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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
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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 toruloidesas the model yeast.......................................17
2.3.1. Considerations in choosingR. toruloidesas the model yeast............17
2.3.2. Life cycle ofR. Toruloides............................................................19
2.3.3.
Growth kinetics ofR. toruloides........................................................202.3.4. Biochemistry of lipid accumulation inR. 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 ofR. Toruloides ................................................29
2.4.4.1.Effect of salinity on Growth phase ofR.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
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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
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5.2. Growth ofR. toruloideson agar support under high salinity condition......52
5.3. Comparison of growth ofR. toruloidesin 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 ofR. toruloideswith varied C/N ratio............................................58
5.6. Growth ofR. toruloidesin 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 ofR. toruloides.........................................................20
Figure 2.2: Growth pattern ofR. toruloidesin 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
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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 ofR. toruloides48 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 ofR. toruloidesin the two set of medium condition
at different time interval.............................................................................................53
Figure 5.2: Plot of optical density at A600on 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 A600obtained from cultures ofR. 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.
toruloidesin medium with varying C/N ratios...........................................................60
Figure 5.7: Microscopic picture of a sample of R. toruloides taken at 48th
hour of
inoculation showing difference in cell sizes...............................................................61
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Figure 5.8 (a): Scatter diagram of optical densityat A600with 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 A600obtained from culture of R. toruloides
in the medium with various C/N ratios. All readings were taken at 48th
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 48th
hour of inoculation.....................................................................................................64
Figure 5.10: Plot of optical density at A600 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 andS2 are duplicates of same concentration whose reading was taken at same time......69
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Nomenclature
Specific growth rate (h-1
)
max Maximum specific growth rate (h-1
)
S Limiting substrate concentration (g/L)
Ks 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
A600 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
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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. toruloidesas 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 ofR. toruloideswas 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.
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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.
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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.
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Introduction
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 toseek 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 effectofhigh 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 beenutilized in this research.
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Literaturereview 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. toruloidesgrowth 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
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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 theproduction of bio-fuel mainly bio-diesel through process of transesterification or
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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
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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-algaeOleaginous 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 (Menget 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
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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
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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 toruloidesas the model yeast
2.3.1. Considerations in choosingR. toruloidesas 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 limitingcondition 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
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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. toruloideshas 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. toruloidescould 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.
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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
EthanolL-
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% FermentorFed batch 144 Li et al.(2008)
R.
glutinis
Glucose 180 72% C16:0=18%,C18:1=60 %
C18:2=12 %
FermentorFed 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 ofR. toruloides
A Sexual and diploid self-sporulating cycles are the two main cycles that makes up
the life cycle ofR. 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.
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Figure 2.1: Proposed life cycle ofR. toruloides
Source: Abe et al. (1986)
2.3.3.
Growth kinetics ofR. toruloides
Growth curve of R. toruloideshas 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. toruloideshas 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.
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Figure 2.2: Growth pattern ofR. toruloidesin 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 (Kovarovaet 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)
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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 (Qp): 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/ maxand the intercept gives
Ks/ max(Leesing et al., 2011).
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2.3.4. Biochemistry of lipid accumulation inR. 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 carboninto 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 lipidto the medium, decreasing the level of lipid accumulation within the cell
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(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 affectingR. toruloidesgrowth, 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. toruloidesY4 was grown in a media with different concentrations of glucose, it
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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 30C 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 5C 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 23C to 30
C but
was inhibited above 34C.
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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 inR. 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. glutiniswas 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 ofR. toruloides forit 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
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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
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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 (Ratledgeet 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
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(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 ofR. 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
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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 ofR.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
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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
culturingR. toruloidesin 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.
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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 thatbrackish 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 usingR. toruloidesas the model yeast for the project, as
discussed previously in this chapter, its growth kinetics has been studied in high
salinity condition.
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Objectives Chapter 3
The economy of the world to some extend is affected by its transportation, which inturn 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 ofRhodosporidium toruloideshas
been studied in high salinity condition which is almost equivalent to salinity in sea
water.
Oleaginous yeast in particular R. toruloideshas 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
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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.
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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 ofR.toruloidescould 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.
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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 ofR. toruloidesgrown 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 4oC to avoid cell death.
4.2.2.1. Working cell Bank or vials
Under aseptic condition, colonies ofR. toruloidesfrom 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 30C 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 4oC and
were sub-cultured every 15 days to maintain the activity of the cells.
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Colonies from the slants were then used to prepare a suspension culture of 150mL
with sterilized (autoclaved at 121C at 15 psi for 15 minutes) basal media in aseptic
condition. The suspension culture was then incubated at 30oC 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 -4oC.
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 109
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.
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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 30oC 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 whetherR. 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.
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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 ofR. toruloideswas 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 ofR. 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
(NH4)2SO4 - - - 12 Varied -
MgSO4.7H2O - - - 1.5 1.5 -
KH2PO4 - - - 1.0 0.2 -
Tween 20 - - - - - Varied
NaCl 35 - 35 35 35 35
Medium name and composition (g/L)
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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 ofR. 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.
toruloideshad 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.
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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 ofR. toruloideswith 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.
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Table 4.2: Composition of Synthetic sea water
Nutrients Composition
(g/L)
Nutrients Composition
(g/L)
a. NaF 0.003 g. Na2S04 4.0
b. SrCl2.6H20 0.02 h. MgCl2.6H20 10.78
c. H3B03 0.03 i. NaCl 23.5
d. KBr 0.1 j. NaSi03.9H20 0.02
e. KCl 0.7 k. Na4EDTA 0.001
f. CaCl2.2H20 1.47 l. NaHCO3 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, SrCl2.6H20 and KBr. 0.01 g/L of Silica was used instead of NaSi03.9H20. 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