fermentation of omega-3 and carotenoid producing marine...
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Fermentation of Omega-3 and Carotenoid Producing Marine Microorganisms
by
Tamilselvi Thyagarajan
MSc
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Deakin University
January, 2015
Acknowledgements
No one who does good work will ever come to a bad end;
Neither here, nor in the world to come
-The Bhagavad Gita
This thesis appears in its current form due to the assistance and guidance of several
people. I would therefore like to offer my sincere thanks to all of them.
I would like to extend my cordial thanks to principal supervisor, Professor Colin J.
Barrow for accepting me as a PhD student, his supervision, guidance, unflinching
encouragement, and support for extending all the facilities throughout the work. I
would also like to thank him for his time and patience towards the revisions of thesis
report and publications.
I want to express my deep thanks to esteemed co-supervisor Associate Professor
Munish Puri for the trust, the insightful discussion, valuable advice in designing
experiments and support during the whole period of the study, and especially for his
guidance during the thesis revisions and publications of the work.
I would like to express my sincere thanks to Emeritus Professor Andrew Sinclair for
his insightful suggestions towards the revision of literature review of this thesis
report. I would also like to thank Professor Marcel Klaassen, Dr. Jacqui L. Adcock,
Dr. Jitraporn Vongsvivut, Dr. Madan Verma, Dr. Bo Wang and Dr. Andrew Sullivan
for their time and expertise towards the use of various equipments and techniques
important for my project work.
Thanks to Dr. Serena Wilkens at National Institute of Water and Atmospheric
Research (NIWA), New Zealand for the training program that benefitted my project.
I would like to express my gratitude to all technical staff at LES, Waurn Ponds for
their continuous support towards lab facilities. A special mention for Mrs. Elizabeth
Laidlaw, whose tremendous energy always motivated me. I would like to thank
Rachel Shanahan for her support during my conference travel and other
administrative issues. I would also like to thank Deborah Scarce and Helen Nicholls
Stary for their support.
I greatly appreciate all my lab-mates and colleagues; Reinu, Adarsh, Avinesh,
Halima, Taiwo, Tim, Avinesh, Dilip, Parveen, Shailendra, Parminder and Lovis for
their constant enthusiasm in the lab and insightful comments in lab presentations. My
heartfelt thanks to my friends; Selva, Radhika, Sudhir, Ashwantha, and Venki for
their continuous encouragement and support.
I cannot finish without thanking my family. I warmly thank and appreciate my father
Thyagarajan, mother Bhanumathi Thyagarajan, brother Mohan and my mother-in-
law Saraswathi Krishnan for their material and spiritual support in all aspects of my
life.
I want to express my gratitude and deepest appreciation to my lovely son, Kavin, for
his great understanding and showers of love that helped me to come out of all the
pain and difficulties during the experiments and thesis writing. And finally, Words
fail to express my gratitude to my husband, Karthikeyan. His sacrifice and support to
build my career especially to complete this thesis was tremendous. He moved with
me to Australia for my PhD and started his career all from the start. I could not have
finished this work without his support and encouragement. My heartfelt thanks and
prayers to god to give him all the best in return.
I would like to acknowledge the financial, academic and technical support of Deakin
University, Waurn Ponds campus, particularly in the award of a PhD scholarship to
pursue research in the areas of algal/marine biotechnology.
Lastly and most important, I praise God, the almighty for being kind to me in
providing this opportunity and granting me the capability to proceed successfully.
Dedicated To my lord Guruvayurappan
v
List of publications
Published
Thyagarajan, T.; Puri, M.; Vongsvivut, J.; Barrow, C., Evaluation of Bread Crumbs as a Potential Carbon Source for the Growth of Thraustochytrid Species for Oil and Omega-3 Production. Nutrients 2014, 6 (5), 2104-2114.
Submitted but not yet published
1. Tamilselvi Thyagarajan, Munish Puri, Colin Barrow; Supplementation of medium with flaxseed oil, but not pure ALA, increases lipid, DHA and astaxanthin accumulation in Schizochytrium sp. S31.
2. Tamilselvi Thyagarajan, Jacqui L. Adcock, Munish Puri*, Colin J. Barrow*Isolation and characterisation of a new Thraustochytrium strain (DT8) for co-production of squalene, astaxanthin, polyunsaturated fatty acids and biodiesel
Other publications/Book chapter
1. Puri, M., Thyagarajan, T., Gupta, A. and Barrow, C. J. 2014, ‘Marine sources for the production of Omega-3 Fatty Acids’, SK Kim (ed.), Springer Handbook for Marine Biotechnology, Springer, 1800 p.
2. Vongsvivut, J, Heraud, P, Gupta, A, Thyagarajan, T, Puri, M, McNaughton, D & Barrow, CJ 'Synchrotron-FTIR Microspectroscopy Enables the Distinction of Lipid Accumulation in Thraustochytrid Strains Through Analysis of Individual Live Cells', Protist, no. 0. http://dx.doi.org/10.1016/j.protis.2014.12.002
Oral Presentations
1. Tamilselvi Thyagarajan*, Halima Mokthar, Colin J. Barrow, Munish Puri; Evaluation of Victorian marine isolate Schizochytrium sp. DT9 for biodiesel production; Global biotechnology congress 14-19 June 2014, Boston.
2. Tamilselvi Thyagarajan*, Munish Puri, Colin J. Barrow; Fermentation of omega-3 and carotenoid producing marine microorganisms; AISRF meeting 12th March 2014, Deakin, Australia.
3. Tamilselvi Thyagarajan*, Munish Puri, Colin J. Barrow; Fermentation of omega-3 and carotenoid producing marine microorganisms; IFM conference, 2nd November 2013, Deakin, Australia.
vi
Sequence deposited to GenBank database
1. DT2 - Thraustochytriidae sp. -KF682137
2. DT3 - Schizochytrium sp. -KF682125
3. DT7 - Schizochytrium sp. -KF682129
4. DT8 - Thraustochytrium sp. -KF682130
5. DT9 - Schizochytrium sp. -KF682131
6. DT13 - Schizochytrium sp. -KF682135
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Table of contents
Acknowledgements.............................................................................................................. iii
List of publications ............................................................................................................... v
Sequence deposited to GenBank database ........................................................................... vi
Table of contents................................................................................................................. vii
List of Tables ....................................................................................................................... xi
List of Figures.................................................................................................................... xiii
List of abbreviations .......................................................................................................... xvi
Abstract.............................................................................................................................. xix
Chapter 1: Introduction and Literature review..................................................................... 2
1.1 Importance of unsaturated fatty acids ............................................................................ 3
1.1.1 Unsaturated fatty acids – focus on marine origin ................................................... 5
1.1.2 Potential alternative sources for the production of EPA and DHA ........................ 6
1.2 Marine microalgae ......................................................................................................... 8
1.2.1 Fermentation of microalgae .................................................................................. 10
1.2.2 Isolation of microalgae for the production of PUFA ............................................ 15
1.2.3 Biosynthesis of omega-3 fatty acids in marine algae............................................ 17
1.3 Microalgae fermentation for the production of PUFAs ............................................... 18
1.3.1 Industrial process of Fermentation for the production of PUFAs......................... 20
1.3.2 Submerged fermentation (SMF) of Schizochytrium sp for the production of DHA............................................................................................................................... ........ 20
1.3.3 Solid Substrate fermentation (SSF) of PUFAs ..................................................... 22
1.4 Extraction of Lipids from Microalgae ......................................................................... 22
1.5 Conclusion ................................................................................................................... 25
1.6 Aims of the study ......................................................................................................... 26
Chapter 2: Evaluation of Bread Crumbs as a Potential Carbon Source for the Growth of Thraustochytrid Species for Oil and Omega-3 Production ................................................ 27
2.1 Introduction.................................................................................................................. 28
2.2 Experimental section.................................................................................................... 29
2.2.1 Preparation of seed culture ................................................................................... 29
2.2.2 Submerged liquid fermentation with glucose medium ......................................... 30
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2.2.3 Preparation of bakery waste bread Crumbs (BC) for Static Fermentation ........... 30
2.2.4 Scanning Electron Microscopy (SEM) ................................................................. 30
2.2.5 Fatty Acid extraction and gas Chromatography (GC) analysis ............................ 31
2.2.6 Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopic analysis.................................................................................................... 31
2.2.7 Statistical analysis................................................................................................. 32
2.3 Results and discussion ................................................................................................. 32
2.3.1. Fermentation growth using bread crumbs as the carbon source .......................... 33
2.3.2 Scanning Electron Microscopy (SEM) observation of cell growth ...................... 35
2.3.3. ATR-FTIR spectroscopy analysis of cell lipid content........................................ 36
2.4 Conclusions.................................................................................................................. 38
Chapter 3: Supplementation of medium with flaxseed oil, but not pure ALA, increases lipid, DHA and astaxanthin accumulation in Schizochytrium sp. S31............................... 39
3.1 Introduction.................................................................................................................. 40
3. 2 Experimental section................................................................................................... 41
3.2.1 Chemicals ............................................................................................................. 41
3.2.2 Fermentation medium preparation ........................................................................ 42
3.2.3 Growth profile study ............................................................................................. 42
3.2.4 Cell dry weight, total lipid and fatty acid study.................................................... 42
3.2.5 Nile red staining.................................................................................................... 43
3.2.6 Fatty acid extraction and Gas chromatography (GC) analysis ............................. 43
3.2.7 Astaxanthin extraction and HPLC analysis .......................................................... 44
3.3. Results and discussion ................................................................................................ 44
3.3.1 Impact of flaxseed oil and ALA on the fermentation profile of Schizochytrium sp. S31. ............................................................................................................................... . 44
3.3.2 Fatty acid profile of Schizochytrium sp. S31 ........................................................ 47
3.3.3 Carotenoid Profile................................................................................................. 51
3.4 Conclusions.................................................................................................................. 52
Chapter 4: Isolation and characterisation of a new squalene producing Thraustochytrium strain (DT8) ....................................................................................................................... 53
4.1 Introduction.................................................................................................................. 54
4.2 Experimental section.................................................................................................... 57
4.2.1 Chemicals ............................................................................................................. 57
4.2.2 Isolation of thraustochytrids ................................................................................. 57
4.2.2.1 Direct plating technique..................................................................................... 57
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4.2.2.2 Pollen baiting technique..................................................................................... 58
4.2.3 Microscopy studies ............................................................................................... 58
4.2.4 DNA extraction and 18S rRNA sequencing ......................................................... 58
4.2.5 Phylogenetic analysis............................................................................................ 59
4.2.6 Microbial growth for squalene production ........................................................... 59
4.2.7 Comparison of glycerol and glucose as a carbon source for squalene production 60
4.2.8 Squalene estimation .............................................................................................. 60
4.2.9 Effect of carbon sources glucose and glycerol on astaxanthin accumulation ....... 61
4.2.10 Lipid extraction and fatty acid profile of strain DT8 .......................................... 61
4.2.11 Gas chromatographic analysis ............................................................................ 61
4.2.12 Astaxanthin extraction and HPLC quantification ............................................... 62
4.3 Results and discussion ................................................................................................. 62
4.3.1 Isolation and identification of thraustochytrid isolates ......................................... 62
4.3.1 Squalene content of Victorian mangrove isolates grown with glucose ................ 65
4.3.2 Squalene productivity for strain DT8 using glycerol as carbon source ................ 66
4.3.3 Optimisation of squalene production with glucose or glycerol as carbon source. 67
4.3.3 Fatty acid profile of DT8 grown in glycerol ......................................................... 68
4.3.4 Carotenoid accumulation ...................................................................................... 69
4.4 Conclusions.................................................................................................................. 70
Chapter 5: Evaluation of Victorian marine isolate Schizochytrium sp. DT9 for biodiesel production .......................................................................................................................... 71
5.1 Introduction.................................................................................................................. 72
5.2 Experimental section.................................................................................................... 74
5.2.1 Chemicals ............................................................................................................. 74
5.2.2 Thraustochytrid isolate and growth conditions..................................................... 74
5.2.3 Transesterification process ................................................................................... 75
5.2.3.2 Direct transesterification via a single step process ............................................ 75
5.2.4 Fatty acid analysis of biodiesel using gas chromatography .................................. 76
5.3 Results and Discussion ................................................................................................ 76
5.3.1 Fermentation profile of DT9................................................................................. 76
5.3.2 Biodiesel formation through transesterification.................................................... 78
5.3.3 Optimisation of solvent and acid catalyst for direct transesterification................ 80
5.4 Conclusions.................................................................................................................. 82
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Chapter 6: Evaluation of Victorian marine isolate Schizochytrium sp. DT13 grown using various carbon sources for the co-production of DHA and biodiesel................................ 83
6.1 Introduction.................................................................................................................. 84
6.2 Materials and methods ................................................................................................. 85
6.2.1 Chemicals ............................................................................................................. 85
6.2.2 Thraustochytrid isolate and growth conditions..................................................... 85
6.2.3 Comparison of glucose and glycerol as a carbon source for DHA production..... 86
6.2.4 Thraustochytrid isolate DT13 growth conditions for biodiesel production .......... 86
6.2.5 Fatty acid analysis of using gas chromatography ................................................. 86
6.2.6 Astaxanthin extraction and HPLC analysis .......................................................... 87
6.2.7 Transesterification process ................................................................................... 87
6.3 Results and discussions:............................................................................................... 88
6.3.1 Phylogenetic analysis and growth profile of Schizochytrium DT13 ..................... 88
6.3.2 Fermentation profile of Schizochytrium DT13 with various carbon sources........ 89
6.3.3 Optimisation of the carbon sources glucose and glycerol for DHA production ... 91
6.3.4 Carotenoid profile of Schizochytrium DT13 with various carbon sources ........... 92
6.3.5 Transesterification of Schizochytrium DT13 biomass for biodiesel production ... 93
6.4 Conclusions.................................................................................................................. 95
Chapter 7: Screening of Lipases for the selective hydrolysis of DHA from Schizochytrium oil. ............................................................................................................................... ....... 96
7.1 Introduction.................................................................................................................. 97
7.2 Materials and methods ................................................................................................. 98
7.2.1 Materials ............................................................................................................... 98
7.2.2 Enzymatic hydrolysis............................................................................................ 98
7.2.3 Analysis of lipid classes by capillary chromatography (Iatroscan) ...................... 99
7.2.4 Analysis of fatty acid composition by gas chromatography ................................. 99
7.3 Results and discussion ............................................................................................... 100
7.3.1 Screening of various lipases for hydrolysis of Schizochytrium oil ..................... 100
7.4 Conclusion ................................................................................................................. 102
Chapter 8: Summary and future directions ...................................................................... 103
Bibliography .................................................................................................................... 107
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List of Tables
Chapter 1:
Table 1.1 Potential biological benefits of omega-3 fatty acids……………………...5
Table 1.2 Fish and Seafood Sources of DHA and EPA…………………………......7
Table 1.3 Microalgae and their wide range of applications…………………………9
Table 1.4 Microalgae and their omega-3 fatty acid content………………………..13
Table 1.5Lipid extraction methods of microalgae……………………………….....24
Table 1.6 Strength and weakness analysis of thraustochytrids as a potential source
forPUFA production………………………………………………………………...25
Chapter 2:
Table 2.1 Fermentation profiles for Thraustochytrium sp. AH-2…………………..32
Table 2.2 Fatty acid profile 1 (mg/g) of unfermented bread crumbs, submerged liquid
fermentation and static fermentation with bread crumbs, for Thraustochytrium sp.
AH-2………………………………………………………………………………...33
Chapter 3:
Table 3.1 Profile of Schizochytrium sp. S31 in a medium containing flax seed oil
and ALA……………………………………………………………………………..47
Table 3.2 Comparison of fatty acid profile on feeding flaxseed oil and -linolenic
acid to Schizochytrium sp. S31……………………………………………………..47
Chapter 4:
Table 4.1 Comparitive squalene productivity for various microorganisms, including
Thraustochytrium DT8……………………………………………………………...54
Table 4.2 Effect of carbon source on biomass and squalene production of
Thraustochytrium DT8 at various glucose and glycerol concentrations…………...65
Table 4.3 Fatty acid profile of Thraustochytrium DT8 with 3% glycerol as carbon
source………………………………………………………………………………..66
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Chapter 5:
Table 5.1 Fatty acid profile of isolate DT9 after three days of fermentation using
glycerol as the carbon source………………………………………………………..75
Table 5.2 Comparison of conventional and direct transesterification for isolate
DT9………………………………………………………………………………….76
Table 5.3 Biodiesel and FAME profile of isolate DT9 based on direct
transesterification method…………………………………………………………...79
Chapter 6:
Table 6.1 Effect of carbon source on fatty acid profile of Schizochytrium DT13 with
various carbon sources……………………………………………………………....88
Table6.2 Biodiesel profile of Schizochytrium DT13………………………………..90
Chapter 7:
Table 7.1 Fatty acid profile of Schizochytrium oil on hydrolysis with lipases for 12 h
at 40 °C and pH7.0…………………………………………………………………..97
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List of Figures
Chapter 1:
Figure 1.1 Marine Foodweb of omega3 fattyacids…………………………………..5
Figure 1.2 (a) desert algae production facility, (b) Photo bioreactor , (c)Transparent
aquariums (closed bio-reactors), (d) open-pond system, (e) Flat panel photo
bioreactor, (f) Tubular algae bioreactor……………………………………...……...12
Figure 1.3 Differential interference contrast (DIC) image of Schizochytrium
limacinum, thraustochytrid strain exposing oil vacuoles (Scale bar 20 m)………..16
Figure 1.4 Biochemical pathways for the production of DHA and EPA (Figure
reproduced and modified from venegas-caleron et al., 2010)………………………18
Chapter 2:
Figure 2.1 Fermentation profile of Thraustochytrium sp. AH-2 under submerged
liquid fermentation with 3% glucose as the carbon source…………………………34
Figure 2.2 SEM images of freeze-dried cells for (a) freeze-dried cells of
Schizochytrium sp. SR21 grown under submerged liquid fermentation; (b)
Schizochytrium sp. SR21 cells grown with 1% BC as alternate carbon source; (c)
Thraustochytrium sp. AH-2 cells grown with 1% BC as alternate carbon source; (d)
Thraustochytrium sp. AH-2 grown under submerged liquid fermentation…………35
Figure 2.3 ATR-FTIR spectra of: (a) Thraustochytrium sp. AH-2; and (b)
Schizochytrium sp. SR21. Black line—submerged liquid fermentation; Blue line—
unfermented BC; Red line—static fermentation with BC…………………………..37
Chapter 3:
Figure 3.1 chemical structure of astaxanthin…………………………………….....41
Figure 3.2Growth profile of Schizochytrium sp. S31……………………………....45
Figure 3.3 Fermentation profile of Schizochytrium sp. S31exhibiting biomass and
lipid content………………………………………………………………………..45
Figure 3.4 Nile red staining of Schizochytrium sp. S31……………………………46
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Figure 3.5 Fatty acid profile of Schizochytrium sp. S31(a) Fatty acid profile of
control, (b) Fatty acid profile of medium with C18:3n3, (c) Fatty acid profile of
medium with flaxseed oil, (d) DHA profile on fermentation ……………………….48
Figure 3.6 Astaxanthin and lipid yields in control, flaxseed oil added and ALA added
fermentations. ……………………………………………………………………….50
Chapter 4:
Figure 4.1 chemical structure of squalene………………………………………......54
Figure 4.2 Thraustochytrids; Marine microbial cell factory of nutraceuticals &
bioactives……………………………………………………………………………55
Figure 4.3 Australian Victorian mangrove isolates (a)Pine pollen attached with
thraustochytrid colonies, (b) to (f) pure cultures of isolates, isolated from Victorian
mangroves…………………………………………………………………………...57
Figure 4.4 Squalene productivity of Australian mangrove isolates, compared with
ATCC controls (Asterics indictaes standard ATCC cultures used in this
study)………………………………………………………………………………...59
Figure 4.5 Fermentation profile of Thraustochytrium DT8 with 2% glucose or
glycerol as carbon source.Dotted line with diamond datapoints represents biomass
profile of glycerol, dotted line with square datapoints indicates biomass profile of
glucose, solid line with circle datapoints represents growth curve on fermentation
with glycerol and solid line with triangle datapoints indicates growth curve of
glucose………………………………………………………………………………63
Figure 4.6 Squalene productivity of Thraustochytrium DT8 with 2% glucose or
glycerol as carbon source. Dotted orange square bar represents sqaulene composition
for fermentation with glucose and dotted blue square represents sqaulene compostion
for fermentation with glycerol………………………………………………………64
Chapter 5:
Figure 5.1 Fermentation profile of Isolate DT9……………………………………..74
Figure 5.2 Direct transesterification yields for isolate DT9 using various solvents .77
Figure 5.3 Fatty acid profile after direct transesterification of isolate DT9 with
different solvents…………………………………………………….........................78
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Chapter 6:
Figure 6.1 Fermentation profile of Schizochytrium DT13 with 2% glucose ……….86
Figure 6.2 Fermentation profile of Schizochytrium DT13 with various carbon
sources at 1% w/v concentration………………………………………….................87
Figure 6.3 Fermentation profile of Thraustochytrium DT13 with carbon sources
glucose and glycerol at varying concentration…………………………………........89
Figure 6.4 Astaxanthin profile of Schizochytrium DT13 with various carbon sources
at 1% w/v concentration………………………………..............................................92
Chapter 7:
Figure 7.1 Capillary chromatography (Iatroscan) profile of Schizochytrium oil after
hydrolysis by lipases T.lanuginosus lipase (TLL), Burkholderia cepacia lipase (BCL)
and Rhizomucar meiehei lipase (RML) for 24 h at 40°C and pH 7.0……………...96
Figure 7.2 Fatty acid profile of important fatty acids of Schizochytrium oil after
hydrolysis by lipases T.lanuginosus lipase (TLL), Burkholderia cepacia lipase (BCL)
and Rhizomucar meiehei lipase (RML) for 12 h at 40°C and pH 7.0……………...98
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List of abbreviations
°C - Degree Celsius
% - Percentage
cm-1 - per centimetre
m - Metre
m - Micrometre
nm - nanometre
mg - milligram
g - Microgram
mL - Millilitre
L - Microlitre
L - Litre
g L-1 - Grams per litre
mg L-1 - Milligrams per litre
g L-1 - Micrograms per litre
h - Hours
min - Minutes
- Alpha
- Gamma
- Omega
- Delta
- Beta
C - Carbon
GPS - Global positioning system
DHA - Docosahexaenoic acid
EPA - Eicosapentaenoic acid
ARA - Arachidonic acid
OA - Oleic acid
GLA - Gamma-linolenic acid
DGLA - Di-homo Gamma-linolenic acid
ALA - Alpha-linolenic acid
LA - Linoleic acid
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SFAs - Saturated fatty acids
MUFAs - Monounsaturated fatty acids
PUFAs - Polyunsaturated fatty acids
FA - Fatty acids
TFA - Total fatty acids
TAGs - Triacylglycerols
FAS - Fatty acid synthase
PKS - Polyketide synthase
DNTPs - Deoxynucleotide triphosphates
BHT - Butylated hydroxytoluene
GYP - Glucose, Yeast extract, Peptone
DNA - Deoxyribonucleic acid
rRNA - Ribosomal ribonucleic acid
PCR - Polymerase chain reaction
Kb - Kilo base
bp - Base pairs
UV - Ultraviolet
sp. - Species
FTIR - Fourier Transform Infrared
SEM - Scanning electron microscope
GC - Gas Chromatography
FID - Flame Ionisation Detector
HPLC - High-performance liquid chromatography
RP-HPLC - Reverse phase HPLC
CDW - Cell dry weight
v/v - Volume by volume
w/v - Weight by volume
w/w - weight by weight
SD - Standard deviation
AU - Absorbance Units
OD - Optical density
BC - Bread crumbs
FFA - Free fatty acid
xviii
FISH - Fluorescence in-situ hybridization technique
AFDD - Acriflavine direct detection method
ER - Endoplasmic reticulum
NADPH - Nicotinamide adenine dinucleotide phosphate
Acetyl Co-A - Acetyl co-enzyme A
FAMEs - Fatty acid methyl esters
EPS - Extracellular polysaccharides
ATCC - American Type Culture Collection
xix
Abstract
Omega-3 fatty acids are widely applied as either nutritional or pharmaceutical
products for the benefit of human health. Omega-3 fatty acids are primarily derived
from fish and these fatty acids are accumulated in them through microorganisms in
their diet. To meet increasing market demand, alternative sources to fish and fish oil
are being explored. Among microorganisms, marine phytoplanktons are good
producers of omega-3 fatty acids. Thraustochytrids are marine microbes that can
accumulate 60% of their dry weight as lipids, of which up to 25 to 40%can be DHA.
Thraustochytrids are heterotrophs and can produce co-products such as squalene,
exopolysaccharides, carotenoids and biodiesel. However, for microbial omega-3
production to be competitive with fish oil for high volume markets such as
nutritional supplements, production costs need to be reduced through improved
fermentation strategies and cheaper raw materials. The utilization of food waste by
microorganisms to produce omega-3 fatty acids or biofuel is a potentially low cost
method with positive environmental benefits.
In the work described in the second chapter of this thesis the marine microorganisms
Thraustochytrium sp. AH-2 and Schizochytrium sp. SR21 were used to evaluate the
use of breadcrumbs as an alternate carbon source for the production of lipids under
static fermentation conditions. For Thraustochytrid sp. AH-2, submerged liquid
fermentation with 3% glucose produced 4.3 g/L of biomass and 44.16 mg/g of
saturated fatty acids after seven days. Static fermentation with 0.5% and 1%
breadcrumbs resulted in 2.5 and 4.7 g/L of biomass, with 42.4 and 33.6 mg/g of
saturated fatty acids, respectively. Scanning electron microscopic (SEM) studies
confirmed the growth of both strains on breadcrumbs. Attenuated total reflection
Fourier transform infrared (ATR-FTIR) spectroscopy for both strains was consistent
with the utilization of breadcrumbs for the production of unsaturated lipids, although
at relatively low levels. The total lipid yield for static fermentation with bread
crumbs was marginally lower than that of fermentation with glucose medium, while
the yield of unsaturated fatty acids was considerably lower, indicating that static
fermentation may be more appropriate for the production of biodiesel than for the
production of omega-3 rich oils in these strains.
xx
In the work described in the third chapter of this thesis Schizochytrium sp. S31 strain,
which accumulates 40% of its biomass as lipids with docosahexaenoic acid (DHA)
as a major constituent, was used to examine the effect of the addition of flaxseed oil
or -linolenic acid (ALA) (18:3n3) to the growth medium. Addition of flaxseed oil at
0.04% increased total lipid yield to 45.2% (w/v), DHA yield to 364.9 mg/L and
astaxanthin accumulation to 42.51 g/g. Addition of ALA into the fermentation
medium resulted in suppression of lipid, DHA and astaxanthin production, indicating
that the flaxseed oil is not simply acting as a source of ALA. Adding flaxseed oil or
-linolenic acid (ALA) (18:3n3) to the growth medium impacted lipid and carotenoid
accumulation in this strain.
In work described in the fourth chapter of this thesis new thraustochytrid strains were
isolated from Australian mangrove sites and were screened for their potential for the
production of omega-3 fatty acids along with co-production of squalene, astaxanthin
and biodiesel. The isolate DT8, identified as a Thraustochytrium sp. strain by 18s
rRNA sequencing, was shown to be a promising candidate for coproduction of
squalene, astaxanthin, omega-6 docosapentaenoic acid (DPA), omega-3
docosahexaenoic acid (DHA) and biodiesel. Carbon source optimisation studies with
glucose and glycerol showed that glucose at 7% (w/v) in medium resulted in
maximal squalene yield, with 2.52 g/L biomass and 16.92 mg/L squalene. On
fermentation with glycerol, the major fatty acids produced by strain DT8 was
palmitic acid (C16:0; 42%), DPA (21%) and DHA (11%) with accumulation of
astaxanthin as the major carotenoid. Fermentation with glycerol, in comparison to
glucose, resulted in a two-fold increase in astaxanthin production. Thraustochytrium
sp. DT8 is a good producer of squalene and has potential for the coproduction of
squalene, astaxanthin, DPA, DHA and biodiesel. In-house isolate Schizochytrium sp.
DT9 was explored for its suitability for biodiesel production. Fermentation with 2%
glycerol produced high yields of biomass with 7.1 g/L on day 3 of fermentation, and
lipid yields of 881 mg/g on day 2 of fermentation. Transesterification of freeze dried
biomass was compared using a conventional two-step process and a direct single step
process. Direct transesterification produced higher yields of total methyl esters (363
mg/g) than yields from the conventional method (259 mg/g). Direct
transesterification was further optimised by investigating the impact of different
solvents and acid catalysts. Among various solvents, toluene resulted in maximal
xxi
extraction yields of crude biodiesel with a 57% yield obtained from biomass.
Hydrochloric acid (HCl) and sulphuric acid (H2SO4) behaved similarly as acid
catalysts. Thus, HCl can be substituted for H2SO4 to address the problem of sulphur
content in biodiesel. The study indicated that isolate DT9 may have potential for
producing biodiesel.
Among all the Victorian isolates, DT13 was identified as having potential for the
production of biomass, lipid and DHA with maximal values 6.5 g/L, 3 g/L and 623
mg/L, respectively, after 5 days of fermentation. DT13 was also screened with
various carbon sources such as olive oil, flaxseed oil and glycerol for the production
of DHA and biodiesel. Biodiesel production with isolate DT13 using direct
transesterification was a more efficient method than the two-step process for
conversion to methyl esters. Schizochytrium sp DT13 may be comparable to
commercial strain Schizochytrium limacinum SR21 in terms of DHA productivity,
although this needs to be confirmed at larger scale. Sodium butyrate was shown be to
a good carbon source for astaxanthin accumulation in DT13.
Application of lipase enzyme for the concentration of DHA from Schizochytrium oil
was investigated in chapter 8. The three lipases Thermomyces lanuginosus lipase
(TLL), Burkholderia cepacia lipase (BCL) and Rhizomucar meiehei lipase (RML)
were tested for their selectivity for the concentration of DHA. After 12 hours of
hydrolysis, all three lipases partially concentrated DPA and DHA into the glycerol
portion. TLL and BCL concentrated higher levels of saturated fatty acid into FFA
than did RML. Fatty acid selectivities were similar for TLL and BCL. Selectivity of
RML for DHA was lower than for the other two lipases, in that RML was less able to
concentrate DHA on the glycerol backbone.
Chapter 1
2
Chapter 1: Introduction and Literature review
Chapter 1
3
1.1 Importance of unsaturated fatty acids
The wealth of the ocean is nearly infinite in terms of the discovery of biologically
active compounds which might be potential nutraceuticals. One of the major groups
of marine compounds of interest are the omega-3 fatty acids. Omega-3 fatty acids are
naturally occurring polyunsaturated fatty acids (PUFAs) that include mainly alpha-
linolenic acid (ALA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA)
and docosahexaenoic acid (DHA) (Raghukumar et al. 1995) with EPA, DPA and
DHA being the most well known in the marine world. PUFAs play multiple roles in
cell function and biology including as precursors for various regulatory molecules
such as prostaglandins, thromboxanes, resolvins, maresins and neuroprotectens.
Some recent advances on the biological function of omega-3 fatty acids are presented
in Table 1.1
Awareness among consumers and dietary recommendations from United Nations
Food and Agriculture Organization (FAO) and the World Health Organization
(WHO) has driven PUFAs, as one of the most important functional food ingredients
in the market. The National Health and Medical Research Council (NHMRC)
recommended intake of long chain omega-3 fatty acids (DHA/EPA) in order to
lower the risk of chronic diseases is 610 mg/day for men and 430 mg/day for women
(Grosso et al. 2014).
The sale of PUFAs as a finished product reached US$24.5 billion for 2011 and it is
expected to reach US$34.5 billion by 2016. The target market for omega-3 fatty
acids is diverse in terms of product type, from fortified food, beverages and dietary
supplements, to infant food and pet food (Shanahan 2012). Oily fish such as
sardines, salmon, tuna and herring are considered to be the best dietary sources of
EPA and DHA. Global aquaculture production was 547 million tonnes in 2003 and it
is increasing by an average of 9% every year.
Chapter 1
4
Table 1.1 Potential biological benefits of omega-3 fatty acids.
Potential biological benefits of omega-3 fatty acids Reference
May play a role in the bone formation and may play role in reducing
incidence of chronic inflammatory diseases, rheumatoid arthritis and
inflammatory bowels disease.
Watkins et al. 2003
DHA plays significant role in the development of central nervous
system (brain and visual function).
Innis 2007
Reducing the incidence of primary and secondary cardiovascular
(CV) diseases.
Lavie et al. 2009
Dietary intake may play a role in Alzheimer’s disease and
schizophrenia.
Boudrault et al. 2009;
Sethom et al. 2010
Reduces bronchial allergic inflammation associated with asthma. Kitz et al. 2010
May play a role in reducing severity of symptoms of arthritis Knott et al. 2011
May play a role in gastrointestinal tract cancer and as chemo
preventive agent with respect to colorectal cancer.
Hull 2011
Supplement to the patients with severe hypertriglyceridemia are
reported with decrease in triglyceride levels and cardiovascular
disease risks.
Samuel et al. 2011
Supplement can decrease plasma homocystine, independent risk
factor for cardiovascular diseases.
Huang et al. 2011
Supplement of EPA and DHA are reported to be beneficial for
decreasing the risk of depression leads to suicide and delaying the
onset of the neurological degeneration of aging.
Deckelbaum &
Torrejon 2012
PUFAs can help to reduce inflammation in overweight, middle-aged
and older adults, and thus promote broad health benefits.
Kiecolt-Glaser et al. 2012
Omega-3 fatty acids prevent inflammation and metabolic disorder
through inhibition of NLRP3 inflammasome activation.
Yan et al. 2013
Omega-3 PUFA supplementation reduces obesity-associated adipose
tissue inflammation.
Chung et al. 2014
Chapter 1
5
Although the global demand is growing, fish as a source of PUFA production is a
declining resource, the fact of exploitation to meet the market demand along with
other disadvantages such as lack of environmental sustainability and increasing
global demand which, ultimately forced a search for alternative PUFA sources
(Atalah 2007).
A major focus of current research is the screening of efficient microbial sources that
produce both EPA and DHA. In this literature review, I will discuss the origin of
omega-3 fatty acids, their biosynthesis and fermentation optimisation for their
production from marine microbes.
1.1.1 Unsaturated fatty acids – focus on marine origin
ALA is the biosynthetic precursor to EPA and DHA. The rate of conversion from
ALA into EPA and DHA is low in humans and there are no other means of PUFA
synthesis in the human body. Vegetable oil is the primary source of ALA, whereas
DHA and EPA are predominantly found in fish and fish oil (Hull 2011; Monica et al.
2010; Simopoulos 2005). In these oily fish EPA and DHA are primarily derived from
microorganisms through their food web (Figure 1.1) (Lavie et al. 2009).
Figure 1.1 Marine Food web of omega-3 fatty acids.
Chapter 1
6
Marine phytoplanktons are considered as ocean factories of omega-3 fatty acids, as
they are the primary producers in the marine food web (Sijtsma & de Swaaf 2004).
One of the major industrially useful sources of PUFA are microorganisms, as they
are renewable and ecofriendly in terms of extraction and refining compared with
animal fatty acids, that are associated with odour almost absence of omega-3 fatty
acids and presence of cholesterol (Cheng et al. 1999).
Microorganisms carry an advantage over traditional energy crops as they have higher
growth rates and biomass production in a shorter time span, without competing for
land space (Harun et al. 2010). Tools of biotechnology may contribute towards
industrial production of fatty acids to serve nutritional demands for DHA and EPA,
by producing feed enriched with PUFAs that increases the content of these essential
omega-3 fatty acids in meat, milk and eggs (Rasmussen & Morrissey 2007;
Varfolomeev & Wasserman 2011).
1.1.2 Potential alternative sources for the production of EPA and DHA
Industrially, omega-3 fatty acids are extracted mainly from fish oil. Various fish
sources and their DHA and EPA levels are shown in Table 1.2 There are limitations
in the use of fish oil due to the contamination with heavy metals and other
environmental pollutants (Colombo et al. 2006). Microbial sources such as a few
strains of bacteria and yeast have been identified from the intestine of deep sea fish.
Bacterial PUFAs are found in the phospholipid fraction, which is 10-15% of dry
weight, compared with PUFAs produced by microalgae that are 80% by dry weight
in triacylglycerol (William et al. 2005). Among the phytoplanktons, microalgae and
marine diatoms are considered to play a significant role in the food web. In
aquaculture, microalgae serve as feed for zooplankton and molluscs, which in turn
serves as feed for fish and fish-eating sea animals. The marine microalgae, Isochrysis
and Nannochloropsis are used as feed for rotifers that are in turn supplied to fish to
enrich their diet with EPA and DHA (Hemaiswarya et al. 2011). Food webs
associated with omega-3 PUFAs are inter-related (Lee et al. 2010). Humans obtain
DHA and EPA mainly by consuming fish (Figure 1. 1). However, microalgae
derived DHA and EPA can be used directly as a source of EPA and DHA for people
who don’t consume fish and sea foods (Kang 2011). Food web association, led to the
Chapter 1
7
exploitation of marine microalgae to act as major reservoirs of PUFAs. PUFAs from
microalgae are eco-friendly and potentially more stable because of the presence of
natural antioxidant carotenoids and vitamins that are bio-encapsulated in the algal
cell wall (Patil et al. 2007; William et al. 2005). With all the above factors,
microalgae can be considered as a potential source for the production of EPA and
DHA.
Table 1.2 Fish and Seafood Sources of DHA and EPA
Fish and Seafood Sources of DHA and EPA (raw and cooked)
Fish source DHA + EPA
(g /100 g portion)
Carp, cooked, dry heat 0.45
Catfish, channel, farmed, cooked, dry heat 0.17
Cod, Atlantic , cooked, dry heat 0.15
Eel, mixed species, cooked, dry heat 0.18
Flatfish (flounder and sole), cooked, dry heat 0.50
Haddock, cooked, dry heat 0.23
Halibut, Atlantic and Pacific, cooked, dry heat 0.46
Herring, Atlantic , cooked, dry heat 2.01
Mackerel, Pacific and jack, mixed species, cooked, dry heat 1.84
Mullet, striped, cooked, dry heat 0.32
Perch, mixed species, cooked, dry heat 0.32
Pike, northern, cooked, dry heat 0.13
Pollock, Atlantic , cooked, dry heat 0.54
Salmon, Atlantic , farmed, cooked, dry heat 2.14
Sardine, Atlantic , canned in oil, drained solids with bone 0.98
Sea bass, mixed species, cooked, dry heat 0.76
Shark, mixed species, raw 0.84
Snapper, mixed species, cooked, dry heat 0.32
Swordfish, cooked, dry heat 0.81
Trout, mixed species, cooked, dry heat 0.93
Chapter 1
8
Tuna, skipjack, fresh, cooked, dry heat 0.32
Whiting, mixed species, cooked, dry heat 0.51
Crustaceans
Crab, Alaska king, cooked, moist heat 0.41
Shrimp, mixed species, cooked, moist heat 0.31
Spiny lobster, mixed species, cooked, moist heat 0.48
Molluscs
Clam, mixed species, cooked, moist heat 0.28
Conch, baked or broiled 0.12
Mussel, blue, cooked, moist heat 0.78
Octopus, common, cooked, moist heat 0.31
Oyster, eastern, farmed, cooked, dry heat 0.44
Scallop, mixed species, cooked, breaded and fried 0.18
Table 1.2 Fish and Seafood Sources of DHA and EPA was reproduced from website (http://www.dhaomega3.org/Overview/Dietary-Sources-of-Omega-3-Fatty-Acids)
1.2 Marine microalgae
Algae are spread in diversified ecosystems that include marine, freshwater, desert
and hot springs and even snow and ice environments. Their diversity in habitat is
achieved by their ability to adapt their growth to the local environment. Algae are
classified as multicellular large sea weeds (macro-algae) or unicellular microalgae
(Gualtieri 2001).Based on their pigment composition they are further classified in to
nine divisions. Some of the largest groups include Chlorophyceae (green algae), Phae
ophyceae (brown algae), Pyrrophyceae (dinoflagellates), Chrysophyceae (golden bro
wn alga),Bacillariophyceae (diatoms) and Rhodophyceae (red algae) (Matsunaga et
al. 2005).
A total of at least 50,000 microalgae species have been reported, but not more than
30,000 species have been studied. Microalgae have been used for a wide range of
human applications and some of these are summarised in Table 1.3. The market for
microalgal products is 5000 tonnes per year and the annual sales value is around $6
Billion with productivity of 75 million tonne per year (Varfolomeev & Wasserman
Chapter 1
9
2011). The market for marine microalgae derived omega-3 grown to $2 billion
revenue in 2012, and is expected to increase by double digits up to at least
2016, primarily due to high growth in the Asia pacific market (Shanahan
2012). Microalgae are effective in the conversion of carbon dioxide to carbohydrates,
proteins and lipids.
Table 1.3 Microalgae and their wide range of applications.
Microalgae Application Reference
Chlorella, Dunaliella,
Isochrysis,Nannochloris,
Nannochloropsis,
Neochloris,
Nitzschia, Phaeodactylum
and Porphyridium spp
Biofuel
Xavier Malcata 2011
Arthrospira, Chlorella,
Nannochloropsis oculata,
D. salina
Cosmetics Spolaore et al. 2006; Stolz &
Obermayer 2005
Chlorella, Scenedesmus,
Phormidium, Botryococcus,
Chlamydomonas and
Spirulina
Domestic water
treatment
Chinnasamy et al. 2010;
Olgu n 2003; Rawat et al.
2011; Wang et al. 2010
Chlorella, Tetraselmis,
Isochrysis, Pavlova,
Phaeodactylum, Chaetocero,
Nannochloropsis,
Skeletonema and
Thalassiosira
Fish feed
Borowitzka 1997; Muller-
Feuga 2000; Spolaore et al.
2006; Yamaguchi 1996
Dunaliella salina,
Haematococcus pluvialis
Food industry
Lorenz & Cysewski 2000;
Metting 1996
Arthrospira, Chlorella,
D.salina and Aphanizomenon
Human and animal
nutrition
Borowitzka 1999; Soletto et
Chapter 1
10
1.2.1 Fermentation of microalgae
Microalgae are exploited for the industrial production of various metabolites such as
carbohydrates, lipids, enzymes, polymers, toxins, antioxidants, pigments and others
(Hempel et al. 2011). Cultivation of microalgae involves two basic processes; these
are autotrophic and heterotrophic mode of growth. Based on the requirement of
microalgae and the demands of the process, autotrophic fermentation processes are
further categorised as open system and closed system (Matsunaga et al. 2005). An
open system is the simplest method of cultivation with the advantage of low
production costs, and this culture system requires large surface area and shallow
depth. Various models of the open pond system are in use that includes natural or
artificial ponds, raceways, tanks, circular ponds with impellers, rotating arms or
flos-aquae al. 2005; Spolaore et al. 2006
Chlorella, Ankistrodesmus
and
Scenedesmus species
Phyco-remediation
Kulkarni & Chaudhari 2006;
Lima et al. 2003; Pinto et al.
2003
Arthrospira and rhodophyte
Porphyridium
Phycobiliproteins
Bermejo Román et al. 2002;
Viskari & Colyer 2003
Chlorella pyrenoidosa Production of
immunostimulatory
polysaccharides
Kralovec et al. 2007
Arthrospira (GLA),
Porphyridium(AA),
Nannochloropsis,
Phaeodactylum, Nitzschia
(EPA), Crypthecodinium,
Schizochytrium (DHA)
Production of
PUFAs
Jiang et al. 1999; Ratledge
2004; Spolaore et al. 2006;
Ward & Singh 2005
Chapter 1
11
paddle wheels (Figure 1.2). In Western Australia, Dunaliella is used for the
production of -carotene using artificial shallow pond system (Brennan & Owende
2010; Chisti 2014). Growth in open pond system can be enriched by supplying CO2
and mixing when required. Limited control on cultivation conditions and
contamination of other microorganisms, which means that this type of system is
restricted for microalgae which are tolerant to extreme conditions. Open pond
systems are mainly suitable for the cultivation of Spirulina and Chlorella which is
applied in countries such as Japan, Thailand, California, Hawaii, Taiwan, India and
China. To overcome contamination problems for less robust species, closed photo
bioreactors are designed in various sizes and shapes to meet the requirement of
microalgae growth and development. In this process plexiglas, acrylic, glass tubes
and flexible plastic coils are used as solar collectors, whereas the microalgal
suspension is circulated continuously through rows of connected transparent tubes or
coils (Octavio et al. 2011). A horizontal tubular photo bioreactor in Florence, Italy
led to high productivity for Spirulina (Masojídek & Torzillo 2008).
Heterotrophic microalgae are less abundant than autotrophic species and they require
the addition of organic sources in the growth medium for energy generation and the
production of various metabolites in the process. Growth is independent of light
energy which allows simpler scale up processes to overcome the disadvantage of
large investment in infrastructure and it also supports reproducible biomass yield in
heterotrophic mode of growth (Brennan & Owende 2010). Heterotrophic microalgae
such as Haemotococcus pluvialis, Chlamydomonas sp and Phaeodactylum sp, can
utilize organic compounds to produce energy. They are commercially cultivated with
the help of fermenters of various scales. In Japan it was reported that Chlorella sp
produced by heterotrophic fermentation had a biomass of 500 ton, which amounts to
50% of total Japanese production of this algae (Octavio et al. 2011).
Chapter 1
12
The mixotrophic microalgae Arthrospira platensis can produce their growth essential
compounds either in the presence of light or organic compounds based on the
available conditions (Mata et al. 2010). Photobioreactors and commercial fermenters
share the common purpose of cultivation but the difference lies in the source of
energy, oxygen supply, pH control, light illumination and the process of sterilisation
(Masojidek & Torzillo 2008).
To make the process of fermentation cost effective both the autotrophic and
heterotrophic processes are mixed to achieve maximum algal biomass and product
yield. Chlorella sorokiniana is fermented in a two stage heterotrophic and
phototrophic culture strategy that allows heterotrophic seed culture in autotrophic
Figure 1.2 Various forms of bioreactors used for algal biomass production. (a) Desert algae production facility, (b) Photo bioreactor, (c) Transparent aquariums (closed bio-reactors), (d) open-pond system, (e) Flat panel photo bioreactor, and (f) Tubular algae bioreactor.
Chapter 1
13
open algal culture system resulting in higher growth rate, cell density and
productivity (Chen 2012; Zheng et al. 2012). Photoheterotrophic fermentation
method of Chlorella minutissima has been reported to lead to 11.9 times higher lipid
content than autotrophic condition reported earlier (ZhaoSheng et al. 2011).
Table 1.4 Microalgae and their omega-3 fatty acid content.
Microalgae DHA 22:6(n-3) (%)
EPA 20:5(n-3) (%)
Reference
Amphora sp NT13 0.3 13.9 Renaud et al. 1999
Chaetoceros sp CS256 0.8 16.7
Fragilaria sp GOC1 1 6.8
Nitzschia spp CS258 0.3 8.4
Nitzschia spp BC8 0.3 5
Skeletonema spp GOC27 1.4 12.9
Skeletonema spp GOC36 1.8 13
Cryptomonas sp CRFI01 6.6 12
Rhodomonas sp NT15 4.6 8.7
Nephroselmis sp GOC52 3 2.4
Tetraselmis spp NT18 0.1 4.3
Tetraselmis spp TEQL01 0.1 3.9
Isochrysis sp NT14 9.9 0.9
Rhodosorus sp CS249 0.4 7.8
Navicula like diatom 0.4 29.6 Mansour et al. 2005
Skeletonema sp 4.2 20.2
Thalassiosira pseudonana 1.3 10.2
Naviculajeffreyi 2.1 20.1
Chapter 1
14
Proteomonas sulcata 5.3 3.3
Rhodomonas salina 8.9 17.6
Pavlova pinguis 10.9 27.1
Heterocapsa 16.5 Traces
Amphidinium sp 24.7 23.9
Schizochytrium limacinum SR21
33.5 ND* Morita et al. 2006
Aurantiochytrium mangrovei MP2
3 ND* Wong et al. 2008
Thraustochytrium sp ONC-T18
1.77 36.05 Scott et al. 2011
Amphidinium sp S1 26.33 17.07 Makri et al. 2011
Amphidinium sp S2 24.58 17.41
Amphidinium sp S3 20.41 15.23
Amphidinium sp S4 22.31 15.29
Asterionella sp S1 8.65 25.01
Asterionella sp S2 8.89 26.43
Prorocentrum. minimum S1 20.87 2.91
P. minimum S2 20.06 3.66
P. triestinum S1 21.97 1.72
P. triestinum S2 20.39 1.92
P. parvum S1 8.63 0.45
P. parvum S2 11.18 0.52
Amphora exigua ND 4.98 Chen 2012
Amphora bigibba 0.03 4.29
Caloneis platycephala ND 7.08
Chaetoceros muelleri 0.37 2.96
Chapter 1
15
Cocconeis scutellum 0.06 10.1
Cylindrotheca sp 0.04 0.48
Melosira nummuloides 0.39 1.69
Navicula lyra 1.06 8.4
Nitzschia panduriformis 0.05 6.52
Nitzschia grossestriata 1.27 7
Seminavis gracilenta 0.05 14.17
Skeletonema costatum 2.68 7.47
Thraustochytrium sp. AMCQS5-5
35.7 7.1 Gupta et al. 2013
ND*- Not determined; Values reported in the table are reported as per the reference.
1.2.2 Isolation of microalgae for the production of PUFA
Isolation of microalgae for the production of PUFA plays a major role in laboratory
scale fermentation design, particularly when industrial scale fermentation is targeted.
In general microalgae can be isolated from freshwater and marine water by following
the traditional micropipette washing technique, centrifuge washing technique and
streak plating technique. Microalgae rich in PUFA content can be isolated with the
help of high-throughput cell sorting coupled with flow cytometry. Photosynthetic
pigments in microalgae emit various fluorescence and this is applied as the principle
behind the flow cytometry. Chlorophyll auto-fluorescence (CAF) of eukaryotic
photosynthetic phytoplankton and green auto-fluorescence of dinoflagellates along
with red and orange auto-fluorescence to differentiate between algal strains is used in
the process of identification and isolation of microalgae. Furthermore, the isolated
microalgae are cultivated to screen the strains with respect to high biomass yield and
lipid content (Doan et al. 2011).
Chapter 1
16
Adaptability to diversified environmental conditions is reflected by the composition
of fatty acids in algae, with very long chain (VLC) (> 18C) fatty acids and PUFA
more abundant in eukaryotic algae than cyanobacteria. The PUFA are about 20% of
lipid content in algae and include, arachidonic acid (ARA), EPA and DHA, and
biotechnological approach can be applied to increase these levels (Harwood &
Guschina 2009).
Thraustochytrids are microalgae like microorganisms that can accumulate 60% of
their dry weight as lipids, of which more than 25% is normally DHA. An
ultrastructure study with transmission electron microscopy illustrated that the cells
are heterogeneous in size, approximately 6–21 m in diameter (figure 1.3) with a
granular cytoplasm, containing oil micelles (Gupta et al. 2012). The high lipid
content of thraustochytrids makes them useful for the commercial production of
omega-3 fatty acids. In general, they are isolated with pine pollen technique (Gupta
et al. 2013; Jakobsen et al. 2008; Raghukumar 2002) and acriflavine direct detection
(AfDD) techniques (Kimura et al. 1999).
Figure 1.3 Differential interference contrast (DIC) image of Thraustochytrium sp. AH-2, thraustochytrid strain exposing oil vacuoles (Scale bar 20 m).
Chapter 1
17
1.2.3 Biosynthesis of omega-3 fatty acids in marine algae
Microalgae are known to produce large amounts of C20-C22 PUFA (Klok et al. 2014).
Polyunsaturated fatty acids can be synthesised by aerobic and anaerobic pathways.
The aerobic pathway involves desaturases and elongases enzymes whereas, the
anaerobic pathway uses polyketide synthase enzymes (Uttaro 2006). PUFA
biosynthesis starts with double bond formation in the aliphatic chain of the fatty acid
and the elongation by two carbon units of the acyl chain, hence in this biosynthetic
pathway alternative action of two enzymes desaturase and elongases contributes to
the synthesis of PUFAs (Certik & Shimizu 1999). Various biochemical pathways for
the production of EPA and DHA are represented in Figure 1. 4
1.2.3.1 Synthesis of EPA
The red alga Porphyridium cruentum has been used extensively to study the
biosynthetic pathway for the production of EPA with the help of exogenously
supplied radiolabelled fatty acids, to reveal the omega-3 and omega-6 pathways for
the synthesis of EPA. In the omega-6 pathway with the action of desaturase, -
linolenate was formed from linoleic acid followed by elongation to dihomo- -
linolenate which was further de-saturated in to arachidonate followed with EPA
formation. In the case of the omega-3 pathway, linoleic acid was desaturated to -
linolenate which was further converted to EPA from the intermediate fatty acids
18:4n-3 and 20:4n-3. Among the two pathways, the omega-6 pathway is considered
to be the more active route (Harwood & Guschina 2009; Pereira et al. 2004).
1.2.3.2 Synthesis of DHA
Biosynthesis of DHA is assumed to involve 6 desaturation of C18:3n3 to form
C18:4n-3 which is then further elongated to C20:4n-3 that undergoes desaturation to
form EPA, which is then converted to DPA, followed by DHA formation.
Conversion of DPA to DHA by 4 desaturase has been identified in
Thraustochytrium sp. This is different from the mammalian path way of DHA
Chapter 1
18
synthesis, which involved the Sprecher pathway with two consecutive elongations
steps (Lippmeier et al. 2009).
In the case of Aurantiochytrium sp, and Schizochytrium sp. the biosynthesis of DHA
is reported by the alternative pathway known as the polyketide synthase pathway
(PKS). Biochemical experiments indicated that desaturase and elongase enzymes are
not involved in the PKS pathway but showed the PKS protein complex on molecular-
genetic analysis. Though the exact sequence of PKS pathway remains to be
determined, identification of protein domain provide evidence for the mechanism of
cis double bond insertion through the action of dehydratase/2-trans,3-cis isomerase
(DH/2,3I) (Nagano et al. 2011;Wallis et al. 2002).
1.3 Microalgae fermentation for the production of PUFAs
Microalgae can be cultivated in natural lagoons or lakes or highly sophisticated
bioreactors. Bioprocess engineering has contributed in the process optimisation of
various parameters for ensuring large scale microalgae production (Makri 2011).
Process optimisation depends on the growth requirements of microalgae.
Autotrophic, heterotrophic or a mixture of both these processes is applied based on
the product yield and also aimed at low production cost. Heterotrophic based
microalgal production systems can lead to a higher magnitude of PUFA production
than those of outdoor autotrophic pond systems (Octavio et al. 2011; Wen 2003).
Autotrophic growth of Porphyridium cruentum reported less total fatty acid
accumulation when compared with heterotrophic growth with glucose and glycerol
(Oh et al. 2009). Microalgae Pavlova lutheria, grown under photoheterotrophic
conditions of high and low light intensity, produced high levels of PUFAs, with high
light intensity favouring higher DHA yield and low light intensity favouring higher
EPA yield (Guiheneuf et al. 2009).
Chapter 1
19
Ani
ma
Conventional 6-pathway
Plan 18:0Stearic acid (SA)
18:1n9
Oleic acid (OA)
gas
9 DES
18:1n9Oleic acid (OA)
18:0Stearic acid (SA)
9 DES 12 DES
18:2 9,12Linoleic (LA)
3Des 18:3 9,12,15Linolenic acid(ALA)
Plants
Animals
Alternative 8 pathway
Microbial 4 pathwayMammalian “Sprecher” pathway
Alternative 8 pathway
Figure 1. 4 Biochemical pathways for the production of DHA and EPA. 4-19 desaturase (Des) enzymes and 5-9 elongase (Elo) enzymes (Figure reproduced and modified from venegas-caleron et al., 2010).
Heterotrophically grown Crypthecodinum sp, and Chlorella sp. are rich in DHA and ARA.
The cells were broken by mechanical and enzyme hydrolysis methods to extract oil and the
resulting algal biomass was freeze dried and used as feed for rotifers and Artemia, which
were then used in formulated brood stock diets of fish larvae (Harel et al. 2002). A list of
microalgae involved in PUFA production is given in Table 4. Microalgae Crypthecodinium
cohinii or microalgae like microorganism Schizochytrium sp are used industrially for the
fermentative production of DHA primarily for the enrichment of food products and infant
formula (Atalah 2007; Fan & Chen 2007).
1.3.1 Industrial process of Fermentation for the production of PUFAs
Microorganisms used in the industrial fermentation for the production of PUFAs are listed below (Ratledge 2004).
1. Crypthecodinium cohnii and Schizochytrium sp in the production of docosahexaenoic acid (DHA) by DSM (formerly Martek Biosciences).
2. Mortierella alpina in the production of arachidonic acid, (ARA) by DSM (formerly Martek Biosciences).
3. Ulkenia sp., in the production of docosahexaenoic acid (DHA) by Nutrinova GmbH, Frankfurt, Germany.
Industrial fermentation is carried in two different processes, (1) Submerged fermentation
(SMF), (2) Solid state fermentation (SSF) (Certik & Shimizu 1999). Here, I describe one
example of a marine microbe that is used commercially for the production of omega-3 fatty
acids such as EPA and DHA.
1.3.2 Submerged fermentation (SMF) of Schizochytrium sp for the production of DHA
Industrial production of DHA usually requires two prerequisites, low cost and high
productivity. High productivity can be achieved only with high cell density and high DHA
content. Fermentation in submerged level is of two types, one stage fermentation and fed
batch fermentation or two stage fermentation; the difference in the process lies with the
supply of carbon source. The carbon source needs to be supplied in fed batch fermentation as
it decreases the negative effect of substrate inhibition. High cell density can be achieved
through fed-batch fermentation (Lee Chang et al. 2013; Ren et al. 2010).
Chapter 1
21
Fermentation processes for PUFA production require isolation and identification of natural
over producers of omega-3 fatty acids. A second important property is the flexibility of the
strain to various fermentation parameters that favour maximum production of PUFAs at low
cost. The ability of the selected strain to grow well in low salt is also important, to avoid
rapid corrosion of the stainless steel fermentation vessels. Martek Biosciences developed a
fermentation process, which included a bio-rational approach to technology development that
involved a package of desirable upstream and downstream process for the production DHA
from Schizochytrium by heterotrophic fermentation in stainless steel fermenters with glucose
as main carbon source (Allison & Morrow 2003; Barclay 2006; Cohen & Ratledge 2005).
Initial lab scale productivity at the 14L stage was achieved in 48 h using 40g/L glucose with
the medium optimized for nitrogen source and at low chloride levels. In scale up to 150,000 L
200g/L biomass and 20% DHA was achieved. The final cell dry weight improved from 21 to
170-210g/L with DHA productivity from 0.05 to 0.45-0.55 g/L/h in going from 14L to 150,
000 L scale. A 10 fold increase from lab scale to final scale up was achieved by optimising
the process of fermentation by fed-batch technology (Barclay 2006; Cohen & Ratledge
2005).
Downstream processes included harvesting of the biomass by initially drying the
fermentation broth using drum driers. Drying is an important step in the process of storing the
sample for longer time without causing damage to the biomass by microbial, chemical, or
sensory deterioration, and after drying the sample target moisture needed to be maintained at
4-6%.
Parameters of SMF that includes specific growth rate, specific glucose consumption, specific
lipid accumulation, cell yield coefficient, lipid yield coefficient, and DHA yield coefficient
are studied to understand kinetic parameters of the fermentation process. In general cell dry
weight is measured by a gravimetric method and the fatty acid profile is analysed using liquid
chromatography (Chin et al. 2006). Extraction of lipids involves transfer of the fermentation
broth to a high pressure homogenizer, crushing the algal cells and extracting the lipids by
using a solvent mixture of n-hexane/ethanol (2:1 v/v) solution (Charlotte Vallaeys et al.
2008). Hexane can be removed by distillation using oilseed processing equipment that
enables filtration, separation and distillation (Kyle 2007).
Oil extraction includes biomass treatment with solvent followed by cell disruption that allows
the separation of oil from the cell, followed by a miscella winterization process that removes
Chapter 1
22
high melting point saturated fatty acyl residues and other impurities. Miscella winterization is
an important step in maintaining clear oil when stored under refrigeration. DHA rich oil
obtained after winterization is further processed by degumming, caustic refining, bleaching
and deodorization. Deodorized oil is blended with high oleic sunflower oil to standardize the
DHA concentration and DHA is then stabilised by adding antioxidants, mainly ascorbyl
palmitate and tocopherols (Cohen & Ratledge 2005; Kyle 2007; Ratledge 2004).
1.3.3 Solid Substrate fermentation (SSF) of PUFAs
Mortierella alpina, a fungal strain, has been grown using solid substrate fermentation for
PUFA production, using rice bran as substrate. It was reported that after 12 days of
incubation 127 mg of total PUFAs were produced per gram of substrate carbon utilised. The
production process was carried out in a solid state column reactor (Jang & Yang 2008).
Fermentation using SSF decreases the waste water output and minimizes fermentation costs
by using cheaper carbon and nitrogen sources including industrial waste. Examples include
rice bran and wheat bran utilised by Mortierella alpina (Certik & Adamechova 2009), okara
powder, bread crumbs and brewers spent grains utilised by Schizochytrium mangrovei (Fan et
al. 2000), and Pear pomace utilised by Mortierella isabellina for the production -linolenic
acid (GLA) (Fakas et al. 2009). SSF with initial moisture content of 60-65% is suitable for
omega-3 fatty acids production, whereas moisture content of 70-75% favours omega-6 fatty
acids production. SSF is lower cost than SMF and so SSF is favoured for waste management
and production of value added products such as PUFAs. Studies have reported that after
feeding freeze dried cells of Schizochytirum, PUFA content increased in rotifers like Artemia
nauplii and Penaeus monodon. A Schizochytrium strain utilised low cost carbon sources such
as breadcrumbs and okara powder to produce lipid (Fan et al. 2001; Stark et al. 2007;
Thyagarajan et al. 2014; Yamasaki et al. 2007).
1.4 Extraction of Lipids from Microalgae
Extraction of omega-3 rich lipids from single cells of microalgae is an important step in
obtaining high oil recovery due to the need to disrupt the cell wall in these organisms
(Ryckebosch et al. 2012). Extraction requires disruption of cells to release oil from oil
vacuoles. Mechanical disruption involves the process of pressing, bead milling or
homogenisation. Pre-treatment of the biomass with acid /alkali and enzymes can decrease the
volume of solvent needed for oil extraction. The objective of mechanical disruption is to
Chapter 1
23
break the cell wall of algae to enable extraction of the oil. In the process of pressing high
pressure is applied, whereas in bead milling agitation with beads damages the algal cells,
after which solvents like benzene, cyclohexane, hexane, acetone, chloroform, methanol, and
ethanol are used in combinations or individually to extract the maximum oil from the algae.
Combinations of cell disruption and solvent extraction methods were used depending on the
microalgae species and the scale up requirements of the fermentation process. Ultrasonication
coupled with hexane extraction has been applied to some microalgae including
Crypthecodinium cohnii. However, although high oil yields were obtained, scale up of the
technology in industrial fermenters requires intensive power consumption and so is not
economically viable (Cravotto et al. 2008). In general physical or mechanical disruption
paired with solvent extraction gives maximum yields, but solvent extraction is not
environmental ideal and also can lead to food grade approval problems (Cohen & Ratledge
2005; Mercer & Armenta 2011). In the solvent extraction process, solvent needs to
effectively penetrates into the algal biomass as demonstrated by Shen et al, where
Scenedesmus dimorphus (autotrophic algae) and Chlorella protothecoides (heterotrophic
algae) are processed initially with mechanical disruption methods followed by solvent
extraction to extract maximum amounts of oil (Shen et al. 2009).
To replace the standard solvent extraction methods, supercritical fluid extraction has been
applied. Some chemicals behave as liquid and gas with increased solvent power when raised
above their critical temperature and pressure points, known as supercritical fluids. Carbon
dioxide (CO2) with combinations of ethanol as co-solvent has been used for extraction as a
supercritical fluid. CO2 can be removed easily as it is gas at room temperature. A limiting
factor of the technique is the high moisture content and so samples need to be dried before
supercritical fluid extraction (Sahena et al. 2009). Table1.5 gives a list of various microalgae
and method used in extraction of lipids.
Origin oil developed an efficient method for microalgal lipid extraction for biodiesel
production process. Dewatering, cell disruption and lipid extraction were performed in a
single step followed by exposure of the algal concentrate to quantum fracturing that involved
pulsed electromagnetic field treatment with various pH modifications, where intracellular
lipids were released from the algal biomass. In the process, cell debris will settled in the
bottom of the gravity clarifier, whereas lipid raised to the top for skimming. Lipid
fractionation followed by trans-esterification resulted in biodiesel production. This method
Chapter 1
24
reduces the energy expenditure and is ecofriendly as it does not use hazardous solvents in the
process of extraction as it can be used directly on wet fermented biomass (Halim et al. 2012).
Three-Phase partitioning of Spirogyra sp with the use of papain as biocatalyst can be a very
effective tool for the extraction of algal oil yield up to 27% (w/w, dry algae) (Reddy &
Majumder 2014). In Rhodosporidium toruloides, biomass pre-treated with microwave and
enzymatic treatment with the recombinant -1,3-glucomannanase, plMAN5C, and extraction
with ethyl acetate achieved 96.6% of the total lipids yield (Jin et al. 2012).
Table 1.5 Lipid extraction methods of microalgae.
Extraction method Microalgae Reference
Bligh and dyer & supercritical fluid extraction with CO2 Spirulina, Nannocloropsis Mercer & Armenta 2011
Wet milling followed with hexane extraction Scenedesmus dimorphus Shen et al. 2009
Bead beater followed with Hexane extraction Chlorella protothecoides Shen et al. 2009
Microwave assisted solvent extraction Crypthecodinium cohnii Mercer & Armenta 2011 supercritical fluid extraction with CO2 Crypthecodinium cohnii Cohen & Ratledge 2005 Solvent/saponification Porphyridium cruentum Mercer & Armenta 2011 Three phase partitioning with use of papain enzyme
Spirogyra sp Reddy & Majumder 2014
Biomass pre-treated with microwave and enzymatic treatment with the recombinant
-1,3-glucomannanase, plMAN5C, and extraction with ethyl acetate Rhodosporidium toruloides Jin et al. 2012 Direct saponification with hexane and ethanol & miniaturized Bligh dyers method
Thraustochytrium sp. ONC-T18 Burja et al. 2007
Chapter 1
25
1.5 Conclusion
Marine microalgae and thraustochytrids are useful sources of bioactive lipids. However, a
complete shift from fish oil to microbial omega-3 production is only possible if production
costs are further reduced through improved fermentation strategies and cheaper raw
materials. Screening of new strains that can naturally produce high levels of PUFAs,
preferably with valuable co-products, will make marine microbial sources more economically
viable. The SWOT (Strength, weaknesses, opportunities and threats) analysis of
thraustochytrids as a potential source for commercial PUFA production is given in Table 1.6.
Table 1.6 Strength and weakness analysis of thraustochytrids as a potential source for PUFA
production.
Strengths
More than 60% of their dry weight can accumulate as lipids.
Able to produce PUFAs along with other value added products such as carotenoids, squalene, enzymes, polysaccharides and biodiesel.
Remarkable presence in the industrial revenue with their presence as ingredient in infant food formula to fortified food of human diet.
Clinical studies on Schizochytrium sp PUFAs proved to be safe for human consumption.
Opportunities
Increasing market demand of PUFA Alternative for Fish oil to meet the
environmental sustainability issues. Industrial interest will be more on
thraustochytrids compared to other autotrophic marine micro algae as they carry the advantage of heterotrophic fermentation set up at cheaper production cost.
Aggressive current research with tools of biotechnology and molecular biology, chances of more attraction to the market are bright.
Weaknesses
Continuous development in process of fermentation is required to meet the challenges of market.
Scale- up process of the technology outcomes from lab scale to Industrial scale.
Threats
Genetically engineered strains may be a possible question from the consumer’s point of view to commercialize the product.
Process of Isolation and the optimisation study on the strain for the production of PUFAs is time taking where there is a chance for industry to deviate its interest to other microbes which takes less time for the process.
Chapter 1
26
1.6 Aims of the study
Omega-3 fatty acids used in food and supplements are currently produced mainly from fish
oil. To meet a growing market demand marine microbes are a potentially sustainable
alternative source for the commercial production of omega-3 fatty acids. The Primary
objective of this study was to isolate novel marine microbes from the Victorian marine
environment and assess their ability to produce PUFAs and other nutritional products,
particularly omega-3 co-products.
The research aims of this project were as follows:
1. To test the ability of thraustochytrid strains to grow on low cost solid state
medium (breadcrumbs).
2. To improve lipid and DHA yields through improved fermentation strategies,
including the addition of flaxseed oil into the fermentation medium.
3. To isolate novel thraustochytrid strains from the local Victorian marine
environment and screen them for their potential for the production of various
metabolites including PUFA, squalene, biodiesel and carotenoids.
4. To test lipases, for their ability to selectively hydrolysis and concentrate omega-3
fatty acids from thraustochytrid oil.
Chapter 2
Chapter 2: Evaluation of Bread Crumbs as a Potential Carbon Source for the Growth of Thraustochytrid Species for Oil and Omega-3 Production
Chapter 2
28
2.1 Introduction
Thraustochytrids are marine protists that belong to Labyrinthulomycetes and were first
reported by Sparrow in 1936. These microorganisms are epibiotic in nature and represent a
diverse group of organisms living in marine and estuarine habitats throughout the world and
exhibiting a saprotrophic mode of nutrition (Raghukumar 2002). Due to their ability to
produce a large amount of oil, including polyunsaturated fatty acids (PUFAs), research has
focused on lowering their cost of production using low-cost carbon and nitrogen sources,
particularly for large-scale industrial fermentation (Gupta et al. 2012). Biomass produced
through heterotrophic fermentation of thraustochytrids is a potentially sustainable approach to
the production of PUFAs for food, feed and supplement applications and oil for biofuel
applications (Lee Chang et al. 2012).
Thraustochytrids can act as microbial cell factories for the production of omega-3 PUFAs,
squalene, and other secondary metabolites such as carotenoids and sterols, along with
enzymes and extracellular polysaccharides (Gupta et al. 2012). For commercial utility in the
production of these materials, thraustochytrid growth should be low-cost, particularly to
compete in the food market as a replacement for fish oils. Because heterotrophic organisms
require a carbon source for bioconversion into oil, they are more expensive to grow in terms
of consumables than autotrophic organisms. Heterotrophic fermentation has some advantages
over autotrophic fermentation, particularly the ability to use standard industrial fermentation
equipment at scale and to get much higher cell density than can be achieved with autotrophic
organisms, which often need complex and expensive equipment to enable light to reach the
cells while preventing contamination (Brennan & Owende 2010; Johnson & Wen 2009). The
cost of glucose as a carbon source in the growth medium to produce biomass can account for
up to 30% of the overall production cost and so commercial biofuel production using
heterotrophic fermentation requires the use of low-cost carbon sources such as glycerol or
food waste (Abad & Turon 2012; Pleissner et al. 2013). Food wastes generated worldwide are
about 1.3 billion tons (Ferreira et al. 2013). Management of food wastes through landfill
dumping is common and is environmentally problematic. Food in landfills can rot and release
methane gas, which is a major contributor to carbon emissions worldwide (Anikwe &
Nwobodo 2002).
Research on using microbial fermentation to convert food waste into value added products is
limited (Rawat et al. 2013). Schizochytrium mangrovei and Chlorella pyrenoidosa have been
Chapter 2
29
grown using food waste obtained by fungal hydrolysis and were reported to produce biomass
potentially useful as a feed supplement or for biodiesel production (Pleissner et al. 2013).
Schizochytrium mangrovei KF6 was also reported to utilize processed bread crust to produce
docosahexaenoic acid (DHA) from shake flask fermentation at 200 rpm under fluorescent
light for eight days (Fan et al. 2000). Polyunsaturated fatty acids were produced by
Mortierella alpina utilizing rice bran as the carbon source in a solid-state column reactor
under static conditions. Static conditions, where there is no agitation during the growth phase,
are often used in solid substrate fermentation, as opposed to submerged liquid fermentation
which requires higher energy inputs for continuous shaking of the flask at constant speed
(Jang & Yang 2008). Other inexpensive carbon sources derived from food wastes that were
studied includes okra powder (Fan et al. 2001), residues from beer and potato processing
(Quilodran et al. 2009), sweet sorghum juice (Liang et al. 2010), coconut water (Unagul et al.
2007), marine aquaculture waste water (Jung & Lovitt 2010) and crude glycerol (Pyle et al.
2008).
The objective of the study described in this chapter was to investigate the ability of some
thraustochytrid strains to utilize bakery waste, specifically breadcrumbs (BC), as an alternate
carbon source in the fermentation medium to produce lipids under static conditions. Scanning
electron microscopy (SEM) was used to investigate the growth pattern of these
microorganisms during static fermentation, and attenuated total reflection Fourier transform
infrared (ATR-FTIR) spectroscopic analysis was performed to observe the production of
unsaturated fatty acids during fermentation. ATR-FTIR spectroscopy and SEM were shown
to be useful for monitoring static fermentation.
2.2 Experimental section
All chemicals including fatty acid methyl ester standards used in this study were procured
from Sigma-Aldrich (Sydney, Australia) and Merck Chemicals (Victoria, Australia) and were
of analytical grade.
2.2.1 Preparation of seed culture
Thraustochytrid strains used in the study were Thraustochytrium sp. AH-2 (ATCC® PRA-
296™, Manassas, VA, USA) and Schizochytrium sp. SR21 (ATCC® MYA-1381™,
Manassas, VA, USA) and these strains were procured from the American Type Culture
Collection (ATCC, Manassas, VA, USA), and grown in liquid medium containing 1 g yeast
Chapter 2
30
extract, 15 g peptone, 20 g glucose (1 g yeast extract, 1 g peptone, 5 g glucose for
Schizochytrium sp. SR21) in 1 L of artificial sea water (ASW) at 70% strength, according to
ATCC product information sheets. In brief, the cultures were grown at 20°C with shaking
speed of 120 rpm for 96 h. Seed culture of 5% (v/v) culture was used in subsequent
submerged liquid glucose fermentation and static fermentation with breadcrumbs as an
alternate carbon source.
2.2.2 Submerged liquid fermentation with glucose medium
Cultures were grown in 250 mL flasks with 50 mL of medium altered and adopted from Li et
al. 2009. Flasks were kept at 20°C with shaking speed of 120 rpm for 7 days with medium at
a starting pH of 6.0 (prior to autoclaving) and the pH was not controlled over the
fermentation period. Flasks were collected for the dry weight determination and fatty acid
estimation, each for 24 h.
2.2.3 Preparation of bakery waste Bread Crumbs (BC) for Static Fermentation
Breadcrumbs (crude powder; non-uniform size) were purchased from a local bakery
(Geelong, Australia) for evaluating their utilization under static fermentation. BC powder was
used for carrying fermentation experiments. Static fermentation was carried with the same
medium as submerged fermentation but with BC (at 0.5% and 1%) substituted for glucose in
the medium. Flasks were kept under static conditions in an incubator at 20°C for 7 days, with
pH of medium adjusted at 6.0 prior to autoclaving but not adjusted during the fermentation
phase. Flasks were inoculated with 5% (v/v) seed culture (Section 2.2.1) under aseptic
conditions. Samples were collected for the cell dry weight and fatty acid estimation after 24
h. Freeze-dried BC were analysed using an EuroEA elemental analyser (Euro Vector, Milan,
Italy) to determine the percentages of carbon and nitrogen content.
2.2.4 Scanning Electron Microscopy (SEM)
A small flake of freeze-dried cells was mounted onto carbon tape on an aluminium stub and
air dried, after which 60 nm of gold was deposited on its surface using a sputter coater. The
cells were examined under a scanning electron microscope (SEM Supra 55 VP, Zeiss, Berlin,
Germany) at accelerating voltage 3–5 KV using secondary electron detector.
Chapter 2
31
2.2.5 Fatty Acid extraction and gas Chromatography (GC) analysis
Fatty acid extraction was performed as previously described with some modifications (Lewis
et al. 2000). In brief, 10 mg of freeze-dried cells were used for lipid extraction. Fatty acids
were extracted with a mixture containing a 2:1 ratio of chloroform to methanol and repeated
3 times. For trans-esterification, 1 mL toluene was added followed by addition of 200 μL of
internal standard, methyl nonadecanoate (C19:0) and 200 μL of butylated hydroxytoluene
(BHT). Acidic methanol (2 mL) was also added to the tube and kept for overnight incubation
at 50°C. Fatty acid methyl esters (FAMEs) were extracted into hexane. The hexane layer was
removed and dried over sodium sulphate. FAMEs were concentrated using nitrogen gas prior
to GC analysis (Christie & Xianlin 2010). The samples were analysed using a GC-FID
system (Agilent Technologies, 6890N, Santa Clara, CA, USA). The GC instrument was
equipped with a capillary column (Suplecowax 10, 30 × 0.25 mm, 0.25 μm thickness).
Helium was used as the carrier gas at a flow rate of 1.5 mL min 1. The injector was
maintained at 250°C and a sample volume of 1 μL was injected. Fatty acids were identified
by comparison to external standards (Sigma-Aldrich, Sydney, Australia). Peaks were
quantified with Chemstation chromatography software (Agilent Technologies, (Agilent
Technologies, Santa Clara, CA, USA) and corrected using theoretical relative FID response
factors (Ackman 2002). Samples are analysed in duplicate and compared to external
standards.
2.2.6 Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR)
spectroscopic analysis
ATR-FTIR measurements of the freeze-dried samples were conducted using an Alpha FTIR
spectrometer (Bruker Optik GmbH, Ettlingen, Germany) equipped with a deuterated
triglycine sulfate (DTGS) detector and a single-reflection diamond ATR sampling module
(Platinum ATR QuickSnap™, Ettlingen, Germany). The ATR-FTIR spectra were acquired at
4-cm 1 spectral resolution with 64 co-added scans within the 4000–400 cm 1 spectral region.
Blackman-Harris 3-Term apodization, power-spectrum phase correction, and zero-filling
factor of 2 were set as default acquisition parameters using OPUS 6.0 software suite (Bruker
Optik GmbH, Ettlingen, Germany). Background spectra of a clean ATR surface were
acquired prior to each sample measurement using the same acquisition parameters.
Chapter 2
32
2.2.7 Statistical analysis
Data were statistically compared using one-way analysis of variance (Anova), and the
significant difference was identified using Tukey’s and Scheffe tests. The analysis was
carried out using SPSS software (IBM® SPSS® Statistics 20, New South Wales, Australia).
2.3 Results and discussion
Thraustochytrium sp. AH-2 was previously isolated from coastal and mangrove habitats of
Goa and further studied for its extracellular alkaline lipase production (Kanchana et al. 2011).
In the present study, control fermentation profiles were obtained using submerged liquid
fermentation with 3% glucose as the carbon source. Under glucose conditions, biomass of 4.3
g/L and total lipid yield of 941 mg/L were achieved (Table 2.1). The biomass and lipid yield
for our species is similar to that reported for T. aureum ATCC 34304 (Bajpai et al. 1991),
although optimization of controlled fermentation conditions would probably enable higher
biomass and lipid production for strain AH-2. Oleic acid (C18:1n9) was the major fatty acid
at 63.19 mg/g, followed by palmitic acid (C16:0) 32.33 mg/g, DHA (C22:6n3) 23.74 mg/g
and stearic acid (C18:0) 11.82 mg/g. C14:0, C15:0. C16:1n7, C17:0, C17:1n7 were present in
lower amounts. DHA (C22:6n3) was the major PUFA, followed by docosapentaenoic acid
(DPA) (C22:5n6) at 4.32 mg/g and eicosapentaenoic acid (EPA) (C20:5n3) at 3.03 mg/g.
Table 2.1 Fermentation profiles for Thraustochytrium sp. AH-2.
Fermentation type Biomass(mg/L)
Lipids(mg/L)
Saturatedfatty acids 1
(mg/g)
Mono-unsaturatedfatty acids 2
(mg/g) Submerged liquid fermentation (3% glucose)
4300 941.32 44.16 63.19
Static fermentation (0.5% bread crumbs)
2530 260.0 42.4 29.00
Static fermentation (1% bread crumbs)
4760 390.0 33.6 22.6
1 Palmitic acid (C16:0); stearic acid (C18:0); 2 Oleic acid (C18:1n9).
Chapter 2
33
2.3.1. Fermentation growth using bread crumbs as the carbon source
Elemental analysis of the freeze-dried BC revealed approximately 40.95% carbon and 3.23%
nitrogen. The fatty acid analysis of unfermented BC is presented in Table 2.2 and shows oleic
acid as the major fatty acid in the profile, followed by palmitic acid (C16:0), stearic acid
(C18:0), linoleic acid (18:2n6) and other fatty acids were also present. The polyunsaturated
fatty acids EPA, DPA and DHA were not detected in the fatty acid profile of BC.
Table 2.2 Fatty acid profile 1 (mg/g) of unfermented bread crumbs, submerged liquid fermentation and static fermentation with bread crumbs, for Thraustochytrium sp. AH-2.
C16:0 C18:0 C18:1n9 C18:2n6 C18:3n3 C20:5n3 C22:5n3 C22:6n3 others
Unfermented bread crumbs fatty acid profile
2.20 1.50 3.10 1.00 0.00 0.00 0.00 0.00 1.50
Submerged liquid fermentation fatty acid profile 2
32.33 a,4 11.82 a 63.19 a 2.76 a 0.00 a 3.03 4.32 23.74 33.35
Static fermentation fatty acid profile 3
25.9 b,5 16.5 b 29.0 b 12.9 b 1.2 b 0.00 0.57 2.40 14.40
20.4 b,6 13.2 b 22.6 b 11.2 b 1.2 b 0.00 0.00 1.30 11.50 1Palmitic acid (C16:0); stearic acid (C18:0); oleic acid (C18:1n9); linoleic acid (C18:2n6); linolenic (C18:3n3); EPA (C20:5n3); DPA (C22:5n3); DHA (C22:6n3); 2 Submerged liquid fermentation medium with 3% glucose incubated for 7 days at 20°C with shaking speed of 120 rpm; 3 Static fermentation with same medium composition as submerged liquid fermentation, with breadcrumbs as 5 and 10 g substituted for glucose in 1litre of 70% ASW at pH 6; 4 Different superscript (a,b) in a column indicates statistically significant difference (p < 0.05); 5 Static fermentation with 0.5% BC, incubated at 20°C for 7 days; 6 Static fermentation with 1% BC, incubated at 20°C for 5 days.
Thraustochytrium sp. AH-2 was grown using BC as the carbon source and the results
compared with those obtained using glucose. BC is known to contain primarily complex
carbohydrate in the form of starch (Scanlon & Zghal 2001). BC was used at levels of 0.5%
and 1%. Higher levels of 3% and 5% BC gave no improvement in cell growth and made lipid
extraction difficult and were not pursued further. Fermentation with 0.5% BC gave 2.5 g/L of
biomass and 260 mg/L of total lipid yield. Fermentation with 1% BC gave 4.7 g/L of biomass
and 390 mg/L of total lipid yield (Figure 2.1). Compared to liquid fermentation with glucose,
the total lipid yield was relatively low for static fermentation.
Chapter 2
34
Figure 2.1 Fermentation profile of Thraustochytrium sp. AH-2 under submerged liquid fermentation with 3% glucose as the carbon source.
Under static fermentation, unsaturated fatty acids were poorly synthesized, although the
presence of C18:3n3 (linolenic acid), EPA and DHA were clearly observed. Previous
study on Schizochytrium mangrovei KF6 under heterotrophic conditions with processed
bread crust also reported low levels of unsaturated fatty acids (Fan et al. 2000). In general,
monosaccharide sugars in the medium result in the production of higher levels of PUFAs
compared to di- and poly-saccharides (Shene et al. 2010). As BC contains mainly starch,
the observed poor synthesis of unsaturated fatty acids is not unexpected. In this study, the
maximum fatty acid profile with 1% BC was observed on day 5, after which fatty acid
content decreased, probably due to lipid consumption by the organism. Lipids and fatty
acids accumulated in oleaginous microorganisms can act as energy sources for growth
that are utilized when there is a lack of available carbon in the medium (Holdsworth &
Ratledge 1988).
The total amount of saturated fatty acids, which was primarily C16:0 and C18:0, were
42.4 mg/g with 0.5% BC and 33.6 mg/g with 1% BC, whilst submerged liquid
fermentation with glucose as the carbon source gave an only slightly higher level of
saturated fatty acids at 44.1 mg/g. Since PUFA production is low and unsaturated
production is relatively high, fermentation with BC provides a fatty acid profile more
consistent with that of biofuel, than did submerged liquid fermentation. Static
0
10
20
30
40
50
60
70
80
90
100
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
2 3 4 5 6 7 9
DH
A (
mg/
L)
Bio
mas
s (m
g/L)
L
ipid
(m
g/L)
No.of days of fermentation
Biomass mg/L lipid (mg/L) DHA (mg/L)
Chapter 2
35
fermentation may be a useful method for converting BC to oil, since parameters are
readily standardized at industrial scale.
2.3.2 Scanning Electron Microscopy (SEM) observation of cell growth
The fermentation growth for Thraustochytrium sp. AH-2 and Schizochytrium sp. SR21
were compared for BC and glucose as the carbon source using SEM. The morphology of
freeze-dried unfermented BC was observed using SEM as a control material. The SEM
images of cells grown using submerged liquid fermentation with glucose show spherical
cells that are clumped together (Figure 2.2a). When grown in the presence of BC, cell
clusters are attached to the BCs, confirming that cells do grow on this complex carbon
source (Figure 2.2b,c).
Figure 2.2 SEM images of freeze-dried cells for (a) freeze-dried cells of Schizochytrium sp. SR21 grown under submerged liquid fermentation; (b) Schizochytrium sp. SR21 cells grown with 1% BC as alternate carbon source; (c) Thraustochytrium sp. AH-2 cells grown with 1% BC as alternate carbon source; (d) Thraustochytrium sp. AH-2 grown under submerged liquid fermentation.
Chapter 2
36
2.3.3. ATR-FTIR spectroscopy analysis of cell lipid content
ATR-FTIR spectroscopic measurement of Schizochytrium sp. SR21 was performed to
confirm the production of unsaturated fatty acids when BC was used as an alternative carbon
source, in comparison to the submerged liquid fermentation with glucose of the same strain.
Figure 3 shows the comparison of the ATR-FTIR spectral features of the raw unfermented
BC, the static fermented 1% BC and glucose fermented cells. In particular, the olefinic C=CH
stretching vibration found at ~3014 cm 1 is commonly known as a representative band for
unsaturated fatty acids (Vongsvivut et al. 2013; Vongsvivut et al. 2012). This band is clearly
observed in the freeze-dried cells that were grown in 1% concentration of BC under static
fermentation and with glucose, suggesting that observable amounts of unsaturated fatty acids
were produced in the cells grown by both fermentation methods. The triplet bands found in
the range of 3000–2800 cm 1, on the other hand, are attributed to C-H stretches of lipids and
proteins (Vongsvivut et al. 2013). At the low wavenumber region, the strong bands centred at
1650 and 1545 cm 1 known as amide I and II bands, respectively, occurred due to the protein
moieties in the BC and the cells. The sharp band at 1725 cm 1, on the other hand, represents
(C=O) stretches of ester functional groups from lipids and fatty acids, and is therefore
indicative of total lipids produced by the cells (Guillen & Cabo 1997; Vongsvivut et al. 2013;
Vongsvivut et al. 2012). According to the intensities of this band, fermentation with glucose
led to a substantially higher amount of total lipids produced in the microorganisms. However,
the ratios of unsaturated fatty acids per total lipids (i.e., I3014/I1725) were found to be
comparable between both fermentation approaches, suggesting that similar yields of
unsaturated fatty acid can be achieved using BC as a carbon source under static fermentation
of Schizochytrium sp. SR21. Therefore, growth of these strains on BC is potentially useful
both for the utilization of food waste and the production of lipid.
Chapter 2
37
(b) Figure 2.3 ATR-FTIR spectra of: (a) Thraustochytrium sp. AH-2; and (b) Schizochytrium sp. SR21. Black line—submerged liquid fermentation; Blue line—unfermented BC; Red line—static fermentation with BC.
(a)
Chapter 2
38
2.4 Conclusions
Thraustochytrium sp. AH-2 and Schizochytrium sp. SR21 were tested for their ability to
utilize BC during static fermentation as a low-cost and environmental friendly carbon source
for producing oil for either biofuel (saturated fatty acid rich) or food (PUFA rich). The fatty
acid profiles from Thraustochytrium sp. AH-2 indicated low levels of PUFA and higher
levels of saturated oil. ATR-FTIR spectroscopy of Schizochytrium sp. SR21 was also
consistent with higher levels of saturated fatty acids. Fermentation on BC containing complex
carbohydrate appears to be more appropriate to the production of biofuel from these
organisms than for the production of high levels of PUFA for food applications, due to the
suppression of PUFA when BC is used as the carbon source.
Chapter 3
Chapter 3: Supplementation of medium with flaxseed oil, but not pure ALA, increases lipid, DHA and astaxanthin accumulation in Schizochytrium sp. S31
Chapter 3
40
3.1 Introduction
Thraustochytrids are marine phytoplanktons that produce both omega-3 fatty acids and
carotenoids (Gupta et al. 2012). Omega-3 fatty acids and carotenoids impact positively on
cell function and biology, accounting for their wide range of health benefits (Aki et al. 2003;
Deckelbaum & Torrejon 2012). Zooplankton consume phytoplankton in their diet and
concentrate omega-3 fatty acids and carotenoids, and then become a food source for salmon,
trout and other aquatic animals, enriching them in these compounds (Kitahara 1984; Steven
1948). Thraustochytrids have a range of industrial applications that include omega-3
production for food and supplements, aqua feed and biofuels (Lee Chang et al. 2012). Of
particular interest is the genus Schizochytrium, which is consumed by humans via a food
chain including mussels and clams (Hammond et al. 2001). The addition of Schizochytrium
mangrovei freeze dried biomass, that is rich in DHA, to the diet of Drosophila melanogaster
was reported to produce anti-ageing effects (Huangfu et al. 2013). Freeze dried
Schizochytrium has been used as a feed for hens to produce omega-3 enriched eggs.
Commercial large scale fermentation of Schizochytrium sp. has been carried out for the
production of DHA for human nutritional purposes (Fedorova-Dahms et al. 2011; Ward &
Singh 2005). Astaxanthin is used in both human and fish nutrition, with the majority used in
aquaculture feed to colour salmon and trout (Lorenz & Cysewski 2000). Natural astaxanthin
content in Schizochytrium species is potentially useful as an animal feed (Chatdumrong et al.
2006), although increased levels are required for commercial viability.
Figure 3.1: Chemical structure of Astaxanthin (Figure reproduced and modified from Kitahara 1984)
Chapter 3
41
To enhance growth and productivity in Schizochytrium strains, the impact of carbon and
nitrogen sources have been studied. D-glucose, DL-malic acid, D-fructose, D-xylose, fumaric
acid, D-cellobiose, pyruvic acid, -D-lactose, 5-keto-D-gluconic acid and glycerol are all
utilized and reported in good yields of biomass, lipid and DHA content in a range of
Schizochytrium and related Thraustochytrium species (Shene et al. 2010). Tween 80 has
been added to medium and reportedly increased biomass, total lipid and DHA yield during
fermentation of Thraustochytrium aureum ATCC 34304 (Taoka et al. 2011). Fatty acid shifts
and metabolic activity changes along with glycerol dissimilation were studied in
Schizochytrium sp.S31 during batch fermentation, with glycerol as the sole carbon source
(Chang et al. 2013). Addition of trace elements such as manganese, iron, cobalt, nickel,
copper, zinc and molybdenum reportedly improved growth for some thraustochytrid species
(Nagano et al. 2013). The use of bread crumbs as an alternate carbon source for the
production of lipids under static fermentation conditions was studied for Thraustochytrium
sp. AH-2 and Schizochytrium sp. SR21 (Thyagarajan et al. 2014). DHA production was
observed to increase in Schizochytrium sp. S31, with high oxygen transfer on fermentation
with glycerol (Chang et al. 2013). An aeration-enhanced membrane reactor (AEMR) was
built and used to control the dissolved oxygen for the fermentation of Schizochytrium sp. S31
in a two-stage mixed culture mode to increase productivity of DHA (Zhang et al. 2013).
Extensive research exists on the various carbon sources and additives available for the
production of DHA, whereas the impact on carotenoid yield has received relatively little
attention. In the present study, we studied the effects of flaxseed oil and -linolenic acid
(ALA) (C18:3n3) on the fermentation profile of Schizochytrium sp. S31, particularly lipid
biosynthesis and carotenoid accumulation.
3. 2 Experimental section
3.2.1 Chemicals
All chemicals used in the study were of analytical and HPLC grade. Fatty acid methyl ester
used in the experiment C14:0, C16:0, C18:0, C18:1n9, C18:3n3 and C19:0 were procured
from Nu-Chek-Prep, Inc (Minnesota, USA) and Sigma-Aldrich (Sydney, Australia).
Fermentation medium components including glucose, mycological peptone and yeast extract
were obtained from Sigma-Aldrich (Sydney, Australia), while sea salt was obtained from
Chapter 3
42
Instant Ocean (Virginia, USA). Flaxseed oil was purchased from a local market (Geelong,
Australia). Methanol, ethyl acetate and acetone used for carotenoid extraction and HPLC
analysis were procured from Fischer and Honeywell (Victoria, Australia). Astaxanthin
standard was procured from CaroteNature, Switzerland.
3.2.2 Fermentation medium preparation
Schizochytrium sp. S31was procured from the American Type Culture Collection (ATCC®
20888™, Manassas, VA, USA). The culture was grown and retrieved in liquid medium (790
By+ Medium) specified by the ATCC. Cultures were maintained in agar plates and
subcultured every 15 days. Growth medium consisted (g/L) 10 of glucose, 1 of mycological
peptone, 1 of yeast extract dissolved in 70% artificial sea water (ASW), at pH 7 (prior to
autoclaving). Medium was autoclaved at 121°C for 20 mins. Optimum growth temperatures
of 20ºC were maintained with 150 rpm shaking speed. Fermentation medium components
were inoculated with agar plate culture and grown for 72 h as seed culture. Seed culture
inoculum (5% v/v) was used to inoculate the fermentation medium. The medium components
without either flaxseed oil or ALA were used as control medium. Flaxseed oil and ALA are
used at concentration of 0.04% (w/v) in the medium. Results are reported as the average of
triplicate experiments along with standard deviation (SD).
3.2.3 Growth profile study
The fermentation was conducted over 7 days. At each 24h interval, growth of the organism
was measured at 660 nm. Rate of glucose depletion was measured using 3,5dinitrosalicylic
acid by measuring the optical density at 540 nm. .
3.2.4 Cell dry weight, total lipid and fatty acid study
To monitor the fermentation profile, the culture was harvested at 24 h intervals and
centrifuged at 4000 rpm for 20min to obtain cell pellet, followed by distilled water washing
to remove medium components. The cell pellet was freeze dried and stored at -20ºC before
fatty acid extraction. Biomass was estimated after freeze drying the thraustochytrid cells.
Chapter 3
43
Total lipid content was measured after extraction of lipids from the cell (as explained in the
GC procedure).
3.2.5 Nile red staining
Axio-imager, Zeiss (Oberkochen, Germany) was equipped to conduct fluorescence
microscopy studies, with an epi-illumination using an HBO 200 high pressure mercury light
source. Nile red fluorescence was viewed using a 450-500 nm band pass exciter filter. Nile
red dye was prepared at 10 g/ml concentration in acetone. Staining was carried out with
defined cell concentrations to observe golden yellow colour fluorescence from the cells that
corresponds to the lipid content (Chen et al. 2009).
3.2.6 Fatty acid extraction and Gas chromatography (GC) analysis
Fatty acid extraction was performed as previously described with some modifications
(Christie & Xianlin 2010). In brief, 10 mg of freeze dried cells were used for lipid extraction.
Fatty acids were extracted with a mixture containing a 2:1 ratio of chloroform to methanol
and repeated 3 times. For trans-esterification, 1ml toluene was added followed by addition of
200 μl of internal standard, methyl nonadecanoate (C19:0) and 200 μl of butylated
hydroxytoluene (BHT). Acidic methanol (2ml) was also added to the tube and kept for
overnight incubation at 50°C. Fatty acid methyl esters (FAMEs) were extracted into hexane.
The hexane layer was removed and dried over sodium sulphate. FAMEs were concentrated
using nitrogen gas prior to GC analysis (Ackman 2002). The samples were analysed by a GC-
FID system (Agilent Technologies, 6890N, Santa Clara, CA, USA). The GC was equipped
with a capillary column (Suplecowax 10, 30 m x 0.25 mm, 0.25 μm thickness). Helium was
used as the carrier gas at a flow rate of 1.5 ml/min. The injector was maintained at 250°C and
a sample volume of 1 μl was injected. Fatty acids were identified by comparison to external
standards (Sigma-Aldrich, Sydney, Australia). Peaks were quanti ed with Chemstation
chromatography software (Agilent Technologies, Santa Clara, CA, USA) and corrected using
theoretical relative FID response factors (Kitahara 1984).
Chapter 3
44
3.2.7 Astaxanthin extraction and HPLC analysis
Carotenoids were extracted by adding acetone as previously described (Armenta et al. 2006),
with slight modifications. To 25 mg of freeze dried cells, 1ml of acetone was added and
vortexed for 5 mins, followed by centrifugation at 4000 rpm for 15 mins. This step was
repeated until the biomass became colourless. All supernatant was collected and stored at
15°C in dark conditions. Final extract volumes were noted for total carotenoid estimation. 1
ml acetone extract was removed and evaporated under nitrogen gas followed by addition of 1
ml mobile phase and subsequently syringe filtered (0.45 m) prior to HPLC analysis of the
extracts. The Agilent 1200 Series HPLC system with the Agilent 1200 Series photodiode
array detector was used for carotenoid analysis. Carotenoids were analysed at 477 nm using 5
m Luna C18 reversed-phase column, 4.6 mm x 250 mm (Phenomenex, Torrance, USA), and
a security guard column C18, 3.0 mm x 4.0 mm, (Phenomenex, Torrance, USA). This
column was equilibrated with mobile phase A consisting of methanol, ethyl acetate and water
(88:10:2, v/v/v) in a gradient mode at a flow rate of 0.75 mL min-1. This flow rate was
maintained for 10 min. Mobile phase composition was changed to 2:50:48 (mobile phase B)
between 10 and 30 min and the flow rate was adjusted to 1.5 mL/min. These conditions were
maintained for a further 5 min and then set for column washing with mobile phase A for 10
min. The calibration curve for astaxanthin was prepared with the standard and used for
identification from different extracts.
3.3. Results and discussion
3.3.1 Impact of flaxseed oil and ALA on the fermentation profile of Schizochytrium sp.
S31.
To determine the impact of added flaxseed oil and ALA on the fermentation profile of
Schizochytrium sp. S31, cultures were grown for 7 days in fermentation medium containing
glucose as the sole carbon source (control), and growth was compared with that of this strain
in medium with added flaxseed oil and ALA. Control and flaxseed oil combinations showed
similar rates of glucose depletion, whereas medium with ALA showed slower rates of
glucose depletion from day 1 to day 3, but showed no difference from the control or flaxseed
oil fermentations after day 4 (Figure 3.2). Fermentation for five days has been shown to result
Chapter 3
45
in maximum glucose depletion previously for this strain (Mendes-Pinto et al. 2001; Wu et al.
2005). For all three cases biomass, as measured by optical density at 660 nm, increased from
day 3 to day 7, with added ALA slightly inhibiting biomass production, particularly at day 6
and 7 (Figure 3.3).
Schizochytrium sp. S31 grown with ALA showed increased rate of lipid accumulation as the
biomass increased, whereas with added flax seed oil resulted in reductions in lipid content but
increases in biomass. Oleaginous organisms utilise their own lipid reserves under carbon
limitations and so this is likely to be occurring from day 4 with the S31 strain (Holdsworth &
Ratledge 1988). In a previous study, when Schizochytrium sp. S31 was supplemented with
ALA in the free fatty acid form, growth was increased at least for the single time point
analysed in that study (Lippmeier et al. 2009).
Figure 3.2 Growth profile of Schizochytrium sp. S31 Optical density is shown with closed squares and glucose consumption with closed triangles.
0.00
2.00
4.00
6.00
8.00
10.00
12.00
0
2
4
6
8
10
12
0 1 2 3 4 5 6 7
OD@
660n
m
Glucose(g/L)
Incubation time (days)
Control
ALA
Flax seed oil
Control
ALA
Flax seed oil
Chapter 3
46
In our investigation ALA appears to inhibit both biomass and lipid production in the first 2
to 3 days of fermentation, but doesn’t result in lower biomass or lipid after day 5 (Figure
3.3). Addition of either ALA or flaxseed oil resulted in higher lipid levels after day 5
(Table 3.1). A previous study with Schizochytrium limacinum SR21 indicated that flaxseed
oil when used as the primary carbon source increased both biomass and total lipid content
during fermentation (Yokochi et al. 1998). Lipid staining with Nile red dye was used to
visualize lipid changes via direct microscopic observations of lipids in the cells. Flaxseed
oil resulted stronger cell fluorescence than cells fermented with ALA, indicating a higher
level of cellular lipid due to addition of flaxseed oil (Figure 3.4).
Figure 3.3 Fermentation profile of Schizochytrium sp. S31exhibiting biomass and lipid content. Biomass is shown with closed triangle and lipid content with closed diamonds.
0
500
1000
1500
2000
2500
3000
3500
4000
4500
0 1 2 3 4 5 6 7
Biom
ass(mg/L)
Lipid(m
g/L)
Incubation time (days)
Biomass control
Biomass flax seed oil
Biomass ALA
Lipid control
Lipid flax seed oil
Lipid ALA
Chapter 3
47
Table 3.1 Fermentation profile of Schizochytrium sp. S31 in a medium containing flax seed oil and ALA .
Control Flaxseed oil ALA
Day Biomass (mg/L)
Lipid(mg/L)
DHA (mg/L)
Biomass (mg/L)
Lipid(mg/L)
DHA (mg/L)
Biomass (mg/L)
Lipid(mg/L)
DHA (mg/L)
0 146±9.4 10±2.0 6±1.9 246±18.5 44±3.7 6±1.2 346±67.9 84±14.8 10±4.4
1 920±56.5 101±6.1 25±2.2 1026±37.7 93±12.9 26±2.8 513±60 56±7.6 7±1.4
2 1840±58.8 573±18.4 169±7.5 1893±33.9 518±35.7 160±13.7 806±270 79±1.6 10±5.0
3 2966±33.9 1237±105 263±3.9 2813±18.5 950±81.9 211±19.6 2333±120 300±19.2 70±20.4
4 3506±33.9 1472±3.7 326±2.6 3426±92.85 1551±74.1 364±9.4 3660±141.2 1014±54.2 259±4.9
5 3533±33.9 628±20.4 127±7.2 3426±94.2 958±102.3 213±25.9 3866±18.5 1270±50.5 247±10.9
6 3800±48.9 871±102 185±25.7 3686±18.8 949±213.4 245±34.6 3922±68.61 1393±74.4 295±11.1
7 3386±41.0 816±54 176±10.5 3433±47.1 1383±62.28 324±14.9 3713±57.6 1514±42.4 293±3.3
3.3.2 Fatty acid profile of Schizochytrium sp. S31
The fatty acid profile of Schizochytrium sp. S31 fermented with control medium had C16:0 as
the major fatty acid and DHA as the second most abundant fatty acid. Lower amounts of C18
fatty acids and traces (less than 1%) of C15:0 and C20:5n3 were also present. Maximum
DHA accumulation was observed on day 4. The fatty acid profile of the flaxseed oil was
analysed prior to adding it to the fermentation medium. Raw flaxseed oil used in this study
contained C16:0 at 5.16%, C18:1n9 at 18.80%, C18:2n6 at 15.14%, C18:3n3 at 52.58% and
other fatty acids at 8.32%.
Chapter 3
48
Figure 3.4 Nile red staining of Schizochytrium sp. S31 Scale bar 20 m.Images were recorded on Axio imager microscope. Nile red fluorescence from microbial cells was viewed using 450-500 nm band pass exciter filter. Schizochytrium sp. S31 cells were grown in medium containing flaxseed oil (a) and ALA (b) for 4 days. Table 3.2 Comparison of fatty acid profile on feeding flaxseed oil and ALA to Schizochytrium sp. S31.
C14:0, myristic acid; C16:0, palmitic acid; C18:0, stearic acid; C18:1n9t, elaidic acid; C18:1n9c oleic acid; C18:2n6c, linoleic acid; C18:3n3, ALA; C20:5n3, EPA; C22:5n6, DPA; C22:6n3, DHA. The fatty acid content was recorded on day 4 of fermentation.
FA Control Flax seed oil ALA
mg/L mg/g mg/L mg/g mg/L mg/g
C14:0 277±0.7 79.2±1.0 295.4±18.8 86.2±5.4 178.2±33.2 47.4±8.8
C15:0 7.9±0.2 2.3±0.1 8.3±0.6 2.4±0.2 3.9±1.0 1±0.3 C16:0 348.2±3.2 99.3±1.5 389.4±18.1 113.7±5.7 246.4±37.3 65.5±9.9 C16:1n7 272±0.7 77.6±0.6 292.4±18.0 85.4±5.2 163.4±33.8 43.4±9.0 C18:0 11±0.2 3.2±0.1 12.8±0.6 3.7±0.2 7±1.1 1.9±0.3 C18:1n9 t 0.00 0.0 9.1±1.1 2.7±0.4 0.0 0.0 C18:1n9 c 70.3±1.5 20±0.5 76.1±5.0 22.2±1.4 41.4±8.2 11±2.2 C18:2n6c 0 0 6.4±1.9 1.9±0.3 0 0 C18:3n3 0 0 21.4±3.7 6.3±1.3 23.9±14.0 6.4±3.7 C20:5n3 9.4±0.1 2.7±0.2 10.8±0.3 3.1±0.2 6.2±0.8 1.7±0.2 C22:5n6 58.2±0.9 16.6±0.3 65±1.8 19±0.9 39.3±7.5 10.4±2.0 C22:6n3 326.5±2.7 93.1±1.5 364.9±9.4 106±4.4 259±4.9 55.9±11.7
a b
Chapter 3
49
After fermentation in the presence of flaxseed oil both C18:2n6 and C18:3n3 were present in
the fatty acids extracted from the cells, and these were absent from the control fermentation
(Table 3.2). Other fatty acid levels were also generally increased for the flaxseed oil
fermentation, particularly DHA which increased from 326 to 364 mg/L (Table 3.2). In
contrast, fermentation with medium containing ALA showed a generally lower level of fatty
acids, as compared to the control at day 4. However, with ALA fermentation ALA was
observed in the cells, although DHA levels decreased versus the control (Table 3.2).
A previous study with Schizochytrium limacinum SR21 reported that flaxseed oil as a carbon
source promoted growth without any significant increase in DHA (Yokochi et al. 1998),
while a different study on Schizochytrium sp. SP1 showed that flaxseed oil increased DHA in
that strain (Gaffney et al. 2014). In the current study the addition of flaxseed oil increased
total lipid and fatty acid content. As an adaptive response to growth conditions, the organism
appears to incorporate preformed C18:1n9t, C18:2n6, C18:3n3 from flaxseed oil. The fatty
acid profiles for the control, the flaxseed oil and the ALA added samples are shown in Figure
3.4 for days 0 to 7.
Experiments were also carried out with other free fatty acid including C14:0, C16:0, C18:0,
C18:1n9 added into the fermentation medium (data not shown). In all cases, we observed
increased biomass and corresponding increases in lipid and DHA, but no increase in lipid as a
percentage of biomass. The fatty acid profiles were similar to those observed without addition
of these fatty acids, indicating that they are being utilized as a carbon source.
These fatty acids are probably not incorporated into the PUFA biosynthetic pathway in
Schizochytrium sp. S31 since the PUFA synthase pathway does not normally released into the
medium or utilize them from the medium, so as to improve energy efficiency during PUFA
biosynthesis (Nagano et al. 2011; Ratledge 2004). ALA was also not directly incorporated as
precursor for PUFA biosynthesis and actually inhibited fermentation in the earlier stages.
Flaxseed oil appears to act both as a carbon source and a source of ALA for growing cells.
Chapter 3
50
Figure 3.5 Fatty acid profile of Schizochytrium sp. S31 (a) Fatty acid profile of control, (b) Fatty acid profile of medium with C18:3n3, (c) Fatty acid profile of medium with flaxseed oil, (d) DHA profile on fermentation C14:0, myristic acid; C16:0, palmitic acid; C18:0, stearic acid; C18:1n9t, elaidic acid; C18:1n9c oleic acid; C18:2n6c, linoleic acid; C18:3n3, -linolenic acid; C20:5n3, EPA; C22:5n6, DPA; C22:6n3, DHA.
Chapter 3
51
3.3.3 Carotenoid Profile
Schizochytrium sp. S31 produces significant quantities of astaxanthin (Gupta et al. 2013).
Astaxanthin is normally produced commercially from the microalgae Haematococcus
Pluvialis, where fatty acid levels correlated with astaxanthin accumulation in oil globules
(Zhekisheva et al. 2005), indicating that increased oil levels in other organisms may also
result in increased carotenoid levels. In Chlorella zofingiensis astaxanthin accumulation also
correlated positively with oil levels (Liu et al. 2011). In a study of the microalgae Dunaliella
bardawil -carotene levels decreased when triacylglycerol synthesis was blocked (Rabbani et
al. 1998). In our study, the control, flax seed oil and ALA medium resulted in 42.38 g/g,
42.51 g/g and 42.03 g/g of astaxanthin, respectively. Astaxanthin levels correlated with lipid
levels (Figure 3.6). Flax seed oil increased lipid levels, promoting astaxanthin accumulation
in the oil globules.
Figure 3.6 Astaxanthin and lipid yields in control, flaxseed oil added and ALA added fermentations.
250
300
350
400
450
500
40.5
41
41.5
42
42.5
43
ALA control Flaxseed oil
Lipid(m
g/g)
Astaxanthin(g/g)
Astaxanthin profile Lipid profile
Chapter 3
52
3.4 Conclusions
Flaxseed oil added in the fermentation medium for Schizochytrium sp. S31fermentation
resulted in increased lipid productivity, higher DHA levels and increased astaxanthin
accumulation. However, addition of ALA alone inhibited lipid, DHA and astaxanthin
production. The addition of flaxseed oil at low levels in the fermentation of Schizochytrium
sp. S31 is an effective method to enhance total lipid and DHA production, while also
increasing astaxanthin accumulation in this strain.
Chapter 4
Chapter 4: Isolation and characterisation of a new squalene producing Thraustochytrium strain (DT8)
Chapter 4
54
4.1 Introduction
Squalene is a bioactive triterpene formed from six isoprene units, with the chemical formula
C30H50 (Kim & Karadeniz 2012). Structurally, squalene is similar to carotenoids, vitamin A,
vitamin K, vitamin D, vitamin E, tocopherols and other cyclic terpenoid compounds, sharing
prenyl units in the structure as a common feature for structural similarity (Kelly 1999).
Squalene is widespread in nature, with major sources including deep sea shark liver oil and
vegetable oil. Among vegetable sources olive oil is the richest source of squalene (Kim &
Karadeniz 2012).
Figure 4.1 Chemical structure of squalene (Figure modified and reproduced from Kelly 1999)
Squalene intake is associated with various beneficial effects on human health. Its tumour
inhibiting activity is documented in human and animal studies (Rao et al. 1998). Ancient
Japanese called shark liver oil “Samedawa” or cure-all (Solomon & Joelsson 1997;
Vannuccini 1999). They used it as a medicine, considering the oil to be a rich healing
substance for a wide range of diseases ranging from constipation to cancer. China recorded
the medicinal benefits of shark liver oil in their ancient pharmaceutical book
“Honzukomuko” (Solomon & Joelsson 1997; Vannuccini 1999). Dr Mitsumaro Tsujimoto
first purified squalene from the liver of shark Squalus spp (Kim & Karadeniz 2012). In 1936,
Nobel prize winner Paul Karrer described the structure of squalene, which was similar to the
strong antioxidants vitamins E and A and helped to elucidate the antioxidant activity of the
compound (Das 2000; Ramirez-Torres et al. 2011). The ability to attack pathogens and leave
skin flora microbes intact led to its use in the treatment of bacterial and fungal infections of
the skin (Reddy & Couvreur 2009). The cosmetic industry is a major consumer of squalene,
using it as a moisturising agent and emollient (Kim & Karadeniz 2012). Industrial potential
Chapter 4
55
and limitations on the availability of shark liver oil has directed research towards alternative
sources for squalene production (Bhattacharjee et al. 2001).
Figure 4.2 Thraustochytrids; Marine microbial cell factory for nutraceuticals & bioactives production
The production of squalene from oleaginous microorganisms is a potentially sustainable and
economical alternative to extraction from shark liver. The biosynthetic pathway for squalene
varies with producing organism, but farnesyl pyrophosphate (FPP) is the starting compound
in the synthesis of squalene in all organisms (Naziri et al. 2011). The yeast Pseudozyma sp.
JCC207 was reported to accumulate squalene at 340.52 mg/L (Chang et al. 2008).
Thraustochytrids have been recently identified as one of the most promising microbes for the
production of squalene, which can be a co-product in the production of polyunsaturated fatty
acids and carotenoids (Li et al. 2009). Culture conditions that influence squalene and sterol
Extracellular polysaccharides(EPS)
Enzymes (Cellulase, Lipase)
Carotenoids
Squalene
Omeg-3 fatty acids Biodiesel
Chapter 4
56
production were studied in some thraustochytrid strains (Lewis et al. 2001).
Aurantiochytrium sp. 18W-13a was found to accumulate 171 mg/g squalene (Nakazawa et al.
2012). Table 4.1 summarises squalene production reported for various microorganisms. In the
present study described in this chapter, we carried out the isolation of thraustochytrids from
Australian mangrove sites and the isolates were screened for squalene production and
glycerol was investigated as a carbon source to replace glucose, for squalene, oil and
astaxanthin production.
Table 4.1 Comparative squalene productivity for various microorganisms, including Thraustochytrium DT8.
Strain Squalene content Reference
Aurantiochytrium sp. BR-MP4-A1 0.18 a Li et al., 2009
Schizochytrium mangrovei FBI 0.162 a Jiang et al., 200
Aurantiochytrium sp. 18W-13a 171 a Nakazawa et al., 2012 Thraustochytrid strain ACEM 6063 (Identified as genus Schizochytrium) 1.8 a Lewis et al., 2001
Thraustochytrium ATCC 26185 0.2 a Lu et al., 2003
Our Isolate Thraustochytrium sp DT8 6.7 a 16.92 b Present study
Saccharomyces cerevisiae 41.16 c Bhattacharjee et al., 2001
Torulaspora delbrueckii 237.25 c Bhattacharjee et al., 2001
Pseudozyma sp. JCC207 340.52 b Chang et al., 2008
Schizochytrium sp. PQ6 33.04 a Hoang et al., 2014
a mg/g ; b mg/L ; c g/g
Chapter 4
57
4.2 Experimental section
4.2.1 Chemicals
All chemicals used were of analytical and HPLC grade. Medium components including
glucose, glycerol, yeast extract, and mycological peptone, together with squalene and
astaxanthin standards, were purchased from Sigma-Adrich (Sydney, Australia) and sea salt
from Instant Ocean (Blacksburg, USA). Chloroform, methanol, hexane and acetone were
purchased from Merck (Melbourne, Australia), while acetonitrile, methanol and ethyl acetate
were purchased from Fischer and Honeywell (Melbourne, Australia).
4.2.2 Isolation of thraustochytrids
Thraustochytrid isolates designated as DT2, DT3, DT7, DT8, and DT13 were isolated from
Barwon mangrove site in Victoria, Australia. The GPS positions (S 38°15.922´ E
144°29.771´) were recorded. The samples were placed in sterile 50 mL falcon tubes
containing 500 mg L-1 of penicillin and streptomycin. The tubes were moderately agitated
for uniform dispersal of the antibiotics. Sample tubes were placed in an ice box and
transported to the laboratory and stored at 4°C until further use. Thraustochytrids were
isolated using the following techniques.
4.2.2.1 Direct plating technique
Collected seawater (3 mL) and sediment (1 g) samples, were suspended in 20 mL of yeast
extract-peptone medium (YP) (1 g L-1 yeast extract, 1 g L-1 peptone), To the autoclaved and
cooled medium, antibiotics and antifungal agents (500 mg L-1penicillin, 500 mg L-1
streptomycin, 50 mg L-1 rifampicin and 10 mg L-1 nystatin in100 % sterile seawater) were
added after pre-filtering using a 0.22 m sterile filter just before pouring of the agar plates. A
portion of the suspension culture, with appropriate antibiotic dilutions, was spread plated on
YP plates containing the above mentioned antibiotic concentration and incubated at 20°C for
5 days. The leaf samples were washed with sterile sea water to remove any particulates,
which can be a source of contaminants. The leaf sample was then inoculated onto the YP
plates containing the same antibiotic concentrations. Growth on the plates was monitored at
Chapter 4
58
24 hour intervals. Different colonies, identified by their morphology as thraustochytrid-like,
were taken using a sterile loop and re-grown in a 50 mL Erlenmeyer flask with 10 mL of
fresh medium. Spherical, uneven, and slimy colonies along with some translucent colonies
were selected for further study. Pure colonies were obtained by the streak plate method.
4.2.2.2 Pollen baiting technique
The falcon tubes containing the seawater samples were pollen-baited with sterile pine pollen
grains (Allife, Silverwater, NSW, Australia). The same concentrations of antibiotic and
antifungal agents were added to the tubes as in the direct plating method. The tubes were
incubated at 20°C for 2 weeks. Sub-samples (10 L) were removed from the water surface
and observed under the microscope at 24 h intervals to check for the appearance of
thraustochytrid colonies growing on the pollen grains. Tubes showing a thin film over the
water level were checked for contamination under a microscope and when microorganisms
other than thraustochytrids were observed, they were discarded. Subsamples (20 L) of
thraustochytrid cells and pine pollen were spread-plated on YP agar medium containing 1 g
L-1 yeast extract, 1 g L-1 peptone, 10 g L-1 agar and antibiotic and antifungal agents in 100 %
sterile seawater. Plates were incubated at 20°C for one week. Thraustochytrid colonies were
sub-cultured on YP agar medium containing antibiotics to obtain pure isolates.
4.2.3 Microscopy studies
The isolates were observed using differential interference contrast (DIC) and Nomarski
microscopy using an Axio-imager. A1 microscope equipped with Axiovision software (Zeiss,
Oberkochen, Germany). Thraustochytrid cells were fixed on a slide and air-dried before
observation under the microscope.
4.2.4 DNA extraction and 18S rRNA sequencing
Thraustochytrid-like isolates identified by microscopical examination were further confirmed
by DNA extraction and 18S rRNA sequencing. 3 mL sub-sample of a 7 day old
thraustochytrid culture was centrifuged at 8,000 x g for 10 min at 4°C to obtain a cell pellet.
The total genomic DNA of the isolates was isolated and purified using PureLink Genomic
Chapter 4
59
DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions.
The extracted DNA was kept at -20°C for further use. DNA was amplified by polymerase
chain reaction (PCR) on a Thermal cycler (Eppendorf Master Cycler, Foster city, CA, USA).
The PCR strategy was modified from Mo et al. 2002. The amplification of 18S rRNA gene
from the extracted DNA was performed with the primers F-5'-
CAACCTGGTTGATCCTGCCAGTA-3' andR-5'TCACTACGGAAACCTTGTTACGAC-3'
(Burja et al. 2006). A 25 L PCR reaction mixture containing 200 M of master mix (ready
to use solution containing Taq DNA polymerase, deoxynucleotide triphosphates (dNTPs),
MgCl2, Promega, Madison, WI, USA) and 1.25 M of forward and reverse primers was used
to amplify 200 ng of DNA template. The conditions for the PCR amplification were initial
denaturation (3 min at 94°C), final denaturation (45 s at 94°C) with annealing (30 s at 64°C),
extension at 72°C for 2 min followed by final extension at 72°C for 10 min. PCR was run for
30 cycles. The amplified DNA was purified from the agarose gel using the Wizard PCR kit
(Promega, Madison, WI, USA) according to manufacturer’s instructions. Collected DNA was
stored at -20°C for further use. DNA concentration was measured by Nano drop 1000
Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The purified DNA of the
selected isolates DT2, DT3, DT7, DT8 and DT13 were sequenced using a DNA sequencer
(Applied Biosystems, Foster city, CA, USA).
4.2.5 Phylogenetic analysis
The five potential thraustochytrids isolated in the lab were selected for molecular
characterization followed by phylogenetic analysis. PCR amplified 18S rRNA was sequenced
and validated after BLAST analysis (Altschul et al. 1990) and found to be closely related to
thraustochytrid species. All sequences were submitted to Genbank with accession numbers.
18S rRNA sequences from related species were outsourced/retrieved from Genbank
databases and used as reference sequences for phylogenetic studies. The phylogenetic tree
was constructed using MEGA5 software (Tamura et al. 2011).
4.2.6 Microbial growth for squalene production
Thraustochytrid in-house isolates DT2, DT3, DT7, DT8 and DT13 in addition to American
Type Culture Collection (ATCC, Manassas, VA, USA) cultures, Schizochytrium limacinum
Chapter 4
60
SR21 (ATCC® MYA-1381™, Manassas, VA, USA) and Schizochytrium S31 (ATCC ®
20888™, Manassas, VA, USA) as standard cultures were selected and used in the study.
Isolates used in the study were maintained on GYP medium consisting of (g L-1); glucose (5),
yeast extract (2), mycological peptone (2), agar (10) and artificial sea water (50%) at 25°C.
Strains were subcultured after 15 days. All the isolates were cultivated in the seed medium
(1% glucose, 0.1% yeast extract, 0.1% mycological peptone, ASW 50%) for 3 days and
production medium (2% glucose, 0.2% yeast extract, 0.2% mycological peptone, ASW 50%)
for 5 days. Seed medium was inoculated from agar plates and grown in a shake flask at 25°C
and 200 rpm shaking speed. Inoculum 5% (v/v) was used to inoculate production medium,
which were then cultured for 5 days in a shake flask at 25°C at 200 rpm. The resultant
biomass was harvested by centrifugation (10,000 x g, 15 mins) and freeze dried until further
screened for squalene production.
4.2.7 Comparison of glycerol and glucose as a carbon source for squalene production
Thraustochytrid isolate DT8 was grown with glucose or glycerol medium to compare relative
squalene production levels. Medium composition of seed medium and production medium
was as described above, except that the carbon source was either glucose or glycerol at
concentrations of 3%, 5% and 7% in the starting medium. The resultant biomass after 7 days
fermentation was harvested by centrifugation (10,000 x g, 15 mins) and freeze dried to enable
quantitation of squalene.
4.2.8 Squalene estimation
Freeze dried cells (30 mg) were saponified in 0.5 ml of 50% (w/v) KOH and 2 ml of 95%
(v/v) ethanol at 60°C for 1 h and extracted three times with 2 ml hexane. The extract was
dried under nitrogen and the residue dissolved in the mobile phase (acetonitrile) and filtered
before HPLC injection (Kovacs et al. 1979). An Agilent 1260 Infinity Series HPLC system
with a photodiode array detector was used for squalene analysis. The analyses were
performed using a Kinetex 2.6u XB-C18 100A column (150 × 4.6 mm) procured from
Phenomenex, Australia. Acetonitrile (100%) was used as the mobile phase at a flow rate of
1.5 ml min-1. Column temperature was maintained at 30°C. Analytes were monitored with a
UV detector at a wavelength of 195 nm. Identification of squalene was based on UV spectra
Chapter 4
61
scanned in the 190-400 nm range at 1.2 nm sampling intervals and compared to a known
standard. The samples were filtered through a 0.45 m Millipore filter membrane before
injection (Pereira et al. 2004).
4.2.9 Effect of carbon sources glucose and glycerol on astaxanthin accumulation
Thraustochytrid isolate DT8 was grown with the medium composition as described in section
2.2, with glucose or glycerol at 2% (w/v) as carbon source. Cultures were grown for 5 days in
a shake flask at 25°C at 200 rpm. The resultant biomass was harvested by centrifugation
(10,000 x g, 15 mins) and freeze dried before being analysed for astaxanthin content.
4.2.10 Lipid extraction and fatty acid profile of strain DT8
Strain DT8 was cultured with production medium (3% glycerol, 0.3% yeast extract, 0.3%
mycological peptone, ASW 50%) for 6 days. The resultant biomass was harvested by
centrifugation (10,000 x g, 15 mins) and freeze dried before determination of fatty acid
profile. Fatty acid extraction was performed following the method of Lewis et al. with some
modifications (Lewis et al., 2000). 10 mg of freeze dried cells were used for lipid extraction.
Fatty acids were extracted with a mixture containing a 2:1 ratio of chloroform to methanol
and this was repeated 3 times. For trans-esterification, 1 ml toluene was added followed by
addition of 200 μl of the internal standard, methyl nonadecanoate (C19:0; 5mg/ml), and 200
μl of butylated hydroxytoluene (BHT; 1mg/ml). Acidic methanol (2 ml) was also added to
the tube and this was incubated overnight at 50°C. Fatty acid methyl esters (FAMEs) were
extracted with hexane and the hexane layer removed and dried over sodium sulphate. FAMEs
were concentrated using nitrogen prior to GC analysis (Christie and Han, 2010).
4.2.11 Gas chromatographic analysis
The samples were analysed using a GC-FID system (Agilent Technologies, 6890N,
Australia). The GC was equipped with a capillary column (Suplecowax 10, 30 m × 0.25 mm,
0.25 μm film thickness). Helium was used as the carrier gas at a flow rate of 1.5 ml min-1.
The injector was maintained at 250°C and a sample of 1 μl was injected. Fatty acids were
identified by comparison to external standards (Sigma-Aldrich, Sydney, Australia). Peaks
Chapter 4
62
were quanti ed with Chemstation chromatography software (Agilent Technologies) and
corrected using theoretical relative FID response factors (Ackman 2002).
4.2.12 Astaxanthin extraction and HPLC quantification
Carotenoids were extracted by adding acetone as per the method of Burja et al. with slight
modifications (Burja et al. 2006). 1 ml of acetone was added to 25 mg of freeze dried cells,
which were then vortexed for 5 mins, followed by centrifugation at 4000 rpm for 15 mins.
This step was repeated until the biomass became colourless. All supernatant was collected
and stored at 15°C in the dark. Final extraction volumes were noted for total carotenoid
estimation. 1 ml of acetone extract was removed and evaporated under nitrogen followed by
addition of 1 ml mobile phase and this was syringe filtered (0.45 m) prior to HPLC analysis.
The Agilent 1260 Infinity Series HPLC system with photodiode array detector was used for
carotenoid analysis. Carotenoids were analysed at 477 nm using a 5 m Luna C18 reversed-
phase column, 4.6 mm × 250 mm (Phenomenex, Lane Cove, Australia), and a security guard
column C18, 3.0 mm × 4.0 mm, (Phenomenex). The column was equilibrated with a mobile
phase consisting of methanol, ethyl acetate and water (88:10:2, v/v/v) at a flow rate of 0.75
mL min-1. 10 μL of sample was injected, and this mobile phase composition was maintained
for 10 min. Mobile phase composition was changed to 48:50:2 between 10 and 30 min and
the flow rate was adjusted to 1.5 mL min 1 (Armenta et al. 2006). These conditions were
maintained for a further 5 min. After each analysis the column was re-equilibrated at the
initial mobile phase composition for 10 min. The calibration curve for astaxanthin was
prepared with appropriate standards and used for identification and quantification in different
extracts.
4.3 Results and discussion
4.3.1 Isolation and identification of thraustochytrid isolates
Mangrove marine water and sediment samples were processed by direct plating and pine
pollen techniques to isolate thraustochytrids under optimum conditions and they were
identified by 18S rRNA sequencing. Isolates designated as DT2, DT3, DT7, DT8 and DT13
sequences were deposited in the GenBank database and have been assigned the accession
numbers, DT2 (Genbank accession number KF682137), DT3 (Genbank accession number
Chapter 4
63
KF682125), DT7 (Genbank accession number KF682129), DT8 (Genbank accession number
KF682130), and DT13 (Genbank accession number KF682135). Isolates such as DT8, DT9,
and DT13 were studied in this work. The reference sequences to the 18S rRNA sequence of
the isolates were obtained from NCBI database and a phylogenetic tree was constructed with
MEGA 5.2 software using the neighbour-joining method (Tamura et al. 2011). Isolate DT81
(see Figure 4.4) studied in the present study was genetically identified as a Thraustochytrium
sp.
Figure 4.3 Australian Victorian mangrove isolates shown at scale bar 20 μm (a) Pine pollen attached with thraustochytrid colonies, (b) & (C) mature thraustochytrid cells releasing spores, (d) & (e) cells representing oil vacuoles, and (f) in-house axenic culture isolated from Victorian mangroves.
.
Chapter 4
64
Figure 4.4 Phylogenetic trees showing the close relationship of the isolates DT8, DT9 and DT13 to related thraustochytrid sequences. 1A joint-collection trip to Barwon heads, Victoria, Australia was carried in September 2012 and help from Adarsha Gupta, Dilip Singh and Avinesh Byreddy in collecting Australian isolates is greatly acknowledged.
Aurantiochytrium sp. 18W-6a (AB811025.1)
Schizochytrium sp. ATCC 20888 (DQ367050.1)
Aurantiochytrium sp. 18W-16a (AB811030.1)
Aurantiochytrium sp. 9W-3a (AB810991.1)
Ulkenia profunda (AB022114.1)
Thraustochytrium sp. AR2-19 (AB810951.1)
DT13
Thraustochytrium aureum (AB022110.1)
Thraustochytrium striatum (AB022112.1)
Thraustochytrium sp. BL3 (FJ821484.1)
Thraustochytrium sp. ONC-T18 (DQ374149.1)
Schizochytrium sp. KH105 (AB052555.1)
DT8
DT9
Aurantiochytrium sp. SEK 218 (AB290573.1)
Aurantiochytrium sp. SEK 218 (AB290573.1)
0.69
0.12
0.26
0.38
0.21
0.00
0.43
0.00
0.00
0.00
0.10
0.12
0.10
0.10
0.00
0.000.00
0.71
0.01
0.29
0.00
0.00
0.23
0.27
0.72
0.22
0.01
0.01
0.05
0.16
0.1
Chapter 4
65
4.3.1 Squalene content of Victorian mangrove isolates grown with glucose
The five isolates from Australian mangrove together with the ATCC culture controls were
grown using heterotrophic fermentation for 5 days with glucose as the carbon source in the
medium to enable comparison of squalene productivity in these strains. All isolates were
found to contain squalene, with strain DT8 showing the highest squalene productivity at 0.8
mg/g (Figure 4.2). The levels for the remaining strains were much lower with DT2 at 0.03
mg/g, DT3 at 0.15 mg/g, DT7 at 0.03 mg/g, DT13 at 0.01 mg/g, SR21 at 0.02 mg/g and S31
at 0.01 mg/g. The productivity of DT8 is higher than that previously reported from related
strains, including Aurantiochytrium sp. BR-MP4-A1 (0.18 mg/g) (Li et al. 2009),
Schizochytrium mangrovei FBI (0.162 mg/g) (Jiang et al. 2004), Thraustochytrium ATCC
26185 (0.2 mg/g) (Lu et al. 2003), Saccharomyces cerevisiae (41.16 g/g) and Torulaspora
delbrueckii (237.25 g/g) (Bhattacharjee et al. 2001). Strain DT8 was selected for further
optimisation studies to maximise sqaulene production.
Figure 4.5 Squalene productivity of Australian mangrove isolates, compared with ATCC controls (Asterics indicates standard ATCC cultures used in this study).
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
DT2 DT3 DT7 DT8 DT13 SR21 S31
Squa
lene
(mg/g)
Thraustochytrid isolates
Chapter 4
66
4.3.2 Squalene productivity for strain DT8 using glycerol as carbon source
To determine whether glucose can be replaced by the lower cost carbon source glycerol,
squalene yields were compared for strain DT8 grown in medium containing glucose or
glycerol. Glucose has been shown to maximise squalene productivity in other strains
(Nakazawa et al. 2012). S. mangrovei PQ6 showed maximum squalene productivity of
33.04 mg/g after fermentation with 9% glucose for 120 h (Hoang et al. 2014).
Thraustochytrids have been shown previously to grow well on glycerol and so this carbon
source was chosen as a low cost source for strain DT8 (Gupta et al. 2013).
Glycerol resulted in maximal biomass of 2.3 g/L on day 7, whilst glucose yielded lower
biomass of 1.3 g/L on day 4 and this level declined until day 7 (Figure 4.3). Squalene
productivity was monitored daily up to day 7 for both glycerol and glucose medium.
Maximum squalene productivity was found to be 2.0 mg/g on day 6 of fermentation with
glucose, and 0.59 mg/g on day 2 with glycerol, decreasing from day 3 to day 7 (Figure 4.4).
A similar trend was previously seen for Aurantiochytrium sp. BR-MP4-A1, where maximum
squalene production occurred at 36 h and then levels declined (Li et al. 2009).
Figure 4.6 Fermentation profile of Thraustochytrium DT8 with 2% glucose or glycerol as carbon source.Dotted line with diamond datapoints represents biomass profile of glycerol, dotted line with square datapoints indicates biomass profile of glucose, solid line with circle datapoints represents growth curve on fermentation with glycerol and solid line with triangle datapoints indicates growth curve of glucose.
0.000.200.400.600.801.001.201.401.601.80
0
0.5
1
1.5
2
2.5
3
0 1 2 3 4 5 6 7
Opticalde
nsity
@66
0nm
Biom
ass(g/L)
Incubation time (Days)
Biomass glycerol Biomass glucoseGlucose growth curve Glycerol growth Curve
Chapter 4
67
Figure 4.7 Squalene productivity of Thraustochytrium DT8 with 2% glucose or glycerol as carbon source. Dotted orange square bar represents sqaulene composition for fermentation with glucose and dotted blue square represents sqaulene compostion for fermentation with glycerol.
4.3.3 Optimisation of squalene production with glucose or glycerol as carbon source
To maximum squalene yield, glucose and glycerol concentrations were optimised in the
fermentation medium for strain DT8. Concentrations at 3%, 5%, and 7% were compared for
glucose and glycerol on day 6 of fermentation. Fermentation with 7% glucose resulted in
biomass of 2.52 g/L and squalene content of 6.7 mg/g (16.92 mg/L). Fermentation with 5%
glycerol gave maximum biomass of 3.5 g/L with squalene content of 0.34 mg/g (Table 4.2).
0.00
0.50
1.00
1.50
2.00
2.50
0 1 2 3 4 5 6 7
Squa
lene
(mg/g)
Incubation time (Days)
Glycerol Glucose
Chapter 4
68
Table 4.2 Effect of carbon source on biomass and squalene production of Thraustochytrium DT8 at various glucose and glycerol concentrations.
Carbon source(%)
Biomass(g/L)
Squalene(mg/g)
Squalene(mg/L)
Glucose 3 1.3±0.0 0.7±0.0 1.0±0.0
5 2.0±0.0 0.9±0.4 1.8±0.5
7 2.5±0.3 6.7±0.0 16.9±1.6
Glycerol 3 3.5±0.3 0.3±0.0 1.1±0.1
5 3.6±1.1 1.0±0.3 3.8±0.2
Glycerol at 7% concentration was not given in the table as the biomass levels were estimated as 0.35g/L which was lower to estimate the squalene accumulation.
A further increase in glycerol concentration did not increase biomass levels, where 7%
glycerol resulted in 0.35 g/L of biomass. Our isolate DT8 exhibited the highest squalene
content among strains from the Thraustochytrium genera. DT8 also showed a higher yield of
squalene as compared to the self-cloned yeast strains Saccharomyces cerevisiae (AM63),
Saccharomyces cerevisiae (AM64) (Mantzouridou & Tsimidou 2010) (Table 4.1).
4.3.3 Fatty acid profile of DT8 grown in glycerol
The fatty acid profile of DT8 was determined after 6 days fermentation with 3% glycerol as
the carbon source. In this study 3% glycerol was chosen for fatty acid analysis as the biomass
levels were comparable to growth achieved using 7% glucose as the carbon source (Table
4.3). The major fatty acids identified in the lipid extract from this sample were C16:0, C18:0,
C18:1n9, C20:4n6, C20:5n3, C22:5n6 and C22:6n3. C16:0 was the major fatty acid at 41.7%
of TFA. C22:5n6 was second most abundant at 20.6% of TFA and C22:6n3 the third most
abundant at 14.4% of TFA (Table 4.3). Strain DT8 was a good PUFA producer. The related
strains Aurantiochytrium sp. BR-MP4-A1 profile (Li et al. 2009) and Schizochytrium sp. PQ6
(Hoang et al. 2014) both produced squalene and had C16:0 and C22:6n3 as the major fatty
acids in their profiles. High levels of C16:0 may have application in biodiesel production
(Gupta et al. 2013), although the presence of PUFA can be problematic due to potential
Chapter 4
69
oxidation and polymerisation. However, if these PUFA can be separated from the saturated
and monounsaturated fatty acids then coproduction of PUFA and biodiesel is possible. Strain
DT8 produces higher levels of both omega-3 DPA and omega-6 DPA, with the level of
omega-6 DPA higher than that observed in other thraustochytrid strains.
Table 4.3 Fatty acid profile of Thraustochytrium DT8 with 3% glycerol as carbon source.
Fatty acid TFA% 1 mg/L
C16:0 41.7±1.2 198±3.0
C18:0 7.8±0.3 36.3±0.0
C18:1n9 8.5±0.6 39.5±0.9
C20:4n6 3.9±0.3 18.1±0.6
C20:5n3 3.2±0.1 14.8±0.0
C22:5n6 20.6±0.3 96.0±3.0
C22:6n3 14.2±2.8 66.7±16.5 Palmitic acid (C16:0); stearic acid (C18:0); oleic acid (C18:1n9); arachidonic acid (C20:4n6); EPA (C20:5n3); DPA (C22:5n6); DHA (C22:6n3),1Total fatty acid percentage (TFA%)
4.3.4 Carotenoid accumulation
Carotenoid analysis of the biomass, from fermentation of DT8 with 2% glucose or 2%
glycerol for 5 days, showed the presence of astaxanthin and traces of zeaxanthin.
Fermentation with glucose gave 44.9 μg/g of astaxanthin, whereas glycerol resulted in a
higher level at 98.2 μg/g astaxanthin. We have previously shown that glycerol can increase
carotenoid levels in other thraustochytrid strains (Gupta et al. 2013). High astaxanthin content
on fermentation with glycerol was achieved because of increased supply of acetyl-CoA or
NADPH. Glycerol as a carbon source metabolised prior to entry in to the glycolytic pathway.
Thus it increases lipid biosynthesis which simultaneously enhances accumulation of
carotenoids as they share same precursor for production (Ratledge 2004; Scott et al. 2011).
Chapter 4
70
4.4 Conclusions
Our isolated Thraustochytrium strain DT8 was found to produce the highest squalene content
among documented Thraustochytrium species, although a Schizochytrium and a
Aurantiochytrium strain have been previously reported that produce higher squalene levels.
Maximum squalene productivity was achieved with medium containing glucose at 7% (w/v).
The use of glycerol increased biomass and fatty acid levels, but resulted in much lower
squalene production. Astaxanthin levels in this strain were increased with glycerol. However,
these are initial studies that needs further process optimisation at bioreactor level to further
improve upon squalene production. This strain may be useful for coproduction of squalene,
astaxanthin, PUFA and biodiesel.
Chapter 5
Chapter 5: Evaluation of Victorian marine isolate Schizochytrium sp. DT9 for biodiesel production
Chapter 5
72
5.1 Introduction:
Biofuel is a sustainable alternate fuel to replace or augment fossil fuel sources (Teo et al.
2014). Urbanisation and global economic development has resulted in increased production
and utilisation of fossil hydrocarbons for lighting, heating and transportation. Fossil fuel
consumption is a requirement for a modern basic standard of living (Chatterjee & Bal 2014).
About 80% of energy requirements through fossil fuels are fulfilled through utilising
petroleum, coal and natural gas (Demirbas & Fatih 2011). Fossil fuel combustion is believed
to be a major contributor to global warming as they release a large amount of carbon dioxide
into the atmosphere (Kirtay 2009). Global demand of fossil fuel, for transportation in
particular, has been increasing year on year, and is expected to increase by an average of
1.8% per year between 2005 to 2035. By 2030 to meet the increasing demand of energy, an
additional 50% supply of energy over the current supply will be required to meet global
demand (Atabani et al. 2012). To address this growing energy need, alternate sustainable
renewable fuel sources need to be identified and developed.
Biofuels are solid, liquid or gaseous fuels that are derived from renewable sources (Demirbas
2009a). These renewable sources include biomass from plants and microorganisms, animal
fats or oil wastes (Demirbas 2009b). Biofuels are nontoxic and carbon neutral fuels as they
release less carbon and other exhaust gases than do fossil fuels (Cucek et al. 2012). Biofuels
are divided in to four generations based on their feed stock material, although in all cases
biodiesel and ethanol are the major biofuels produced (Puri et al. 2012). First generation
biodiesel, was produced through transesterification of oleaginous oil crops or oil waste
(Singh et al. 2014). Oil waste is an inexpensive feed stock for biodiesel production (Demirbas
2009a). A transesterification process was carried through base-catalysed, acid-catalysed,
enzymatic and or supercritical transesterification to convert the triacylglycerides into methyl
esters for biofuel use. Although base catalysis in the presence of methanol is the most
common method used for methyl ester formation the process can result in some free fatty
acid (FFA) and soap formation, which can make the purification process more complex
(Zhang et al. 2003). Acid catalysis helps to prevent FFA formation, but is a more expensive
process (Demirbas 2009a). Continuous transesterification processes can improve cost
effectiveness and require less solvent (Lee et al. 2011).
Chapter 5
73
Fermentable sugars from sugar cane or starch from corn can be used as feed material in the
first generation bioethanol production (Von Blottnitz & Curran 2007). These sugars are
converted into bioethanol and water using yeast enzymes (Patni et al. 2013). Second
generation biofuels are produced from lignocellulose biomass taken directly from agro-forest
residues, grasses and aquatic plants (Limayem & Ricke 2012). Processing of this primary
biomass results in the formation of secondary biomass (Abraham et al. 2013). Post-consumer
residues such as oil lubricants and animal fats are referred to as tertiary biomass. All these
types of biomass require pre-treatment with subsequent physical, thermochemical or
biological conversion to produce biofuel (Naik et al. 2010).
First and second generation biofuel production has the disadvantages of land competition,
biodiversity loss and ecological imbalance (Mata et al. 2010). Feed stock price generally
accounts for about 60-75% of total biofuel cost (Canakci & Sanli 2008). Although low cost
feed stock such as, non-edible oil, domestic cooking oil waste, animal fats, soap- stocks and
greases are cheaper alternative feed stock, they are not available in enough quantities to meet
the required demand (Demirbas & Fatih 2011). Third generation biofuel utilises oleaginous
microalgae which can potentially be grown in almost unlimited amount using fermentation.
The oil is extracted from the biomass and transesterified to produce biodiesel (Mutanda et al.
2011; Varfolomeev & Wasserman 2011). Third generation biofuel has the advantages of easy
cultivation, higher energy yields per hectare, minimal utilisation of fertile land and
production of value added co-products. If large scale fermentation methods can be developed
cost effectively then microalgae offer a better biofuel production method than conversion of
lignocellulose biomass, or food and oil crops (Zhu et al. 2014). In the early 1970s with the oil
crisis Japan started to cultivate the phototrophic microalgae Chlorella, which can convert
solar energy in to chemical energy (Spolaore et al. 2006). The U.S. National Renewable
Energy Laboratory (NREL) played a major role in further development of research from
1978 to 1996 through their Aquatic Species Program (ASP). They substantiated microalgae
as potentially low cost alternative energy sources to fossil fuels. However, further research is
still required to make the process economically competitive (Mata et al. 2010).
The microalgae, Botryococcus braunii (Khatri et al. 2014), Chlorella sp. (Li et al. 2011),
Crypthecodinium cohnii (Couto et al. 2010), Dunaliella sp. (Tang et al. 2011), Isochrysis sp.
(Feng et al. 2011), Nannochloris sp. (Brown et al. 2010), Neochloris oleoabundans (Li et al.
2008), Nitzschia sp. (Ryu & Rorrer 2010), Pavlova lutheri (Meireles et al. 2003),
Phaeodactylum tricornutum (Yu et al. 2009), Porphyridium cruentum (Oh et al. 2009),
Chapter 5
74
Scenedesmus obliquus (Mandal & Mallick 2009), Skeletonema sp. (Popovich et al. 2012) and
Schizochytrium (Johnson & Wen 2009) are some of the oleaginous organisms that have been
explored for the development of cost effective biofuel production. These organisms have oil
accumulation between 20 and 80% of dry biomass. Medium macro and micro nutrients and
fermentation parameters can be optimised to increase total lipid levels in these strains (Mata
et al. 2010; Rawat et al. 2013; Zhu et al. 2014).
Thraustochytrids produce omega-3 fatty acids and other nutraceuticals as co-products, which
can offset the costs of biofuel production (Gupta et al. 2014 ; Lee Chang et al. 2014). To
improve cost and biomass levels, various carbon sources such as bread crumbs (Thyagarajan
et al. 2014), tween 80 (Taoka et al. 2011), glycerol (Lee Chang et al. 2013; Scott et al. 2011),
sorghum juice (Liang et al. 2010) and hydrolysed seed cakes of jatropha plant (Liang et al.
2010b) were tested in the fermentation process. In the work described in this chapter we
investigated the growth of our in-house isolate DT9 by fermenting with glucose and glycerol
as the carbon sources. We also optimised the transesterification process for oil obtained from
this strain, by investigating the effects of solvent and acid catalyst on yields.
5.2 Experimental section
5.2.1 Chemicals
The chemicals used in the study were of analytical grade. The other medium components
including glucose, glycerol, yeast extract and mycological peptone (Sigma-Aldrich, Sydney,
Australia) and sea salt (Instant Ocean, Virginia, USA) were used for biomass production
while solvents including chloroform, methanol, toluene, hexane, acetone and acids such as
hydrochloric acid and sulphuric acid (Merck, Victoria, Australia), were used for lipid
extraction, transesterification process and fatty acid determination.
5.2.2 Thraustochytrid isolate and growth conditions
Thraustochytrid isolate designated as DT9 (Genbank accession number KF682135) was
isolated from Victorian mangroves, Australia. DT9 was identified as thraustochytrid based on
colony morphology, appearance, reproduction pattern and molecular identification as
described in Chapter 4 (Sections 4.2.6 and 4.2.7). The culture was maintained on GYP
medium consisting of (g L-1 ) glucose (5), yeast extract (2), mycological peptone (2), agar
Chapter 5
75
(10) and artificial sea water (50%) at 25°C and subcultured after 15 days. All the isolates
were cultivated in the seed medium (1% glucose, 0.1% yeast extract, 0.1% mycological
peptone, ASW 50%) for 3 days and production medium (2% glucose, 0.2% yeast extract,
0.2% mycological peptone, ASW 50%) for 5 days. Seed medium (50 ml) was inoculated
from agar plates and grown in a shake flask at 25°C and 200 rpm of shaking speed. Inoculum
5% (v/v) was used to inoculate the production medium, cultured for 5 days in a shake flask at
25°C at 200 rpm. The resultant biomass was harvested by centrifugation (10,000 x g, 15
mins) and freeze dried until further use.
5.2.3 Transesterification process
5.2.3.1 Fatty acid extraction and transesterification using a conventional two-step process
In the conventional two-step process 1g of freeze dried cells were extracted with a mixture
containing a 2:1 ratio of chloroform and methanol. The process was repeated 3 times and all
of the extracted material was pooled and the weight of lipids determined using gravimetric
analysis (Lewis et al. 2000). Transesterification was carried out on extracted biomass by the
addition of 3.4 ml of methanol, 0.6 ml of acid catalyst H2S04 and 4ml of chloroform. The
mixture was heated to 90°C for 40 mins with continuous stirring. The reaction mixture was
then cooled to room temperature to stop the reaction. Two ml of distilled water was added
and the mixture was mixed for 45 sec. If required for phase separation the mixture was
centrifuged at 8000 rpm for 10mins. The solvent layer was collected in a preweighed vial and
the weight of extracted biodiesel was determined gravimetrically (Johnson & Wen 2009).
5.2.3.2 Direct transesterification via a single step process
Direct transesterification was performed directly on the biomass. To 1 g of freeze dried cells
3.4 ml of methanol, 0.6 ml of acid catalyst H2S04 and 4ml of chloroform was added. The
mixture was heated to 90°C for 40 mins with continuous stirring. The reaction mixture was
then cooled to room temperature to stop the reaction. Two ml of distilled water was added
and the mixture was mixed for 45 sec. If required for phase separation the mixture was
centrifuged at 8000 rpm for 10mins. The solvent layer was collected in a preweighed vial and
the weight of the extracted biodiesel was determined gravimetrically (Johnson & Wen 2009).
Direct transesterification was repeated using different solvents (hexane, toluene, chloroform
or acetone) and yields were determined for each reaction.
Chapter 5
76
5.2.4 Fatty acid analysis of biodiesel using gas chromatography
Fatty acid content of Crude extracted biodiesel was analysed using GC. Ten milligrams of oil
was dissolved in 1 ml of toluene followed by addition of 200 μl of internal standard, methyl
nonadecanoate (C19:0; 5mg/ml) and 200 μl of butylated hydroxytoluene (BHT; 1mg/ml).
Acidic methanol (2 ml) was added to the tube and the sample incubated overnight at 50 °C.
Fatty acid methyl esters (FAMEs) were extracted into hexane. The hexane layer was removed
and dried over sodium sulphate. FAMEs were concentrated using nitrogen gas prior to GC
analysis (Destaillats 2010). The samples were analysed using a GC-FID system (Agilent
Technologies, 6890N, Santa Clara, CA, USA). The GC was equipped with a capillary column
(Suplecowax 10, 30 m × 0.25 mm, 0.25 μm film thickness). Helium was used as the carrier
gas at a flow rate of 1.5 ml min-1. The injector was maintained at 250°C and a sample volume
of 1 μl was injected. Fatty acids were identified by comparison to external standards (Sigma-
Aldrich, Sydney, Australia). Peaks were quanti ed with Chemstation chromatography
software (Agilent Technologies, Santa Clara, CA, USA) and corrected using theoretical
relative FID response factors (Ackman 2002).
5.3 Results and Discussion
5.3.1 Fermentation profile of DT9
Isolate DT9 was identified as thraustochytrid based on colony morphology, appearance and
reproduction pattern. Molecular identification identified the strain as Schizochytrium sp
(Phylogenetic tree analysis was discussed in detail in chapter 4). DT9 was fermented using
either glucose or glycerol as the carbon source and biomass, lipid and fatty acid productivity
were compared. Glycerol resulted in the maximum biomass of 7.1 g/L, while glucose resulted
in 5.1 g/L, after 3 days of fermentation. Lipid accumulation followed the same trend, with
fermentation using glycerol resulted in maximum lipid of 881 mg/g, and glucose resulted in
834 mg/g, after 2 days of fermentation (Figure 5.1).
Chapter 5
77
Figure 5.1 Fermentation profile of Isolate DT9
Glycerol appears to result in overall higher biomass and lipid productivity, compared with
glucose, for isolate DT9. Some reported related strains also grow better in glycerol then in
glucose, including Thraustochytrium sp. ONC T18, Thraustochytrium sp. AMCQS5-5
and Aurantiochytrium sp. strain TC 20 (Chang et al. 2013; Gupta et al. 2013; Scott et al.
2011). The major components of the fatty acid profile of DT9 were C14:0, C15:0, C16:0,
C16:1n7, C17:0, C18:0, C18:1n9, C20:5n3, C22:5n6 and C22:6n3, which is comparable
to the profile from Schizochytrium mangrovei FB3 (Fan & Chen 2007). Palmitic acid
(C16:0) and DHA (C22:6n3) are the most abundant fatty acids in the profile of DT9 at
36% and 23% on TFA, respectively. The saturated or mono unsaturated fatty acids C16:0,
C16:1n7 and C18:1n9 were 825 mg/L, 353 mg/L and 109 mg/L, respectively. DPA
(C22:5n6) and DHA (C22:6n3) were 178 mg/L and 528 mg/L, respectively (Table 5.1).
0
100
200
300
400
500
600
700
800
900
1000
0
1
2
3
4
5
6
7
8
9
0 1 2 3 4 5
Lipid(m
g/g)
Biom
ass(g/L)
Incubation days
Glucose(Biomass) Glycerol(Biomass)
Chapter 5
78
Table 5.1 Fatty acid profile of isolate DT9 after three days of fermentation using glycerol as the carbon source.
Myristic acid (C14:0); palmitic acid (C16:0); palmitoleic (C16:1n7), stearic acid (C18:0); oleic acid (C18:1n9); eicosapentaenoic acid (EPA) (C20:5n3); docosapentaenoic acid (DPA) (C22:5n6); docosahexaenoic acid (DHA) (C22:5n6).
5.3.2 Biodiesel formation through transesterification
Biodiesel was produced from the freeze dried biomass through extraction followed by either
conventional transesterification or direct transesterification. The resultant biodiesel profiles
and yields are compared for these two methods. Direct transesterification resulted in a 52%
yield (w/w) of biodiesel per dry biomass, and 36% yield (w/w) of total fatty acid per dry
biomass. Conventional transesterification resulted in 35% yield (w/w) of biodiesel per dry
biomass, and 26% yield (w/w) of total fatty acid per total lipid, respectively. Myristic acid,
palmitic acid, DPA and DHA were the most abundant fatty acids in the resulted biodiesel
obtained by either method. Direct transesterification resulted in a higher yield of methyl
esters at 363 mg/g, than that obtained from the conventional method (259 mg/g) (Table 5.2).
FAMEs content of biodiesel was 70% by direct transesterification and 73% from the
conventional method
Fatty acid profile TFA (mg/L) TFA (%)
C14:0 238 ± 13 10 ± 0.1
C15:0 8 ± 0.2 0.3 ± 0.0
C16:0 825 ± 30 36 ± 0.2
C16:1n7 353 ± 16 15 ± 0.1
C18:0 27 ± 1.3 1.2 ± 0.0
C18:1n9 c 109 ± 7.3 4.7 ± 0.1
C20:5n3 14 ± 0.5 0.6 ± 0.0
C22:5n6 178 ± 7.1 7.8 ± 0.0
C22:6n3 528 ± 22.4 23 ± 0.0
Chapter 5
79
Table 5.2 Comparison of conventional and direct transesterification for isolate DT9.
Biodiesel profile(mg/g of TFA) a Conventional method a Direct transesterification c Amount of crude extract 35 ± 4.6 52 ± 0.3 c Amount of biodiesel 26 ± 3.8 36 ± 1.6 C14:0 42± 5 55±2.5 C15:0 1.2 ± 0.2 1.6±0.1 C16:0 91 ± 10.9 122±5.8 C16:1n7 20 ± 4.4 31±1.3 C18:0 3.4 ± 0.4 4.5±0.3 C18:1n9 c 13 ± 2.3 19±0.8 C22:5n6 25 ± 3.9 36±1.6 C22:6n3 57± 10.2 86±3.8
Fame content in biodiesel (%) 73 ± 1.3 70 ± 2.8 b Degree of FAME unsaturation 2 ± 0.06 2.14 ± 0.01
a Chloroform was used in both the transesterification method. Data represent the mean values of three replicates ± standard deviations. b Degree of FAME unsaturation = [1.0 (% monene) + 2.0 (% diene) + 3.0 (% triene) + 4.0 (%tetraenes) + 5.0 (% pentanes) + 6.0 (% hexane)]/100 (Chen & Johns 1991) Myristic acid (C14:0); palmitic acid (C16:0); palmitoleic (C16:1n7), stearic acid (C18:0); oleic acid (C18:1n9); eicosapentaenoic acid (EPA) (C20:5n3); docosapentaenoic acid (DPA) (C22:5n6); docosahexaenoic acid (DHA) (C22:5n6). c Amount of crude extract and biodiesel are given by % wt of dry biomass.
Direct transesterification resulted in higher overall yields than conventional two-step
transesterifcation, although the fatty acid profiles were similar in both cases. Direct
transesterification also used less solvent and was a more rapid process, and so has both
product and cost advantages over conventional transesterification. Previous work on a
related strain (Schizochytrium limacinum) also showed advantages of direct
transesterification (Johnson & Wen 2009).
Chapter 5
80
5.3.3 Optimisation of solvent and acid catalyst for direct transesterification
To determine the most suitable solvent to treat the biomass together with methanol for acid
catalysed direct transesterification, chloroform, hexane, toluene and acetone were tested.
Toluene resulted in maximal extraction yields of 57% (w/w) biodiesel and 33% (w/w) fatty
acid content. Chloroform resulted in the second highest yield and hexane the lowest (Table
5.2). Total FAME content on direct transesterification for toluene was 330 mg/g, chloroform
was 320 mg/g and hexane 323 mg/g. Transesterification yields of DT13 was comparable to
Aurantiochytrium sp. TC 20 (Chang et al. 2013; Cho et al. 2012). Microalgae Pavlova lutheri
was reported in the literature with increased lipid content using methanol and toluene as
solvents compared to chloroform and ethanol in combination with methanol (Carvalho &
Malcata 2005). However to our knowledge, this is the first study where hexane, acetone,
toluene and chloroform were tested as solvents for direct esterification for a Schizochytrium
isolate.
Figure 5.2 Direct transesterification yields for isolate DT9 using various solvents
0
100
200
300
400
500
600
700
Hexane Acetone Toluene Chloroform
Lipid(m
g/g)
TFA mg/g of crude biodiesel Crude Biodiesel (mg/g) of biomass (Graviometric)
Chapter 5
81
Figure 5.3 Fatty acid profile after direct transesterification of isolate DT9 with different solvents.
The fatty acid profiles were similar for all extraction solvents, although acetone appears to
result in lower levels of the major fatty acids (Figure 5.3). Acid catalysts such as HCl and
H2SO4 are studied in the experiment as these acid catalysts can produce more biodiesel yield
Compared to base catalysts (Lotero et al. 2005). Acid catalysts are tolerant to high moisture
and FFA content of the biomass hence they can act as better catalyst for biodiesel production
through direct transesterification method. HCl has been suggested as a substitute to H2SO4 in
the direct transesterification process, resulting in lower sulphur content (He et al. 2009). Use
of these strong acids leads to biodiesel with high sulphur content which is out of specification
for biodiesel production. In our hands, replacing sulphuric acid with HCl resulted in slightly
higher yields, although these were not significantly different between the two acids (Table
5.3).
0
20
40
60
80
100
120
140
160
180
C14:0 C15:0 C16:0 C16:1n7 C18:0 C18:1n9 c C20:5n3 C22:5n6 C22:6n3
Fattyacid
(mg/g)
Hexane Chloroform Acetone Toluene
Chapter 5
82
Table 5.3 Biodiesel and FAME profile of isolate DT9 based on direct transesterification method
a Amount of crude extract and biodiesel are given by % wt of dry biomass. Degree of FAME unsaturation = [1.0 (% monene) + 2.0 (% diene) + 3.0 (% triene) + 4.0 (%tetraenes) + 5.0 (% pentanes) + 6.0 (% hexane)]/100 (Chen & Johns 1991). Myristic acid (C14:0); palmitic acid (C16:0); palmitoleic (C16:1n7), stearic acid (C18:0); oleic acid (C18:1n9); eicosapentaenoic acid (EPA) (C20:5n3); docosapentaenoic acid (DPA) (C22:5n6); docosahexaenoic acid (DHA) (C22:5n6). Data represent the mean values of three replicates ± standard deviations.
5.4 Conclusions
In summary, this work shows the potential of in-house isolate DT9 for biodiesel production.
Direct transesterification resulted in higher yields of fatty acid methyl ester (FAME) than the
conventional two-step method. This is the first study of our knowledge to address the
optimisation of direct transesterification process in a thraustochytrid isolate. Toluene resulted
in a higher yield of crude biodiesel from direct transesterification of biomass than did
chloroform, hexane or acetone solvents.
Chapter 6
Chapter 6: Evaluation of Victorian marine isolate Schizochytrium sp. DT13 grown using various carbon sources for the co-production of DHA and biodiesel
Chapter 6
84
6.1 Introduction
Thraustochytrids are members of the phytoplankton group and have been extensively studied
for the production of nutraceuticals and valuable compounds (Lee Chang et al. 2014; Gupta
et al 2012). Omega-3 fatty acid production from thraustochytrids has been extensively
studied and they can accumulate more than 70 % of their biomass as lipid with high fractions
of DHA (Kim et al. 2013). Along with DHA thraustochytrids can produce other fatty acids
that are appropriate for biodiesel production, such as saturated fatty acids (Hong et al. 2013).
Other compounds of commercial interest from thraustochytrids are squalene, carotenoids,
exopolysacchrides (EPS) and enzymes (Lee Chang et al. 2014).
The global market for nutritional products containing omega-3 oils was more than $2 billion
in 2012. Increasing demand from the Asia-pacific market is leading to continued growth in
the omega-3 category (Shanahan 2012). Thraustochytrids offer a renewable omega-3
production system and have been the subject of research such as improved techniques of
isolation (Gupta et al. 2013) and improved fermentation processes (Zhang et al. 2013). To be
commercially competitive with fish oil for the supplement and functional food market,
omega-3 fermentation methods need to be lower in cost than they currently are. For
heterotrophic fermentation the carbon source is a major cost of production and can contribute
as much as a third of the total production costs. Therefore, work toward using low cost
carbon sources is important for commercial viability of omega-3 oils from thraustochytrids,
particularly for the general nutritional supplement market (Abad & Turon 2012; Pleissner et
al. 2013).
Various carbon sources have been used for DHA and lipid production from Schizochytrium
strains (Liang et al. 2010; Shene et al. 2010; Thyagarajan et al. 2014; Wu et al. 2005;
Yokochi et al. 1998). Fatty acid shifts and metabolic activity changes were studied for
Schizochytrium sp.S31 during batch fermentation, with glycerol used as the sole carbon
source (Chang et al. 2013). Fed-batch fermentation was studied for the biodiesel and DHA
production in Aurantiochytrium sp. KRS101 (Kim et al. 2013). Addition of trace elements
such as manganese, iron, cobalt, nickel, copper, zinc and molybdenum reportedly improved
growth for some thraustochytrid species (Nagano et al. 2013). Schizochytrium mangrovei and
Chlorella pyrenoidosa have been grown using food waste obtained by fungal hydrolysis and
were reported to produce biomass potentially useful as a feed supplement or for biodiesel
Chapter 6
85
production (Pleissner et al., 2013). Other inexpensive carbon sources derived from food
wastes that were studied includes okara powder (Fan et al. 2001b), residues from beer and
potato processing (Quilodran et al. 2009), sweet sorghum juice (Liang et al. 2010a), coconut
water (Unagul et al. 2007), marine aquaculture waste water (Jung & Lovitt 2010) and crude
glycerol (Pyle et al. 2008).
The objective of the work described in this chapter was to investigate the potential of our
isolate DT13 to utilise various carbon sources for the production of DHA, carotenoids and
biodiesel.
6.2 Materials and methods
6.2.1 Chemicals
The chemicals used in this study were of analytical grade. The other medium components
including glucose, glycerol, yeast extract and mycological peptone (Sigma-Aldrich, Sydney,
Australia) and sea salt (Instant Ocean, Virginia, USA) were used for biomass production,
while solvents including chloroform, methanol, hexane, and acids such as hydrochloric acid
(Merck, Victoria, Australia), were used for lipid extraction, transesterification processes and
fatty acid determination.
6.2.2 Thraustochytrid isolate and growth conditions
Thraustochytrid isolate designated as DT13 (Genbank accession number KF682135) was
isolated from Victorian mangroves, Australia. DT13 was identified as thraustochytrid based
on colony morphology, appearance, reproduction pattern and molecular identification, as
described in Chapter 4 (Sections 4.2.6 and 4.2.7). The culture was maintained on GYP
medium consisting of (g L-1) glucose (5), yeast extract (2), mycological peptone (2), agar (10)
and artificial sea water (50%) at 25°C and subcultured after 15 d. All the isolates were
cultivated in the seed medium (1% glucose, 0.1% yeast extract, 0.1% mycological peptone,
ASW 50%) for 3 days and production medium (2% glucose, 0.2% yeast extract, 0.2%
mycological peptone, ASW 50%) for 5 d. Seed medium was inoculated from agar plates and
grown in shake flasks at 25°C and 200 rpm. Inoculum (5% v/v) was used to inoculate the
production medium, cultured for 7 days in a shake flask at 25°C at 200 rpm. The resultant
biomass was harvested by centrifugation (10,000 x g, 15 min) and freeze dried and stored
until further use.
Chapter 6
86
6.2.3 Comparison of glucose and glycerol as a carbon source for DHA production
Thraustochytrid isolate DT13 was grown with glucose or glycerol containing medium to
compare relative DHA production levels. Composition of seed medium and production
medium was as described in section 6.2.2, except the carbon sources source were either
glucose or glycerol at concentrations of 3%, 5% 7% or 10%, along with the nitrogen sources
yeast extract and peptone at 0.3%, 0.5%, 0.7% or 1% in the medium. The resultant biomass
after 5 days fermentation was harvested by centrifugation (10,000 x g, 15 min) and freeze
dried to enable quantitation of lipids.
6.2.4 Thraustochytrid isolate DT13 growth conditions for biodiesel production
Thraustochytrid isolate DT13 was grown with glycerol medium at 2% w/v concentration for
biodiesel production. The remaining medium components and the fermentation procedure are
as described in section 6.2.2. The resultant biomass after 5 d fermentation was harvested by
centrifugation (10,000 x g, 15 min) and freeze dried for conversion to biodiesel by either
conventional or direct transesterification processes.
6.2.5 Fatty acid analysis of using gas chromatography
The fatty acid content of crude extracted biodiesel was analysed using GC. Ten milligrams of
oil was dissolved in 1 ml of toluene followed by addition of 200 μl of internal standard,
methyl nonadecanoate (C19:0; 5mg/ml) and 200 μl of butylated hydroxytoluene (BHT;
1mg/ml). Acidic methanol (2 ml) was added to the tube and the sample incubated overnight
at 50°C. Fatty acid methyl esters (FAMEs) were extracted into hexane. The hexane layer was
removed and dried over sodium sulphate. FAMEs were concentrated using nitrogen gas prior
to GC analysis (Destaillats, 2010). The samples were analysed using a GC-FID system
(Agilent Technologies, 6890N, Santa Clara, CA, USA). The GC was equipped with a
capillary column (Suplecowax 10, 30 m × 0.25 mm, 0.25 μm film thickness). Helium was
used as the carrier gas at a flow rate of 1.5 ml min-1. The injector was maintained at 250°C
and a sample volume of 1 μl was injected. Fatty acids were identified by comparison to
external standards (Sigma-Aldrich, Sydney, Australia). Peaks were quanti ed with
Chemstation chromatography software (Agilent Technologies, Santa Clara, CA, USA) and
corrected using theoretical relative FID response factors (Ackman 2002).
Chapter 6
87
6.2.6 Astaxanthin extraction and HPLC analysis
Carotenoids were extracted by adding acetone as previously described (Armenta et al., 2006),
with slight modifications. To 25 mg of freeze dried cells, 1ml of acetone was added and
vortexed for 5 mins, followed by centrifugation at 4000 rpm for 15 min. This step was
repeated until the biomass became colourless. All supernatant was collected and stored at
15°C in dark conditions. Final extract volumes were noted for total carotenoid estimation. 1
ml acetone extract was removed and evaporated under nitrogen gas followed by addition of 1
ml mobile phase and subsequently syringe filtered (0.45 m) prior to HPLC analysis of the
extracts. The Agilent 1200 Series HPLC system with the Agilent 1200 Series photodiode
array detector was used for carotenoid analysis. Carotenoids were analysed at 477 nm using 5
m Luna C18 reversed-phase column, 4.6 mm x 250 mm (Phenomenex, Torrance, USA), and
a security guard column C18, 3.0 mm x 4.0 mm, (Phenomenex, Torrance, USA). This
column was equilibrated with mobile phase A consisting of methanol, ethyl acetate and water
(88:10:2, v/v/v) in a gradient mode at a flow rate of 0.75 mL min-1. This flow rate was
maintained for 10 min. Mobile phase composition was changed to 2:50:48 (mobile phase B)
between 10 and 30 min and the flow rate was adjusted to 1.5 mL/min. These conditions were
maintained for a further 5 min and then set for column washing with mobile phase A for 10
min. The calibration curve for astaxanthin was prepared with the standard and used for
identification from different extracts.
6.2.7 Transesterification process
6.2.7.1 Fatty acid extraction and transesterification using a conventional two-step
process
In the conventional two-step process 1g of freeze dried cells were extracted with a mixture
containing a 2:1 ratio of chloroform and methanol. The process was repeated 3 times and all
of the extracted material was pooled and the weight of lipids determined using gravimetric
analysis (Lewis et al., 2000). Transesterification was carried out on extracted biomass by the
addition of 3.4 ml of methanol, 0.6 ml of acid catalyst H2SO4 and 4ml of chloroform. The
mixture was heated to 90°C for 40 mins with continuous stirring. The reaction mixture was
then cooled to room temperature to stop the reaction. Two ml of distilled water was added
and the mixture was mixed for 45 sec. If required for phase separation the mixture was
Chapter 6
88
centrifuged at 8000 rpm for 10mins. The solvent layer was collected in a preweighed vial and
the weight of extracted biodiesel was determined gravimetrically (Johnson & Wen 2009).
6.2.7.2 Direct transesterification via a single step process
Direct transesterification was performed directly on the biomass. To 1 g of freeze dried cells
3.4 ml of methanol, 0.6 ml of acid catalyst H2SO4 and 4ml of chloroform was added. The
mixture was heated to 90°C for 40 mins with continuous stirring. The reaction mixture was
then cooled to room temperature to stop the reaction. Two ml of distilled water was added
and the mixture was mixed for 45 sec. If required for phase separation the mixture was
centrifuged at 8000 rpm for 10 mins. The solvent layer was collected in a preweighed vial
and the weight of the extracted biodiesel was determined gravimetrically (Johnson & Wen
2009).
6.3 Results and discussions:
6.3.1 Phylogenetic analysis and growth profile of Schizochytrium DT13
The in-house strain of interest DT13 (Genbank accession number KF682135) was identified
as a Schizochytrium sp (Phylogenetic tree and molecular identification was given in detail in
chapter 4) and it was found similar to the commercial strain Schizochytrium limacinum
SR21. To examine the fermentation profile, isolate DT13 was cultivated in 2% (w/v) glucose
medium from day 1 to day 7. Maximum biomass, lipid and DHA were observed as 6.5 g/L, 3
g/L and 623 mg/L, respectively, on day 5 of fermentation (Figure 6.1). The fermentation
profile was found to be similar to Schizochytrium limacinum SR21 (Chin et al. 2006).
Chapter 6
89
Figure 6.1 Fermentation profile of Schizochytrium DT13 with 2% glucose
6.3.2 Fermentation profile of Schizochytrium DT13 with various carbon sources
DT13 was grown using various carbon sources to investigate its ability to utilise different
carbon sources. Glycerol, glucose, fructose, sodium butyrate, flaxseed oil and olive oil , each
at 1% w/v, resulted in good levels of dry cell weight and lipids. However, cellulose,
cellobiose, starch, xylose, sucrose and bread crumbs were not well utilised, and resulted in
low dry cell weight and total lipid yields. Simple carbon sources were more readily utilised
by DT13 for the production of lipids than were complex carbon sources. Some other related
strains also reported the utilisation of a variety of simple carbon sources, including
Schizochytrium limacinum SR21 and Scenedesmus sp. strain R-16 (Ren et al. 2013; Yokochi
et al. 1998). DHA production was maximal with the carbon sources glycerol, glucose,
fructose, olive oil, sodium butyrate and flaxseed oil, with yield of 344 mg/L, 284 mg/L, 274
0
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1 2 3 4 5 6 7
DHA(m
g/L)
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ass(mg/L)
Lipid(m
g/L)
Incubation (d)
Biomass (mg/L)
total lipid (mg/L)
DHA (mg/L)
Chapter 6
90
mg/L, 222 mg/L, 184 mg/L and 178 mg/L, respectively (Figure 6.2). Good utilisation of olive
oil and flaxseed oil indicates that DT13 may be able to utilise seed cake
waste of olive oil or flaxseed oil for biodiesel production.
Figure 6.2 Fermentation profile of Schizochytrium DT13 with various carbon sources at 1% w/v concentration Gly-glycerol; Glu-glucose; CSL-cornsteep liquor; BC-breadcrumbs; FO-flaxseed oil; OL-olive oil; SB-sodium butyrate
The fatty acid profile of DT13 showed the presence of mainly the saturated fatty acids such
as myristic acid and palmitic acid, the monounsaturated fatty acid oleic acid, and the
polyunsaturated fatty acids EPA, DPA and DHA. Fermentation with glycerol gave 551 mg/L
of saturated fatty acids and 379 mg/L of mono unsaturated fatty acids. Olive oil resulted in
540 mg/L of saturated fatty acids and 3.3 g/L of mono unsaturated fatty acids (Table 6.1).
These carbon sources may be useful for biodiesel production if the higher value co-products
DHA can be successfully separated from the fatty acid mixture (Chang et al. 2013; Dang et
al. 2013; Kim et al. 2013; Lee et al. 2011).
0
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DHA Biomass Lipid
Chapter 6
91
Table 6.1 Effect of carbon source on fatty acid profile of Schizochytrium DT13 with various
carbon sources
Glycerol Glucose Flaxseed oil Olive oil Fatty acid mg/L C14:0 129±10 92±5.8 22±0.3 11±1.1 C15:0 4±0.5 0.00 0.00 0.00 C16:0 398 ±39 340±21 337±12 432±20 C16:1n7 235±25 150±6 28±0.1 38±0.8 C18:0 19±1.8 15±0.4 68±17 96±3.9 C18:1n9t 0.00 0.00 452±95 3227±206 C18:1n9 c 144±13 106±4.0 63±2.1 110±2.7 C18:2n6c 0.00 0.00 416±61 441±6.4 C18:3n3 0.00 0.00 1618±123 128±60 C20:0 1.9±0.5 3±0.1 7±0.8 19±0.2 unknown 2.7±0.2 1.9±0.3 23±0.2 4±0.1 unknown 2.8±0.3 2.4±0.1 11±0.1 5.8±0.1 unknown 3.8±0.4 3±0.1 0.00 0.00 C20:5n3 12±1 10±0.1 12±0.1 11±0.7 C22:5n6 131±13 106±4.6 56±0.6 76±4.8 unknown 6±0.7 4.8±0.4 7±0.4 8.2±0.4 unknown 4±0.2 3±0.1 18±0.1 7±1.2 C22:6n3 344±35 284±13 178±2.3 222±17 Saturated fatty acids 551±53 448±28 427±30 540±25 Mono unsaturated fatty acids 379±38 256±10 545±97 3375±210
Myristic acid (C14:0); pentadecanoic (C15:0); palmitic acid (C16:0); palmitoleic (C16:1n7), stearic acid (C18:0); elaidic (C18:1n9t); oleic acid (C18:1n9c); linoleic (C18:2n6c); -linolenic acid (C18:3n3); arachidic acid (C20:0); eicosapentaenoic acid (EPA) (C20:5n3); docosapentaenoic acid (DPA) (C22:5n6); docosahexaenoic acid (DHA) (C22:6n3).
6.3.3 Optimisation of the carbon sources glucose and glycerol for DHA production
Glucose and glycerol concentrations were optimised to maximise DHA production. 10%
glucose gave 24.6 g/L of biomass, 10.5 g/L of lipid, and 2.6 g/L of DHA (Figure 6.3). These
yields were comparable to those obtained for the commercial strain Schizochytrium
limacinum SR21(Yokochi et al. 1998) and higher than obtained for Schizochytrium sp. S31
(Wu et al. 2005). Glycerol at 5% resulted in 5.2 g/L biomass, 1.5 g/L lipid and 445 mg/L
DHA. Further increasing the glycerol concentration resulted in decreased yields due to
Chapter 6
92
substrate inhibition. With a previous study with Schizochytrium limacinum SR21 also showed
lower yields at higher glycerol concentrations (Ethier et al. 2011; Huang et al. 2012).
Figure 6.3 Fermentation profile of Thraustochytrium DT13 with carbon sources glucose and glycerol at varying concentration. The carbon sources represented in the given figure as Glu-glucose; Gly-glycerol
6.3.4 Carotenoid profile of Schizochytrium DT13 with various carbon sources
DT13 fermentation with glycerol, glucose, sodium butyrate, flaxseed oil, olive oil, corn steep
liquor or fructose showed the presence of the carotenoid astaxanthin. Using the organic acid
sodium butyrate as the carbon source gave maximal astaxanthin production of 44.9 μg/g
(Figure 6.4). Sodium butyrate is as precursor to acetyl CoA and may directly promote lipid
production thus also increasing the accumulation of astaxanthin in the oil globules. A study
with the commercial astaxanthin producing strain Haematococcus Pluvialis also showed that
astaxanthin levels increased as lipid levels increased, due to astaxanthin accumulation in oil
globules (Zhekisheva et al. 2005). Ours is the first study in a thraustochytrid strain to show
that sodium butyrate gives high astaxanthin levels.
0
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Glu 3% Glu 5% Glu 7% Glu 10% Gly 3% Gly 5% Gly 7% Gly 10%
DHA(m
g/L)
Biom
ass(mg/L)
Lipid(m
g/L)
Biomass mg/L Lipid mg/L DHA mg/L
Chapter 6
93
Figure 6.4 Astaxanthin profile of Schizochytrium DT13 with various carbon sources at 1% w/v concentration Gly-glycerol; Glu-glucose; SB-sodium butyrate; FO-flaxseed oil; OL-olive oil; CSL-cornsteep liquor
6.3.5 Transesterification of Schizochytrium DT13 biomass for biodiesel production
Biodiesel was produced from the freeze dried biomass through extraction followed by either
conventional transesterification (conventional method) or direct transesterification. The
resultant biodiesel profiles and yields are compared for these two methods. Direct
transesterification resulted in a 57% yield (w/w) of biodiesel per dry biomass, and 40% yield
(w/w) of total fatty acid per total lipid. Conventional transesterification resulted in 46% yield
(w/w) of biodiesel per dry biomass, and 34% yield (w/w) of total fatty acid per total lipid.
Myristic acid, palmitic acid, palmitoleic acid, oleic acid (cis form), DPA and DHA were the
major fatty acids in the resulting biodiesel obtained by either transesterification method.
Direct transesterification resulted in a higher yield of methyl esters at 401 mg/g, than that
obtained from the conventional method (342 mg/g) (Table 5.2). FAMEs content of biodiesel
was 69% by direct transesterification and 73% from the conventional method. Direct
transesterification resulted in higher overall yields than conventional two-step
transesterification, although the fatty acid profiles were similar in both cases. Direct
transesterification also used less solvent and was a more rapid process, and so has both
product and cost advantages over conventional transesterification. Previous work on a related
strain (Schizochytrium limacinum) also showed advantages of direct transesterification
(Johnson & Wen 2009).
40
41
42
43
44
45
46
Gly 1% Glu 1% SB 1% FO 1% OL 1% CSL 1% Fructose 1%
Astaxanthin(g/g)
Carbon source
Chapter 6
94
Table 6.2 Biodiesel profile of Schizochytrium DT13
c Conventional method c Direct method
C14:0 60±1.1 54±2.1
C15:0 2.1±0.1 4±0.1
C16:0 109±1.4 161±6.8
C16:1n7 33±0.5 19.4±0.6
C18:0 4±0.1 5.8±0.2
C18:1n9 c 22±0.1 14±0.5
C20:5n3 3.5±0.1 3.6±0.1
C22:5n6 32±0.1 42±1.7
C22:6n3 74±0.6 95±3.6
TFA mg/g of biomass 342±1.4 401±16 Lipid / Crude Biodieseld (mg/g) of biomass (Gravimetric) 465±13 575±4 a Amount of crude extract 46±1.3 57±0.4 a Amount of biodiesel 34±0.1 40±1.6
b Fame content in biodiesel (%) 73±2.3 69±2.3 a Myrstic acid (C14:0); pentadecanoic acid (C15:0); palmitic acid (C16:0); palmitoleic acid (C16:1n7); stearic acid (C18:0); oleic acid (cis form) (C18:1n9); EPA (C20:5n3); DPA (C22:5n6); DHA (C22:6n3) b Chloroform was used in both the transesterification method. Data represent the mean values of three replicates ± standard deviations. c Data represent the mean values of three replicates ± standard deviations. d Lipid extracted after direct transesterification reaction (crude biodiesel).
Chapter 6
95
6.4 Conclusions
In summary, this work shows the potential of in-house isolate DT13 for the production of
DHA and biodiesel with a variety of carbon sources. In particular, olive oil, flaxseed oil and
glycerol may be useful for large scale fermentation of the DT13 strain for the commercial
production of biodiesel and DHA. Direct transesterification was a more efficient method than
the two step process for conversion to methyl esters. Schizochytrium sp DT13 may be
comparable to commercial strain Schizochytrium limacinum SR21 in terms of DHA
productivity, although this needs to be confirmed at scale. Sodium butyrate was shown be to
a good carbon source for astaxanthin accumulation in DT13.
Chapter 7
96
Chapter 7: Screening of Lipases for the selective hydrolysis of DHA from Schizochytrium oil.
Chapter 7
97
7.1 Introduction
Omega-3 fatty acids are potential nutraceuticals widely consumed by people around the
world for their health benefits (Doughman et al. 2007). Omega-3 fatty acids are
polyunsaturated fatty acids (PUFAs) that include mainly alpha-linolenic acid (ALA),
eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid
(DHA) (Raghukumar et al. 1995) with EPA and DHA being the most well known in the
marine world. Biological benefits of PUFAs are well known as they play multiple roles in
cell function and biology including as precursors for various regulatory molecules such as
prostaglandins, thromboxanes, resolvins, maresins and neuro protectens. Clinical benefits of
PUFAs in the treatment of Alzheimer’s disease and schizophrenia are benefited by adequate
dietary intake of these fatty acids (Boudrault et al. 2009; Sethom et al. 2010). PUFA
supplements can help in reducing obesity associated inflammation (Chung et al. 2014),
arthritis (Knott et al. 2011), cardiovascular disease (Lavie et al. 2009), depression and age
related neurological disorders (Deckelbaum & Torrejon 2012). The human body cannot de
novo synthesises omega-3 fatty acids and poorly converts short-chain omega-3 fatty acids
into EPA and DHA (Huang et al. 2005). Hence, diet enriched with omega-3 rich foods such
as oily fish acts as an important source of these compounds. But some western diets and
vegetarian diets do not meet the proposed intact levels of these essential fatty acids.
The National Health and Medical Research Council (NHMRC) recommended intake of long
chain omega-3 fatty acids (DHA/EPA) in order to lower the risk of chronic diseases,
recommended levels for men and women is 610 mg/day and 430 mg/day respectively
(Grosso et al. 2014). Most of the commercially used fish oil such as anchovy (Engraulis
ringens) and sardine (Sardinops sagax) oils contain 15-22% of EPA and 9-15 % DHA. To
increase the concentration of DHA and EPA and remove various saturated fatty acids that
were not beneficial for the clinical use, various strategies for manufacturing were developed
(Kralovec et al. 2012).
Lipases are successfully used industrially for the enzymatic processing of fish oil and
concentration of glyceride rich PUFAs, while removing saturated and mono unsaturated fatty
acids in the free fatty acid portion (FFA) (Haraldsson et al. 1997). Enzymatic processes are
greener in terms of utilisation of chemicals and solvents compared to distillation and urea
complexation techniques (Shahidi & Wanasundara 1998). In the process of PUFA
Chapter 7
98
concentration various lipases were studied for fish oil hydrolysis, but studies are very limited
on thraustochytrid oil.
In the present study, we screened Rhizomucar meiehei lipase (RML), Thermomyces
lanuginosus lipase (TLL) and Burkholderia cepacia lipase (BCL) for the hydrolysis of
Schizochytrium oil and their fatty acid selectivity for the concentration of PUFAs. These
lipases were selected as they were previously studied for the concentration of omega-3 fatty
acids from fish oil and concentration of PUFAs in glyceride portion.
7.2 Materials and methods
7.2.1 Materials
Schizochytrium oil was procured from Anhui Minmetals Development (Anhui, China). The
main fatty acids present in the Schizochytrium oil were determined using GC to be myristic
acid 7.8%, palmitic acid 21.8%, DPA 17.54% and DHA 42%. Lipase enzymes such as
Thermomyces lanuginosus lipase (TLL), Burkholderia cepacia lipase (BCL) and Rhizomucar
meiehei lipase (RML) were obtained from Sigma–Aldrich (Castle Hill, Australia). Gas and
thin layer chromatography standards were purchased from Sigma–Aldrich (Castle Hill,
Australia) and Nu-Chek Prep (Elysian, MN, USA). All other chemicals used were of
analytical grade.
7.2.2 Enzymatic hydrolysis
One gram of Schizochytrium oil and 2500 μL of 0.1 M phosphate buffer (pH 7.0), were
mixed in a glass bottle, and to the mixture lipase concentration of 1000U/g were added and
flushed with nitrogen. The reaction mixture at pH7.0 was incubated at 40°C for 24 hours with
magnetic stirring at 200 rpm and samples were withdrawn at different time intervals for
analysis. Glycerol and free fatty acid (FFA) portions of each sample were obtained by solvent
extraction according to a previously reported method (Gamez-Meza et al. 2003). The
percentage hydrolysis was determined from the acid and saponification values of the oil
before and after hydrolysis, as previously reported (Liu et al. 2008). Whole lipid, glycerol and
FFA fractions were kept under nitrogen and stored at 4°C until used.
Chapter 7
99
7.2.3 Analysis of lipid classes by capillary chromatography (Iatroscan)
Both the hydrolysed and unhydrolysed portions of the Schizochytrium oil were analysed by
capillary chromatography with flame ionisation detector (Iatroscan MK5, Iatron Laboratories
Inc., Tokyo, Japan). The Iatroscan settings were: air flow rate; 200 mL/min, hydrogen flow
rate; 160 mL/min and scan speed of 30 s/scan. Under these conditions, the chromarods were
cleaned by scanning twice before applying samples. One microliter of each lipid fraction in
hexane was spotted onto the rods with the aid of an auto pipette along the line of origin on the
rod holder and developed for 22 min in a solvent tank containing hexane/diethyl ether/acetic
acid (60:17:0.2, v/v/v). TLC standards purchased from Nu-Chek Prep were used to identify
each lipid class.
7.2.4 Analysis of fatty acid composition by gas chromatography
Prior to analysis by gas chromatography, fatty acids in both the unhydrolysed and hydrolysed
fish oil fractions were converted to methyl esters following the method of Christie and Han
(Christie & Xianlin 2010) with some modifications. Ten milligrams of oil was dissolved in 1
mL toluene, and 200 μL of internal standard (5 mg/mL methyl nonadecanoate (Sigma–
Aldrich) in toluene) and 200 μL of antioxidant (1 mg/mL 2,6-di-tert-butyl-4-methylphenol
(butylated hydroxytoluene; BHT, Sigma–Aldrich) in toluene) were added. To this, 2 mL of
hydrogen chloride in methanol (prepared by adding 1 mL acetyl chloride (Sigma–Aldrich)
drop wise to 10 mL methanol on ice) was added, the solutions mixed and left overnight at
50°C in a sealed tube. The solution was cooled and 5 mL sodium chloride solution (5% w/v)
was added. The fatty acid methyl esters were extracted twice with hexane (5 mL) and the
hexane layer washed with 5 mL potassium bicarbonate solution (2% m/v). The hexane layer
was dried over sodium sulphate, and most of the hexane was removed by rotary evaporation,
leaving 1.5 mL for analysis. Samples were analysed using an Agilent 6890 gas
chromatograph with flame ionisation detector (FID), equipped with a Supelcowax 10
capillary column (30 m, 0.25 mm i.d., 0.25 lm film thickness; Supelco). The oven was
programmed from 140°C (5 min hold) to 240°C (5 min hold) at a rate of 4°C/min for a total
run time of 35 min. A volume of 1 μL of solution was injected with a split ratio of 50:1
(injector temperature, 250°C). Helium was used as the carrier gas (1.5 mL/min, constant
flow). Detector gases were 30 mL/min hydrogen, 300 mL/min air and 30 mL/min nitrogen.
Peak areas were integrated by ChemStation software and corrected using theoretical relative
FID response factors (Craske & Bannon 1987).
Chapter 7
100
7.3 Results and discussion
7.3.1 Screening of various lipases for hydrolysis of Schizochytrium oil
To determine the effect of various lipases on the degree of Schizochytrium oil hydrolysis, 1 g
of Schizochytrium oil was hydrolysed with each of TLL, BCL and RML. Capillary
chromatography (Iatroscan) was used to profile the Schizochytrium oil after hydrolysis by
each lipase after 24 h. It was observed that RML hydrolysed 80% of oil, TLL 44% of the oil
and BCL 35% of the oil, in the same time period. Previous study on RML with fish oil
supported efficient hydrolysis by this lipase (Bilyk et al. 1991). The degree of hydrolysis of
Schizochytrium oil by TLL was similar to that obtained for fish oil under uncontrolled pH
(Akanbi et al. 2013).
Figure 7.1 Capillary chromatography (Iatroscan) profile of Schizochytrium oil after hydrolysis by lipases T.lanuginosus lipase (TLL), B.cepacia lipase (BCL) and R.meiehei lipase (RML) for 24 h at 40°C and pH 7.0.
Table 7.1 Fatty acid profile of Schizochytrium oil on hydrolysis with lipases for 12 h at 40°C and pH 7.0.
Lipase TFA % C14:0 C16:0 C20:5n3 C22:5n6 C22:6n3
Native oil 7.8 21.8 1.8 17.5 42.0
B.cepacia Glycerol portion 4.6 4.5 1.6 22.6 55.3 FFA portion 9.9 31.5 2.4 11.2 28.7
T.lanuginosus Glycerol portion 4.7 6.0 1.7 21.5 54.5 FFA portion 10.6 31.8 2.0 13.0 30.9
R.meiehei Glycerol portion 4.7 5.7 3.1 23.4 53.5 FFA portion 8.0 24.9 2.2 16.4 40.9
0102030405060708090
0 2 4 8 12 24
Oilhydrolysis
(%)
Time (h)
RML
BCL
TLL
Chapter 7
101
After 12 hours of hydrolysis, all the three enzymes RML, TLL and BCL partially
concentrated DPA and DHA in the glycerol portion. The abundant saturated fatty acids C14:0
and C16:0 were preferentially concentrated into the FFA portion. In particular, TLL and BCL
concentrated higher levels of these saturated fatty acid into FFA than did RML. Fatty acid
selectivities were similar for TLL and BCL. Selectivity of RML for DHA was lower than for
the other two lipases, in that RML was less able to concentrate DHA on the glycerol
backbone. TLL had a similar selectivity profile for Schizochytrium oil as was observed
previously with fish oil, with high DHA content in the glycerol portion and saturated fatty
acids in the FFA portion and less selectivity for EPA than for DHA (Figure 7.2) (Akanbi et
al., 2013).
Figure 7.2 Fatty acid profile of important fatty acids of Schizochytrium oil after hydrolysis by lipases T.lanuginosus lipase (TLL), B.cepacia lipase (BCL) and R.meiehei lipase (RML) for 12 h at 40°C and pH 7.0.
Chapter 7
102
A recent computational search for lipases that can preferentially hydrolyze long-chain omega-
3 fatty acids from fish oil triacylglycerols indicated BCL as a lipase with potentially higher
affinity towards DHA triacyl glycerol (Kamal et al. 2015). Hence BCL, which in our study
show similar selectivity to TLL, may be industrially useful as for the concentration of DHA
from a variety of oil sources, including Schizochytrium oil
7.4 Conclusion
In summary, testing of T.lanuginosus lipase (TLL), Burkholderia cepacia lipase (BCL) and
Rhizomucar meiehei lipase (RML) for the concentration of DHA from Schizochytrium oil
showed that TLL and BCL significantly discriminated against the hydrolysis of omega-3 fatty
acids, particularly DHA. Although, Rhizomucar meiehei lipase (RML) was observed to
concentrate PUFAs, its selectivity to DHA was less than for the other two lipases.
Chapter 8
Chapter 8: Summary and future directions
Chapter 8
104
The objective of the study was to understand and develop the process of fermentation in
marine microorganisms for the production of omega-3 fatty acids and carotenoids. Among
marine microorganisms, thraustochytrids that belong to phytoplankton group was identified
as sources of omega-3 fatty acids and other valuable co-products. Thraustochytrids are
industrially useful for the production of omega-3 rich oils for food and supplements, aqua
feed and biofuels. However, more efficient and cost-effective production of omega-3 oils in
these strains requires:
Fermentation strategy using lower cost raw materials, particularly carbon.
Screening for new strains that can naturally produce high levels of PUFAs together
with useful co-products that can be harvested together with the omega-3 oil.
In the work described in the second chapter of this thesis, the marine microorganisms
Thraustochytrium sp. AH-2 and Schizochytrium sp. SR21 were used to evaluate the use of
breadcrumbs for lipid production. The potential of microbial fermentation to convert bakery
waste into oil either for biofuel or PUFA production was investigated.. Scanning electron
microscopic (SEM) studies confirmed the growth of both strains on breadcrumbs. The fatty
acid profiles from Thraustochytrium sp. AH-2 indicated low levels of PUFA and higher
levels of saturated oil. ATR-FTIR spectroscopy of Schizochytrium sp. SR21 was also
consistent with the utilization of breadcrumbs (BC) for the production of higher levels of
saturated fatty acids. Fermentation on BC appeared to be more appropriate for the production
of biofuel from these organisms.
In the work described in the third chapter of this thesis, Schizochytrium sp. S31 was used to
examine the effect of the addition of flaxseed oil or -linolenic acid (ALA) on PUFA
production. Addition of ALA (0.04%, v/v) to the fermentation medium resulted in decreased
lipid, DHA and astaxanthin production. However, flaxseed oil (0.04%, v/v) when added to
the medium, increased lipid productivity, DHA levels and astaxanthin accumulation. Nile red
staining of Schizochytrium sp 31 cells was used to visualise oil content in the growing cells.
The addition of flax seed oil at low levels during the fermentation of Schizochytrium sp. S31
is an effective method to enhance total lipid, DHA production and astaxanthin accumulation
in this strain.
In the work described in the fourth chapter of this thesis, new thraustochytrid strains were
isolated from Australian mangrove sites and were screened for their potential to produce
omega-3 fatty acids along with co-production of squalene, astaxanthin and biodiesel. Pine
Chapter 8
105
pollen baiting and direct plating techniques were used for the isolation of thraustochytrids.
Some of the collected isolates DT2, DT3, DT7, DT8 and DT13 sequences were deposited in
the GenBank database and were assigned the accession numbers, DT2 (Genbank accession
number KF682137), DT3 (Genbank accession number KF682125), DT7 (Genbank accession
number KF682129), DT8 (Genbank accession number KF682130), and DT13 (Genbank
accession number KF682135). They were identified. In-house isolate DT8 was identified as
Thraustochytrium sp. strain by 18S rRNA sequencing and was shown to be a promising
candidate for squalene production amongst all isolates. Fermentation with 7% glucose
resulted in biomass of 2.52 g/L and squalene content of 6.7 mg/g (16.92 mg/L). Fermentation
with glycerol (2%) produced high yields of biomass, fatty acid yield and astaxanthin levels.
This strain may be useful for the coproduction of squalene, astaxanthin, PUFA and biodiesel.
In-house isolate Schizochytrium sp. DT9 was studied for its potential to produce biodiesel
using glycerol as carbon source and results from this study are discussed in Chapter 5 of this
thesis. Conventional and direct transesterification methods were investiagted for the
conversion of the accumulated lipid in the dry biomass to biodiesel. Direct transesterification
gave a higher total methyl ester yield than the conventional method. Among various solvents
tested,toluene resulted in maximal extraction yields of crude biodiesel with a 57% yield
obtained from biomass. HCl was tested as a substitute for H2SO4 as an acid catalyst, to
address the problems of sulphur content in the biodiesel. HCl and H2SO4 resulted in similar
yields. In Chapter 6, Australian Victorian mangrove isolate DT13 was studied for its ability
to utilise various carbon sources for the fermentation of omega-3 fatty acids and carotenoids.
Isolate DT13 when grown on glucose (2%, w/v) produced DHA(623 mg/L) after 5 days of
fermentation. Fermentation with glycerol gave 551 mg/L of saturated fatty acids and 379
mg/L of mono unsaturated fatty acids. Olive oil resulted in 540 mg/L of saturated fatty acids
and 3.3 g/L of mono unsaturated fatty acids. These carbon sources may be useful for
biodiesel generation if the higher value co-products DHA can be successfully separated from
the fatty acid mixture. Direct transesterification was an efficient method for the production of
biodiesel, with a yield of 57% of crude biodiesel, compared to 46% from the conventional
method.
In Chapter 7 lipases were screened for the selective hydrolysis of DHA from Schizochytrium
oil. Testing of Thermomyces lanuginosus lipase (TLL), Burkholderia cepacia lipase (BCL)
and Rhizomucar meiehei lipase (RML) for the concentration of DHA showed that TLL and
BCL significantly discriminated against the hydrolysis of omega-3 fatty acids, particularly
Chapter 8
106
DHA. Although, Rhizomucar meiehei lipase (RML) was observed to concentrate PUFAs, its
selectivity to DHA was less than that of the other two lipases.
The market for omega-3 finished products is growing and expected to reach US$34.5 billion
by 2016. Also the prevalence of vegetarians and vegans worldwide is increasing and so the
availability of omega-3 fatty acids derived from algae is highly desirable as a vegetarian
replacement for fish oil. Thraustochytrids are currently used for the commercial production of
omega-3 fatty acids for some purposes such as infant formula. However, these oils are too
expensive for the general food market or nutritional supplement market and so existing
processes need improvement to decrease costs and harvest valuable co-products.
Innovation both at the laboratory and Industry level should be aimed in devising upstream
and downstream strategies for cost-effective polyunsaturated and co-product production. New
marine microorganisms from varied habitats need to be continue to be explored in terms of
the isolation of new thraustochytrid strains that have the capacity to accumulate high
concentration of omega-3 fatty acid naturally. Novel marine sources possessing
environmental stress should be explored for the isolation of new thraustochytrid strains.
Various inexpensive carbon sources such as whey from dairy industry, processed fruit pulp
from food processing industry, oil cake, and molasses can be tested for growing
thraustochytrids. Such cost-effective carbon sources may lead to higher or comparative yield
to glucose and glycerol, thus decreasing upstream processing costs. In addition,
thraustochytrids ability as decomposers could be explored by growing then on a variety of
marine waste products, such as lignocellulosic waste and husk from the grain processing
industry
The development of processes for the conversion of thraustochytrid biomass to biodiesel to
achieve cost efficiencies that rival petroleum-based fuels is an ongoing challenge that
demands in-depth understanding of the recovery of lipids and effective marine biomass
utilisation. Development more efficient solvent extraction and drying processes is also
important. Rupturing of biomass cell walls prior to solvent contact can improve lipid
extraction yields. In addition, process optimisation for co-product production during
thraustochytrid fermentation can improve the overall economics of large-scale
thraustochytrid fermentation.
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