Growth Kinetics of oleaginous yeast,

Rhodosporidium toruloides, in high salinity condition

A dissertation submitted to
The University of Manchester for the degree of MSc
In the faculty of Engineering and Physical Sciences

2011


Shimme Sharma





School of Chemical Engineering and Analytical Sciences
2

Contents:
Abstract..............................................................................................................8
Declaration.......................................................................................................9
Copyright statement.........................................................................................9
Acknowledgement............................................................................................10
1. Introduction..........................................................................................................11
2. Literature review..................................................................................................12
2.1. Introduction ......................................................................................12
2.2. Oleaginous Micro-organisms...............................................................12
2.2.1. Oleaginous micro-algae......................................................................15
2.2.2. Oleaginous Bacteria........................................................................15
2.2.3. Oleaginous moulds and yeasts.....................................................15
2.2.4. Oleaginous yeast........................................................................15
2.3. Rhodosporidium toruloides as the model yeast.......................................17
2.3.1. Considerations in choosing R. toruloides as the model yeast............17
2.3.2. Life cycle of R. Toruloides............................................................19
2.3.3. Growth kinetics of R. toruloides........................................................20
2.3.4. Biochemistry of lipid accumulation in R. toruloides ........................23
2.4. Factors affecting R. toruloides growth, lipid accumulation and fatty acid
profiles....................................................................................................24
2.4.1. Mode of operation.......................................................................24
2.4.2. Culture conditions.............................................................................25
2.4.2.1.Temperature and pH..................................................................25
2.4.2.2.Dissolved Oxygen concentration in the culture ........................26
2.4.3. Media Composition....................................................................28
2.4.4. Effect of the presence of high concentration of salt in the media on
the growth kinetics of R. Toruloides ................................................29
2.4.4.1.Effect of salinity on Growth phase of R.toruloides......................30
2.4.5. Role of Surfactants on the growth and lipid yield of the oleaginous
yeast............................................................................................30
2.5. Disadvantages of using oleaginous yeast..................................................31
2.6. Summary..............................................................................................32
3

3. Objectives.............................................................................................................33
4. Materials and Methods.........................................................................................36
4.1. Introduction..................................................................................................36
4.2. Materials.............................................................................................36
4.2.1. Chemicals..................................................................................36
4.2.2. Microorganism.............................................................................36
4.2.2.1.Working cell Bank or vials...........................................................36
4.2.2.2.Cell Count.....................................................................................37
4.2.3. Equipments.........................................................................................37
4.3. Methods.......................................................................................................37
4.3.1. Media composition and preparation...................................................37
4.3.1.1.Inoculums medium.......................................................................37
4.3.1.2.Process medium............................................................................38
4.3.1.2.1. Experiment on slants or petri plates.................................38
4.3.1.2.2. Experiment on Carbon to nitrogen ratios, Tween 20, and
in Bioreactor.....................................................................38
4.3.1.2.3. Synthetic sea water experiment........................................41
4.3.2. Experiment set up and design.............................................................43
4.3.2.1.Design and setup of experiment on petri plates............................43
4.3.2.2.Design and setup of experiments in shake flasks.........................43
4.3.2.3.Design and set up for the Bioreactor experiment.........................44
4.3.3. Analytical methods.............................................................................46
4.3.3.1.Microscopy...................................................................................46
4.3.3.2.Optical density determination.......................................................47
4.3.3.3.Dry cell weight (DCW) measurement..........................................47
4.3.3.4.pH measurement...........................................................................48
4.3.3.5.Residual Glucose analysis............................................................48
4.3.3.6.Lipid extraction............................................................................49
4.3.4. Statistical method...............................................................................51
4.4. Summary......................................................................................................51
5. Results and Discussion.........................................................................................52
5.1. Introduction.................................................................................................52
4

5.2. Growth of R. toruloides on agar support under high salinity condition......52
5.3. Comparison of growth of R. toruloides in basal medium with synthetic sea
water and basal medium with sodium chloride...........................................53
5.4. Experiment on the growth of R. toruloides with Tween 20 in the
medium........................................................................................................55
5.5. Growth of R. toruloides with varied C/N ratio............................................58
5.6. Growth of R. toruloides in Batch mode Bioreactor.....................................65
5.7. Results of pH in the shake flask experiments..............................................68
5.8. Summary......................................................................................................70
6. Conclusion and Further work...............................................................................72
References
Appendix

List of tables
Table 2.1: Lipid content of a number of oleaginous micro-organisms and
conventional crops......................................................................................................14
Table 2.2: The fatty acid profile of some of the oleaginous micro-organisms...........17
Table 2.3: Different species of oleaginous yeast compared for their biomass density
and lipid accumulation...............................................................................................18
Table 4.1: Process media composition.......................................................................39
Table 4.2: Composition of Synthetic sea water..........................................................42


List of Figures
Figure 2.1: Proposed life cycle of R. toruloides.........................................................20
Figure 2.2: Growth pattern of R. toruloides in batch culture.....................................21
Figure 2.3: Stages that lead to lipid accumulation in oleaginous yeast......................24
Figure 2.4 (a) and (b): Oxygen solubility in both fresh water (a) and sea water (b)
with varied temperature and pressure.........................................................................27
5

Figure 4.1: Photograph showing one of the set up of the shake flask experiment
taken at 0 hours of inoculation before sampling.....................................................44
Figure 4.2: Photograph of the inoculum of R. toruloides 48 hours old......................44
Figure 4.3: Photograph of the set-up of 5L volume Bioreactor used for batch
fermentation................................................................................................................45
Figure 4.4: Photograph of one of the samples of R. toruloides taken at 48 hours
viewed under a light microscope from Olympus Japan with JVC TK-C1381 colour
video camera using a 100X objective lens.................................................................47
Figure 4.5: Photograph of a Soxhlet System HT 1043 Lipid Extraction Unit...........49
Figure 5.1: The observed growth of R. toruloides in the two set of medium condition
at different time interval.............................................................................................53
Figure 5.2: Plot of optical density at A
600
on a logarithmic scale with different time
interval, comparing the growth of R. toruloides in the two different media. A
positive control without salinity is also compared.....................................................54
Figure 5.3: Scatter diagram of OD on logarithm scale versus fermentation time,
comparing correlation of medium with synthetic sea water and with NaCl .............55
Figure 5.4: Plot of OD on logarithmic scale with fermentation time for different
concentration of Tween 20. S1 and S2 stand for duplicates that are flask 1 and flask
2 whose readings were taken at the same time...........................................................56
Figure 5.5: Plot of OD on logarithmic scale versus different concentration of Tween
20................................................................................................................................58
Figure 5.6 (a): Plot of optical density at A
600
obtained from cultures of R. toruloides
in medium with varying C/N ratios. Duplicates from flasks 1 and 2 were also taken
at the same time..........................................................................................................59
Figure 5.6 (b): Plot of dry biomass weight in g/L obtained from cultures of R.
toruloides in medium with varying C/N ratios...........................................................60
Figure 5.7: Microscopic picture of a sample of R. toruloides taken at 48
th
hour of
inoculation showing difference in cell sizes...............................................................61
6

Figure 5.8 (a): Scatter diagram of optical density at A
600
with varied C/N ratios......61
Figure 5.8 (b): Scatter diagram of dry biomass with varied C/N ratios.....................61
Figure 5.9 (a): Plot of optical density at A
600
obtained from culture of R. toruloides
in the medium with various C/N ratios. All readings were taken at 48
th
hour of
inoculation..................................................................................................................63
Figure 5.9 (b): Plot of dry biomass concentration in g/L of the culture of R.
toruloides in the medium with various C/N ratios. All readings were taken at 48
th

hour of inoculation.....................................................................................................64
Figure 5.10: Plot of optical density at A
600
on logarithm scale taken at different
fermentation times......................................................................................................66
Figure 5.11: Plot of dry Biomass weight on Logarithmic scale taken at various
fermentation times......................................................................................................66
Figure 5.12: Plot of optical density at 600nm, dry biomass weight, and residual
glucose concentration with increasing fermentation time..........................................67
Figure 5.13: Plot of optical density and dry biomass weight of the batch culture in
the bioreactor with respect to fermentation time........................................................68
Figure 5.14 (a): Plot of pH measured in the two different medium conditions of
synthetic sea water and sodium chloride, combined with the pH of medium without
salinity at different time interval................................................................................69
Figure 5.14 (b): Plot of pH measured in all the different the medium with different
Tween 20 concentration, combined positive control, at different time interval. S1 and
S2 are duplicates of same concentration whose reading was taken at same time......69





7

Nomenclature
Specific growth rate (h
-1
)

max
Maximum specific growth rate (h
-1
)
S Limiting substrate concentration (g/L)
K
s
Saturation constant (g/L)
x
t
Biomass concentration after time interval of t hours
x
o
Initial biomass concentration at time zero
t Fermentation time in hours

Standard deviation
Mean of the observed values
N Sample size
x
1
Observed values of samples
A
600
Absorbance at 600 nm
ATP Adenosine tri-phosphate
NADPH Nicotinamide Adenine dinucleotide phosphate
C/N Carbon to Nitrogen ratio
Ppm Parts per million
Rpm Revolution per minute
PUFA Poly-unsaturated fatty acid
OD Optical Density








8

Abstract
Microbial oil production from oleaginous yeast has been of great interest for its
ability to accumulate high lipid content and high biomass density in a shorter time.
Rhodosporidium toruloides in particular has been utilized in this study for its ability
to accumulate a very high content of lipid almost more than 70% of its dry cell
weight. The thesis aims at investigating the growth kinetics of R. toruloides in high
salinity conditions in order to exploit the oceanic resources rather than fresh water.
The growth requirement for each organism differs from one another, so in order to
maximise the lipid yield, R. toruloides behaviour in high salinity needs to be studied.
Medium composition such as addition of Tween 20, use of different C/N ratio, and
use of synthetic sea water medium has been carried out. A batch culture was run in
the bioreactor with growth condition of temperature, pH and oxygen saturation being
controlled.
The results show that R. toruloides growth kinetics is not affected by the set of
synthetic sea water or sodium chloride medium. Use of surfactants like tween 20 did
not make a significant increase in the growth of R. toruloides as tween 20 increases
the growth of the cells by increasing the oxygen mass transfer whose effect was not
significant in shake flasks due to lower oxygen saturation level in flasks when
compared to bioreactor. Different C/N ratios were utilized in the medium to observe
the growth of R. toruloides. The pattern showed that increasing the glucose
concentration increased the inhibitory effect on the growth of the cells. However,
increasing nitrogen concentration increased the growth rate of the cells. A batch
bioreactor was run using the initial glucose concentration of 100g/L and after 76
hours, the specific growth rate of R. toruloides was 0.15 0.05 h
-1
, lipid content was
29.73% (w/w) and lipid was 3.92 g/L. The dry biomass concentration increased from
0.13 g/L to 10.54 g/L and glucose concentration decreased to 47 g/L. The lipid
content was lower than expected after 76 hours, although, if the bioreactor was run
for more number of hours, the yield could have been improved.

Keywords: R. toruloides, Growth kinetics, Salinity, C/N ratio, Tween 20,
Bioreactor, Synthetic sea water.
9

Declaration
Any piece of the work referred to this dissertation has not been submitted in support
of an application for another degree or qualification of this or any other University or
other Institute of learning.
Copyright Statement

1. Copyright in text of this dissertation rests with the author. Copies (by any
process) either in full, or of extracts, may be made only in accordance with
instructions given by the author. Details may be obtained from the relevant
Programme Administrator. This page must form part of any such copies made.
Further copies (by any process) of copies made in accordance with such
instructions may not be made without the permission (in writing) of the author.
2. The ownership of any intellectual property rights which may be described in
this dissertation is vested in the University of Manchester, subject to any prior
agreement to the contrary, and may not be made available for use by third
parties without the written permission of the University, which will prescribe
the terms and conditions of any such agreement.
3. Further information on the conditions under which disclosures and exploitation
may take place is available from the Academic Dean of the Faculty of
Engineering and Physical Sciences.









10

Acknowledgement
I would like to take the opportunity to give my sincere gratitude to my supervisor,
Professor Colin Webb for his constant support and guidance without which this
dissertation would not have been completed successfully.
I would also like to express my sincere thanks to Dr Antoine Trzcinski, for his
throughout supervision and training that lead to the successful accomplishment of
objectives within this short period of time.
I thank all members of Professor Colins group especially, Wang Xi, Apiliak
Salakkam, Esra Uckun, Musaalbakri Manan and Ernesto Hernsndez, whose
motivation and guidance helped me achieve success in this project.
Finally, I would like to thank my parents and friends for their love and support
throughout the course of this project.













11

Introduction Chapter 1
The current situation related to the depletion of fossil fuel resources and its related
adverse environmental effect due to toxic gases emissions, has led researchers to
seek for a sustainable alternative for the production of bio-fuels especially from
organic sources or biomass. The increase in the demand for fossil fuel consumption
basically for transportation and relating it to the current situation of its depletion
requires an urgent need to seek for an alternative for bio-fuel production.
Many oleaginous micro-organism like yeasts, fungi, bacteria and micro-algae have
been utilized in the past as a feedstock for bio-fuel production because of their non-
competitive nature for arable land and food, presence of renewable long-chained
poly-unsaturated fatty-acid profile similar to composition of conventional fuels,
faster growth rate, and more sustainable when compared to oil crops. However, the
fermentation cost for production of microbial oil is high, which requires a need to
optimize the process. As the growth requirement for every species varies, therefore,
the process could be optimized applying different culture conditions and medium
composition in order to maximise the productivity.
Most of the research done until today utilized fresh water for the production of
microbial oil, other than in some species of micro-algae such as Dunaliella
primolecta that accumulates lipid in salinity conditions (Chisti, 2007). However,
micro-algae require larger area for cultivation and higher doubling time than
oleaginous yeast. In this research, the effect of high salinity on the growth and lipid
production in oleaginous yeast was investigated by studying its growth kinetics. This
study could help in the optimization of the culture conditions and medium
composition for maximal productivity in order to obtain a more feasible and
sustainable commercial process for the production of bio-diesel. It also provides an
insight on the use of oceanic resources for the production of microbial lipid rather
than restricting to use of fresh water, which could be used for human consumption.
R. toruloides has an ability to produce higher biomass density and lipid content of
more than 70% of its total dry biomass (Li et al., 2007). It also has a mechanism to
tolerate extreme conditions of high salinity and variation in pH, therefore, it has been
utilized in this research.
12

Literature review Chapter 2
2.1. Introduction
In the recent years, there has been an increase in the demand of the use of bio-fuel
mainly bio-diesel, due to their potential benefits over the use of other conventional
fuels. Bio-fuel is mainly renewable as the feedstock used for its production is mainly
organic sources. The composition of the fatty-acids in the bio-diesel mainly depends
on the source of feedstock as stated by Canakci and Sanli (2008). For this purpose,
oil from crops like palm, sunflower, among others has been utilized but due to their
high demand as food for human consumption has lead to a food versus fuel
competition. The quest to seek for a sustainable, renewable and environment friendly
alternative source for bio-fuel production has been of great importance. Currently,
the use of oleaginous micro-organisms for the production of bio-diesel has been of of
great interest for its ability to accumulate lipid more than 20% of its dry (Meng et al.,
2009). Rhodosporidium toruloides, oleaginous yeast, has been utilized by many
researchers for its ability to accumulate lipid more than 75% of its dry biomass by
utilizing only a carbon and a limiting nitrogen source (Ratledge and Wynn, 2002).
Leesing and Baojungharn (2011) has cited some of the benefits of using oleaginous
yeasts as they bear no competition with food-crops, are not affected by any seasonal
change as in case of food-crops, they produce a high biomass density and lipid
content, similar fatty-acid profile with the oil produced from the crops like jatropha,
sunflower, among others. R. toruloides growth kinetics has been studied by varying
the medium composition in order to obtain best suitable condition for the production
of high biomass density as well as lipids.
High salinity conditions has been utilized in our study in order to explore the use of
oceanic resources or brackish water rather than being restricted to use of fresh water
sources, hence, reducing the competition with fresh water that could be used for
human consumption.

2.2. Oleaginous Micro-organisms
Microorganisms that have the ability to accumulate lipids more than 20% of the
organisms total dry biomass weight are known as oleaginous micro-organisms
(Ageitos et al., 2011) which is higher than most of the organisms that have the
13

ability to synthesize a minimum amount of lipid, although, the lipid produced by
most of these organisms may not be used for bio-diesel production. These micro-
organisms could be certain species of bacteria, yeasts, moulds and algae (Zhao et al.,
2011), whose lipid profile could be compared with the lipid profiles of many
conventional energy crops like jatropha, sunflower, or palm oil (Vicente et al., 2010)
as majority of the lipids are poly-unsaturated of omega-3 and omega-6 series
(Amaretti et al., 2010 ) triacylglycerols bearing long-linear chain fatty acids usually
with 14- 20 carbon atoms (Zhao et al., 2011), hence, showing resemblance to the
lipid profiles used for bio-fuel production. Non-oleaginous yeast organisms usually
produces lower level of lipids even if the culture conditions are same as that with
oleaginous micro-organisms and at nitrogen limiting condition, their growth almost
ceases (Ageitos, et al., 2011).
The microbial fatty acids with omega-3 and omega-6 series also have nutritional
importance as they contain linoleic, alpha-linolenic and gama-linolenic,
arachidonic,eicosapentaenoic, docosapentaenoic and docosohexaenoic acids
(Ratledge et al., 2002; Amaretti et al., 2010). For example docosapentaenoic and
docosohexaenoic acids could be used for brain development in infants,
arachidonic acid on hepatic fuel utilization in infant, among others.
Lipids produced from microbial cells are also known as single cell oil used for bio-
diesel production. They are non-food sustainable feedstock with a much higher yield
and less requirement of cultivation area than when compared with energy crops,
making it a more potential feedstock for bio-fuel production. The oleaginous
microorganisms are chemoheterotrophs that requires organic carbon and nitrogen for
their growth and lipid accumulation. They bear little or no competition with
conventional food-crops for the bio-fuel production and the product produced are
mainly renewable making it a more environment friendly process. However, the
production cost might be considerably higher due to the fermentation cost being
included.
Several authors, Ratledge (2004); Ageitos et al. (2011); Chisti (2007); Meng et al.
(2009), have found that a good number of oleaginous yeasts and micro-algae are
capable of accumulating a significant amount of lipids which could be used for the
production of bio-fuel mainly bio-diesel through process of transesterification or
14

other petroleum refinery operations (Zhang et al., 2011). Despite of this the amount
of lipid may vary depending on the species, strain, culture conditions (For example
pH, temperature, culture time, oxygen saturation, presence of salt and presence of
surfactants likes Tween 20) and media composition. A comparison of lipid content
of some of the oleaginous micro-organisms is shown in the table 2.1.
Table 2.1: Lipid content of a number of oleaginous micro-organisms and
conventional crops

Microorganism Species Oil content
(% w/w )
Reference
Yeast Rhodosporidium toruloides 66

Meng et al. (2009);
Ratledge (1991)


Candida curvata 58
Lipomyces starkeyi 63
Cryptococcus albidus 65
Rhodotorula glutinis 72
Mould Mucor mucedo 51 Meng et al. (2009);
Ratledge (1991)


Aspergillus nidulans 51
Aspergillus oryzae 57
Mortierella isabellina 86
Humicola lanuginosa 75
Mortierella vinacea 66

Bacterium Arthrobacterium sp. >40 Meng et al. (2009)

Acinetobacter calcoaceticus 27-38
Rhodococcus opacus 24-25
Bacillus alcalophilus 18-24
Algae



Botryococcus braunii 25-75 Meng et al. (2009);
Chisti (2007)

Neochloris oleabundans 35-54
Cylindrotheca sp. 16-37
Nitzschia sp. 45- 47
Schizochytrium sp. 50-77
Tetraselmis sueica

15-23
Crops Jatropha seed 30-50 Pramanik (2003)
Soy bean 19 Chisti (2007)

Canola 33
15


Most of the oleaginous micro-organisms as seen from the table 2.1 have higher lipid
content than the conventional crops.
2.2.1. Oleaginous micro-algae
Oleaginous micro-algae are potential sun-light driven cell factories capable of
accumulating very high lipid content with high cell density (Chisti, 2007) when
compared to photosynthetic crops (Patil et al., 2010). They are capable of converting
carbon-dioxide and light energy phototrophically or heterotrophically in presence of
organic carbon and limiting organic nitrogen sources into lipid contents mainly
between 1 90% of its dry biomass (Meng et al., 2009) with a doubling time of less
than 24 hours. The fatty acid composition of the produced microbial lipid varies
depending on the nitrogen limitation and temperature variation that is low
temperature promotes high poly unsaturated fatty acids (PUFA). The composition of
lipid mainly is triacylglyceride with fatty acid profile of mostly C16 and C18 which
is similar to fatty acid composition of many oil based crops (Meng et al., 2009).
However, micro-algae require a larger cultivation land and a longer fermentation
time (Chisti, 2007) than bacteria and yeast, hence, making it less good choice as an
alternative.

2.2.2. Oleaginous Bacteria
Bacteria on the other hand use simple carbon sources with very simple cultivation
method to produce higher biomass density within a doubling time of nearly 12-24
hours (Meng et al., 2009). However, a very few number of species accumulates
lipids, and of what they accumulate is in less amount for commercialisation. Also,
the lipid extraction is complicated in case of bacteria when compared with other
oleaginous micro-organisms as shown in Table 2.1. Hence, they bear no much
significance to industries for lipid production
2.2.3. Oleaginous moulds and yeasts
Oleaginous moulds and yeasts are considered to have a major contribution in the
production of lipids or single cell oils (Meng et al., 2009). They have similar
substrate requirement as that with the micro-algae, that is requires only organic
carbon and limiting nitrogen source for the biomass production and lipid
accumulation, respectively. The lipid produced by the moulds depends on several
16

factors such as higher temperature promotes higher rate of PUFA, also depends on
strain, that is thermophiles produces mostly saturated fatty acid than mesophiles, and
lastly it depends on the fermentation condition that could be batch or fed-batch or
continuous culture (Papanikolaou et al., 2001).

2.2.4. Oleaginous yeast
Among 600 species of oleaginous yeast, only about thirty species (Beopoulos et al.,
2009) such as Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkeyi,
Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulan, and
Yarrowia lipolytica, (Ageitos et al., 2011; Li et al., 2007) are capable of
accumulating very high rate of intracellular lipid and some have accumulation rate of
more than 70% of its total dry biomass weight (Li et al., 2007).

Both yeasts and moulds produces lipids that are majorly triacylgycerols and have
fatty acid profiles that are long-chained PUFA with mainly oleic (18:1) and linoleic
(18:2), palmitic (16:0) and palmitoleic (16:1) acids (Meng et al., 2009). The more
the microbial oil is unsaturated the lower would be the clouding and gel point in the
bio-diesel and increasing the stability of the fuel. R. toruloides Y4 is the most
common producer of single cell oil and has been well characterized in the literature
to have a fatty acid profile with mainly C 16 and C18 long chained PUFA (Meng et
al., 2009).

There are several advantages of using oleaginous yeasts over other microbial sources
that they have a doubling time of less than 1 hour, their growth is not easily affected
by varying climatic change, scaling up of the culture is easier as compared to other
microbes, and also they have an extensive species diversity with numerous
biochemical and physiological properties (Papanikolaou et al., 2001) and culture
conditions (Ageitos et al., 2011) providing an opportunity to produce various
profiles of long changed PUFA (Papanikolaou et al., 2001; Iassonova et al., 2008;
Ageitos et al., 2011).

One of the disadvantages of using this system is the expense added due to the
fermentation process. To make the process more feasible, the cost of the raw-
materials usually calculated from the lipid produced per unit of carbon source, and
17

the cost related to the process of fermentation usually calculated through amount of
lipid produced per unit volume per unit time, should be catered for by keeping the
cost of the carbon source to as minimum as possible (Ageitos et al., 2011; Ykema et
al., 1988).
Another problem using yeast is that they have a low growth rate at the initial stage,
so measures should be taken to maximise the specific growth rate in order to enhance
the lipid and biomass production in a limited time (Jacob et al., 1990).
Table 2.2: The fatty acid profile of some of the oleaginous micro-organisms


Micro-organism

Lipid composition (weight/total lipid)
C16:0 C16:1 C18:0 C18:1 C18:2 C18:3
Microalage 12-21 55-77 1-2 58-60 4-20 14-30
Yeast 11-37 1-6 1-10 28-66 3-24 1-3
Fungi 7-23 1-6 2-6 19-81 8-40 4-42
Bacteria 8-10 10-11 11-12 25-28 14-17 -

Source: (Meng et al., 2009)

2.3. Rhodosporidium toruloides as the model yeast

2.3.1. Considerations in choosing R. toruloides as the model yeast
Rhodosporidium toruloides also known as Rhodotorula gracilis is deep orange
coloured halotrophic oleaginous yeast found in the phylum basidiomycetes (Ageitos
et al., 2011). They are capable of accumulating lipids more than 75% of its total dry
weight with a high cell density of over 100 g/L under one of the substrate limiting
condition mainly nitrogen (Wu et al., 2010). They are also capable of withstanding
high salinity and changes in pH conditions and still grow and accumulate lipids and
other products like carotenoids. They are capable of growing in various growth
media with little difference in the percentage of biomass and lipids according to the
culture conditions, hence could easily be used for experiment on high salinity. The
size of the lipid bodies accumulated intracellularly under nitrogen limited condition,
ranges from 0.5 m to 2 m but once the carbon source becomes limited, the
18

organism has a mechanism to disintegrate the lipids to use it as an ATP source for
mitosis and meiosis (Ratledge et al., 1987).
Their lipid accumulated is generally of triacylglycerol class consisting of long
chained PUFA profiles that are similar to the lipid extracted from the conventional
oil bearing crops (Li et al., 2007), hence giving a sustainable, inexpensive, non-
competitive, renewable feedstock for feasibility of bio-diesel production in
industries. The higher the PUFA in the microbial lipid, the more stable the bio-diesel
could be.
R. toruloides has been cultured in batch, fed-batch and continuous cultures. It has
been found that they are capable of adapting to different fermentation process with a
simple glucose and nitrogen source and gave good results in varied process. For
example in multiple fed-batch mode with varied glucose feeding interval, it gave 79
g/L of lipid in 140 hours and with glucose, peptone and yeast extract in single fed
batch, it gave around 67.5 g/L of lipids in 134 hours (Ageitos et al., 2011). This
shows that substrate as well as substrate feeding strategies makes a significant
difference in the biomass density and lipid accumulation capacity of the yeast.
Hence, R. toruloides could be chosen as the model yeast for the experiment (Zhao et
al., 2011).
Some of the oleaginous yeasts are compared in Table 2.3 to show the level of
biomass density and lipid accumulation in a respected time.










19

Table 2.3: Different species of oleaginous yeast compared for their biomass density
and lipid accumulation

Oleagi-
nous
yeast
Carbon
source
Biomass
concentr
-ation
(g/L)
Lipid
content
(w/w)
Fatty acid
composition
Culture

Cultur
-e time
In
hours
Source
C.
curvatus
glycerol 118 69% C16:0 =28%
C18:0 =15%
C18:0 =48%
Fermentor
Fed-batch


50 Iassonova
et al.,
(2008)
L.
starkeyi
Xylose
Ethanol
L-
arabinose
Glucose
20.5 61.5% C16:0=33%,
C18:1=55%

flask

120

Li et al.
(2008)

R.
toruloide
s

Glucose 100 76% C16:0=24%,
C18:1=55%
Fermentor
Fed batch

144
Li et al.
(2008)
R.
glutinis

Glucose 180 72% C16:0=18%,
C18:1=60 %
C18:2=12 %
Fermentor
Fed batch
-

Li et al.
(2008)

Y.
lipolytica

Animal
fat
15 44 C16:0 =11%
C18:0 =28%
C18:2 =51%
Fermentor
120
Beopoulos
et al.
(2009)

2.3.2. Life cycle of R. toruloides
A Sexual and diploid self-sporulating cycles are the two main cycles that makes up
the life cycle of R. toruloides as shown in Figure 2.1.
According to Abe et al. (1986), considering two kinds of cells type A and type Aa
when brought in contact in respective media, they conjugates to form Dikaryotic
hyphae which in turn leads to the growth of teliospores where the nuclei of the two
cells fuse together. The spores then germinate leading to the isolation of the alleles
of the two cells to form sporidia that propagates further by budding into haploids or
aneuploids yeasts.
The next sexual cycle again begins with conjugation but with opposite mating type
and the cycle continues as the first cycle but the diploid sporidia of both cell type
produced are non-isolative at this stage. The life-cycle indicates the diversity of this
yeast and hence, studying growth kinetics in more details is very important.
20














Figure 2.1: Proposed life cycle of R. toruloides
Source: Abe et al. (1986)
2.3.3. Growth kinetics of R. toruloides
Growth curve of R. toruloides has a similar growth pattern and phases as that with
other micro-organisms like bacteria under a given physiological conditions. As given
by Stanbury et al. (1995), R. toruloides has a lag, log, and a stationary phase whose
time interval varies according to the culture conditions like temperature, pH,
inoculums age, culture time, media composition, and the type of fermentation
process that could be batch , fed-batch or continuous. In a batch culture, the lag
phase length usually depends on the inoculums age, the growth phase of the
inoculums, the media to which it is added and the contamination level as well, as the
culture takes time to get adapted to a new environment, hence could have a longer
lag phase.
The cells then enter the exponential phase where it multiplies rapidly through
process of mitosis and meiosis, with a doubling time of less than an hour. The
exponential phase reaches a maximum and then the cells become stationary that is,
death rate of the cells is equivalent to the birth rate, once one of the main substrate
becomes limited in the medium. Most of the intracellular accumulation of lipids
takes place at this stage making the cells thicken at times. A growth curve of R.
toruloides in batch condition has been shown in figure 2.2.
21


Figure 2.2: Growth pattern of R. toruloides in batch culture

Source: Stanbury et al. (1995)

Experimental approach to find parameters of growth kinetics
Micro-organisms in its natural habitat grows in various mixtures of substrates and
the growth may be controlled by more than a single substrate .Hence, the kinetic
properties of the micro-organism may change according to the variation in the media
and according to how well they adapt to the new culture conditions. This might make
it difficult for the researcher to determine experimentally what effect each of the
substrate in the media may have on the growth.
Monod model is used to get a relationship between the growth-limiting substrate (S)
and the specific growth rate () with parameters of the maximum specific growth
rate (
max
), and the Monods constant or saturation constant (K
s
) that are used to
describe microbial growth kinetics (Kovarova et al., 1998). The equation describing
the relationship is given below:


=


(2.1)
At exponential stage in the batch culture, the equation used to get the maximum
growth rate of the yeast in per hour is given by;



=x (2.2)

On integration and taking natural logarithm the equation becomes;
ln x
t
= ln x
o
+ t (2.3)
22

Thus, a plot of natural logarithm of biomass concentration (x
t
) against fermentation
time gives a straight line whose slope is the specific growth rate ()

of the yeast in
the culture (Stanbury et al., 1995).

Determination of growth kinetics through experiment
Leesing et al. (2011) gave a way of determining growth kinetics by calculating
various parameters from the experimental results.
a. Volumetric lipid production rate (Q
p
): Determined from the plot of lipids
produced against fermentation time.
b. Product yield (Y
P/S
): Determined by calculating amount of lipid produced per
substrate concentration (d
P/dS
)
c. Specific product yield (Y
P/X
): Determined by calculating amount of lipid
produced in g/L per amount of biomass produced in g/L
d. Volumetric rate of substrate consumption (Q
S
): Determined from the plot of
substrate (g/L) present in the fermentation medium versus fermentation time.
e. Volumetric cell mass production rate (Q
X
): Determined from a plot of dry
cells (g/L) versus fermentation time.
f. Specific growth rate, () is determined from the plot of natural logarithm of
biomass versus times, the slope of which gives .
g. Specific rate of lipid production (q
P
): Determined by multiplying and Y
P/X
.

Determined specific growth rate and substrate concentration are then used to
determine other parameters of
max
and K
s
with Hanes linear plot given in the
equation below:
(2.4)
Plot of S/ verses S is plotted and the slope gives 1/
max
and the intercept gives
K
s
/
max
(Leesing et al., 2011).


23

2.3.4. Biochemistry of lipid accumulation in R. toruloides
In most of the lipid accumulating micro-organisms, the maximum amount of lipid
that the organism can accumulate varies even if all the physiological conditions are
kept constant. For a cell to accumulate lipid, the carbon source should be in excess
and one of the other substrate especially nitrogen should be limiting (Ratledge and
Wynn, 2002). Once the culture become nitrogen deficient (Botham and Ratledge,
1979), the yeast tends to cease their growth and enter the stationary phase where it
converts excess carbon into lipid bodies and initiate the synthesis of triaclyglycerol
in the endoplasmic reticulum and lipid bodies of the cell. Evans and Ratledge, (1984)
suggested that for the lipid accumulation to begin, a significant concentration of
intracellular ammonium ions should be accumulated within the cell, which in turn
would trigger the synthesis of lipid.
Lipid accumulation mostly depends on the time of exhaustion of nitrogen from the
medium, the level of cellular metabolites mainly citric acid and malic acid in the cell
and energy in form of ATP which is required at all stages for fatty acid synthesis
(Beopoulos et al., 2009). The lipid accumulation process takes place in both
cytoplasm and mitochondria of the cell. As the nitrogen gets exhausted, there is a
significant decrease in AMP which in turn hinders the function of NAD
+
dependent
Isocitrate dehydrogenase (ICDH). This increases the levels of isocitrate which gets
converted rate into citrate by an enzyme aconitase in the mitochondria. Due to low
level of AMP, tricarboxylic (TCA) cycle is detained at this stage, thus increasing the
level of citrate in the cell. The citrate is then directed to the cytoplasm with the help
of citrate transporter where it is converted into Acetyl- Co A by ATP citrate lyase
(ACL) (Ageitos et al., 2011). Acetyl Co A is then directed for the biosynthesis of
fatty acid by the enzyme fatty acid synthetase (FAS) which in turn synthesises
triacylglycerol.
Citrate and malate are the main precursors for the production of acetyl Co-A and
NADPH (Ratledge and Wynn, 2002) and the accumulation of lipid mainly depends
on their cellular level and their ability to metabolise it (Ratledge, 2004). The
concentration of the metabolites mainly citric and malic acid should be maintained at
a certain level because a very high cellular concentration of them may direct the lipid
to the medium, decreasing the level of lipid accumulation within the cell
24

(Papanikolaou et al., 2001). For this reason a proper carbon to nitrogen ratio (C/N)
should be optimized for higher levels of lipid production (Beopoulos et al., 2009).

CYTOPLASM MITOCHONDRIA

Figure 2.3: Stages that lead to lipid accumulation in oleaginous yeast

Source: Ratledge and Wynn (2002)

2.4. Factors affecting R. toruloides growth, lipid accumulation and
fatty acid profiles

Many authors Wu et al., (2010 and 2011); Li et al., (2007) and Granger et al.,
(1992), have shown how different media composition and physiological conditions
vary the biomass density, lipid accumulation and fatty acid profiles in the yeast.
There are several factors like culture conditions, inoculums time, media composition,
oxygen availability, presence of salts, presence of surfactants, among others, that
could affect the product yield of the culture and some of it is explained below:

2.4.1. Mode of operation
Conditions like type of fermentation either batch; fed-batch, or continuous process in
a single or multiple stages are also one of the factors that affect the biomass density,
lipid profiles and lipid accumulation in the yeast. Li et al. (2007) reported that when
R. toruloides Y4 was grown in a media with different concentrations of glucose, it
25

had less inhibitory effect with high level of glucose concentration until an optimum
of 150 g/L. When the experiment was carried out in flasks fed-batch and up-scaled
fed-batch it gave results of 151.5 g/L with lipid content 48% (w/w) and 106.5 g/L
with lipid content 67.5% (w/w), respectively. Also, the lipid profile was similar with
oleic, palmitic, stearic and linoleic acid in fed-batch but there was a slight increase in
stearic acid in fed-batch. Similarly, Hassan et al. (1993) reported that lipid
accumulation was better in continuous culture than in batch with an amount of
45.6% (w/w). Hence, the mode of fermentation operation also has an effect on the
product yield.
2.4.2. Culture conditions
For the growth and proliferation of R. toruloides, several considerations like
temperature, pH, atmospheric pressure, dissolved oxygen, among others, are required
in order to maintain the culture.
2.4.2.1. Temperature and pH
Every strain has a particular optimum temperature and pH outside which it becomes
inactive. R. toruloides requires an optimum temperature of about 30

C with an
optimum pH of 5.5 for its growth and proliferation, but can survive a pH range of 3.0
to 6.0. Temperature also has an effect on the oxygen dissolution in the media in
presence of salinity, as higher temperature leads to the evaporation of oxygen to the
atmosphere decreasing the oxygen saturation in the medium for the growth of the
cells. Hence, there might be inhibitory effect on the growth and lipid production of
the cells with variation in temperature. Granger et al. (1992) reported the effect of
temperature on PUFA production and degree of saturation under nitrogen limiting
condition. He reported that lower temperatures lead to reduced specific growth rate
and lipid content but with every 5

C reduction, accumulation of -Linoleic acid

increased thrice together in case of R. glutinis and also there was a significant
increase in the degree of un-saturation as the activity of the enzyme desaturase only
works up to an optimum temperature (Saxena et al., 1998). Almagro et al. (2000),
reported the effect of temperature variation on Debaryomyces hansenii that is, its
growth in presence of salinity highly depends on the temperature, a growth
improvement was observed when temperature was increased from 23

C to 30

C but
was inhibited above 34

C.
26

The pH of the media is one of the most important parameter for growth and lipid
production in oleaginous yeasts. It depends on the medium composition (Angerbauer
et al., 2008), media conditions and mode of operation. For instance pH in seawater is
normally around 8 and may need to be adjusted according to the yeasts optimum.
Ageitos et al. (2011) reported that in R. glutinis, pH has a great influence on the lipid
yields with percentages of 12, 48 and 44 (w/w) at a pH of 3, 5, and 6 respectively.
Saenge et al. (2011) reported that when R. glutinis was cultured in a pH controlled
environment, a good amount of lipid was accumulated whereas, in an uncontrolled
pH environment, the pH dropped due to acid formation leading to poor growth and
lipid production. Hence, proving that R. glutinis has an optimum that gives highest
lipid yield and deviation in the pH might have inhibitory effects on the growth and
lipid yield. Therefore, it is more likely that every micro-organism has an optimum
pH for their growth and product yield and pH needs to be adjusted accordingly.
2.4.2.2.Dissolved Oxygen concentration in the culture
The availability of dissolved oxygen is also important as it may vary with the change
in media composition, salinity, temperature, pressure, among others. Oxygen is a
very important factor for the growth of R. toruloides for it being an aerobic microbe,
hence, its availability affects the growth and proliferation of the cell and in most
cases, the complete cut of oxygen supply for longer time may lead to death of the
microbe. The amount of dissolved oxygen in the media could be directly
proportional to the lipid accumulation in most cases (Li et al., 2008).
Saturation of dissolved oxygen in fresh water and sea water depends on the
temperature and pressure as seen in the figure 2.4 (a) and (b). In fresh water at sea
level the oxygen saturation is14.6 mg/L at 0

C and 8.2 mg/L at 25

C (Viklund, 2011)
and below concentration of 5 mg/L has adverse effect on the biological activity of
the micro-organism. A concentration less than 2 mg/L might lead to death of the
micro-organism. The amount of dissolved oxygen in freshwater is generally 6 mg/L
and not less than 4 mg/L for growth of living organism.
Normal salinity in the deep sea water is approximately 35000 mg/L or 35 g/L and the
dissolve oxygen concentration in it might range from 0 20 mg/L depending on the
location and the available aquatic organisms in the sea. The dissolved oxygen
27

saturation decreases with increase in salinity and usually a higher concentration of
oxygen level is observed in fresh water when compared with sea water as shown in
the figure 2.4 (a) and (b).

Figure 2.4 (a)

Figure 2.4 (b)
Figure 2.4 (a) and (b): Oxygen solubility in both fresh water (a) and sea water (b)
with varied temperature and pressure.
Source: Oxygen Solubility in Fresh and sea water: Engineering ToolBox
Figure 2.4 (a) and (b) explains that at lower temperature, saturation of dissolved
oxygen in mg/L was found to be higher in fresh water than in sea water at all
28

variation in pressure. However, in both sea water and fresh water, dissolved oxygen
saturation was directly proportional to the increase in pressure and inversely
proportional to the increase in temperature. Therefore, it can be suggested that for a
proper growth and lipid production in oleaginous yeast, an optimum temperature and
pressure is required for maximum oxygen saturation in saline water.
2.4.3. Media Composition
In natural condition, the media in which the culture grows could be mixed and more
than one substrate could be simultaneously responsible for the growth and
proliferation of the oleaginous yeast. Varying media composition has varied effect in
the growth and lipid production of different species and strains of the oleaginous
yeast. It has been reported by Beopoulos et al. (2009) that substrate limitation mainly
nitrogen has a major effect in the lipid content and lipid profile of the oleaginous
yeast due to high charge ratio of ATP: AMP (Ratledge et al., 2002), although other
substrate limitation like phosphate (Wu et al., 2010) and sulphate (Wu et al., 2011)
has been proven to accumulate high quantity of lipid regardless of the presence of
rich sources of nitrogen. Lipid production and fatty acid profile in the yeast requires
excess of carbon to be assimilated and converted into lipids under nitrogen limited
condition and has a different level depending on the strain of oleaginous yeast. Also,
a very high carbon concentration may bring substrate inhibition to the culture and the
growth may cease (Ageitos et al., 2011). Hence, for an effective process, an
optimum carbon to nitrogen ratio plays an important role in determining the
potentiality of the oleaginous yeast. Hassan et al. (1993) reported that an optimum
C/N ratio for most of the oleaginous yeast ranges from 20 to 30 (w/w) when cocoa
butter as a substrate was used in his case. Also, Granger et al. (1993), reported that
for C/N ratio range from 30 to 100 (w/w), the lipid yield increased up to a maximum
of C/N ratio of 60, after which it decreased due to prolonged depletion of substrate
from the medium eventually leading to loss of cell activity. Hence, it is suggested
that although nitrogen depletion is required for onset of lipid production, it
prolonged depletion from the medium may lead to loss of cell activity.
Carbon limited condition of the media tends to produce more unsaturated fatty acid
than in nitrogen limited condition as reported by Ratledge and Wynn (2002),
although, at low carbon concentration, the oleaginous yeast tends to produce more of
biomass than lipids (Holdsworth and Ratledge, 1988). Holdsworth and Ratledge
29

(1988) suggested the reason for this behaviour of oleaginous yeast as at low substrate
concentration, the cell starves, and in most of the oleaginous yeast, they adapt to this
situation by converting the intracellular accumulated lipid into biomass for the
growth and proliferation of the cells.

The type of carbon source used is another factor that affects the growth, lipid yield
and fatty acid profile of the oleaginous yeast. A number of carbon sources has been
used such glucose, L-arabinose, xylose, mannitol, among others, although glucose
has been mostly employed as the lipid composition produced from glucose is mainly
the triacylglycerol. For example, Ageitos et al. (2011) reported that using xylose as
carbon source, the lipid profile was 15% of C18:0 and 4% of C18:2 while using
ethanol, the result gave 51% C18:1 and 25% C16:0. Also, Saxena et al., (1998)
reported that a very high specific growth rate of 0.34 h
-1
was observed when
oleaginous yeast, R. minuta, was grown on glucose but had different specific growth
rate of 0.11 h
-1
,0.3 h
-1
, and 0.36 h
-1
, and when grown on carbon sources of
galactose, fructose, and sucrose, respectively.
The experiment conducted in this project deals with the growth kinetics at high
salinity, so type of carbon source and optimum C/N ratio is very important in order
to increase the product yield as R. toruloides may have different effects with
different carbon source or C/N ratio in high salinity condition.
2.4.4. Effect of the presence of high concentration of salt in the media on
the growth kinetics of R. toruloides
Salinity is defined by unit mass of dissolved salt per unit mass of the sea water. In
sea water or salty water, the salinity ranges from 30 to 50 parts per thousand but may
vary with the location and depth of the sea. The water that is saline is usually a
mixture of fresh water and sea water and often known as brackish water. The
composition of sea water as given by Anderson (2008) consists of inorganic salts
mainly sodium (10.75 g/L) and chloride (19.35 g/L) in high percentage while trace
amount of other salts like magnesium (1.295 g/L), sulphate (2.7 g/L), calcium (0.416
g/L), potassium (0.39 g/L), bicarbonate (0.145 g/L), bromide (0.066 g/L), borate
(0.027 g/L), strontium (0.013 g/L), fluoride (0.001 g/L) and others in concentration
of 0.001 g/L. Since, most of the inorganic salt present in the sea water is sodium and
30

chloride, therefore, sodium chloride at a concentration of 35000 mg/L equivalent to
salinity of sea water could be used as a substitute to sea water. The fact that seawater
has other salts in different concentration together with 23 g/L of NaCl, therefore, it
might alleviate the stress on the organism when compare to use of only NaCl in a
concentration of 35 g/L in the medium.
2.4.4.1. Effect of salinity on Growth phase of R.toruloides
According to Hosono (1992), Zygosuccharomyces rouxii when grown with 15%
(w/v) NaCl had a relative effect on the fatty acid profile with a small increase in C
16:1 and C 18:1 and a small decrease in C 18:0 and C 18:2 than in the media without
salt due to increase in the membrane fluidity at high salinity to equilibrate the
cytoplasm osmotically with the saline environment. Membrane lipid plays a very
important role in order to maintain the integrity of the cell.
Every strain of yeast may have a different behaviour with the saline condition and
each may have different stress control mechanism in order to withstand the osmotic
stress at high salinity. Therefore, the time interval between each phase in the growth
phase may vary depending on the strain and other culture conditions as reported by
Almagro et al. (2000) for the case of Debaryomyces hansenii and Saccharomyces
Cerevisiae. He reported that the growth and thermal death varied for each species
when were investigated with potassium and sodium ions in the medium.

2.4.5. Role of Surfactants on the growth and lipid yield of the oleaginous
yeast
Surfactants are surface active agents that lower the surface tension of the liquid
media and also decrease the interfacial tension between the media and the yeast cells
in the culture, hence, may increase the oxygen mass transfer from the media and
make it more available for the cells. They could be responsible for altering the
physiological characteristics of the oleaginous yeast, re-arranging the structure and
permeability of cell membrane, stimulating growth and cellular respiration,
improving both primary and secondary metabolites production, and may help in the
enhancement of lipid accumulation in the yeast based on the principle of membrane
fluidity. Every surfactant has different affect on the lipid accumulation and growth of
the yeast, hence among various surfactants like Tween 20, Tween 80 and gum
31

arabic, the best possible surfactant should be chosen for investigating a purpose, for
instance growth kinetics in this project.
Saenge et al. (2011) reported the use of Tween 20 on R. glutinis that showed an
increase in substrate consumption, glycerol in his case, with a result of 48.21 %,
lipid content of 35.22% (w/w), and 5.47 g/L of biomass density.
He suggested that the reason behind this phenomenon could be that the surfactants
being emulsifiers can breakdown organic substrates into droplets that could be more
accessible for the yeast to assimilate, improving their growth rate, and product
excretion. However, Li et al. (2006) reported that Tween 20 enhanced lipid
production but decreased cell growth rate.

Tween 20, also known as polysorbate 20 is a polysorbate surfactant that has long
chain of stable and relatively non-toxic ployoxyethylene, which makes it useful as an
emulsifier when added to the culture of oleaginous yeast or other micro-organisms
with organic based media composition. Hence, when used with basal media for
culturing R. toruloides in high salinity condition, it may enhance the growth rate and
lipid yield of the yeast by altering the membrane fluidity and increasing the oxygen
mass transfer rate.
.
2.5. Disadvantages of using oleaginous yeast
Although oleaginous yeast has many advantages over other micro-organisms, there
are some limitations that need to be considered before using oleaginous yeast as a
model. One of the limitations is that the fermentation cost of the process makes the
use of the use oleaginous yeast a little expensive. Secondly, the microbial oil has to
compete with the commercially available high valued oil produced by the oily- crops
because of the quality issue. Also, even a little variation in the media composition or
culture condition may bring huge changes in the fatty acid profile of the
triacylgylcerol making it difficult to control. Another factor could be even with the
capacity of the oleaginous yeast to product high value product, to develop a more
integrated and novel process for commercial production, may take a longer time.
Oleaginous yeasts have thicker cell wall that makes the permeability of many
solvents difficult; hence, cell disruption and lipid extraction could be a more a
difficult process with reduced yield.
32


2.6. Summary
From the literature review, it is found that oleaginous yeast could be an alternative
source of feedstock for the bio-diesel industries and there could be a possibility that
brackish water could be useful for this purpose. Different oleaginous yeasts have
different growth requirements, therefore, in order to obtain a maximum growth and
lipid productivity in high salinity conditions, the medium needs to be optimized with
a suitable carbon to nitrogen ratio. Carbon and nitrogen sources are required for the
growth of oleaginous yeast, although nitrogen depletion is important for the onset of
lipid production. As discussed earlier in this chapter, surfactants like Tween 20 could
be used in order to enhance the oxygen mass transfer rate for better growth and lipid
accumulation, as salinity decreases the dissolution of oxygen in the medium.
Considering the benefits of using R. toruloides as the model yeast for the project, as
discussed previously in this chapter, its growth kinetics has been studied in high
salinity condition.


















33


Objectives Chapter 3
The economy of the world to some extend is affected by its transportation, which in
turn increases the demand of petroleum based products. The depletion of fossil fuel
reserves and its adverse impact issues on the environment has diverted the subject of
interest on seeking for a sustainable alternative feedstock for bio-fuel production.
The driven interest on microbial based oil is due to the fact that they are non-
competitive with the food crops and the produced lipids are triacylglycerol that has
long chained poly-unsaturated fatty acid profile similar to that required for bio-diesel
production.
Most of the microbial oil produced in the past has utilized fresh water for the growth
and lipid production of the micro-organism. In order to exploit the use of oceanic
sources, especially sea water, the growth kinetics of Rhodosporidium toruloides has
been studied in high salinity condition which is almost equivalent to salinity in sea
water.
Oleaginous yeast in particular R. toruloides has been studied for the production of
microbial oil for its ability to produce high biomass density and accumulate high
lipid content. It is also a diverse species that can tolerate osmotic stress and variation
in pH, hence, chosen to study its growth kinetics in high salinity condition. One of
the major limitations of the process of microbial oil production is its fermentation
cost that in turn may increase overall production cost. For this purpose, optimization
of the culture media and conditions are very important in order to maximise the
yield. Use of low priced renewable raw materials would also make a huge difference
in the production cost, contributing in the development of a process that is more
economical for commercialisation.
One of the objectives of this study is that to grow R. toruloides in high salinity
condition as that with the salinity in deep sea using the organic medium and
investigate its behaviour by studying its growth kinetics. The parameters studied in is
the biomass production, lipid yield, residual glucose concentration, pH changes,
optical density, specific growth rate and oxygen saturation in high salinity when
34

compared to fresh water which would later help to improve the process for higher
lipid productivity.
The objective to begin with in this study is the use of sodium chloride over synthetic
sea water in order to provide high salinity conditions to the oleaginous yeast. The
study of the behaviour of R. toruloides is investigated in the medium with two
different salinity composition that may help in saving time, effort and cost of
preparing synthetic sea water in the laboratory. Production cost related to chemicals
used for preparing synthetic sea water would be however, ignored as if the objective
of this study is fulfilled, the sea water could be used directly to serve the purpose of
high salinity condition for the oleaginous yeast.
From the literature review, it is clear that every organism has different growth
requirement in different set of conditions. Mostly, oleaginous yeasts require a carbon
and a nitrogen source for their growth and proliferation. Lipid accumulation only
begins once the nitrogen source is completely exhausted from the medium. The
preference of nitrogen and carbon source and their optimum ratio required for their
biomass production and lipid accumulation, and varies from species to species.
Glucose has been the most common carbon source utilized by oleaginous yeast,
although nitrogen sources could be varied depending on the requirement of the
process and from culture to culture. In this study, the growth of R. toruloides on
various carbon to nitrogen ratios (C/N) has been carried out in order to find an
optimum that could provide maximum yield.
Surfactants as seen from the literature, has been useful in increasing the permeability
of the cells in the culture medium, hence, increasing the net oxygen mass transfer
rate. The oxygen saturation usually drops when salinity is increased, hence, in order
to improve the oxygen saturation, study on surfactants were carried out. Use of
surfactants in the culture medium was believed to increase the lipid productivity and
biomass density of R. toruloides by increasing oxygen mass transfer rate in the
culture. Its use might also help the cells to maintain their membrane integrity when
grown under high osmotic stress condition. In this study, Tween 20 has been
exploited for this purpose as its composition is organic and the results obtained from
it as seen from the past studies, has been better than the use of other surfactants.
35

The above studies were carried out in form of experiments in order to explore the
prospects of producing maximum microbial lipid in high salinity conditions. The
growth kinetics was therefore studied on the basis that variation in biomass
production, lipid accumulation and specific growth rate of R. toruloides could be due
to change in medium composition and culture conditions, and would produce
different results when the culture of R. toruloides was grown in high salinity
conditions. This study would therefore provide an insight on varying the culture
conditions in order to optimizing the process and maximise the lipid yield.





















36


Materials and Methods Chapter 4
4.1. Introduction
This chapter accounts for all the materials used during the course of the project such
as chemicals and the equipments with their basic principle of operation citing visual
images when required. It also describes the procedures such as experimental and
analytical that has been applied in order to carry out the experiments leading to the
results.
4.2. Materials
4.2.1. Chemicals
Fisher Scientific Chemicals was the company from where all the chemicals used for
the experiment were bought. Although, there were few exceptions such as
Magnesium sulphate bought from Analar and Malt extract, yeast extract, glycerol
from Sigma-Aldrich.
4.2.2. Microorganism
Mother culture of R. toruloides grown on a YMY media slants containing 1% (w/v)
of glucose, 0.3% (w/v) malt extract, 0.3% (w/v) yeast extract and 0.5% (w/v)
peptone and agar but without salinity, was obtained from Shell Company, and was
stored in the same form at 4
o
C to avoid cell death.
4.2.2.1. Working cell Bank or vials
Under aseptic condition, colonies of R. toruloides from the mother culture were used
to prepare slants with basal media including salt of concentration of 3.5% (w/v) and
1.5 % (w/v) agar. A pH of 5.5 was maintained using 0.1% (v/v) phosphoric acid. The
composition of the media is described later in this chapter. The slants were incubated
at 30

C for 4 to 5 days until good growth of the culture with orange colonies was
observed on the slants without contamination. The slants were stored at 4
o
C and
were sub-cultured every 15 days to maintain the activity of the cells.
37

Colonies from the slants were then used to prepare a suspension culture of 150mL
with sterilized (autoclaved at 121

C at 15 psi for 15 minutes) basal media in aseptic

condition. The suspension culture was then incubated at 30
o
C for 40 hours on an
incubator shaker at 200 revolutions per minute (rpm). In aseptic condition, 50mL of
media was transferred to each of the three 50mL sterilized centrifuge tubes and
centrifuged at 10000 rpm for 10 minutes. The supernatant was carefully discarded in
the laminar flow to avoid contamination. The cells from one of the centrifuge tube
were re-suspended in 30mL fresh autoclaved basal media with 10% (v/v) glycerol
and vortexed. The suspension was then transferred to the second tube with the cells
aseptically, vortexed and the process repeated with the third tube.
1mL each from this suspension culture was transferred aseptically using a sterilized
micropipette tip to 30 cryopreservation tubes, parafilmed and stored at -4
o
C.
4.2.2.2. Cell Count
One of the vials from the working cell bank was used for cell counting. One drop of
diluted cells was used on a haemocytometer and cells were counted using 100X
objective lens. The cell concentration in each tube was found to be 1.49 x 10
9

cells/mL.
4.2.3. Equipments
Eppendorf Centrifuge 5804, pH meter pHep

by Hanna H198108, light microscope
from Olympus Japan with JVC TK-C1381 colour video camera, Shimadzu UV- Vis
Spectrophotometer uv mini 1240, Rotary shaker, Analar Glucose analyzer, Vortex
by Merck Ltd PAT 02956, Weighing scale by Sartorius Analytic and Swiss Quality
Precisa 125A, and a Soxhlet System HT 1043 lipid extraction unit were used during
the course of the experiments.
4.3. Methods
4.3.1. Media composition and preparation
The media composition for different sets of experiments and there preparation has
been described below. All media were dissolved in distilled water according to the
volume required. Each time before the medium was prepared and used for culturing
with R. toruloides; it was autoclaved at 121

C, 15 psi for 15 minutes and cooled to

room temperature in the laminar air-flow chamber maintaining aseptic conditions.
38

4.3.1.1. Inoculums medium
The medium used for inoculum preparation was Basal with high salinity obtained by
adding a concentration of 3.5 % (w/v) of sodium chloride as depicted to be present in
sea level. The medium contained 2% (w/v) glucose, 1% (w/v) peptone, 1% (w/v)
yeast extract and 0.3% (w/v) malt extract as given by Wu et al. (2011).
The volume and composition of the inoculums medium depended on the volume of
the process medium that is 2% (v/v) for 50mL medium for the flask experiment, and
5% (v/v) for 4L process media. The composition of the nutrients to be added was
calculated accordingly. For experiment conducted on petri-plates, 1.5% (w/v) agar
was used for solidification of the medium.
Before conducting any experiment, one of the vials from working cell bank was
thawed by leaving it in room temperature in laminar air-flow cabinet for 10 minutes,
after which it was used to inoculate 50mL of fresh sterilized basal medium with
3.5% (w/v) sodium chloride. The medium was then incubated for 48hours at 30
o
C on
a rotary shaker at 200 rpm.
4.3.1.2. Process medium
All the process media were prepared in duplicates whenever the experiment was
conducted in flasks. Readings were taken from both duplicates at the same time
interval for a particular set of experiment.
4.3.1.2.1. Experiment on slants or petri plates.
As given in the table 4.3, E1 and E2 media were used for this experiment. 1.5% of
agar was used in each case for solidifying the media on petri plate. In both cases the
nutrients were dissolved in distilled water and pH adjusted to 5.5 using 0.1%
phosphoric acid. This experiment was conducted to investigate whether R. toruloides
was able to grow in high salinity or not. Slants from mother culture were used for
this purpose.
4.3.1.2.2. Experiment on Carbon to nitrogen ratios, Tween 20, and in
Bioreactor
Summary of the media composition for different sets of experiments has been
summarised in table 4.1.

39



Table 4.1: Process media composition

For each separate experiment, media E1, E2, E3, E4, E5 and E6 were prepared by
dissolving the nutrients in distilled water and the pH in each case was adjusted to 5.5
using 0.1% phosphoric acid.
Culture of R. toruloides was grown on media E3, E4, E5, and E6 for the experiments
conducted on batch growth in the bioreactor, with varied carbon concentration over
fixed nitrogen concentration (varied C/N ratios), with varied nitrogen concentration
over fixed carbon concentration and with Tween 20, respectively. These studies were
done to investigate the difference in growth pattern of R. toruloides in different sets
of medium compositions. For each of the flasks experiments, a negative control was
prepared, which was the medium without any inoculation with R. toruloides whose
result analysis was done in the same way as other media which were inoculated the
analysis but at the end of the experiment.
Medium E2 was simple basal medium without salinity while E1 was basal media
with salinity. E1 and E2 in most of the experiments have been used as positive
control against the experiment conducted.


Nutrient


E1


E2


E3


E4


E5


E6
Glucose 20 20 100 Varied 40 20
Peptone 10 10 10 - - 10
Yeast extract 10 10 10 0.5 0.5 10
Malt extract 3 3 3 - - 3
(NH
4
)
2
SO
4
- - - 12 Varied -
MgSO
4
.7H
2
O - - - 1.5 1.5 -
KH
2
PO
4
- - - 1.0 0.2 -
Tween 20 - - - - - Varied
NaCl 35 - 35 35 35 35
Medium name and composition (g/L)
40

Medium E3 was used to investigate the growth kinetics and lipid accumulation of R.
toruloides in batch culture performed in a bioreactor where most of the culture
conditions could be easily controlled.
Carbon to Nitrogen ratio experiment
Medium E4 was used to investigate the effect of varying the concentration of
glucose (used as the carbon source) over a fixed concentration of nitrogen source
(from ammonium sulphate and yeast extract) on the growth of R.toruloides. For this
purpose different concentration glucose was prepared in duplicates keeping other
nutrients in the media fixed. The concentrations of glucose used were 10, 20, 40, 60,
100, 120, 150, 200, 250, and 300 g/L.
Medium E5 was used to investigate the effect of varying nitrogen concentration over
a fixed concentration of glucose on the growth of R. toruloides. The concentration of
glucose was chosen from the experiment conducted with E4 medium. The
concentration of yeast extract was fixed at 0.5 g/L while ammonium sulphate was
varied with concentrations of 2, 6, 10, 12, and 14 g/L where one value of 12 g/L was
common in both experiment with E4 and E5 media.
Each of the concentrations of either varied glucose in E4 or varied nitrogen
concentration in E5 were prepared in duplicates.
One of the limitation in both experiments were, the time parameter had to be kept
fixed at 48hours to reduce the load of handling a large number of flasks at the same
time. Hence, the time fixed at 48 hours was chosen because it was assumed that R.
toruloides had grown to a maximum but still in its exponential phase as seen from
the growth curve of the experiment conducted with synthetic sea water.
The total nitrogen present in yeast extract was assumed to be 10.5% as given on the
cover of the company, Sigma Aldrich, from where the chemical was bought. The
percentage of nitrogen was calculated from both yeast extract and ammonium
sulphate and added up for each of the concentration of nitrogen used in E5 medium.
The same was calculated for percentage of carbon in each of the concentration of
glucose used in E4 medium.
For E4 and E5 media C/N ratios were calculated.
41

Tween 20 experiment
Medium E6 was used for this experiment to investigate the effect on Tween 20 on
the growth of R. toruloides. Different concentration of Tween 20 was used and
readings were taken at an interval of 24 hours until 96 hours. The concentrations of
Tween 20 used were 0.5, 1.5, and 3.0 g/L and in each case it was prepared in
duplicates for each time interval. Medium E1 was also prepared to compare the
growth of R. toruloides with and without Tween 20 and the reading were taken from
the culture with medium E1 together with cultures with media E6 at each time
interval. Only a single set of flasks with E1 medium was prepared that is not in
duplicates for the reading to be taken at each time interval.
For all experiments conducted with E3, E4, E5 and E6 media respectively, there
were certain limitations that needed to be considered. High concentration of glucose
deepens the colour of the media and at times may react with the nitrogen source
present in the media when the temperature is increased in the autoclave providing
variation in the result. Also, high concentration of glucose sometimes has an effect
on the yeast, as at this stage, its metabolism switches to anaerobic even in the
presence of air, a process known as crab effect.
4.3.1.2.3. Synthetic sea water experiment
The synthetic sea water experiment was a flask experiment. The composition of
synthetic sea water used in the experiment has been summarised in the Table 4.2.
This experiment was conducted to investigate whether R. toruloides was able to
grow equally well in basal medium with sodium chloride as that in basal medium
with synthetic sea water.









42

Table 4.2: Composition of Synthetic sea water
Nutrients Composition
(g/L)
Nutrients Composition
(g/L)
a. NaF 0.003 g. Na
2
S0
4
4.0
b. SrCl
2
.6H
2
0 0.02 h. MgCl
2
.6H
2
0 10.78
c. H
3
B0
3
0.03 i. NaCl 23.5
d. KBr 0.1 j. NaSi0
3
.9H
2
0 0.02
e. KCl 0.7 k. Na
4
EDTA 0.001
f. CaCl
2
.2H
2
0 1.47 l. NaHCO
3
0.2

Source: Eaton et al. (2005)
Some of the chemicals were not available in the laboratory and since their use was in
very small quantity and would not make much difference in the composition, hence,
were ignored when preparing synthetic sea water medium. These chemicals include
NaF, SrCl
2
.6H
2
0 and KBr. 0.01 g/L of Silica was used instead of NaSi0
3
.9H
2
0. Also,
one of the limitations of using this medium is that it cannot be used with medium
containing other trace elements which if used might lead to toxicity of the culture.
All the salts given in the Table 4.2 were mixed step by step in distilled water only
after when the former gets dissolved, and the volumes made up to the required mark.
The pH of the medium was 8.0 0.2 and the salinity was reported to be as 34 0.5
g/Kg by Eaton et al. (2005).
E2 process medium nutrients (whose composition could be referred from the Table
4.1) were then dissolved in synthetic sea water medium and the volume was made
according to the requirement for the experiment.
Another set of medium of the same volume as that with synthetic sea water, was
prepared with basal nutrients and sodium chloride (E1 process medium from Table
4.1). The volume of the medium in both cases of synthetic sea water and sodium
chloride was made in duplicates.
43

One more set of medium with only basal medium nutrients was prepared which
stood as a positive control against the other two medium. Only a single set of
medium was prepared in this case, that is flasks were not prepared in duplicates.
The pH was adjusted to 5.5 using 0.1 M phosphoric acid in each set of medium.
4.3.2. Experiment set up and design
4.3.2.1. Design and setup of experiment on petri plates
The two media E1 and E2 were autoclaved at 121

C, 15 psi for 15 minutes and

carefully poured into petri dishes in laminar air flow cabinet maintaining aseptic
conditions. It was left to cool for 45 minutes after which using a loop and the
streaking technique, the media was inoculated using R. toruloides mother culture in
aseptic conditions. It was incubated at 30

C for 4 to 5 days until proper growth was

observed without contamination. Triplicates were done for each culture medium.
Results were compared by observing the colonies formed in both cases, noting their
density and thickness. For negative control, to check for any contamination, one
plate without the inoculum was prepared and observed towards the end of the
experiment.
4.3.2.2. Design and setup of experiments in shake flasks
All shake flask experiments were conducted in 250mL Erlenmeyer flasks containing
20% (v/v) of process media (50mL) to provide space for exchange of respiratory
gases required for the growth of R. toruloides. In each case culture medium was
inoculated with R. toruloides in aseptic conditions. The flasks were then incubated at
30
o
C in a rotary shaker at 200 rpm for proper mixing of the suspension culture to
provide enough oxygen for their growth. The readings were taken at time interval of
24 hours until 96 hours in case of synthetic sea-water and Tween 20 experiment. For
C/N ratio experiment the reading was taken at 48 hours as previously stated.
Sampling was done from the respective flasks at a particular time allocated for each
experiment. pH, optical density, microscopy, was analysed for all the shake flask
experiments including glucose analysis done for experiment with varied carbon to
nitrogen ratios. In all sampling cases, the flasks were shaken well before sample
could be taken to ensure proper mixing of the cells in the culture. Photographs of one
44

of the set up of the shake flask experiment taken at 0 hours of inoculation and 48
hour old inoculum are shown in the figure 4.1 and 4.2 respectively.

.

4.3.2.3. Design and set up for the Bioreactor experiment
200mL of 48 hours old inoculum of R. toruloides was used to culture 3.8L of
sterilized E1 media to make up a working volume of 4L in a 5L bioreactor. The
mode of operation of the bioreactor is batch and it was run for 76 hours. Before the
media together with the bioreactor reservoir was autoclaved, with the help of the
controller (4), the oxygen probe was calibrated using 2% sodium bisulphite and pH
probe was calibrated using buffer solution of pH 4 and 7 step by step and both
probes were inserted inside the reservoir through their respective ports (5) and (6).
Bottles with 100mL of 10% concentrated sulphuric acid (10); 100mL of 2M KOH
(11) and 20mL of antifoam (in ratio 1:1) (12) were connected to the bioreactor
reservoir through ports (7), (8), and (9) respectively and were autoclaved all together
with sample collecting bottles. After autoclaving, the medium was cooled down and
inoculated through 1L inoculum bottle (2) in the laminar air-flow cabinet. Inoculum
bottle was connected to the inoculum port (13) on the reservoir through which the
medium was inoculated. The set up was connected and temperature was set at 30
o
C
using the controller connected to the temperature probe (3) attached by a rope to the
bioreactor reservoir (1). The pH was also set at 5.5 and the starting pH was noted.
Any changes in pH of the media were adjusted automatically using acid and base
through the pump. The oxygen level was set to 100% saturation at the start of the
experiment and the drop in its level was noted at various times interval. The image of
the setup is given in figure 4.3 below.
Figure 4.1: Photograph showing one of the
set up of the shake flask experiment taken at
0 hours of inoculation before sampling
Figure 4.2: Photograph of the
inoculum of R. toruloides 48
hours old
45


Figure 4.3: Photograph of the set-up of 5L volume Bioreactor used for batch
fermentation
The media was stirred using a stirrer (14) connected to the Rushton impellor inside
the bioreactor. The speed of the impeller was set to 600 rpm of the power of the
motor and was adjusted later during the course of the experiment depending on the
foaming of the culture. Air flow was started and adjusted to 1.67 vvm (vvm is the
effective volume of the reactor/ volumetric flow rate of air) using a rotameter (16)
and was introduced into the reservoir through the sparger (18). A water outlet and
inlet was connected to the condenser (15) for maintaining the temperature and heat
inside the bioreactor to an optimum. During the course of the experiment, acid, base
and antifoam was added to the bioreactor through pumps whenever any change in
pH or excess foaming was observed. A filter (17) was connected to the bioreactor in
order to maintain the pressure of the culture to an optimum. The filter was used such
that no contamination from the atmosphere could affect the culture growth. Samples
through the sample port (19) were taken under aseptic conditions at particular time
intervals and an injection at (20) is attached to cater for any pressure difference
during sampling.
1
3
2
11
10
14
19
15
5
6
12
4
8
20
9
16
18
8
7
13
17
46

Samples were collected and analysed for their optical density, dry cell weight and
contamination, soon after the samples were collected at each time interval. For
carrying out some of the analysis that could not be carried out instantly such as
residual glucose analysis and lipid extraction, fermentation broth from samples of
each time interval were centrifuged at 10000 rpm for 10 minutes and 1mL of
supernatant were stored at 4
o
C in a sterilized eppendorf tube.
One of the limitations associated with this experiment was the foaming during the
culture growth which lead to the oxygen level fall to nearly zero at time 22 hours
from the time of inoculation. The reactor was left in that position for 3 hours until
the antifoam was autoclaved and under aseptic conditions was added to the
bioreactor. Second reading after 22 hours was then taken at 26
th
hour. The culture
was then checked for contamination. Despite of this fact, the growth of the R.
toruloides did not get hindered to a greater extent. The impeller speed had to be
adjusted each time during excessive foaming.
4.3.3. Analytical methods
4.3.3.1. Microscopy
Each time the samples were collected from shake flasks or bioreactor, it was viewed
under the a light microscope from Olympus Japan with JVC TK-C1381 colour video
camera, at a magnification of 100X to observe for any contamination. The sample
was first well shaken and using a sterilized tip, a drop of it was transfer on a clean
slide. A cover slip was carefully placed on it and a small drop of oil was place on its
top. It was then observed under the microscope for any contamination and pictures
were taken each time. A photograph of R. toruloides cells when viewed under the
microscope at magnification of 100X has been shown in Figure 4.4.


47


Figure 4.4: Photograph of one of the samples of R. toruloides taken at 48 hours
viewed under a light microscope from Olympus Japan with JVC TK-C1381 colour
video camera using a 100X objective lens.

4.3.3.2. Optical density determination
Cell concentration in a sample is a measure of its optical density as stated by Beer-
Lamberts law. Also, cell concentration is directly related to it cell mass. Hence, cell
growth of R. toruloides could be observed by measuring the optical density at a
particular wavelength of 600 nm using a Shimadzu UV- Vis Spectrophotometer uv
mini 1240. The fermentation broth from each sample was centrifuged at 10000 rpm
for 10 minutes and the supernatant discarded. The cells were then washed by adding
distilled water to the cells, vortexed, followed by centrifugation at 10000 rpm for 10
minutes. The washing procedure was repeated twice before the cells were re-
suspended in 4mL of distilled water and vortexed again for proper mixing of the
cells. Distilled water was used as the blank and the optical density of the cell
suspension was measured by adding 4mL of cell suspension in the cuvette. Dilutions
were made if the optical density was greater than the range of 1.5.
Plot of natural log of optical density against fermentation times gave the growth
curve from which specific growth rate per hour and doubling time was calculated.
4.3.3.3. Dry cell weight (DCW) measurement
Dry cell weight (DCW) in g/L was carried out to get a more accurate result on the
growth of R. toruloides. It was measured to find the volumetric cell mass production
rate (Q
x
) which is the plot of biomass verses time. Dry centrifuge tubes of 10mL
capacity were labelled according to the number of samples and weighed noting down
the weight of each tube. 5mL of fermentation broth from each sample was poured
R. toruloides cells in Basal media with
sodium chloride
48

into the respective centrifuge tubes and centrifuged at 10000 rpm for 10 minutes.
The supernatant was discarded and the tubes were kept in the oven at 60
o
C for
overnight. It was allowed to cool in the desiccator for a minimum of 1 hour before
the final weights were measured. The actual weight of the biomass obtained was
calculated by subtracting the weight of the respective centrifuge tubes (W1) in grams
from the final weight of the dry biomass and the centrifuge tube (W2) in grams and
multiplied by 100 to get the percentage of biomass.
DCW (g/L) = (W2 W1) X 100% (4.1)
A 5- 13 m sized filter was used to filter the biomass from the fermentation broth
left towards the end of the experiment with bioreactor. The cells were filtered with
the help of a vacuum suction pump. Each filter was weighed before filtration and dry
biomass weight was calculated using equation 4.1. The filters with biomass were left
overnight at 60
o
C before measurement was taken.
4.3.3.4. pH measurement
A pH meter pHep

by Hanna H198108 was used for all pH measurements. It was
calibrated each time before use. The pH of the un-autoclaved fermentation medium
was measured and adjusted to 5.5 using 0.1% phosphoric acid. The pH at the time of
sampling was obtained for each of the flasks experiment. The pH in the bioreactor
was maintained using the controller, hence, pH of the *samples from the bioreactor
was not carried out.
4.3.3.5. Residual Glucose analysis
Residual glucose concentration is measured to know the amount of substrate
consumed at different time interval. Volumetric rate of substrate consumption (Q
s
)
was obtained from the plot of substrate present at different fermentation time. The
residual glucose concentration in g/L was measured using a Glucose Analyzer
bought from Analox Instrument, UK.
The working principle of the Glucose Analyser is that under an optimum culture
condition, the concentration of glucose in the supernatant or fermentation broth is
directly proportional to the uptake of oxygen by the cells in the culture. Therefore,
glucose analysis is based on the principle of oxygen uptake. The equation below
shows the reaction that takes place in presence of oxygen and -D glucose.
49

D-glucose + Oxygen
Glucose

oxidase
Gluconic acid + Hydrogen peroxide
(4.2)
D-glucose reacts with the dissolved oxygen present in the fermentation medium in
presence of the enzyme glucose oxidase to form gluconic acid and hydrogen
peroxide
The equipment before being used was calibrated using a standard glucose solution of
4.5 g/L. Sample fermentation broth was diluted according to the initial concentration
of glucose to bring the range of the values between 0 and 4.5 g/L. The samples were
mixed with the help of a vortex and 5L of it was injected into the injection port and
the reading was noted at each time. The values were then multiplied with its dilution
factor.
One of the limitation of this technique is that the analyser needs to be calibrated
every after 10 readings to reduce the chances of errors. Cells and other suspension in
the fermentation broth should be removed by centrifugation before the analysis.
4.3.3.6. Lipid extraction
Lipid extraction was performed using a Soxhlet extractor. The lipid was extracted to
find the lipid yield, specific product yield (Y
P/X)
, and specific rate of lipid
production (q
P
). The Figure 4.5 shows a labelled image of the Soxhlet System HT
extraction Unit whose operating procedure has been discussed.

Figure 4.5: Photograph of a Soxhlet System HT 1043 Lipid Extraction Unit
7
4
5
2
9
8
1
3
6
10
11
50

6 Metals cups (1) were washed and kept in the oven overnight. It was then kept in
the desiccator for at least an hour before their weight could be recorded. The cups
were then placed in sequence on the metal cup holder (2). Dry biomass of R.
toruloides obtained from the Bioreactor was grounded using liquid nitrogen at -
196
o
C for disrupting the cells. A known weight of not more than 0.5 0.05 g of
these disrupted cells were placed on 6 separate thimbles (size of 26 x 60 mm) noting
the weight in each case. Thimble adapter was placed on their top and then these
thimbles were placed inside the respective metal cups. The metal cups were then
placed on the heating plate (3) under the extraction unit (4). The temperature and
water flow were adjusted to 140
o
C and 2L/minute respectively using the controller
(11) to prevent solvent evaporation. Air pump and circulating pump was started.
Chloroform and methanol were mixed in the ratio 1:1 and 30mL of this mixture was
added to each of the metal cups. The lever (5) was pulled down for the extraction
unit to come in contact with biomass in the thimbles and knob (6) on the extraction
unit was turned vertical. The knob (8) on the condenser unit (7) was lifted upwards
to rising position and the system was left to run for 2 hours.
After about 2 hours, the knob (8) was pulled down from boiling to rising position
and the system was left in that position was about half an hour for the lipid/oil to
settle down in the metal cup. The extraction was done after this period of time.
For extraction, the temperature of the system was reduced to 100
o
C, the knob (6) was
turned in horizontal position. The evaporation knob (9) was pulled upwards and the
system cover (10) was also opened. The system was left was 10 minutes for the
extraction to complete.
The lever (5) was pulled upwards freeing the metal cups from the thimbles and
extraction unit. The metals cups was then placed in the oven for overnight at 70
o
C
using the holder (2). Before taking the final reading of the metal cups, they were kept
in the desiccators for a minimum of 1 hour and the reading were then taken. The
percent weight of the lipid was got by subtracting the metal cup weight (W1) in
grams from the weight of metal cup and the oil (W2) in grams and multiplying by
100.
The lipid content was calculated from the formula;
% Lipid content =

x 100 (4.3)
Where; Ws = weight of dry biomass used for extraction (g)
51

One of the limitations of this experiment was that it was very difficult to remove the
dry biomass from the filter paper and for this reason the biomass should not be kept
for drying more than overnight in the oven and the temperature should be kept lower
to around 60

C.
4.3.4. Statistical method
Experiments may have variations in the results obtained, so in order to cater for this
deviation, minimum and maximum error associated with any experiment needs to be
estimated. Systematic errors were minimized through calibration of equipments.
Since most of the flask experiments were carried out in duplicates, hence, random
errors could be estimated manually by estimating the difference in the two results
and comparing it with the expected result, but for some cases, standard deviation had
to be calculated.
Standard deviation is the measure of the deviation from the expected or mean value.
It was calculated using the formula given in equation 4.4.

) (4.4)
Standard deviation was then used to cater for errors in the results that were obtained.

4.4. Summary
The chapter summarises the materials and the methods used for carrying out the
experiments towards fulfilling the aim of the project. The principle operating
procedure for most of the equipments used for the analysis of data has also been
described in this chapter together with any assumptions and limitation taken while
conducting the experiments. These methods lead to the analysis of the result which is
later described in the next chapter.





52

Results and Discussion Chapter 5
5.1. Introduction
In this chapter, the results for each of the experiments performed are discussed citing
any agreement or dissimilarity when compared with the past work by other
researchers. Most of the experiments were successful, but some failed due to
contamination. The results obtained from these successful experiments have been
discussed in this chapter. For all the flask experiments, each time the sampling was
done from a separate flask which was later discarded once its purpose was served.
This was a measure to avoid any contamination which could occur if sampling was
done from the same flask at various time intervals.

5.2. Growth of R. toruloides on agar support under high salinity
condition
A preliminary test to check for the growth of R. toruloides in high salinity conditions
was performed on petri-dishes or slants. Two different set of medium conditions, one
with basal medium with high salinity of 3.5% (w/v) and the other set was with only
basal medium without salinity. The growth of R. toruloides was then observed
constantly on all the petri-dishes with the two sets of medium condition. The result
showed that the culture of R. toruloides on petri-dish with saline medium took a
longer time to grow than on medium without salinity, but later grew equally well in
both media. The reason for this was that R. toruloides has a longer lag phase in
media with salinity than without salinity, as the inoculum used was from mother
culture that had no salinity. Therefore, the cells have a longer lag phase in case of
medium with salinity .This progression of growth is shown in the figure 5.1.







53

Time in hours Basal medium without
salinity
Basal medium with
salinity
0


48


120



Figure 5.1: The observed growth of R. toruloides in the two sets of medium
condition at different time interval.
5.3. Comparison of growth of R. toruloides in basal medium with
synthetic sea water and basal medium with sodium chloride
The aim of this experiment as discussed in the previous chapter was to compare the
growth of R. toruloides in both basal medium with synthetic sea water and basal
medium with sodium chloride. In both medium the salinity was estimated to be 3.5
% (w/v) and hence, the experiment was performed to predict whether R. toruloides
would be able to grow equally well in both medium. This will eradicate the need to
prepare synthetic sea water for each experiment with high salinity, hence, saving
54

time and effort used for the preparation of synthetic sea water medium in laboratory.
The results obtained from the experiment have been shown in the figures 5.2 and 5.3.
Figure 5.2 is a bar graph showing the growth of R. toruloides by plotting an optical
density at A
600
of the culture from the two medium together with their duplicates at
different fermentation time. The basal medium without salinity was the positive
control whose optical density was also combined with respective fermentation time.
The cell concentration was equivalent to optical density of R. toruloides. There was
no significant difference observed in the growth of R. toruloides between the media
with synthetic sea water and with sodium chloride.

Figure 5.2: Plot of optical density at A
600
on a logarithmic scale with different time
interval, comparing the growth of R. toruloides in the two different media. A
positive control without salinity is also compared.
A scatter diagram in the Figure 5.3 was drawn for a better understanding of the
comparison of the growth of R. toruloides in three media. The optical density of R.
toruloides in all three media were coinciding with each other, depicting a more
distinct similarity in the growth of R. toruloides.
0.3703704
1
2.7
7.29
19.683
53.1441
0 24 48 72 96
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Basal with Synthetic
Seawater-flask 1
Basal with Synthetic
Seawater-Flask 2
Basal with NaCl-flask1
Basal with NaCl-flask
2
Positive control
Fermentation time (hours)
55


Figure 5.3: Scatter diagram of OD at A
600
on logarithm scale versus fermentation
time, comparing the correlation of medium with synthetic sea water and with NaCl
The result was confirmed from the approximate specific growth rate of R. toruloides
in synthetic sea water medium, sodium chloride medium and in the positive control
with only basal medium, which were 0.07 0.003 h
-1
, 0.068 0.003 h
-1
, and 0.069 h
-
1
respectively.
Hence, for the rest of the experiments with high salinity, sodium chloride was used,
saving ample of time and effort that could be used for carrying out other
experiments. The cost of preparing synthetic sea water in laboratory was also
eradicated, although, on large scale sea water could be used directly for serving the
purpose of salinity.
5.4. Experiment on the growth of R. toruloides with Tween 20 in
the medium
The aim of this experiment was to investigate the effect of the surfactant, Tween 20
on the growth of R. toruloides. As from the literature, Saenge et al. (2011) reported
the effects of Tween 20 on the growth of oleaginous yeast. As proposed, Tween 20
enhances the permeability of the cells and in turn may increase the oxygen mass
transfer rate that could eventually increase the growth rate of R. toruloides in the
culture medium. Three concentration of Tween 20 were chosen which were 0.5, 1.5,
and 3.0 g/L as Saenge et al. (2011) reported the optimum concentration between 0.5
0.3703704
1
2.7
7.29
19.683
53.1441
0 24 48 72 96
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Fermentation time in hours
Positive control
Basal with Synthetic
Seawater-flask 1
Basal with Synthetic
Seawater-Flask 2
Basal with NaCl-
flask1
Basal with NaCl-flask
2
56

1.5 g/L. Therefore for better understanding of this study the experiment was also
performed with a higher concentration of 3.0 g/L Tween 20.
The results obtained are given in the Figures 5.4 and 5.5. Combination of natural
logarithm of optical density for different concentration of Tween 20 used including
their duplicates, with respect to change in fermentation time has been summarised in
the figure 5.4. A positive control with only basal medium without Tween 20 has also
been plotted in the figure 5.4.

Figure 5.4: Plot of OD on logarithmic scale with fermentation time for different
concentration of Tween 20. S1 and S2 stand for duplicates that are flask 1 and flask
2 whose readings were taken at the same time.
Explanation of the Figure 5.4 is that at the time of inoculation (at 0 hours), the
growth of R. toruloides was found to be similar in all the flasks but after 24 hours,
there was an increase in growth of R. toruloides in medium with low concentration
of Tween 20 that was 0.5 g/L, however the fastest growth was obtained in the
medium without Tween 20 (positive control). The growth pattern after 24 hours
showed that the cells were decreasing with increasing concentration of Tween 20
that is with concentrations of 1.5 and 3.0 g/L. After 48 hours of inoculation, it was
found that the growth of R. toruloides increased in all the flasks with no much
significant difference in their growth pattern, hence, similar growth was observed.
The growth rate at this time was still slightly in a decreasing order with increasing
0.3703704
1
2.7
7.29
19.683
53.1441
0 24 48 72
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Fermentation time in hours
0.5g/l(S1)
0.5g/l (S2)
1.5g/l(S1)
1.5g/l(S2)
3.0 g/l(S1)
3.0g/l(S2)
Positive control
without tween 20
57

Tween 20 concentrations. There was a slight increase observed in the growth of R.
toruloides from 48 to 72 hours in all the flasks.
The reasons probably for the growth pattern seen in figure 5.4, could be that the
culture without Tween 20 had a shorter lag phase when compared to the medium
with Tween 20 because the inoculum medium and the process medium was similar
in case of culture medium without Tween 20. Hence, the cells in the medium without
Tween 20 reached exponential phase within 24 hours whereas the culture with
Tween 20 in the medium took time to adapt to the change in the environment
condition, therefore had a decreasing growth with increasing concentration of Tween
20. Therefore, the culture showed to adapt faster to a lower concentration of Tween
20 and reach exponential phase within 24 hours with concentration of 0.5 g/L. After
a certain time of 48 hours until 72 hours, the cells probably get adapted to the
environment and the effect of Tween 20 could not be noticed at a concentration of
3.0 g/L on the 72
nd
hours of inoculation.
The result show that Tween 20 had a detrimental effect during the first 24 hours but
after a certain time they were able to grow equally well in all concentrations of
Tween 20. This is in agreement with Saenge et al. (2011), who reported that Tween
20 was able to increase the growth rate and lipid production with an optimum of 1.5
g/L for the concentration between 0.5 1.5 g/L. Our study showed that even on
increasing Tween 20 concentration above 1.5 g/L, the growth of R. toruloides did not
get inhibited, hence, it was proven that Tween 20 had no inhibitory effect on the
growth of R. toruloides and increasing its concentration may increase the growth rate
if the experiment would be performed in a bioreactor with controlled oxygen
saturation. The effect of Tween 20 on oxygen mass transfer could not be well studied
in shake flask experiment due to low concentration of dissolved oxygen in flasks,
hence, probably if this experiment would be carried out in a bioreactor changing the
mode of operation from batch, fed batch or continuous, better results could be
observed by. Saenge et al. (2011) also proposed that Tween 20 increases the lipid
production, but due to the experiment being performed in shake flasks with only
50mL sample, it was difficult for the lipid to be extracted. Hence, if the process
could be done on a larger scale in the bioreactor, the effect of Tween 20 on lipid
production could be investigated.
58

Figure 5.5 was plotted to give a rough estimation of the specific growth rate of R.
toruloides in the three different medium with different concentration of Tween 20.
The specific growth rate of R. toruloides in the medium with Tween 20 of
concentration 0.5, 1.5, and 3.0 g/L are 0.13 0.0002 h
-1
, 0.079 0.0226 h
-1
, and
0.135 0.0045 h
-1
and that with the positive control is 0.145 h
-1
.
The overall result from the estimated specific growth rate of the three medium
conditions showed that the maximum specific growth rate was obtained in the
medium with 3.0 g/L concentration of Tween 20 which was still lower than that with
the positive control. The result showed that the effect was Tween 20 had no much
significant change in shake flasks; hence, bioreactor experiment with controlled
oxygen saturation needs to be carried out for better results.

Figure 5.5: Plot of OD on logarithmic scale versus different concentration of Tween
20
5.5. Growth of R. toruloides with varied C/N ratio
The aim of this experiment was to investigate the effect of varying carbon to
nitrogen ratios on the growth of R. toruloides. Optimum carbon to nitrogen ratio is a
very important factor in maximising the growth rate and lipid production of R.
toruloides, as different organism has different growth requirement in different set of
conditions. Hence, since the projects deals with growth kinetics of R. toruloides in
high salinity, carbon to nitrogen ratio in the medium should be optimized.
0.0188168
0.0508053
0.1371742
0.3703704
1
2.7
7.29
19.683
53.1441
0 24 48 72
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Fermentation time in hours
0.5g/l(S1)
0.5g/l (S2)
1.5g/l(S1)
1.5g/l(S2)
3.0 g/l(S1)
3.0g/l(S2)
Positive control
without tween 20
59

This was done with two set of experiments, first with varying carbon concentration
and keeping nitrogen concentration fixed, and the other set was varying nitrogen
concentration while keeping the carbon concentration fixed. In both sets of
experiment one carbon to nitrogen ratio, 6.29, was same which was got from first set
of experiment with optimum carbon to nitrogen ratio.
Experiment 1: In the first set of experiment, the various C/N ratio used was 1.54,
3.08, 6.29, 9.25, 15.42, 18.50, 23.13, 30.84, 38.55, and 46.26 calculated from the
glucose concentration of 10, 20, 40, 60, 100, 120, 150, 200, 250 and 300 g/L.
The results of the optical density and dry biomass weight obtained from the culture
of different C/N ratio medium from the first set of experiment was plotted which are
shown in figure 5.6(a) and (b).

Figure 5.6 (a): Plot of optical density at A
600
obtained from cultures of R. toruloides
in medium with varying C/N ratios. Duplicates from flasks 1 and 2 were also taken
at the same time.
0
1
2
3
4
5
6
7
8
1.54 3.08 6.29 9.25 15.42 18.5 23.13 30.84 38.55 46.26
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C/N ratios
OD Flask -1
OD Flask-2
60


Figure 5.6 (b): Plot of dry biomass weight in g/L obtained from cultures of R.
toruloides in medium with varying C/N ratios.
In both figures 5.6 (a) and (b) the concentration of carbon was varied and
concentration of nitrogen was fixed. All readings were taken at 48 hours after
inoculation. The optimum C/N ratio could not be clearly estimated from the figure
5.6 (a) and (b), although it was showing a decline in growth of R. toruloides with
increasing concentration of glucose as shown from the figure 5.6 (a). However, the
same tendency was not observed with the biomass concentration data in Figure 5.6
(b) which makes the conclusions difficult to draw. The reason for this difference
between Figures 5.6 (a) and (b) could be due to the fact that, different cells in the
medium could be at different stage of cell division at a particular time, and that some
cells could be thicker because of the more accumulation of lipid than the other cells.
This may bring variation in the turbidity of the medium giving a difference in optical
density, hence showing a decline in growth with increasing glucose concentration. In
case of dry biomass density, it is a possibility that more of the lipids were
accumulated at a higher glucose concentration due to inhibitory effects of glucose
that builds stress conditions in the culture; therefore, the sample with more thickened
cells due to accumulated lipid, will show a difference in dry biomass weight and
optical density. Figure 5.7 shows the photograph of the cells of R. toruloides in a
particular sample that shows observable difference in the sizes of the cell at a
particular time of 48hours. Hence, the difference in the growth pattern of R.
toruloides with analytical measurement of optical density and dry biomass
concentration is explained. A Scatter diagram to show the correlation of optical
0
5
10
15
20
25
1.54 3.08 6.29 9.25 15.42 18.5 23.13 30.84 38.55 46.26
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C/N ratios
Dry Biomass Flask-1 Dry Biomass Flask-2
61

density and dry biomass weight has been shown in figure 5.8 (a) and (b)
respectively.

Figure 5.7: Microscopic picture of a sample of R. toruloides taken at 48
th
hour of
inoculation showing difference in cell sizes.

Figure 5.8 (a): Scatter diagram of optical density at A
600
with varied C/N ratios


Figure 5.8 (b): Scatter diagram of dry biomass with varied C/N ratios.
Flask 1 y = -0.0819x + 6.7361
R = 0.8931
Flask 2 y = -0.0784x + 6.9773
R = 0.9276
0
1
2
3
4
5
6
7
8
0 10 20 30 40 50
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OD Flask -1
OD Flask-2
Linear (OD Flask -
1)
Linear (OD Flask-
2)
Flask-1 y = -0.041x + 13.217
R = 0.0213
Flask-2 y = -0.0377x + 14.051
R = 0.0165
0
5
10
15
20
25
0 10 20 30 40 50
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Dry Biomass Flask-1
Dry Biomass Flask-2
Linear (Dry Biomass
Flask-1)
Linear (Dry Biomass
Flask-2)
Thicker R.
toruloides cells
Smaller sized R.
toruloides cell
62

Figure 5.6 (a) showed that up to C/N ratio of 9.25 (glucose concentration of 60 g/L),
the growth of R. toruloides were similar, but as the concentration of the glucose was
increased, it showed a decline in the growth. This was contradicting with the results
reported by Li et al, (2007) who reported that the growth of R. toruloides increased
until a glucose concentration of 150 g/L, after which the growth declined due to
inhibitory effects of excess glucose on the cells when nitrogen was already
exhausted. The difference in the result could be probably due to the variation in the
culture conditions and the sampling timing. As vials were directly used to inoculate
the flasks, the cells might have a different lag phase with different concentrations of
glucose at a particular time which was 48 hours for this experiment. Glucose at times
reacts with nitrogen source at high temperatures, when autoclaved together, this
might change the media conditions for the growth of R. toruloides, hence, the higher
glucose concentration, the more the effect of the reaction in the medium and thus on
the growth of the culture. At higher salinity, the oxygen level of the medium
decreases, hence, less oxygen available for the glucose catabolism, leaving excess
glucose in the medium un-metabolised, decreasing the growth rate of the cells.
Another reason as suggested by Granger et al. (1993) could be due to prolonged
depletion of nutrients in the medium, the cell might lose its activity, slowing down
the growth rate.
Better results could be obtained if glucose and the nitrogen source in the medium
were separately autoclaved and fed-batch could be carried out for better
understanding of effect of increasing glucose concentration at various fermentation
times. Although, the growth rate of R. toruloides decreased with increased
concentration of glucose, the lipid yield would probably increase due to the stress on
the cells by inhibitory effects of glucose. Lipid extraction although, could not be
carried out due to low volume of medium that is 50mL for each sample.
As seen from the figure 5.6 (b) C/N ratio of 6.29 (glucose concentration of 40 g/L)
gave a higher dry biomass concentration in both the duplicates; hence this ratio was
used for the next set of experiment with varying nitrogen concentration and keeping
carbon concentration fixed.
Experiment 2: A second set of experiment was performed to investigate the
difference in the growth of R. toruloides when the amount of nitrogen source
63

(ammonium sulphate) was decreased with a fixed optimum glucose concentration
assumed from the results of first set of experiment. The amount of yeast extract was
fixed because it was used in a small quantity. To check for the effect of increasing
the concentration of nitrogen (lowering C/N ratio), one set of flask with a higher
concentration of ammonium sulphate above the optimum which was 12 g/L in first
set of experiment was used. The C/N ratios calculated was 5.40, 6.29, 7.55, 12.59,
and 37.77 for ammonium sulphate concentrations of 14, 12, 10, 6, and 2 g/L.
The cell concentration obtained after a growth period of 48 hours is shown in the
figure 5.9 (a) and (b).

Figure 5.9 (a): Plot of optical density at A
600
obtained from culture of R. toruloides
in the medium with various C/N ratios. All readings were taken at 48
th
hour of
inoculation.
An increase in the growth of R. toruloides was observed with increasing
concentration of nitrogen source (ammonium sulphate) as shown in the figure 5.9
(a). This was due to the fact that nitrogen together with carbon is required for the
initial growth of the cells. Therefore, increasing its concentration increased the
growth rate of the cells, despite the C/N ratio was decreased with increasing nitrogen
source. This phenomenon was coinciding with the result reported by Li et al. (2007)
who suggested that for an efficient growth of the culture, a higher nitrogen
concentration is required for the initial growth of the oleaginous yeast, although lipid
accumulation only begins once the nitrogen source is exhausted. For better
understanding of the effect of nitrogen source on the growth of R. toruloides, fed-
batch could be performed feeding nitrogen source at different time interval.
5
5.2
5.4
5.6
5.8
6
6.2
6.4
6.6
6.8
7
37.77 12.59 7.55 6.29 5.4
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64

On the other hand, the dry biomass weight of the cultures slightly decreased with
increasing nitrogen concentration as shown in the figure 5.9 (b).

Figure 5.9 (b): Plot of dry biomass concentration in g/L of the culture of R.
toruloides in the medium with various C/N ratios. All readings were taken at 48
th

hour of inoculation.
This phenomenon shown in figure 5.9 (b) differed from the observed growth pattern
in figure 5.9 (a). Figure 5.9 (b) shows that increasing the concentration of
ammonium sulphate decrease the biomass density, which contradicts with the result
obtained for optical density. This could probably be explained by the fact that, cells
quickly metabolises lower concentration of nitrogen, and begins accumulating lipid
in the stationary phase. Thus, time for the onset of lipid production is increased with
increase in nitrogen concentration. For the cells with higher amount of lipid has more
biomass weight than with lower amount of lipid as biomass weight includes the
weight of the cell components including the lipid content. Hence, lower nitrogen
concentration; the cells might accumulate more lipids giving a higher biomass
production than with increasing nitrogen concentration. At C/N ratio of 6.29 there
was no consistency in observed results of optical density and dry biomass in the two
sets of experiment, therefore, useful conclusions on choosing an optimum C/N ratio
were difficult to draw. An optimum carbon to nitrogen ratio could be better studied if
the sampling was conducted at various fermentation times
The process could be better understood if lipid extraction would be carried out, but
as for this experiment, due to lower volume of culture medium, lipid extraction could
not be performed.
0
1
2
3
4
5
6
7
37.77 12.59 7.55 6.29 5.4
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65

As no optimum C/N ratio was obtained from the result, a C/N ratio of 20 was chosen
from previous work of Akuagbazie (2010) for investigating the growth kinetics of R.
toruloides in the bioreactor. For the bioreactor, the initial glucose concentration of
100 g/L was chosen and the rest of the concentrations of the other nutrients in the
basal medium were kept same as that used for the inoculum medium under high
salinity.
5.6. Growth of R. toruloides in Batch mode Bioreactor
The aim of this experiment as discussed earlier was to find the growth kinetics of R.
toruloides in high salinity condition. During the process, growth conditions of the
medium was maintained at the pH (5.5 to 6) and the temperature (at 30

C) using the
controller as discussed in chapter 4. The initial oxygen concentration was set to
100% before autoclaving the media, and its level was constantly checked during the
course of the experiment. The C/N ratio of the medium used was 20 chosen from
previous work of Akuagbazie (2010). An inoculum 48 hours old was used because
the cells have a longer lag phase when inoculated with vials stored at 4

C due to its
reduced activity at lower temperature as reported in the previous work done by
Akuagbazie (2010). The reactor was run for 76 hours and sampling was done at a
frequent time interval for observing the change in optical density, dry cell weight,
residual glucose concentration and check for contamination in the culture during the
course of the experiment. Lipid extraction was done from the biomass collected after
76 hours (after shutting down the bioreactor).
The results obtained gave the specific growth rate of R. toruloides, specific lipid
yield, volumetric cell biomass production and amount of lipid produced.
Figure 5.10 shows the optical density at A
600
with increasing fermentation time and
figure 5.11 gives the dry biomass weight in g/L on a logarithmic scale with
increasing fermentation time. The two graphs depicts that R. toruloides enters the
exponential phase after 8 hours of inoculation and reached a maximum at 30
th
hour
of inoculation, after which it entered the stationary phase.
66


Figure 5.10: Plot of optical density at A
600
on logarithm scale taken at different
fermentation times.

Figure 5.11: Plot of dry Biomass weight on Logarithmic scale taken at various
fermentation times.
The results obtained from the experiment gave specific growth rate of R. toruloides
as 0.15 0.05 h
-1
, lipid content was 29.73% (w/w) and lipid was 3.92 g/L. The dry
biomass concentration increased from 0.13 g/L to 10.54 g/L. The glucose
concentration decreased from the initial concentration of 100 g/L to 47 g/L as shown
in the figure 5.12. The results obtained were lower than expected probably because
glucose tends to react with the nitrogen source at high temperatures. A better result
could be obtained if the glucose and nitrogen source are autoclaved separately and
cooled before they are mixed together on the time of inoculation. The lipid content
0.1371742
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19.683
53.1441
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0.0508053
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0.3703704
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7.29
19.683
0 4 8 22 26 28 30 32 34 36 38 46 48 50 52 54 56 76
N
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Fermentation time in hours
67

could be increased if the bioreactor was run for longer time, probably up to 144
hours as in reported by Akuagbazie (2010). The process could be improved if the
mode of operation could be continuous or fed-batch, as in accordance to results
reported by Li et al. (2007), whose work on fed-batch increased both the specific
growth rate and lipid production of the culture.
From the literature, it is seen that the dissolved oxygen concentration decreases with
increase in salinity. In the bioreactor the dissolved oxygen concentration was
maintained at not less than 86 % saturation throughout the process except at 22
nd

hour of inoculation where due to excessive foaming the mixing was temporarily
stopped and the concentration of oxygen decreased to nearly zero. However, on
addition of antifoam, the dissolved oxygen concentration was reached back to 86 %
saturation and was maintained not lower than this level throughout the course of
fermentation.

Figure 5.12: Plot of optical density at 600nm, dry biomass weight, and residual
glucose concentration with increasing fermentation time.
The specific growth rate and the lipid content were unexpectedly low but the dry
biomass weight was similar when compared to the previous work done by
Akuagbazie (2010). The rest of the medium conditions other than high salinity in the
bioreactor were kept similar with her work in the bioreactor. The residual glucose
concentration although was 47 g/L after 76 hours which differed from her results that
gave 60 g/L of residual glucose after 144 hours. It is difficult to conclude on the
0
20
40
60
80
100
120
140
160
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Fermentation time (hours)
OD Dry Biomass in g/L Residual glucose concentration in g/L
68

difference in the residual glucose concentration from both cases, as it could have
been better understood if the bioreactor was run up to 144 hours.
The correlation of optical density with the dry biomass weight obtained at various
fermentation time intervals was shown in Figure 5.13. The plot suggested that the
culture of R. toruloides was able to grow well in high salinity conditions as result of
specific growth rate from both analyses of optical density and dry cell weight
showed similarity.


Figure 5.13: Plot of optical density and dry biomass weight of the batch culture in
the bioreactor with respect to fermentation time.
The growth kinetics of R. toruloides in batch mode bioreactor was studied in this
experiment and respective growth parameters as mentioned above were calculated.
The results showed that R. toruloides was able to grow well in high salinity
conditions with a lag phase of 8 hrs and that the lipid content might be improved if
the bioreactor was run for more number of hours.
5.7. Results of pH in the shake flask experiments
The results of the changes in pH obtained from the culture medium at different
fermentation time were plotted for the experiment 5.3 with synthetic sea water and
experiment 5.4 with Tween 20.
0
2
4
6
8
10
12
0
5
10
15
20
25
0 20 40 60 80
D
r
y

b
i
o
m
a
s
s

w
e
i
g
h
t

i
n

g
/
L
O
p
t
i
c
a
l

d
e
n
s
i
t
y

a
t

6
0
0

n
m
Fermentation time (hours)
OD Dry Biomass in g/L
69

A bar graph for experiment with synthetic sea water was plotted showing the pH
obtained in all three media which was combined together for each time interval as
given in the figure 5.14 (a). Another bar graph was plotted for experiment with
Tween 20, where the pH from all the flasks with varied medium Tween 20
concentration including duplicates and positive control were combined for different
fermentation time as shown in figure 5.14 (b).

Figure 5.14 (a): Plot of pH measured in the two different medium conditions of
synthetic sea water and sodium chloride, combined with the pH of medium without
salinity at different time interval.

Figure 5.14 (b): Plot of pH measured in all the different the medium with different
Tween 20 concentration, combined positive control, at different time interval. S1 and
S2 are duplicates of same concentration whose reading was taken at same time.
0
1
2
3
4
5
6
7
8
9
0 24 48 72 96
p
H
Fermentation time in hours
Positive Control
Basal with Synthetic
sea water-Flask 1
Basal with Synthetic
sea water-Flask 2
Basal with NaCl-
Flask 1
Basal with NaCl-
Flask 2
0
1
2
3
4
5
6
7
8
9
0 24 48 72
p
H
Fermentation time in hours
0.5g/l(S1)
0.5g/l (S2)
1.5g/l(S1)
1.5g/l(S2)
3.0 g/l(S1)
3.0g/l(S2)
Positive control without
tween 20
70

It was found that the pH in both the experiment had a steep increase after a time
interval of 48 hours. This increase had a deviation from the expected result as during
growth of R. toruloides, it produces by-products like citric acid and malic acid that
leads to a decrease in the pH of the medium as suggested by Ageitos et al. (2011).
Also, Papanikolaou et al. (2006) reported that during fermentation of glucose by
oleaginous yeast, glucose is broken down into products like citric acid in the Krebs
cycle which is accumulated on nitrogen depletion, eventually decreasing the pH.
This result shows that the pH of the culture differed from the expected result. The pH
of the media set as negative control (without inoculation) for both media being
tested, showed no increase from the initial pH of 5.8, hence, it could be suggested
that the yeast might produce some products into the medium after a certain time
interval that would lead to an increase in the pH of the media in the both the
experiments. Also, it could be suggested that most of the citric acid was converted in
acetyl Co A, which was eventually converted into lipid, hence, the effect of citric
acid on the pH was not observed to a greater extent. This phenomenon also
explained that despite of the deviation of pH in the medium from the optimum, R.
toruloides, was still able to tolerate and grow in a pH as high as 8.0, as shown in
figure 5.14 (a) and (b). This makes R. toruloides a considerably excellent micro-
organism that can tolerate extreme conditions and hence, could be used as a model
organism for various experiments.
5.8. Summary
The results obtained from all the experiment in this chapter showed that R.
toruloides, was able to grow well even in high salinity, although there was a longer
lag phase observed when compared to the condition without salinity.
The growth of R. toruloides was observed to be similar in the medium with synthetic
sea water and in medium with sodium chloride with a specific growth rate of 0.07
0.003 h
-1
, and 0.068 0.003 h
-1
, respectively. The results verified that for further
experiments with high salinity, sodium chloride alone could serve the purpose of
salinity and synthetic sea water need not to be prepared each time. This could reduce
time and effort required for preparing synthetic sea water in laboratory.
71

The effect of Tween 20 did not show much significant change on the growth of R.
toruloides in shake flasks because of the lower oxygen saturation in shake flasks
when compared to bioreactor with controlled parameters. However, the result
deduced that R. toruloides that had a longer lag phase for increasing concentration of
Tween 20, although after a certain time interval, the growth of R. toruloides
improved in medium with higher Tween 20 concentration as final dry cell weight
was similar to the flask with no Tween 20. The effect of Tween 20 could be more
prominently observed when experiment in the bioreactor could be carried out in
proper oxygen maintained condition.
Optimum carbon to nitrogen ratio is a growth requirement for every organism. The
growth of R. toruloides was observed to increase up to a certain glucose
concentration but on increasing its concentration, the growth of R. toruloides was
observed to decrease due to inhibitory effects of excess glucose on the cells whereas,
the growth improved with increasing concentration of nitrogen source. However, an
optimum C/N ratio could not be deduced. The process needs to be studied at
different time interval and with different modes of operation like fed-batch in order
to better understand the effect on carbon to nitrogen ratios on the species growth.
The growth kinetics of R. toruloides obtained from running a batch bioreactor for 76
hours, showed that, R. toruloides was able to grow with a specific growth rate of
0.15 h
-1
at 100 g/L of glucose without much inhibitory effects, although after 76
hours 47 g/L of unconsumed residual glucose still remained, which brings in a
requirement to optimize the carbon to nitrogen ratio in high salinity, and also
increase the number of hours of run of the bioreactor, to obtain a high cell density
and lipid yield. The lipid yield obtained was 29.73% (w/w) and lipid as 3.92 g/L.
The dry biomass increased from 0.13 g/L to 10.54 g/L which was very low and
hence, could be improved if the mode of operation of the bioreactor would be
continuous or fed-batch.
In all shake flasks experiments, the pH considerably increased after a time period of
48 hours. The result showed that R. toruloides was able to tolerate and grow even at
a pH of 8.0, hence, could be an excellent model organism to be used for further
work.
72

Conclusion and Further work Chapter 6
All the experiments performed whose results have been discussed in chapter 5,
showed that R. toruloides was able to grow well even in conditions with high
salinity, but the process needs to be optimized in order to increase the lipid yield and
biomass density. The carbon to nitrogen ratio plays a very important role for the
improvement of the process as carbon and nitrogen are the main source of nutrient
for the growth of R. toruloides and lipid accumulation begins on the depletion of
nitrogen. The effects of varying carbon and nitrogen concentration could be better
studied in fed- batch mode of operation, as either carbon or nitrogen could be made
limiting and added to the medium after a certain time interval.
The ability for the R. toruloides to tolerate high salinity and produce a decent amount
of biomass and lipid without much optimization of the process, makes it a good
feedstock for the bio fuel production from oceanic resources rather than using fresh
water that could be utilized for human consumption. Hence, by optimization of the
process, the bio-fuel production could be taken to oceanic level rather than
restricting it on the land which could be used for other agricultural purposes.
The effect of surfactants like Tween 20 in this study could be shown better in
bioreactor where the level of oxygen could be maintained rather than in shake flasks
where the oxygen level is low, so net oxygen mass transfer was observed to improve
the yield. Future work recommended could be a more insight study on the carbon to
nitrogen ratios in high salinity conditions by performing the experiment in fed-batch
conditions. Extensive study should be carried out on the effect of Tween 20 on the
growth and lipid production by R. toruloides in the bioreactor, which due to time
constrain and number of contaminations could not be carried out for this project.
Different carbon and nitrogen sources in high salinity could also improve the process
for better yield and cost reduction by using cheaper bio-degradable raw materials.
In addition, gas chromatography could be carried out in order to compare the fatty-
acid profile obtained in high salinity and non- salinity conditions as fatty acid
obtained in high salinity could be higher due to high osmotic stress and this stress
may also lead to the production of other secondary metabolites that could be good
marketable by-products for many industries.
73

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78

Appendix
1. Results with synthetic sea water and sodium chloride experiment

Table 1.1: Optical density with time
Time in
hours
Positive
control
Basal with
Synthetic
Seawater-
flask 1
Basal with
Synthetic
Seawater-
Flask 2
Basal with
NaCl-
flask1
Basal with
NaCl-flask
2
0 0.538 0.537 0.557 0.604 0.571
24 3.912 3.7 3.834 3.73 3.912
48 13.89 14.78 12.94 13.47 13.89
72 17.94 17.88 18.66 19.7 17.7
96 21 23.25 23.52 23.7 22.65

Table 1.2: Variation in pH with Time in all media
Positive Control Basal with
Synthetic sea
water-Flask 1
Basal with
Synthetic sea
water-Flask 2
Basal with
NaCl-Flask 1
Basal with
NaCl-Flask 2
5.93 5.89 5.91 5.79 5.8
5.95 5.87 5.9 5.89 5.87
5.56 5.98 5.89 5.74 5.74
7.84 7.62 7.67 7.61 7.53
8.27 7.83 7.72 7.97 7.97

2. Experiment with Tween 20

Table 2.1: Optical density for various concentration of Tween 20
time in
hours
0.5g/L
(S1)
0.5g/L
(S2)
1.5g/L(S1) 1.5g/L(S2) 3.0
g/L(S1)
3.0g/L(S2) Positive
control
0 0.604 0.648 0.618 0.599 0.583 0.621 0.622
24 13.48 14.32 2.52 1.48 1.4 1.16 19.96
48 20.28 20.55 17.1 17.25 16.74 15.69 20.31
72 26.64 25.64 27.56 22.32 28.96 28.76 28.61
S1 and S2 are Flasks 1 and 2 for same concentration of Tween 20 whose readings
were taken at the same time.

Table 2.1: pH for various concentration of Tween 20
time in
hours
0.5g/L(S1) 0.5g/L
(S2)
1.5g/L(S1) 1.5g/L
(S2)
3.0
g/L(S1)
3.0g/L(S2) Positive
control
0 5.7 5.7 5.7 5.7 5.7 5.8 5.7
24 5.5 5.6 5.8 5.8 5.7 5.7 5.6
48 6.5 5.9 5.7 5.7 5.6 5.6 6.6
72 8 8 7 7 7 7.3 8



79

3. Experiment on C/N ratios
Table 3.1: Optical density and dry biomass weight of R. toruloides in medium with
different C/N ratio for first set of experiment
C/N ratio OD Flask -1 OD Flask-2 Dry Biomass
Flask-1
Dry Biomass
Flask-2
1.54 6.35 7.08 6.82 6.74
3.08 6.56 6.44 10.68 13.68
6.29 6.66 6.24 20.12 21.92
9.25 6.1 6.84 14.9 15.24
15.42 5.84 5.56 9.34 9.82
18.5 4.94 5.36 18.5 17.9
23.13 4.68 5.42 10.78 9.88
30.84 3.26 4.12 13.06 12.6
38.55 3.74 4.32 8.32 15.4
46.26 3.44 3.28 11.74 10.06

Table 3.2: Optical density and dry biomass weight of R. toruloides in medium with
different C/N ratio for second set of experiment
C/N ratio OD Flask -1 OD Flask-2 Dry Biomass
Flask-1
Dry Biomass
Flask-2
37.77 5.72 6.1 5.92 5.66
12.59 6.56 6.5 5.6 5.7
7.55 6.34 6.54 5.36 5.46
6.29 6.55 6.72 4.88 5.48
5.4 6.7 6.84 5 5.12

4. Experiment in the bioreactor
Table 4: OD, Dry biomass and residual glucose obtained in the bioreactor
Time in hours OD Dry Biomass in
g/L
Residual glucose
concentration in g/L
0 0.254 0.13 130
4 0.381 0.65 150
8 0.404 0.83 120
22 1.71 1.7 120
26 2.14 3.16 120
28 6.12 5.57 110
30 10.73 7.11 107
32 15.22 8.03 107
34 16.88 8.6 103
36 17.98 8.88 90
38 19.44 8.98 90
46 19.46 9.14 90
48 20.04 9.24 80
50 20.34 9.36 77
52 20.52 9.44 77
54 20.62 9.64 53
56 20.88 9.87 53
76 22 10.54 47