growth kinetics of oleaginous yeast, rhodosporidium toruloides, in high salinity condition

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The thesis discusses the use of oleaginous yeast for the production of lipid in saline condition. The process has been optimized in order to get the highest yield.

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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