2 + (4.871 … )2 + (22.23 … )2 = √517.9 ≈ 22.76 hours
1.1952 … 2.948 … 0.8920 …

16
D. Gustav Christensson

8 VISUALISED DATA
Graph 1: Correlation between Temperature and Doubling Time of P. chrysogenum samples. Error bars show 1 standard deviation from the mean.

Effect of Temperature on Doubling Time of Penicillium chrysogenum


250.00

200.00
Doubling Time (hours)

150.00

100.00

50.00

0.00
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0
Temperature (°C)

17
The Effect of Temperature on Fungal Growth

9 RESULTS
The graphed results show that as the temperature increases from 11.3°C to 44.1°C, the
doubling time rises and falls twice. From 11.3°C to 16.6°C, the growth rate slows down
(doubling time rises from 41.16 hours to 119.4 hours). At room temperature, the doubling
time returns below 100 hours. When approaching mammal core temperature at 35.3°C,
growth slows to an all-time maximum doubling time of 141.3 hours, yet from 35.3°C to
44.1°C, the doubling time falls to 20.04 hours (the all-time minimum). Although warm
temperatures feature the lowest doubling time, the doubling time decreases at either
extreme of the graph, so no conclusion can be drawn on whether hot or cold temperatures
favour growth more.

An inverse quartic function (plotted above) was chosen to model this trend due to its
resemblance to observed patterns:

1
𝑡𝑑 =
(7.76×10−7 )(𝑇 4 ) − (7.98×10−5 )(𝑇 3 ) + (2.93×10−3 )(𝑇 2 ) − (4.53×10−2 )(𝑇) + 0.266

The equation models an increase in doubling time between 0°C and 16.2°C, from 3.8 hours
to 65.51 hours. Although the model underestimates the doubling time by 53.9 hours at the
first peak, the predicted temperature for this maximum is within 0.4°C of the data, indicating
a good fit. As the temperature increases, the doubling time reaches a local minimum of
47.60 hours at 25.3°C, before rising again to the all-time maximum of 78.88 hours at 35.7°C.
This is only 0.4°C warmer than the recorded temperature (even though the estimated
doubling time is 62.5 hours lower than the measurements). Afterwards, it decreases past
14.07 hours at the upper limit for this experiment (45°C). Extrapolating the data indicates
that the doubling time asymptotically approaches (but never reaches) zero hours both for
warm and cold temperatures.

One argument in favour of this mathematical model is that the projected line passes within
1 standard deviation of all the measurements for both variables. Moreover, the correlation
coefficient between the quartic function and the reciprocals of the data is 𝑅 2 = 0.931 ,
suggesting an accurate fit. The model also predicts the peak temperatures within 0.5°C and
foresees the impossibility of reaching a doubling time of zero hours. However, an imperative
limitation is that there are no data points between 18.5°C and 28.6°C to confirm the local
minimum predicted by the equation. Secondly, the model underestimates the doubling time
in all but two cases. Furthermore, since all extrapolation is speculative, it is only possible to

18
D. Gustav Christensson

validate this equation from 11.3°C to 44.1°C. The relatively large error bars could also
undermine any attempt at a regression line, hindering further inferences.

10 DISCUSSION
To evaluate the reliability of the results, one can compare the predicted local minimum
doubling time at 25.3°C with the optimum conditions for P. chrysogenum growth identified
in scientific literature. For example, one study found that P. chrysogenum grew best in
glycerol at 23.4°C, and best in sorbitol at 23.6°C (Sautour, Rouget, Dantigny, Divies, &
Bensoussan, “Application of Doehlert design to determine the combined effects of
temperature, water activity and pH on conidial germination of Penicillium chrysogenum,”
2001). The study also found slowest germination times at 16°C, which is consistent with a
local maximum doubling time in my results. Similarly, a previous study by the same authors
(“Prediction of conidial germination of Penicillium chrysogenum as influenced by
temperature, water activity and pH,” 2001) found that increasing temperature from 15°C to
25°C increased germination rate between 232% and 288%; my decrease in doubling time
corroborates this. Although P. chrysogenum produces the most penicillin at 30°C, if the
same temperature is kept for both synthesis and growth, then the optimal temperature varies
between 23°C and 28°C (Owen & Johnson, 1955, p. 377), corresponding to the minimum
on the graph. This is a common temperature for optimal P. chrysogenum growth (Gonzàlez,
Resnik, & Vaamonde, 1988), which reinforces the validity of the data.

On the other hand, most species of Penicillium do not grow successfully in extreme
temperatures, unlike the results. For instance, P. glabrum cannot survive beyond 33.8°C
(Nevarez, et al., 2009). Moreover, neither P. digitatum nor P. italicum can grow above 37°C
(Plaza, Usall, Teixidò, & Vinas, 2003, p. 552). P. marneffei is limited to below 39.8°C (Cao,
et al., 2007, p. 403); likewise, temperatures higher than 40°C are considered “extreme” for
P. digitatum (Carrillo-Inungaray, et al., 2014, p. 933). Even the thermophilic P. citrinum and
P. urticae, which thrive between 30°C and 37°C, fail to grow above 40°C (Mislivec, Dieter,
& Bruce, 1975, p. 1188). In contrast, the lowest doubling time for my species occurred
beyond the conventional maximum temperatures for growth. A similar problem appears with
cold temperatures: at 10°C, P. roqueforti usually demonstrates the “slowest start of growth”
(Li, Wadsö, & Larsson, 2009, p. 1497), but this is incompatible with my findings.

Overall, most Penicillium species grow best at room temperature and not at extreme
temperatures but my data cannot confirm this, both because there was no trial at 25°C and
because doubling time tends to zero at extreme temperatures. Few theories can explain

19
The Effect of Temperature on Fungal Growth

why my samples grew fastest above 40°C, where molecular vibrations typically disrupt
intramolecular forces in polypeptide chains and denature enzymes such as kinases. As
scientific literature demonstrates that Penicillium is not a thermophilic genus, a proposed
explanation is that the heat may have denatured the casein in the agar, changing its optical
properties and rendering it less transparent. Similarly, cold conditions may have made the
agar denser and raised its absorbance; the spectrophotometer may have misinterpreted this
as fungal growth.

It seems unusual that microorganisms, which can reproduce in less than one hour, should
have an average doubling time of 72.82 hours. However, a similar study which used
spectrophotometry to examine the effect of temperature on Escherichia coli DH5α
discovered doubling times from 25 hours to 80 hours, which are comparable to this
investigation (Nguyen, 2006, p. 92). It is therefore probable that spectrophotometry
underestimates doubling time, limiting its usefulness to a relative indication of the optimum
temperature.

11 EVALUATION OF METHOD
The most significant weakness of the method was that the absorbance of the P.
chrysogenum samples increased beyond the “critical value” of 0.4, where absorbance and
cell concentration cease to be proportional (Widdel, 2010). The large size of fungal cells
compared to pigments normally examined with spectrophotometry means that an
anisotropically scattered photon can be scattered a second time towards the photometer,
which receives more light, underestimates the absorbance and overestimates the doubling
time (University of Colorado, 2005). Because all final absorbance recordings were above
0.4, they were not accurate measures of cell concentration. To address this issue, the
samples should’ve been diluted with water in new test-tubes to lower the absorbance below
0.4; these values could then have been corrected with the dilution factor. This would greatly
increase the accuracy of the results.

Another significant methodological issue was that it was difficult to distinguish P.


chrysogenum from other Penicillium species by colour. The challenge persists in scientific
contexts: “many commercial labs are incapable of correctly identifying Penicillium to species
level” (Kung'u, 2012), therefore, “DNA sequences are essential for robust identification of
Penicillium species” (Visagie, et al., 2014, p. 343). For our purposes, observing spores
released by the sample under a light microscope would suffice, since spores from different

20
D. Gustav Christensson

Penicillium species form different branching patterns. This would guarantee that the correct
microorganisms are observed.

A further weakness of the method was that the environment temperature fluctuated beyond
the sample measurements. When the water bath was busy for other purposes, the 40°C and
45°C trials had to be temporarily relocated to containers filled with water. Despite manually
supplying warm water periodically, temperatures sometimes fell by 7°C. To overcome this
issue, a second, unused water bath should be acquired so that the temperature could be
controlled effectively for a constant growth rate and more accurate data.

It is also possible that the agar may have been contaminated by undesired airborne
microbes during the experiment, because the test-tubes were never sealed to guarantee
indefinite oxygen for aerobic respiration. To prevent other microorganisms for interfering
with the growth of P. chrysogenum and the absorbance, the test-tubes should be sealed
with stoppers and ventilated daily (such as while measuring absorbance).

Another drawback was that the light shining on the samples was not monochromatic. Instead
it consisted of several wavelengths which would’ve been scattered or absorbed differently
by the fungal samples. Even though the incandescent light-bulb was never changed, it may
have radiated light of different wavelengths as it got hotter. To eliminate this problem, a
monochromatic source of light such as a laser pointer should be used instead. This would’ve
lessened systematic error.

Finally, controlling the fungi’s starting mass was problematic. A small mass was chosen
deliberately to allow more space for growth, but as the starting mass equalled the digital
weighing scale’s resolution, the percentage uncertainty was 100%. To solve this problem, a
more sensitive weighing scale or more spacious test-tubes should be procured.

12 CONCLUSION
In conclusion, the data demonstrates that P. chrysogenum reproduced fastest at 44.1°C,
followed by 11.3°C. The slowest doubling times (at 16.6°C and 35.3°C) feature faster growth
in-between, which to a large extent refutes my hypothesis that 37°C would have induced
maximum growth. Although the mathematical model gives a local minimum doubling time at
25.3°C, extrapolation asserts that the doubling time is lower in extremely cold or hot
environments, suggesting that P. chrysogenum is an ‘extremophile’. Nevertheless, further
experiments based on direct measurements of cell growth should seek to verify the two
peaks at 16.6°C and 35.3°C, which are rarely mentioned in scientific literature. Given the

21
The Effect of Temperature on Fungal Growth

many sources of error, the alleged thermophilic and cryophilic properties of P. chrysogenum
should also be validated by further research.

13 ACKNOWLEDGEMENTS
The author is grateful to Miglena Shabanska for guidance in choosing the topic; Marco
Montorsi for lessons in uncertainty propagation calculations; Andy Stabellini for granting
access to the digital sensors used and to the school for providing other equipment. The
author also thanks those who supported this investigation by offering comments on the draft.

14 REFERENCES
Cao, C., Li, R., Wan, Z., Liu, W., Wang, X., Qiao, J., . . . Calderone, R. (2007, August). The
effects of temperature, pH, and salinity on the growth and dimorphism of Penicillium
marneffei. Medical Mycology, 45(5), 401-407. doi:10.1080/13693780701358600

Carrillo-Inungaray, M. L., Hidalgo-Morales, M., Rodríguez-Jimenes, G. d., García-Alvarado, M.


Á., Ramírez-Lepe, M., Munguìa, A. R., & Robles-Olvera, V. (2014, September). Effect
of Temperature, pH and Water Activity on Penicillium digitatum Growth. Journal of
Applied Mathematics and Physics, 2, 930-937. Retrieved July 17, 2016, from
http://file.scirp.org/pdf/JAMP_2014092411165685.pdf

Clark, J. (2013, September). The Beer-Lambert Law. Retrieved May 8, 2016, from
Chemguide: http://www.chemguide.co.uk/analysis/uvvisible/beerlambert.html

Clark, J., & Gunawardena, G. (n.d.). The Beer-Lambert Law. Retrieved May 8, 2016, from UC
Davis ChemWiki:
http://chemwiki.ucdavis.edu/Core/Physical_Chemistry/Spectroscopy/Electronic_Spectro
scopy/Electronic_Spectroscopy_Basics/The_Beer-Lambert_Law

Gonzàlez, H., Resnik, S., & Vaamonde, G. (1988, March). Influence of temperature on growth
rate and lag phase of fungi isolated from Argentine corn. International Journal of Food
Microbiology, 6(2), 179-83. Retrieved July 27, 2016, from
http://www.ncbi.nlm.nih.gov/pubmed/3275297

Kung'u, J. (2012, May 2012). Penicillium. Retrieved July 18, 2016, from MBL:
http://www.moldbacteria.com/mold/penicillium.html

Li, Y., Wadsö, L., & Larsson, L. (2009, February 4). Impact of temperature on growth and
metabolic efficiency of Penicillium roqueforti – correlations between produced heat,
ergosterol content and biomass. (A. Gilmour, Ed.) Journal of Applied Microbiology,
106(5), 1494–1501. doi:10.1111/j.1365-2672.2008.04110.x

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D. Gustav Christensson

Mislivec, P., Dieter, C., & Bruce, V. (1975, November-December). Effect of Temperature and
Relative Humidity on Spore Germination of Mycotoxic Species of Aspergillus and
Penicillium. Mycologia, 67(6), 1187-1189. Retrieved July 17, 2016, from
http://www.jstor.org/stable/3758839

Nevarez, L., Vasseur, V., Le Madec, A., Le Bras, M., Coroller, L., Leguérinel, I., & Barbier, G.
(2009, April 15). Physiological traits of Penicillium glabrum strain LCP 08.5568, a
filamentous fungus isolated from bottled aromatised mineral water. International
Journal of Food Microbiology, 130(3), 166-71. doi:10.1016/j.ijfoodmicro.2009.01.013

Nguyen, M. T. (2006, May). The effect of temperature on the growth of the bacteria
Escherichia coli DH5α. Saint Martin’s University Biology Journal, 1, 87-94. Retrieved
July 17, 2016, from
http://homepages.stmartin.edu/fac_staff/molney/website/SMU%20Bio%20Journal/Nguy
en%202006.pdf

Owen, S., & Johnson, M. J. (1955). The Effect of Temperature Changes on the Production of
Penicillin by Penicillium chrysogenum W49-1331. College of Agriculture, Department of
Biochemistry. Madison, Wisconsin: University of Wisconsin. Retrieved July 17, 2016,
from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1057142/pdf/applmicro00302-
0061.pdf

Plaza, P., Usall, J., Teixidò, N., & Vinas, I. (2003, March 12). Effect of water activity and
temperature on germination and growth of Penicillium digitatum, P. italicum and
Geotrichum candidum. (A. Gilmour, Ed.) Journal of Applied Microbiology, 94(4), 549-
554. doi:10.1046/j.1365-2672.2003.01909.x

Sautour, M., Rouget, A., Dantigny, P., Divies, C., & Bensoussan, M. (2001, November 23).
Application of Doehlert design to determine the combined effects of temperature, water
activity and pH on conidial germination of Penicillium chrysogenum. (A. Gilmour, Ed.)
Journal of Applied Microbiology, 91(5), 900-906. doi:10.1046/j.1365-2672.2001.01449.x

Sautour, M., Rouget, A., Dantigny, P., Divies, C., & Bensoussan, M. (2001, March). Prediction
of conidial germination of Penicillium chrysogenum as influenced by temperature, water
activity and pH. Letters in Applied Microbiology, 32(3), 131-134. doi:10.1046/j.1472-
765x.2001.00872.x

University of Colorado. (2005, March 19). Measuring bacterial growth. Retrieved May 8, 2016,
from virtuallaboratory.net, inc.: http://virtuallaboratory.colorado.edu/BioFun-
Support/labs/EColi%20introduced/section_04.html

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The Effect of Temperature on Fungal Growth

Visagie, C., Houbraken, J., Frisvad, J., Hong, S., Klaassen, C., Perrone, G., . . . Samson, R.
(2014, June). Identification and nomenclature of the genus Penicillium. Studies in
Mycology, 78, 343-371. doi:doi:10.1016/j.simyco.2014.09.001

Widdel, F. (2010, June 5). Theory and Measurement of Bacterial Growth. Max-Planck-Institut
fuer Marine Mikrobiologie. Bremen, Germany: Universität Bremen. Retrieved May 8,
2016, from Max-Planck-Institut für Marine Mikrobiologie: http://www.mpi-
bremen.de/Binaries/Binary13037/Wachstumsversuch.pdf

24
D. Gustav Christensson

15 APPENDICES

15.1 AGAR COMPOSITION


The nutritive agar used in this experiment consisted of the following substances:

Table 9: Composition of Saboraud Dextrose Agar

Substance Concentration (g dm–3)


Pancreatic Digest of Casein 5
Peptic Digest of Animal Tissue 5
Glucose 40
Agar 15
15.2 CALCULATING SAMPLE ABSORBANCE

Figure 16: Attenuation of Light through Fungal Sample and Nutritive Agar

The absorbance of the Penicillium sample was calculated with Beer-Lambert’s Law, which
states that
𝐼0
𝐴 = log ( )
𝐼
where 𝐴 is the absorbance of the sample (proportional to its concentration), 𝐼0 is the
incident light intensity (measured in lux) and 𝐼 is the transmitted light intensity (also
measured in lux). However, because the fungal sample rested above a thick layer of
nutritive agar which was not to be measured, this law had to be modified. An assumption
was made that the incident light (of intensity 𝐼0 ) would first be attenuated by the fungal

25
The Effect of Temperature on Fungal Growth

sample so that its intensity decreased to 𝐼Sample . Then, the intensity would’ve been
attenuated from 𝐼Sample to 𝐼Total as it passed through the nutritive agar (Figure 16). According
to Beer Lambert’s Law, the absorbance of the fungal sample can be written as
𝐼0
𝐴Sample = log ( )
𝐼Sample
while the absorbance of the nutritive agar can be written as
𝐼Sample
𝐴Agar = log ( )
𝐼Total
Finally, the absorbance of the entire system can be written as

𝐼0
𝐴Total = log ( )
𝐼Total
Adding the absorbance of the sample and the nutritive agar yields
𝐼0 𝐼Sample
𝐴Sample + 𝐴Agar = log ( ) + log ( )
𝐼Sample 𝐼Total

𝐼0 𝐼Sample
𝐴Sample + 𝐴Agar = log (( )( ))
𝐼Sample 𝐼Total
𝐼0
𝐴Sample + 𝐴Agar = log ( )
𝐼Total
𝐴Sample + 𝐴Agar = 𝐴Total
This shows that the overall absorbance is the sum of the individual absorbance from each
part.

Before the experiment began, the absorbance of the agar was measured as shown in
Figure 17.

Figure 17: Attenuation of Light through Nutritive Agar Only

26
D. Gustav Christensson

In this configuration, the following equation is true:


𝐼0
𝐴Agar = log ( )
𝐼Agar

Substituting this into the previous equation of absorbance sums, one obtains:
𝐴Sample + 𝐴Agar = 𝐴Total
𝐴Sample = 𝐴Total − 𝐴Agar
𝐼0 𝐼0
𝐴Sample = log ( ) − log ( )
𝐼Total 𝐼Agar

𝐼0 𝐼Agar
𝐴Sample = log (( )( ))
𝐼Total 𝐼0
𝐼Agar
𝐴Sample = log ( )
𝐼Total

This yields an effective way to calculate the absorbance of the sample without interference
from the agar solution beneath it.

15.3 FINDING THE DOUBLING TIME


It was assumed that the Penicillium followed exponential growth that could be modelled by
the equation
𝑡
𝐴 = 𝐴0 (2𝑡𝑑 )

where 𝐴 is the final absorbance, 𝐴0 was the starting absorbance, 𝑡 is the time elapsed and
𝑡𝑑 is the doubling time. Rearranging for 𝑡𝑑 yields
𝑡
𝐴 = 𝐴0 (2𝑡𝑑 )

𝑡
log 2 𝐴 = log 2 (𝐴0 (2𝑡𝑑 ))

𝑡
log 2 𝐴 = log 2 (𝐴0 ) + log 2 (2𝑡𝑑 )

𝑡
log 2 𝐴 = log 2 (𝐴0 ) + log 2 (2)
𝑡𝑑
𝑡
log 2 𝐴 = log 2 (𝐴0 ) +
𝑡𝑑
𝑡
log 2 𝐴 − log 2 (𝐴0 ) =
𝑡𝑑
𝐴 𝑡
log 2 ( ) =
𝐴0 𝑡𝑑

27
The Effect of Temperature on Fungal Growth

1 𝑡𝑑
=
𝐴 𝑡
log 2 (𝐴 )
0
𝑡
= 𝑡𝑑
𝐴
log 2 (𝐴 )
0

which can be applied to the collected data. The Standard Deviation for this formula can be
found using partial derivatives:

2 2 2

ln 2 𝑡 ln 2 𝑡 ln 2
𝜎𝑡𝑑 = (𝜎𝑡 ) + 2 (𝜎𝐴 0 ) + − (𝜎𝐴 )
𝐴
ln (𝐴 ) 𝐴 𝐴 2
0
𝐴0 ln (𝐴 ) 𝐴 ln (𝐴 )
0 0
√( ) ( ) ( )

28