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Electron microscopy of docosahexaenoic acid-rich oil bodies in Schizochytrium sp. and other organisms producing oil bodies may visually reveal triglyceride structure

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Title:
Electron microscopy of docosahexaenoic acid-rich oil bodies in Schizochytrium sp. and other organisms producing oil bodies may visually reveal triglyceride structure
Creator:
Ashford, Amy Lynn
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English
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x, 114 leaves : illustrations ; 28 cm

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Subjects / Keywords:
Electron microscopy ( lcsh )
Docosahexaenoic acid ( lcsh )
Triglycerides ( lcsh )
Thraustochytriales ( lcsh )
Oils and fats ( lcsh )
Docosahexaenoic acid ( fast )
Electron microscopy ( fast )
Oils and fats ( fast )
Thraustochytriales ( fast )
Triglycerides ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 106-114).
General Note:
Department of Integrative Biology
Statement of Responsibility:
by Amy Lynn Ashford.

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University of Colorado Denver
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Auraria Library
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
42611936 ( OCLC )
ocm42611936
Classification:
LD1190.L45 1999m .A74 ( lcc )

Full Text
ELECTRON MICROSCOPY OF DOCOSAHEXAENOIC ACID-RICH
OIL BODIES IN SCH1ZOCHYTRIUM SP.
AND OTHER ORGANISMS PRODUCING
OIL BODIES MAY VISUALLY REVEAL
TRIGLYCERIDE STRUCTURE
by
Amy Lynn Ashford
B.A., University of Colorado at Boulder, 1993
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
Biology
1999


This thesis for the Master of Arts
degree by
Amy Lynn Ashford
has been approved
by
Linda Dixon
Date


Ashford, Amy Lynn (M.A., Biology)
Electron Microscopy of Schizochytrium sp. and Other Closely Related Organisms
Producing DHA-Rich Oil Bodies May Visually Reveal Triglyceride Structure
Thesis directed by Professor Linda Dixon
ABSTRACT
Schizochytrium sp. produces oil bodies containing large amounts of highly
unsaturated fatty acids, particularly the omega-3 fatty acid docosahexaenoic acid
(DHA 22:6n-3). Various methods of preparation for electron microscopy were used
to investigate the formation of DHA-containing oil bodies in Schizochytrium. No
plastid-like organelles were observed in the cells. In many instances, the oil bodies
appeared adjacent to endoplasmic reticulum (ER) membranes. Based on these
structural observations, it appears that the biosynthesis of oil bodies occurs in a
manner similar to that which has been postulated in higher plants. The results suggest
that the oil body lipids accumulate within the ER bilayer, giving rise to the round,
blister-like oil body structures. In addition, electron microscopic analysis of
Schizochytrium sp. employing sample preparation by high-pressure freeze
substitution may reveal both secondary and tertiary structures of the triacylglycerols
within oil bodies. A fine secondary structure consisting of alternating light- and dark-
staining bands was observed inside the oil bodies. The dark and light bands were
in


28.78 + 1.35 A and 21.56 + 1.35 A in width, respectively. The tertiary structure
appears to be a ribbon-like structure coiled and interlaced within the oil body.
Freeze-fracture photomicrographs exhibited fracture planes with terraces which
averaged 52.2 + 6.8 A in height and measurements correspond to the combined width
of two halves of two light bands and one dark band observed in the high-pressure
freeze substitution photomicrographs. The results suggest the triacylglycerols within
Schizochytrium sp. oil bodies may be organized in a triple chain length semi-
crystalline structure. Photomicrographs of other algal or algae-like organisms with
known fatty acid compositions were compared employing the same high-pressure
freeze substitution technique. Although staining patterns were not observed in all
organisms investigated, the tertiary pattern was also observed in Isochrysis galbana.
The 3-dimensional pattern as well as the secondary were also apparent in
Thraustochytrium sp. ATCC 20890. These results strongly suggest that the semi-
crystalline triple chain length conformation and resulting staining pattern may result
from the specific fatty acid composition and ratio of the triacylglycerols occuring in
some thraustochytrids.
This abstract accurately represents the content of the candidates thesis. I recommend
its publication.
Signed
IV


CONTENTS
FIGURES....................................................... ix
CHAPTER
1. INTRODUCTION..................................... 1
Rationale.................................. 2
Hypotheses................................. 3
2. REVIEW OF THE LITERATURE......................... 5
Algal Evolution............................ 5
Schizochytrium sp.......................... 9
Oil Body Formation.............................. 13
Fatty Acid Biosynthesis......................... 13
Electron Microscopy............................. 15
The Omega-3 Fatty Acid DHA...................... 19
How Was The Nutritional Importance Of
Omega-3 Fatty Acids Discovered?........... 19
Fatty Acid Nomenclature................... 20
Omega-6 Fatty Acids....................... 21
Omega-3 Fatty Acids....................... 25
The Omega-3 Fatty Acid DHA................ 26
DHAs Unique Physical Properties
In Membranes.............................. 31
v


3. ELECTRON MICROGRAPHIC ANALYSIS OF
DOCOSAHEXAENOIC ACID-RICH OIL BODY
FORMATION IN THE THRAUSTOCHYTRID,
SCHIZOCHYTRIUM SP..................................... 34
Abstract........................................ 34
Introduction.................................... 35
Materials and Methods........................... 36
Cell Culture.............................. 36
Lipid Analysis............................ 37
Sterol Composition Analysis............... 37
Electron Microscopy....................... 38
Results......................................... 40
Discussion...................................... 42
Acknowledgements................................ 50
References...................................... 50
4. ELECTRON MICROSCOPY MAY REVEAL STRUCTURE OF
DOCOSAHEXAENOIC ACID-RICH OIL WITHIN
SCHIZOCHYTRIUM SP..................................... 54
Abstract........................................ 54
Introduction.................................... 55
Materials and Methods........................... 57
Cell Culture.............................. 57
vi


Lipid Analysis.......................... 57
13C NMR Spectroscopy.................... 58
High Pressure Freeze Substitution....... 59
Freeze Fracture......................... 59
Results and Discussion........................ 60
Acknowledgements.............................. 70
References.................................... 70
5. HIGH PRESSURE FREEZE SUBSTITUTION
ELECTRON MICROSCOPY MAY REVEAL
TRIACYLGLYCEROL STRUCTURE IN
ALGAE-LIKE ORGANISMS................................. 73
Abstract...................................... 73
Introduction.................................. 74
Materials and Methods......................... 75
Cell Culture............................ 75
High Pressure Freeze Substitution....... 77
Lipid Analysis.......................... 77
Results....................................... 78
Discussion.................................... 89
References.................................... 95
6. CONCLUSION........................................... 96
7. REFERENCES.............................................. 105
vii


FIGURES
Figure
2.1 Light Micrograph Of Schizochytrium sp................... 10
2.2 Life Cycle Of Schizochytrium sp......................... 12
2.3 Eicosanoid Formation In The Blood Stream................ 24
2.4 Biosynthesis Pathway Of Polyunsaturated
Fatty Acids............................................. 28
3.1-3.3 Glutaraldehyde Fix Of Schizochytrium sp................. 47
3.4-3.6 High Pressure Freeze Substitution Fix Of
Schizochytrium sp....................................... 48
3.7 Oil Body Formation In Schizochytrium sp................. 48
3.8 Basal Bodies Of Two Flagella............................ 48
3.9 Whole Cell Freeze Fracture of
Schizochytrium sp....................................... 48
3.10 Scale In-Transit To The Plasma Membrane................. 48
3.11 Scales On Outer Surface Of
Schizochytrium sp....................................... 48
3.12 Possible Sterol Crystals Within An Oil
Body of Schizochytrium sp. As Visualized
By Glutaraldehyde Fixation.............................. 49
3.13 Possible Sterol Crystals Within A Freeze Fractured
Oil Body of Schizochytrium sp........................... 49
viii


4.1 13C NMR of DHA and DP An-6 Positions On
Triacylglycerol Molecules.............................. 66
4.2 Tertiary Structure Within Oil Bodies
Of High Pressure Freeze Substituted
Schizochytrium sp. Cells............................... 67
4.3 Secondary Structure Within Oil Bodies
Of High Pressure Freeze Substituted
Schizochytrium sp. Cells............................... 68
4.4 Whole Cell Schizochytrium sp. As
Visualized By Freeze Fracture.......................... 69
4.5 Proposed Triple Chain Length Model For
Oil Within Schizochytrium sp........................... 70
5.1-5.3 Whole Cells And Close-Up Of An Oil Body In
Crypthecodenium cohnii................................. 80
5.4-5.5 Tertiary Structure Within Oil Bodies
Of Isochrysis galbanci................................. 83
5.6-5.7 Thraustochytrium sp. And Oil Bodies With
Light And Dark Bands................................... 85
5.8-5.12 Whole Cell And Oil Bodies Of
Schizochytrium sp...................................... 87
IX


TABLES
Table
2.1 Summary Of International Organizations
With Established Daily Recommended
Values For Omega-3 Fatty Acids............................... 33
3.1 Fatty Acid Composition Of Schziochytrium sp.................. 40
4.1 Fatty Acid Composition Of Schizochytrium sp.................. 61
5.1 Fatty Acid Composition Of Various Algal And
Algae-Like Microorganisms.................................... 88
x


CHAPTER 1
INTRODUCTION
Schizochytrium sp. is a unicellular algae-like organism that is currently used
for the production of docosahexaenoic acid- (DHA)-rich oil. Either the dried algal
product or extracted oil may serve as a source of DHA for use in dietary supplements
or functional foods and food ingredients. DHA is important for infant nutrition and
the maintenance of normal cardiovascular health as well as providing for maintenance
of health in several other areas including inflammation, and neurological disorders2.
Schizochytrium, and thraustochytrids in general, have had a rocky taxonomic
history. Thraustochytrids were first classified as fungi because of their heterotrophic
nature and relative lack of pigmentation. Later they were classified as algae due to
their cellular components and growth characteristics. To this day, scientists disagree
on whether this group of microorganisms are more algae-like or more fungi-like3.
Yet, modem genetic techniques, such as 5s rRNA analysis, strongly suggest that
Schizochytrium is most closely related to the red and brown algae. For the purposes
of this thesis Schizochytrium will be considered to be algae-like3,4.
The phylogenetic question is of importance because oil production in this
algae-like organism is not completely understood. Species of Schizochytrium have
never been reported to possess chloroplasts or plastid-like structures and only a few
1


strains in this species are known to make significant quantities of lipids5. Oil
production, and specifically biosynthesis of shorter chain fatty acids, (some of which
are precursors to DHA) takes place in chloroplasts in most plants and algae6. I
thought that the unique oil producing strains of Schizochytrium, now utilized as a
commercial oil production organism achieving 60-70% dry weight as oil
(unpublished data), might possess chloroplasts or at least remnant chloroplasts.
Rationale
The results of the experiments discussed in this thesis will hopefully aid in the
increased understanding of the phylogeny, ultrastructure and lipid production in the
algae-like microorganism Schizochytrium sp. Clarification of the phylogenetic
relationship of this organism may aid with increased safety of the use of this organism
for the production of the omega-3 fatty acid DHA. Better understanding of the
cellular organelles involved in lipid synthesis in this microorganism might suggest
new ways to continue to improve the economics of lipid production with this
organism. Finally, elucidation of the triacylglycerol structure and resulting staining
pattern may help others to understand how triacylglycerols may be organized in the
semi-crystalline state. The research presented in this thesis examines a core set of
hypotheses identifying and exploring the cellular organelles involved in lipid
production in Schizochytrium sp.
2


Hypotheses
The first question examined was to determine if rare oil producing strains of
thraustochytrids such as Schizochytrium possessed chloroplasts or plastid-like
structures. Any of the following, including chloroplasts, degenerated chloroplasts, or
any organelle containing thylakoid membrane structures would be considered to fall
into the category of plastid-like structures. Secondly, if plastids were present, was oil
body formation is associated with chloroplasts or plastid-like structures in
Schizochytrium sp. Third, if no plastid-like structures or thylakoid-containing
organelles were to be found, I hypothesized that oil body formation would be
associated with endoplasmic reticulum (ER) membranes.
While exploring the first three questions, I observed a unique secondary
fingerprint-like structure of alternating light and dark staining bands within oil bodies.
I therefore conducted additional experiments to try to identify and explain this
secondary fingerprint-like pattern presumably within the triacylglycerols.
Following Chapter 2 (Review Of The Literature), three papers are presented
which summarize the research I conducted on these hypotheses. The first paper
entitled Electron micrographic analysis of docosahexaenoic acid-rich oil body
formation in the thraustochytrid, Schizochytrium sp. presents data on the mechanism
of oil body formation obtained using three different electron microscopy preparation
techniques. The second paper entitled Electron microscopy may reveal structure of
docosahexaenoic acid-rich oil within Schizochytrium sp. describes a tertiary ribbon
3


like pattern inside oil bodies as well as a secondary alternating light and dark
fingerprint pattern within the ribbon-like bands. A model is proposed for the
secondary triacylglycerol structure and the resulting secondary light and dark banding
pattern. The third paper entitled Electron microscopy may reveal triacylglycerol
structure and staining patterns in algae-like organisms examines the secondary and
tertiary triacylglycerol structure within oil bodies of several microalgal species to
determine whether this pattern was real or an artifact of the sample preparation
procedure for high pressure freeze substitution electron microscopy.
4


CHAPTER 2
REVIEW OF THE LITERATURE
Algal Evolution
It has been hypothesized that algae have evolved several times by way of
separate endosymbiotic events. These events are specifically those that include a
photosynthetic prokaryote (cyanobacterium) being phagocytosized by a non-
photosynthetic eukaryote and with symbiotic existence thereafter13. These events
have led to the existence of several types of algal and algae-like organisms14.
According to relatedness trees derived from phylogenetic analysis there are two
distinct evolutionary patterns between heterotrophic and photosynthetic eukaryotes.
The first pattern suggests that the green algae and red algae are not to be closely
affdiated with any heterotrophic taxa of organisms and their radiation began after
successful symbiosis with another photosynthetic organism. The other pattern
includes radiation of eukaryotic algae that contain chlorophyll a + c (the
chromophytes). These organisms are closely related to many heterotrophic taxa and
include organisms such as the alveolates and stramenopiles15 of which
Schizochytrium sp. is a member16,17.
Currently, Schizochytrium sp. is considered to be a member of the golden
algae most closely related to the red and brown algae4. However, the taxonomy of
5


this particular group of organisms is in a constant state of flux18. Schizochytrium was
first considered to be a fungal organism based on its relative lack of pigmentation and
heterotrophic nature18. Following ultrastructure and growth cycle studies, researchers
placed them in the heterokont algae19, and some even placed them in their own
phylum with the labyrinthulids Modem molecular genetic techniques such as 18s
rRNA analysis3 and 5s RNA sequences evaluated by correspondence analysis4 have
provided definitive evidence that thraustochytrids (and Schizochytrium) are not fungi
and should be placed in the Chromophyta (now called stramenopiles) with the
heterokont algae.
The most recent taxonomic placements of Schizochytrium is summarized as
follows21:
Kingdom: Chromophyta
Phylum: Heterokonta
Order: Thraustochytriales
Family: Thraustochytriaceae
Genus: Thruastochytrium
The chromophytes have been placed within a larger taxonomic group called the
stramenopiles based on the distinguishing characteristics of tubular mitochondrial
cristae and tripartite tubular flagellar hairs16,17. Schizochytrium, a thraustochytrid, has
also been placed within the phylum Labyrinthulomycota (labyrinthulids) by
6


Porter20. This taxonomic placement would read as follows17:
Kingdom: Protista
Something: Stramenopiles
Phylum: Labyrinthulomycota
Class: Labyrinthulea
Family: Thraustochytriidae
Genus: Thraustochytrium
Schizochytrium sp., based on being considered a Labyrinthulid, is
hypothesized to have been one of the earliest stramenopiles. There are two general
hypotheses about how stramenopiles evolved. The first hypothesis is that the
common ancestor among stramenopiles may have been a flagellated heterotroph that
acquired its plastid DNA independently from other groups. The alternative
hypothesis is that colorless stramenopiles may have descended from the chromophyte
algae (those that contain chlorophyll a + c) and secondarily lost their plastid DNA17.
Liepe et al. performed an analysis of parsimony, neighbor joining and maximum
likelihood analyses and suggested that the last common ancestor of the stramenopiles
was a heterotrophic flagalleted organism. Additionally, their results supported the
hypothesis that this heterotrophic protist with two flagella and tubular mitochondrial
cristae developed tripartite tubular hairs. This adaptation gave rise to what we today
call the labyrinthulids, which includes the Thraustochytrids. Furthermore, the authors
deduce that autotrophy within the stramenopiles would have arisen independently of
the other major photosynthetic organisms and that independent loss of chloroplasts in
labyrinthulids and other heterotrophic stramenopiles is not the most parsimonious
7


explanation17. From this data, the authors suggested the possibility that
Schizochytrium sp. may be an early stramenopile prior to the acquisition of
chloroplasts17. Other studies have confirmed this conclusion suggesting that the
heterokont algae are monophyletic and represent some of the oldest stramenopiles .
The first focus of the present experiment was to look for chloroplasts or
plastid-like organelles in Schizochytrium sp. using various techniques of cell
preparation for electron microscopy. Chloroplast development requires the synthesis
of chlorophyll, yet chlorophyll production and membrane assembly can take place
either in the presence or absence of light for most algal species. With the availability
of light, chloroplasts can simply enlarge and divide as the cell grows and divides.
Therefore, chloroplasts are maintained in a relatively highly developed state whether
the cell is grown in the dark or light ". In this way, if Schizochytrium sp. does contain
chloroplasts, they should be visible in some form in electron micrographs.
Chloroplasts can be observed in several forms in an electron photomicrograph
because there are several pathways and intermediate steps of chloroplast development
in cells. At the earliest point in development, the chloroplast begins as a proplastid
which is a double or triple membrane bound organelle with just a few, one or two,
lamallae inside of the organelle. If the cell is subjected to intermittent light, the
proplastid will develop into a protochloroplast. This organelle looks similar to the
proplastid except that it will have developed more prominent lamellar membraneous
structures which will soon develop into thylakoids. If at this point light is abundant,
8


full thylakoids and granal stacks develop and the organelle looks like a true
chloroplast '. The other pathway of development that proplastids can take occurs
when they are grown in the dark. The proplastid develops into what is called an
etioplast which contains a prolamellar body' which is a extensive and
organized network of intersecting tubules that resides in etioplast23. As development
of the etioplast progresses and light intensity increases, prothylakoid membranes
begin to protrude from the prolamellar body. The development of prothylakoids
proceeds into thylakoid membranes and granal stacks as the chloroplast matures23,25.
The observation of any of these structures in electron photomicrographs of
Schizochytrium sp. should provide evidence for the existence of chloroplasts in this
organism.
6
Schizochytrium sp. Life Cycle
As observed in a light micrograph, Schizochytrium sp. cells are approximately
pm in diameter and can achieve 60-70% of their dry weight as lipid26 (Fig. 2.1).
9


Figure 2.1. Light micrograph of Schizochytrium sp. This photograph clearly shows
numerous oil bodies within individual cells growing in the tetrad stage of the life
cycle.
10


When Shizochytrium sp. is grown in culture medium representative of nearly full
strength sea water, the organism can be observed as it passes through its entire life
cycle21. Schizochytrium sp. grows by dividing asexually by successive bi-partition20
with resulting formation of tetrad cell cultures. Upon nitrogen deprivation, the cells
tend to produce large amounts of storage oils (personal observation). Over 60% of
the weight of the cells can be oil and DHA can comprise over 35% of the oil .
Under continuing conditions of nutrient limitation, each Schizochytrium sp. cell
divides and forms a sporangium containing many (16-24) zoospores. When nutrients
are abundant, a hole forms within the encasement of the sporangium and motile
zoospores are released. The zoospores are motile for a few hours at most, then they
lose their flagella and become single cells and the cell cycle begins over again
(personal observation) (Fig. 2.2). Most of the stages of the life cycle are presented in
electron photomicrographs of Schizochytrium sp. in later chapters.


Figure 2.2 Life Cycle of Schizochytrium sp.
Zoospore
=c> o
Sporangium
Cell
Division
03
Tetrad -
Oil
Production


Oil Body Formation
The mechanisms of oil body formation have been extensively studied in plants
and oil seeds partially due to the profitable nature of the oleochemicals market27. In
most plants and seeds, storage lipids are in the form of triacylglycerols10. Although
similarities exist in plant cells between the steps from fatty acid biosynthesis up to oil
body formation, there is one fundamental difference. In most plant and algae cells,
fatty acid biosynthesis takes place in chloroplasts10. However, some algal and algal-
like organisms do not have chloroplasts, but still produce oil .
Fatty Acid Biosynthesis
Fatty acid biosynthesis begins with the major carbon source substrate acetyl-
CoA which is a product of the glycolysis pathway. The next step is the conversion of
acetyl-CoA into malonyl-CoA by the enzyme acetyl-CoA carboxylase. This
conversion is considered a rate-limiting step or first committed step of fatty acid
biosynthesis and is intimately involved with regulation of the rate of fatty acid
biosynthesis. Further, malonyl-CoA serves as a substrate for fatty acid elongation in
two carbon units10.
The fatty acid synthetase system is typically located exclusively in plastids as
a dissociable complex of polypeptide units. Each of these units act as either catalysts
or acyl carrier molecules. Fatty acids are elongated from malonyl-CoA and acetyl-
13


CoA by the sequential addition of two-carbon units to a growing acyl chain. The
growing acyl chains are, meanwhile, attached to acyl carrier proteins. Elongation of
fatty acids up to 18 carbons long typically takes place in the chloroplast10. Elongation
beyond 18 carbon fatty acids, which are characteristic of many storage oils, occurs in
extraplastidic regions, possibly in the ER. It is suggested that desaturation and
elongation in the ER is accomplished by enzymes in the Kennedy pathway10.
The earliest studies of the mechanisms of oil body formation were done by
Frey-Wyssling et al.29 and Wanner and Theimer65 who developed the spherosome
theory. Though the use of electron microscopy, Frey-Wyssling et al. proposed that
the origin of shperosomes and oil droplets was associated with ER membranes in com
plant cells. The formation of oil bodies was described as occurring as accumulations
of granular material (possibly lipoproteins) within the lipid bilayer of ER membranes
and subsequent budding off of the granular vesicles into the cytoplasm. The
researchers then report a clearing of the vesicle from the center out. The resulting
droplet was filled with refractory material which was most likely oil" .
The study of oil bodies has progressed along with the improvement of electron
microscopy techniques. More recent descriptions of oil body formation do not differ
significantly from those of Frey-Wyssling et al. and Wanner and Theimer10. Murphy
reports in an extensive review10 two theories that cover observations of oil body
formation in rapeseeds and maize to developing seeds of mustard, safflower, castor
bean and crambe10. The first theory of oil body formation in rapeseeds and maize is
14


that triacylglycerols would accumulate within the phospholipid leaflets of the ER
bilayer. This would force apart the bilayer to produce a swelling which would
eventually pinch off to form an oil body. Surrounding the oil body would then be a
phospholipid monolayer which would carry with it an appendix of the ER
membrane10.
The alternate theory that describes oil body formation in plants such as
mustard and safflower. It is thought that oil bodies arise from the cytoplasm from a
membraneous matrix of lipid and protein. This is the mechanism by which oil bodies
may arise in fungi10.
Because thraustochytrids are not known to be good oil producers and
mechanisms of lipid production have not been widely studied in this group of
organisms28, it is unclear how oil bodies form in Schizochytrium sp. Hopefully the
following experiments will aid in the understanding of the mechanism of oil body
formation in this microorganism.
Electron Microscopy
Successful preparation of cells for electron microscopy involves preservation
of all of the components of the cells in the sample which is as close to their native
state as possible30. Fixation should enable the cell sample to withstand the process of
preparative techniques such as sectioning and being irradiated by the electron beam in
the electron microscope31.
15


Commonly, cells are fixed for electron microscope viewing using chemical
fixitaves such as OSO4, glutaraldehyde or formaldehyde that produce cross-links
between molecules and precipitation of proteins which arrests all cellular processes31.
Glutaraldehyde fixation is widely used in the preparation of plant tissues with
relatively good results of fixation. 0s04 is a chemical that is also frequently used as a
stain because it stains double bonds in molecules. The limitation of chemical
fixatives include the slow rate of fixation relative to the speed of cellular reactions
and possible resulting morphological changes30. Additionally, chemical fixatives can
induce changes in osmotic pressures that can shrink and alter the structures of
organelles that are particularly sensitive to osmotic stability32. Due to these
limitations, different types of electron microscopy sample preparation techniques
have been developed.
A large improvement over chemical fixation can be achieved with freezing of
cell samples. This technique offers the advantage of a much faster rate of fixation
and simultaneous stabilization of all cellular components. However, freezing
techniques such as dipping a sample into very cold liquid such as liquid propane or
liquid nitrogen is restricted to very small cells, smaller than 10 pm. Additionally,
freezing of cell samples adds the hazard of ice crystal formation and damage of
internal organelle structures30. Cryoprotectants can be used to avoid the formation of
ice crystals. Cryoprotectants such as dextran and mannitol interact with water
molecules and greatly reduce the ability of water to provide nucleation sites for
16


31
formation of ice crystals' Various freezing techniques are currently used in
conjunction with cryoprotectants including plunge-freezing, spray freezing, metal
mirror freezing31, double propane jet freezing32, and high-pressure freezing33.
The real advance in freezing techniques for smaller and larger cells and
sample sizes came with the development of high-pressure freezing. Freezing
techniques such as plunge dipping and propane jet freezing provided ice damage-free
freezing in biological material up to 10-40 pm thick. Beyond that, ice crystal damage
could be observed at the microscopic level30. This amount of ice crystal formation
can be overcome with the application of high-pressure to the sample prior to and
during rapid freezing (on the order of -5,000C to -500C/sec)30.
The theory behind high-pressure freezing is related to Le Chatliers
principle:30 When water freezes, its volume increases. High-pressure inhibits the
expansion of water as it undergoes crystallization by modifying the following three
physical parameters of water: 1) lowering of waters freezing point, 2) reduction in
the rate of ice crystal nucleation, and 3) slowing the growth of ice crystals.
Moreover, the reduction in ice crystallization reduces the amount of heat caused by
crystallization and, therefore reduces the amount of heat that has to be extracted from
the sample during rapid freezing .
Furthermore, extremely high freezing rates such as these can arrest water
molecules instantly so that they will not be able to substantially rearrange
themselves31. I have assumed that the same time scale applies to that of lipids and
17


that arrest of movement of triacylglycerol molecules will also be instantaneous and
preserve lipids in their native structure. The utilization of the high-pressure freezing
technique allows for ice crystal damage-free freezing in samples up to 0.6 mm
thick30.
The other part of the technique that is critical to the success of good sample
preparation is associated with the substitution of water with acetone after the high
pressure freeze has taken place. It is important to extract the water out of the cell
samples and replace it with resin so that the sample will be strong enough to handle
the rigors of sectioning. Acetone is a very good dehydration agent as it will replace
the water endogenous to the cells and thus reduce the chance of ice crystal formation.
The only problem associated with dehydration is possible extraction of lipids. Use of
osmium tetroxide (0s04), which also stains double bonds, aids in reducing the rate of
lipid extraction as does lowering the temperature to 4C31. Since Os04 stains double
bonds34, and the DHA contained within the oil of Schizochytrium sp. is highly
unsaturated (having many double bonds), it will stain well and be highly visible in the
electron photomicrographs.
Finally, the third technique used in the present experiment for electron
microscopy sample preparation was freeze fracture which provides 3-dimensional
visualization of cellular structure. This technique can be done after high pressure
freezing, but the way it was done for this experiment was that the sample was plunge-
frozen, then freeze-fractured on a freeze-etch machine. Samples are loaded onto a
18


cold stage and a cold knife (such as a razor blade) is used to fracture the sample.
Then a replica is make of the fracture plane of the frozen specimen. A combination
of metal, usually a platinum-carbon mixture is sprayed onto the sample (Pt-C
shadowing) at an oblique angle (usually around 45) to the average plane of the
fracture plane then a final layer of carbon is applied. Organic material is cleaned off
of the replica samples and they are then viewed by an electron microscope31,35.
The Omega-3 Fatty Acid DHA
How Was The Dietary Importance Of Omega-3
Fatty Acids Discovered?
The link between dietary fat and heart disease has been well established since
the 1950s36. In the late 1970s and early 1980s two Danish researchers, Dyerberg
and Bang, studied epidemiological data and found that Greenland Eskimos had the
lowest rate of heart disease in the world, yet they consumed very high amounts of fat
in their diet37. Upon closer examination of the Eskimo diet, the researchers found that
this population of people ate large amounts of fish, seal and whale meat. These
particular types of fish and marine mammals contain omega-3 fatty acids, specifically
eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3).
These long chain, polyunsaturated fatty acids (PUFA) have since been identified as
the cardio-protective nutrients found in fish38. Since this revolutionary discovery,
19


much research has taken place that suggests that the omega-3 fatty acids EPA and
DHA are important in many other areas of nutrition and health.
Fatty Acid Nomenclature
There are three basic types of fatty acids including saturated,
monounsaturated, and polyunsaturated fatty acids. Saturated fatty acids are those that
contain no double bonds and are, therefore, saturated with hydrogen molecules on
each available carbon molecule. Monounsaturated fatty acids contain one double
bond, and polyunsaturated fatty acids contain one or more double bonds' The
various types of fatty acids are named according to the number of carbon atoms and
double bonds they posses. For example, a fatty acid with 18 carbons and three double
bonds would be accordingly named 18:3. Additionally, the position of the double
bonds within the carbon chain plays a part in the naming of different fatty acids as
well. The carboxyl end of the fatty acid chain is referred to as the alpha end, and the
final carbon in the fatty acid chain is the omega end. If the first double bond is three
carbons in from the omega end, then the fatty acid is referred to as an omega-3 fatty
acid. The same nomenclature holds for fatty acids in which the first double bond
from the omega end is six carbons in. This would be called an omega-6 fatty acid40.
An example of the notation for omega-6 or omega-3 fatty acids would be as follows:
18:2n-6 would indicate that this fatty acid contains 18 carbons, 2 double bonds, and
the first double bond is six carbons in from the omega end of the molecule.
20


Therefore, 18:2n-6 represents an omega-6 fatty acid. Omega-6s and omega-3s are
two biologically important types of polyunsaturated fatty acids.
Omega-6 Fatty Acids
It is inappropriate to talk about omega-3 fatty acids without first mentioning
the omega-6 fatty acids. Omega-6 fatty acids are all derived from linoleic acid (LA,
18:2n-6). Linoleic acid is considered to be an essential fatty acid. This means that it
cannot be synthesized in the body from other fatty acids, and therefore humans and
other mammals need to obtain it in their diet. When deficiency conditions exist for
this essential fatty acid, resulting skin lesions and scaly skin are observed39.
Linoleic acid is the precursor of other fatty acids (Figure 23). The body uses
several enzymes to convert LA to longer and more unsaturated fatty acids which
serve different purposes in human health The first of these longer chain omega-6
fatty acids is y-linolenic acid (GLA, 20:3n-6). GLA is typically found in high
concentrations in evening primrose oil and is converted in the body to the series-1
eicosanoids39. The next omega-6 fatty acid along the biosynthetic pathway that plays
an important role in human health is arachidonic acid (ARA, 20:4n-6). ARA is fairly
abundant in the human diet and can be obtained through foods like red meats.
Additionally, a large portion of LA in our diet is converted to ARA. ARA is
important for infant growth during early life. Additionally, ARA is converted to the
series-2 eicosanoids40. In fact, ARA has 20 carbons, and the prefix eicosa- means 20.
21


Finally, an omega-6 fatty acid that has gained attention in the past few years
of research is docosapentaenoic acid (DPA, 22:5n-6)41. This fatty acid is similar in
structure to DHA, except that it is from the omega-6 family. In cases of DHA
deficiency, as in infants that have not been fed DHA through the mothers placenta or
breast milk, DP An-6 is found to be in the place of DHA in the infants brain
composition42. In other words, DHA-deficiency conditions result in increased
concentrations of DPAn-6.
ARA-Derived Eicosanoid Production ARA can be converted into three
general types of eicosanoids. The first group is the prostaglandins consisting of the
main players PGE2, and PGF2 and PGE (prostacyclin)39. The overall effects of the
prostaglandins PGE2 and PGF2 are to constrict blood vessels and cause contractions
in smooth, involuntary muscles. Prostacyclin, PGE, however, is active in dilating
blood vessels and reducing the tendency of blood to clot. The action of PGE is
relatively weak40'43. The actions of the series-2 prostaglandins are very strong. In
other words, these eicosanoids are potent and have strong effects on various tissues at
very low concentrations .
The second type of eicosanoid formed from ARA is the thromboxanes (TX)
which are strong blood clotting agents43. Upon injury to a tissue, collagen is usually
exposed in the basal layer. Collagen exposure triggers the production of
thromboxanes in the area of the injury44. Thromboxane production (as well as other
eicosanoid production) occurs by clipping fatty acids out of nearby cell membranes
22


by the enzyme phospholipase A2. (Fig. 2.3) When ARA is abundant in cell
membranes, as they are in the human body, large amounts of thromboxanes can be
produced. The other function of thromboxanes is to constrict blood vessels in the
area of injury. Altogether the functions of thromboxanes contribute in an effort to
close a wound. The first step in conversion of ARA to both prostaglandins (including
prostacyclin) and thromboxanes involves a single enzyme called cyclooxygenase 2
(COX2)40.
The third type of eicosanoid formed from ARA are the leukotrienes. These
eicosanoids serve to increase inflammation and immune response when present.
Specifically, the results of high concentrations of ARA-derived leukotrienes are
increased mucus production, local inflammation, bronchial tube constriction, in
general, an increased immune response to allergy, irritation or injury40. The
conversion of ARA to leukotrienes is performed by an enzyme called lipoxygenase' .
23


Figure 2.1 Eicosanoid Formation in the Blood Stream
Inside the blood vessel
Cell membrane
fk
Activation
Reaction
Free Fatty Acid
Fatty Acid
in a
Phospholipid
Blood
Vessel
3-0 Cell
Eicosanoid


Omega-3 Fatty Acids
Omega-3 fatty acids are the family of fatty acids derived from a-linolenic acid
(LNA, 18:3n-3). LNA is considered to be an essential fatty acid as well because it
also cannot be synthesized in the body from other fatty acid precursors Some
researchers feel that LNA plays a role in maintaining skin and cell membrane
integrity39. Although LNA is converted in the body to EPA and DHA, the conversion
is known to be inefficient due to increased enzyme competition for unsaturation with
omega-6 fatty acids45. In a metabolic study done by Emken45, total LNA conversion
to the omega-3 fatty acids EPA, DPAn-3 and DHA combined amounted to 15%. In
terms of conversion to DHA, only 2-3% conversion was reported45. In fact, the
conversion is understood to be so inefficient that the United States Food and Drug
Administration has limited their definition of omega-3 fatty acids to only include
EPA and DHA and exclude LNA46.
EPA, the 20-carbon metabolite of LNA, is converted to the series-3
eicosanoids in the human body39. EPA is typically found in cold water fatty fish
along with DHA which is the most unsaturated fatty acid in nature. DHA can be
found in the body in relatively high concentrations in the brain and retinas47.
EPA-Derived Eicosanoid Production EPA is the precursor to eicosanoids of
the 3-series which are generally less potent than those of the 2-series40. The
formation of eicosanoids from EPA follows the same biosynthetic pathway scheme as
ARA. EPA is converted to prostaglandins (including prostacyclin) and thromboxanes
25


of the 3-series by the cyclooxygenase enzyme. These eicosanoids have similar
actions to the series-2 eicosanoids, yet they exert their effects more weakly39. Such is
the case for EPA-derived leukotrienes. Persistance of these weaker leukotrienes tends
to calm the immune response so that it does not over-react to insult or injury44.
Balance Is Important. If the balance is maintained between the omega-6 and
omega-3 fatty acids in our diet, and therefore the composition of our cell membranes
and therefore the available phospholipid pools, then resulting eicosanoid production
would also be balanced Currently, the concentration of omega-6 fatty acids in the
human diet (particularly in the Western diet) is so high49, that this imbalance of
eicosanoids to the ARA-derived side, may cause predisposition to chronic conditions
such as arthritis50, increased incidence of asthma51, and cardiovascular disease52.
The Omega-3 Fatty Acid DHA
Two theories of how the biosynthesis of DHA occurs have been proposed.
Originally, it was thought that conversion of DP An-3 (22:5n-3) to DHA (22:6n-3)
took place by way of a A4 desaturase inserting a double bond in the 4 position of the
hydrocarbon chain''. It is possible that this biosynthetic pathway exists in
microalgae, however, there is not much evidence for its existence in humans54. The
alternative pathway of DHA biosynthesis that is independent of a A4 desaturase has
been proposed by Sprecher et al53. They propose that DPAn-3 (22:5n-3) is first
elongated to 24:5n-3, then desaturated by the A6 desaturase enzyme to 24:6n-3. This
26


compound is then converted by (3-oxidation to DHA 22:6n-3. Evidence for the
predominance of the Sprecher pathway of DHA biosynthesis in humans is growing
due to experiments with skin fibroblasts from Zellweger syndrome patients. These
patients are unable to convert EPA to DHA because they lack the ability to perform
peroxisomal (3-oxidation, the final step in the conversion of 24:6n-3 to DHA 22:6n-3
in the proposed Sprecher pathway. Consequently, a small build-up of 24:6n-3 is
observed and while DHA could be detected in control fibroblasts, none could be
detected in Zellweger fibroblasts. Conversion of LNA to EPA was 50-60% in
Zellweger fibroblasts, while it was only 30% in control fibroblasts54. This evidence
supports the theory that EPA is converted to DHA independent of a A4 desaturase in
human cells.
27


Figure 2.4. Biosynthetic pathway of polyunsaturated fatty acids.
Omega-6 Family Omega-3 Family
18:1 18:2n-6 18:3n-3
oleic acid linoleic acid a-linolenic acid
X A6 desaturase
18:3n-6
y-linolenic acid
18:4n-3
octadecatetraenoic acid
I
I
20:3n-6
dihomo-y-linolenic acid
20:4n-3
eicosatetraenoic acid
I
I
20:4n-6
arachidonic acid
I
22:4n-6
docosatetraenoic acid

22:5n-6
docosapentaenoic acid
20:5n-3
eicosapentaenoic acid
I
22:5n-3
docosapentaenoic acid
24:5n-3
|A6
* 24:6n-3
AT
22:6n-3
docosahexaenoic acid
28


Utilization of the alternate pathway involving the A6 desaturase enzyme may
explain the depression in DHA conversion from EPA as reported by Grimsgaard et
al55. Ethyl esters of either purified EPA or DHA were administered to healthy non-
smoking men aged 36-56 years in a randomized, double-blind, placebo controlled
trial. Those fed DHA experienced a 69% increase in DHA and a 29% increase in
EPA in serum phospholipids, while those fed EPA showed a 297% increase in serum
phospholipid EPA, wherease DHA decreased by 15%. The authors of the study
suggested that EPA may not be elongated to DHA in humans55. This set of data also
brings up another important point related to long chain PUFA metabolism. Because
the group of young men fed purified DHA showed increases in both serum
phospholipid DHA and EPA, the results indicated retroconversion of DHA to EPA.
Conquer and Holub have reached similar conclusions about human
retroconversion of DHA to EPA and state a 9-12% rate of retroconversion in healthy
subjects56,57. It is possible that DHA can serve as a source of EPA in the body.
Wash-out studies have been preformed in which healthy volunteers consumed ethyl
ester capsules containing EPA + DHA for 18 weeks and allowed to wash-out of the
body for 24 weeks. Analysis of blood plasma samples revealed a four week washout
period for EPA while DHA did not return to baseline levels even at 24 weeks58. In
this way, DHA can serve as a storage reservoir to help compensate for sporadic DHA
and/or EPA consumption.
29


DHA, being a very long-chain and highly unsaturated molecule contributes to
membrane fluidity especially in cold environments like cold Atlantic waters. It is
thought that this is one of the reasons why polyunsaturated fatty acids are so
important for the survival of organisms like algae and fish in cold environments59. In
fact, algae and cold water fatty fish such as herring, salmon and mackerel are the best
food sources of DHA60.
Fish do not synthesize DHA de novo in their bodies61, they obtain it through
their diet. Fish eat smaller marine animals like shrimp that have eaten smaller marine
organisms like rotifers that have fed on marine microflora such as single cell algae
which are the original source of DHA in the food chain62.
In the human body, DHA is found in high concentrations in the brain and
retina of the eye63. Forty percent of the polyunsaturated fatty acids in the human
brain are DHA47. DHA is found primarily in neural cell membranes and myelin
sheathes around axons. Morover, 60% of the total fat in the retina of the eye is
DHA47. A main component of the cell membranes of the cones and rods is the
rhodopsin molecule, each of which is surrounded by six DHA molecules. This highly
fluid membrane environment aids in rapid transmission of the rhodopsin-derived
photo-chemical messages to the brain62.
While in a membrane, DHA has been proposed to naturally fold into an a-
helix which can compress and decompress like a spring in response to differing
temperatures and pressures at the lipid-water interface. Using computer modeling
30


studies, DHA has been estimated to be of a length between 29.03 to 24.85A
depending on whether it resides in the s/z-l or sn-2 position of the phospholipid
molecule63. Additionally, temperature is believed to play a role in the length of the
DHA molecule because temperature affects compression and decompression of the
helix shape of the molecule. In general, the length of the DHA molecule increases as
the temperature decreases64. Because of its helical shape, DHA can be easily
incorporated into both membrane phospholipids and triglycerides such as storage
oils63.
The Unique Physical Properties Of
DHA In Membranes
DHA plays a very important role in every cell of the human body. Because
animals, including humans, typically incorporate DHA into the phospholipids in cell
membranes rather than into storage oil triclycerides, DHA is poised and available for
release into the blood stream, retroconversion to EPA, and subsequent conversion to
series-3 eicosanoids40. Since DHA is in every cell membrane in the body, it
contributes to the maintenance of membrane fluidity59. This is particularly important
in several areas including the synapse region of neuron cell membranes in the brain.
A highly fluid nerve cell membrane environment aids with the rapid secretion,
reuptake or interaction of neurotransmitters such as dopamine with receptors on post-
synaptic connections62.
31


It has been hypothesized that increased dietary DHA and EPA from fish was
instrumental in the evolution of intelligence in humans. Many early Homo sapien
fossils have been found in certain areas of Africa adjacent to ancient seas and mini
oceans. Because fish are found in these seas, they were believed to be a large part of
the early Homo diet. The authors of the study suggest that DHA is a limiting nutrient
for brain growth and that the DHA from the fish the early Homo sapiens were eating
lead to brain growth and our ability of complex thinking today65.
Worldwide, the Western populations, especially the United States, are
reported to have the lowest concentrations of DHA66. Many countries, including
Japan67, Canada68 and several European governments69'75, have established
recommended daily levels of intake of omega-3 fatty acids. These recommendation
levels range from 200 to 2000 mg/day (Table 2.1). The United States Food and Drug
Administration has not yet established a daily recommended level of omega-3 fatty
acids despite extensive scientific evidence supporting various health benefits due to
dietary intake76.
Once an understanding of the important and beneficial role DHA can play in
human nutrition is gained, it is easy to visualize a need for alternative food and
supplement sources. The algae-like microorganism, Schizochytrium sp. can provide a
concentrated and pure, food grade source of DHA for use in the creation of DHA-
enriched foods and food ingredients.
32


Table 2.1. Summary Of International Organizations With Established Daily
Recommended Values For Omega-3 Fatty Acids
Or2anization Daily Recommended Values
British Nutrition Foundation EPA/DHA 0.5% energy (l-2g/day)
COMA Long chain n-3s 0.1-0.2g/day
Health & Welfare Canada 1.0 1.8 g n-3/day
ISSFAL Infants: 35-40 mg/day DHA
Ministry of Health Sweden/Finland Omega-3 0.5% energy (or l-2g/day)
National Nutritional Council Norway Omega-3 0.5% energy (orl-2g/day)
NATO Workshop on n-3/n-6 FAs 0.8g/day EPA/DHA (0.27% calories)
World Health Organization Infants: 40 mg DHA per kg body weight
33


CHAPTER 3
ELECTRON MICROGRAPHIC ANALYSIS OF DOCOSAHEXAENOIC ACID-
RICH OIL BODY FORMATION IN THE THRAUSTOCHYTRID,
SCH1ZOCHYTR1UM SP.
Abstract
Schizochytrium sp., a thraustochytrid included among the heterokont algae,
produces oil bodies containing large amounts of highly unsaturated fatty acids,
particularly the omega-3 fatty acid docosahexaenoic acid (DHA 22:6n-3). Using this
organism, OmegaTech, Inc., and Kelco (a Unit of Monsanto Company) have
developed a fermentation technology that yields DHA-rich dry cells and oil for use as
dietary supplements and function food and feed ingredients. Various methods of
preparation for electron microscopy were used to investigate the formation of DHA-
containing oil bodies in Schizochytrium. No plastid-like organelles were observed in
the cells. In many instances, the oil bodies appeared tightly appressed against regions
of ribosome-deficient endoplasmic reticulum (ER) membranes. Based on these
structural observations, we propose that the biosynthesis of these oil bodies occurs in
a manner similar to that which has been postulated in higher plants. In particular, we
propose that the oil body lipids accumulated within the ER bilayer, giving rise to
round, blister-like oil body structures. The results of the present study add to
34


evidence that thraustochytrids (including Schizochytrium sp. ATCC 20888) are
stramenopiles (chromophytes) and are most closely related to other algal species.
Introduction
Several types of microorganisms have been utilized for the heterotrophic
production of specialty oils. Microbial oil production systems have been developed
using both fungi and algae13. Recently, a member of the thraustochytrids,
Schizochytrium sp. (ATCC 20888), was isolated that produces high levels of oil rich
in the long chain omega-3 fatty acid docosahexaenoic acid (DHA, C22:6n-3)4. DHA
C £ O
is important for human nutrition the development of infants and the maintenance
of normal cardiovascular health in adults9,10. A commercial scale production
technology for DHA-rich algae has now been developed based on propagation of this
unique strain in conventional fermenters11.
Prior to isolation of this strain, thraustochytrids had not been known to
accumulate significant amounts of lipids. The earliest research on the
thraustochytrids classified them as fungi because of their heterotrophic nature and
relative lack of pigment12. Later, other researchers classified them as members of the
heterokont algae or as protists13 or in their own phylum with the labyrinthulids14.
Current analyses using molecular biology techniques have definitively demonstrated
that thraustochytrids are not fungi and should be placed in the Chromophyta with the
heterokont algae15'16. The chromophyte algae themselves have been placed in a larger
35


taxonomic group, the stramenopiles, based on possession of the unique cellular
characteristics of tubular mitochondrial cristae and tripartite tubular flagellar
hairs Confusion still exists, however, as to whether thraustochytrids represent
autotrophic stramenopiles which have lost their chloroplasts or whether they represent
some of the earliest heterotrophic stramenopiles prior to acquisition of chloroplasts.
As oil production in microalgae is generally associated with chloroplasts19, we
felt that a study of oil body formation in a unique oil-producing strain of
thraustochytrid might provide some additional evidence as to the placement of
thraustochytrids in the evolution of stramenopiles. Despite their heterotrophic nature,
light is known to stimulate growth in some thraustochytrids and oil production in
Schizochytrium sp. is increased by approximately 10% in cultures exposed to light
when compared to cultures grown in the dark (Barclay, unpublished data). The
purpose of this study was to utilize electron micrographic techniques to analyze oil
body formation in Schizochytrium sp. and determine whether the origin of oil body
formation occurs in chloroplasts, remnant plastids, or in the endoplasmic reticulum.
Materials and Methods
Cell Culture
Schizochytrium sp. (ATCC 20888) was cultured in 250 ml shake flasks in 50
ml of a medium containing either a low carbon to nitrogen ratio (approximately 10:1)
to produce low oil cells or a high carbon to nitrogen ratio of approximately 30:1 to
stimulate oil production. Monosodium glutamate or ammonium sulfate was utilized
36


as the nitrogen sources. The other culture media components were used as outlined in
the low chloride medium described by Barclay21. The flasks were shaken at 220 rpm
and maintained at 29C. Cultures from these flasks were used to inoculate 1 L
(working volume) fermenters. Cultures were grown in an Omni-Culture fermenter
produced by the Viritis Company, Gardina, N.Y. Fermentation conditions were set to
operate at 29C, sparging at 1 vvm air, mixing at 450 rem, and pH control at 6.5.
Lipid Analysis
Fatty acids in whole cells were methylated in 4% sulfuric acid in methanol
(100C for lh). The fatty acid methyl esters were then separated and quantified on a
Varian 3500 gas liquid chromatograph equipped with a flame ionization detector and
a 30 m x 0.25 mm (ID) RTX 2330 fused silica capillary column (Restek, Bellefonte,
PA). NuCheck-Prep (Ellysian, MN) fatty acids were employed as standards in the
analysis. Fatty acid standards were reported as percent of total fatty acid methyl
esters (FAME).
Sterol Composition Analysis
Algal samples were saponified using KOH and EtOH. Non-saponifiable
fractions were extracted in hexane and analyzed by high-performance liquid
chromatography using a method similar to that described by Rodrfguez-Palmero et
al22.
37


Electron Microscopy
Glutaraldehyde Fixation. Schizochytrium sp culture was grown in a 250 ml
shake flask in a growth medium containing a carbon to nitrogen ratio of
approximately 10:1. The flask was placed on a rotary shaker (220 rpm, 29C) and
exposed to a light intensity of 100pE*m'*sec' These conditions might favor the
induction of chloroplasts or plastid-like structures if they exist in Schizochytrium sp.
After 24 hours of growth, a sample was collected for electron microscopic analysis to
observe the effects of illumination on any potential plastid-like cellular organelles.
The remainder of the culture was used to inoculate a non-illuminated 1 L fermenter.
The fermentation medium was prepared with a carbon to nitrogen ratio of
approximately 30:1 to favor oil formation in the cells. Samples were subsequently
taken at 24 and 48 h into the fermentation (48 h and 72 h samples, respectively). All
samples were washed in a 2.0 g/L Na2S04 solution and immediately fixed in 2%
glutaraldehyde solution containing 2.0 g/L NaiSCL. The fixed samples were washed
three times in 10 mM sodium phosphate buffer (pH 7.0) and suspended in 2%
osmium tetroxide (Os04) for approximately 1 h. Samples were washed thoroughly
with deionized water and stored overnight at 4C. Samples were then run through a
dehydration series of 20, 40, 60, 80, and 100% acetone at 10 minute intervals then left
in 100% acetone for approximately 1.5 h. Subsequently, the samples were embedded
38


in Spurrs resin, sectioned and viewed with a Philips CM 10 electron microscope
(Philips Model CM 10).
High-Pressure Freeze Substitution. Schizochytrium sp culture was grown in a
250 ml shake flask in a growth medium containing a carbon to nitrogen ratio of
approximately 10:1. The flask was placed on a rotary shaker (220 rpm, 29C) and
exposed to a light intensity of 100pE*m'2sec-1. After 24 hours of growth, a sample
was collected for electron microscopic analysis. The remainder of the culture was
used to inoculate a non-illuminated 1 L fermenter. The fermentation medium was
prepared with a carbon to nitrogen ratio of approximately 30:1 to favor oil formation
in the cells. Samples were subsequently taken at 24 and 39 h into the fermentation
(48 h and 63 h samples, respectively). Samples were prepared using vacuum
filtration and washed briefly in 15% w/v dextran (avg MW 36 kD), a non-permeating
cryoprotectant, in growth medium. The samples were then frozen in a BAL-TEC
HPM-010 high-pressure freezer (Technotrade International, Manchester, NH) and
stored under liquid nitrogen. The samples were freeze-substituted in 2% Os04 in
acetone at -80C following the procedures outlined by Staehelin et al23.
Freeze-fracture. Samples from shake flask cultures were collected at 48 h
when oil bodies are their biggest and glutaraldehyde was added to the sample over 15
min to a final concentration of 1%. The cells were then washed and resuspended in a
solution of 5 g/L Na2S04. Glycerol, the cryoprotectant, was added over a period of
20 min to a final concentration of 30% (v/v). Samples were frozen in liquid propane
39


and freeze-fractured at -113C in a BAL-TEC BAF-060 freeze-etch system
(Technotrade International, Manchester, NH). Replicas were cleaned on bleach
followed by a 7.8% w/v potassium dichromate, 33% v/v sulfuric acid solution, then
picked up on specimen grids for viewing by transmission electron microscopy.
Results
The fatty acid profile of Schizochytrium sp. consists primarily of seven fatty
acids (Table 1). DHA (C22:6n-3) and docosapentaenoic acid (DPA, C22:5n-6) are
the predominant polyunsaturated fatty acids comprising approximately 30% and 10%
total fatty acids, respectively. Myristic (04:0), palmitic (06:0) and palmitoleic
(06:1) are the predominant saturated and monounsaturated fatty acids, respectively.
Together, these five fatty acids comprise over 80% of the total fatty acids in
Schizochytrium sp. Lipid fractions from Schizochytrium sp. biomass contain
approximately 10% sterols.
Table 3.1
Fattv Acid % Total Fattv Acids
14:0 17.6
14.1 0.7
16:0 26.4
16:1 13.9
18:1 3.6
20:4n-6 0.1
20:5n-3 0.4
24:1 0.2
22:5n-6 8.9
22:5n-3 0.2
22:6n-3 27.6
40


Schizochytrium sp. cells sampled from illuminated shake flask cultures
contained no plastid-like structures (Figs. 1,4). The rapidly dividing cells did contain
a normal compliment of eukaryotic organelles including: mitochondria, nuclei,
ribosomes, peroxisomes, ER and Golgi bodies (Figs. 1,4).
Schizochytrium sp. cells sampled at three points during a fermentation cycle
(glutaraldehyde-fixed and OsCVstained) exhibited oil bodies as large homogeneous,
electron-dense structures which increased in size throughout the fermentation. No
chloroplasts or plastid-like structures were observed within the cells (Figs. 1-3).
Cells of Schizochytrium sp. preserved by freeze substitution also displayed oil
bodies which increased in size throughout the fermentation (Figs. 4-6). Plastid-like
structures were not observed in these cells either, but clues to the formation of
triacylglycerol-containing oil bodies were revealed. Though not associated with
plastid-like structures, many of the oil bodies in Schizochytrium sp. appeared to be
tightly appressed against regions of ribosome-deficient ER membranes (Fig. 7).
Additionally, structures resembling peroxisomes were often observed in close
physical association with oil bodies, especially during early formation of oil bodies
(Fig. 4).
Sagenogenetosomes, the cellular organelle associated with formation of the
ectoplasmic net (a network mesh of extracellular material connecting groups of
individual cells), were not observed in our micrographs.
41


All of the cells observed in these electron micrographs exhibited features
which are characteristic of thraustochytrids and their stramenopile (chromophyte)
ancestry. Numerous mitochondria are observed containing tubular cristae (e.g., Figs.
1,4). The biflagellate nature of a developing Schizochytrium zoospore is observed in
Figure 8. Figure 10 revealed a scale encapsulated in a vesicular membrane in transit
to the surface of the cell as visualized by freeze substitution electron microscopy.
The freeze-fracture technique also confirms the presence of scales forming an outer
covering of individual Schizochytrium sp. cells (Fig. 11). An example of a freeze-
fractured cell is displayed in Figure 9. Freeze-fractured Schizochytrium sp. cells
contain oil bodies which exhibit layered fracture plane terraces (Fig. 9).
Within the oil bodies of 72 h glutaraldehyde-fixed cells were non-staining
angular regions (Fig. 12). These angular, crystal-shaped structures were also apparent
in oil bodies of freeze-fractured cells (Fig. 13).
Discussion
Previous electron micrographic studies of thraustochytrids have indicated a
lack of plastid-like structures in these microorganisms, but these studies only
investigated strains of thraustochytrids which do not produce large lipid inclusions24.
Until recently, species of thraustochytrids that can accumulate commercially
significant amounts of lipids and oils were unknown. The present study focused on a
recently isolated, high lipid producing strain of Schizochytrium that is being utilized
42


to commercially produce DHA by fermentation4. Since lipid production is known to
be a chloroplast-mediated process in microalgae and plants19, and since
Schizochytrium sp. accumulates very high amounts of lipid in laboratory fermenters
(60-70% of dry weight), this strain appeared to be an ideal organism in which to
assess the status of chloroplasts in thraustochytrids.
No chloroplasts or plastid-like structures were detected in Schizochytrium sp.
in illuminated or non-illuminated cultures during the present study. If such structures
exist at all in this species, they are apparently few in number and play a minor role in
the organisms physiology. These observations suggest that the moderate
enhancement of lipid production that we, Weete ", and Goldstein have observed in
illuminated fermenters cannot be occurring by way of plastid-mediated biochemical
processes. Enhancement of lipid production with light stimulation has been noted in
other strains of non-chloroplast-containing lipid-producing microbes. This process
appears to occur in strains which also produce carotenoids' so there may be some
link between carotenoid and lipid production. Correspondingly, Schizochytrium sp.
contains small amounts of two carotenoids, canthaxanthin and (3-carotene.
Although the location of fatty acid biogenesis in Schizochytrium sp. is still
unclear, the oil bodies themselves seemed to form along tightly appressed regions of
ribosome-deficient ER in a manner similar to that postulated to occur in higher plants
yn -jo
and oil producing fungi During exponential phase growth conditions, sections of
ER can be seen appressed against almost every oil body in Figure 4. This
43


phenomenon can also be observed in stationary phase cells at 48 and 63 h, although
not as frequently. These observations suggest that oil bodies in Schizochytrium sp.
appeared to form following the endoplasmic reticulum half-unit membrane
hypothesis29'31. This hypothesis proposes that the triacylglycerols, rather than being
expelled from the lipid producing membranes, or collecting as oil droplets coalescing
in the cytoplasm, form an accumulation between phospholipid layers inside the ER
membrane. This accumulation grows into an oil body that is surrounded by a half-
unit phospholipid membrane derived from the ER. We propose that this process
occurs at several or many independent locations at a time (Fig. 4). Oil body
formation is clearly pictured in Figure 7.
Peroxisomes were present in Schizochytrium sp. and were most easily
observed in exponential growth phase cells (Fig. 4). In mammals, peroxisomes are
the site of P-oxidation. An important step in the synthesis of DHA via the recently
described Sprecher pathway is p-oxidation from C24:6n-3 to C22:6n-3 DHA32. Prior
to elucidation of this pathway, it was thought that DHA is converted directly from
docosapentaenoic acid (C22:5n-3) by utilizing a A4 -desaturase32. The Sprecher
pathway has recently been demonstrated to occur in other microalgae (Jon Sargent,
personal communication) and involves elongation of C22:5n-3 to C24:6n-3,
desaturation to C24:6n-3, and finally p-oxidation (in the peroxisomes) to form
C22:6n-3 DHA. Research is currently underway to elucidate the pathway of DHA
biosynthesis in Schizochytrium sp.
44


Features such as flagella, oil bodies, and scales which are normally associated
with Schizochytrium sp. were apparent in electron micrographs. Interestingly,
sagenogenetosomes, an ectoplasmic net-producing organelle, were not observed in
Schizochytrium sp. cells during the present study. Most thraustochytrids produce an
ectoplasmic net or rhizoid which interconnects clumps of cells by a mesh of
extracellular material33. Schizochytrium sp. is unique in that ectoplasmic net
production is relatively weak when grown in production fermentation medium
(Barclay, unpublished data). This may explain the inability to detect
sagenogenetosomes in our study.
During log phase growth, as are the inocula of fermentation runs, cultures pass
through a phase where individual cells may undergo sporulation34. Sporangia
develop containing a large number of immature cells. Shortly thereafter, a hole forms
in the outer encasement of the sporangia. Motile, biflagalette zoospores swim out
through the hole24,3536. Figure 8 clearly shows the basal bodies of the two flagella
most likely originating from a developing zoospore.
Scales have been proposed to reside in the outer covering of thraustochytrids
such as Schizochytrium. The scale structure observed in high-pressure freeze
substituted and freeze-fractured cells of Schizochytrium sp. (Figs. 10,11) is similar to
scales shown on the surface of Schizochytrium aggregatum37.
Glutaraldehyde-fixed 72 h cells exhibited oil bodies which contained non-
staining angular regions (Fig. 12). These angular, crystal shaped structures were also
45


apparent in oil bodies of freeze-fractured cells (Fig. 13). The shape of these regions
is reminiscent of the shape of sterol crystals. This same feature has been described in
glutaraldehyde-fixed oil bodies of Thraustochytrium sp. by Weete et al.25 Although
we observed no evidence of internalization of these structures during oil body
enlargement as postulated by Weete et al., we can confirm the presence of these
similar structures in Schizochytrium sp. These photographs, together with the known
sterol composition of Schizochytrium sp., suggest that these angular structures within
oil body inclusions may be sterol crystals.
In conclusion, this study evidences that oil body formation in Schizochytrium
sp. originates from extensions of ribosome-deficient ER membranes in a manner
similar to that which occurs in higher plants and fungi and that this organism does not
contain structures which resemble chloroplasts
46


Figures 3.1-3.3 Glutaraldehyde Fix Of Schizochytrium sp. Cultures of Schizochytrium sp. grown for 24 h, 48 h and 72 h
Figures 3.4-3.6 High Pressure Freeze Substitution Fix Of Schizochytrium sp. Cultures of Schizochytrium sp. grown for
24 h, 48 h and 65 h.


Figure 3.7 Oil Body Formation In Schizochytrium sp. 3.8 Basal Bodies Of Two
Flagella. 3.9 Whole Cell Freeze Fracture of Schizochytrium sp. 3.10 Scale In-Transit
To The Plasma Membrane. 3.11 Scales On Outer Surface Of Schizochytrium sp.
48


Figure 3.12 Possible Sterol Crystals Within An Oil Body Of Schizochytrium sp As Visualized By
Glutaraldehyde Fixation.
Figure 3.13 Possible Sterol Crystals Within A Freeze Fractured Oil Body of Schizochytrium sp.


Acknowledgements
I would like to thank Dr. L. Andrew Staehelin for his assistance in
interpretation of photographs and preparation of the manuscript.
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15. Cavalier-Smith, T., Allsopp, M.T.E.P., and Chao, E.E. (1994) Thraustochytrids
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16. Manella, C.A. Frank, J., and Delihas, N. (1987) Interrelatedness of 5s RNA
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18. Leipe, D.D., Wainright, P.O., Gundrerson, J.H. Porter, D., Patterson, D.J., Valois,
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19. Kentrick, A.J., and Ratledge, C. (1992) Microbial polyunsaturated fatty acids of
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20. Goldstein, S. (1963) Studies of a new species of thraustochytrium that displays
light stimulated growth. Mycologia. 55:799-811.
21. Barclay, W.R. (1994) Process for growing Thraustochytrium and Schizochytrium
using non-chloride salts to produce a microfloral biomass having omega-3
highly unsaturated fatty acids. U.S. Patent No. 5,340,742.
22. Rodriquez-Palmero, M., de la Presa-Owens, S., Castellote-Bargallo, A.I., Lopez
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Determination of sterol content in different food samples by capillary gas
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23. Staehelin, L.A., Giddings, T.H., Kiss, J.Z., and Sack, F.D. (1990)
Macromolecular differentiation of Golgi stacks in root tips of Arabidopsis and
Nicotiana seedlings as visualized in high-pressure frozen and freeze-
substituted samples. Protoplasma. 157:75-91.
24. Perkins, F.O. (1974) Phylogenetic considerations of the problematic
thraustochytriaceous labrinthulid Dermocystidium complex based on
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5:45-63.
25. Weete, J.D., Kim, H., Gandhi, S.R., Wang, W., and Dute, R. (1997) Lipids and
ultrastructure of Thraustochytrium sp. ATCC 26185. Lipids. 32:839-845.
26. Cerda-Olmedo, E., and Lipson, E.D. (1987) A biography of Phycomyces. In
Cerda-Olmedo, E., and Lipson, E.D. [Ed.] Phycomyces. Cold Spring Harbor
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27. Huang, A.H.C. (1992) Oil bodies and oleosins in seeds. Annu. Rev. Plant.
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28. Murphy, D.J. (1993) Structure, function and biogenesis of storage lipid bodies
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29. Schwartzenbach, A.M. (1971) Observations on spherosomal membranes.
Cytobiologie. 4:145-147.
30. Wanner, G., and Theimer, R.R. (1978) Membraneous appendicies of
spherosomal (oleosomes). Possible role in fat utilization in germinating
oilseeds. Planta. 140:163-169.
31. Wanner, G., Formanek, H., and Theimer, R.R. (1981) The ontogeny of lipid
bodies (spherosomes) in plant cells. Planta. 151:109-123.
32. Moore, S.A., Hurt, E., Yoder, E., Sprecher, H., and Spector, A.A. (1995)
Docosahexaenoic acid synthesis in human skin fibroblasts involves
peroxisomal retroconversion of tetracosahexaenoic acid. J. Lipid Res.
36:2433-2443.
33. Moss, S.T. (1980) Ultrastructure of the endomembrane sagenogenetosome -
ectoplasmic net complex in Ulkenia visurgensis (Thraustochytriales).
Botanica Marina. 23:73-94.
34. Kazama, F. (1980) The zoospore of Schizochytrium aggregatum. Can. J. Bot.
58:2434-2446.
35. Gaertner, A. (1981) A new marine Phycomycete, Schizochytrium minutum sp.
nov. (Thraustochytriacae) from saline habitats. Verojf. Inst. Meeresgorsch.
Bremerh. 19:61-69.
36. Raghu-Kumar, S. (1988) Schizochytrium octosporum sp. nov. and other
thraustochytrids from the North Sea (Rosfjord, Norway). Trans. Br. Mycol.
Soc. 90(2):273-278.
37. Darley, W.M., Porter, D., and Fuller, M.S. (1973) Cell wall composition and
synthesis via Golgi-directed scale formation in the marine eucaryote,
Schizochytrium aggregatum, with a note on Thraustochytrium sp. Arch.
Mikrohiol. 90:89-106.
53


CHAPTER 4
ELECTRON MICROSCOPY MAY REVEAL
STRUCTURE OF DOCOSAHEXAENOIC ACID-RICH
OIL WITHIN SCHIZOCHYTRIUM SP.
Abstract
Schizochytrium sp. is an algae-like microorganism utilized for the commercial
production of docosahexaenoic acid (DHA)-rich oil and biomass for use as food and
feed ingredients such as dietary supplements and DHA for infant formulae. Electron
microscopic analysis of whole cell preparations of Schizochytrium sp. employing
sample preparation by high-pressure freeze substitution may reveal both secondary
and tertiary semi-crystalline/crystalline structures of the triacylglycerols within the
microbial oil bodies. Following freeze substitution using osmium tetroxide, a fine
secondary structure consisting of alternating light- and dark-staining bands was
observed inside the oil bodies. The dark bands were 28.78 + 1.35 A in width and
light bands were 21.56 + 1.35 A in width. The tertiary (three-dimensional) structure
appears to be a multi-layered ribbon-like structure which appears to be coiled and
interlaced within the oil body. Freeze-fracture photomicrographs exhibited fracture
planes with terraces which averaged 52.2 + 6.8 A in height and correspond to the
combines width of two halves of two light bands and one dark band observed in the
54


high-pressure freeze substitution photomicrographs. The results suggest the
triacylglycerols within Schizochytrium sp. oil bodies may be organized in a triple
chain length semi-crystalline structure. These results are also compared to oil bodies
in electron micrographs of other comparable oil-producing species of algae or algae-
like protists with known fatty acid compositions employing the same high-pressure
freeze substitution technique.
Introduction
Triacylglycerols in various types of biological systems have been proposed to
occur in semi-crystalline states1. Confirmation of these semi-crystalline
triacylglycerol structures has only been observed indirectly by use of freeze-fracture
electron microscopy. From this analysis, triacylglycerol structure is seen as a
terrace-like structure in the freeze fracture planes. Schizochytrium sp. is an algae-like
microorganism that is commercially utilized to produce docosahexaenoic acid (DHA)
by fermentation3. The triacylglycerols can comprise up to 70% of the biomass of
Schizochytrium sp., and 20-40% of the fatty acids in the triacylglycerols are DHA4.
Recently, a study was conducted investigating the formation of oil bodies in
Schizochytrium sp. in an attempt to identify the location of oil production in this
algae-like organism. During this study high-pressure freeze substitution was utilized,
a relatively new type of cell preparation that theoretically preserves very fine
structures within the cells. High-pressure prevents the formation of ice crystals in the
55


cells during freezing. The cells are then treated with an acetone/osmium tetroxide
(OSO4) solution which removes intracellular water by successive dehydration and
stains any double bonds present within molecules inside the cells5. Analysis of
microbial oil bodies has historically been conducted by glutaraldehyde fixation with
OSO4 staining wherein the DHA-containing oil bodies appear as homogeneous dark-
staining inclusions6. When the electron micrographs produced using the high-
pressure freeze substitution technique were observed, a fine structure was revealed
within the DHA-rich oil bodies of Schizochytrium sp. This fine secondary structure
seemed to be the result of an alternating light and dark staining pattern and may be
related to the particular fatty acid composition of each triacylglycerol molecule. In
addition, the photomicrographs revealed a tertiary structure that appeared to be a
three- dimensional ribbon-like band of light and dark staining triacylglycerols
interlaced throughout the oil body.
This paper illustrates the structures observed within the oil bodies and
proposes a model that may describe the triacylglycerol structure of this unique DHA-
rich oil in Schizochytrium sp. Other comparable oil-producing algal or algae-like
strains with known fatty acid profiles were investigated. Some of these algal species
contained long-chain omega-3 polyunsaturated fatty acids. I employed the same
high-pressure freeze substitution preparation process to investigate triacylglycerol
structure within oil bodies.
56


Materials and Methods
Cell Culture
Schizochytrium sp. (ATCC 20888) was cultured in 250 ml shake flasks in a
Na2S04-based medium as described by Barclay4. Isochrysis galbana (UTEX LB
987), Nannochloropsis occulatci (UTEX LB 2164), and Neochloris oleoabundans
(UTEX 1185) strains were obtained from UTEX and grown at 25C with light.
Nannochloropsis occulata and Isochrysis galbana were grown with light (approx.
100pE/m2/sec) in f/2 medium7 containing 60% strength sea water, KNO3, KH2PO4,
trace metals, vitamins and supplemented with soil extract for three to four weeks.
Q
Neochloris oleoabundans was grown in Bold Basal Medium containing DI water,
NaNO.3, CuCl^fLO, MgSCL, H3BO3, Bolds Basal EDTA mix, and soil extract under
light (approx. lOOpE/m /sec) for three to four weeks. Crypthecodenium cohnii
ATCC 30543 was obtained from the American Type Culture Collection (ATCC) and
grown in ATCC Culture Medium 460 (A2E6 Medium) in the dark at 25C for
approximately 72 h. Cells were harvested for sample preparation by gentle vacuum
filtration.
Lipid Analysis
Fatty acids in whole cell samples were transesterified using 4% sulfuric acid
in methanol (100C for 1 h). The fatty acid methyl esters were then separated and
quantified on a Varian 3500 gas liquid chromatograph equipped with a flame
57


ionization detector and a 30 m x 0.25 mm (ID) Rtx 2330 fused silica capillary column
(Restek, Bellefonte, PA). NuCheck-Prep (Ellysian, MN) fatty acid standards were
employed in the analysis. Fatty acid standards were reported as % of total fatty acid
methyl esters (FAME).
13C NMR Spectroscopy
Quantitative 13C spectra were obtained on the purified triacylglycerol fraction
from Schizochytrium sp. microalgae (approximately 50-100 mg dissolved in 0.6 ml
CDCI3 in 5 mm tubes) at a frequency of 75 MHz with the NOE-suppressed, inverse-
gated, proton-decoupled technique. The free induction decay (FID) was acquired
with a pulse delay of 40 s as described by Aursand et al9. The chemical shifts were
referenced indirectly to tetramethylsilane (TMS) by using the central peak of CDCI3
(5=77.08 ppm). In this analysis, assignments of the downfield resonance at 173.2,
172.8 ppm, based on literature precedent9 were attributed to carboxyl carbons of all
fatty acids except DHA/DPA (fatty acids that contain a A4 unsaturation) located at
positions sh-1,3 (a-position) and sn-2 (P-position) on the glycerol backbone,
respectively. Assignments of the downfield resonance at 172.5 and 172.1 ppm were
attributed to carboxyl carbons of DHA/DPA (fatty acids that contain a A4
unsaturation) located at positions sn-1,3 (a) and sn-2 (P) on the glycerol backbone,
respectively. Integration of carboxyl groups was used to estimate the amount of DPA
and DHA esterified to the sn-2 and .sh-1,3 positions (quantitative analysis).
58


High-Pressure Freeze Substitution
Cell samples were collected at 0, 24, and 63 h into the fermentation cycle and
washed briefly in 15% w/v dextran (avg. MW 39 kD), a non-permeating
cryoprotectant, in growth medium. The samples were then frozen in a BAL-TEC
HPM-010 high-pressure freezer (Technotrade International, Manchester, NH) and
stored under liquid nitrogen. The samples were freeze-substituted in 2% 0s04 in
acetone at -80C following the procedures outlined by Staehelin et al.]0.
Freeze-Fracture
Samples were collected at 48 h and glutaraldehyde was added to the sample
over 15 min to a final concentration of 1%. The cells were then washed and
resuspended in a solution of 5 g/L NaiSCL. Glycerol, which was used as a
cryoprotectant, was added over a period of 20 min to a final concentration of 30%.
Samples were frozen in propane and freeze-fractured at -113C in a BAL-TEC BAF-
060 freeze-etch system (Technotrade International, Manchester, NH). Replicas were
cleaned on bleach followed by a 7.8% w/v potassium dichromate, 33% v/v sulfuric
acid solution, then picked up on specimen grids for viewing by transmission electron
microscopy. Crude lipid widths and terrace heights were measured on resulting
photomicrographs using a Bausch & Lomb dissecting microscope with a calibrated
eyepiece.
59


Results and Discussion
The fatty acid profile of Schizochytrium sp. is relatively simple consisting of
seven fatty acids (Table 1). DHA (C22:6n-3) and DP An-6 (docosapentaenoic acid,
C22:5n-6) are the primary polyunsaturated fatty acids and myristic (C 14:0), palmitic
(C16:0) and palmitoleic (C16:1) acids are the major saturated and monounsaturated
fatty acids. Together, these five fatty acids comprise over 80% of the total fatty acids
in the triacylglycerols of Schizochytrium sp. The results of C NMR analysis
indicated that DHA and DP An-6 are preferentially (71-75%) esterified in the sn-2
position of the glycerol with the remainder (25-29%) linked in the sn-1,3 position
(Fig. 1). Given this and the total fatty acid composition of the oil, essentially all of
the sn-2 positions would be occupied by DHA or DPA. This stereospecific
distribution of DHA and DPAn-6 fatty acids is similar to that reported for other
highly unsaturated fatty acid oils of marine, algal, and microbial origin11.
60


Table 4.1
Fatty Acid_____________% Total Fatty Acids
14:0 17.6
14.1 0.7
16:0 26.4
16:1 13.9
18:1 3.6
20:4n-6 0.1
20:5n-3 0.4
24:1 0.2
22:5n-6 8.9
22:5n-3 0.2
22:6n-3 27.6
When the Schizochytrium sp. cells were prepared by the high-pressure freeze
substitution technique, patterns within the oil bodies were suggestive of both
secondary and tertiary structures (Figs. 2,3). The fine pattern of alternating light and
dark staining bands may be indicative of differential staining of the various saturated
and unsaturated fatty acids that comprise the triacylglycerol molecules. OsC>4 reacts
with the double bonds of unsaturated fatty acids making them appear dark in the
electron micrographs. The dark and light bands measured 28.78 + 1.35 A and 21.56
+ 1.35 A (n=10), respectively. Larsson2 provides a diagram drawn to scale of
trilaurin as viewed along the shortest axis of the triacylglycerol unit. The length from
the alpha carbon to the end of the 12-carbon fatty acid chain is depicted as 11.7 A.
Correspondingly, Gawrisch et al.13 report that the length of 16:0 should fall within the
range of 14-15 A. Applying this assumption, the width of the dark bands corresponds
very closely to the reported length of DHA in the crystalline state, 24.85 A12. The
61


width of the light bands should be approximately twice the length of crystalline
myristate (Cl4:0) and/or palmitate (C 16:0) as can be calculated from the data of
Larsson and Gawrisch et al. '
The fine secondary structures within the oil bodies cannot be thylakoid
membranes because the laminar structures are much thinner (56 60.2 A) than the
o
thickness of other known bilayers in the electron photomicrographs (83.33 A per
thylakoid bilayer) which comprise the thylakoid membranes14. Also, the abundant
triacylglycerol content versus the modest phospholipid content of these cells could
not account for the amount of lipid observed inside the oil bodies4. The oil bodies
comprise approximately 41 % of the cell volume at 48 h as determined by electron
micrographs. Additionally, the freeze-fracture technique revealed that there are no
proteins present on the surfaces of these laminar structures as would normally be
found on thylakoid membranes5 (Fig. 4).
Larsson has previously proposed that crystalline triacylglycerol structures can
occur in one of two alternative chain layer configurations based on segregation of
similar fatty acid tails2. The banding pattern observed in Schizochytrium sp. oil
bodies may best be described using Larssons triple chain length model suggesting
that the triacylglycerols in the oil bodies are organized in a semi-crystalline state by
segregation of the different fatty acid types into separate layers.
I propose that the unsaturated fatty acid chains containing DHA, and possibly
DPAn-6, may be segregated into one interlocking dark staining layer and the
62


saturated fatty acid chains may be segregated end to end to form light staining layers.
This type of arrangement could be facilitated by the predominance of the long chain
highly unsaturated fatty acids DHA and DPAn-6 in the sn-2 position of the
triacylglycerol. Thus, following Larssons triple chain length model for mixed
triacylglycerols2, the suggested secondary light and dark banding structure of the
triacylglycerols in Schizochytrium sp. may be illustrated by the model presented in
Fig. 5. Analysis of the freeze-fracture photomicrographs appears to confirm this
layered structure in oil bodies as evidenced by terraced fracture planes (Fig. 4).
Terrace heights averaged 51.2 + 6.8 A which, in the proposed model, could
correspond to the distance from one fracture plane between two saturated lipid layers,
including one interlocked dark staining layer, to the next fracture plane between the
next two saturated lipid layers (Fig. 5).
Larssons model has been previously confirmed indirectly by freeze-fracture
techniques2. The use of the high-pressure freeze substitution technique in the present
study may provide for the first time direct visual confirmation of the organized state
of triacylglycerols due to the unique composition (and resulting staining pattern) of
the particular triacylglycerols which accumulate in the oil bodies of Schizochytrium
sp. Cell sample preparation procedures could possibly affect the conformation of the
triacylglycerols within the oil bodies. However, the high-pressure freeze substitution
technique rapidly freezes the cells within approximately 10 msec by exposure to
liquid nitrogen under pressure5. Segregation of the fatty acid chains would most
63


likely not occur in such a short amount of time and, thus, the triple chain length
structure may be the native conformation of the triacylglycerols in the cells prior to
freezing.
The tertiary (three-dimensional) structure that may be revealed in the high-
pressure freeze substitution photomicrographs appears to be a ribbon-like multi-layer
structure of triacylglycerols coiled within the oil body. These ribbon-like bands of
triacylglycerols average approximately 500 A in width. Their relatively constant
width is surprising and a model has not yet been developed to explain this structure.
In order to address the possibility that the observed secondary and tertiary
structures in Schizochytrium sp. were not artifacts of preparation, oil body structure
was analyzed in other microorganisms known to contain related fatty acid profiles.
The high-pressure freeze substitution technique was used to prepare cell samples of
the algae Neochloris oleoabundans, Nannochloropsis occulata, Crypthecodenium
cohnii, and Isochrysis galbana. Electron micrographs from this experiment were
compared with micrographs of Schizochytrium sp. Except Neochloris oleoabundans,
all of these microorganisms are known to contain significant amounts of either the
long chain omega-3 fatty acids eicosapentaenoic acid (C20:5n-3) or DHA1619.
Electron microscope photographs did not reveal the secondary fingerprint-like pattern
in any of the algal strains. The micrographs did, however, reveal a similar tertiary
ribbon-like banding pattern of oil body lipids in Isochrysis galbana (Fig. 6).
64


Similar studies with other algal strains including protists producing oils
enriched with highly unsaturated fatty acids in the sn-2 position are needed. Further
study should provide additional support for the proposed secondary fingerprint-like
structure model and provide insight to aid in the development of a model explaining
the tertiary ribbon-like banding of the lipids in the oil bodies.
65


(N
cn
r--
T1 M 1 I 1 i 11 M 1 1 1 1 i 1 i 1 1 i 1 f i i | I t~*~ 1 i 1 1 1 | 1 i M 1 i i | i i m | i i i i | i i i i i i ti i | i i i i | i rri | i i i i | i i i rr t i i i | i i i i |
200 180 160 140 120 100 80 60 40 20 PPM 0
13C NMR of DHA and DP An-6 Positions On Triacylglycerol Molecules



4.2
Figure 4.2 Tertiary Structure Within Oil Bodies Of High Pressure Freeze
Substituted Schizochytrium sp. Cells
67


i
4.3
Figure 4.3 Secondary Structure Within Oil Bodies Of High Pressure Freeze
Substituted Schizochytrium sp. Cells
68


4.4
Figure 4.4 Whole Cell Schizochytrium sp. As Visualized By Freeze Fracture
69


Fracture plane
i
£ 0< !
w> 00 j
5 .n SO 1
o C3 oc j
Uh
D (N !
H
Dark band 0
28.78 + 1.35 A
Fracture plane
Light band 0
21.56+ 1.35 A
Fracture plane
Fracture plane
4.5 Proposed Triple Chain Length Model For Oil Within Schizochytrium sp.
70


Acknowledgements
I would like to thank Dr. Ruben Abril, Patricia Abril, Dr. L. Andrew
Staehelin, Dr. Klaus Gawrish, and David Underwood for their assistance in the
preparation of this manuscript.
References
1. Peck, M.D. (1994) Interaction of lipids with immune function I: Biochemical
effects of dietary lipids on plasma membranes. Journal of Nutritional
Biochemistry. 5:466-478.
2. Larsson, K. (1994) Lipids Molecular Organization, Physical Functions and
Technical Applications. The Oily Press, Dundee, p. 7-81.
3. Barclay, W.R., Meager, K.M., and Abril, J.R. (1994) Heterotrophic production of
long chain omega-3 fatty acids utilizing algae and algae-like microorganisms.
Journal of Applied Phycology. 6:123-129.
4. Barclay, W.R. (1992) Process for the heterotrophic production of microbial
products with high concentrations of omega-3 highly unsaturated fatty acids.
U.S. Patent No. 5,130,242.
5. Staehelin, L.A., Giddings, T.H., and Moore, P.J. (1988) Structural organization
and dynamics of the secretory pathway of plant cells. Curr. Top. Plant
Biochetn. Physiol. 7:45-61.
6. Weete, J.D., Kim, H., Gandhi, S.R., Wang, Y., and Dute, R. (1997) Lipids and
ultrastructure of Thraustochytrium sp. ATCC 26185. Lipids. 32:839-845.
7. Solar Energy Research Institute (1984) Microalgae Culture Collection 1984-1985.
Prepared by the Microalgal Technology Research Group, f/2 Medium
according to F. Haxo, Scripps Institute of Oceanography.
71


8. Bischoff, H.W., and Bold H.C. (1963) Phycological studies. IV. Some algae from
Enchanted Rock and related algae species. The University of Texas. Publ.
6318. pp. 95.
9. Aursand, M., Rainuzzo, J.R., and Grasdalen, H. (1993) Quantitative high-
13 1
resolution C and H nuclear magnetic resonance of co-3 fatty acids from
white muscle of atlantic salmon (Salmo salar), JAOCS. 70:971-981.
10. Staehelin, L.A., Giddings, T.H., Kiss, J.Z., and Sack, F.D. (1990)
Macromolecular differentiation of Golgi stacks in root tips of Arabidopsis and
Nicotiana seedlings as visualized in high-pressure frozen and freeze-
substituted samples. Protoplasma. 157:75-91.
11. Myher, J., Kuksis, A., Geher, K., Park, P., and Diersen-Schade, D. (1996)
Stereospecific analysis of triacylglycerols rich in long-chain polyunsaturated
acids. Lipids. 31:207-215.
12. Applegate, K.R., and Glomset, J.A. (1991) Effect of acyl chain unsaturation on
the conformation of model diglycerols: a computer modeling study. J. Lipid
Res. 32:1635-1644.
13. Holte, L.L., Peter, S.A., Sinnwell, T.M., and Gawrisch, K. (1995) 2H nuclear
magnetic resonance order parameter profiles suggest a change of molecular
shape for phosphatidylcholines containing a polyunsaturated acyl chain.
Biophysical Jorunal. 68:2396-2403.
14. Lichtle, C., Arsalane, W., Duval, J., and Passaquet, C. (1995) Characterization of
the light harvesting complex of Giraudyopsis stellifer (Chrysophyceae) and
effects of light stress. J. Phyc. 31: 380-387.
15. Alberts, B., Bray, D., Lewis, J., Raff, M., Roberts, K., and Watson, J. (1989)
Molecular Biology of the Cell, 2nd Edition. Garland Publishing, Inc. New
York & London, p. 432.
16. Arredondo-Vega, B.O., Band-Schmidt, C.J., and Vasquez-Duhalt, R. (1995)
Biochemical composition of Neochloris oleoabundans adapted to marine
medium. Cytobios. 83:201-205.
17. Sukenik, A., Zmora, O., and Carmeli, Y. (1993) Biochemical quality of marine
unicellular algae with special emphasis on lipid composition. II.
Nannochloropsis sp. Aquaculture. 117:313-326.
72


18. Henderson, R.J., and MacKinlay, E.E. (1991) Polyunsaturated fatty acid
metabolism in the marine dinoflagellate Crypthecodenium cohnii.
Phytochemistry. 30(6): 1781-1787.
19. Sukenik, A., and Wahnon, R. (1991) Biochemical qualith of marine unicelluar
algae with special emphasis on lipid composition. I. Isochrysis galbana.
Aquaculture. 97:61-72.
73


CHAPTER 5
HIGH PRESSURE FREEZE SUBSTITUTION ELECTRON
MICROSCOPY MAY REVEAL TRIACYLGLYCEROL
STRUCTURE IN ALGAE-LIKE ORGANISMS
Abstract
Previous experiments have suggested that triacylglycerols within oil bodies of
Schizochytrium sp. may be organized in a triple chain length structure. Because of
the unique fatty acid composition of the oil produced by this organism, a tertiary 13-
dimensional) ribbon-like pattern as well as a light and dark secondary staining pattern
is revealed in electron micrographs. In order to address the question of whether the
staining patterns were due to the unique saturated and highly unsaturated fatty acid
composition of the oil or if the pattern was an artifact of preparation, similar electron
microscope studies were carried out investigating oil bodies of other strains of algal
and algae-like microorganisms utilizing high pressure freeze substitution. Although
staining patterns were not observed in all organisms investigated, the tertiary pattern
was seen in lsochrysis galbana. The tertiary ribbon-like pattern as well as the
secondary light and dark staining pattern were apparent in both Thraustochytrium sp.
ATCC 20890 and again in Schizochytrium sp. These results strongly suggest that the
staining pattern is not an artifact of preparation and the triple chain length
74


conformation may be facilitated by a 2/3 to 1/3 or 1/3 to 2/3 saturated plus
monounsaturated fatty acid to highly unsaturated fatty acid ratio.
Introduction
Previous experimentation has suggested that oil bodies in Schizochytrium sp.
may contain triacylglycerols that are structured in a semi-crystalline or liquid-
crystalline state. The fatty acid composition of the storage oil in Schizochytrium sp. is
unique in that over 80% of the triacylglycerols can be accounted for by only five
different fatty acid types. Approximately two-thirds of the triacylglycerol fraction
consists of the saturated fatty acids 14:0 (myristic acid), 16:0 (palmitic acid) and the
monounsaturated fatty acid 16:1 (palmitoleic acid). Since osmium tetroxide (0s04)
stains double bonds, these particular fatty acids would be expected to either stain very
lightly or not at all. The remaining one-third of the oil is composed of the
polyunsaturated fatty acid docosahexaenoic acid (DHA, C22:6n-3) and a small
amount of docosapentaenoic acid (DPAn-6, C22:5n-6). These fatty acids would most
likely stain dark with Os04 because they each contain several double bonds.
An alternating light and dark secondary fingerprint-like pattern was seen
inside oil bodies in electron phytomicrographs of Schizochytrium sp. Additionally, a
larger tertiary 3-dimensional ribbon-like structure could be seen interlaced within oil
bodies. This structure had a relatively constant width of approximately 500 A. The
slight possibility still remains that the fine secondary pattern is an artifact of electron
75


microscopy preparation using the high pressure freeze substitution technique. The
purpose of this experiment is to address this question by using the high-pressure
freeze substitution technique for electron microscopy preparation on several other
algal and algae-like microorganisms. Several organisms were chosen because they
were known to produce amounts of highly unsaturated fatty acids, especially
eicosapentaenoic acid (EPA, C20:5n-3) and/or DHA. Included among chosen
microorganisms to study were Crypthecodenium cohnii, Isochrysis galbana,
Thraustochytrium sp. ATCC 20890, Schizochytrium sp. derivative SD104, and a
repeat of the experiment with Schizochytrium sp. ATCC 20888.
Materials and Methods
Cell Culture
Schizochytrium sp. ATCC 20888. Schizochytrium sp. ATCC 20888 was
grown in 250 ml shake flasks in 50 ml of a high chloride medium containing a low
carbon to nitrogen ratio of approximately 10:1 (molar concentrations) to produce low
oil cells. Monosodium glutamate was used as the nitrogen source. The flasks were
shaken at 220 rpm and maintained at 29C and exposed to light at an intensity of
approximately 100 pE/m2/sec. After 24 hours a small volume of this flask was used
to inoculate a separate flask containing growth medium with a high carbon to
nitrogen ratio of approximately 30:1 to stimulate oil production. Ammonium sulfate
was used as the nitrogen source. The other culture medium components were used as
76


outlined in the low chloride medium described by Barclay1. This culture was grown
for 48 hours. Both cultures were sampled (at 24 and 48 hours, respectively) for
electron microscopy preparation.
Schizochytrium sp. SD104 and Thraustochytrium sp. ATCC 20890. These
algae-like microorganisms were grown separately in 250 ml shake flasks on a rotary
shaker at 220 rpm and maintained at 29C. The full strength sea water culture
medium contained a high carbon to nitrogen ratio using monosodium glutamate and
yeast extract as the nitrogen sources as well as low concentrations of trace metals and
vitamins. Cultures were sampled at 48 h.
Crypthecodenium cohnii ATCC 30334 sibling species G. Crypthecodenium
cohnii was obtained from ATCC and grown in 150 ml flasks containing 50 ml of
culture medium. Cultures were grown in Porphyridium medium from UTEX The
Culture Collection of Algae at the University of Texas at Austin catalog, which
appeared as a supplemental issue to the Journal of Phycology2. This medium
contained 50% strength sea water, 10% soil extract, 0.1 g/L yeast extract, 0.1 g/L
tryptone, 3 g/L glucose and supplemented with 0.001 mg/L biotin and 0.1 mg/L
thiamine. The cultures were grown in the dark. After seven days, 100 ml of fresh
Porphyridium medium containing one-tenth of the normal concentration of yeast
extract and tryptone was added to the 50 ml of culture to nitrogen-stress the culture
for oil production. The Crypthecodenium cohnii culture was grown in this manner for
six days and was then sampled for electron microscopy preparation.
77


Isochyrsis galbana. Cultures were grown in 150 ml flasks containing 50 ml of
f/2 culture medium The medium contained full strength sea salts, 0.3 g/L NaN03,
0.02 g/L KH2PO4, f/2 vitamin mix, trace metals and 5 ml of soil extract. After
growing for 22 days, 100 ml of fresh f/2 medium lacking NaNCL was added to the 50
ml of culture to nitrogen-stress the culture for oil production. The culture was grown
for 6 days and was subsequently sampled for electron microscopy preparation.
High-pressure Freeze Substitution
Crypthecodenium cohnii cell samples were washed briefly in 20% w/v dextran
(avg. MW 39 kD), a non-permeating cryoprotectant, in 5 g/L Na2S04. The other
algal and algae-like microorganism cell samples were washed briefly in 0.15M
mannitol which was used as the cryoprotectant. The samples were then frozen in a
BAL-TEC HPM-010 high-pressure freezer (Technotrade International, Manchester,
NH) and stored under liquid nitrogen. The samples were freeze-substituted in 2%
OsC>4 in acetone at -80C following the procedures outlined by Staehelin et at.
Lipid Analysis
Fatty acids in whole cells were transesterified using 4% sulfuric acid in
methanol (100C for 1 h). The fatty acid methyl esters were then separated and
quantified on a Varian 3500 gas liquid chromatograph equipped with a flame
ionization detector and a 30 m x 0.25 mm (ID) Rtx 2330 fused silica capillary column
78


(Restek, Bellefonte, PA). NuCheck-Prep (Ellysian, MN) fatty acid standards were
employed in the analysis. Fatty acid standards were reported as % of total fatty acid
methyl esters (FAME). Lipid widths were measured on resulting photomicrograph
contact sheets using a Bausch & Lomb dissecting microscope with a calibrated
eyepiece.
Results
Electron photomicrographs of each of the algal and algae-like microorganisms
showed fairly successful freezing of all samples. Crypthecodenium cohnii cells
exhibited a wide range of organelles. The most interesting organelle was the nucleus
or nuclear region of the cell displayed several dark-staining inclusions reminiscent of
polytene chromosomes. This organism also displayed oil bodies that were
homogeneous and stained light gray. Neither the interlaced, ribbon-like tertiary
structure or the light and dark fingerprint-like pattern were observable in the oil
bodies. The fatty acid profile of Crypthecodenium cohnii consisted of (% total fatty
acids) 23.4% lauric acid (Cl2:0), 42.1% myristic acid (C14:0), 14.1% palmitic acid
(Cl6:0), 0.7% stearic acid (Cl8:0), 4.8% oleic acid (Cl8:1), 0.3% (C20:l), and
13.2% DHA (C22:6n-3) Figs. (5.1-5.3).
79


80


I
Figure 5.1-5.3 Whole Cells And Close-Up Of An Oil Body From
Crypthecodenium cohnii.
81


Schizochytrium sp. SD104 did not section well even after three experimental
tries. It has relatively thin cell walls compared to Schizochytrium sp. ATCC 20888
which may contribute to difficulty in preparation for electron microscopy preparation.
Oil bodies in Schizochytrium sp. SD104 did not stain, therefore, no patterns were
distinguishable. It is possible that the oil bodies did not stain with 0s04 because they
may contain primarily pigments such as p-carotene and canthaxanthin. The fatty acid
profile of Schizochytrium sp. SD104 is similar to that of Schizochytrium sp. ATCC
20888.
Electron photomicrographs of Isochrysis galbana exhibited oil bodies that
contained the tertiary, 3-dimensional interlaced pattern but the secondary fingerprint
pattern was not visible. The tertiary ribbon-like bands measured an average of
o
187.5A (n=10). The fatty acid profile of Isochrysis galbana is as follows: (% total
fatty acids) 15.1% myristic acid (Cl4:0), 12.4% palmitic acid (C16:0), 4.0%
palmitoleic acid (Cl6:1), 20.6% oleic acid (Cl8:1), 4.0% linoleic acid (C18:2n-6),
20.8% (Cl8:4), and 17.6% DHA (C22:6n-3). Interestingly, one of the pictures shows
a chloroplast with fully developed thylakoid membranes adjacent to an oil body
displaying the tertiary interlaced pattern (Figs. 5.4,5.5).
82


Figure 5.4-5.5 Tertiary Structure Within Oil Bodies Of Isochrysis galbana
83


Thraustochytrium sp. ATCC 20890 which is closely related to Schizochytrium
sp. ATCC 20888 displayed both the tertiary, 3-dimensional interlaced pattern and the
secondary light and dark fingerprint-like pattern (Figs. 5.6,5.7). The fatty acid profile
of Thraustochytrium sp. ATCC 20890 is as follows: (% total fatty acids) 21.4%
palmitic acid (16:0), 1.4% arachidonic acid (C20:4n-6), 18.9% EPA (C20:5n-3), 5.0%
DPAn-3 (C22:5n-3), 43.5% DHA (C22:6n-3), and 9.9% other fatty acids (Table 5.1).
84


Figure 5.6-5.7 Thraustochytrium sp. And Oil Bodies With Light & Dark Bands
85


Schizochytrium sp. ATCC 20888 results were repeated. Both the interlaced
tertiary pattern as well as the secondary light and dark fingerprint-like pattern were
visible in most or all oil bodies (Figs. 5.8-5.10). As stated in the previous chapter, the
fatty acid profile consists primarily of five fatty acids including myristic (C14:0),
palmitic (C16:0), palmitoleic (C16:l), docosapentaenoic (DPAn-6, C22:5n-6) and
docosahexaenoic (DHA, C22:6n-3) acids.
86


I
Whole Cell And Oil Bodies From Schizochytrium sp.
87
Figure 5.8-5.12


88


Table 5.1 Fatty Acid Composition Of Various Algal and Algae-Like Microorganisms
Fatty Acid Crypthecodenium cohnii Schizochytrium sp. SD104 Isochrysis galbana Thraustochytrium so. ATCC 20890 Schizochytrium so. ATCC 20888
12:0
14:0 41.2 1.5 15.1 17.6
14.1 0.7
16:0 26.8 33.3 12.4 21.4 26.4
16:1 0.7 4.0 13.9
18:0 2.1 9.3
OO vo 18:1 4.1 31.4 20.6 3.6
18:2n-6 4.0
18:4 20.8
20:1 2.0
20:4n-6 0.7 1.4 0.1
24.1
20:5n-3 2.3 18.9 0.4
22:5n-3 0.4 1.4 5.0 0.2
22:5n-6 0.7 8.9
22:6n-3 2.3 11.2 17.6 43.5 43.5


Discussion
A previous electron microscope study of Schizochytrium sp. ATCC 20888
utilizing the high-pressure freeze substitution electron microscopy technique revealed
an interesting staining pattern within oil bodies of the cells. The oil bodies exhibited
a tertiary, 3-dimensional ribbon-like banding pattern which seemed to be interlaced
and coiled within the oil body. Observable within the ribbon-like bands was a
secondary alternating light and dark fingerprint-like staining pattern. It has been
hypothesized that the secondary light and dark pattern corresponds to the 0s04
staining pattern of double bonds in similar individual fatty acid chains of the
triacylglycerol molecules that have segregated into various layers. Specifically, the
highly unsaturated fatty acid chains, DHA and DPAn-6, from the sn-2 position of the
triacylglycerol which may be segregated into one interlocking layer, tend to stain
darkly. The remaining saturated fatty acid chains from the sn-1,3 positions, primarily
myristic (04:0), palmitic (06:0), and palmitoleic (06:1) acids, are segregated into
end to end layers and either do not stain at all or only stain lightly. Together, these
layers may form what may be best described as Larssons triple-chain length structure
model of mixed triacylglycerols5.
It is possible that this secondary light and dark staining pattern is due to the
unique fatty acid composition of the oil produced by Schizochytrium sp. This unique
oil can be generalized as containing two-thirds saturated plus monounsaturated fatty
90