1

SUMMARY
This lab report examines whether it is possible to produce new strains of Rhodotorula yeast
using mutagenisis which are able to over-produce pigments. The mutagenesis of two species
of this carotenoid producing yeast, R. glutinis and R. minuta, was performed by exposing the
cultivated yeast to UV light in order to cause mutations in the DNA of the strains.

A YMEA medium was prepared and inoculated with the parent strains of the 2 species of
yeast. Dilutions of these parent strains was performed, and a series of plating procedures were
carried out in order to measure the number of colony-forming units in the parent strains. An
average of 208 x 108 CFU/mL and 6.28 x 108 CFU/mL were obtained for R. glutinis and R.
minuta respectively. Four samples, two with a dilution factor of 10 3 and two with a factor of
104, were subsequently exposed to UV light for ten and twenty minutes in order to perform
the mutation procedure. The plating procedure of these mutated strains was performed. An
average of 559 x 103 CFU/mL was found for the R. glutinis mutant strains exposed to 10
minutes, corresponding to a survival rate of 0.00269% with respect to the parent strains. In
contrast, for the R. minuta cells exposed to 10 minutes of UV light, an average of 228 x 10 3
CFU/mL was found with a corresponding survival rate of 0.0363%

Both mutated species of R. glutinis and R. minuta showed a change in the colour of the cells,
producing a stronger pink pigmentation when compared to the parent strain. Both parent and
mutant strains were centrifuged, freeze dried and milled to break the cells of the yeast so that
pigment extraction could be performed. Acetone was used to perform the pigment extraction
from these strains. High-performance liquid chromatography (HPLC) analysis was
subsequently performed to evaluate the changes in pigment concentration of the parent and
mutated cells. This was done to confirm the mutagenesis of the yeast from the parent strains.
Changes in concentration of the pigments β-carotene, γ-carotene, torulene and torularhodin
were successfully evaluated via HPLC analysis. UV light exposure reduced the overall
pigment concentration of both parent strains, a reduction from 31.8 µg/g yeast to 14.5 µg/g
yeast at a 10 minute UV exposure time and 20.09 µg/g yeast at a 20 minute UV exposure
time for R. minuta and from 13.3 µg/g yeast to 14.5 µg/g yeast at 10 minute UV exposure
time for R. glutinis. HPLC analysis confirmed the metabolic pathway of both strains of
Rhodotorula, and the influence in concentration change of its main carotenoid products. Due
to mutagenisis resulting from the UV light exposure Β-Carotene was found to be the highest
concentrated pigment present in both parent and mutated strains of R.minuta. This equated to
an average of 45% in each strain with γ-Carotene, torulene and Torularhodin carotenoids
making up the remaining 55%. When considering R.glutinis, torulene was found to be the
highest concentrated pigment in the parent strain and mutated strain, having a value of 45%
and 65% concentration in the parent and mutated strain respectively. Thus the other pigments
made up the remaining 55% and 35% respectively.

Overall, the mutants obtained provided a variation in pigments, and thus the colour differed
slightly to that of the parent strains. Additionally, the mutants were composed of different
amounts of carotenoids to the parent strains. There was a general trend of less pigmentation
present in the mutated strains than the parent strains, with R. glutinis strains being more
dominant in torulene than β-carotene, with the converse being true for R. minuta strains.

2
TABLE OF CONTENTS
SUMMARY........................................................................................................................................2
TABLE OF CONTENTS........................................................................................................................3
1. INTRODUCTION............................................................................................................................4
2. METHOD.......................................................................................................................................6
2.1 GROWTH OF PARENT STRAINS.............................................................................................................6
2.2.1 MUTAGENESIS PROCEDURE.............................................................................................................6
2.2.2 PLATING PROCEDURE.....................................................................................................................7
2.3.1 IDENTIFYING MUTANTS...................................................................................................................8
2.3.2 GROWTH OF MUTANTS..................................................................................................................8
2.4 PIGMENT EXTRACTION PROCEDURE......................................................................................................9
2.5 EXTRACTION PROCESS.......................................................................................................................9
3. RESULTS......................................................................................................................................10
3.1 SURVIVAL OF CELLS THROUGH MUTAGENESIS.......................................................................................10
3.1.1 Rhodotorula glutinis..........................................................................................................10
3.1.2 Rhodotorula minuta...........................................................................................................12
3.2 ANALYSIS OF HPLC DATA................................................................................................................13
4. DISCUSSION................................................................................................................................16
4.1 COMPARISON OF MUTANTS TO PARENT CULTURE.................................................................................16
4.2 POTENTIAL IMPROVEMENTS.............................................................................................................17
5. CONCLUSION AND RECOMMENDATIONS...................................................................................19
6. REFERENCES................................................................................................................................20
7. APPENDIX...................................................................................................................................22
7.1 APPENDIX A: AREA OF PIGMENTS IN RHODOTORULA FROM HPLC ANALYSIS.............................................22
7.2 APPENDIX B: HPLC DATA FOR STRAINS OF RHODOTORULA IN ΜG OF PIGMENT / G OF YEAST.......................26
7.3 APPENDIX C: COUNT OF MUTANT UNITS FOR EACH AGAR PLATE..............................................................26

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1. INTRODUCTION
Rhodotorula is a pigmented basidiomycete 1 yeast which has applications in multiple sectors
with different species producing four major pigments: β-carotene, γ-carotene, torulene and
torularhodin (Moliné et al, 2012). In addition to having a high growth rate, Rhodotorula is
able to scavenge a wide variety of nutrients from its surroundings, allowing it to survive in a
variety of environments including air, soil, lakes, ocean water, and even various dairy
products (Ellis, 2016; Wirth et al, 2012). Some specific species of Rhodotorula have also
emerged as opportunistic pathogens, containing the ability to colonise and infect susceptible
patients. Of these species, R. mucilaginose, R. glutinis, and R. minuta are known to cause
disease in humans, recognised as emerging yeast pathogens in the last two decades. It is
important to note however that biopigments produced by yeast species are harmless to human
health, while, in contrast, filamentous fungi derived biopigments do present health concerns
(Oz et al, 2019).

Different strains of Rhodotorula (such as R. glutinis and R. minuta) have certain key
characteristics including the ability to: produce large amounts of carotenoids, produce single-
cell proteins from ethanol, and produce acetic acid and acetaldehyde (Wirth et al, 2012). The
carotenoids produced by Rhodotorula protect the cells against the effect of excessive
radiation from the visible and UV light spectrum. The resultant pigmentation is of particular
interest due to the variety of colours produced: from cream to yellow, salmon, pink, orange,
coral red and blood red (Hernández-Almanza et al. 2014). The pigments are produced via the
mevalonate pathway2 which produces isopentyl pyrophosphate units which are successively
condensed to form phytoene. Subsequently, the aforementioned pigments are produced, the
proportion of each carotenoid differing depending on the yeast strain type and conditions
(Moliné, 2012).

1
Member of a large group of fungi which produces spores on basidia. This includes shelf
fungi, rusts, smuts and multiple mushroom species (Ellis, 2016).
2
Also known as the “isoprenoid pathway” or “HMG-CoA reductase pathway”

4
 Figure 1. Metabolic pathway of carotenoids biosynthesis in Rhodoturula (Moliné, 2012).

The ability of various Rhodotorula species to produce interesting biochemical products such
as carotenoids has been cultivated for use in the pharmaceutical, chemical, food and feed
industries (Larone, 1995; Wirth et al, 2012). By using microbial pigments produced by yeasts
such as Rhodotorula, it is possible to obtain controllable and predictable yields (Lau, 2018).
These carotenoids are thus used as colouring agents in the food industry, in addition to use as
vitamin A precursors. For example, colonies of R. glutinis are often coral red to salmon or
orange coloured, and can be smooth to wrinkled, or highly glossy to semi-glossy (Ellis,
2016). Due to the seasonal and geographical variability in the marketing and production of
some pigments of plant origin, carotenoids from Rhodotorula present an alternative microbial
route. This route is low-cost and utilises natural carbohydrate sources as a substrate to
provide economic advantages. While the use of Rhodotorula to produce pigments such as
torulene and torularhodin is currently limited due to the cost of production, it has potential as
there is currently a high demand for using microbial fermentation to produce natural
carotenoids in industries due to public concern regarding food safety and health (Lau et al,
2018; Tang et al, 2019). Carotenoids produced from various Rhodotorula strains thus have
vast potential in the food, feed, health products and cosmetics industries, with a market value
estimated to reach over $2 billion by 2022 (Lyman, 2019).

Additionally, Rhodotorula yeast is utilized in biofuels, enzyme production, biosurfactants,

and as an antagonistic yeast. Rhodotorula species in biodiesel production are classified as
“second generation” biofuels, meaning they are produced using non-food/non-edible sources.
In this case, the species used are able to accumulate single-cell oils which are used. Enzymes
produced from Rhodotorula (mainly D-Amino acid oxidase) have advantages in industries
due to their higher turnover rate and increased stability of FAD binding (Lyman, 2019; Tang
et al, 2019). They are thus used in the production of cephalosporin, a widely sold antibiotic.
In addition, enzymes produced are used in the cosmetic, health and flavouring industries.
Biosurfactants produced by species of the yeast are able to effectively reduce the surface
tension between oil and water mixtures (Wirth et al, 2012). Due to this, they are used in

5
industries such as cosmetics, food, explosive, pharmaceutical, detergents and paints. As these
resultant biosurfactants are biodegradable (in contrast to chemical/synthetic surfactants
currently produced which pose significant environmental concerns) they have great potential
as society moves towards a more sustainable future. Finally, Rhodotorula species are able to
act as antagonistic yeasts, preventing rot in various agricultural produce. Due to their
potential, they are an active area of research when considering reducing the losses in
harvested fruit occurring due to decay from filamentous fungi (Lyman, 2019).

The production of Rhodotorula yeasts is a growing area of opportunity and growth due to its
potential in various sectors and areas. However, when considering the carotenoid
concentration of Rhodotorula, when compared to other microorganisms, the amount
produced is relatively low. Thus, through the process of mutagenesis, it is possible to
examine whether new strains of Rhodotorula can be generated which are able to over-
produce pigments.

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2. METHOD
Utilising mutagenesis an experiment was carried out to determine if new strains of
Rhodotorula could be produced which are able to over-produce pigments. UV light was used
as a mutagen to cause mutations in the DNA of the yeast strains, and these were subsequently
analysed. The procedure was carried out over 2 weeks, in three different sessions, with tutors
completing additional preparation and analysis between sessions. The method is as follows:

2.1 Growth of parent strains

Figure 2: Conical flask with YMEA and strain

Prior to the first session, the parent strains of the yeast, R. glutinis and R. minuta, were
prepared. 50mL of YMEA medium3 was added to a 250 mL conical flask, and a cotton bung
was added before it was covered with aluminium foil. The flask was autoclaved, and then
inoculation from the parent flask took place using a sterile loop. It was then placed in an
incubator (25 ˚C, 48 hours, 237rpm).

2.2.1 Mutagenesis procedure

During the first session, serial dilution was used to dilute the R. glutinitis strain obtained by a
factor of 102 and 103 using a sterile saline solution and a pipette as shown in Figure 3. The
two 103 diluted solutions were then transferred into quartz tubes 4. These tubes were placed in
a sealed box and were exposed to UV light exposure, for 10 minutes and 20 minutes
respectively.

After UV exposure the 103 dilution factor solutions were diluted again to a 104 dilution factor
using a pipette and saline solution. 500 µL of all 4 solutions thus obtained 5 were transferred
and spread onto separate agar plates using a disposable spreader. The plates were then sealed
and covered in aluminium foil6 and incubated for 48 hours.

3
YMEA medium was made by combining yeast extract (3g/L), malt extract (3g/L), peptone
(5g/L) and glucose (10g/L) to water
4
Quartz tubes were used in order to ensure that UV light was not absorbed by the tube
medium (as is the case when using glass and plastic tubes)
5
The four solutions obtained were: 1 - DF1 (dilution factor 103) 10 min. 2 – DF1 20 min. 3 –
DF10 (dilution factor 104) – 10 min. DF10 20 min.

7
 Figure 3. Dilution of R. glutinis by factor of 102 and 103

2.2.2 Plating Procedure

During session 2, the solution diluted by a factor of 10 2 was subsequently used in order to
dilute samples by a factor of 104, 106, 107 and 108 using sterile saline solution and a pipette to
ensure accurate measurements (see Figure 4). Each solution of R. glutinis was mixed for a
few seconds in order to ensure homogeneity.

Figure 4. Dilution procedure of R. glutinis parent strain by factors of 104, 106, 107, and 108.

6
This provided a sealed environment, ensuring the samples were not exposed to additional
light

8
500 µL of the 106, 107 and 108 dilution factor solutions was subsequently spread onto an agar
plates, with three plates prepared per dilution factor 7. This was carried out under a
temperature controlled fume hood to minimize contamination with total of 9 agar plates was
obtained. These plates were then incubated at 25˚C for 48 hours.

2.3.1 Identifying mutants

During session two the colony forming units (CFU) were measured visually after the agar
plates had been incubated for minimum 48 hours. The number of CFU’s present varied from
very few to hundreds. The CFU on the parent plate were counted as well as the mutant plates.
A colony which was strong in pigment was chosen (R.Glutinis), and the number of CFU/mL
was calculated based on the visual analysis of the agar plates, and the corresponding volume
of solution used.

Figure 5. Example of the growth of mutants on agar plates

2.3.2 Growth of mutants

During session two, a flask of autoclaved YMEA medium was prepared per mutant using 50
mL of solution in a 250 mL conical flask. The pigmented mutant colony was transferred to
the flask using a sterile loop. The flask was incubated for 48 hours at 25 ˚C at 225rpm.

7
A disposable spreader was used and discarded in an autoclave bag for each agar plate

9
 Figure 6. YMEA medium prepared for Rhodotorula mutants obtained

2.4 Pigment extraction procedure

Prior to the third session, a centrifuge tube was filled with 15 mL of the sample from the flask
prepared in section 2.3.2. This sample was then spun down for 3 min at 4000 RCF. The liquid
was disposed in a bio-waste container and the cells were resuspended in 15 mL of deionized
water and centrifuged again. The water was poured off and the samples were sent to freeze
drying.

Figure 7. Pigment extraction procedure of mutated strain

2.5 Extraction process

During session three, once the strains were freeze-dried, 5 different samples were weighed
out exactly into a bead beater tube. These weighed 0.0444, 0.0328, 0.0319, 0.0338 and
0.0303g respectively. 1mL of acetone was added to the samples, before being introduced to
the bead beater for 60 s at 4000 rpm. Subsequently, the samples were then spun down in a
minifuge for 60 s. The top layer of the coloured liquid was removed and placed in a separate
amber vial. The same procedure was executed 3 times with the precipitate from the

10
centrifugation process until the supernatant liquid was colourless. The samples in the amber
vials where then analysed via High Pressure Liquid Chromatography (HPLC) in order to
measure the concentration of the different pigments present in both the parent and mutated
strains obtained during the procedure.

Figure 8. Pigment extraction procedure for dry samples

3. RESULTS

3.1 Survival of cells through mutagenesis

3.1.1 Rhodotorula glutinis

During session two of the lab, the parent cells of R. glutinis (consisting of a dilution factor of
106, 107 and 108) were examined in order to determine the amount of colony forming units
(CFU). A dilution factor of 108 was used as the basis to count the CFU per mL of R. glutinis
as this sample had between 30 and 300 CFU, which is considered a reliable count number of
colonies. The samples with dilution factors of 106 and 107 were not used to calculate the CFU
because they exceeded the value to be considered in a reliable count (over 300). Table 1
below shows the CFU count per 500 µL of solution for each 108 sample.

11
 Figure 9. Parent R. glutinis cells with a dilution factor 106, 107, and 108 after 48hour
incubation period

Table 1: CFU count of parent at dilution factor 108

Sample No. CFU count per 500 µL CFU count of 1 mL
1 105 210
2 109 218
3 98 196
Average 104 208

The average number of colonies in each plate was found to be 104. As this number lies within
the reliable count number (30<104<300), this value is valid. Thus, from samples with a 108
dilution factor, the average CFU is calculated to be 208 ×108 CFU/mL.

This CFU counting procedure was then repeated for the dilution factor 1 and 10 (DF1 and
DF10) of the mutated strains which were exposed to 10 minutes and 20 minutes of UV light.
It was chosen to examine the strains exposed to 10 minutes of UV light, as there were clear
colonies which had grown (as evident in Figure 10). Table 2 thus shows the average colony
forming units of the mutant strains of R. glutinis obtained in 5 samples with dilution factors
of 103 and 104 respectively. Using the samples with a dilution factor of 103, the average CFU
was calculated to be equal to559 ×103 CFU /mL .

The mutant units after an incubation period of ~96 hours were around 2-4mm in diameter and
had a circular form. Due to their small size, the elevation of the mutants was not observable.

12
The colours of each mutant, for dilution factors of 10 3 (DF1) and 104 (DF10) were consistent,
being a salmon toned pink-orange colour, and they were opaque in terms of opacity.

Figure 10. Mutant R. glutinis cells exposed to 10 minutes of UV light (a) DF1 (b) DF10

Table 2: CFU count of mutant strains at dilution factor 103 and 104
UV Exposure Dilution Average CFU Average CFU Average number
(min) factor in 500 µL in 1 mL of cells in mutant
1 (10 )
3
172 344 x 103
10 559 x 103
10 (10 )
4
38.67 77 x 103

The samples exposed to 20 minutes of UV light were not counted as, at the time of analysis,
there were no colonies observed to be growing. This is likely due to two factors;
contamination of bacteria on the agar plates and/or the bacteria in the plates still existing in a
dormant state8.
Although the amount of CFU for mutants exposed to 20 minutes of UV light is not available,
an estimate of the percentage of R. glutinis cells which survived exposure to 10 minutes of
UV light was calculated.

3
CFU parent 559 ×10
% survived= ×100 %= 8
× 100 %=0.00269 % Eq. 1
CFU mutant 208 ×10

8
The bacteria were not growing, but still trying to adapt after exposure to 20 minutes of UV
light. It is possible if they were incubated for longer (ie. >96 hours) that some growth would
be evident.

13
It was thus determined that only 0.00269% of the initial R. glutinis parent cells survived in
UV light.

3.1.2 Rhodotorula minuta

A similar analysis of results as identified in Section 3.1.1 was carried out for samples of R.
minuta. This data was provided by Group 12 to enable a comparison between the different
strains of Rhodotorula yeast observed. The average number of CFU found in the parent
strains of R. minuta is shown in Table 3 for dilution factors of 106, 107 and 108. From these
samples an average number of CFU was calculated to be 6.28 x 108 CFU/mL.

Table 3: CFU count of parent R. minuta strain at dilution factor 106, 107, and 108
Dilution Average CFU in Average CFU in
factor 500 µL 1 mL
10 6
276 552
107 43 86
10 8
2 4
Average - 6.28 x 108

Subsequently samples exposed to 10 minutes of UV light with a dilution factor of 1 and 10

respectively were used to perform the CFU counting procedure for the mutant strains. Table
2 shows the CFU of the mutant strains of R. minuta obtained from 3 samples of solutions
with a dilution factor of 103 and 104. Thus, based on these samples, an average number of
CFU was calculated to be equal to 228 ×103 CFU /mL. The number of colonies formed in
samples exposed to 20 minutes of UV light were not counted as at the time of analysis, there
were no units observed to be growing.

Similar to the mutants of R. glutinis, the mutants observed from R. minuta were circular in
shape, and existed in singular units. They were additionally a salmon toned pink-orange
colour, and were opaque in terms of opacity.

Table 4: CFU count of mutants of R. minuta at dilution factor 103 and 10 minute UV
exposure
UV Exposure Dilution Average CFU Average CFU Average number
(min) factor in 500 µL in 1 mL of cells in mutant
1 (103) 123 246 x 103
10 228 x 103
10 (10 )
4
21 210 x 103

An estimate of the percentage of R. minuta cells which survived 10 minutes of UV light

exposure was subsequently calculated.
CFU mutant 228 ×103
% survived= ×100 %= × 100 %=0.0363 % Eq. 2
CFU parent 6.28 ×108

As a result, only 0.0363% of the initial R. minuta parent cells survived in UV light.

14
3.2 Analysis of HPLC data

Using high-performance liquid chromatography (HPLC), the pigment concentrations of the

parent and mutated strains of yeast were analysed. In order to identify each pigment present,
the retention times of each specific pigment (as shown in Table 5) were used in conjunction
with the values obtained by the chromatogram.

Table 5: Average pigment retention time on chromatogram

Retention time (min) 13 – 13.5 15.5 -15.7 15.7 -16 15.8 – 16.3
Pigment Torularhodin Torulene γ- Carotene β- Carotene

The area of the pigments in each of the identified retention times was determined from the
HPLC data (see Appendix A) and then used to calculate concentration of each pigment in
µg/g yeast using the standard equation below where concentration is found in mg/L and the
area is given in mAU:

concentration=6.132×10 × area
−6
Eq. 3

The area of β-Carotene is unnecessary for evaluation as the concentration can be found in
HPLC data (see Appendix A: Table D). The concentration of each pigment according to their
strain and UV exposure time was subsequently calculated in units of µg of pigment/g of yeast
(see Appendix B). Subsequently, the concentration in mg/g of yeast of each pigment was
calculated using Equation 4 where the mass of colonies which survived in yeast is given in
µg/g of yeast, the concentration in mg/L as given in Equation 3, the solvent volume in mL
and the sample size in mg (as identified in Section 2.5).

Colonies survived = 1000×concentration × solvent volume × sample size Eq. 4

An average was taken to compare the difference in pigment concentrations as shown in

Figure 11 and Figure 12. From these figures it can be observed that colony concentration is
generally higher in the parent strains than it is in the mutant strains. Figure 13 and Figure 14
additionally show the different pigment compositions in terms of carotenoid percentage
present of the parent and mutated strains for both Rhodotorula strains. These figures thus
clearly show the variance of pigment composition after being subjected to radiation.

When considering R. glutinis, the mutant strains exposed to 20 minutes of UV light UV were
not considered for analysis as the agar plates had been contaminated by bacteria. However, it
is evident that there is an increase in the pigment torulene in the mutant strain. In contrast, β-
Carotene experiences the greatest decrease, with a decrease of about 75% from the parent
strain. When considering the R. minuta samples, there is around a 50% decrease in the
pigment concentrations for all pigments after 10 minutes of UV light exposure. Although the
concentration of pigments decreased at 10 minutes of UV exposure, it further increases after
20 minutes of UV light mutation as evident.

15
Figure 11: Bar graph of pigment concentrations of R.Glutinis in mutant and parent strains

Figure 12: Bar graph of pigment concentrations of R. Minuta in mutant and parent strains

16
Figure 13: Bar graph of pigment composition of R. glutinis in mutant and parent strains

Figure 14: Bar graph of pigment composition of R. minuta in mutant and parent strains

17
4. DISCUSSION

4.1 Comparison of mutants to parent culture

The production of carotenoids by yeasts such as Rhodotorula varies depending on the species
examined, the medium constituents and the environmental conditions in which the yeast is
cultivated. The number of carotenoids produced by each genus can be classified as low (less
than 100 μg g-1), medium (101 to 505 μg g-1) or high (more than 500 μg g-1) (Kobayashi et al,
1991; Davoli, 2004). Focusing on yeasts of the genus Rhodotorula, there are multiple species
that can be used as carotenoid producers including by not limited to Rhodotorula glutinis,
Rhodotorula mucilaginosa, Rhodotorula acheniorum, Rhodotorula minuta and Rhodotorula
graminis. These are the known best for the production of β –carotene which is why they have
been extensively studied. These yeasts are also known to produce other carotenoids such as
torulene, torularhodin and γ –carotene. (Maldonade, 2008)

In this study, the species R. glutinis and R. minuta are examined. The main carotenoid likely
produced is usually β –carotene (about 25-43% of total carotenoids present) and torulene is
expected to be the second largest (about 28-30% of total carotenoids present) (Rucker, 2007).
The study carried out in this report was the use of mutagenesis via exposure of the culture to
UV radiation to yield a mutated cell culture which would generate a higher yield of
carotenoids than the parent culture. This is a difficult task as the success rate of achieving a
mutated cell that would achieve the desired result has an average success rate of lower than
1% (Cordova, 2018). The UV radiation used for this process was UV-B at a wavelength of
312nm. As is evident from the results (see section 3), the majority of the colony forming units
that were present in the parent culture were killed when the culture was exposed to the UV
light. For both R. glutinis as well as R. minuta the survival rate of the colony forming units
was below 0.1% with the survival rate of each culture being 0.00269% and 0.0363%
respectively. Both of these survival rates are less than desirable. However, the R. minuta cells
had a much larger survival rate in comparison with the survival rate of R. glutinis, being
better by more than 10-fold. This can be explained as the R. minuta culture was seen to have
a larger concentration of the carotenoid torularhodin, which is known to improve the survival
rate of cells in yeasts. (Moliné et al, 2010).

The results also show that the concentration of the carotenoids after being subjected to UV
light dropped regardless of the time duration in which the culture was exposed to radiation.
However, considering the strain of R. minuta in which the cells were exposed to UV light for
a period of twenty minutes, the concentration of torulene was higher than that of the cells
which were subjected to the light for ten minutes. This shows some improvement in
carotenoid production. Unfortunately, the same cannot be said for the R. glutinis strains
examined, as cultures of this species were not tested at UV radiations of twenty minutes due
to contamination of the samples.

Additionally, contrary to literature, the species both in the parent culture as well as the
mutated cell cultures had similar concentrations of β–carotene and torulene. The average
concentration of the carotenoid torulene in the R. minuta culture was about 43% of total

18
carotenoids present. In comparison, the average concentration of β–carotene was 46%. It
must be noted that exposure of the culture to UV light did have the bonus of increasing the
yields of these two carotenoids and reduced the yield of γ – carotene which could have
certain benefits. There was one positive result in that for the R. glutinis culture exposed to
UV light for ten minutes, the concentration of torulene increased in comparison to the parent
culture with concentrations of 13.8 μg/g yeast and 17.4 μg/g yeast respectively. However, the
overall concentrations for both the species did not show an improvement of total carotenoid
concentration.

4.2 Potential Improvements

Radiation at a wavelength of 320–400 nm (UV-A) causes only indirect damage to DNA,

proteins, and lipids. In contrast, radiation at a wavelength of 280-320 nm (UV-B) causes
damage to DNA by generating two types of mutagenic lesions (Moliné et al, 2010). In this
laboratory the mutation procedure was completed utilising a UV intensity of 312nm, which is
in the UV-B range. However, testing the cultures at different intensities could help improve
the mutation procedure. It may be also be worthwhile varying the distance the cultures are
from the source of the radiation as well as trialling various exposure times. This will enable
the ideal intensity and exposure time for maximum effect to be determined in order to obtain
mutated cultures which produce desirable results.

Furthermore, different methods such as exposure to LED light could be an alternate approach
to improve the mutation process. This hypothesis has been previously tested and presents
additional possibilities for obtaining mutated cells and corresponding results (Yen and Zhang,
2011). A less harsh mutagen procedure may result in more of the cells surviving, allowing for
a wider range of mutants which are able to over-produce the desired pigments, or produce
them in different ratios to what is currently known.

Improvements could be made to the laboratory methods used for this experimental analysis.
The first possible improvement that could be made would be to try testing the other species of
the Rhodotorula yeast strain in addition to R. glutinis and R. minuta. Strains such as
Rhodotorula mucilaginosa have already shown positive results in varying mutations and may
provide additional mutated strains which are able to over-produce pigments (Wang, 2017).
Having a wider range of strains which to test from would allow more results which can be
used to determine which strains best survive mutation and provide usable pigments.

Another key improvement that could be made to the experiment is to increase the number of
samples tested to get a larger sample array and improve the chances of successful mutation. It
has to be noted that this would be more labour intensive but would still be beneficial. This
would be done in addition to completing the experiment as a whole several times, each time
using the previously gained mutant strains as parent strains for the next starting point (ie.
further mutating these strains). This will allow mutants to be generated which differ greatly
from the initial parent strains. Thus, they may have more desirable results in terms of pigment
production and percentage of carotenoids.

19
Finally, it must be noted that some of the agar plates used for this experiment were subject to
contamination (ie. undesired bacteria growth on the R. glutinis 20minute mutation strains).
This was due to various factors including the use of agar plates which had frosted over in the
fridge forming ice particles which then melted resulting in some of the agar plates used being
contaminated beforehand with moisture. This could affect the success of the experiment.
Furthermore, completing all procedures under the fume hood, in a temperature controlled
climate will also serve to limit contamination from outside sources, ensuring all
experimenting is contained and out-side factors are eliminated.

20
5. CONCLUSION AND RECOMMENDATIONS
The mutagenesis of two strains of Rhodotorula yeast, R. glutinis and R. minuta, was
performed via exposure to UV light with the aim to produce a mutated strain that would
successfully generate a higher yield of carotenoids than the parent culture.

Utilising UV light with a wavelength of 312nm as the mutagen, parent strains of R. glutinis
and R. minuta were exposed for a 10 and 20minute interval in order to induce mutagenesis in
the DNA of the yeast. Samples were obtained and plated utilising agar plates before being
incubated. A visual analysis of the cell units produced was carried out in order to determine
the amount of colony forming units per strain and the survival rate of the mutated cells.
Furthermore, an HPLC analysis was carried out to examine the concentrations of the
pigments produced by both parent and mutated strains.

The results obtained showed that the majority of the parent culture cells were killed during
the radiation exposure, equating to a survival rate of less than 0.1% for all samples with
respect to the original parent colony forming units. However, both mutated species of R.
minuta and R. glutinis showed a change in the colour of the cells, producing a stronger
salmon pink colour compared to the parent strains. Overall, the concentration of the
carotenoids produced by the mutated strains decreased in comparison to the parent strains,
regardless of the exposure duration or sample concentration. In contrast, the mutated strains
of R. glutinis produced a higher concentration of torulene with respect of its parent strain.
This suggests possible mutation of the species by means of changes in the composition of one
carotenoid. With regards to both the parent and mutant strains, R. glutinis produced more
torulene than β–carotene whilst R. minuta produced more β–carotene than torulene.

Due to the cells having such a low survival rate (<0.1%), in addition to mutant strains failing
to over-produce pigments this mutation process was not as effective as it could be.
Improvements could be made to the mutagenesis of the species by varying the wavelength of
the UV light strains were exposed to, decreasing the exposure time, or changing the distant
the samples were placed at from the light. Potential improvements can also be made to the
methods used for this experimental analysis, including expanding the number of Rhodotorula
species analysed and completing the whole laboratory under a fume hood, thus limiting the
potential for contamination and ensuring a temperature controlled environment. It is
additionally recommended that the laboratory be repeated multiple times, utilising the mutant
strains obtained the previous time as the new parent strain starting points. This will enable
mutants to be generated which differ greatly from the initial parent strains, increasing the
possibility of producing mutated strains which are able to successfully over-produce
pigments as desired.

21
6. REFERENCES
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Carotenoids: Pathways and Regulation. In Progress in Carotenoid Research. IntechOpen.
Davoli, P., Mierau, V., & Weber, R. W. S. (2004). Carotenoids and fatty acids in red yeasts
Sporobolomyces roseus and Rhodotorula glutinis. Applied Biochemistry and
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Ellis, D. (2016). Rhodotorula. Mycology Online. Retrieved from
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Hernandez-Almanza, A., Montanez, J. C., Aguilar-Gonzalez, M. A., Martinez-Avila, C.,
Rodríguez-Herrera, R., & Aguilar, C. N. (2014). Rhodotorula glutinis as source of
pigments and metabolites for food industry. Food Bioscience, 5, 64-72.
Kobayashi, M., Kakizono, T., & Nagai, S. (1991). Astaxanthin production by a green alga,
Haematococcus pluvialis accompanied with morphological changes in acetate media.
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Larone DH. (1995). Medically Important Fungi—A Guide to Identification. 3rd edition.
Washington, DC, USA: American Society for Microbiology.
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Abdullah, MO., Toh, SC., Lihan, S. (2018). Production of pigments by Rhodotorula
mucilaginosa. Malaysian Journal of Microbiology Vol 14(4) (pp. 344-350).
Lyman, M., Urbin, S., Strout, C., & Rubinfeld, B. (2019). The Oleaginous Red Yeast
Rhodotorula/Rhodosporidium: A Factory for Industrial Bioproducts. In Yeasts in
Biotechnology. IntechOpen.
Maldonade, I. R., Rodriguez-Amaya, D. B., & Scamparini, A. R. (2008). Carotenoids of
yeasts isolated from the Brazilian ecosystem. Food Chemistry, 107(1), 145-150.
Moliné M., Libkind D., van Broock M. (2012). Production of Torularhodin, Torulene, and β-
Carotene by Rhodotorula Yeasts. In: Barredo JL. (eds) Microbial Carotenoids From
Fungi. Methods in Molecular Biology (Methods and Protocols), vol 898. Humana Press,
Totowa, NJ
Moliné, M., D. Libkind, and M. van Broock. (2012). Production of Torularhodin, Torulene,
and β-Carotene by Rhodotorula Yeasts, in Microbial Carotenoids From Fungi: Methods
and Protocols. J.-L. Barredo, Editor. Humana Press: Totowa, NJ. (pp. 275-283).
Moliné, M., Flores, M. R., Libkind, D., del Carmen Diéguez, M., Farías, M. E., & van
Broock, M. (2010). Photoprotection by carotenoid pigments in the yeast Rhodotorula
mucilaginosa: the role of torularhodin. Photochemical & Photobiological Sciences, 9(8),
1145-1151.
Oz, S., Mariano-Silva, G., Leite, M.O., Mura, F.B. (2019). Production of fungal and bacterial
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327-360). Elsevier.

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Rucker, R. B., Zempleni, J., Suttie, J. W., & McCormick, D. B. (2007). Handbook of
vitamins. CRC Press.
Tang, W., Wang, Y., Zhang, J., Cai, Y., He, Z. (2019). Biosynthetic Pathway of Carotenids
in Rhodotorula and Strategies for Enhanced Their Production. J. Microbiol Biotechnol
(pp. 507-517).
Wang, Q., Liu, D., Yang, Q., & Wang, P. (2017). Enhancing carotenoid production in
Rhodotorula mucilaginosa KC8 by combining mutation and metabolic engineering.
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cultivation of Rhodotorula glutinis. Bioresource technology, 102(19), 9279-9281.

23
7. APPENDIX

7.1 Appendix A: Area of pigments in Rhodotorula from HPLC Analysis

Table A: Tabulation of area and calculation of Torularhodin concentration
Concentration mg (g- µg (g-
Area (e.g.
File name torularhodin torularhodin)/g torularhodin)/g
torularhodin)
(mg/L) yeast yeast
A-1 2146.000 0.013159 0.001 0.731071
A-2 2295.000 0.014073 0.001 0.668548
A-3 4778 0.029299 0.002 1.562597
A-4 0.000 0 0.000 0
A-5 1473.000 0.009032 0.001 0.586522
A-6 1961.000 0.012025 0.001 0.809754
A-7 0.000 0 0.000 0
A-8 0.000 0 0.000 0
A-9 0.000 0 0.000 0
A-10 4577.000 0.028066 0.001 1.272842
A-11 5897.000 0.03616 0.002 1.959913
A-12 3335.000 0.02045 0.001 0.921181
A-13 0.000 0 0.000 0
A-14 3057.000 0.018746 0.001 1.175268
A-15 3514.000 0.021548 0.001 1.4223
A-16 6152.000 0.037724 0.002 2.232193
B-1 0.000 0 0.000 0
B-2 6673.000 0.040919 0.003 2.986776
B-3 4937.000 0.030274 0.002 1.904005
B-4 12773.000 0.078324 0.007 6.931331
B-5 2104.000 0.012902 0.001 0.691782
B-6 - 0 0.000 0
B-7 3452.000 0.021168 0.001 0.707949
B-8 2740.000 0.016802 0.001 0.760257
B-9 9489.000 0.058187 0.005 4.952047
B-10 - 0 0.000 0
B-11 - 0 0.000 0
B-12 - 0 0.000 0
B-13 - 0 0.000 0
B-14 - 0 0.000 0
B-15 2251.000 0.013803 0.001 1.061779
B-16 8316.000 0.050994 0.002 2.007626
B-17 6537.000 0.040085 0.002 1.597007
B-18 4542.000 0.027852 0.002 1.875525
B-19 16664.000 0.102184 0.010 9.505456
B-20 6695.000 0.041054 0.004 4.390774

24
Table B: Tabulation of area and calculation of Torulene concentration
Concentration mg µg
File name Area (e.g. torulene) torulene (torulene)/g (torulene)/g
(mg/L) yeast yeast
A-1 23905.000 0.14658546 0.008 8.143637
A-2 27149 0.166477668 0.008 7.908678
A-3 29977 0.183818964 0.010 9.803678
A-4 8066 0.049460712 0.003 3.34194
A-5 14853 0.091078596 0.006 5.914195
A-6 18032 0.110572224 0.007 7.445941
A-7 10931 0.067028892 0.004 4.215654
A-8 11341 0.069543012 0.005 4.651706
A-9 17294 0.106046808 0.006 5.974468
A-10 47467 0.291067644 0.013 13.20035
A-11 38401 0.235474932 0.013 12.76287
A-12 40472 0.248174304 0.011 11.17902
A-13 10699 0.065606268 0.004 4.000382
A-14 24052 0.147486864 0.009 9.246825
A-15 29686 0.182034552 0.012 12.01548
A-16 44585 0.27339522 0.016 16.17723
B-1 12536 0.076870752 0.006 5.823542
B-2 0 0 0.000 0
B-3 36220 0.22210104 0.014 13.96862
B-4 0 0 0.000 0
B-5 21977 0.134762964 0.007 7.225896
B-6 - 0 0.000 0
B-7 35936 0.220359552 0.007 7.369885
B-8 47593 0.291840276 0.013 13.20544
B-9 92439 0.566835948 0.048 48.24136
B-10 - 0 0.000 0
B-11 - 0 0.000 0
B-12 - 0 0.000 0
B-13 - 0 0.000 0
B-14 - 0 0.000 0
B-15 20826 0.127705032 0.010 9.823464
B-16 93636 0.574175952 0.023 22.60535
B-17 50011 0.306667452 0.012 12.21783
B-18 32113 0.196916916 0.013 13.2604
B-19 - 0 0.000 0
B-20 - 0 0.000 0

25
Table C: Tabulation of area and calculation of γ-Carotene concentration
Concentration
mg (g- µg (g-
Area (e.g. Gamma gamma
File name carotene)/g carotene)/g
carotene) carotene
yeast yeast
(mg/L)
A-1 2384.000 0.014618688 0.001 0.812149
A-2 2684.000 0.016458288 0.001 0.781866
A-3 3053.000 0.018720996 0.001 0.998453
A-4 0.000 0 0.000 0
A-5 1448.000 0.008879136 0.001 0.576567
A-6 1613.000 0.009890916 0.001 0.666055
A-7 1041.000 0.006383412 0.000 0.401472
A-8 1051.000 0.006444732 0.000 0.431086
A-9 2005.000 0.01229466 0.001 0.692657
A-10 5075.000 0.0311199 0.001 1.411333
A-11 9946.000 0.060988872 0.003 3.30563
A-12 4434.000 0.027189288 0.001 1.224743
A-13 1343.000 0.008235276 0.001 0.502151
A-14 7265.000 0.04454898 0.003 2.793039
A-15 11717.000 0.071848644 0.005 4.742485
A-16 13406.000 0.082205592 0.005 4.864236
B-1 1440.000 0.00883008 0.001 0.668945
B-2 0.000 0 0.000 0
B-3 3251.000 0.019935132 0.001 1.253782
B-4 0.000 0 0.000 0
B-5 2495.000 0.01529934 0.001 0.82034
B-6 - 0 0.000 0
B-7 3611.000 0.022142652 0.001 0.740557
B-8 4949.000 0.030347268 0.001 1.37318
B-9 7769.000 0.047639508 0.004 4.054426
B-10 - 0 0.000 0
B-11 - 0 0.000 0
B-12 - 0 0.000 0
B-13 - 0 0.000 0
B-14 - 0 0.000 0
B-15 1826.000 0.011197032 0.001 0.86131
B-16 10256.000 0.062889792 0.002 2.475976
B-17 1964.000 0.012043248 0.000 0.479811
B-18 2617.000 0.016047444 0.001 1.080636
B-19 - 0 0.000 0
B-20 - 0 0.000 0

26
Table D: Tabulation and calculation of β-Carotene concentration

Concentration beta mg (beta µg (beta

File name
carotene (mg/L) carotene)/g yeast carotene)/g yeast

A-1 0.165 0.009 9.17

A-2 0.181 0.009 8.60
A-3 0.208 0.011 11.09
A-4 0.064 0.004 4.32
A-5 0.09 0.006 5.84
A-6 0.106 0.007 7.14
A-7 0.091 0.006 5.72
A-8 0.068 0.005 4.55
A-9 0.127 0.007 7.15
A-10 0.279 0.013 12.65
A-11 0.269 0.015 14.58
A-12 0.233 0.010 10.50
A-13 0.081 0.005 4.94
A-14 0.207 0.013 12.98
A-15 0.255 0.017 16.83
A-16 0.328 0.019 19.41
B-1 0.1 0.008 7.58
B-2 0.01 0.001 0.73
B-3 0.203 0.013 12.77
B-4 0.023 0.002 2.04
B-5 0.151 0.008 8.10
B-6 - 0.000 0.00
B-7 0.191 0.006 6.39
B-8 0.206 0.009 9.32
B-9 0.39 0.033 33.19
B-10 - 0.000 0.00
B-11 - 0.000 0.00
B-12 - 0.000 0.00
B-13 - 0.000 0.00
B-14 - 0.000 0.00
B-15 0.116 0.009 8.92
B-16 0.3302 0.013 13.00
B-17 0.211 0.008 8.41
B-18 0.141 0.009 9.49
B-19 0.025 0.002 2.33
B-20 0.012 0.001 1.28

27
7.2 Appendix B: HPLC data for strains of Rhodotorula in µg of pigment / g of yeast

Table E: Minuta HPLC data in µg/g yeast

Minuta Parent 10 min 20 min
Torularhodin 1.721858286 0.7113 0.9874
Torulene 12.9337113 6.5771 8.6187
γ-carotene 2.946835662 0.7098 0.8642
β-carotene 14.30 6.55 9.62

Table F: Glutinis HPLC data in µg/g yeast

Glutins Parent 10 min 20 min
Torularhodin 2.033553 1.802317 NO DATA
Torulene 15.02599 17.41159 NO DATA
γ-carotene 1.72175 1.477893 NO DATA
β-carotene 13.13 10.70 NO DATA

7.3 Appendix C: Count of mutant units for each agar plate

Table G: Mutant number of colonies count under 10 minutes UV

Samples No. DF1 DF 10
1 (AK) 171 54
2 (AA) 183 48
3 (AJ) 85 30
4 (JS) 229 46
5 (SS) 192 54

28